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,3 LIBRARY
Michigan State
Ul liversity

 

 

 

This is to certify that the
dissertation entitled

INFLAMMATORY SIGNALING IN MACROPHAGES: REGULATION
BY G-PROTEIN COUPLED RECEPTOR KlNASE-2 AND 5

presented by
SONIKA PATIAL

has been accepted towards fulfillment
of the requirements for the

Ph.D. degree in Cell and Molecular Biology

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M 'or Professor’s Signature

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Date

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INFLAMMATORY SIGNALING IN MACROPHAGES: REGULATION BY G-
PROTEIN COUPLED RECEPTOR KlNASE-2 AND 5

By

Sonika Patial

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Cell and Molecular Biology

2010

ABSTRACT

INFLAMMATORY SIGNALING IN MACROPHAGES: REGULATION BY G-
PROTEIN COUPLED RECEPTOR KINASE-Z AND 5

By

Sonika Patial

G-protein coupled receptor kinases (GRKs) are serine-threonine protein kinases
which phosphorylate agonist bound G-protein coupled receptors (GPCRs) leading to their
desensitization. Although originally discovered with regard to GPCR desensitization,
recent studies have shown that GRKs play much wider roles than previously appreciated.
In this regard, studies have shown that GRKs can phosphorylate non GPCR receptor as
well as non receptor substrates. There is an ample amount of evidence in the literature
showing that the expression and activity of GRKs is altered in several inflammatory
disease conditions. For instance, the activity and expression levels of GRK2 were found
to be significantly decreased in peripheral blood mononuclear cells (PBMC) of patients
with rheumatoid arthritis whereas the expression levels of GRK2 and GRKS were found
to be enhanced in the neutrophils of sepsis patients. Moreover in our previous studies we
found that stimulation of primary peritoneal macrophages with LPS under in vitro
conditions causes an increase in the expression of GRK2. These studies point to a crucial
role of GRKs in inflammatory signaling, however, the physiological importance of
changes in the expression levels of GRKs is not well established. To enhance our
understanding of the role of GRK2 and 5 in inflammatory signaling, we first investigated
the mechanism by which GRK2 and 5 regulate TNFa-induced inflammatory signaling in

mouse macrophage cell line.

 

Our results in this study demonstrated that both GRK2 and 5 positively regulate
TNFa-induced NFKB signaling. Knockdown of GRK2 and 5 inhibited TNFa-induced
NF KB signaling whereas overexpression of GRK2 and 5 substantially enhanced TNFa-
induced NFKB activity. GRK2 and 5 were found to interact with and phosphorylate IKBa,
an inhibitor of NF KB and this was found to be the biochemical mechanism of regulation
of NFKB signaling pathway by GRK2 and 5.

To further elucidate the role of GRKs in inflammation under in vivo conditions,
we utilized mice with homozygous GRKS gene deletion. Primary peritoneal macrophages
from GRK5"' mice stimulated with LPS showed an inhibition of NFKB activity as
compared to cells from GRKSH+ mice. Secretion of several LPS-induced inflammatory
cytokines was found to be reduced in cell culture supernatants of GRK5"' mice. Plasma
levels of cytokines and chemokines were also found to be reduced which was associated

+/+

with reduced liver injury in GRK5'/' mice compared to GRKS mice suggesting that
GRKS positively regulates LPS-induced inflammatory signaling in vivo by modulating
transcription factor NFKB. Since homozygous gene deletion of GRK2 is lethal in mice,
we utilized Cre-loxP system to achieve a cell specific deletion of GRK2 whereby GRK2
was specifically deleted in the cells of myeloid lineage. LPS injection into these mice
caused an increased expression of cytokines / chemokines in the plasma as well as in the
primary peritoneal macrophage cell culture supematants. GRK2 deficient mice also
exhibited an increased lung and liver injury. Mechanistically, IKKB-NFKBI p105 f1“ PL2-
MEK-ERK pathway was found to be negatively regulated by GRK2 which resulted in an
increased expression of cytokines / chemokines in GRK2 deficient mice. Taken together,

these results show that both GRK2 and 5 play a crucial role in LPS-induced inflammatory

signaling under in vivo conditions by regulating NFKB and ERK signaling pathways.

ACKNOWLEDGMENTS

I would like to express my profound gratitude to my advisor Dr. Nara
Parameswaran who gave me the opportunity to be his student. I want to thank him for
his generous time, patience, sincere guidance, continuous encouragement and support.
His precious scientific advice and great attitude towards science compelled me to
adopt those elite qualities in my deve10ping scientific career which I will take with me
through my entire career. I feel extremely fortunate to have Dr. Parameswaran as my
major advisor for whom no words of praise are sufficient.

I also deeply acknowledge my guidance committee members; Dr. Karl Olson
Dr. Kathleen A Gallo, Dr. Andrea Amalfitano and Dr. William Spielman for their
great help, insightful comments, constructive discussions and continual support. I am
indebted to them for their valuable suggestions and continued inputs to improve my
research progress as well as my scientific career. I am also highly thankful to Dr.
Patricia Senagore for her help in pathology studies.

I am thankful to the Cell and Molecular Biology Program, Dr. Susan Conrad
(Director, Cell and Molecular Biology Program) and Dr. Barbara Sears (Director,
Genetics Program) for accepting me into their programs and giving me this great
opportunity to enhance my scientific career. I sincerely thank Becky Manse], Jeannine
Lee and Christine VanDeuren for all their help and making every administrative work
look so simple starting from the day I submitted my application to the graduate school.

I am deeply obliged to Shipra, Katie and Sita for their help in my research

work. I would like to thank all the lab members from Dr. Amalfitano’s laboratory,

especially Daniel Appledom, Sergey Seregin and Sarah Godbehere for their endless
help during my research work.

My sincere thanks are extended to all current and former colleagues in Dr.
Parameswaran’s laboratory, especially Katie Porter, Shipra Shahi, Megan Hull, Wen
Qin, Nandita, Taehyung, Sita Ram and Babu Gonipeta for all their kind help,
cooperation, and friendly atmosphere that they provided me. I also want to thank all
my friends and colleagues in Cell and Molecular Biology Program, Physiology
department and Genetics Program. I am greatlyappreciative of my friends here at
MSU specially Stancy, Madalina, Neli, Navneet, Hey-jin, Michelle, Vishal, Dorothy,
Krista, Scott, Nitin, Chris, Fei, Amal, Miha, Sunetra, Aparajita, Ram and Anita for
their kind help and support during all these years.

I greatly appreciate Josselyn, Barb, Carolyn, Richard and Kim for their support
and help throughout my stay in the physiology department and making me feel at
home.

I greatly appreciate ULAR staff and Human Histopathology Division specially
Amy and Kathy for their help in my research work.

I am thankful with all my heart to my parents for their encouragement, love
and support which motivated me to accomplish my educational goals. Finally, my
special thanks go to my dear husband, Yogesh who has been a source of motivation
during all those tough moments. It is a very special time of my life as we were blessed

with our baby boy, Krish on November IS, 2009.

TABLE OF CONTENTS

LIST OF TABLES ............................................................................... ix
LIST OF FIGURES .............................................................................. x
KEY TO ABBREVIATIONS ................................................................. xiii

CHAPTER 1: ...................................................................................... 1

Introduction ............................................................................. 1

CHAPTER 2: ..................................................................................... 3

Literature Review ..................................................................... 3

2.1: G protein coupled receptor kinases (GRKs) ................................ 3

2.1.1: Historical perspectives of GRK discovery .................................. 3

2.1.2: GRK mediated desensitization of GPCRs ................................... 4

2.1.3: Structure and distribution of GRKs .......................................... 5

2.1.4: Regulation of GRKs by other proteins ..................................... 12

2.1.4.1 Regulation of GRKS by Caveolin, Clathrin and a-actinin ............ 12

2.1.4.2 Regulation of GRKs by Calcium binding proteins ..................... 13

2.1.4.3 Regulation of GRKs through phosphorylation by other kinases.....14

2.1.4.4 Regulation of GRKs by GM subunits and phospholipids ............ 16

2.1.4.5 Regulation of GRKs by interaction with agonist activated GPCRs.17
2.1.5: Other novel interactions of GRKs and their physiological effects......18

2.2 Physiological and Pathological roles of GRKs ............................. 22
2.2.1: Physiological roles of GRKs as determined by knockout mice ......... 23
2.2.2: Pathological roles of GRKs ................................................... 25
2.3: GRKs and inflammation ....................................................... 29
2.3.1: GRKs expression and inflammation ......................................... 29
2.3.2: GRKs deletion and inflammation ............................................ 30
2.3.3: GRKs regulation of chemokine signaling and inflammation ............ 31
2.4: LPS and TNFa induced inflammation ...................................... 31
2.4.1 LPS induced inflammation ................................................... 31
2.4.1.1: Toll like receptor 4 (TLR4) signaling .................................... 34
2.4.2 TNFa-a potent pro-inflammatory cytokine .................................. 36

vi

2.5: N F-KB transcription factor in inflammation ............................... 41

2.6: MAPIQ in inflammation ....................................................... 48
2.6.1: ERK1/2 kinase ................................................................. 48
2.6.2: P38 MAPK ..................................................................... 49
2.6.3: JNK kinase ..................................................................... 51
2.7: Septic shock ...................................................................... 51
2.7.1 Animal models of sepsis ...................................................... 52
2.7.2 Different animal species as models for sepsis ............................. 56
2.7.3 Neutrophils and Monocyte / macrophages in sepsis ...................... 56
2.7.4 Coagulation defect in sepsis .................................................. 57
2.7.5 Multiple organ failure ........................................................ 58
Hypothesis and Specific Aims ....................................................... 60
References .............................................................................. 62
CHAPTER 3:
G—protein coupled receptor kinases mediate TNFa—induced NFKB signaling via
direct interaction with and phosphorylation of IKBa .................................... 80
Abstract ................................................................................ 81
Introduction ............................................................................ 82
Material and Methods ................................................................ 85
Results .................................................................................. 91
Discussion ............................................................................ 128
References ............................................................................. 1 3 3
CHAPTER 4:

G-protein coupled receptor kinase 5 (GRKS) mediates Toll-like receptor-4
-induced NFKB pathway in macrophages and is necessary for the production of

inflammatory cytokines and chemokines in viva ........................................ 139
Abstract ............................................................................... 140
Introduction ........................................................................... 141
Material and Methods ............................................................... 144
Results ................................................................................. 147
Discussion ............................................................................ 1 77
References ............................................................................. 1 82

CHAPTERS:

vii

Myeloid specific G-protein coupled receptor kinase 2 (GRK2) is a negative
regulator of NFchl p105-ERK pathway and limits endotoxemic shock in mice..186

Abstract ................................................................................ 187
Introduction ............................................................................ 1 88
Material and Methods ............................................................... 191
Results ................................................................................. 195
Discussion ............................................................................ 240
References ............................................................................. 248

CHAPTER 6:

Summary and Conclusions ................................................................... 252
6.1 Specific aims and results of the study ..................................... 252
6.2 Limitations of the study ....................................................... 254
6.3 Positive outcomes of the study ............................................. 255
6.4 Future directions ............................................................... 257

viii

Table 2.1

Table 2.2
Table 2.3

Table 2.4

LIST OF TABLES

Classification of G-Protein coupled receptor kinases (GRKs)............7

Phenotypic characteristics of GRK Knockout/Transgenic mice ....... 27
Involvement of GRKs in diseases ............................................ 32

Animal models of sepsis and their advantages verses
disadvantages .................................................................. 54

Figure 2.1
Figure 2.2

Figure 2.3

Figure 2.4

Figure 3.1

Figure 3.2

Figure 3.3

Figure 3.4

Figure 3.5

Figure 3.6

Figure 3.7

Figure 3.8

Figure 3.9

Figure 3.10

Figure 3.11

LIST OF FIGURES

Images in this dissertation are presented in color

Comparison of Linear Structure of GRK5 ................................. 9
LPS activates NF KB and MAPK pathways ............................... 37
TNFa stimulates NFKB and MAPK pathways ............................ 39

Mammalian NFKB and IKE family members.................................43

GRK2 knockdown in macrophages inhibits TNFa-induced

IKBO. phosphorylation and degradation ..................................... 93
Effect of different GRK2 siRNA oligos on

TNFa-induced IKBa phosphorylation ....................................... 96
GRK2 overexpression enhances TNFa-induced Icha

phosphorylation in macrophages ............................................ 98
GRK5 knockdown inhibits TNFa-induced IKBO.

phosphorylation and degradation in macrophages ...................... 101
Effect of a different GRK5 siRNA oligo on

TNFa-induced IKBa phosphorylation ..................................... 104
Role of GRKs is specific for TNFa-

NFKB pathway .................................................................. 107

Expression of macrophage inflammatory protein-113 (MIPI B),
an NFKB-regulated gene, is inhibited by GRK2 or GRK5
knockdown in macrophages ................................................ 109

Kinase activity of GRK2 and GRK5 is essential for

mediating TNFa-induced NFKB transcriptional activation ............ 111
GRK2 interacts with the N-terminus of IKBO. .......................... 115
Direct interaction between GRK5 and IKBO. ............................ 117
IKBa is a substrate for GRK2 and GRK5 in vitro ....................... 120

Figure 3.12

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Figure 4.5

Figure 5.1

Figure 5.2

Figure 5.3

Figure 5.4

Figure 5.5

Figure 5.6

Figure 5.7

Figure 5.8

Figure 5.9

IKKB knockdown does not inhibit TNFa-induced IicBa
phosphorylation but inhibits LPS-induced phosphorylation ........... 125
GRK5 null peritoneal macrophages show reduced TLR4-induced

NFKB activation .............................................................. 149
GRK5 has no effect on TLR4—induced MAPK activation ............. 154
LPS induction of cytokines and chemokines in peritoneal macrophages
from GRK5“+ and GRK5"' mice .......................................... 159
Profile of plasma cytokines after LPS challenge in GRK5”+ and
GRK5"’ mice .................................................................. 167

GRK5"' mice show reduced LPS-induced liver injury.................. 1 74

mye

. . . A .
Generation and characterization of GRK2 mice .................. 197

Complete blood count of cells from GRK2Amye verses GRK2fl/fl

mice ........................................................................... 199

LPS injection causes an enhanced secretion of pro and anti-
ye

inflammatory cytokines in the plasma of GRK2Am mice .......... 204
Amye . . . . .
GRK2 mice show enhanced tissue injury and mortality as

fl/fl .
compared to GRK2 mice ............................................... 211

. Am e ,
Peritoneal macrophages fi'om GRK2 y mice secrete enhanced

amounts of pro-inflammatory cytokines and chemokines in response to
LPS stimulation .............................................................. 216

. A .
Neutrophils from GRK2 mye mice secrete enhanced amounts of pro-

inflammatory cytokines and chemokines in response to LPS
stimulation .................................................................... 219

GRK2 deficiency causes an enhanced LPS stimulated ERK1/2

activation in peritoneal macropahges ..................................... 224
GRK2 deficiency does not affect LPS stimulation of Akt and GSK3B
activation in peritoneal macrophages ..................................... 227

The existence of IKKB-NFKBI p105/Tp12-MEK-ERK pathway in
peritoneal macrophages confirmed by using an IKKB inhibitor...... 230

xi

Figure 5.10

Figure 5.11

GRK2 deficiency causes an enhanced LPS stimulated NFKBI p105
activation in peritoneal macrophages ..................................... 233

Pharmacological inhibition of ERK and IKKB causes a significant
inhibition of LPS-induced secretion of cytokines and chemokines in

A .
peritoneal macrophages from GRK2 mye mice ....................... 236

xii

ANOVA

ATCC

ATP

cDNA

CK

CMV

CRE

DTT

EDTA

EMSA

GPCR

GRK

GRK2

GRK5

GST

HA

HEK

HEPES

HRP

IAPI

KEY TO ABBREVIATIONS

Analysis of Variance

American Type Culture Collection
Adenosine triphosphate
Complimentary DNA

Casein Kinase

Cytomegalovirus

Cyclization recombination
Dithiothreitol
Ethylenediaminetetraacetic acid
Electrophoretic mobility shift assay
G-Protein coupled receptor
G-Protein coupled receptor kinase
G-Protein coupled receptor kinase-2
G-Protein coupled receptor kinase-5
Glutathione S-transferase
Hemaglutinin

Human Embryonic Kidney
N-2-Hydroxyethylpiperazine-N'-2-Ethanesulfonic Acid
Horseradish peroxidase

Inhibitor of apoptosis 1

xiii

IKKB
IL
IPTG
IKBa
KCI
LB
LoxP
LPS
MgC12
ml
mM
mRNA
NaCl

NFKB

PBS

RIP 1
RT
RT-PCR

SDS-PAGE

IKB kinase B

Interleukin

Isopropyl B-D-l-thiogalactopyranoside
Inhibitor of NFKB

Potassium Chloride

Luria Bertani

Locus of crossover in P1
Lipopolys‘accharide

Magnesium Chloride

Milliliter

Millimolar

Messenger RNA

Sodium Chloride

Nuclear factor-kappa B

Nanomolar

Phosphate Buffered Saline

Presence of active hydrogen

Receptor interacting protein 1

Reverse Transcriptase

Reverse transcriptase-polymerase chain reaction

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

xiv

SEM Standard error of mean

TAD Transactivation domain

TBS-T Tris—Buffered Saline-Tween 20

TNF R TNF receptor

TNFa Tumor necrosis factor-alpha

TRADD Tumor necrosis factor receptor 1 associated death domain protein
TRAF TNF receptor associated factor

UV Ultraviolet

pg Micro gram

pl Microliter

um . Micrometer

XV

CHAPTER 1

INTRODUCTION

G protein coupled receptor kinases (GRKs) are serine threonine kinases which
play an important role in the desensitization of G protein coupled receptors (GPCRs).
Agonist bound GPCRs are a target for phosphorylation by GRK5, thus leading to their
rapid homologous desensitization. Although originally discovered with regard to this
classical role of GPCR desensitization, recent studies have shown that GRK5 can
phosphorylate non-GPCR receptor as well as non-receptor substrates. Moreover,
GRKs have also been shown to be involved in other phosphorylation-independent
protein-protein interactions. In this regard, GRKs have been shown to regulate Toll '
like receptor 4 (TLR4) induced inflammatory signaling in macrophages [1]. Thus, it
was hypothesized that GRK5 are important regulators of inflammatory signaling in
macrophages and therefore GRK5 might play essential roles in the regulation of
inflammatory diseases. This thesis research investigates the role of GRKs in
inflammatory signaling mediated by TNF receptors in macrophages under in vitro
conditions as well as the role of GRKs in TLR4-induced inflammatory signaling under
in vivo conditions in a mouse model of sepsis.

This brief introduction provides an overview of the hypothesis tested in this
thesis with a glance on the focus of subsequent chapters. Chapter two provides a
comprehensive literature review which provides an in-depth discussion of the
scientific literature surrounding the focus of this thesis project, including the classical
and novel roles of GRK5 as well as the involvement of GRK5 in inflammation and

immune response. Chapters three through five are organized based on the specific

aims of this thesis research project, each consisting of an abstract, introduction,
materials and methods, results and discussion relevant to the individual aim. Chapter
three focuses on the role of GRK5, in particular GRK2 and GRK5, in TNFa induced
inflammatory signaling particularly in mouse macrophages under in vitro conditions.
Chapter four investigates the role of GRK5 at the in vivo level in a mouse model of
LPS induced sepsis using a homozygous GRK5 gene deletion. Chapter five tests the
role of GRK2 in inflammatory signaling at the in vivo level whereby the focus was to
generate a myeloid cell specific genetic deletion of GRK2 and investigating the role of
this conditional deletion in a mouse model of LPS induced sepsis. Chapter six
highlights the results obtained in this thesis project with a discussion on how these
findings contribute to our current understanding of the novel roles of GRK5 as well as
the important role of GRK5 in LPS mediated sepsis. Also, discussed are some of the

limitations of this project as well as future directions.

CHAPTER 2

LITERATURE REVIEW

2.1: G protein coupled receptor kinases (GRK5)

G-protein coupled receptor kinases (GRK5) are serine/threonine kinases which
were originally discovered with regard to G-protein coupled receptor (GPCR)
desensitization [2]. They are the key modulators of GPCR signaling and play an
important role in fine tuning the GPCR signaling mechanism preventing the

overstimulation of GPCRs even in the presence of continuous agonist stimulation.

2.1.]: Historical perspectives of GRK discovery

Studies of mechanisms involved in the homologous desensitization of [32-
adrenergic receptor (BZAR) and rhodopsin (the prototypic light receptor) led to the
discovery of GRK5. In particular, rhodopsin played an important role in our current
understanding on the desensitization mechanism of GPCRs by GRKs due to its
availability in greater quantities. Rhodopsin undergoes light dependent
phosphorylation, first discovered in vivo in the 1970’s [3, 4]. Subsequently, it was
shown that the kinetics of rhodopsin phosphorylation correlate with the quenching of
cGMP phosphodiesterase activity, suggesting that rhodopsin phosphorylation has a
role in its desensitization [5]. Rhodopsin kinase (GRKl) was then purified and shown
to phosphorylate the receptor at multiple serine and threonine residues.

The occurrence of agonist-induced phosphorylation and desensitization of

BZAR even in the absence of protein kinase A (in kin' S49 lymphoma cells) and alpha

subunit of Gs (in cyc' cells) suggested the presence of another kinase, subsequently
identified as B-adrenergic receptor kinase (BARK) [6, 7]. In the mid 1980’s, BARK
was also discovered as a functional homologue to rhodopsin kinase and this further
suggested the existence of a multigene family of protein kinases which phosphorylate
agonist bound GPCRs [8]. Subsequently, crude BARK preparations were made and
were shown to mediate agonist dependent phosphorylation and uncoupling of the
receptor in vitro. A cDNA encoding BARK (or GRK2) was subsequently sequenced
and cloned, which expressed an enzyme that preferentially phosphorylates the agonist-
bound B2AR [9].

Pure preparations of BARK however did not lead to a significant loss of Gs
coupling. This suggested that an essential cofactor needed for BARK mediated
desensitization was lost during the purification process. This essential cofactor was
subsequently discovered as an additional protein called arrestin. Four distinct members
of arrestins are now known. Two out of these are restricted to phototransduction
pathways (visual arrestinl and visual arrestin 2). The other two i.e beta arrestin 1
(arrestin 2) and beta arrestin 2 (arrestin 3) are expressed ubiquitously and regulate the
internalization of many GPCRs as well as also play an important role in cellular

signaling [10].

2.1.2: GRK mediated desensitization of GPCRs
GRKs cause homologous desensitization of the receptors whereby only the
agonist activated receptors are phosphorylated and desensitized [2],[11]. Agonist-

induced desensitization of a GPCR is a multistep process. GRKs first phosphorylate

agonist bound GPCRs on serine and threonine residues located in the carboxy-terminal
tail region and/or the third cytoplasmic loop. This process enhances the affinity of
agonist bound-GPCR for binding to arrestins which then sterically inhibit the
interaction of the receptors with the G-proteins [12] leading to rapid homologous
desensitization. Finally, the GRK-arrestin system induces the internalization of
inactivated receptors to endosomal compartments via endocytosis.

Endocytosis of receptors has a fundamental role in receptor biology although
this process is not necessary for homologous desensitization [13]. Intemalization of
receptors can have three consequences viz. (1) Dephosphorylation of the receptor,
leading to resensitization and recycling back to the cell surface (2) Targeting of the
receptor to lysosomes leading to degradation (3) or activation of other signaling
pathways. Furthermore, endocytosis can occur via clathrin coated pits, caveolea or
other uncoated vesicles.[14, 15]. For clathrin dependent internalization,
phosphorylation of receptors by GRKS and subsequent binding by arrestins appears to
be essential [12]. Moreover, arrestin and clathrin dependent endoycytosis is used by a

majority of GPCRs.

2.1.3: Structure and distribution of GRK5

The GRK family consists of seven different genes and based on sequence and
functional similarities they are divided into three subfamilies [16]. The rhodopsin
kinase subfamily comprises GRKI (rhodopsin kinase) and GRK7 (cone opsin kinase)

that are found exclusively in retinal cells. The GRK2 subfamily comprises pleckstrin

homology domain containing GRK2 (B-ARKl) and GRK3 (B-ARKZ). Their

membrane recruitment depends on interaction with the GBy subunits of G proteins and
phosphatidyl-inositol 4,5—biphosphate. The GRK4 subfamily comprises GRK4, GRK5
and GRK6 which are predominantly localized at the membrane either due to
palmitoylation on C-terminal cysteine residues (GRK4/6) or interaction between a
positively charged domain at the C-terminus with the negatively charged membrane
phospholipids (GRK5) [17]. GRK4 is predominantly found in testes [18] and to a
lesser extent also in brain and kidney [19] whereas GRK2, GRK3, GRK5 and GRK6
are widely expressed (Table 2.1).

All GRK5 have a similar structural organization possessing an N-terminal
domain, a central catalytic domain (with homology to other serine-threonine kinases)
and a C-terminal domain. The N-terminal domain of 183-188 amino acids includes a
region of homology to regulators of G-protein signaling (RGS) proteins that are
known to act as GTPase activating proteins for many Ga subunits. The carboxyl
terminal domain is of variable length and contains key determinants important for
localization and translocation to the membrane either by post translational
modifications or by sites of interaction with lipids or membrane proteins. In this
regard, GRKl and GRK7 are isoprenylated, GRK4 and GRK6 are palmitoylated at
one or more cysteine residues clustered within the last 15-20 amino acids of the C-
terminus, which is responsible for their membrane localization whereas GRK5 is

localized in the membrane by virtue of a phosphatidyl inositol 4,5 biphosphate binding

 

Table 2.1: Classification of G protein coupled receptor kinases (GRK5)

GRK5 have been subdivided into three subfamilies based on the sequence and
functional similarities. 1. Rhodopsin kinase subfamily which comprises GRKI
(Rhodopsin kinase ) and GRK7 (cone opsin kinase). 2. GRK2 subfamily
which comprises GRK2 (B-ARKl) and GRK3 (B-ARKZ). 3. GRK4 subfamily
which comprises GRK4, GRK5 and GRK6.

 

Rhodopsin kinase subfamily

GRKI

 

 

 

 

 

 

GRK7
GRK2 (B-ARKI)
BARK subfamily GRK3 (B-ARKZ)
GRK4
GRK4 subfamily GRK5

 

 

GRK6

 

 

 

Figure 2.1: Comparison of Linear structure of GRKs

The seven isoforms of GRKs share a number of structural and functional
similarities. The amino terminal domain (183-188 amino acids) includes a
region of homology to regulators of G protein signaling (RGS) proteins. The
central catalytic domain has a homology to other serine threonine kinases. The
carboxyl terminal domain is of variable length and contains several key
determinants for the localization and translocation of kinases to the membrane.
This occurs either through post translational modifications or the presence of
sites of interaction with lipids or membrane proteins. As shown in the figure,
GRKI and GRK7 undergo isoprenylation; GRK4 and GRK6 undergo
palmitoylation: GRK5 has positively charged amino acid clusters at the C-
terminus through which it binds to membrane phospholipids: GRK2 and
GRK3 bear an extended C-terminus known as Pleckstrin homology domain
(PH domain). This domain modulates the membrane targeting of GRK2 and
GRK3.

PL, phospholipids; auto (:h), stimulatory or inhibitory autophosphorylation

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domain at the N-terminus and a polybasic region (amino acids 547—560) close to the
C-terminus [20]. GRK2 and GRK3 hear an extended C-terminus which is involved in
the modulation of the kinase targeting to the membrane. GRK2 and GRK3 are
cytoplasmic proteins that translocate to the membrane upon receptor stimulation. Their
C-terminus harbors a pleckstrin homology (PH) domain (residues 561-655) that
partially overlaps with the GBy-binding region. It has been shown that free GBy sub-
units can bind to GRK2 with a high affinity. Furthermore it has been shown that GBy
is required for association of GRK2 to lipid vesicles in reconstituted systems [21].

Hence, it is believed that the interaction of GBy with GRK2 and GRK3 targets
them to membranes at the site of GPCR activation. Binding of GBy also enhances the
activity of GRK2 by causing allosteric activation of this kinase. The PH domain of
GRK2 and GRK3 also interacts with phosphatidylinositol 4,5-biphosphate (PIP2) and
other acidic phospholipids such as phosphatidyl serine (PS) which regulate its kinase
activity [22]. Thus, GBy and lipids contribute synergistically to membrane localization
and activation of GRK2 (Figure 2.1).

The GRK4-6 family of GRKs has been shown to contain a nuclear localization
sequence (NLS). It was found that the nuclear localization of GRK5 is regulated by
GPCRs. Binding of an agonist onto a GPCR leads to an elevation of intracellular
calcium levels and activates a calcium sensor protein, calmodulin. Furthermore, GRK5
was found to bind to DNA under in vitro conditions [23]. More recently, it was shown
that GRK5 can act as a histone deacetylase (HDAC) kinase independent of its actions

on GPCRs [24].

ll

2.1.4: Regulation of GRKs by other proteins

2.1.4.1 Regulation of GRK3 by Caveolin, Clathrin and a—actinin

Caveolins control the basal kinase activity of GRKs: Caveolin is a 22-24
kDa integral membrane protein and is a prominent structural component of caveolae.
Caveolae are specific cholesterol and glycosphingolipid-enriched plasma membrane
structures in cells. A 20 a region within the N-terminal domain of caveolin (known as
scaffolding domain) has been found to mediate the association of caveolin with other
proteins. Caveolins serve as scaffolding proteins for some GPCRs as well as certain
mitogen activated protein kinases (MAPKs) and G proteins and help
compartrnentalize signaling pathways. Recent studies have shown that GRK2 is
present in caveolin rich fractions of cellular membranes. GRK2 contains caveolin
binding motifs in the PH domain (residues 567-584) as well as in the N-terminal
domain (residues 63-71) while GRK3 and GRK5 can bind to caveolin only through N-
terminal motifs. GRK2 binding to caveolin 1 or 3 inhibits GRK2 mediated
phosphorylation suggesting caveolins can control the basal kinase activity of GRK2.
In addition, it is possible that GRK-caveolin interactions may facilitate GRK5
interactions with other signaling molecules [25].

Clathrin promotes arrestin independent internalization: Clathrin is a
protein present in the internalized vesicles containing GPCRs. GRK2 can also interact
with clathrin protein via a clathrin box located in the carboxyl terminal region of
GRK2 [26]. B-arrestins bind to phosphorylated GPCRs and promote the internalization

of the receptors by interacting with clathrin. Certain GPCRs, however, are only

12

slightly internalized due to their low affinity binding to B-arrestins. GRK2 bound to
such receptors can facilitate GPCR internalization due to its ability to bind clathrin.
This internalization is however, B-arrestin independent but depends on the presence of
dynamin. [27].

Alpha-actinin inhibits GRKs: a-actinin is a protein that falls under the
spectrin superfamily of actin crosslinking proteins. It is found most abundantly in
muscle cells. It binds and crosslinks actin as well as lipid, creating a submembraneous
meshwork consisting of crosslinked actin. a-actinin also binds various ion channels,
cell adhesion molecules as well as transmembrane receptors, thereby incorporating
them into the actin network creating signaling domains. It has been found that all
GRK5 can interact with a-actinin. a-actinins can completely inhibit GRK3, or

modulate their activity, as well as substrate specificity [28].

2.1.4.2 Regulation of GRK5 by Calcium binding proteins
Recoverin inhibits GRK]: Recoverin is a protein present in vertebrate
photoreceptors, certain retinal cone bipolar cells and pineal glands, exhibiting a

distribution quite similar to GRK]. Recoverin binds directly to GRKI and inhibits it in

2+ . . . . . .
a Ca -dependent fashion. Calcrum levels are very high during dark conditions and
fall substantially under light conditions in the vertebrate photoreceptor systems. Hence,
GRK] would be complexed to recoverin and inactive when rhodopsin is in its inactive

state under dark conditions. However, following rhodopsin activation, a drop in

intracellular calcium levels causes the release of GRK] from recoverin. GRKI is now

13

disinhibited and can facilitate rapid phosphorylation and desensitization of rhodopsin
[29].

Calmodulin inhibits GRKs: Calmodulin is a universal mediator of calcium
signaling, which has been shown to inhibit the activity of GRK2, 3, 4, 5, and 6 albeit
with different potencies [30]. Calmodulin binds directly to both GRK2 and GRK5,
with different affinities, inhibiting the interaction of these enzymes with their agonist
occupied receptors. GRK5 is highly sensitive to the presence of calcium bound
calmodulin (IC50 ~ 50nM), whereas GRK2 is affected only at higher concentrations

(IC50 ~ ZuM) [31]. Thus GRK2 and GRK5 mediated desensitization of GPCRs would

+
be predicted to be inhibited in the presence of Ca2 /calmodulin. GRK2 has a

relatively low affinity for calmodulin suggesting that inhibition of GRK2 would occur
only at sites highly enriched in calmodulin. However, the relatively high affinity of
GRK5 for calmodulin suggests that this regulatory mechanism might be present at

multiple locations.

2.1.4.3 Regulation of GRK5 through phosphorylation by other kinases

Protein Kinase C (PKC) activates GRK2 but inhibits GRK5: GRK5
activity, the protein stability as well as their ability to interact with other proteins is
highly regulated by their own phosphorylation by other protein kinases. In this regard
protein Kinase C (PKC) has been shown to phosphorylate both GRK2 and GRK5 in
intact cells as well as under in vitro conditions. PKC phosphorylation of GRK2
enhances the translocation and interaction of GRK2 with the plasma membrane

causing an increased activity towards receptor substrates. It does not, however, affect

14

the catalytic activity of GRK2 since GRK2 cannot phosphorylate soluble peptides
under similar conditions. PKC phosphorylates GRK2 at serine 29 within the
calmodulin binding motif of GRK2 [32]. It has been suggested that the inhibitory
effect produced by calmodulin binding to GRK2 is abolished after phosphorylation by
PKC, allowing the binding of GRK2 to the receptor substrate. In contrast to this,
phosphorylation of GRK5 by PKC leads to the inhibition of its activity towards both
receptors and soluble substrates suggesting a direct effect on the catalytic activity of
GRK5. There are two major sites of phosphorylation by PKC in the C-terminal 26
amino acids of GRK5. The inhibitory autophosphorylation sites also reside in this
region which are targeted in the presence of calmodulin. Thus Ca/Calmodulin and
PKC both act as inhibitors by targeting identical inhibitory sites on GRK5. [20].

Protein kinase A (PKA) activates GRK2: Protein kinase A (PKA) is another
kinase that can phosphorylate GRK2 at serine 685. This site is located near the GBy
binding domain of GRK2. PKA phosphorylation of GRK2 at this site enhances the
binding of GRK2 with the GBy subunits, facilitating the membrane targeting of GRK2
and further the interaction of GRK2 with activated receptors. This leads to an
enhanced GRK2 activity towards B2-adrenergic receptors [33].

C-Src activates GRK2: c-Src, a tyrosine kinase has been shown to directly
phosphorylate GRK2 under in vitro conditions as well as it promotes tyrosine
phosphorylation of GRK2 upon stimulation of B2-adrenergic receptors [34, 35]. This
process is however dependent on the ability of B-arrestins to recruit c-Src since B-
arrestin mutants defective in c-Src binding are defective in this process. [36]. Tyrosine

phosphorylation of GRK2 directly enhances its catalytic activity as is seen by its

15

increased activity towards both soluble and membrane bound substrates. Hence,
recruitment of c-Src by B-arrestins to the activated GPCR results in GRK2
phosphorylation at tyrosine residues and increases its catalytic activity.

Extracellular signal regulated kinase 1 (ERKl) inhibits GRK2: ERKI, a
MAPK, has also been shown to phosphorylate GRK2 at serine 670 under in vitro and
in situ conditions [37, 38]. This phosphorylation site is present within the GBy binding
domain of GRK2. Hence, ERKl phosphorylation of this site impairs the interaction of
GRK2 with GBy, inhibiting GRK2 translocation to the activated membrane receptors
thus inhibiting its catalytic activity. This process triggers a negative feedback loop that
prevents accumulation of an active pool of GRK2. Moreover, both kinases have been

found to co-immunoprecipitate in an agonist dependent manner [3 8].

2.1.4.4 Regulation of GRK5 by GBy subunits and phospholipids

GRK2 and GRK3 contain a PH domain (residues 561-655) in the carboxy-
terminal region that partially overlaps with the GBy-binding region. Free GBy subunits
bind to GRK2 with high affinity, and is required in reconstituted systems for
association of GRK2 to lipid vesicles and GPCR phosphorylation [21]. This is
important since both GRK2 and GRK3 are cytoplasmic proteins that transiently
translocate to the membrane upon GPCR stimulation. Hence, it is suggested that the
interaction of GBy with GRK2 and GRK3 targets these kinases to membrane sites
where receptors are activated and GBy dimers are released. GBy binding increases the

activity of GRK2 by promoting GPCR-mediated allosteric activation of GRK2.

16

PH domains of GRK2 and GRK3 can also interact directly with PIP2 and other
acidic phospholipids, affecting their kinase activity [22, 39]. GRK2 phosphorylation of
membrane receptors is enhanced by atleast two to three fold by binding of
phosphatidyl serine (PS) and PIPZ. Among these, PS directly affects the catalytic
activity of GRK2 since PS also enhances the phosphorylation of soluble substrates.
PIP2 appears to bind to the amino-terminus of the PH domain suggesting that GBy and
lipids have a synergistic role in GRK2 translocation and activation. GRK5 is also
regulated by lipids despite lacking the PH domain. GRK5 possesses two different
regions rich in positively charged amino acids (amino terminal residues 22-29 and
carboxyl terminal residues 547-560) that can bind to lipids [40]. The amino terminal
binding site exhibits a high specificity for PIP2 in contrast to the carboxyl terminal
binding site which does not exhibit a stringent specificity for binding to lipids. PIP2
binding onto the amino terminal binding site increases GRK5 phosphorylation of
receptor substrates but does not affect phosphorylation of soluble peptides or GRK5
autophosphorylation. On the other hand, lipid binding onto the carboxyl-terminal
binding site stimulates autophosphorylation of GRK5 and its activity towards different
substrates. These lipid binding domains are highly conserved in the GRK4 subfamily

and binding of PIPZ enhances receptor phosphorylation by GRK4 and GRK6 as well.

2.1.4.5 Regulation of GRK3 by interaction with agonist activated GPCRs
The activity of GRKs is also regulated by interaction with agonist stimulated
GPCRs. Activity of GRKI towards a peptide substrate increases in the presence of

activated rhodopsin receptor. This is because the association of GRKl with the third

17

intracellular loop of rhodopsin causes kinase activation [41]. Similarly, the catalytic
activity of GRK2 is regulated in the presence of either agonist activated B2-AR and
rhodopsin, or synthetic peptides derived from intracellular loops. These interactions
appear to occur through the N—terminal region of GRKs [42]. It is thought that
interaction of the GRKs with the receptor causes a conformational change in the
GRK5 thus releasing an autoinhibitory constraint and causing kinase activation.

GRK2 and GRK3 has also been shown to interact to activated Gaq subunits
with high affinity, leading to a reduced Gaq mediated phospholipase C-B activation
both in vitro and in intact cells [43, 44]. Hence the interaction of GRKs with the
GPCRs and Gaq subunits may serve two functions, 1) translocation of GRKs to the
membrane and 2) phosphorylation independent termination of signal transduction
mediated by GPCRs by interfering with the binding of stimulated GPCR and Gaq with

their effectors [45].

2.1.5: Other novel interactions of GRK5 and their physiological effects

Over the past few years it has become evident that the role of GRK3 is not
limited to GPCR phosphorylation, desensitization and internalization. Apart from
these classical functions, GRKs have now been shown to interact to non GPCR
receptor substrates and to a variety of signaling proteins, regulating cell signaling in a
phosphorylation dependent as well as independent manner. In this regard, GRKs have
been shown to phosphorylate other non GPCR membrane receptors such as Platelet
derived growth factor (PDGF) receptor [46] as well as non receptor substrates such as

tubulin [47, 48], synnucleins [49], phosducin [50], ribosomal protein P2 [51], ezrin-

18

radixin-moesin (ERM) family protein ezrin [52] and NFKBI P105 [1]. In addition,
GRK5 have been shown to regulate several signaling pathways in a phosphorylation
independent manner by interacting with a variety of signaling as well as trafficking
proteins. The novel interactions of GRKs along with their functional consequencies
are summarized:

PDGFRB receptor: The platelet-derived growth factor receptor-B (PDGFRB)
plays a crucial role in regulating the proliferation and survival of mesenchymal cells.
It has been found that GRK2 phosphorylates PDGF RB receptor on Ser1104 which is
responsible for its desensitization in physiological systems. GRK2 mediated

phosphorylation of Ser1104 on PDGFRB promotes its dissociation from the PDZ

+ +
domain containing NHERF protein (Na /H exchange regulatory factor) which is

involved in potentiating PDGF RB dimerization and activation, thus causing receptor
desensitization [46].

Tubulin: In a quest to search for other proteins that might interact with GRK2,
a GST fusion protein containing the C-terminus of GRK2 was used. GRK2 was found
to bind to the cytoskeletol protein tubulin through its carboxy terminal domain
(residues 467-689) by this method. Furthermore, GRK2 can phosphorylate tubulin
and phosphorylation of rhodopsin is inhibited by tubulin in a dose dependent manner
[47]. However, the physiological relevance of this interaction / phosphorylation is not
very clear yet although it is possible that the GRK2-tubulin interaction might have a
role in regulating the desensitization of GPCRs by GRK2. Furthermore, it is also
speculated that GRK5 might play a role in regulating microtubule dynamics and

cytoskeletol reorganization.

19

 

Synuclein: To identify new substrates for GRK3, bovine calf brain extracts
were prepared and were used in phosphorylation reactions. Synuclein, a protein highly
expressed in brain was identified as a substrate by this method. Lipids and GBy
subunits, which stimulate phosphorylation of GPCRs by GRK3, also activate
synuclein phosphorylation by GRK3. Furthermore, all GRKs can phosphorylate
synuclein. Phosphorylation of synuclein by GRKs blocks the interaction of synuclein
with phospholipids. This in turn reduces the inhibitory effect on Phospholipase D2
(PLD2), an enzyme involved in regulating vesicular trafficking. Hence, it is possible
that agonist bound GPCRs activate GRKs which then phosphoryate GPCRs targeting
them for endocytosis as well as also phosphorylate synuclein thus activating PLD2
which is involved in vesicle formation needed for endocyotsis as well as recycling of
receptors.

Raf kinase inhibitor protein (RKIP): RKIP is a protein that belongs to a
family of phosphatidylethanolamine-binding proteins (PEBPs) that serve as inhibitors
of kinase signaling pathways. RKIP is a physiological inhibitor of Ras-Raf-MEK-
ERK pathway involved in several cellular processes such as differentiation,
proliferation, cell survival and cell transformation. RKIP has been shown to interact
with GRK2 [53]. GPCR stimulation leads to the activation of PKC which can
phosphorylate RKIP on serine 153. Phosphorylation of RKIP leads to an increase in its
affinity towards GRK2 which then leads to its dissociation from its target Rafl,
prolonging the activation of ERK kinase. The interaction of GRK2 with RKIP blocks
the kinase activity of GRK2, impairing the desensitization process, and leading to a

prolonged signaling.

20

Phosphoinositide 3-kinase (PI3K): PI3Ks are a conserved family of lipid
kinases whose function is to catalyze the addition of a phosphate on the third position
of inositol ring. Stimulation of GPCRs causes activation of PI3Ks increasing the levels
of D-3 phosphatidyl inositols which are potent signaling molecules. A direct protein-
protein interaction has been shown for PI3Ky and GRK2 through 197 amino acid
residues PIK domain in PI3K [54]. GRK2 and PI3Ky form a cytosolic complex and
translocate to the membrane in an agonist dependent manner. This interaction plays an
important role in receptor sequestration as it has been shown that inhibition of kinase
activity of PI3K results in attenuation of B-adrenergic receptor sequestration.
Furthermore, cardiac specific overexpression of the PIK domain causes disruption of
the GRK2-PI3K interaction under in vivo conditions, preserving B-adrenergic receptor
signaling even after prolonged catecholamine administration. This helps restore
myocardial contractibility to normal in heart failure conditions [55].

Akt: Akt is a serine threonine kinase which directly interacts with GRK2. The
region of interaction has been determined to be GRK2 C-terminus (aa 492-689). This
interaction leads to an inhibition of Akt phosphorylation. A study using rats with
portal hypertension showed an enhanced expression of GRK2 in their sinusoidal
endothelial cells. Enhanced GRK2 was found to lead to an inhibition of Akt
phosphorylation. Since Akt phosphorylation is required for the production of NO by
eNOS, inhibition of NO production was found to lead to portal hypotension. [56].

GPCR-kinase interacting proteins (GIT proteins): The GIT family of
proteins were found to be binding partners of GRKs [57]. Their structure consists of a

zinc-finger motif and several ankyrin repeats in the N-terminal portion with a GAP

21

 

(GTPase activating protein) domain in the first 45 amino acids. The GIT proteins are
active as GAPS on ARF 6 protein present on the plasma membrane. Yeast two hybrid
screen has identified GITl and GIT2 as binding partners for GRK2, 3, 5 and GRK6
[57]. However, the functional significance of this interaction is not very clear as yet.

MEK: GRK2 and MEKl has also been shown to be present in the same
multimolecular complex. Furthermore, it was found that elevated levels of GRK2
inhibit chemokine mediated induction of ERK whereas decreased levels of GRK2
promote chemokine mediated ERK activity. MEK activity was not found to be
affected though. Furthermore, neither the kinase activity nor the interaction of GRK2
to G protein subunits is necessary for this effect. [58].

Heat shock protein 90 (Hsp 90): Hsp 90 is a highly conserved chaperone
protein that interacts with a wide variety of signaling proteins. GRK2 has been found
to interact with Hsp 90 and it has been shown that the disruption of this interaction
causes an increased degradation of GRK2 via the proteasome pathway. This suggests
that the interaction of GRKs with the heat shock proteins plays an important role in the

folding and maturation of GRKs [59].

2.2 Physiological and Pathological roles of GRKs

Development of genetically modified mouse models with either a targeted
deletion of a particular GRK or overexpressing a GRK transgene has given us a great
insight into the roles of individual GRKs in various signaling pathways as well as in

an intact animal.

22

2.2.1: Physiological roles of GRKs as determined by knockout / transgenic mice
GRK] is required for rhodopin desensitization: GRKl or rhodopsin kinase

was the first GRK to be discovered [60] and was found to phosphorylate light

activated rhodopsin at C-terminal residues. It is expressed in retina and in mammalian

pineal gland. A better idea of the role of GRKl under in vivo conditions came with the

-/- .
generation of mice deficient in GRKl (GRKI ) [61]. Deficrency of GRK] led to an

elimination of the light dependent phosphorylation of rhodopsin causing the single
photon response to become larger and longer than normal. Also, a day of constant
light caused the rods in these mice to undergo apoptotic degeneration. Furthermore,
the cone response recovered around 30-50 times slower than normal when the mice
were exposed to a bright conditioning flash.

GRK2 plays an essential role in embryonic development: Similar to GRKl,

studies with GRK2 knockout mice confirmed an important role of GRK2 in

. . , -/-
embryonic cardiac development and function. GRK2 (GRK2 ) homozygous

knockout mice embryos die during gestation. Examination of these embryos revealed
that there was a pronounced hypoplasia of the ventricular myocardium as well of the
interventricular septum due to which the atrial and ventricular cavities appeared to be
unusually large [62]. Furthermore, it was found that there was a 70% decrease in left
ventricular ejection fraction suggesting an impaired heart function. These mice,
however, developed normally with no apparent adult cardiac phenotype at baseline
except a modestly enhanced contractile function when GRK2 was specifically deleted

in cardiac myocytes [63]. This study suggests that GRK2 does not play a specific role

23

in cardiac development, rather it might have a much more broader role in embryonic
development. A specific role for GRK2 in embryonic development has not been
identified yet though. Contractile response to B-adrenergic receptor and angiotensin II
receptor stimulation is attenuated by myocardical overexpression of GRK2 [64].
Vascular smooth muscle (VSM) specific overexpression of GRK2 similarly attenuates
B-adrenergic receptor signaling resulting in increased resting blood pressure [65].
GRK3 knockout mice exhibit supersensitivity to olfactory stimuli: GRK3

knockout mice have a normal embryonic as well as postnatal development. GRK3

. -/- . .
deletion (GRK3 ) has shown that GRK3 plays a role in the regulation of olfactory

. . . -/- . . .
receptors. Cilia preparations from GRK3 mice do not show a fast agonist induced

desensitization which is normally seen after stimulation with odorants [66].

GRK4 regulates dopamine-l receptor: Transgenic mice overexpressing
GRK4 were generated whereby it was found that the transgenic mice overexpressing
wild type GRK4y showed a normal phenotype in contrast to the mice carrying a
naturally occurring polymorphism A142V in GRK4y which showed increased kinase
activity and hypertension. The diuretic and natriuretic effects of D1 agonist were also
found to be impaired in these mice suggesting that GRK4 regulates dopamine-1
receptor in kidney [67].

GRK5 is required for M2 muscarinic receptor desensitization: GRK5

. -/- . . . . .
deletion (GRK5 ) caused an increase in cholrnergic responses such as hypothermia,

hypoactivity, tremor, salivation and antinociception [68]. Furthermore, central M2

muscarinic receptors have been found to be resistant to desensitization in the absence

24

of GRK5. It was also found that B2-adrenergic receptor induced airway smooth

. . -/- . . . . .
muscle relaxation was reduced in GRK5 mice [69]. Transgenic mice wrth cardiac

specific overexpression of GRK5 show enhanced B-adrenergic receptor desensitization
in comparison to non-transgenic control mice. However, there was no change in the
contractile response to angiotensin II receptor stimulation [70]. Vascular smooth
muscle (VSM) specific overexpression of GRK5 showed an increase in blood pressure
which was also found to be gender dependent i.e. male mice showed a much larger
increase in blood pressure as compared to that in female mice [71]. However, unlike
the VSM specific overexpression of GRK2, overexpression of GRK5 in VSM did not
cause vascular or cardiac hypertrophy.

GRK6 plays an essential role in desensitization of chemokine receptors: T
cells from GRK6 knockout mice (GRK6’l') show an impaired chemotactic response to
CXCL12 (ligand for CXC chemokine receptor 4 CXCR4) [72] whereas neutrophils
from the same mice showed an enhanced chemotactic response to CXCL12 [73] as
well as to leukotriene B4. Furthermore, acute migration of neutrophils from the bone
marrow in response to G—CSF has been found to be impaired suggesting that this
might be due to CXCR4-mediated retention of neutrophils in the bone marrow [73]

(Table 2.2).

2.2.2: Pathological roles of GRK5
Change in GRK5 expression and activity have been found in several
pathologies suggesting an important role of GRKs in particular disease conditions.

Reduced response to B-receptor agonist and a loss of cardiac contractility in human

25

chronic heart failure has been shown to be linked to an increased expression of GRK2
and reduced expression of B1 receptor [74, 75]. Furthermore, the activity as well as the
expression of GRK2 in lymphocytes is significantly enhanced in hypertensive subjects
as compared to normotensive controls [76]. This increase negatively correlates with B-
agonist stimulated lymphocyte adenylyl cyclase activity [77]. Reduced adenylyl
cyclase activity correlates with a reduced vasodilator response to B-adrenergic agonist
stimulation of vascular smooth muscle receptors in hypertensive state [78].

A significant decrease in expression as well as activity of GRK2 and GRK6
has been shown in peripheral blood mononuclear cells (PBMCs) of patients with
rheumatoid arthritis as compared to those from healthy individuals [79]. This reduced
expression and activity of GRKs correlates with an increased sensitivity to B2-
adrenergic stimulation as B-agonist induced CAMP production has also been found to
be increased in leucocytes from these patients. Furthermore, it was found that beta
blockers and receptor antagonists for a chemokine, MCPl (Monocyte Chemoattractant
Protein 1) can reduce the severity of disease in chronic arthritis [80, 81]. This is
important since chemokine receptors which fall under classical GPCRs, as well as
other GPCRs such as those for prostaglandins and substance P play an important role
in inflammation in Rheumatoid arthritis. This suggests that cell type specific decrease
in GRKs activity might be responsible for sustained activation of G—protein coupled

pro-inflammatory receptors and hence defects in cellular metabolism. Rat models of

26

Table 2.2: Phenotypic characteristics of GRK Knockout/Transgenic mice

Modified from:
Premont, RT and Gainetdinov, RR. Physiological roles of G-protein coupled
receptor kinases and arrestins. Annual Review of Physiology 2007 69:511-534

Metaye, T., Gibelin, H., Perdrisot, R., Kraimps, GL. Pathophysiological roles
of G-protein coupled receptor kinases. Cellular Signaling 2005 17:917-928

27

 

Knockout

Phenotype

 

GRKl

Prolonged response and light-induced apoptosis in
rods
Slow resensitization in cones

 

GRK2 double allele deletion

GRK2 single allele deletion

GRK2 cardiac specific deletion

VSM specific overexpression of
GRK2

VSM specific deletion of GRK2

GRK2 ablation in myeloid cells

Embryonic lethal, thin myocardium syndrome in
embryos

Enhanced myocardial contractile response to a B-
agonist

Enhanced chemotaxis of T-lymphocytes

Altered progression of experimental autoimmune
encephalomyelitis

Enhanced inotropic sensitivity to isoproterenol

VSM BAR signaling attenuated
Diminished BAR mediated dilation
High blood pressure

Enhanced BAR mediated dilation
Does not rescue high blood pressure
Increased an) AR stimulation

Earlier onset of H1 (Hypoxia-Ischemia) brain
injury

 

 

 

GRK5 myocardial overexpression

GRK3 Enhanced airway response to a cholinergic agonist
Loss of kappa opioid tolerance
Lack of olfactory receptor desensitization

GRK4 Normal fertility and sperm function. No obvious
phenotype

GRK5 Excessive opposition of airway smooth muscle

relaxation after B—agonist administration
Enhanced hypothermia, hypoactivity, tremor, and
salivation by oxotremorine

Attenuation of contractility and heart rate in
response to a B—agonist

 

_G_RK6

 

 

Enhanced locomoter- stimulating effect of cocaine
and amphetamine

Impaired chemotaxis of T-lymphocytes

Enhanced chemotaxis of bone-marrow-derived
neutrophils and impaired acute neutrophil
mobilization in response to G-CSF

Altered central dopamine receptor regulation

 

28

 

arthritis also showed that changes in GRK2 and GRK6 levels occur after induction of
arthritis. GRK2 has also been found to be reduced in platelets from patients with
depression whereby treatment with antidepressants has been found to cause an
upregulation in GRK2 levels [82]. Lung homogenates from Cystic fibrosis patients
show a decreased B-agonist stimulated adenylyl cyclase activity and a simultaneous
increase in GRK activity as well as mRNA and protein for both GRK2 and GRK5 [83]
suggesting that an increase in GRKs might be responsible for alteration in B2-

adrenergic receptor density.

2.3: GRKs and inflammation

GRKs play an important role in inflammation and disease. Firstly, GRK2, 3, 5
and 6 have been found to be expressed at particularly high levels in immune cells [84]
and their levels in immune cells are dynamically regulated in response to
inflammation suggesting an important role for these kinases in immune activity.
Secondly, targeted deletion of GRKs in vivo affects the progression of various acute as
well as chronic inflammatory diseases again suggesting an important role for GRKs in
disease. Furthermore, chemokine receptor signaling has also been shown to be tightly
regulated by GRKs under in vitro conditions suggesting that GRKs play an important

role in inflammation.

2.3.1: GRKs expression and inflammation: Mak et al., showed an increased

expression of GRK2 and GRK5 in the lungs of rats treated with IL-IB, a pro-

29

 

inflammatory cytokine, and this effect was found to be abolished when the rats were
treated with an anti-inflammatory steroid, dexamethasone [85]. Pro-inflammatory
cytokines as well as oxygen radicals have also been found to reduce the levels of
GRK2 under in vitro conditions [79, 86]. LPS has been shown to downregulate
chemokine induced expression of GRK2 and GRK5 in neutrophils (PMNs) [87].
Moreover, pro-inflammatory cytokines such as IL-1, TNF-a and IFN-y regulate GRK2
expression at the transcriptional level by regulating the GRK2 promoter [88]. In one
study, our lab found that stimulation of mouse peritoneal macrophages with ligands
for TLR2, TLR3, TLR4 and TLR7 caused an upregulation in the levels of GRK2.
Conversely, stimulation of mouse peritoneal macrophages with ligands for TLR2,
TLR4 and TLR7 caused a significant decrease in the GRK5 and GRK6 protein levels
[89]. In another study, it was found that Lipoteichoic acid (LTA), a known TLR2
agonist, increased the expression of GRK2 in neutrophils. This corresponded with a
downregulation in the expression of chemokine receptor CXCR2 in neutrophils in,
sepsis and an impaired migration of neutrophils into an infectious focus in vivo and
reduced chemotaxis in vitro [90]. These studies suggested that GRKs have an
important role in inflammation since TLRs are important mediators of inflammatory

response.

2.3.2: GRKs deletion and inflammation: Heterozygous GRK2 knockout mice

. +/-
(GRK2 ) have been found to have an advanced onset of multiple sclerosis with

increased numbers of inflammatory cells in the spinal cord as compared to control

mice [91]. However, overexpression of GRK2 in vascular smooth muscle cells has

30

been found to induce hypertension and cardiac hypertrophy [65]. Another finding

shows that arachidonic acid induced acute ear inflammation but not PMA-induced

. -/- —/+ . .
inflammation increased substantially in GRK6 and GRK6 mice suggesting a

specificity towards an agent causing inflammation rather than a general effect of

GRK6 deficiency on the chemotactic activity of neutrophils [92].

2.3.3: GRKs regulation of chemokine signaling and inflammation: GRKs can also
regulate chemokine responses in inflammation. Increased signaling by chemokine

receptors plays an important role in disease pathology, regulating the migration of

. +/- .
neutrophils, monocytes and T-cells. In this regard, T-cells from GRK2 mice have

been found to have enhanced CCRS agonist induced calcium mobilization, PKB and

ERK1/2 signaling and migration towards CCRS agonist under in vitro conditions [93]

+
Spleenocytes from GRK2 mice were also found to have an enhanced CCR2

mediated signaling to ERKl/Z [5 8].

Hence, it is clear that the alterations in expression and activity of GRKs as well
as the role of GRKs in regulating chemokine mediated responses play an important
role in inflammatory diseases and should be taken into account when developing

therapeutic strategies for such conditions (Table 2.3).

2.4: LPS and TNFa induced inflammation

2.4.1 LPS induced inflammation

31

 

Table 2.3: Involvement of GRKs in diseases

Slightly modified from
Metaye, T., Gibelin, H., Perdrisot, R., and Kraimps, IL (2005) Cellular
signaling 17(8), 927-928

32

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

GRK Disease GRK modification
GRKI Oguchi disease Genetic mutation associated with a loss of
GRK activity
GRK2 Chronic heart Increased expression of mRNA and protein
failure
GRK4y Hypertension Genetic variants with a marked stimulation
of GRK4]! activity
GRK2 Hypertension Increased protein expression
GRK2/GRK6 Rheumatoid Decreased protein expression
arthritis
GRK2/GRK6 Opiate addiction Decreased protein expression
GRK2 Differentiated Increased enzymatic activity
thyroid carcinoma
GRK2/GRK4y/5 Ovarian cancer Increased protein expression
GRK3 Hyperfunctioning Increased protein expression
thyroid nodule
GRK2 Depression Increase in membrane fixation
GRK3 Bipolar disorder Single nucleotide polymorphism in the
promoter region of GRK3
GRK2/GRK5 Cystic fibrosis Increased expression of mRNA and protein
GRK2/GRK3/ Left ventricular Increased expression of mRNA and protein
GRK5 overload diseases
GRK2/GRK6 Cardiopulmonary Decreased protein expression

 

bypass in cardiac
diseases

 

 

33

 

The mammalian innate immune system defends against bacterial or viral
infection and essentially forms the first line of host defense. Toll like receptors (TLR)
are crucial components of the innate immune system, and recognize a wide variety of
microbial products known as pathogen associated molecular patterns (PAMPs) present
in bacteria, viruses and other pathogens. Of the 10 human TLRs, the best characterized
is TLR4, which recognizes gram negative bacterial outer membrane structural
component known as lipopolysaccaride (LPS) activating immune cells such as

monocytes and macrophages leading to the induction of endotoxic shock in mammals.

2.4.1.1: Toll like receptor 4 (TLR4) signaling

TLR4 works in concert with a coreceptor protein CD14 plus a secreted protein
MD2 to transmit cell signals whereby CD14 is required for presentation of LPS to
TLR4-MD2 [94]. Formation of LPS—TLR4-MD2 complex is required for the
activation of downstream signals. LPS recognition causes TLR4 to undergo
oligomerization and recruitment of downstream adapter proteins through interaction
with the TIR domain (Toll-interleukin-l receptor). The TIR domain is highly critical
for signal transduction since it has been found that a single point mutation in the TIR
domain can abolish the response to LPS [95]. TLR4 utilizes five TIR domain
containing adapter proteins. These are MyD88 (Myeloid differentiation primary
response gene 88), TIRAP (TIR domain-containing adapter protein, also known as
Mal or MyD88-adapter-like), TRIF (TIR domain- containing adapter inducing [FN-B),
TRAM (TRIP-related adapter molecule), and SARM (Sterile alpha and HEAT-

arrnadillo motifs-containing protein) [96].

34

 

TLR4 signaling has been divided into MyD88 dependent and MyD88-
independent (TRIP-dependent) pathways. Under MyD88 dependent signaling, upon
LPS stimulation, MyD88 activates a death domain-containing kinase IRAK-4 (IL-1
receptor-associated kinase-4). IRAK-4 leads to the activation of TRAF6 (TNF
receptor associated factor 6). TRAF6 activates TAKl (Transforming growth factor-B-
activated kinase 1) which then activates IKK (IKB kinase) and MAPK pathways [97,
98]. IKB kinase finally leads to the activation of NFKB which controls the expression
of pro—inflammatory cytokines and chemokines as well as various other genes
involved in innate and adaptive immunity. Activation of MAPK causes induction of
AP-l, another transcription factor which is also involved in the expression of pro-
inflammatory cytokines and chemokines [99]. It has been found that NFKB and
MAPK are activated through MyD88 independent pathway as well.

MyD88 independent signaling is carried on by TRIF which is another TIR-
domain containing protein. MyD88-independent pathway also leads to the activation
of NFKB, however, this activation occurs later than the MyD88-dependent activation.
It was shown by Covert et al. that NFKB activation by MyD88-independent pathway
requires protein synthesis which leads to a delay in activation. It was later discovered
that TRIP-dependent pathway activates TNFa expression and secretion in an NFKB-
independent manner through a TRIP-dependent pathway specific transcription factor
IRF3. The secreted TNFa then binds onto its receptors and activates NFKB. This
SUggested that the activation of NFKB by TRIP-dependent pathway occurs by a
secondary response through TNFa, thus resulting in an autocrine pathway for delayed

NFKB activation [100] (Fig. 2.2)-

35

LPS challenge leads to a quick production of TNFa peaking at 1.5 hours.
Excessive production of pro-inflammatory cytokines such as TNFa not only enhances
immune responses required for fighting against invading pathogens but can also
produce deleterious effects that perturb regular hemodynarnic and metabolic balances
in the system. TNFa is produced in very high levels during the early phase of the
response to LPS induced endotoxemia. TNFa in turn can affect the expression levels

of TLR4 suggesting some feedback regulation [101].

2.4.2 TNFa-a potent pro-inflammatory cytokine

Tumor necrosis factor-alpha (TNFa) is a pro-inflammatory cytokine primarily
secreted by activated macrophages and monocytes. It is expressed as a 26kDa type II
membrane-bound protein that self-associates into a bioactive homotrimer [102, 103]
Normally TNFa is kept in balance by other anti-inflammatory factors. But in case of
inflammation, this balance is shifted and TNFa levels increase which in turn cause
upregulation of adhesion molecules on the endothelium (such as ICAMl and VCAMI)
and cause stimulation of fibroblasts leading to their proliferation, and recruitment of
leukocytes to the site of inflammation. TNFa also stimulates the production and
release of other cytokines and chemokines from macrophages.

TNFa mediates its diverse effects through two cell surface receptors:
p55TNFR1 and p75TNFR2. TNFRl is constitutively expressed on almost all
nucleated cells whereas TNFR2 is expressed mainly on immune and endothelial cells

[104]. The TNFRl contains a death domain (DD) which is lacking in TNFR2 [105].

36

Figure 2.2: LPS activates NFKB and MAPK pathways

Stimulation of TLR4 receptor by LPS causes TLR4 to dimerize and recruit
MyD88. Myd88 binds IRAK4 which in turn phosphorylates IRAKl. IRAKI
binds TRAF6 which in turn binds a preformed complex bound to the
membrane consisting of TABl/TAKl/TABZ. Active TAKl then
phosphorylates and activates the IKK complex which in turn phosphorylates
and activates IKBa which undergoes proteasomal degradation releasing NFKB
subunits (p50 and p65) which then translocate to the nucleus to initiate gene
transcription. LPS also activates MAP kinases namely c-Jun N-terminal
kinase (INK), p38 and ERK1/2 MAP kinase.

In macrophages, however, stimulation with LPS leads to H(KB mediated
phosphorylation and degradation of NFicBl p105 releasing associated p50
subunit which translocates to the nucleus to modulate gene transcription. p105
degradation also liberates the associated MAP3 kinase (TPL-2) which then

activates ERK kinase. “Images in this dissertation are presented in color”.

37

 

  
  
 
  
     

.i ,1 j,_P\b p

Proteasomal
degradation

 

NFKB target .
- ene transcription

Transcription

38

Figure 2.3: TNFa stimulates NFKB and MAPK pathways

Binding of TNFa onto TNF receptor causes receptor trimerization, which
facilitates recruitment of adapter protein TRADD. TRADD leads to the
recruitment of RIPl to the signaling complex. RIP] acts as an adaptor that
facilitates recruitment of IKK complex to TNFR. H(KB then phosphorylates
and activates IKBa which undergoes proteasomal degradation releasing NFKB
subunits (p50 and p65) which then translocate to the nucleus to initiate gene
transcription. TNFa also activates MAP kinases namely c—Jun N-terminal
kinase (JNK), p38 and ERKl/2 MAP kinase.

In macrophages, however, stimulation with TNFa leads to IKKB mediated
phosphorylation and degradation of NFKBl p105 releasing associated p50
subunit which translocates to the nucleus to modulate gene transcription. p105
degradation also liberates the associated MAP3 kinase (TPL-2) which then

activates ERK kinase. “Images in this dissertation are presented in color”.

39

 

  
  
 
 
  
 
   

Proteasomal
degradation

 

 

» NFKB target
gene transcription

Transcription

40

 

Stimulation of TNFRI leads to the recruitment of DD-containing adapter
molecule, Tumor necrosis factor receptor 1 associated death domain protein (TRADD)
[106] followed by binding of DD-containing Ser/Thr kinase, Receptor interacting
protein-1 (RIPl) [107]. This signaling complex is then bound by other adapter proteins
such as TNF receptor associated factor 2/5 (TRAF2/5) and Inhibitor of apoptosis 1 (c-
IAPl) which subsequently leads to the activation of NFKB and MAPK pathways (Fig.

2.3) [108].

2.5: NFKB transcription factor in inflammation

NFKB regulates transcription of genes involved in inflammation, the innate and
adaptive immune responses, cell proliferation, cell adhesion, genes involved in
controlling programmed cell death (apoptosis), and genes involved in cellular stress
response and tissUe remodeling [109-113]. It was discovered as a transcription factor
that binds to the intronic enhancer of the kappa light chain gene (the KB site) in B cells
around 20 years ago. The NFKB family of transcription factors consists of five
members viz. NFKB-l (p105 precursor and p50), NFKBZ (p100 precursor and p52),
Rel A (p65), c-Rel and Rel B. All of these possess a Rel homology domain (RHD) at
the N-terminus which is required for homo and hetero dimerization as well as for
sequence specific DNA binding. Rel A, Rel B and cRel also contain a transcription
activation domain (TAD) at their C-terminus which is absent in the p50 and p52
subunits. Hence, p50 and p52 interact with other factors to positively regulate
transcription. NF KB is sequestered in the cytoplasm in the form of an inactive complex

with IKB family of inhibitory protein under unstimulated conditions [114]. The IKB

41

family consists of three classical members viz. IKBa, IKBB, and IicBe all of which are
characterized by the presence of multiple ankyrin repeats that are important for
binding to NFKB dimers and interfere with the nuclear localization signals present in
the NFKB. NFicBl (p105) and NFKBZ (p100) are synthesized as large precursors that
contain RHD at their N-terminal and multiple ankyrin repeats in their C-terminal
halves due to which they function as IKB like proteins. Proteolysis of the C-terminus
of these precursors yields mature P50 and P52 subunits [115].

Classical or canonical pathway of NFKB activation typically involves IKKB (subunit
of IKB kinase complex)-dependent phosphorylation and subsequent 26S proteasomal

degradation of IKBa, IKBB, and 168. The kinetics of phosphorylation and degradation

of IKBB and IKBe are however much slower than that of IKBO. (Fig. 2.4). The
proteasomal degradation leads to the release and translocation of NFKB (most
commonly the P50-Rel A dimer) to the nucleus [116] where it causes an increased
transcription of genes encoding for cytokines, chemokines, adhesion molecules
(ICAMl or Intercellular adhesion molecule 1, VCAMl or vascular cell adhesion
molecule 1, ELAM or endothelial leucocyte adhesion molecule 1), matrix
metalloproteinases (MMPs), cyclo-oxygenase 2 (COX2), inducible nitric oxide
synthase (iNOS) as well as inhibitors of apoptosis all of which are important part of an
innate immune response [117]. As further elaborated in chapters 3 and 4, our studies
both in cultured cell line as well as primary cells implicate GRK5 as an important
mediator of NFKB pathway by potentially phosphorylating the same residues as that of

IKKB. These results suggest that GRK5 might be a better and alternative target to

42

Figure 2.4: Mammalian NFKB and IKB family members

The Nuclear factor KB (NFKB) family has five members. These are Rel A
(P65), c-Rel, Rel B, p105/p50 (NFKBI) and p100/p52 (NF KB2). At the amino
terminal, there is a structurally conserved Rel homology domain (RHD). RHD
contains the dimerization, nuclear localization (N) and DNA-binding domains.
Rel A, Rel B and c-Rel also have a carboxy terminal non homologous
transactivation domain (TAD). Rel B also contains a leucine zipper motif (LZ).
Inhibitor of NFKB (IKB) family has IKBa, IKBB, IKBe and Bcl3 as members all
of which have multiple ankyrin repeats (ANK). p105 and p100 contain RHDs
at the amino terminus and ANK repeats at the carboxyl-terminus due to which
they act like IKB proteins. P105 and p100 undergo proteolytic processing to
generate p50 and p52 as NFKB proteins. The number of amino acids in each
protein are shown on the right. “Images in this dissertation are presented in

color”.

43

 

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44

that of IKKB, since IKBO. phosphorylation and NFkB activation is only partially
affected.

However, there is a more recently discovered alternative pathway of NFKB
activation which involves IKKa (subunit of IKB kinase complex)—dependent
phosphorylation and degradation of P100 and subsequent activation of p52:RelB
dimers [118]. Furthermore, IKKB dependent degradation of IKBa has been found to
occur within minutes whereas the kinetics of IKKo. dependent degradation of p100 is
slower and requires several hours.

IKBa is the best studied IKB family member and is known to regulate the
classical RelAszO heterodimers. In response to inflammatory stimuli such as LPS or
TNFa, it undergoes rapid degradation and is finally resynthesized in an NFKB
dependent fashion which constitutes a negative feedback loop whereby newly
synthesized IKB enters the nucleus and associates with deacetylated RelAszO
heterodimers shuttling them back to the cytoplasm [119, 120].

LPS and TNFa act as important stimulators of NFKB signaling pathway. NFKB
transcription factor is activated in tissues such as liver, lungs and spleen within 4 hours
of intra-peritoneal challenge of LPS [121]. It activates more than 150 genes, most of
which are pro—inflammatory in function. Deficient NFKB activation in intestinal
epithelium has been linked to an increased inflammation under in vivo conditions
suggesting that a defect in NFKB signaling can lead to immunosuppression which
triggers and also maintains inflammation [122].

NFKB has been found to be highly activated in several inflammatory disease

conditions such as sepsis, rheumatic diseases, inflammatory bowel disease, multiple

45

sclerosis etc. as well as in other diseases such as cancer and diabetes as detected by
electrophoretic mobility shift assays as well as tissue staining using NFKB specific
antibodies of biopsies from these patients. These changes are in turn accompanied by
an increased recruitment of inflammatory cells to the tissues. Hence, although a
prompt activation of NFKB is required for a good immune response, it should also be
terminated properly to prevent from reaching a stage of tissue damage, organ failure
and finally death.

Inhibition of NFKB activation has been shown by several studies to be
effective in controlling inflammatory disease conditions [123]. Several drugs used to
treat inflammatory disease conditions have effects on NFKB activity. Anti TNFa drugs
have been found to be highly effective in controlling inflammation and are widely
used against rheumatoid arthritis. Moreover, anti-inflammatory drugs such as
corticosteroids, aspirin and other non-steroidal anti-inflammatory drugs (NSAIDS)
although do not directly target NFKB, they do control the expression of genes
regulated by NFKB [124].

In sepsis, inhibition of NFKB activation prevents LPS-induced iNOS and
COX2 mRNA and protein expression and activity. NFKB inhibition also corrects
cardiovascular functional abnormalities as well as help restore systemic hypotension
in sepsis [125]. Inhibition of IKK activity also reduces cytokine production and
decreases infiltration of neutrophils in lungs, liver and colon thus improving the rate of
survival in polymicrobial model of sepsis [126]. Inhibition of NFKB activation further
reduces LPS induced increases in microvascular permeability. In liver, inhibition of

NFKB activation has been found to suppress LPS induced cytokine and adhesion

46

molecule expression, reduce influx of neutrophils as well as prevent LPS induced
increase in microvascular endothelial permeability [127].

Although NFKB is an attractive target for therapeutic inhibition under certain
disease conditions, considering its role in normal cellular physiology as well as in
mounting effective immune response, its inhibition might have serious effects. Thus,
although it has been suggested by several studies that inhibition of NFKB activation in
viva leads to reduced inflammatory responses in sepsis, it also favors apoptosis in
immune cells which may lead to immunosuppression and fatal outcome in severe
sepsis.

As for instance, it was found that inhibiting NFKB exacerbated acute
inflammation but helped attenuate chronic inflammation in the intestinal tract. NFKB
protects against epithelial cell apoptosis which was reduced leading to an aggravation
of an acute inflammatory response. In chronic inflammation however the risk of
epithelial cell apoptosis is absent, thus NFKB inhibition is beneficial under such
conditions [128]. In another study, it was found that mice with a targeted deletion of
IKKB in myeloid cells are more susceptible to endotoxic shock due to an increased
production of plasma IL-lB suggesting the possible complications that could arise
from IKKB inhibition [129]. Blockage of NFKB could also compromise normal host
defenses. It was shown that mice were unable to clear opportunistic infections such as
those involving Listeria monocytogenes after NFKB inhibition [130]. Another study
showed that blocking NFKB pathway in LPS model of sepsis inhibited inflammatory
as well as injury promoting responses thereby improving survival. On the other hand,

blocking NFKB pathway in a bacterial model of sepsis inhibited host defense

47

responses in addition to inhibiting inflammatory and injury promoting responses. Thus
the beneficial effects of inhibiting inflammatory responses would be compromised by
the impairment of bacterial clearance capacity. Hence, the best approach would be to
identify specific targets in the NFKB signaling pathway in different disease conditions

for therapeutic targeting rather than considering global inhibition of NFKB.

2.6: MAPKs in inflammation
Cells continuously respond to signals in the extracellular environment by sensing
through cell surface receptors and further transmitting the signals to the cytosol and
nucleus by activating signal transduction pathways. MAPKs constitute a family of
highly conserved serine-threonine kinases that play a role in several cellular processes
such as cell proliferation, cell survival/apoptosis, differentiation as well as cell stress
and inflammatory conditions.
2.6.1: ERKl/Z kinase

ERK1/2 is one of the three MAP kinases that is induced under inflammatory
conditions by LPS as well as TNFa. Activation of ERK cascade may be responsible
for monocyte and macrophage reprogramming and thus dysregulation of cytokine
cascade. It has been suggested that MEK-ERK1/2 pathway is activated in a Raf-1
dependent manner by LPS as demonstrated by the inhibition of LPS induced TNFa
production upon repressing Ras or Raf-l [131]. Furthermore, inhibition of MEK in
monocytes causes reduction in the LPS induced production of certain pro-
inflammatory cytokines such as We, IL-1, IL-8 and PGE2 which suggests that ERK

plays a crucial role in inflammatory gene expression [132]. ERK inhibitors have been

48

found to be useful in reducing inflammation in an ear edema model in mice as well as
experimental osteoarthritis model in rabbits [133].

In macrophages however, activation of ERK1/2 MAP kinase requires a serine-
threonine kinase TPL2 (Tumor progression locus 2) also known as COT. TPL2 in turn
is present in a complex with NFKBI p105. Stimulation with LPS causes
phosphorylation and proteasomal mediated degradation of NF KBl p105. This releases
p50 as well as TPL2. In unstimulated macrophages, TPL2 MEK kinase activity is
blocked due to its association with p105. Released TPL2 fimctions as a MAP3 kinase
which phosphorylates MAP2 kinase MEK1/2 which in turn phosphorylates and
activates MAP kinase ERK1/2 [134]. It has been found that LPS upregulation of
TNFa and COX2 is reduced in TPL2 deficient macrophages due to a defective
ERKl/Z activation suggesting an important role of this pathway in mediating an
immune response [135, 136]. Thus, in macrophages LPS stimulation of IKK complex
plays a role in activating both NFKB and ERK pathways via NFKB] p105 regulation
(Figure 2.2). Interestingly, as described in the studiesin chapter 5, GRK2 appears to
be a negative regulator of this pathway in primary peritoneal macrophages. Previous
studies in Raw264.7 macrophage cell line however have shown that NFKB] p105-
ERK pathway is regulated by GRK5. The implications of our current findings in

primary cells are further discussed in chapter 5.
2.6.2: P38 MAPK

The p38 mitogen activated protein kinase is activated by diverse stimuli such

as UV light, heat shock, certain mitogens, LPS as well as pro—inflammatory cytokines

49

such as IL-1 and TNFa. It has been shown that TNFa stimulation of neutrophils
causes an enhanced p38-MAPK dependent phosphorylation and activation of PLA2
which is a primary regulator of arachidonate signaling [137]. Hence, p38 directly
affects arachidonate signaling which can both stimulate and suppress inflammatory
responses. P38 also plays a role in the production and release of cytokines such as
TNFa and IL-1 [138]. P38 also plays a major role in the movement of neutrophils to
inflammatory sites. P38 is involved in increasing the expression of ICAM-1 (adhesion
molecule) on vascular endothelium which is required for binding of neutrophils
through L-selection in the initial steps in the migratory process. Furthermore, p38 is
required in the efficient generation of reactive oxygen species by NADPH oxidase
activity in response to multiple agonists once the neutrophils reach their destination
[139]. P38 has also been suggested to play a role in some chronic neuronal diseases
such as Alzheimer’s where it is found to be activated in association with the
neurofibrillary tangle-bearing neurons containing the aggregated paired helical
filament protein tau [140]. P38 is also required for the angiogenesis in inflammation in
a murine model of collagen induced arthritis [141]. P38 is required for the
upregulation of Bl bradykinin receptors. These receptors are present at very low levels
in most tissues. However, they are strongly upregulated after various types of tissue
injury. It acts as a potent vasodilator resulting in increased capillary permeability
which causes accumulation of extracellular water bed in conditions such as acute
respiratory distress syndrome [142]. Several studies have shown the role of p38
MAPK in sepsis. In one such studies, it was shown that the p38 MAPK activity was

markedly increased in splenic and peritoneal macrophages and inhibition of p38

50

MAPK markedly improves survival in a polymicrobial ceacal ligation and puncture

(CLP) model of sepsis [143].

2.6.3: JN K kinase

JNK is the third important MAP kinase that is activated under inflammatory
stimuli. LPS has been shown to activate JNK kinase in THPl monocytic and Raw
264.7 cells as well as other cell types. JNK kinase induces transcription factors namely
AP-l, c-Jun, ATP-2, and Elk-1, all of which are important mediators of inflammatory
gene transcription. JNK activation of AP-l is important for synthesis of TNFOL, as well
as proliferation and differentiation of T-cells. JNK is found to be activated in joint
synoviocytes suggesting an association to rheumatoid arthritis possibly due to an
enhanced production of TNFOL. However, JNK deficiency has been found to reduce
the progression of experimental autoimmune encephalitis (EAE), due to an increase in

the expression of an anti-inflammatory cytokine IL-10 by macrophages [144].

2.7 : Septic shock

Encounter with microbes or their components such as LPS causes the body to
initiate an innate immune response, the purpose of which is to defend the body against
infection. Sepsis or septic shock is a very complex clinical condition which results
when the normal host response to infection goes unchecked leading to tissue damage
and ultimately multiple organ failure. Sepsis is a leading cause of death amongst
critically ill patients [145]. In United States, it accounts for nearly 250,000 deaths

annually [145]. The initial clinical features are characterized by fever, transient

51

hypotension, decreased urine output and thrombocytopenia which progresses into
profound hypotension, coagulation abnormalities and multiple organ failure.
Mononuclear cells play a key role in this process by releasing an array of pro-
inflammatory cytokines and chemokines such as IL-1, IL-6, TNFa as well as other
molecules such as reactive oxygen species (ROS), platelet activating factor (PAF) and
nitric oxide (NO). These pro-inflammatory cytokines play an important role in sepsis

by inducing a complex network of secondary responses to fight infection.

2.7.1 Animal models of sepsis

There are three primary methods of inducing sepsis in animals: a) By injection
of an exogeneous toxin such as LPS, b) By altering the animal’s endogenous
protective barrier. This involves methods such as inducing an intestinal leakage as is
done by ceacal ligation and puncture (CLP) or by colon ascendens stent peritonitis
(CASP) and o) By infusing exogenous bacteria.

Endotoxin (LPS) is a component of the outer membrane of gram negative
bacteria and plays an essential role in the pathogenesis of sepsis. LPS administration /
injection induces systemic inflammation with increase in pro-inflammatory cytokines
such as TNFa and IL-1 but without bacteremia. The CLP model involves performing a
surgery whereby ceacum is ligated distal to the ileocecal valve and then punctured
using a needle which leads to the release of fecal contents into the peritoneum causing
polymicrobial bacteremia and sepsis. The severity of sepsis produced can be adjusted

based on the length of the ligated cecum as well as the size / number of punctures. The

52

 

bacterial infusion model of sepsis can approximate introducing a single pathogen in a
controlled manner thus allowing reproducible infection.

Each of these methods has its advantages and disadvantages as listed in Table
2.4. For example, LPS injection model is very simple and also a sterile method.
Furthermore the dose of LPS can be titrated. This model can be used to mimic early
sepsis as is seen in human patients where there is little hemodynarnic compromise.
This is very useful to study systemic and renal responses during initial phases of sepsis
due to the fact that a lower dose of LPS does not cause any systemic hypotension
despite decreasing glomerular perfusion. CLP model has an advantage that it shows a
similar cytokine profile as is seen in human sepsis. Furthermore, multiple bacterial
species are observed in circulation similar to human sepsis. The disadvantage being
the strain variability and also that the standard CLP model does not develop
reproducible acute lung or kidney injury as seen in human sepsis.

Despite the fact that sepsis is mostly characterized by the presence of multiple
species of bacteria, human sepsis can also be caused by a single species. Bacterial
infusion model can be used to study the sepsis caused by a single species as well as for
mimicking pneumonia and other nosocomial infections. The single pathogen can be
introduced in a controlled manner. This model essentially provides complimentary
information in a pathogen specific manner.

Out of these three different models of sepsis, LPS injection or infusion has
been widely used model in sepsis research. Infusion or injection of LPS induces a

systemic inflammatory response that resembles several initial clinical

53

Table 2.4: Animal models of sepsis and their advantages verses

disadvantages

Slightly modified from

Doi, K., Leelahavanichkul, A., Yuen, PS, and Star, RA. (2009) The Journal
of clinical investigation 119(10), 2868-2878

54

 

Animal model

Advantage

Disadvantage

 

LPS injection

Simple and sterile with some
similarities to human sepsis
pathophysiology

Early and transient increase in
inflammatory mediators more
intense than in human sepsis

 

Early silent period;

Age and strain variability;

 

 

CLP or CASP moderate and delayed peak of early hemodynarnic period in
mediators; some models
multiple bacterial flora
Infusion or No change in intrarenal
instillation of Early hyperdynamic state microcirculation;
exogenous need large animals;
bacteria labor intensive

 

 

 

55

 

characteristics of sepsis although there is no bacteremia. As compared to human sepsis,
LPS injection/infusion however causes an earlier induction of cytokines. The cytokine
levels are also higher in this case as compared to that observed in human sepsis.
However, an exception to this is meningococcal sepsis as cytokine levels observed in
LPS injection/infusion model are comparable to what is seen in meningococcal sepsis

[146, 147].

2.7.2 Different animal species as models for sepsis

In general small animals are preferred for sepsis research due to the fact that
they can be generated as genetically similar, are relatively inexpensive and can be
maintained as pathogen free. However, large animals have also been utilized for this
purpose. For example, pig model has been used due to the similarity of its
cardiovascular, renal and gastrointestinal anatomy and physiology to that of humans.
Primates, in particular baboons are immunologically similar to humans thus making
them good models to study the cytokine response[l48]. There are species differences
in the sensitivity to endotoxins. Moreover, within a particular species also there are
differences in response based on sex, maturity levels, diet as well as estrus states.
Furthermore, transgenic animal models with either deletions or overexpression of
SPecific gene products are being widely used today to study the roles of particular

genes.

2.7.3 Neutrophils and Monocyte/ macrophages in sepsis

56

Cells of myeloid lineage are known to play an important role in sepsis.
Neutrophils are the first and most abundant cells which arrive at the infection site.
These cells have large stores of proteolytic enzymes as well as have a machinery to
generate reactive oxygen species (ROS) and reactive nitrogen species (RNS) which
can degrade internalized pathogens. Damage to the host tissues in sepsis can also
occur due to premature neutrophil activation during migration and by extracellular
release of cytotoxic molecules as well as amplification of acute inflammatory
responses. Neutrophils constitutively favor apoptosis which is important for resolution
of inflammation and cell turnover. It is important for neutrophils to undergo apoptosis
soon after they kill microbes using ROS, RNS and proteolytic enzymes since delayed
clearance of neutrophils can also contribute to organ injury. Apoptotic clearance of
cells induces anti-inflammatory effects in tissues. Neutrophils from patients with
sepsis has been shown to have a prolonged in vitro survival as well as increased
cellular activation [149]. Although molecules such as ROS and RNS have beneficial
physiologic functions such as their involvement in intracellular signaling for cytokines,
redox regulation as well as defense mechanisms against pathogens, overproduction or
reduced scavenging of these molecules can cause oxidative / nitrosative stress which

plays a key role in enhancing sepsis [150].

2.7.4 Coagulation defect in sepsis
One of the hallmark clinical features of sepsis is the disorders of coagulation.
Pro-inflammatory cytokines, in particular IL-1 and IL-6 induce coagulation during

sepsis. IL-10, on the other hand regulates coagulation by inhibiting the expression of

57

tissue factor (TF) on monocytes. A more severe form of coagulation is Disseminated
intravascular coagulation (DIC) which is seen in 30-50% of the cases. Coagulation is
initiated by the microbial components by inducing the expression of tissue factor on
mononuclear and endothelial cells. The tissue factor then activates a series of
proteolytic signaling cascades resulting in the formation of thrombin from
prothrombin ultimately generating fibrin from fibrinogen. Furthermore, high plasma
concentrations of Plasminogen-activator inhibitor type-1 (PAI-l) prevent formation of
plasmin from plasminogen thereby impairing the normal regulatory fibrinolytic
mechanisms (breakdown of fibrin by plasmin). Also, it has been found that a state of
sepsis induces downregulation of antithrombin, protein C and tissue factor pathway
inhibitor, all of which are naturally occuring anticoagulants. These proteins also
possess anti-inflanunatory properties: For eg. activated protein C directly inhibits the
production of TNFa by inhibiting the transcription factors NFKB and AP-l in
monocytes [151]. Hence, there is an increased production and reduced removal of
fibrin which causes the deposition of fibrin clots in small blood vessels, impairing
tissue perfusion and ultimately resulting in an organ failure. In our sepsis studies, we
observed that the blood in our myeloid specific GRK2 deleted mice starts undergoing
coagulation after around 12 hours of LPS injection. Complete blood count for the
blood showed no significant differences except eosinophilia in GRK2 deleted mice as
compared to control mice (Discussed in chapter five). However, the importance of this

observation is not clear as yet.

2.7.5 Multiple organ failure

58

The end stage in sepsis is characterized by multiple organ failure leading
ultimately to death. However, the pathogenesis of multiple organ failure is highly
complex and incompletely understood. Tissue hypoxia and hypoperfiision play an
important role. In addition to hypoxia, dysoxia is also seen which is characterized by a
state of inability to utilize the available oxygen. Furthermore, adequate oxygenation of
tissues is also compromised due to the development of tissue exudates in sepsis.
Microvascular occlusion occurs due to a widespread deposition of fibrin leading to
tissue hypoperfusion.

Cellular infiltration of tissues, in particular by neutrOphils damage tissues by
releasing lysosomal enzymes as well as superoxide derived free radicals. TNFa and
some other cytokines such as IL-1 causes an increased expression of inducible nitric
oxide synthase (iNOS) leading to an enhanced production of nitric oxide (NO) which
contributes further to vascular instability and also causes direct myocardial depression
seen in sepsis [152]. Sepsis is characterized by high cardiac output and a reduced
peripheral resistance due to dilatation of systemic resistance vessels which causes a
progressive systemic hypotension leading finally to organ dysfunction due to impaired
organ perfusion [153]. It is believed now that multiple organ dysfunction occurs due to
a combination of factors such as NO overproduction, antioxidant depletion, decreased

ATP concentration, and mitochondrial dysfunction [154].

59

Hypothesis and Specific Aims

GRKs, in particular GRK2 and GRK5 are highly expressed in immune cells.
Furthermore, several studies have established that the expression levels of GRKs
change in the immune cells such as neutrophils and macrophages under inflammatory
disease conditions such as rheumatoid arthritis and sepsis. However, the physiological
importance of such changes in the expression levels of GRKs is not well established.
To understand the physiological importance of GRKs in immune cell signaling as well

as their role in inflammatory diseases, I propose the following overall hypothesis:

Hypothesis: GRKs are important regulators of inflammatory signaling in
macrophages and therefore GRKs might play essential roles in the regulation of

inflammatory diseases.

Each of the chapters in this dissertation is dedicated to address the following specific

aims:

Aim 1: To determine the biochemical mechanisms by which GRKs regulate

TNFa—induced signaling pathway in mouse macrophages

The role of GRKs in TNFa signaling was examined using siRNA mediated
knockdown as well as by overexpression techniques, using cultured macrophage cell
line

Aim 2: To determine the role and mechanism by which GRKs regulate
inflammation in a mouse model of (_li_sease (Endotoxic shock)

60

Subaim 1: To study the role of GRK5 in inflammation in a GRK5 knockout mice
using endotoxic shock model

GRK5 knockout mice were procured from Jackson labs and the role of GRK5 in
inflammation was investigated by comparing the levels of pro-inflammatory cytokines,
survivability etc. between the knockout and wt control mice

Subaim 2: To generate and characterize a myeloid cell specific GRK2 knockout
mice and to study the role of GRK2 in inflammation using endotoxic shock model
A GRK2 deletion specifically in myeloid cells was achieved using a Cre—loxP
technology and the role of GRK2 in inflammation was investigated in these mice by
comparing the levels of pro-inflammatory cytokines, survivability etc. between the

knockout and littermate control mice.

Importance:

The findings from these studies will have a great impact on our current understanding
on the roles of GRKs in inflammatory signaling. The in vivo studies in knockout mice
will further strengthen our in vitro findings as well as establish a previously

unidentified role of GRKs in sepsis.

61

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79

CHAPTER 3

G-protein coupled receptor kinases mediate TNFa—induced NFKB
signaling via direct interaction with and phosphorylation of IKBa

This chapter represents a manuscript that was published in Biochemical Journal
(2010) 425, 169-178

Authors who contributed towards this study were: Sonika Patial, Jiansong Luo, Katie
J. Porter, Jeffrey L. Benovic and Narayanan Parameswaran

80

ABSTRACT

Tumor necrosis factor-or (TNFOL) is a multifunctional cytokine involved in the
pathophysiology of many chronic inflammatory diseases. TNFOL activation of the
nuclear factor KB (NFKB) signaling pathway particularly in macrophages has been
implicated in many diseases. We demonstrate here that G-protein coupled receptor
kinase-2 and 5 (GRK2 and 5) regulate TNFor-induced NFKB signaling in Raw264.7
macrophages. RNAi knockdown of GRK2 or 5 in macrophages significantly inhibits
TNFa-induced IKBOL phosphorylation and degradation, NFKB activation, and
expression of the NFKB-regulated gene, macrophage inflammatory protein-1B.
Consistent with these results, over-expression of GRK2 or 5 enhances TNFa-induced
NFKB activity. In addition, we show that GRK2 and 5 interact with IKBOt via the N-
terminal domain of IKBOt and that IKBOL is a substrate for GRK2 and 5 in vitro.
Furthermore, we also find that GRK5 but not GRK2 phosphorylates IKBOt at the same
amino acid residues (Ser32/36) as that of IKKB. Interestingly, associated with these
results, knockdown of IKKB in Raw264.7 macrophages did not affect TNFor-induced
IKBOL phosphorylation. Taken together, these results demonstrate that both GRK2 and

5 are important and novel mediators of a non-traditional IKBa-NFKB signaling

pathway.

81

INTRODUCTION

Tumor necrosis factor-alpha (TNFor) is a pleiotropic cytokine secreted by
immune cells, in particular by monocytes and macrophages and mediates a number of
biological activities ranging from cell proliferation, differentiation and death to
inflammation and innate and adaptive immune responses [1]. Irnportantly, TNFOL has
been implicated in the pathogenesis of chronic inflammatory diseases such as
rheumatoid arthritis and Crohn’s disease and several drugs targeting the TNFOL system
are already in clinical practice [2]. TNFa mediates its diverse effects through two cell
surface receptors: p55TNFR1 and p75TNFR2. TNFRl is constitutively expressed in
most nucleated cells whereas TNFR2 is expressed mainly in immune and endothelial
cells. Stimulation of TNFRI leads to the recruitment of several death domain
containing adapter proteins such as TRADD (Tumor necrosis factor receptor 1
associated death domain protein) and RIPl (Receptor interacting protein-1). This
signaling complex interacts with adapter proteins such as TRAF2/5 (TNF receptor
associated factor 2/5) and c-IAPl (Inhibitor of apoptosis 1) which subsequently leads
to the activation of signaling pathways that regulate many of the important biological
activities of TNFa [3]. Especially important from a physiological as well as
pathological perspective is the major role of the “Nuclear factor K B (NFKB)”
signaling pathway in chronic inflammatory disease [4].

The NFKB family of transcription factors regulate genes involved in
inflammation, innate and adaptive immune responses, cell proliferation, cell adhesion,
programmed cell death (apoptosis), and cellular stress response and tissue remodeling

(reviewed in [5]). The NFKB family members include p65 (RelA), p50, RelB, cRel

82

and p52. These NF KB transcription factors are sequestered in the cytoplasm in the
form of homo- or heteromeric complexes with the inhibitory proteins of the IKB
family. The IKB family members include IKBOL, IKBB, IKBe, p105 (NFKBl), and p100
(NFKB2). Activation of the NFKB pathway typically involves the phosphorylation of
an IKB member primarily by the IKB kinase (IKK) complex. The phosphorylated IKB
then undergoes ubiquitination and subsequent proteolysis leading to the release and
translocation of NFKB into the nucleus where it affects gene transcription (reviewed in
[5]. Amongst the various IKB members, IKBOL plays a critical role in the early
activation of NFKB after ligand stimulation [6]. IKBa binds to NFKB subunits p50 and
p65 under unstimulated conditions. Upon stimulation IKBOL undergoes rapid
phosphorylation by IKKB, followed by ubiquitination and degradation. IKBOL
degradation releases the NFKB subunits p50 and p65, which then translocate into the
nucleus to evoke NFKB-dependent gene transcription (reviewed in [5]. In addition to
being physiological substrates for the IKK complex of enzymes, the IKB family
members have also been shown to be substrates for other kinases [7]. In this context,
we demonstrated recently that p105 is a substrate for G-protein coupled receptor
kinase-5 (GRK5) and that GRK5 phosphorylation of p105 regulates Toll-like receptor-
4-induced p105 phosphorylation in macrophages [8].

G-protein coupled receptor kinases (GRKs) are serine/threonine kinases
originally discovered for their role in the phosphorylation of G-protein coupled
receptors (GPCRs) (reviewed in [9]). The seven mammalian GRKs are divided into

three subfamilies based on sequence and functional similarities. The rhodopsin kinase

subfamily (GRKl and GRK7); the GRK2 subfamily (GRK2 and GRK3) and the

83

GRK4 subfamily (GRK4, 5 and 6). All GRKs have a similar structural organization
possessing N-terminal, catalytic and C-terminal domains. Interestingly, recent studies
have shown that GRKs have functions that go beyond their role in GPCR
phosphorylation. For example, GRKs have been shown to phosphorylate a number of
cytoplasmic and nuclear proteins as well as additional classes of membrane-localized
receptors [IO-12] Moreover, yeast-two hybrid analysis identified NFKB] p105 as a
GRK2-interacting protein thttp://www.signalinggateway.orgldata/YZH/cgi-
bin/y2h.cgi) while we found that GRK5 but not GRK2 regulates p105 function in
macrophages [8].

To better understand the role of GRKs in NFKB signaling in macrophages and
to identify the potential role of these kinases in the regulation of other IKB members,
we tested the role of GRK2 and 5 in the regulation of IKBOt in the context of TNFor
signaling in macrophages. Surprisingly, in contrast to the regulation of p105, our
studies reveal that both GRK2 and 5 regulate the TNFor-induced IKBa-NFKB pathway
in Raw264.7 mouse macrophages. We further demonstrate that IKBOL directly interacts
with GRK2 and 5 and is differentially phosphorylated by GRK2 and 5. Our
observations add new insight into NFKB signaling as well as demonstrate novel

GPCR-independent roles for GRKs.

84

MATERIALS AND METHODS:

Materials

Mouse TNFa was from PeproTech Inc. (Rocky Hill, NJ). IRDye® 700

labelled NFKB oligonucleotide probes were from LI-COR Biosciences (Lincoln, NE).
Protease inhibitor cocktail tablets were from Roche Diagnostics (Indianapolis, IN).
Luciferase assay buffer and Substrate were from Promega (Madison, WI). All other
reagents were from Sigma unless otherwise noted.

Antibodies: Anti-P50, P65, Lamin B, Protein A/G PLUS-agarose beads, Actin-HRP
and polyclonal IKBOL antibody were from Santa Cruz Biotechnology (Santa Cruz, CA),
anti-mouse IKBCX and phosphO-IKBCX antibodies were from Cell Signaling Technology
(Boston, MA), HA monoclonal antibody was from Covance and HA polyclonal
antibody from Sigma. GRK2 and 5 monoclonal antibodies were from Upstate
Biotechnology.

Plasmids: pcDNAGRKZ, pcDNAGRK2-K220R, pcDNAGRKS and pcDNAGRKS-
K215R have been described previously [13, 14]. HA-GRK2 was created by excising
GRK2 from pRKS-GRKZ construct with EcoRI and SalI and then inserting into pHA-
CMV vector. Expression plasmid pCMX-IKBa (Addgene plasmid 12331) was from
Dr. I. M. Verrna [15], HA-IKKB (Addgene plasmid # 15470) from Dr. H. Nakano
[16], Flag-IKKy (Addgene plasmid # 11970) from Dr. J. D. Ashwell [17], and Flag-
IKBa from Dr. G. Pei [18]. Adenoviruses expressing GRK2 and GRK5 were kindly
provided by Dr. W. J. Koch.

Cell Culture

85

Raw 264.7 macrophages and HEK293T cells were obtained from ATCC and
were grown in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with

10% fetal bovine serum (Invitrogen) and penicillin (100 units/ml) and streptomycin

(100 pg/ml) at 370 C in 5% C02.

RNA interference

Control siRNA pool (against luciferase gene not expressed in macrophages),
GRK2 siRNA pool (against mouse GRK2), GRK5 siRNA pool (against mouse GRK5)
and IKKB siRNA pool (against mouse IKKB) were purchased from Dharrnacon
(Dharmacon Research Inc., Lafayette, CO). Raw264.7 macrophages were transfected
with siRNA using Amaxa nucleofector (Program D-O32) as described previously [8].
The cells were analyzed for knockdown by Western blotting after 48 hours of
transfection.
Luciferase assay

HEK293T cells were transfected with an NFKB promoter luciferase plasmid
(pELAM luciferase plasmid [19, 20] kindly provided by Dr. E. Latz, University of
Massachusetts) and LacZ-expression plasmid [21] (kindly provided by Dr. Philip B.
Wedegaertner, Thomas Jefferson University) together with either vector (control) or
GRK2/5 wild type or GRK2-K220R/GRK5-K215R kinase deficient expression
plasmids. Cells were stimulated with TNFoc for 8 hours, lysates prepared and analysed
for NFKB luciferase activity using Promega Luciferase Reporter Assay kit according
to manufacturer’s instructions. B-Gal activity was determined as described previously

[21]. NFKB luciferase activity was expressed as a ratio of luciferase to beta-

86

galactosidase activity. All experiments were performed in triplicate and repeated at
least 3-5 times.
Nuclear Extracts

Nuclear extracts were prepared as described in Nuclear extraction kit
(Panomics, CA). Briefly, control or GRK2 knockdown cells in a 60 mm plate were
lysed in 500 pl of the buffer A mix [buffer A (100 mM HEPES, pH 7.9, 100 mM KCl,
100 mM EDTA), 100 mM DTT, 10 pl protease inhibitor cocktail and 0.38% NP-40].

Sonication was done to disrupt the cell clumps. The lysate was then centrifuged at a

maximum speed (15,000 x g) for 3 min at 4°C. Supematants (cytosolic fraction) were

collected and the pellet suspended in 100 pl of buffer B mix [buffer B (100 mM
HEPES, pH 7.9, 2 M NaCl, 5 mM EDTA), protease inhibitor cocktail and 100 mM
DTT). The lysates were kept on a rocking platform for ~2 hours, after which the
lysates were centrifuged at high speed for 20 min and the supematants (nuclear
fraction) were collected for further analysis.

Electrophoretic mobility shift assay (EMSA)

Electrophoretic mobility shift assays (EMSA) were performed using nuclear
extracts [6]. Briefly, ~9 fmol of double stranded oligonucleotide probes corresponding
to the human consensus NFKB sequence end labeled with IRDye® 700 were
incubated with 5 pg of nuclear extracts for 20 min in the dark at room temperature.
Samples were then subjected to electrophoresis using 6% non-denaturing

polyacrylamide gels and then analysed on LI-COR’s odyssey.

Co-immunoprecipitation and Western Blotting

87

HEK293T cells were transiently transfected using Fugene (Roche) with the
indicated expression plasmids in 60-mm cell culture dishes. The cells were washed
twice with ice-cold phosphate-buffered saline after 48 hours and were lysed in 400 pl
of cold lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 10% glycerol,
1% NP-40 containing protease and phosphatase inhibitors). Lysates were then

incubated with antibody and protein A/G PLUS-agarose beads for 1 hour at 4° C. The

beads were washed three times with lysis buffer and the immune complexes eluted
with 2X SDS sample buffer. The eluates were then run on SDS-PAGE and transferred
to nitrocellulose. Western blotting was then performed as described previously with
either fluorescently tagged secondary antibodies using Licor or with HRP-conjugated
secondary antibodies using chemiluminescence [22].
Glutathione S-transferase (GST) protein expression and purification

GST (GE HealthCare Biosciences, NJ), GST-IKBOt and GST-IKBa(Al-75)
[23] (kindly provided by Dr. Junan Li, Ohio State University) in pGEX bacterial
expression plasmids were expressed and purified as described by the manufacturer
(Amersham Biosciences). Briefly, overnight bacterial cultures were diluted 1:100 in
100 ml of fresh LB-ampicillin, grown for 2-3 hours until the OD reached 0.5-0.9. The
culture was then induced with IPTG (1 mM) and incubated for another 3 hours after
which the cells were pelleted (6000 x g for 10 min at 4° C) and resuspended in ice
cold PBS. Suspended cells were then disrupted using a sonicator and solubilized in 1%

Triton-X-IOO. The cells were then centrifuged at 12,000 x g for 10 min at 4° C and the

supematants collected. These supematants were incubated with 2 ml of the 50%

slurry of Glutathione Sepharose 4B equilibrated with PBS for 30 min and the resin

88

was transferred to a column (Empty Disposable PD-IO column). The column was
washed 3 times with PBS after which the fusion proteins were eluted by adding 1 ml
of elution buffer (50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0). The purity
and integrity of the fusion proteins were analysed and confirmed by SDS-PAGE and
Coomassie blue staining.
Overlay assay

Interaction between GST fusion proteins and GRK2 or 5 were determined
using an overlay assay [24]. Briefly, GST-fusion proteins (5 pg) were resolved on a

10% SDS-PAGE gel and transferred to nitrocellulose membranes. Membrane was then

blocked with 5% w/v fat-free milk in TBS-T and incubated overnight at 4° C in lysates

(~400 pg total protein) of HEK293T cells expressing vector, HA-GRKZ, or GRK5.
Blots were then washed three times with TBS-T (Tris- Buffered saline-Tween 20) and
immuno blotting performed using appropriate antibodies. Blots were then stained with
Ponceau to confirm equivalent amount of GST-fusion protein loading.
Phosphorylation assay

In vitro phosphorylation reactions were performed using purified GRK2 and
GRK5 with IKBa as a substrate [8]. Purified GST-IKBa or GST (~200 nM each) were
incubated with 25 nM GRK2 or GRK5 at 30° C for 15 min in the presence or absence

of 2 mg/ml soybean phosphatidylcholine and 60 nM purified GBy in 20 mM Tris-HCI,

pH 7.5, 2 mM EDTA, 5 mM MgC12, 0.2 mM ATP, 1—2 pCi of [32P]ATP in a final

volume of 20 pl. Reactions were stopped by the addition of 5 pl of SDS sample buffer

and incubation at room temperature for 30 min. Samples were then electrophoresed on

a 10% SDS-polyacrylamide gel, the gel was dried, and 32P-labeled IKBO. was

89

visualized by autoradiography and quantified by excising the bands and scintillation
counting.
Real time Q-RT-PCR

RNA extraction and real-time Q-RT-PCR were performed as described
previously [22]. Briefly, total RNA was extracted using TRIzol reagent (Invitrogen).
After sodium acetate—ethanol precipitation and several ethanol washes, the RNA
integrity was verified by formaldehyde-agarose gel electrophoresis. Synthesis of
cDNA was performed with reverse transcriptase (RT) with the total RNA using the
superscript II kit with Oligo-dt (12—18) primers as recommended by the manufacturer
(Invitrogen). cDNA was amplified by PCR in a final reaction volume of 25 pl using
SYBR Green Supermix (Invitrogen) with 10 pmol of each primer for MIPIB and
cyclophilin (for normalization) (primers obtained from IDT DNA Technologies,
primer sequence available upon request). Real-time PCR was performed using
MX3000P (Stratagene) thermocycler and data analyzed using MX3000P software.
Statistical analysis

All values are represented as meaniSEM. Data were analyzed. and statistics
performed using GRAPHPAD PRISM software (San Diego, California) using
student’s t-test (for comparing two groups) and AN OVA (for comparing three or more

groups). P value of less than 0.05 was considered significant.

90

RESULTS
GRK2 mediates TN For-induced NFKB activation in Raw264.7 macrophages
Previous studies have shown that of the seven GRKs, GRK2 and 5 are
expressed at relatively high levels in immune cells including macrophages [25, 26].
Furthermore, recent studies have shown that GRKs can regulate GPCR as well as non-
GPCR signaling in macrophages [8]. Because TNF receptor-induced NFKB signaling
plays a major role in macrophage biology and has been implicated in many chronic
inflammatory diseases [4, 27], we explored the role of GRKs in TNFa-induced NFKB
signaling in macrophages. For this, we first tested the effect of GRK2 knockdown
using siRNA pool, on TNFor-induced IKBOL phosphorylation and degradation.
Treatment of control macrophages with TNFor caused a time-dependent increase in
IKBOL phosphorylation (at serines-32/36) and a subsequent decrease in IKBOL levels
(Fig. 3.1A). IKBa phosphorylation was maximal at 5 min after TNFor stimulation,
whereas, the consequent decrease in IKBOL levels was maximal at 15 min after
stimulation in control cells. However, the TNFor-mediated increase in IKBOt
phosphorylation and decrease in IKBa levels were both significantly blocked in GRK2
knockdown macrophages. Compared to the maximal stimulation of 95i5% at 5 min in
control cells, IKBO. phosphorylation reached only 40:l:2% in GRK2 knockdown cells
(Fig. 3.1B). Consistent with the effects on IKBOt phosphorylation, IKBOL degradation
was also inhibited in GRK2 knockdown cells compared to control cells. At 15 min
after treatment IKBOL levels reached 62:l:7% of untreated levels (=100%) in control
cells, whereas it reached only 942E5% in GRK2 knockdown cells (Fig. 3.1C).

Importantly, IKBOL levels did not differ between control and GRK2 knockdown cells in

91

the absence of TNFoc treatment (0136:0057 in control cells v/s 01341-0057 in
GRK2 knockdown cells), suggesting that the effect of GRK2 knockdown on IKBa
levels is specific for stimulated conditions.

Because I'KBa phosphorylation and degradation leads to subsequent release
and translocation of the NFKB subunits, primarily p50 and p65, we next examined the
nuclear levels of the NFKB subunits in control and GRK2 knockdown cells before and
after TNFor treatment. As with IKBa phosphorylation/degradation, TNFor-induced p5 0
and p65 nuclear translocation were significantly inhibited in GRK2 knockdown cells
compared to control cells (Fig. 3.1D). These effects of GRK2 are specifically due to a
decrease in nuclear translocation and not a decrease in expression levels of these
proteins since the cytosolic expression of p50 and p65 did not differ between control
and GRK2 knockdown cells. Associated with these findings, we also observed that
the NFKB binding activity (assessed by EMSA) was inhibited in GRK2 knockdown
macrophages (Fig. 3.1E).

Ftuthermore, to rule out the non-specific effects of the siRNA, we repeated
these experiments using individual siRNAs and obtained similar results (Fig. 3.2A).
Interestingly, the effect of GRK2 knockdown on IKBa was specific for TNFoc-induced
pathway because LPS-induced IKBfX degradation was not inhibited by GRK2
knockdown (Fig. 3.2B).

Over-expression of GRK2 causes an increase in TNFor induced IKBa
phosphorylation

To further rule out the possibility of non-specific effects of RNAi, we tested

the effects of GRK2 over-expression in TNFor-induced IKBOL phosphorylation in

92

Figure 3.1: GRK2 knockdown in macrophages inhibits TNFa—induced
IKBa phosphorylation and degradation

A, B and C. Raw 264.7 macrophages were transfected with either control
siRNA or GRK2 siRNA using Amaxa’s nucleofector. Forty-eight hours after
transfection, cells were serum starved for ~3 hours, and stimulated or not with
TNFa (25 ng/ml) for the indicated time points. Lysates were extracted and run
on SDS-PAGE, transferred to nitrocellulose and then immunoblotted with
primary antibodies against pIKBa, IKBa and tubulin. Secondary antibodies
were fluorescently labeled and the blots were developed using Odyssey’s
Licor as described before [8]. Representative blots for pIKBa, IKBOL, tubulin
and GRK2 are shown in (A). Quantitation is shown in (B) and (C).
***p<0.001 compared to control; *p<0.05 compared to control. N=7.

D. Raw264.7 macrophages with control or knockdown levels of GRK2 were
treated with TNFor as indicated. Nuclear lysates were then extracted and
immunoblotted for NFKB P50 and P65 (Rel A) as shown. Nuclear protein
lamin B is shown as a loading control. A representative experiment is shown.
E. Nuclear extracts (from D above) were incubated with IR700 dye-labeled
NFKB oligonucleotide probes and electrophoretic mobility-shift assay was
performed as described in the Materials and Methods. Gels were scanned on
Licor Odyssey (Licor Biosciences) to detect binding. A representative

experiment is shown.

93

 

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94

Figure 3.1. Continued. . ..

 

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95

Figure 3.2: Effect of different GRK2 siRNA oligos on TNFor-induced

IKBa. phosphorylation ,

A. Raw 264.7 macrophages were transfected with either control or GRK2
specific individual oligos (siRNA GRK2 #1 (top panel) and siRNA GRK2 #2
(bottom panel) using Amaxa’s nucleofector. Forty-eight hours after
transfection, cells were serum starved for ~3 hours, and stimulated or not with
TNFa (25 ng/ml) for the indicated time points. Lysates were extracted and run
on SDS—PAGE, transferred to nitrocellulose and then immunoblotted for
plea and tubulin. Representative blots from three similar experiments are
shown.

B. Raw 264.7 macrophages were transfected with either control siRNA or
GRK2 siRNA using Amaxa’s nucleofector. Forty-eight hours after
transfection, cells were serum starved for ~3 hours, and stimulated or not with
LPS (1 pg/ml) for the indicated time points. Lysates were extracted and run on
SDS-PAGE, transferred to nitrocellulose and then immunoblotted with
primary antibodies against IKBa and tubulin. Secondary antibodies were
fluorescently labeled and the blots were developed using Odyssey’s Licor as

described before [8]. Quantitation is shown as percent control.

96

 

 

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% control

 

 

 

Time (min)

97

 

 

Figure 3.3: GRK2 over-expression enhances TNFa-induced IKBa
phosphorylation in macrophages

Raw264.7 macrophages were infected with adenoviruses encoding empty
vector or GRK2 at 50,000 viral particles/cell. Forty-eight hours after infection,
cells were semm starved for 3-4 hours, and stimulated with TNFa (25 ng/ml)
for indicated times. Lysates were immunoblotted for pIKBa, IKBO. and tubulin.
A representative blot is shown in (A) and quantitation in (B). *p<0.05

compared to vector. N=3.

98

 

 

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99

 

macrophages. Raw264.7 macrophages were transfected with adenoviruses expressing
vector or GRK2 and the effect of TNFor-induced IKBa phosphorylation tested as
described earlier. As predicted, over-expression of GRK2 significantly enhanced TNFor-
induced IKBOL phosphorylation compared to vector controls (Fig. 3.3).
Role of GRK5 in TNFor-induced IKBa phosphorylation and degradation

We next tested whether other GRKs can regulate IKBa
phosphorylation/degradation. We especially focused on GRK5 since we previously
showed that GRK5 inhibits LPS-induced p105 phosphorylation in macrophages [8].
Interestingly, similar to the effects of GRK2, knockdown of GRK5 using siRNA pool
(Fig. 3.4A) significantly inhibited TNFoc-induced IKBa phosphorylation and degradation
(Fig. 3.4B, C & D). TNFa-stimulated IKBOL phosphorylation (Ser32/36) in GRK5
knockdown cells reached only 27i5% of the maximal response after 5 min compared to
100% in control cells (Fig. 3.4C). Similarly, IKBa levels after 15 min of TNFOt
stimulation reached 622t6% of untreated levels in control cells but only 99:5% in GRK5
knockdown cells (Fig. 3.4D). Unlike GRK2 knockdown, basal levels of IKBOL were
somewhat elevated after GRK5 knockdown (0.361i0.080 in control vs. 0.563i0.071 in
GRK5 knockdown cells). Similar to GRK2 siRNAs, individual siRNAs against GRK5
also gave similar results to that of the pool (Fig. 3.5A). Interestingly, over-expression of
GRK5 using adenovirus only modestly enhanced IKBOt phosphorylation (Fig. 3.58).

Role of GRKs is specific for IKBa-NFKB pathway

100

Figure 3.4: GRK5 knockdown inhibits TNFoi-induced IKBa
phosphorylation and degradation in macrophages

Raw 264.7 macrophages were electroporated with either control or GRK5
siRNA as indicated. Forty-eight hours after electroporation, cells were serum
starved for ~3 hours, and stimulated with TNFa (25 ng/ml) for the indicated
times. Lysates were immunoblotted for pIKBa, IKBO. and tubulin and blots
developed using Licor’s Odyssey. Representative blots for GRK5 knockdown
is shown in (A), pIKBa and IKBOt in (B). Quantitation for IKBOt
phosphorylation and IKBOL degradation are shown in (C) and (D) respectively.
***p<0.001 compared to control. N=6.

101

 

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0 5 15.30 0 5 15 30(Time,min)

 

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102

 

Figure 3.4. Continued....

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103

Figure 3.5 Effect of a different GRK5 siRNA oligo on TNFa—induced

IKBa phosphorylation

A. Raw 264.7 macrophages were transfected with either control or GRK5
specific individual oligo (siRNA GRK5 #1) using Amaxa’s nucleofector.
Forty-eight hours after transfection, cells were serum starved for ~3 hours,
and stimulated or not with TNFa (25 ng/ml) for the indicated time points.
Lysates were extracted and run on SDS-PAGE, transferred to nitrocellulose
and then immunoblotted for pIKBa and tubulin. Representative blots from
three similar experiments are shown.

B. Raw264.7 macrophages were infected with adenoviruses encoding empty
vector or GRK5 (kindly provided by Dr. W. Koch, Thomas Jefferson
University) at 1000 viral particles/cell. Forty-eight hours after infection, cells
were serum starved for 3-4 hours, and stimulated with TNFa (25 ng/ml) for
indicated times. Lysates were immunoblotted for pIKBa, and tubulin. A

representative blot from two such experiments is shown.

104

 

 

Control siRNA GRK5 siRNA1
0 5 15 30 0 5 1.5. 30(Time,min)

 

 

 

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105

 

 

In addition to IKBa phosphorylation, TNFor treatment also induces NFKBl p105 (another
member of the IKB family) phosphorylation at Ser932 [28]. Previous studies have shown
that GRK2 and 5 can interact with p105 and that GRK5 negatively regulates LPS-
induced p105 phosphorylation [8]. To determine whether the observed effects of GRK2/5
knockdown are specific for TNFoc-induced IKBOt phosphorylation or whether p105
phosphorylation can also be regulated in a similar manner, we examined TNFor-induced
p105 phosphorylation in control and GRK2/5 knockdown macrophages. Interestingly,
unlike IKBa phosphorylation, GRK2/5 knockdown did not affect TNFor—induced p105
phosphorylation (at Ser932) (Fig. 3.6A and 3.6B). This suggests that the role of GRK2/5
is specific for TNFor-induced IKBa-NFKB pathway.
Regulation of NFKB-dependent gene expression by GRK2 and Sin macrophages
IKBa-NFKB pathway was recently shown to be critical for the expression of
MIPlB (macrophage inflammatory protein 1B), one of the major chemokines expressed
by macrophages [29]. Therefore, we tested whether the role of GRK2 and 5 on the
TNFor-induced NFKB pathway extended downstream of NFKB activation to MIPlB
mRNA expression. Treatment of control macrophages with TNFoc induced MIPlB
mRNA expression by ~4-5-fold. This increase in MIPlB expression was significantly
blocked in both GRK2 and 5 knockdown macrophages (Fig. 3.7A and 3.7B). These
results demonstrate that GRK2 and 5 regulate TNFor-induced IKBOi-NFKB pathway as
well as its physiological gene expression target in macrophages.

Kinase activity of GRK2 and 5 is required for TNFa-induced NFKB-dependent gene

transcription

106

Figure 3.6 Role of GRKs is specific for IKBa-NFKB pathway

A. Raw 264.7 macrophages were transfected with either control siRNA or
GRK2 siRNA using Amaxa’s nucleofector. Forty-eight hours after
transfection, cells were serum starved for ~3 hours, and stimulated or not with
TNF or (25 ng/ml) for the indicated time points. Lysates were extracted and run
on SDS-PAGE, transferred to nitrocellulose and then immunoblotted with
primary antibody against phospho-p105. For quantitation, p-p105 bands were
normalized as described before [8]. Quantitation is shown as percent maximal
response. N=4.

B. Raw 264.7 macrophages were transfected with either control siRNA or
GRK5 siRNA using Amaxa’s nucleofector and treated as described above in

A. Quantitation is shown as percent maximal response. N=4.

107

10 20 30
Time (min)

-A- GRK2 siRNA

 

0

 

 

 

 

1251 -I—Control siRNA
125' -I—Contro| siRNA

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Time (min)
108

 

Figure 3.7 Expression of Macrophage inflammatory protein-1B (MIPl B),
an NFKB-regulated gene, is inhibited by GRK2 or GRK5 knockdown in
macrophages

A. Control or GRK2 knockdown Raw264.7 macrophages were treated or not
with TNFor for 24 hours and RNA extracted as described in the Methods.
Quantitative real-time RT-PCR was performed using MIPIB specific primers.
MIPlB mRNA levels were normalized to cyclophilin levels. ** p<0.01. N=3.
B. Control or GRK5 knockdown Raw264.7 macrophages were treated with
TNFor and mRNA levels of MIPl B and cyclophilin determined as described in
(A) above. *** p<0.001; **p<0.01. N=3.

109

EControl siRNA
-GRK2 siRNA **,
[—

A

O

O
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MIP1B mRNA

% maximal response
01 \I
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Time (hr)

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110

Figure 3.8 Kinase activity of GRK2 and 5 is essential for mediating
TNFa-induced NFKB transcriptional activation

A. HEK293T cells were transfected with vector, GRK2 wild type or GRK2-
K220R (kinase deficient mutant) expression plasmids along with pELAM-
luciferase and LacZ. Forty hours after transfection, cells were stimulated (in
triplicates) with different concentrations of TNFa (as indicated) for 8 hours.
Lysates were prepared and analysed for luciferase and B-galactosidase
activities. Quantitation was performed after normalizing luciferase activity to
B-galactosidase activity. Data is expressed as fold basal of the vector
transfected cells. Immunoblots showing expression levels of GRK2 wild type
and GRK2 kinase deficient mutant are shown in the top panel. *P<0.05. N=4.
B. HEK293T cells were transfected with vector, GRK5 wild type or GRK5-
K215R (kinase deficient mutant) expression plasmids along with pELAM-
luciferase and LacZ. Cell treatments, activity assays and quantitation were
performed as described in (A) above. Irnmunoblots showing expression levels
of GRK5 wild type and GRK5 kinase deficient mutant are shown in the top
panel. **P<0.0l N=4.

111

NFKB-luciferase activity
(Fold basal)

NFKB-lucrferase actrvrty
(Fold basal)
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112

To further explore the biochemical mechanism by which GRK2 and 5 mediate
TNFor-induced NFKB activity, we next tested whether the kinase activity is essential for
the observed effects of the GRKs. For this, we over-expressed vector or wild type or
kinase dead GRK2 or GRK5 in HEK293T cells along with an NFKB reporter plasmid
(pELAM luciferase) and LacZ (for transfection normalization). Forty-hours after
transfection, cells were serum starved (~3 hours) and were stimulated (or not) with TNFor
for 8 hours and luciferase and B-galactosidase activity determined as described in the
methods. In vector transfected cells, TNFa stimulation significantly increased NFKB-
luciferase activity (Fig. 3.8). As predicted, over-expression of wild type GRK2 or GRK5
significantly enhanced TNFa-induced NFKB activity while over-expression of kinase-
inactive GRK2 or GRK5 (GRK2-K220R or GRK5-K215R) had no effect (Fig. 3.8A and
3.8B). This demonstrates that the kinase activities of GRK2 and 5 are required for the
observed effects of GRKs in TNFor-induced NFKB signaling. Interestingly, as observed
with IKBa phosphorylation in macrophages, over-expression of GRK2 but not GRK2-
K220R enhanced NFKB-luciferase activity even in the absence of ligand stimulation (1.0
i 0.51 activity in vector cells compared to 2.35 at 1.07 in GRK2 and 0.70 :t 0.32 in
GRK2-K220R expressing cells). In contrast, over-expression of GRK5 or GRK5—K215R
did not significantly affect basal NFKB activity (1 i 0.14 activity in vector cells
compared to 1.51 :t 0.17 in GRK5 and 0.86 i 0.04 in GRK5-K215R expressing cells).
Interaction of GRK2 and 5 with NBC:

To define the biochemical mechanisms that mediate GRKs’ actions, we first
tested whether GRKs affect H(KB expression or activity. For this purpose, we knocked

down and over-expressed GRK2/5 in Raw264.7 macrophages and HEK293T cells

113

respectively and examined the levels of IKKB. Neither knockdown nor over-expression
of GRK2/5 affected IKKB levels in the presence or absence of ma (data not shown).
To further rule out the effect of GRKs on H(KB activity, we performed an in vitro IKKB
kinase assay using IKKB immunoprecipitated from cells over-expressing GRK2/5. In
vitro phosphorylation of GST-IKBOL by immunoprecipitated IKKB was not affected by
over-expression of either GRK2 or GRK5 (data not shown). Similarly, the interaction of
IKBor and IKKB was not affected by over-expression of GRKs in HEK293T cells (data
not shown). Based on these results we hypothesized that GRKs might mediate their
effects via directly interacting with and phosphorylating IKBa. To first examine if GRK2
or 5 can interact directly with IKBOL, we tested the ability of GST or GST-IKBa to bind
GRK2 and 5 using an overlay blot assay. For this, bacterially expressed and purified GST
or GST-IKBOL or GST-IKBa(76-302) were run on SDS-PAGE, and transferred to
nitrocellulose. The membranes were then incubated with HEK293T lysates over-
expressing either vector, GRK2 or GRK5 and tested for the ability of GRKs to
specifically bind to GST-IKBOL. As hypothesized, both GRK2 (Fig. 3.9A and 3.9B) and
GRK5 (Fig. 3.10A and 3.10B) bound to GST-IKBOL with no significant binding to GST.
In addition, neither GRK2 (Fig. 3.9A) nor GRK5 (Fig. 3.10A) interacted appreciably
with an N-terminal deletion mutant of IKBa [IKBa(76-302)], suggesting that both GRKs
primarily interact with IKBa at the N-terminus. We also tested the ability of GRKs to
interact with IKBa in intact cells and found that immunoprecipitation of IKBa co-
immunoprecipitated both GRK2 and 5 (Fig. 3.9C and 3.10C) in HEK293T cells; Fig.

3.9D in human monocytic cells THPl). Taken together, these results suggest that IKBCX is

114

Figure 3.9 GRK2 interacts with the N-terminus of IKBa

A. Purified GST-IKBa (full length), GST-IKBa (76-302) and GST alone, (5 pg
each) were resolved on a SDS-PAGE and transferred to a nitrocellulose
membrane. The membrane was incubated overnight in HEK293T lysates
over-expressing HA-GRK2 (A) or vector (B). Membrane was washed and
probed for HA-GRK2 binding to GST-IKBOL by immunoblotting using anti-
HA antibody. Western blot of the overlay assay is shown in the top panel and
a Ponceau stain of the overlay blot is shown in the bottom. Blots are
representative of at least four individual experiments.

C. GRK2 and IKBa were co-expressed in HEK293T cells and the lysates were
immunoprecipitated using IKBa polyclonal antibody and the blots were then
subjected to immunoblotting as indicated.

D. Lysates from THPl monocytic cells were immunoprecipitated with IKBa
polyclonal antibody. Immunoprecipitates were then denatured using sample
buffer, boiled and separated on a 10% SDS-PAGE gel. Irnmunoblotting was
performed for GRK2 as shown.

115

 

 

 

 

 

 

 

 

 

 

 

 

 

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116

 

Figure 3.10 Direct interaction between GRK5 and IKBa

A. Purified GST-IKBa (full length), GST-IKBa (76-302) and GST alone, (5 pg
each) were resolved on a SDS-PAGE and transferred to a nitrocellulose
membrane. The membrane was incubated overnight in HEK293T lysates
over-expressing GRK5 (A) or vector (B). Membrane was washed and probed
for GRK5 binding to GST-IKBOL by immunoblotting using anti-GRK5
antibody. Western blot of the overlay assay is shown in the top panel and a
Ponceau stain of the overlay blot is shown in the bottom. Blots are
representative of at least four individual experiments.

C. GRK5 and IKBa were co-expressed in HEK293T cells and the lysates were
immunoprecipitated using IKBa polyclonal antibody and the blots were then

subjected to immunoblotting as indicated.

117

 

 

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a direct interaction partner for both GRK2 and GRK5.

Phosphorylation of IKBa by GRK2 and 5

Experiments using kinase-dead mutants of GRK2 and 5, as well as the experiments
described above, suggest that IKBa may be a substrate for GRKs in TNFa-induced NF KB
signaling. To directly test this, we performed in vitro phosphorylation assays using
purified GRK2 and 5 with IKBa as the substrate. GRK5 effectively phosphorylated IKBa
to a stoichiometry of ~0.75 mol/mol (Fig. 3.11B) and interestingly, phosphorylation was
effectively attenuated by the addition of phospholipids, which normally activate GRK5
(Fig. 3.11D) [30]. In contrast, IKBa was a relatively poor substrate for GRK2 (Fig.
3.11A), although the phosphorylation was enhanced by the addition of GBy subunits and
phospholipids, known activators of GRK2 (Fig. 3.11C) [31]. Taken together, these
results demonstrate that IKBa is an in vitro substrate for both GRK2 and GRK5.

To identify the GRK phosphorylation sites in IKBct and to examine if IKBa is
differentially phosphorylated by GRK2 and 5, we first tested whether the known IKKB
phosphorylation sites (Ser32/36) are also phosphorylated by GRK2 or GRK5. Indeed,
previous studies have shown that these two residues are targeted by additional kinases
such as ribosomal S6K and CK 11, especially in NFKB pathways that are largely IKKB-
independent [32, 33]. To test this, we assessed the ability of GRK2 and GRK5 to
phosphorylate a GST-IKBa S32/36A mutant. Our results show that GRK2 mediated
phosphorylation of wild type and mutant GST-IKBoi was comparable, suggesting that
these sites are not phosphorylated by GRK2 (Fig. 3.11E). In contrast, GRK5 mediated

phosphorylation of the IKBO. mutant was decreased ~60% compared to wild type IKBa

119

Figure 3.11 IKBa is a substrate for GRK2 and GRK5 in vitro

A and B. Purified GRK2 and GRK5 were prepared as described before [30].
200 nM purified GST-IKBa or GST alone were incubated with 25 nM GRK2
or GRK5 in buffer containing 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 0.1
mM [y32P] ATP, 5 mM MgC12 and the phosphorylation reactions were done
for various time points as indicated. Reaction was stopped with SDS buffer,
electrophoresed on a 10% SDS-PAGE, the gel dried and subjected to
autoradiography. Phosphorylated IKBd bands were then counted on a liquid
scintillation for quantification.

C and D. The reactions were incubated in the absence or presence of
liposomes as indicated and phosphorylation reactions performed for 30 min
and stoichiometry quantified as described above. GRK2 phosphorylation
samples also contained GBy in addition to liposomes.

E and F. GRK2 and GRK5 were incubated with 200 nM purified GST-IKBa
(wild type or mutant S32A/S36A) in a kinase reaction and phosphorylated
IKBa quantified using liquid scintillation as described in the methods.

G. Purified GRK2, GRK5 or IKKB were incubated with GST-IKBa as a
substrate as described above without [y3zP]ATP. Reaction was stopped with
SDS buffer, electrophoresed on a 10% SDS-PAGE and transferred to a
nitrocellulose membrane. Immunoblotting was performed using an antibody

that specifically detects phospho-IKBOL(ser32).

120

mol Pi/mol IKBO.

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123

(Fig. 3.11F), suggesting that GRK5 phosphorylates one or both of these sites. This result
was confirmed using an antibody that recognizes phosphoSer32 in IKBa. For these
studies, GST-IKBa was initially phosphorylated in vitro using GRK2, GRK5 or IKKB and
the samples were run on SDS PAGE and immunoblotted using anti-IKBa-phospho-Ser32.
These results show that Ser32 is selectively phosphorylated by GRK5 but not GRK2 and
that GRK5 mediated phosphorylation of IKBa is comparable to that seen with IKKB (Fig.
3.11G). Overall, these results reveal that Ser32 is phosphorylated by GRK5 and that
GRK2 and GRK5 phosphorylate distinct residues.
IKKB knockdown does not inhibit TNFor-induced IKBa phosphorylation in
Raw264.7 macrophages

Similar to GRK5, other kinases such as casein Kinase II and ribosomal S6 kinase
have been shown to phosphorylate IKBa at Ser32/36. Interestingly, these kinases were
shown to selectively regulate IKBOt-NFKB pathway in an IKKB-independent manner.
Therefore, we hypothesized that because of the role of GRK2 and 5 as “IKBa kinases”,
IKKB may be dispensable in Raw264.7 cells, particularly for TNFa-induced IKBa
phosphorylation. To test this hypothesis, we knocked down IKKB in Raw264.7
macrophages and tested the effect of TNFor on IKBOL phosphorylation (Ser32/36). As
predicted, we found that TNFor-induced IKBa phosphorylation is not significantly
affected by knockdown of IKKB, suggesting that H(KB may be redundant in this system
(Fig. 3.12A). However, it is possible that the level of knockdown is not sufficient to
inhibit IKBa phosphorylation because of the residual IKKB kinase activity present. To
rule out this possibility, we further tested the ability of IKKB knockdown to inhibit LPS-

induced IKBa phosphorylation. Interestingly, our results demonstrate that LPS-induced

124

Figure 3.12 IKKB knockdown does not inhibit TNFa-induced IKBa
phosphorylation but inhibits LPS-induced phosphorylation

A. Raw 264.7 macrophages were electroporated with either control or IKKB
siRNA as indicated. Forty-eight hours after electroporation, cells were serum
starved for ~3 hours, and stimulated with TNFa (25 ng/ml) for the indicated
times. Lysates were innnunoblotted for pIKBa (Ser32/36) and tubulin for
normalization. Representative blots are shown at the top and quantitation at
the bottom. N=4.

B. Raw 264.7 macrophages were electroporated with either control or IKKB
siRNA as indicated. Forty-eight hours after electroporation, cells were serum
starved for ~3 hours, and stimulated with LPS (1 pg/ml) for the indicated
times. Lysates were immunoblotted for pIKBa (Ser32/36) and tubulin for

normalization. Quantitation is shown. N=4.

125

 

 

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126

 

IKBa phosphorylation (at Ser32/36) is inhibited by IKKB knockdown and this effect was
particularly evident at later time points (Fig. 3.12B). These results suggest that the IKKB
plays a crucial role in LPS-induced IKBa phosphorylation, but not in TNFor-induced
IKBOi-NFKB pathway in Raw264.7macrophages. Taken together, our results demonstrate
a critical role for GRK2 and 5 in the regulation of TNFor-induced IKBOt—NFKB pathway

in Raw264.7 macrOphages and suggest that IKBa phosphorylation by GRKs might be an

essential step in this regulation.

127

DISCUSSION

G-protein coupled receptor kinases were first discovered for their role in GPCR
phosphorylation and desensitization [34, 35]. Recent studies, however, have revealed a
number of non-GPCR substrates for GRKs [10, 11]. Although GRKs mediate their
cellular effects for the most part through their catalytic activity, recent studies have also
proposed a kinase-independent role for GRKs in cellular signaling (via protein-protein
interaction with the RH domain). In this regard, GRK2 has been shown to interact with
MEKl and regulate ERK activation in a kinase-independent manner [36]. GRK2 has also
been shown to interact with other proteins such as PI3K [3 7], Akt [3 8], and GIT [39] and
regulate a number of cell biological effects. In addition to the receptor and cytosolic
substrates, GRKs, have also been found to phosphorylate nuclear proteins. A
physiologically important role for the nuclear localized GRK5 was recently identified by
Martini et al [40] who showed that the nuclear GRK5 is a HDAC kinase the mediates the
epigenetic regulation of gene expression in cardiomyocytes. Taken together these studies
suggest that the role of GRKs is much broader than previously appreciated.

Studies have just begun to emerge on the potential role for GRKs in the regulation
of various components of the NFKB pathway. In this regard, we demonstrated that GRK5
stabilizes LPS-stimulated p105 levels in macrophages [8]. More recently, while this
manuscript was in preparation, a similar stabilizing role for GRK5 in maintaining IKBOL
levels in endothelial cells was shown [41]. Surprisingly in the present study we find that
GRK2 and GRK5 are important regulators of IKBa-NFKB signaling and mediate TNFa-

induced IKBa phosphorylation. In addition, in contrast to the findings of Sorriento et al

[41] in endothelial cells, our results demonstrate that GRK2 and GRK5 mediate TNFor-

128

induced NF KB-dependent gene transcription in macrophages. Our results further suggests
that even with in a given cell type, the role of GRKs is selective for a particular ligand.
Our data further demonstrate that the role of GRK2 and GRK5 on TNFa-induced
NFKB signaling is dependent on the kinase activities because the kinase-deficient GRKs
failed to mediate NFKB activation. Also, in vitro kinase assays indicate that IKBOL may be
differentially phosphorylated by GRK2 and 5. Our results show that the presence of GBy
subunits and liposomes significantly enhances the phosphorylation of IKBOt by GRK2.
GBy subunits have clearly been demonstrated to be important in the translocation of
GRK2 to the plasma membrane for GPCR phosphorylation. Whether a similar role for
GBy subunits in TNFa signaling exists, is presently not known. However, Kawamata et
al [42] showed recently that TNFor signaling is mediated by activation of G-proteins in
adipocytes. In addition, TNFor treatment of THP-l monocytic cells has been shown to
mediate GRK2 translocation to the membrane and affect B-adrenergic receptor
desensitization [43], suggesting possible regulation of TNFor-induced GRK2 activity by
G-proteins. In contrast to the role of lipids in GRK2 activity, GRK5 phosphorylation of
IKBa appears to be effectively inhibited in the presence of lipids even though previous
studies have clearly shown that lipids activate GRK5 [30]. Thus it is possible that GRK5
phosphorylation of IKBOt is regulated by biochemical mechanisms that are distinct from
its phosphorylation of other substrates such as synucleins [30] as well as from that of
IKBa phosphorylation by GRK2. Whether these differences in the phosphorylation of
IKBa by GRK2 and 5 translate into regulation of IKBOL in different sub-cellular

enviromnents in not known and will be tested in future studies. Other studies have clearly

129

shown that IKBa-NFKB complexes can be present in different sub-cellular environments
[44, 45] and therefore, these complexes could be potentially regulated by GRK2 or 5
depending on the local cellular environment.

IKKB has been identified as the primary kinase that phosphorylates IKBOL.
However, there is now extensive evidence that other kinases including IKKa, CK II and
ribosomal S6K can phosphorylate IKBO. at the same sites as that of IKKB. This
redundancy can in part be explained by the receptor- and cell type-specific regulation of
IKBor-NFKB pathways [7, 32, 46]. For example, studies have shown that UV light-
induced IKBOL degradation is mediated by phosphorylation of IKBoc by CK II [7]. Also,
PMA-induced IKBor phosphorylation has been shown to involve ribosomal S6K [32].
Similarly, recent studies have shown IKKB-dependent and —independent pathways that
regulate IKBOL-NFKB pathway in human macrophages in response to specific ligands
[47]. Our studies clearly suggest that GRKs while necessary for TNFor-induced IKBa-
NFKB pathway, are not involved in LPS-induced IKBa-NFKB signaling. Interestingly,
knockdown of IKKB does not affect TNFoc-induced IKBa phosphorylation (at Ser32/36),
but does inhibit LPS-induced IKBOL phosphorylation, suggesting that in Raw264.7
macrophages, GRKs play the role of IKBa kinases selectively for TNFoc signaling. Our in
vitro kinase reactions further support our findings in macrophages in that, GRK5 is able
to phosphorylate some residues in IKBOL that are similar to that of IKKB. Thus it is
possible that GRK5 specifically might function in a similar capacity to that of IKKB. If
GRK5 can function as an IKBOL kinase, then what is the role of GRK2? It appears from

our macrophage experiments that GRK2 is also necessary for TNFor-induced IKBOL

130

phosphorylation (Ser32/36). However, in vitro GRK2 does not phosphorylate Ser32/36
and therefore appears to phosphorylate a different set of residues. In cells, GRK2
phosphorylation of these unidentified residues appears to be necessary for
phosphorylation of Ser32/36 since knockdown of GRK2 inhibits IKBa phosphorylation
at these two sites. It is also possible the level of IKKB knockdown obtained might not be
sufficient to define IKKB-independent regulation because of the residual kinase activity
present in the knockdown cells. If this is the case, GRKs might work co-operatively with
IKKB in the phosphorylation of IKBa. In this regard, although Ser32/36 in IKBOt are the
well-characterized IKK phosphorylation sites, there is strong evidence that IKK also
phosphorylates less well-characterized sites at the c-tenninus [48]. Therefore, in our
experiments, we carmot rule out the role of IKKB on phosphorylation of these other sites.
Our results, however, indicate that H(KB knockdown can certainly inhibit LPS-induced
IKBa phosphorylation (on Ser32/36) and therefore, suggest that LPS and TNFa signal to
NFKB activation via different mechanisms in Raw264.7 macrophages.

In conclusion, our studies unravel important biochemical roles for GRK2 and 5 in
TNFor-induced IKBOL phosphorylation and NFKB signaling. Because regulation of the
NFKB pathway appears to be receptor-specific as well as cell type-specific, further broad
and unbiased proteomic approaches are necessary to identify macrophage-specific and
TNFR-specific signaling complexes that mediate NFKB activation. These studies will

undoubtedly identify therapeutic targets for inhibiting TNFa signaling in chronic

inflammatory diseases.

131

Acknowledgement: We would like to thank Dr. Christina Pao and Ms. Michelle Kratz
for preparing purified GRKs. This work was funded in part by grants from National

Institutes of Health AR055726 and HL095637 (to NP.) and GM44944 (to J LB.) and

grant-in-aid from the Midwest affiliate of American Heart Association (to NR).

132

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138

CHAPTER 4

G-protein coupled receptor kinase 5 (GRK5) mediates Toll-like
receptor-4-induced NF-KB pathway in macrophages and is necessary
for the production of inflammatory cytokines and chemokines in viva

The authors include:

Sonika Patial, Shipra Shahi, Yogesh Saini, Daniel M. Appledom, John J. Lapres,
Andrea Amalfitano, Narayanan Parameswaran

139

ABSTRACT

G-protein coupled receptor kinase-5 (GRK5), a serine-threonine kinase is one of the
seven GRK family members, the primary function of which is to regulate the
desensitization of G-protein coupled receptors. Recent studies have shown that GRK5
also regulates NFKB pathway stimulated by non-GPCRs. This study was undertaken

to determine the potential role of GRK5 in inflammatory signaling in primary

. -/- . . . . . .
macrophages and in vivo usrng GRK5 mice. Consrstent With our prevrous findings in

macrophage cell lines, we demonstrate here that TLR4-induced IKBa-NFKB pathway

is inhibited in primary macrophages from GRK5-L mice compared to cells from

/ . . . . . .
GRK5+ + mice. Our results also indicate that this role of GRK5 1S specrfic for IKBa-

NFKB pathway because activation of other signaling pathways, including ERK, JNK
and p38 are similar between the two genotypes. Consistent with the effects on the

NFKB pathway, LPS-induced cytokine/chemokine production was broadly inhibited in

-/- . . , . .
the cells from GRK5 mice. To examrne the m vzvo relevance of these findings, we

injected GRKSJ- and GRK5“+ mice with LPS and measured plasma cytokine levels

at various times after injection. Confirming the in vitro data, LPS-induced
cytokines/chemokines were significantly inhibited in vivo, in the GRK5"' mice.
Associated with these effects LPS-induced liver injury is also decreased in the GRK5"'
mice. Taken together, our findings demonstrate that GRK5 acts as a positive regulator
of LPS-induced inflammatory signaling and further suggest that these findings could

have potential implications for drug development in inflammatory diseases.

140

INTRODUCTION

G-protein coupled receptor kinases (GRKs) are serine-threonine protein
kinases that regulate the phosphorylation and desensitization of G-protein coupled
receptors [1]. GRK family (seven members identified, GRKl-7) is subdivided into
three main groups on the basis of sequence homology viz. rhodopsin kinase (GRKI
and GRK7), B-adrenergic receptor kinase (GRK2 and GRK3), and the GRK4 kinase
(GRK4, GRK5 and GRK6) subfamilies. Although these seven members share certain
characteristic features, they are distinct enzymes with specific properties. GRK5 is the
best characterized member of the GRK4 subfamily of GRKs which is expressed
ubiquitously in all mammalian tissues. It is a membrane associated protein which has
been shown to be selectively required for muscarinic receptor desensitization [2].
Recent studies have also shown a nuclear role for the GRK4 family of kinases, owing
to the presence of a “Nuclear localization signal” in their sequence[1, 3].

Apart from their role in receptor desensitization, recent studies showed that
GRKs also perform other cellular functions by phosphorylating non-receptor
substrates such as tubulin, and synucleins [4, 5]. GRKs also interact with a variety of
other cellular proteins such as caveolin, calmodulin and actin [6-8]. Along the same
lines, recently, GRK5 was also found to interact with members of the IKB (Inhibitor of
KB) family. In this regard, GRK5 was shown to interact with and phosphorylate
NFKBl p105 as well as IKBa [9, 10]. The fimctional outcomes of these studies were
primarily studied using macrophage cell line. Role of GRK5 in regulating NFKB

pathway in primary macrophages and the functional relevance of this regulation in

vivo, however, is presently not known.

141

NF-KB family of transcription factors (NFKBlpSO, NFKB2p52, RelA(p65),
RelB, c-Rel) play an essential role in regulating both innate and adaptive immunity.
These proteins are held in the cytoplasm under unstimulated conditions in association
with an inhibitory protein family called IKB (0t, B, 8, p100, p105, Bel-3) {reviewed in
[11]}. In the canonical NFKB pathway, stimulation with inducers such as LPS or
TNFa activates IKB kinase complex, which, phosphorylates IKBa as well as other
IKBs including p105. Phosphorylated IKBa undergoes proteasomal degradation thus
allowing NFKB factors to translocate to the nucleus and bind onto their cognate DNA
binding sites thus initiating the transcription of a wide array of genes including those
of cytokines and chemokines. NFKB, although essential for regulating both innate and
adaptive immune responses, its constitutive activation is often associated with several
inflammatory diseases such as sepsis, rheumatoid arthritis, inflammatory bowel
disease, multiple sclerosis, asthma and many more. Because of this role in
inflammatory diseases, IKKB has been targeted for drug development, but has faced
some pitfalls because inhibition of IKKB not only prevents the deleterious effects of
NFKB pathway, it also blocks the protective effects of the NFKB pathway, thereby
tipping the balance between inflammatory and antiinflammatory effects of the NFKB
pathway towards the harmful outcomes. Thus further understanding of the regulators
of the NFKB pathway is an important area being actively pursued by many research
laboratories.

Based on our previous studies, we hypothesized that deficiency of GRK5 in
mice would result in inhibition of NFKB pathway in response to inflammatory stimuli

and that would be associated with inhibition of several cytokines and chemokines both

142

from macrophages and in vivo in mice. We demonstrate here that GRK5 is a critical
mediator of TLR4-induced NFKB pathway in primary macrophages and in vivo.
Importantly, our results also demonstrate a crucial role for GRK5 in inflammation

induced by TLR4 activation in vivo in mice.

143

MATERIALS AND METHODS
Materials

Protease inhibitor cocktail tablets were from Roche Diagnostics (Indianapolis,
IN), Phospho ERK, Phospho p38, Phospho JNK, JNK, NFKBI Phospho P105,
phospho-IKBa antibodies were from Cell Signaling Technology (Boston, MA). ERK,
NF KBl P105 were from Santa Cruz Biotechnology. Tubulin antibody was from sigma.
Monoclonal GRK5 antibody was from Upstate Biotechnology. E. coli LPS (0111:B4)
from Sigma was used for mice injections. Ultra pure LPS from Invivogen was used for
in vitro peritoneal macrophage stimulation.
Animals

Heterozygous GRK5 mice (backcrossed to C57BL6 background for at least 5
generations) were purchased from Jackson labs. Heterozygous mice were bred to
obtain wild type and homozygous GRK5 knockout mice. The litter mate wild types
and knockouts were fiirther bred and the F1 and F2 wild type and knockout mice were
used for the experiments. Animals were housed four to five mice per cage at 22—24°C
in rooms with 50% humidity and a 12-h light—dark cycle. All animals were given
mouse chow and water ad libitum. All animal procedures were approved by the
Michigan State University Institutional Animal Care and Use Committee and
conformed to NIH guidelines. Tail tips were used for isolating genomic DNA and
genotyping performed by PCR. All experiments were performed on female mice, 6-8
weeks of age.

Peritoneal Macrophage isolation

144

To isolate peritoneal macrophages, mice were injected by intra-peritoneal
injection with lml of 4% thioglycollate. Peritoneal macrophages were collected by
performing a peritoneal cavity lavage after 4 days of thioglycollate injection in
Dulbecco’s phosphate buffered saline (DPBS). Cells were washed atleast three times
and then counted and plated on cell culture plates in RPMI 1640 media supplemented

with 10% fetal bovine serum (Invitrogen) and penicillin (100 units/ml) and

streptomycin (100 pg/ml) at 37° C in 5% C02. After around 18 hours of plating, cells

were serum starved for ~ 3-4 hours and stimulated with LPS (1 pg/ml) for the indicated
time points.
Cytokine analysis

A mouse 23-plex multiplex based assay was used to determine the
cytokine/chemokine concentrations according to manufacturer’s instructions via
Luminex 100 technology as described previously [12]. Plasma from LPS injected mice
collected at different time intervals and supematants from peritoneal macrophages
stimulated with LPS for different time points were used to assess the
cytokine/chemokine levels.
Western blot analysis

Cells were lysed in lysis buffer (20mM Tris-HCl (pH 7.4), lmM EDTA,
150mM NaCl) containing 1% Triton X-100 with protease inhibitors. Lysed cells were
then centrifuged at a maximum speed (13,000 X g) for 10 min at 4°C and protein
concentration of the supematants determined by Bradford assay. Western blotting was
performed as described previously [9]. Briefly, equal amounts of protein were run on

polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes

145

were then blocked in Licor blocking buffer (Licor Biosciences) or 5% w/v skimmed
milk for 1 hour after which the membranes were incubated in primary antibodies
overnight. Secondary antibodies used were either fluorescently tagged or HRP-
conjugated. Blots were developed either on Licor odyssey or using
chemiluminescence.
Statistical analysis

All values are represented as mean 5: SEM. Data were analyzed and statistics
performed using GRAPHPAD PRISM software (San Diego, California). The student’s
t-test was used to compare mean values between two experimental groups and
Analysis of Variance (ANOVA) with Bonferroni post test was used to compare more

than two groups. P value of less than 0.05 was considered significant.

146

RESULTS
Reduced NFKB activation in GRK5 knockout macrophages

Stimulation of cells with LPS and other cytokines particularly TNFa and IL-lB
is known to activate IKBOt-NFKB pathway, which plays an essential role in controlling
both innate and adaptive immune responses by regulating the transcription of a large
number of genes including cytokines and chemokines. As described before, under
resting cellular conditions, NFKB is retained in the cytosol by the inhibitory protein
IKBa. However, stimulation with LPS triggers the phosphorylation of IKBoi by IKB
kinase-B (H{KB). Phosphorylated IKBa then undergoes proteasomal degradation
releasing NFKB subunits p50 and p65, which now translocate into the nucleus, bind to
its cognate DNA sequence and evoke gene transcription. Previous studies from our
laboratory have shown that IKBa interacts with GRK5 and that GRK5 phosphorylates
IKBa at least at one of the sites (serine32) phosphorylated by IKKB. In Raw264.7
macrophages cell line, we demonstrated that depletion of GRK5 levels significantly
blocks TNFa-induced IKBOL phosphorylation [9]. Based on these results, we proposed
that GRK5 interaction with and phosphorylation of IKBOL mediates TNFa-induced
NFKB activation in this cell line model. Sorriento et al, at the same time published that
GRK5 interaction with IKBOL in endothelial cells, negatively regulates LPS-induced
IKBa phosphorylation. These studies found that this effect of GRK5 was independent
of its kinase activity [13]. In previous studies, LPS-induced p105 phosphorylation was
shown to be negatively regulated by GRK5 in Raw264.7 macrophages cell line [10].

Because of these various observations, we set out to examine the role of GRK5 in

NFKB signaling in physiologically relevant cells. For that, we obtained thioglycollate-

147

elicited primary peritoneal macrophages from GRK5“+ and GRK5-L mice and

initially tested the effect of LPS on NFKB signaling. We first determined

phosphorylation of IKBa in GRK5+/+ and GRK5-L macrophages in response to LPS.

IKBa was not phosphorylated either in GRKSJr/+ and GRKSJ- macrophages under

basal conditions. Treatment with LPS however caused a marked increase in

phosphorylation of IKBa, which peaked at 60 min post stimulation in GRK5“+ cells.

Consistent with our previous findings, in GRK5-L cells, IKBa phosphorylation was

significantly inhibited (WT:100i0.000%; KO:49.7423:10.907%) (p < 0.001) as

compared to GRKSH+ control cells (Fig. 4.1A). To test the significance of this in

terms of NFKB activation, we examined the nuclear translocation of NFKB subunit

p65 in response to LPS in GRK5+/+ and GRK5-L macrophages. Confirming our

finding on IKBO. phosphorylation, nuclear translocation of p65 was significantly
inhibited in the knockout cells compared to the wild type macrophages (Fig. 4.1B).

Furthermore, electrophoretic mobility shift assays demonstrate greatly attenuated
NFKB binding to its consensus sequence in GRK5-L cells compared to the GRKSIL/+
macrophages (Fig. 4.1C). Taken together, these results suggest that GRK5 positively
regulates LPS induced NF KB signaling. Interestingly, LPS-induced phosphorylation of
NFKB p105 (another IKB protein) was ~20% higher at all time points in the GRK5

knockout macrophages compared to the wild type cells, but was not statistically

significant (Fig. 4.1D).

148

Figure 4.1 GRK5 null peritoneal macrophages show reduced NFKB

activation

A. Thioglycollate elicited peritoneal macrophages from both GRK5+/+ and

GRK5-L mice were stimulated with LPS (lpg/ml) for various time points as

indicated. Cell lysates were extracted and separated by SDS/PAGE,
transferred to nitrocellulose membrane and immunoblotted with primary
antibodies against phospho-IKBa and tubulin. Secondary antibodies were
either HRP labeled or fluorescent tagged and the blots were developed by
chemiluminescence or using LI-COR Biosciences Odyssey system
respectively. Representative blots for PIKBa and tubulin and quantification is
shown in A. ***p<0.001, N=4.

B. Peritoneal macrophages were stimulated with LPS as above and nuclear
extracts were prepared and immunoblotted for NF KB p65 (Rel A) as shown.
Actin is shown as a loading control. A representative blot from three such

experiments is shown.

C. Nuclear extracts were also incubated with IRDye 700-labelled NFKB

oligonucleotide probes and an EMSA was performed as described in the
materials and methods section. Gels were then scanned on a LI-COR
Biosciences Odyssey system to detect binding. A representative gel fi'om three
such experiments is shown.

D. Lysates fi'om peritoneal macrophages stimulated with LPS (lpg/ml) were
also immunoblotted using primary antibody against NF KBl p105 (another IKB
protein). Representative blot and quantification is shown. Tubulin was used as

a loading control.

149

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151

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152

GRK5 does not affect TLR4-induced MAPK activation

LPS stimulation of TLR4 also leads to the activation of MAP kinases in
addition to NFKB. Because MAPKs (ERK, JN K and p3 8) are also important regulators
of inflammatory response and to further determine whether our observed findings on
GRK5 regulation of NFKB was specific for that pathway, we sought to examine the

LPS-induced activation status of the three important MAPKs in primary macrophages

-/- . . .
from GRK5 mice and GRK5”+ control mice. Peritoneal macrophages were

stimulated with LPS (lpg/ml) for various time points and immunoblotting performed
to determine the phosphorylation of MAPKs at different time points. Irnmunoblot
analysis however revealed no difference in the kinetics or magnitude of
phosphorylation of ERK, JNK and p38 between wild type and GRK5 deficient
macrophages suggesting that the GRK5 signaling in response to LPS is selective for
IKB-NFKB pathway (Fig. 4.2A, 4.28, 4.2C).
Impaired LPS-induced cytokine and chemokine production in GRKS'” peritoneal
macrophages

To further understand the physiological relevance of our findings, we
examined the role of GRK5 in LPS-induced inflammatory cytokine/chemokine
production. We hypothesized that because NFKB regulates a number of inflammatory

genes, deficiency of GRK5 would broadly inhibit several cytokines/chemokines

induced by LPS. For this, we treated primary macrophages from GRK5“+ and GRK

5 mice wrth LPS for various time pornts and collected the cell culture supematants.

Cytokine/chemokine levels in the supematants were measured using 23-plex Biorad’s

153

Figure 4.2 GRK5 has no effect on TLR4-induced MAPK activation

+/+
Thioglycollate elicited peritoneal macrophages from both GRK5 and

GRK5 - mice were stimulated with LPS (lpg/ml) for various time points as

indicated. Cell lysates were extracted and separated by SDS/PAGE,
transferred to nitrocellulose membrane and immunoblotted with primary
antibodies against phospho-ERK, ERK, phospho-JNK, JNK, phospho-p38 and
tubulin. Secondary antibodies were fluorescently labeled and the blots were
developed using LI-COR Biosciences Odyssey system. (A) P-ERK, ERK and
its quantitation is shown. (B) shows a representative blot and quantitation for
p-JNK, JNK. (C) show a representative blot and quantitation for p38 and
tubulin. N=4.

154

 

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157

bioplex assay. As predicted, our results indicate that LPS-induced production of
several inflammatory cytokines/chemokines is inhibited in the GRK5 knockout mice
macrophages. In particular, after 12 hours. of LPS treatment the following
cytokines/chemokines were markedly inhibited: IL-Z (Fig. 4.33, WT: 3.975i0.727pg;
KO: 2.285i0.189pg), IL-3 (Fig. 4.3b, WT: 1.680i0.370pg; KO: 0.934i0.136pg), IL-4
(Fig. 4.3c, WT: 5.654i1.233pg; KO: 3.226:I:O.4l9pg), IL-S (Fig. 4.3d, WT:
6.216251.120pg; KO: 3.559zt0.344pg), IL-12p70 (Fig. 4.3c, WT: l9.521d:3.439pg; KO:
11.656zt1.014pg), IL-12p40 (Fig. 4.3f, WT: 195.308zt30.828pg; KO:
105.707i8.911pg), IL-l7 (Fig. 4.3g, WT: 8.6302t2.15213g; KO: 3.129iO.425pg),
MCPl (Fig. 4.3b, WT: 691.623i115.893pg; KO: 447.905:l:44.838pg), IFNy (Fig. 4.3i,
WT: 12.667:l:2.670pg; KO: 6.710i0.927pg), KC (Fig. 4.3j, WT: 211.4963c40.370pg;
KO: 96.345i6.589pg), GM-CSF (Fig. 4.3k, WT: 11.271:I:2.138pg; KO:
6.378i0.570pg), Eotaxin (Fig. 4.31, WT: 91.907i15.865pg; KO: 57.302:Iz5.426pg). An
important point to note is that some cytokines/chemokines were similar between the
wild type and KO suggesting that GRK5 does not regulate all cytokine/chemokine
production en masse. These included TNFa, IL-lB, lL-6, IL-9, IL-13, MIPlB, IL-la,
IL-10, GCSF, MIPla, RANTES (data not shown).
Impaired LPS signaling in viva in GRK5 knockout mice

The secretion of cytokines and chemokines into the plasma is an essential step
which orchestrates the anti-infection process. Cytokines and chemokines contribute by
enhancing the microbicidal activities of phagocytosing cells such as monocytes and
macrophages as well as by recruiting leukocytes to the site of infection. Although

contributing towards the anti-infectious process, their excessive production can cause

158

Figure 4.3 LPS induction of cytokines and chemokines in peritoneal
macrophages from WT (GRK5+/+) and GRK5 null (GRK5-F) mice

Thioglycollate elicited peritoneal macrophages were stimulated with lpg/ml
LPS in cell culture plates. Cell culture supematants were then collected at
various time intervals and secretary levels of cytokines / chemokines were
determined using Biorad’s 23 plex Luminex based assay. Cells were lysed and
protein concentration was determined by Bradford assay. Levels of cytokines /
chemokines were normalized to total protein concentration and shown in

picograrns. Cytokines and chemokines shown include IL-2 (a), IL-3 (b), IL-4,
(c), IL-5 (d), IL-12p70 (e), IL-12p40 (f), IL-l7 (g), MCPl (h), IFNY (i), KC

(j), GM-CSF (k) and Eotaxin (I). Data was analyzed by Two way ANOVA
followed by a Bonferroni posttest for each group. N=6. *p<0.05; **p<0.01;
***p<0.001.

159

U'

lL- 3 secretion (pg)

lL- 2 secretion (pg)
b.’ ‘1’ '1‘

A
J

 

 

   
 
 
  
 
 

 

Time (Hours)

*GRK5*’*

. -A-GRK5"'

 

 

 

Time (Hours)

160

Figure 4.3. Continued....

C

3‘ *GRK5*’*
-A-GRK5"'

IL- 4 secretion (pg)

 

 

 

Time (Hours)

8. *GRK5*’*
I vex-GRK5"

IL- 5 secretion (pg)

 

 

 

0 5 1'2 1'3 24
Time (Hours)

161

Figure 4.3. Continued....

+1-1-

25- ‘I'GRKS

    

-A-GRK5"'

*
A

 

 

e

   

my 5 mu 5
2 1 1

63 53812.3 N. 1..

1'5

12
Time (Hours)

 

   

‘—

24

 

 

 

 

H.

+4

55

KK

RR

an

{a

nw n.. no mu n.. mu
4 0 6 2 8 4
2 2 1 1

33 55.58 see 2 1..

 

1'8

12
Time (Hours)

162

Figure 4.3. Continued. . ..

9

14-

*GRK5*’*
-A-GRK54'

IA—‘L
9 '9

 

IL- 17 secretion (pg)

 

 

 

h Time (Hours)
1000' -I- G RK5+I+
900- 'A- G RK5"'
8001
700-
600-
500-
400-
300-
200-
100-

 
  
  
  
  
  
  

MCP1 secretion (pg)

 

 

o is 1'2 1'8 24
Time (Hours)

163

Figure 4.3. Continued. . ..

—.

KC secretion (pg)

lFNy secretion (pg)

18-

15‘

12-

 

3001

250-

200'

1 501

1 00-

50-

 

'I'GRK5""'
’A'GRKSJ'

 

 

 

 

1'3 2'4
Time (Hours)

*GRK5*’*
'A'GRKS'”

 

é 1'2 1'3 2'4
Time (Hours)

164

Figure 4.3. Continued. . ..

4
2

*GRK5*’*
“A'GRKS '

 

‘E__..--........

  
 

1'8

1'2
Time (Hours)

 

 

16-

k

35 559.03 “50.5.6

14+
.1.

‘I'GRKS
'A'GRKS

 

 

 

1251

 

3.3 5:203 Exflom

Time (Hours)

165

severe side effects. Detectable levels of these inflammatory mediators i.e. cytokines
and chemokines in the blood stream are indicative of their exacerbated production.
Plasma is one of the major sources which can be utilized to measure the levels of these
inflammatory mediators. Moreover, evaluation of plasma cytokines over a time course
can provide a better understanding of the nature and severity of the disease process. To
investigate the role of GRK5 in TLR4 signaling in vivo, we sought to determine the

LPS induced production of cytokines and chemokines in mice plasma. For this

purpose, GRKSH+ and GRK5'/' mice were intra-peritoneally injected with LPS

(30pg/gm body weight) and blood collected at 1, 3 and 12 hours after injection. Using
Biorad’s 23-plex cytokine assay, we determined the plasma levels of various cytokines
and chemokines after LPS injection. We rationalized that, because GRK5 mediates a
broad spectrum of cytokine/chemokines in the primary macrophages in vitro, LPS
injection in vivo in mice would result in diminished cytokine responses in the GRK5"'
mice. Consistent with this prediction, LPS-induced cytokine/chemokine levels were
broadly inhibited in the GRK5"' mice compared to GRK5”. Again, as is the case with
the primary macrophages, not all cytokine/chemokines were affected in this manner.
Only a subset of cytokines/chemokines were affected by GRK5 deficiency suggesting
selective regulation of these cytokines/chemokines by GRK5. Cytokines that were
inhibited in the GRK5"’ mice included TNFa (Fig. 4.43, WT: 218576zt35294pg/ml;
KO: 119970i2321lpg/ml at 1 h time point), IL-lB (Fig. 4.4b, WT:
2237.9271648.049pg/ml; KO: 1052.435i108.982pg/ml at 12 h time point ), IL-2 (Fig.
4.4c, WT: 54.615i2.745pg/ml; KO: 36.817i2.053pg/m1 at 12 h time point ), IL-3 (Fig.

4.4d, WT: 37.070i6.248pg/ml; KO: 22.973i2.274pg/ml at 12 h time point), IL-4 (Fig.

166

Figure 4.4 Profile of plasma cytokines after LPS challenge in WT
(GRK5+/+) and GRK5 null (GRK5-F) mice

Plasma cytokines TNFa (a), IL-lB (b), IL-2 (c), IL-3 (d), IL-4 (e), IL-5 (1’),
IL-lO (g), IL-12 (p40) (h), IL-12 (p70) (i), IL-13 (j), IL-17 (k), MCPl (I),
Rantes (m), Eotaxin (n) were determined in WT and GRK5'/' mice after an

intra-peritoneal injection of 30pg/g body weight LPS at specified times viz. 1
hr, 3 hrs and 12 hrs (N=6 per group). Data was analyzed by Two way
ANOVA followed by a Bonferroni posttest for each animal group. *p<0.05;
**p<0.01; ***p<0.001.

167

‘P

0) OJ
C 0|
O O
O O
n n

25004

N
O
O
O
n

1500-
1000-
5004

Plasma IL-1B(pglml) 0' Plasma TNFMPQImI) 9’

 

4.0

J

0

are»
3’

20-
101

Plasma IL- 2 (pg/ml)
h
D

 

§

i

i

'- GRK5 ”t
-A'GRK5 "-

***

 

 

 

Noinjctrl 1'hr 3hrs 12hrs
Time (Hours)

-l- GRK5 *’*
-A- GRK5 "'

 

 

No inj ctrl 1i1rs 3hrs 12'hrs
Time (Hours)

‘- GRK5 *’*
-A- GRK5 "' ***

   
 

“‘~23

 

No in] ctrl 1i1rs sins 12'hrs

Time (Hours)

168

Q.

«5 0|
0 O
l 1

Plasma IL- 3 (pg/ml)
o:
C

Figure 4.4. Continued. . ..

fkoRK5+

 

 

 

CD

12.5-
10.0-
7.5'
5.0'
25*

Plasma IL- 4 (pg/ml)

 

No in] ctrl 1hrs sins 12'hrs
Time (Hours)

'- GRK5 *’*
-A- GRK5 "' ***

  

...“i§

 

0.0

600-
500-
v 400-
300-
200‘
100-

ngmI) '4'

Plasma lL- 5

 

No in] ctrl 1i1rs 3hrs 12'hrs

Time (Hours)
***

  
   

-I- GRK5 t“
-A- GRK5 "-

.---i§

   

 

No inj ctrl 1hrs 31'1rs 12'hrs
Time (Hours)

169

(Q

3'

Figure 4.4. Continued....

20000- *
'- GRK5 ”t

10000-

Plasma IL- 10 (pg/ml)

 

 

 

No inj ctrl 1i|rs 3I'1rs 12'hrs
Time (Hours)

-l- GRK5 ***
-A- GRK5 "'

***

   

N 03
O O
O O
O O
<.= <.=

1ooool

 

 

 

 

Plasma lL- 12 (p40) (pg/ml)

No inj ctrl 1 hrs 3 hrs 12'hrs
Time (Hours)
400-
-I-GRK5 *’* ***

 
  
 

300.. 'A' GRK5 4'

A N
C O
O O
n n

 

 

L

 

Plasma IL- 12 (p70) (pg/ml)

No inj ctrl 1 hrs 3 hrs 12'hrs
Time (Hours)

170

Figure 4.4. Continued. . ..

3000-

2000‘

1000-

Plasma IL- 13 (pg/ml) h.-

 

-I- GRK5 *’+ ***

-A- GRK5 "'

    

   

 

x

6000-
50004
40004
3000-
2000-
1000‘

Plasma IL- 17 (pg/ml)

 

No i'njctrl 1hrs 3hrs 12'hrs
Time (Hours)

**

-I- GRK5 *’*
-A- GRK5 "'

 

No inj ctrl 1 hrs 3 hrs 12 hrs
Time (Hours)

200000-

100000-

Plasma MCP1 (pg/ml)

 

‘- GRK5 *’* ***

fikoRK5+ *

     

 

Noinjctrl1hrs 3hrs 12'hrs

Time (Hours)

171

Figure 4.4. Continued....

 
   
 

 

 

m
‘5 20000-
31 -I- GRK5 +’* ***
3 -A'GRK5 "'
(D
E
2 10000-
:15 ----A
m
E
m
(B
a 04

 

No inj ctrl 1hrs 31'1rs 12'hrs
Time (Hours)

8000- ***

 

Plasma Eotaxin (pg/ml) 3

 

 

No inj ctrl 1 hrs 3 hrs 12'hrs
Time (Hours)

172

4.4e, WT: 8.970i0.571pg/m1; KO: 5.672i0.375pg/ml at 12 h time point), IL-5 (Fig.
4.4f, WT: 417.9932t129.706pg/ml; KO: 98.120i24.171pg/ml at 12 h time point), IL-
10 (Fig. 4.4g, WT: 13648.600i3891.250pg/ml; KO: 5645.438i1033.724pg/ml at 12 h
time point), IL-12p4O (Fig. 4.4b, WT: 22694i3038pg/ml; KO: 5101:}:1129pg/ml at 12
h time point), IL-12p70 (Fig. 4.4i, WT: 288$:40pg/ml; KO: 137i13pg/ml at 12 h time
point) IL-l3 (Fig. 4.4i, WT: 2220i163pg/ml; KO: 11581201pg/ml at 12 h time point),
IL-17 (Fig. 4.4k, WT: 3780i17S6pg/m1; KO: 195i6lpg/ml at 12 h time point), MCP1
(Fig. 4.41, WT: l39016i38025pg/ml; KO: 36490i12175pg/ml at 12 h time point),
Rantes (Fig. 4.4m, WT: 13920i1855pg/ml; KO: 7510i651pg/ml at 12 h time point),
Eotaxin (Fig. 4.4n, WT: 6548i615pg/ml; KO: 4443i471pg/ml at 12 h time point).
Cytokines that were not affected by GRK5 deficiency included GCSF, GM-CSF, lFNy,

IL-la, IL—6, IL—9, KC, MIPla, MIPIB (data not shown).
Diminished liver injury in GRK5-l. mice

Due to the fact that a major group of “pro-inflammatory” cytokines and
chemokines were markedly decreased in GRK5'/' mice, we hypothesized that tissue

injury seen as a result of LPS-induced endotoxemia would be reduced in GRK5"' mice.
To this end, we assessed the extent of liver damage by determining the plasma

concentrations of liver injury marker alanine transaminase (ALT) at 12 hours after

LPS challenge, both in control GRK5"' and GRK5 deficient i.e. GRKS'I' mice. Our
results show that GRK5'/' mice display significantly reduced levels of plasma ALT as

compared to GRK5"+ mice (503:4 U/L in GRK5'/' mice verses 84i10 U/L in GRKS'l'

mice) demonstrating that GRK5 deficiency results in reduced LPS dependent

173

Figure 4.5 GRK5-I. mice show reduced LPS-induced liver injury

GRKS'L/+ and GRK5'/' mice were injected with LPS and plasma levels of liver

injury marker ALT were measured after 12 hours. Data was analyzed by

unpaired t test; N=6. *p<0.05

174

 

EJGRK5”*

I GRK5 "'

100

 

5 O 5
7 5 2

:5. .22 5<

 

175

production of inflammatory mediators which translates into a decreased liver injury
(Fig. 4.5).

Taken together, these results suggest that GRK5 plays an important role in LPS
mediated signaling pathway affecting crucial transcription factors responsible for the
production of a wide array of pro- as well as anti-inflammatory cytokines, not only in

primary macrophages in vitro, but also in vivo.

176

DISCUSSION

LPS binding onto TLR4 induces the activation of NFKB as well MAPK
signaling pathways. In this study we show that GRK5, a GPCR kinase is a regulator of
LPS-induced NFKB signaling pathway. GRK5 null primary macrophages show a
significant reduction in LPS-induced phosphorylation of IKBO. as well as nuclear
translocation of p65, a subunit of NFKB. Furthermore, the binding of NFKB onto its
consensus DNA oligonucleotide was also found to be significantly inhibited in GRKS'
/' macrophages. These findings are consistent with our recent report that siRNA
mediated knockdown of GRK5 in Raw264.7 macrophage cell line significantly blocks
TNFa-induced NFKB signaling. In this study we not only confirm our previous
findings, we also provide evidence that GRK5, by mediating NFKB signaling, has
biologically relevant role, in the production of inflammatory cytokines/chemokines in
both primary macrophages in vitro and in mice in vivo.

Our studies further show that the MAPKs including ERK1/2, JNK and p38 are
not regulated by GRK5. LPS-induced P105-ERK pathway was found to be negatively
regulated by GRK5 in Raw264.7 macrophage cell line using RNAi against GRK5 [10].
Although we observed ~20% enhancement of pPlOS and pERK levels in the GRK5"'
macrophages, it did not reach statistical significance. Thus, Raw264.7 macrophages
are clearly different from primary macrophages with regard to the role of GRK5 in
ERK activation. It is also quite possible that the different results obtained may be
related to the mechanism by which GRK5 was depleted. Germline deletion of a gene
could have different consequences when compared to RNAi knockdown. In particular

other compensatory mechanisms can ceme into play when germline deletion is used.

177

Interestingly, with regard to GRK5 regulation of IKBoc, it appears that both methods
lead to similar effects suggesting that GRK5 is an important if not the only regulator
of IxBa-NFKB signaling. It is important to point out that other kinases including
IKKa, casein kinase and ribosomal S6K have also been shown to regulate IKBOL-
NFKB signaling by phosphorylating IKBG. [14-16]. It is clear in the GRK5 knockout
that other kinases (especially IKKB) are also phosphorylating IKBG. because
phosphorylation in the absence of GRK5 was not completely blocked, but was
inhibited ~50%.

LPS binding onto TLR4 receptor leads to the activation of both NFKB and
MAPK signaling that eventually induces the expression and production of several
cytokines and chemokines. We found that the secretion of several inflammatory
cytokines and chemokines is regulated by GRK5 in peritoneal macrophages as well as
under in vivo conditions. LPS induction of TNFa, IL-IB, IL-2, 3, 4, 5, 10, IL-12p40,

IL-12p70, IL-13, IL-17, MCP1, Rantes and eotaxin were found to be inhibited in

GRK5'/' mouse plasma.

Cytokines are soluble mediators that coordinate inflammation by being
secreted from one cell and activate receptors on other cells. TNFa is the first cytokine
that appears in the blood of experimental animal models after LPS injection as well as
in human volunteers receiving LPS [17]. TNFa causes the induction of a large number
of inflammatory mediators by acting through a signaling cascade as well as by
autocrine mechanisms [18, 19]. This has been shown by blocking TNFOL which caused
a decrease in the levels of other cytokines. For e.g. in baboons infected with E.coli,

blocking TNFa significantly reduced the levels of IL-la, IL-6 and IL-8 [20, 21].

178

Concentration of TNFo. observed in sepsis patients correlates with the severity and
outcome of the disease. For eg., concentrations of TNFa as well as IL-IB and IL-2 in
plasma were found to be higher in patients of septic shock as compared to patients of

sepsis alone [22]. In our studies, LPS injection resulted in the secretion of TNFa in the

plasma of both GRKS'L/+ and GRK5-L mice at 1 hour post injection. The levels

. . . -/- .
observed were, however, srgnlficantly less in GRK5 mice.

IL-l family of cytokines, in particular IL-lB is a key pro-inflammatory
mediator. IL-lB is released in response to an insult and initiates a host defense
response by up-regulating other cytokines, acute phase proteins as well as by acting as
a potent pyrogen [23, 24]. Furthermore, it has been shown to be required for the
clearing of bacterial infections [25]. IL-IB does so by activating MAPK and NFKB
pathways [26]. Excessive activation of IL-lB however, results in multi organ failure

which is commonly observed in sepsis [27]. We found sustained plasma levels of IL-

IB up to 12 hours of LPS injection in GRK5+/+ mice. However, IL-IB levels reach a
peak by 3 hours and thereby start declining in GRK5'/' mice. At 12 hours, the levels
observed in GRK5'/' mice plasma were significantly less as compared to those

observed in GRK5++ mice. Initial increase 1n lL-lB rs important to fight agalnst

infection, however, a sustained activation might lead to endotoxemia. We did not see a
significant difference in the levels of IL-IB in the cell culture supematants of

peritoneal macrophages however, suggesting that GRK5 might regulate the production

179

.4

I.“ W“? ***-m

of IL-lB in cells other than macrophages. It is also possible that the integrative
pathophysiology present in vivo may not be reproducible in vitro.

IL-12p40 is produced by activated macrophages, neutrophils, microglia and
dendritic cells. IL-12 has been shown to be important for the production of IFNy and
leads to lethality in LPS induced septic shock model of mice [28]. IL-17 is secreted
primarily by T lymphocytes and is involved in recruiting neutrophils and acts together
with other cytokines such as TNFa and IL-IB, to further enhance inflammation [29].
Eotaxin and MCP1 act as chemoattractants for eosinphils and leucocytes respectively.

IL-13, IL-4 and IL-10 are anti—inflammatory cytokines which act by inhibiting
the production of pro-inflammatory cytokines. For instance, IL-l3 has been shown to

act by inhibiting the production of IL-IB and TNFa which are induced by LPS. Since

some of the cytokines which were found to be reduced in GRK5'/' mice are primarily

T-cell cytokines, fiiture studies will look into the role of these cell type specific
cytokines in inducing inflammation.

Although secretion of pro-inflammatory cytokines serves as an essential
prerequisite for initiating an effective innate immune response to fight infection, they
are also associated with deleterious effects leading to multi organ failure and
ultimately death. Similarly, anti-inflammatory cytokines despite important for
controlling an exaggerated inflammatory response, also lead to a suppression of the
immune system which is required for a proper functioning of the system. Systemic
inflammatory response syndrome (SIRS) is a consequence of an imbalance between
pro and anti-inflammatory cytokines. Our studies show that absence of GRK5 reduces

the production of several pro as well as anti-inflammatory cytokines. These cytokines

180

 

are still produced to some extent which is crucial for inducing an effective immune
response. However, since exaggerated production of these inflammatory mediators is
modulated by GRK5, we speculate that our results would have important

consequences in inflammatory diseases. In fact, our observations on reduced ALT

levels in the GRK5—l- mice compared to the GRK5+/+ mice suggests that inhibition of

GRK5 in sepsis might mitigate organ injury. Future studies will address whether

GRK5 would be a viable therapeutic target for inflammatory diseases.

181

 

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184

 

29 Yu, J. J. and Gaffen, S. L. (2008) Interleukin-17: a novel inflammatory
cytokine that bridges innate and adaptive immunity. Front Biosci. 13, 170-177

 

185

CHAPTER 5

 

 

Myeloid specific G-protein coupled receptor kinase 2 (GRK2) is a
negative regulator of NFKBlpIOS-ERK pathway and limits
endotoxemic shock in mice

The authors include:

Sonika Patial, Yogesh Saini, Sitaram Parvateni, Daniel M. Appledom, Gerald W.
Dom, John J. LaPres, Andrea Amalfitano, Patricia Senagore, and Narayanan
Parameswaran

186

ABSTRACT

G-protein coupled receptor kinase 2 (GRK2) is a serine-threonine kinase which plays
an important role in phosphorylation and desensitization of G-protein coupled
receptors (GPCRs). It is highly expressed in myeloid cells particularly in macrophages
and its expression levels are altered in a number of inflammatory disorders including
sepsis. Using myeloid cell-specific GRK2 knockout mice (GRKZAmye) we demonstrate
that GRK2 acts as a negative regulator of LPS-induced inflammatory cytokine
production in vivo. Consistent with this effect, LPS-mediated lung and liver injury as
well as lethality are enhanced in the GRK2Alrlrlye mice compared to the wild type litter-
mates (GRKZfl/fl). Similar to the plasma cytokine levels, peritoneal macrophages from
the knockout mice show enhanced secretion of pro-inflammatory cytokines in
response to LPS stimulation. To elucidate the biochemical and signaling mechanisms
mediating these pathophysiological effects of GRK2, we examined the activation of

Am” mice. Our

various signaling pathways in response to LPS in GRKZfl/fl and GRK2
results indicate that disruption of GRK2 in the macrophages selectively enhances the
activation of NFKBl P105-ERK1/2 pathway in response to LPS stimulation.
Importantly, inhibition of the ERK pathway limits the enhanced production of some
LPS-induced cytokines/chemokines in the GRK2 knockout mice, demonstrating the
role of the enhanced ERK activation in the pro-inflammatory phenotype of the GRK2
knockout macrophages. Taken together, our studies reveal previously undescribed role

for GRK2 in LPS-induced p105-ERK pathway and the consequent pro-inflammatory

cytokine production and endotoxemia in mice.

187

 

INTRODUCTION

G—protein coupled receptor kinases (GRKs) are serine-threonine kinases that
recognize and phosphorylate activated G-protein coupled receptors (GPCRs) thus
causing a cessation of G-protein-dependent signaling. GRK2 is one of seven members
of GRKs and is expressed at high levels in immune cells [1, 2]. Furthermore,
expression levels of GRK2 has been found to be altered in immune cells from human
patients with a variety of immunological disorders as well as in a number of animal
disease models [3-7]. In this regard, recent studies have shown that the expression
levels of GRK2 is increased in polymorphonuclear (PMNs) cells from septic patients
[8]. In addition, GRK2 levels were found to be increased in human PMNs in response
to lipopolysaccharide. This increase in GRK2 levels has been postulated to be
important in limiting chemokine receptor (a GPCR)-induced chemotaxis of immune
cells. In fact, neutrophils from human septic patients showed significantly attenuated
chemotaxis of neutrophils [8]. Furthermore, Toll-like receptor-2 induced
polymicrobial sepsis was shown to mediate downregulation of CXCL2 receptor (a
GPCR), as well as CXCL2-induced chemotaxis of neutrophils and this was proposed
to be mediated via upregulation of GRK2 expression in these immune cells. Several
other studies have also determined the role of GRK2 in immune cell chemotaxis
owing to the fact that chemokine receptors belong to the GPCR family and that the
observations are remarkably in-tune with the known classic role for GRK2, that is,
GPCR desensitization. In spite of these seminal advances in GRK2 biology, role of

GRK2 in macrophages particularly in response to non-GPCRs is not well understood.

188

 

More importantly, role of myeloid cell-specific GRK2 in lipopolysaccharide-induced
inflammation and endotoxemia in vivo is not known.

Lipopolysacchrides (LPS) activates a class of innate immune receptors called
the Toll-like receptors (TLRs) which act as the first line of host defense against
bacterial infections. Toll like receptors (TLRs) recognize a wide variety of microbial
products known as pathogen associated molecular patterns (PAMPs) from bacteria,
virus and other pathogens. Among the TLRs, LPS specifically activates TLR4 and
triggers an inflarmnatory response that is necessary to fight against infection. Under
endotoxemic conditions, however, this system is over-stimulated and therefore the
cytokine response elicited by the host is harmful to the host resulting in endotoxemic
shock and eventual death. Activation of TLR4 by LPS triggers the recruitment of
adapter proteins such as TRIF and Myd88 as well as other TIR domain containing
proteins that eventually activates the inhibitor of KB kinase (IKK) complex [9]. The
activated IKK complex then phosphorylates IKBO. (an inhibitor of NFKB) thereby
targeting it for ubiquitination and proteasomal degradation. IKBa degradation then
enables the release and nuclear translocation of NFKB which then regulates the
expression of genes involved in inflammation as well as innate and adaptive immune

responses. In macrophages, activation of IKK complex also phosphorylates NFKBI

p105 (another IKB protein), which under unstimulated conditions is stoichiometrically
bound to a MAP3K called TPL2. LPS stimulation and phosphorylation of p105 leads
to eventual partial degradation of p105 and subsequent release of TPL2. P105-free
TPL2 then activates MEKl/2, which then activates the ERKl/Z pathway. In addition

to these pathways, LPS also mediates the activation of p38, JNK, Akt signaling

189

 

pathways. TLR4—induced activation of these signaling pathways and the subsequent
activation of transcription factors such as NFKB, AP-l and EGR-l mediates the
pathogenesis of inflammation and endotoxemia and shock.

Although chemotaxis of immune cells play a pivotal role in the pathogenesis of
endotoxemia and sepsis, production of inflammatory cytokines and chemokines also
play a requisite role in this pathogenesis. Because cells of the myeloid lineage,
particularly the macrophages express high levels of GRK2 and that TLR4 activation
enhances GRK2 levels significantly in these cells, we hypothesized that myeloid cell-
selective knockout of GRK2 will significantly affect the pathogenesis of TLR4-
induced inflammation and endotoxemia in mice. In this study, we generated a
myeloid-cell-specific GRK2 knockout and demonstrate that GRK2 negatively
regulates TLR4-induced NFKBI p105-ERK pathway, thereby limiting the

pathogenesis of endotoxemic shock in mice.

190

 

MATERIALS AND METHODS
Materials

Protease inhibitor cocktail tablets were from Roche Diagnostics (Indianapolis,
IN), Phospho ERK, Phospho p38, Phospho JNK, JNK, NFKBI Phospho P105,
Phospho AKT, AKT, iNOS, phospho-IKBOL and IKBOL antibodies were from Cell
Signaling Technology (Boston, MA). ERK, NFKBI P105 and HRP (Horseradish
Peroxidase)-conjugated anti-actin antibody were from Santa Cruz Biotechnology.
Tubulin antibody was from sigma. Monoclonal GRK2 antibody was from Upstate
Biotechnology. E. coli LPS (0111:B4) fiom Sigma was used for mice injections. Ultra
pure LPS from Invivogen was used for in vitro peritoneal macrophage stimulation.
Animals

Animals were housed four to five mice per cage at 22—24°C in rooms with
50% humidity and a 12-h light—dark cycle. All animals were given mouse chow and
water ad libitum. All animal procedures were approved by the Michigan State
University Institutional Animal Care and Use Committee and conformed to NIH
guidelines. Tail tips were used for isolating genomic DNA and genotyping performed

by PCR. All experiments were performed on male mice, 6-8 weeks of age.

A
Generation of GRK2 mye mice

GRKZfl/f' mice in which exons 3-6 of GRK2 are flanked by LoxP sites were

fl/fl LysMCre

crossed with LysMCre mice to generate GRK2 mice. A breeding colony

fl/fl LysMCre

was maintained by mating GRK2fl/fl with GRK2 . The mice were

fl/fl LysMCre

generated on a mixed C57BL6/129sv background. GRK2 were used in

191

 

. . . /
experiments and compared only to littermate GRK2fl fl controls. LysMCre and

GRK2fl/f' mice were genotyped as described previously respectively [10, 11]. Myeloid

Amye

cell GRK2 deleted mice (GRK2 ) will be referred to as GRK2 deficient and

. . fl/fl . . . .
littemate control mice (GRK2 ) Will be referred to as control mice in the entrre

manuscript.
Peritoneal Macrophage isolation

To isolate peritoneal macrophages, mice were injected by intra-peritoneal
injection with lml of 4% thioglycollate. Peritoneal macrophages were collected by
performing a peritoneal cavity lavage after 4 days of thioglycollate injection in
Dulbecco’s phosphate buffered saline (DPBS). Cells were washed atleast three times
and then counted and plated on cell culture plates in RPMI 1640 media supplemented
with 10% fetal bovine serum (Invitrogen) and penicillin (100 units/ml) and
streptomycin (100 [lg/H11) at 37° C in 5% C02. After around 18 hours of plating, cells
were serum starved for ~ 3-4 hours and stimulated. with LPS (1 jig/ml) for the indicated
time points.
Cytokine analysis

A mouse 23-plex multiplex based assay was used to determine the
cytokine/chemokine concentrations according to manufacturer’s instructions via
Luminex 100 technology as described previously [12]. Plasma from LPS injected mice
collected at different time intervals and supematants from peritoneal macrophages
stimulated with LPS for different time points were used to assess the

cytokine/chemokine levels.

192

Bronchioalveolar lavage fluid (BALF)

Bronchioalveolar lavage fluid was collected at different intervals after LPS
injection (using 2ml of 0.9% Normal saline). In each mice, around 90% of the total
injected volume was consistently recovered. The BALF was centrifuged at 450g for 10
min. The supematants were used to determine the concentration of total proteins using
Bradford assay with bovine serum albumin (BSA) as a standard.

Western blot analysis

Cells were lysed in lysis buffer (20mM Tris-HCl (pH 7.4), lmM EDTA,
150mM NaCl) containing 1% Triton X-100 with protease inhibitors. Lysed cells were
then centrifuged at a maximum speed (13,000 X g) for 10 min at 4°C and protein
concentration of the supematants determined by Bradford assay. Western blotting was
performed as described previously [13]. Briefly, equal amounts of protein were run on
polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes
were then blocked in Licor blocking buffer (Licor Biosciences) or 5% w/v skimmed
milk for 1 hour after which the membranes were incubated in primary antibodies
overnight. Secondary antibodies used were either fluorescently tagged or HRP-
conjugated. Blots were developed either on Licor odyssey or using
chemiluminescence.

Morphological assessment of liver injury:

Livers from control as well as GRK2 deficient mice, both LPS injected as well
as PBS (vehicle control) injected for 12 hours were fixed in 10% buffered formalin
solution for histological examination after sectioning of tissues and staining with

Hematoxylin and Eosin.

193

rd". w—nh'
, .

Survival study

Six to eight week old mice were challenged via an intra-peritoneal injection of
LPS (20ug/gm body weight) from Escherichia Coli (serotype 01112B4; Sigma-Aldrich,
St. Louis, MO). The mice were monitored for LPS induced lethality every 6 hours for
a period of 48 hours. Differences in survival were analyzed using Kaplan-Meier test
using Prism 5 software (GraphPad Software, La Jolla, CA). A value of less than 0.05
was considered as statistically significant.
Statistical analysis

All values are represented as mean :1: SEM. Data were analyzed and statistics
performed using GRAPHPAD PRISM software (San Diego, California). The student’s
t-test was used to compare mean values between two experimental groups and
Analysis of Variance (ANOVA) with Bonferroni posttest was used to compare more

than two groups. P value of less than 0.05 was considered significant.

194

 

RESULTS
Generation and characterization of myeloid cell deficient GRK2 mice

Generation of myeloid cell specific GRK2 deficient mice was accomplished by
first breeding mice expressing Cre recombinase under the control of Lysozyrne-M

(LysM) promoter (LysM-Cre mice express Cre-recombinase specifically in myeloid

cells) with mice homozygous for Lox-P flanked GRK2 alleles (GRKZH/fl). Double
heterozygous mice obtained from this breeding (GRK2fl/' LysMCre+/') were further

intercrossed to obtain GRK2"/fl LysMCre+/' mice (myeloid -specific GRK2 deficient:

Amye

GRK2 ) and GRKZfUfl (control mice). A breeding colony was then maintained to

breed GRK2fl/f' LysMCre+/' with GRKZ'Vfl to generate both GRK2"/fl LysMCre+/'

Amye

as well as GRKZ'Vfl mice. GRK2 and GRKZfl/f' litterrnates were used throughout

. A . .
this study. GRK2 mye were born at the expected Mendelian ratios. Furthermore,

immunoblot analysis of peritoneal macrophages and bone marrow derived

macrophages showed the absence of GRK2 protein only in macrophages from

GRKZAmye mice but not in GRKZfl/fl mice (Figure 5.1C and 5.1D). However, the

expression of GRK2 was not affected in whole organs such as lungs and spleen as
shown in Figure 5.1E.
Myeloid lineage GRK2 deficiency does not affect the development or
maintenance of myeloid cells

Since GRK2 has been shown to be essential for embryonic development of

mice, we examined the total numbers of myeloid lineage cells in both GRK2 deficient

195

‘1 at E~x'-;~.I_\v- _.
l

and control mice. For this purpose, we performed a complete blood count (CBC) on
both GRK2 deficient and control mice. As shown in Figure 5.2, we did not observe
any difference in the total numbers of RBC’s, platelets, WBCs, neutrophils,
lymphocytes, monocytes, suggesting no phenotypic difference in terms of
development of blood cells in myeloid cell-specific deficiency of GRK2. LPS
treatment however, did cause a significant increase in the number of RBCs as well as
hemoglobin content in both control and GRK2 deficient mice (Figure 5.2A and 5.2B).
Furthermore, there was a significant increase in the number of segmented neutrophils
and a marked decrease in the number of lymphocytes after LPS treatment in both
control and GRK2 deficient mice (Figure 5.2C). This however, was also not different
between the two genotypes. Interestingly, there was a significant increase in the
number of eosinophils in LPS treated GRK2 deficient mice compared to the
corresponding controls (Figure 5.2D). While the significance of this is not clear in
endotoxic shock, it is possible that tissue factor (TF) expression by eosinophils may
play a role in hypercoagulation observed in endotoxic mice and this may be
exaggerated in the GRK2 deficient mice [14].

However, we did not observe any difference in the plasma prothrombin time or
partial thromboplastin time in LPS treated control mice and GRK2 deficient mice
(data not shown). Even though plasma coagulability is not affected in GRK2 knockout,
it is possible that increased eosinophils in LPS treated GRK2 deficient mice might
lead to an increased TF which could then cause increased deposition of fibrin clots in
small blood vessels, thus impairing tissue perfusion and ultimately causing an organ

failure.

196

 

Figure 5.1 Generation and characterization of GRKZAmye mice

A. Schematic representation of the presence of loxP sites surrounding exons
3-6 that were targeted for deletion with Cre recombinase

B. Cre mediated recombination leads to the deletion of exons 3-6 of GRK2
gene

C. Western blot showing the absence of GRK2 protein in thioglycollate

elicited peritoneal macrophages from GRK2Amye (GRK2 deficient mice) mice

fl/fl . . . .
as compared to GRK2 (littermate control) mice. Tubulin is shown as a

loading control.

D. Western blot showing the absence of GRK2 protein in Bone marrow
derived macrophages from GRKZAmye mice as compared to GRKZfl/fl' mice.

Tubulin is shown as a loading control.

E. Western blot showing that GRK2 is normally expressed in organs such as

lungs and spleen in both GRK2Amye as well as GRK2fl/fl mice

197

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1 2 3 4 5 6 7
GRKZflIfl
LysMCre
GRKZAMye
D.
Peritoneal Mac BMDMs
GRKZfl/fl, GRKZAMYe GRKZfl/fl GRKZAMye
.2.-..2... »~.-.-~--» Tubulin "—- “Tubulin
E. GRK2fl/fl GRKZAMye
Lungs Spleen Lungs Spleen
1"" 1"" GRK2

 

 

 

198

 

 

Figure 5.2 Complete blood count of cells from GRK2Amye verses

GRKZfl/f' mice

A. RBC, platelet and WBC counts were performed 12 hours after injection of
LPS. Blood was collected using sodium citrate anticoagulant and the counts
were performed. Cells are represented as per ul.

B. Graph showing the total protein and Hemoglobin content in g/dl in the
blood of both GRKZ'Vfl and GRKZAmye control mice (injected with PBS)

verses LPS injected mice.

C. Graph showing the percent segmented neutrophils, lymphocytes and
monocytes in the blood of both GRKZfl/f' and GRKZAmyc control mice
(injected with PBS) verses LPS injected mice.

D. Graph showing the percent eosinophils in the blood of both GRK2fl/fl and

GRKZAmye control mice (injected with PBS) verses LPS injected mice.

199

 

P

Total cells per ul

gldl

I: GRK2 f" f' ctrl

 

 

 

 

 

 

108'
- . -GRK2 f" f' LPS
_ [1 I1 EIGRKz “We ctrl
107-W ~ I -GRK2 Amye LPS
106= 'I' h
10' RBC Platelet WBC
**
201 III GRK2 “’" ctrl
I GRK2 "’1' LPS I I
III GRK2 “We ctrl-“=l -
- GRK2 “We LPS
10-
l
l
l
(0-

 

 

 

 

 

 

 

Total Protein H93

200

 

Fig 5.2 continued ......

C.

  

   

 

 

 

 

 

 

 

 

 

  
     
     
       
     
      

amt

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90-
80- El GRK2 "1" ctrl
70- I GRK2 "l" LPS
w 60- .. _, .GRKZ AmV°ctrl
g 50- ‘ :=:- Ecnkz AmWLns
f; 40. F 5:5:
° 30- . .. '_:;
. 'u'.
20- j . 15;
10“ :I::
0‘ E '7 ; :3:
Segmented N Lymphocyte Monocyte
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.2
E 5.0-
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39
49° 3
4° 3°
3° 9

201

Deficiency of GRK2 in myeloid cell lineage causes an increased secretion of
cytokines and chemokines in plasma in response to LPS injection

GRK2 is highly expressed in myeloid cells particularly in neutrophils and
macrophages. Furthermore, it has been shown previously that there is an increased
expression of GRK2 in the neutrophils of sepsis patients [8]. Moreover, we have also
found that the expression levels of GRK2 increase over time in primary macrophages
from mice in response to LPS stimulation [15]. Keeping these findings in mind and to

further characterize the role of GRK2 in sepsis, we injected LPS (30ug/g body weight)

in GRK2 deficient (GRKZAmye) and their littermate controls (GRKZfl/fl) by intra-

peritoneal route and collected blood at different time intervals. Plasma levels of
cytokines and chemokines were determined using Biorad’s 23-plex assay. Our results
show that IL-12 (p40), MIPla and IL—10 are significantly enhanced in the GRK2
deficient mice compared to controls. Although plasma IL-12 (p40) levels peaked at 3
hours in the control mice, in GRK2 deficient mice these levels were significantly
elevated at 12 hours post-injection (31363i8069 pg/ml in GRK2 deficient compared
to 1454733071 in control mice). Similar to IL-12 (p40), IL-10 levels were also
significantly enhanced in the GRK2 deficient mice at 12 hours post-injection

(17300i5938 pg/ml in GRK2 deficient compared to 6146i2046 in control mice).
Compared to IL-12 (p40) and IL-10, MIPlcx levels were markedly enhanced in the
GRK2 deficient mice at l and 3 hours post-LPS injection. At one hour, MIPloc levels
were 1695i149 pg/ml in GRK2 deficient compared to 1019i149 pg/ml in control
mice. Similarly, at 3 hour post-LPS injection, MIPla levels 3627i314 pg/ml in GRK2

deficient compared to 2604i330 in control mice. In addition to these three

202

 

inflammatory factors, IL-6 (at 3 hours: levels were 143229140133 pg/ml in GRK2
deficient mice compared to 85806i24085 pg/ml in control mice), MIPIB (at 3 hours:
levels were 22888:|:3736 pg/ml in GRK2 deficient mice as compared to 16646i2952
pg/ml in control mice) and IL-17 (at 12 hours: levels were 1670i621 pg/ml in GRK2
deficient mice as compared to 801:1:245 pg/ml in control mice) were also enhanced in
GRK2 deficient mice, but did not reach statistical significance. Compared to these
factors, a similar trend (enhanced in GRK2 deficient mice compared to controls) was
observed for IL-la, IL-IB, eotaxin, RANTES, MCP1 and IL-9. (Figure 5.3).
Importantly, plasma levels of cytokines/chemokines including IL-2, IL-3, IL-4, IL-5,
IL-12 (p70), IL-l3, TNFa, IFNy, GCSF, GMCSF and KC were not different between
control and GRK2 deficient mice after LPS injection, suggesting selective regulation
of TLR4-induced cytokines/chemokines by myeloid cell-specific’ GRK2. Taken
together, these results suggest that GRK2 somehow acts as a negative regulator of LPS
induced inflammatory response and hence the deficiency of GRK2 can augment the
inflammatory response to LPS. '

Enhanced tissue injury and mortality in GRK2 deficient mice

Lung Injury: Acute lung injury in sepsis is associated strongly with
cytokine/chemokine response and is characterized by an initial recruitment of immune
cells such as neutrophils and macrophages soon after the onset of infection as well as

lung edema with extravasation of plasma proteins due to an increase in vascular

permeability. Hence, detection of plasma proteins serves as one useful indicator of

203

 

Figure 5.3 LPS injection causes an enhanced secretion of pro and anti-

inflammatory cytokines in the plasma of GRK2Amye mice

Mice were injected with LPS (30ug/g body weight) via an intra-peritoneal
injection and blood was collected at different time intervals. Secretion of
cytokines and chemokines was assessed using a Biorad 23 plex multiplex
based assay. Levels of IL-12 (p40), MIPla, IL-10, MIPlB, IL-6, IL-17, IL-la,
IL-lB, Eotaxin, Rantes, MCP1 and 1L-9 are shown in pg/ml. N=11 for 1, 3
and 12 hrs. N=6 for 0, 6 and 18 hrs. *p<0.05; **p<0.01.

204

 

Plasma lL-12 (p40) (pg/ml)

Plasn'a NIP-1a (pgln‘i)

Plasma lL-10 (pg/ml)

50000~

40000'

300004

20000-

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Time (Hours)

205

Figure 5.3. Continued.

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Plasma MIP-1p (pg/ml)

 

  

 

  

 

 

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0 3 6 9 15
Time (Hours)
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*GRKZ fllfl
A -A- Amye
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ill 3 '
E 50000- ;
0 . . . .
0 3 6 9 12
Time (Hours)
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-A- yo
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i
2000-
: l ......... A
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,1 1500-
s 1000-
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500-

 

 

o 3 8 8 1'2 15 18
Time(Hours)

206

Figure 5.3. Continued.

500-
*G RKZ fl/fl

400. 'A'GRKZ Amy" L

   
  
  

Plasrra IL- 1a (ngi)
n w
o o
‘5’ P

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9

 

 

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Time(Hours)

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Time (Hours)

 

12000- 'l-GRKz fl,“

90001

60004

 

3000‘

Plasma eotaxin (pglrrl)

 

 

0888121818

Time (Hours)

207

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. é.

Figure 5.3. Continued.

250001
200001
150001

100001

01
o
o
9

Plasma RANTES (pg/ml)

 

‘I'GRKZ "’fl
1 ’A'GRKZ Am"

   

 

1 20000-

800001

400001

Plasma MCP-1 (pgln'l)

 

3 6 9 12 15 18
Time (Hours)

*GRKZ fllfi

'A'cnkz Am"

 

 

Plasrm lL-9 (pglml)

 

 

3 6 9 12 15 18

Time (Hours)
-.-G R K2 "I"

”A'GRKZ Am”

.1.

a e 81'21'51'8

Time (Hours)

208

-.m__d

Tm_'
I.
l
l.

 

lung injury. We hypothesized that the enhanced cytokine/chemokine response
observed in the GRK2 deficient mice will be associated with exaggerated lung injury
and permeability. Increase in lung permeability in response to LPS administration was
determined by measuring the leakage of total protein into bronchoalveolar lavage
(BAL) fluid. As shown in Figure 5.4A there was a significantly enhanced protein
content in LPS injected GRK2 deficient mice as compared to their littermate controls
at 12 hours post injection (0.060i.004 jig/u] protein in control compared to
0.093i.009 jig/pl protein in the GRK2 deficient mice), suggesting that the lung injury
in response to LPS is worse in the GRK2 deficient mice compared to the control mice.
These results suggest that the enhanced cytokine response observed in vivo may be
associated with the exaggerated lung injury observed in the GRK2 deficient mice in
response to LPS injection.

Liver injury: In addition to the lungs, liver injury is another significant consequence
of endotoxemia. To examine if the enhanced cytokine response also affected liver
pathology, we performed a histopathological analysis of the livers from 12-hour LPS-
injected control and GRK2 deficient mice. As predicted, the inflammatory response to
LPS in the GRK2 deficient mice was found to be considerably enhanced as evidenced
by much more intense accumulation of inflammatory cells and marked edema with
fibrin strands in prominent periportal areas (Figure 5.4B). This suggests that the
inflammatory response to systemic LPS administration develops much more
excessively in mice deficient in myeloid cell GRK2 when compared to control mice.
Endotoxic mortality: Organ failure in response to endotoxemia eventually leads to

mortality in mice. Because lung and liver injury were exaggerated in the GRK2

209

 

 

deficient mice compared to controls, we hypothesized that the mortality of GRK2
deficient mice would be higher than the control mice. As predicted, we found that
50% of the GRK2 deficient mice died within the first 24 hours of LPS injection as
compared to only 20% in the control group. Our results further show that whereas
100% of the GRK2 deficient mice subjected to LPS induced sepsis died within 42
hours of injection, the GRK2 control mice showed a significantly enhanced survival
rate with 30% surviving at 48 hours (Figure 5.4C). These results suggest that myeloid
cell GRK2 plays a protective role in LPS induced endotoxemic death.
Enhanced LPS-induced cytokine response in GRK2 deficient primary
macrophages

Results presented above suggest that the deficiency of GRK2 in myeloid cells
can affect the cytokine/chemokine response in plasma. Although myeloid cells, in
particular monocytes, neutrophils and macrophages are the major cells involved in the
secretion of cytokines and chemokines, other cells such as lymphoid cells (T cells and
B cells) as well as fibroblasts are also involved in this process. To further investigate if
myeloid cells are the major producers of this response in vivo, we determined the
effect of LPS stimulation on the secretion of cytokines and chemokines in cell culture
supematants. For this purpose, we isolated primary peritoneal macrophages from both
GRK2 deficient as well as control mice and stimulated them with LPS (lug/ml) under
in vitro conditions. As was observed in vivo in the GRK2 deficient mice, LPS-induced
cytokine/chemokine responses were significantly enhanced in primary macrophages in

vitro. As shown in Figure 5.5, we observed a markedly enhanced secretion of pro-

210

 

 

Figure 5.4 GRK2Amye show enhanced tissue injury and mortality as

compared to GRKZfllfl mice

A. Mice were injected with LPS (30pg/g body weight) via intra-peritoneal
route and broncho-alveolar lavage fluid was collected after 12 hours. The cells
were pelleted by centrifuging at 450g for 10min and the supernatant was used
to measure the total protein using Bradford assay. Protein is shown in jig/pl.

B. Morphological changes in mouse liver sections of LPS-injected myeloid
cell GRK2 deficient verses control mice. (A) Control mice liver (B) GRK2
deficient mice liver. Arrows show neutrophils, arrowheads show fibrin in
venous spaces. (H&E, original magnification X 10). (C) Control mice liver.
Neutrophils and minimal edema present in periportal areas. (D) GRK2
deficient mice liver. Numerous neutrophils; star shows edema; arrowhead
shows fibrin stands in prominent periportal areas (H&E, original

magnification, X 20).
. . Amye . . fl/fl
C. GRK2 defic1ent mice (GRK2 ) and littermate control mice (GRK2 )

were injected intra-peritoneally with 20ug/gm LPS and their survival was
monitored over a period of 48 hours. Black line shows the survival rate for
control mice (N=10) and dashed line shows the survival rate of GRK2

deficient mice (N=10). Data were analyzed with the Kaplan-Meier test.

211

 

 

Protein concentration

.>

**

 

0.12-
0.10-

)

o
b
8"

 

0.06-

BALF
#9 I Pl

(
o
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0.021

 

 

 

O'OO'GRK'Z "’" GRK2 “We

12 hour LPS injection

212

 

 

 

m
.m
m
c
4
5
n
w.
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F I ..
and?
2..

 

213

Percent survival

Figure 5.4. Continued....

C.

100

 

80-

601

40-

201

 

 

 

 

0 8 1'2 1'8 2'4 3'0 3'6 4'2 48
Time (Hours)

214

ml

Fur—MLJI. "

 

. . . fl
inflammatory cytokines VIZ. TNFa (59221247pg in GRKZAmye; 363i60pg 1n GRKZfl/

at 6 h), IL-6 (185i26pg in Grammy"; 80i19pg in GRKZ'M' at 24 h), IL-la

Amye

(5.8il.lpg in GRK2 ; 3.2i1.1pg in GRK2fl/fl at 24 h), IL-lB (55.2:1:5.3pg in

Amye

GRK2 ; 27.5:l:4.7pg in GRKzfl/fl at 24 h), IL-9 (24i2pg in GRKZAmye; 111:ng in

GRK2fl/fl at 12 h) as well as chemokines viz. MCP1 (155221: 82pg in GRKZAmye;

710i111pg in GRKZfl/fl at 12 h), MIPla (16401178111; in GRKZAmye; 815i136pg in

GRKZfl/fl at 24h) and GCSF (117:1:22pg in Grimm”; 47ilOpg in (3111(szfl at 24 h)

in the GRK2 deficient mice macrophages compared to control cells. Importantly,
cytokines including IL-2, IL-12p40, IFNy and IL-17 did not differ between control
and GRK2 deficient mice macrophages, suggesting selective regulation of TLR4-
induced cytokine responses by GRK2. To determine the differences between
macrophages and neutrophils in terms of the cytokine regulation, we also assessed the
levels of cytokines and chemokines in neutrophils (treated in vitro) from these mice.
Compared to the results obtained with macrophages we observed that neutrophils from
GRK2 deficient mice show an enhanced secretion of only G-CSF and KC compared to
control mice (Figure 5.6). Other cytokines such as IL-la, IL-18, IL-6, IL-12 (p70),
GMCSF, MIPla, and MIPIB showed a similar trend although these were not
statistically significant. Furthermore, some cytokine/chemokines (IL-12p40, RANTES,
IL-17) were not different between control and GRK2 deficient mice suggesting
specific regulation of some cytokines by GRK2 in the neutrophils.

GRK2 deficiency augments LPS-stimulated ERK phosphorylation but not P38

and JNK phosphorylation in primary macrophages

215

 

 

Figure 5.5 Peritoneal macrophages from GRK2Amye mice secrete

enhanced amounts of pro-inflammatory cytokines and chemokines in
response to LPS stimulation

Thioglycollate-elicited peritoneal macrophages were stimulated with LPS
(lug/ml) in 12-well cell culture plates and cell culture medium was collected
at different time intervals. Levels of inflammatory mediators were determined
using biorad 23 plex assay. Cells were lysed to determine the protein
concentration using Bradford method. Levels of TNFa, IL-6, IL-la, IL-18,
IL-9, MCP1, MIPla and GCSF are shown as pg/iig of total cellular protein.
*p<0.05; **p<0.01; ***p<0.001.

216

P2..

V“

 

TNFa secretion in pg

lL-1a secretion in pg

 

 

 

 

 

 

 

 

 

 

   

 

 

q .- fol q
800 -A- SEE; Amye 225 -I- G RKZ fl/fl ***
700 1 * 2001 -A- GRK2 Amye l

: “ o' .E 1 0"
500 2 : 0‘ g 5
: .o . 125-
400 '1 : “ '0' g
: \‘ ',' 8 100‘
300 - : "E ,o' a
: ‘ ' ‘9 75‘
1001 25-
0 I V I W e I I I I
0 6 12 18 24 0 6 12 18 24
Time (Hours) Time (Hours)
7- 'I'GRKZ “’1" * so. *GRKZ W“ ***
'A' GRK2 Amye l 'A' GRK2 Amye I
6" a ' ' o '
a 50' *1: .1"
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m. o
s- 20" '
2- A i
1 . 10" 'I'
c . . . . 0L. . - . .
0 6 12 18 24 0 6 12 18 24

Time (Hours)

217

Time (Hours)

 

Figure 5.5 Continued...

 

 

 

 

 

 

 

Time (Hours)

451 'I'GRKZ W"
4% ‘A'GRKZ Me I
g 35- ' 83
C
.E 30. 'E
c .o
i i
s 20- 8
w .-
o: 151 ii
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c I T I I
0 6 12 18 24
Time (Hours)
225“ 'l'GRKZ "I"
2000- 'A'GRKZ Amye m
83 l 83
.5 ml ------------- .s
C C
a e
2 2
ii i?
2 J 11
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2 :9
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218

*GRKZ fl’" ***
'A'GRKZ We .5

gs

 

 

 

1 501

1 251

1 001

 

 

Time (Hours)

'I'GRK2 W" ***
’A'GRKZ “We 1

 

 

Time (Hours)

 

Figure 5.6 Neutrophils from GRK2Amye mice secrete enhanced amounts

of pro-inflammatory cytokines and chemokines in response to LPS
stimulation

Thioglycollate-elicited neutrophils were stimulated with LPS (lug/ml) in 12-
well cell culture plates and cell culture medium was collected at different time
intervals. Levels of inflammatory mediators were determined using biorad 23
plex assay. Cells were lysed to determine the protein concentration using
Bradford method. Levels of GCSF, KC, IL-la, IL-IB, IL-6, IL-12p70, GM-
CSF, MIPla and MIPlB are shown as pg/pg of total cellular protein.
**p<0.01.

219

 

G-CSF secretion (pg)

lL-1 B secretion (pg)

GM-CSF secretion (pg)

80- *Gszfo'fi'GRKngYG

 

 

 

0 6 1'2 1'8 2'4
Time (Hours)
*GRKzfllfl .A.GRK2AMye

201

 

 

0 0 6 1'2 1'8 2'4
Time (Hours)
17.5-'.'GRK2fl/fl 'A'GRKZAMye
15.0-
12.5-
10.0-
7.5-
5.0-
2.5-

0.0

 

 

 

 

0 6 1'2 1'8 2'4
Time (Hours)

220

 

Figure 5.6 Continued. . ..

IL-6 secretion (99) KC secretion (pg)

MIP-1 a secretion (pg)

1251

100- l

'I'GRKZflm 'A-GRK2AMYS

75- x

50- '0'

25-

 

 

300-

2001

100-

0 6 1'2 1'8 2'4
Time (Hours)
'I'GRKZflm 'A'GRKZAMye

 

 

 

0 6 1'2 1'8 2'4
Time (Hours)

200-

100-

O

 

 

 

0 6 1'2 1'8 2'4
Time (Hours)

221

Figure 5.6 Continued. . ..

lL-12p70 secretion (pg)

NIP-1 (3 secretion (pg)

51 -I-GRK2fl/fl .A.GRK2AMye

lL-1 a secretion (pg)
(.0

 

 

 

0 6 1'2 1'8 2'4
Time (Hours)

17.51 *GRKZ'V'I 'A'GRKZAMye

15.01
12.51
10.01
7.5-
5.0-
2.51
0.0

 

 

 

0 6 1'2 1'8 2'4
Time (Hours)
1751'GRK2fl/fl 'A’GRKZAMye
1501
1251
1001
751
50-
251

 

 

 

0 6 1'2 1'8 24
Time (Hours)

222

‘ . ‘. A" sou-(aw
FA

 

To begin to understand the mechanisms of how GRK2 negatively regulates
LPS-induced cytokine/chemokine response in vivo and in primary macrophages in
vitro, we tested the effect of LPS in GRK2 deficient and control cells on various
signaling pathways. LPS stimulation of TLR4 signaling leads to the activation of

MAPK signaling cascades including ERK, JNK and p38 kinases as well as IKBa-

NFKB signaling pathways. These pathways have been shown to be the central
regulators of inflammatory responses in endotoxemia. Therefore, we assessed the
phosphorylation status of ERK, JNK, p38 and IKBa in the primary macrophages both
in control as well as GRK2 deficient macrophages upon exposure to LPS. Peritoneal
macrophages from GRK2 deficient and control mice were stimulated with LPS for
various time points and immunoblotting performed for pERK1/2, pJNK, pP38 and
pIicBa. Our results suggest an interestingly selective role for GRK2 in LPS-induced
ERK phosphorylation. Thus, although LPS-stimulated pJNK, pP38 and pIKBa levels
were similar between the control and GRK2 deficient macrophages (Fig. 5.7B), pERK
levels were markedly enhanced in the GRK2 deficient cells (Figure 5.7A). Phospho-
ERK levels were 36i11% and 95i3% at 15 and 30 min respectively in GRK2
deficient cells, whereas in control cells the levels reached only 13i3% and 51i8% at
15 and 30 min respectively. We also examined the levels of pAkt and pGSK3 (known
to be regulated by GRK2) after LPS stimulation and did not find any evidence for their
regulation in these cells by GRK2 (Figure 5.8).

GRK2 negatively regulates NF—KBI-plOS-TPLZ-MEK-ERK pathway in primary
macrophages

223

 

 

Figure 5.7 GRK2 deficiency causes an enhanced LPS stimulated ERK1/2

activation in peritoneal macrophages
A. GRK2Amye and control primary peritoneal macrophages were stimulated

with lug/ml of LPS for different time points and the phosphorylation of
MAPKs and IKBO. was determined by western blotting. A representative blot
for phospho ERK and ERK and quantification for the same is shown in (A).
***p<0.001 compared to control. N=6.

B. A representative blot for phospho-p38, actin, phospho-JNK, JNK and
phospho-IKBa, tubulin is shown.

224

 

 

 

 

 

 

 

  
  
   
  

A.
GRKzflm GRKZAmye
0 15 30 60 90 120 180 0 15 30 60 90 120180 (Time, min)
:: $3 1': ' ' 2: 352 '1": pERK
~~~~~~~~ 1- "- 1-- -- - - ERK
125 +6sz "l“
. . Amye
*** A GRK2

—\
O
O

\l
0|

0|
0

25

ERK phosphorylation
(% maximal response)

 

O 30 60 90 120150180
Time (min)

225

 

 

Figure 5.7. Continued....

 

 

GRK2 fllfl GRK2 Amye
0 15 30 60 0 15 30 60 (Time,min)

 

 

I
- I: f. 1:1. .- ., .u. ~ .5_ .1 _ ,. 1... , . ,.. ; 1 ‘ ’13.." ‘ "W . ‘12:, ."‘7"."'-:. ?”.'E:, . '2"
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iii? “1",", “-11 - 111 111° W.._-JNK

111 . _ ..._T‘:'-lP|KBa

’H‘ ' I
*" In.‘ 1...:- 1. F1” mun w “,2... 1"" TUbUIIn

 

 

226

*4?

 

 

Figure 5.8 GRK2 deficiency does not affect LPS stimulation of Akt and

GSK3|1 activation in peritoneal macrophages
GRKZAmye and control primary peritoneal macrophages were stimulated with

lug/ml of LPS for different time points and the phosphorylation of Akt and
GSK3B was determined by western blotting. A representative blot for
phospho-Akt, Akt, phospho-GSK3B and tubulin is shown.

227

 

 

 

 

GRK2 "I" GRK2 Amve

0 15 30 60 0 15 30 60 (Time, min)
pAkt

 

 

~~fl~_~'“”Akt

 

 

 

 

 

GRK2 "m GRK2 MW"

0 15 30 60 0 15 30 60 (Time, min)
“pGSK3

 

 

it.

n— a... :g-mgggm—grubulin

 

 

 

228

To further elucidate the biochemical mechanisms of GRK2 regulation of the
ERK pathway, we examined the upstream regulators of ERK phosphorylation in
primary macrophages. Previous studies have demonstrated that ERK activation in
macrophages in uniquely regulated via LPS-stimulated IKKB-NFKB] p105 pathway.
That is, under unstimulated conditions, p105 (an IKB family member bound to NFKB
p50 subunits) is also stoichiometrically bound to TPL2 (a MAP3K). When activated,
p105 is phosphorylated by IKKB, which then undergoes partial degradation releasing
NFKB p50 subunits as well as TPL2. TPL2 then phosphorylates and activates MEK1/2,
which then activates ERK1/2. We first confirmed the existence of IKKB mediated
ERK signaling pathway in peritoneal macrophages. For this purpose, primary
peritoneal macrophages from GRK2 control mice were stimulated with either LPS
alone or pretreated with BMS345541 (IKKB inhibitor) before stimulation with LPS
and the phosphorylation of ERK was assessed by immumoblotting. As shown in
Figure 5.9, we observed that the pharmacological inhibition of IKKB indeed
attenuated ERK phosphorylation confirming the existence of IKKB-NFKB] p105 /
TPL2-MEK-ERK pathway. To investigate at what level GRK2 regulates this pathway,
we examined the phosphorylation of MEK1/2 and p105 after LPS stimulation in
control and GRK2 deficient macrophages. Our studies reveal that GRK2 negatively
regulates ERK pathway at the level of p105, because LPS-induced MEK1/2 as well as
p105 phosphorylation were both enhanced significantly in the GRK2 deficient
macrophages compared to control cells (Figure 5.10). Phospho-p105 levels were

471:1 5% and 84i9% at 15 and 30 min respectively after LPS treatment in the GRK2

229

 

Figure 5.9 The occurrence of IKKB-NFKBlp105/T plZ-MEK-ERK
pathway in peritoneal macrophages confirmed by using an IKKB
inhibitor

Peritoneal macrophages from control mice were stimulated with either LPS
alone or pretreated with BMS345541 (SpM), a specific inhibitor against IKKB,
30 minutes prior to stimulation with LPS (lug/ml) for various time points as
shown. Irnmunoblotting was performed for phospho-ERK and ERK proteins

using specific antibodies. A representative blot is shown.

230

 

l
[

 

 

 

LPS LPS+IKKi
0 30 60 0 30 60 (Time, min)

5:: ....;: pERK

 

 

231

 

 

deficient cells compared to 21i5% and 5321:10% at 15 and 30 min respectively in
control cells. Because LPS-induced IKKB-mediated IKBO. phosphorylation was not
affected by GRK2, our results indicate that GRK2 likely regulates at the level of p105.
This is further supported by previous studies, which have shown that GRK2 indeed

directly interacts with p105, even though these studies did not find any functional.

significance of this interaction. Taken together our studies indicate that GRK2

interaction with p105 negatively regulates LPS-induced ERK activation, as well as
potentially the p50-mediated NFKB activation.
Role of enhanced p105-ERK activation on exaggerated inflammatory response in
GRK2 deficient mice

Our results so far suggest that deficiency of GRK2 in primary macrophages
results in the enhanced activation of p105-ERK pathway, which is associated with an
enhanced inflammatory cytokine response observed in macrophages as well as in vivo
in mice. To further demonstrate that the effects on the cytokines observed in the
GRK2 deficient mice are a result of enhanced p105-ERK activation, we tested the
effects of LPS on cytokine/chemokine responses in primary macrophages in presence
or absence of an ERK inhibitor. We treated peritoneal macrophages from control and
GRK2 deficient mice with LPS in the presence or absence of PD98059 (a MEK
inhibitor). Cytokine/chemokine secretion was determined using 23-plex Biorad assay
kit. Among the cytokine/chemokines that were enhanced in the GRK2 knockout
macrophages, the ERK inhibitor inhibited IL-la, MIPla, GCSF and MCP1 in the
GRK2 knockout macrophages but not in the control mice macrophages. In the

presence of the ERK inhibitor, the levels cytokines/chemokines returned to the levels

232

 

 

 

.ua‘i’. l

Figure 5.10 GRK2 deficiency causes an enhanced LPS stimulated NFKB]

p105 activation in peritoneal macrophages
GRK2Arnye and control primary peritoneal macrophages were stimulated with

1 jig/ml of LPS for different time points and the activation of NFKB] p105 and
MEK were determined by western blotting. A representative blot for phospho-
NFKBl p105, ERK, phospho-MEK, tubulin and quantification for the same is
shown. ERK and tubulin were used as loading controls. *p<0.05 compared to

control. N=7.

233

 

 

 

 

GRK2 "’1' GRK2 MW"
0 15 30 60 90120180 0 15 30 60 90120 180 (Time,min)

 

 

. 2.... -,.... ~1- "" "*"‘ ‘ ' NFKB1P105
be... -- in“ Inm- ~ ‘ “M 111-11' = pMEK

~~~...-).L.~.~~~~~muTubulin

 

—.— GRK2 fl/fl
.fi. GRK2 Amye

  

NFKB1 p105 phosphorylation
(% maxrmal response)

0
0 30 60 90 120150180

Time (min)

234

 

 

observe
than the
lL-la. l
GRK2

examine
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also Sig
Interest
were bl
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likely i
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genotyp

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observed in the control LPS treated cells (except for MIPla which was slightly higher

than the control LPS) (Fig. 5.11A). This demonstrates that the enhanced secretion of
IL-la, MlPla, GCSF and MCP1 result from an enhanced ERK activation observed in
GRK2 deficient macrophages. Because IKK-p105 pathway regulates ERK, we
examined if the secretion of these inflammatory factors are also affected by inhibition
of IKKB. Except for MCP-l, the other three factors i.e. IL-la, MIPIOL, GCSF were
also significantly blocked with the IKKB inhibitor (BMS345541) (data not shown).
Interestingly the enhanced secretion of IL-6, and IL-18 in the GRK2 deficient cells
were blocked only by the IKKB inhibitor and not by ERK inhibitor, suggesting that
these two cytokines are regulated by GRK2 exclusively via the IKKB-p105 pathway
likely involving the p50-NFKB activation (Figure 5.11B). Although IL-9 was
consistently enhanced in the GRK2 deficient cells compared to the control cells,
neither BMS345541 nor PD98059 efficiently blocked IL-9 secretion in cells from both
genotypes. Thus IL-9 may not be exclusively regulated by IKKB or ERK.

Taken together, our results demonstrate that GRK2 negatively regulates p105-
ERK pathway thereby regulating the production of ILl-a, GCSF, MIPIOL and MCP1

in response to LPS.

235

 

Figure 5.11 Pharmacological inhibition of ERK and IKKB cause a

significant inhibition of LPS-induced secretion of cytokines and

m e .
y mice

chemokines in peritoneal macrophages from GRK2A
A. Peritoneal macrophages were either left unstimulated, stimulated with LPS
or pretreated with PD98059 (lOuM), a MEK inhibitor, 30 minutes prior to
stimulation with LPS (lug/ml) and the cell culture supernatant was collected
24 hours later. The secretion of cytokines and chemokines was assessed as
described before using biorad 23 plex assay. N=5. *p<0.05; **p<0.01;
***p<0.001.

B. Peritoneal macrophages were either left unstimulated, stimulated with LPS
or pretreated with BMS345541 (SliM), a specific inhibitor against IKKB, 30
minutes prior to stimulation with LPS (lug/ml) and the cell culture
supernatant was collected 24 hours later. The secretion of cytokines and

chemokines was assessed as described before using biorad 23 plex assay. N=5.
*p<0.05; **p<0.01; ***p<0.001.

236

 

 

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238

 

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Figure 5.11. Continued....

 

 

 

 

 

 

 

 

 

 

 

 

 

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239

DISCUSSION

It has been well documented that the expression levels of GRK2 are altered in
certain inflammatory disease conditions. For instance, GRK2 levels are significantly
reduced in leukocytes from patients of active relapsing-remitting multiple sclerosis
(MS) or with secondary progressive MS [3]. Peripheral Lymphocytes of patients with
Alzheimers disease have an increased expression both mRNA and protein for GRK2
[16]. GRK expression have also been found to be altered in PBMCs of patients with
rheumatoid arthiritis [5]. Although the significance of these changes is not clear
particularly in these diseases, studies are just emerging on the pathophysiological role
of GRK2 in these diseases in rodent models. For example, in an acute model of
arthritis, GRK2 was shown to be a negative regulator of granulocyte chemotaxis to
LTB4 (a potent Chemoattractant for granulocytes). Heterozygous GRK2 knockout
mice showed an increased weight loss as well as development of arthritis was
significantly enhanced in these mice [17]. It is, however, not clear what signals
regulate the expression levels of GRK2. In a recent study, we demonstrated that TLR4
increases GRK2 levels in primary macrophages [15]. Other studies have demonstrated
a similar role for TLR signaling in regulating GRK2 levels in immune cells. Although
these studies proposed that the consequence of this increase is in limiting chemokine
signaling, our studies presented here demonstrate that GRK2 regulates TLR4 signaling
and LPS-induced cytokine/chemokine production and the consequent endotoxic shock.

We show that GRK2 deficient mice show an enhanced liver and lung injury in
response to LPS injection and are prone to LPS induced septic shock. This is not

surprising in the light of the fact that other studies using heterozygous GRK2 mice

240

 

 

have shown similar results with other disease conditions. For instance, GRK2 +/- mice
were found to be more susceptible for arthritis disease severity [l7]. GRK2 +/-
animals also show an advanced onset of experimental autoimmune encephalomyelitis
in association with an increased early cerebral infiltration of inflammatory cells [3].
Recent studies demonstrated that GRK2 +/- mice also show an increased susceptibility
to neonatal hypoxic-ischemia brain damage [18]. While this manuscript was in
preparation, another study showed the role of cell-type specific GRK2 in regulating
hypoxia-ischemia brain damage [19]. Therefore, in general reduced levels of GRK2
increase the susceptibility to several disease conditions and we demonstrate here that
myeloid cell GRK2 deficient mice are much more prone to LPS induced septic shock.
The migration of neutrophils to the inflammatory sites although important for
host defense, is also in part responsible for tissue damage observed in sepsis. MIPla,
MIPlB, MCP1, Rantes and eotaxin are members of CC chemokine family which serve
as major chemoattractants for neutrophils, mononuclear cells as well as eosinophils
[20-23]. The secretary levels of all these chemokines were found to be enhanced in
GRK2 deficient mice plasma. Out of these however, the levels of MIPla were found
to be significantly increased both in plasma as well as cell culture supematants.
Furthermore, MIPla was found to be specifically regulated by p105-ERK signaling
since ERK inhibitor as well as IKK inhibitor inhibited its increased synthesis. MIPla
is produced by several cell types including lymphocytes, monocytes/macrophages,
mast cells, basophils, epithelial cells and fibroblasts and binds to CC chemokine
receptor 1 (CCR1 and CCR5) to exert its biological actions. MIPla plays a crucial

role in activation and chemotaxis of several populations of macrophages. It stimulates

241

the proliferation of mature tissue macrophages and also has been shown to induce the
secretion of TNFa, IL-6 and IL-la from elicited peritoneal macrophages [24]. It is
highly expressed in acute as well as chronic lung inflammation [25]. MIPla alpha has
been previously shown to be responsible for LPS induced lung capillary leakage and
early mortality in endotoxic shock [23]. We, in our studies also found a substantially
increased lung capillary permeability as assessed by the measurement of total protein
in the broncho-alveolar lavage fluid suggesting a role for increased MIPla in
enhanced lung injury observed in our studies. Furthermore, there is an increased
infiltration of inflammatory cells in the liver resulting in an increased liver damage.
Hence, we propose MIPla to be an important factor responsible for inducing a
substantially increased lung and liver injury in GRK2 deficient mice. Another
important pro-inflammatory cytokine IL-12p40 is also found to be highly secreted in
GRK2 deficient mouse plasma. It is a component of IL-12 cytokine and is an
important mediator of cell mediated immunity [26]. Furthermore, it has been shown to
play an important role in inflammatory diseases such as experimental autoimmune
encephalitis [27]. IL-lO, an important anti-inflammatory cytokine was also found to be
increased in GRK2 deficient mouse plasma. An increase in an anti-inflammatory
cytokine should prevent from leading to endotoxemia in theory. However, studies
have illustrated that although increase in IL-lO prevents an over—exuberant immune
response, it can also lead to immunosuppression shifting the balance toward lethal
consequencies. Since we observe an early lethality in LPS injected GRK2 deficient
mice, it is suspected that the balance is shifted towards an increased inflammatory

state leading to organ injury and mortality.

242

 

 

 

Our results demonstrate that the peritoneal macrophages from GRK2 deficient
mice have an increased ERK activation in response to LPS treatment as compared to
control mice. Inhibitor studies further support our findings that the increased ERK
activity is responsible for the enhanced expression of certain pro-inflammatory
mediators in GRK2 deficient peritoneal macrophages. The effect of GRK2 on ERK
activation has also been shown by other investigators. In HEK 293 cells, increased
GRK2 was found to cause reduced ERK activation in response to C-C chemokine
ligand 2 (CCL2). On the other hand, splenocytes from GRK2+/- mice, were found to
have an enhanced ERK activation in response to chemokines. These studies also
showed that GRK2 and MEK are present in the same multi-molecular complex but
unlike our studies, GRK2 did not affect MEK activity [28]. In another study, pro-
inflarnmatory cytokine IL-lB was found to induce a 2—3 fold increase in the expression
of GRK2 in primary astrocytes and a simultaneous decrease in CCL2-induced ERK1/2
activation whereas astrocytes from GRK2 +/- mice show an increase in ERKl/Z
phosphorylation [29]. While these other studies have focused on chemokine receptor
(a GPCR)-induced ERK activation, our studies address the role of GRK2 in TLR4
signaling, especially in primary macrophages. Thus although GRK2 has very
important roles in GPCR signaling, our studies here demonstrate that GRK2 is equally
important in regulating TLR4-induced ERK activation and the consequent
cytokine/chemokine production.

In this study we have determined that the mechanism by which GRK2
regulates TLR4-induced ERK activation lies at the level of NFKBl p105. In

macrophages, NFKB] p105 lies upstream of MEK1/2 kinase and is present in a

243

 

 

complex with Tp12, a MAP3K. Our studies clearly show that LPS-induced MEK1/2
as well as NFKBI p105 phosphorylation are significantly enhanced in macrophages
from GRK2 deficient mice. We believe that the mechanism lies at the level of NFKB]
p105 for the following reasons: 1. LPS-induced Icha phosphorylation is not affected
by GRK2 deficiency in macrophages. IKBOL is also phosphorylated by IKKB, the same
enzyme that phosphorylates NFKB] p105. Thus if the regulation by GRK2 is at or
above the level of IKKB, then one would expect that IKBa phosphorylation to also be
regulated by GRK2. 2. Previous studies have shown that GRK2 and NFKB] p105
directly interact with each other. GRK2 and NFKB] p105 have been shown to interact
in yeast two-hybrid assays. And the RH domain of GRK2 has been shown to directly
interact with the Carboxy terminus of p105 in direct interaction assays. Previous
studies, however, have also shown that NFKB] p105 is a poor substrate for GRK2,
suggesting that the regulation might be phosphorylation independent. This is not
entirely surprising given the role of RH domain of GRK2 in mediating
phosphorylation-independent cell signaling, protentially as a scaffolding protein. In
studies using Raw264.7 macrophages cell line, however, we did not find any
functional significance of GRK2 in LPS-induced NFKBI p105-ERK pathway, using
RNAi. In the present studies we find that in primary macrophages from genetically
modified levels of GRK2, GRK2 does regulate LPS-induced NFKBI p105-ERK
pathway. Thus the differences between the studies could be related to the differences
in cell types (cell line v/s primary cells) and/or the method of decreasing GRK2 levels

(RNAi v/s genetic).

244

 

 

 

GRK2 has previously been shown to regulate a number of signaling pathways
not necessarily restricted to GPCR signaling. Even with in GPCRs, role of GRK2 has
expanded considerably beyond its role in GPCR phosphorylation. Some of these
signaling pathways are relevant to TLR4 signaling. For example, Liu et al., have
demonstrated that Akt interacts with GRK2 and this interaction inhibits Akt activity,
which then limits the activation of eNOS and the production of NO in sinusoidal
endothelial cells leading to intrahepatic portal hypertension. Correspondingly, GRK2
deficient mice develop less severe portal hypertension after liver injury [30]. Even
though TLR4 activation in primary macrophages induces Akt phosphorylation, we did
not observe any difference in Akt phosphorylation between GRK2 deficient and
control macrophages. One possibility for this difference is the different cell types
examined. In another study, Peregrin et al. showed that GRK2 can interact with and
phosphorylate p38 MAPK and this can lead to the inactivation of p38 MAPK.
Furthermore, peritoneal macrophages from GRK2+/- mice were found to have an
enhanced production of TNFa due to an enhanced activation of p38 [31]. In our
studies, however, we did not observe enhanced activation of p38 in the GRK2
deficient macrophages compared to the control cells. The reason for this difference is
not clear. In our studies, however, we demonstrate that inhibition of enhanced ERK
activation significantly lowers the enhanced cytokine/chemokine levels to that of the
wild type levels, suggesting a clear role for this signaling pathway in mediating
MIPla, IL-loc, GCSF and MCP1. Our studies fiirther demonstrate that the NFKB]

P105-mediated p50-NFKB pathway may also be equally important in mediating the

enhanced secretion of IL-1 B and IL-6 in the GRK2 deficient macrophages because the

245

IKKB inhibitor blocked the production of these two cytokines even though they were
not affected by the ERK inhibitor.

It is not clear why we see a slightly different response in terms of the cytokines
/chemokines which are secreted in plasma verses those in cell culture supematants.
However, one can envision several possible reasons for this discrepancy: 1. A variety
of cell types are involved in producing cytokines under in vivo conditions and the
levels observed in plasma are an overall contribution of these various cell types as
compared to only one cell type used in vitro in our studies. 2. LPS stimulation of
TLR4 onto macrophages is a direct signaling mechanism leading to the expression of
genes for cytokines / chemokines as compared to indirect signaling mechanisms due to
cross talks involved under in vivo conditions. 2. We used only one type of
macrophages i.e. thioglycollated elicited peritoneal macrophages under in vitro
conditions. However, there are a variety of macrophages (tissue specific) which
contribute to the effects seen under in vivo conditions.

Several studies have shown that sepsis patients have increased circulatory
levels of TNFa, MIPla, MIPlB, IL-6 and IL-10. Furthermore, in one study it was
found that neutrophils from septic patients show a reduced chemotaxis due to a failure
to induce an increase in tyrosine phosphorylation and actin polymerization in response
to chemokines. These neutrophils were found to have high expression levels of GRK2
and GRK5. Moreover, neutrophils from healthy individuals treated with LPS and
cytokines were also found to have an increased expression of GRK2 and GRK5. This
suggested that the pro-inflammatory mediators produced during sepsis increase the

expression of GRKs leading to desensitization of neutrophils to chemokines [8].

246

 

 

However, our studies suggest that GRK2 is protective against sepsis in the initial
phase since deletion of GRK2 in myeloid cells increases susceptibility to sepsis. In an
already established septic condition however, the expression levels of GRK2 might
increase in an effort to protect the system from the lethal consequences of sepsis.
Reduced chemotaxis might be detrimental since neutrophils are required to help fight
infection. The system, however, needs to maintain a balance and it is tempting to
hypothesize that this might be mediated by increasing GRK2 levels under septic
conditions. Obviously further studies are necessary to delineate these issues.

In summary, mice with a deficiency of GRK2 in myeloid cells are more
susceptible to LPS-mediated endotoxemia and this is associated with enhanced
secretion of several cytokines/chemokines. We further demonstrate that the enhanced
cytokine/chemokine in the GRK2 deficient mice is related to enhanced activation of
the p105-ERK pathway in macrophages. Our results suggest that the increase in GRK2
levels observed in immune cells under septic conditions might limit the progression of

sepsis.

247

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CHAPTER 6

SUMMARY AND CONCLUSIONS

6.1 Specific aims and results of the study

The major aim of this thesis was to investigate the roles of GRKs in inflammatory
signaling. It was hypothesized that GRKs are important regulators of inflammatory
signaling in macrophages and therefore GRKs might play essential roles in the
regulation of inflammatory diseases. Summarized below are the specific aims along

with the results obtained:

Specific Aim 1: To determine the biochemical mechanisms by which GRKs regulate
TNFa-induced signaling pathway in mouse macrophages
MS;
1. Knockdown of GRK2 and GRK5 inhibits TNFa-induced NFKB signaling in
Raw 264.7 mouse macrophage cell line
2. Overexpression of GRK2 and GRK5 increases TNFa-induced NF KB activity
3. GRK2 and GRK5 interact with and phosphorylate IKBa, an inhibitor of NFKB
4. GRK5 phosphorylates serine 32 on IKBO. which is the same site that is

phosphorylated by IKKB in classical NFKB signaling

Specific Aim 2: To determine the role and mechanism by which GRKs regulate
inflammation in a mouse model of disease (Endotoxic shock)
Subaim 1: To study the role of GRK5 in inflammation in a GRK5 knockout mice

using endotoxic shock model

252

Results:

. . -/- . . . . . ..
1. Primary peritoneal macrophages from GRK5 mrce exhrbrt an rnhrbrtron of

LPS-induced NFKB activation as compared to primary peritoneal macrophages

from GRK5“+ mice

2. GRK5 gene deletion does not affect LPS-induction of MAPKs viz. ERK, JNK

and p38

3. Peritoneal macrophages from GRK5-L mice show reduced secretion of key
. . . . +/+ .
inflammatory cyokrnes in comparison to those from GRK5 mrce

4. LPS-induced secretion of plasma cytokines is also inhibited in GRKSJ- mice
compared to GRKSWL/+ mice

. . . . -/- .
5. Reduced secretion of cytokrnes and chemokines 1n the plasma of GRK5 mrce

was associated with reduced ALT levels (an indicator of liver injury),
suggesting diminished liver injury in GRKJ- mice

Subaim 2: To generate and characterize a myeloid cell specific GRK2 knockout
mice and to study the role of GRK2 in inflammation using endotoxic shock model
EELS;
1. A myeloid cell specific knockout of GRK2 was generated using Cre-LoxP
system
2. Myeloid lineage GRK2 deficiency did not affect the development or

maintenance of myeloid cells

253

3. LPS injection caused an enhanced secretion of cytokines and chemokines in
the plasma of myeloid cell-specific GRK2 deficient mice in comparison to
littermate control mice

4. Primary peritoneal macrophages as well as neutrophils from GRK2 deficient
mice showed an increased secretion of cytokines and chemokines in response
to LPS

5. GRK2 deficient mice exhibited a greater lung and liver injury and were more
susceptible to LPS-induced septic shock

6. LPS induced NFKBI p105 as well as ERK phosphorylation were found to be
enhanced in the peritoneal macrophages from GRK2 deficient mice

7. Pharmacological inhibition of ERK and NFKB] p105 blocked the enhanced
expression of cytokines and chemokines in the peritoneal macrophages from

GRK2 deficient mice

6.2 Limitations of the study

1. Knockdown of both GRK2 and GRK5 inhibited TNFa-induced NF KB activation in
Raw 264.7 mouse macrophage cell line. Primary peritoneal macrophages from a
germline GRK5 deleted mice similarly showed an inhibition of LPS-induced NFKB
activity. Primary peritoneal macrophages from myeloid cell specific GRK2 deleted
mice however, showed no change in LPS-induced NFKB activity. Results from these
three different studies should be compared with some degree of caution due to the

following reasons:

254

a) Three different methods of inhibiting the expression levels of GRKs were used viz.
siRNA, germline deletion and Cre-LoxP mediated gene deletion.

b) Three different types of cells were utilized: Cultured mouse macrophages,
peritoneal macrophages from mice of different genetic backgrounds

2. LPS injection model of sepsis was utilized in this study. Although this is a widely
used model due to its simplicity, it should be noted that it does not mimic human
sepsis which is characterized by bacteremia with multiple species of bacteria.

3. Since these studies were done on mouse macrophages, its relevance to humans is
not known at present.

4. Myeloid cell specific GRK2 deficient mice were used in this study due to the non-
availability of homozygous GRK2 knockout mice. Hence, results obtained in this
study cannot be generalized as negative regulation of sepsis by GRK2. This is because

GRK2 present in non-myeloid cells might regulate sepsis differently.

6.3 Positive outcomes of the study

This thesis research investigated the roles of GRK2 and GRK5 in
inflammatory signaling in macrophages. The three individual studies in this project
characterized the biochemical mechanisms by which GRKs regulate inflammatory
signaling as well as the physiological consequence of such regulation at in viva levels.

This study found that GRK5 can fiinction as an IKB kinase and phosphorylate
serine 32, one of the same sites phosphorylated by IKKB in the classical NFKB
pathway. Although further studies are needed to investigate the phosphorylation of

IKBa by GRK2 and 5 at in vivo levels as well as identify the sites of phosphorylation

255

on IKBa, our studies provide preliminary evidence that GRK5 can act as an IKB kinase
with a capacity similar to IKKB in macrophages and can thereby regulate TNFa and
TLR4-NFKB signaling. Since NFKB signaling plays an important role in several
inflammatory disease conditions, our findings in macrophages could have great impact
in terms of regulation of the pathogenesis of several inflammatory diseases. Indeed,
our in vivo studies using LPS injection mouse model of sepsis validates our in vitro
findings in macrophages.

Studies of modulation of LPS-induced sepsis by GRK5 suggest that GRK5
positively regulates LPS induced signaling under in vivo conditions. Our studies also
show that the regulation of expression of inflammatory mediators by GRK5 occurs at

later time points. This is evident from the fact that the levels of most of the cytokines

. . . . . . . . 4-
and chemokines 1n the plasma are srmrlar at earlier time pomts 1n both GRK5 as

+/+ . . . . . .
well as GRK5 mrce. This suggests that at earlier time pornts cytokrnes are secreted

from stored granules in response to LPS and hence GRK5 does not affect their

secretion. However, by 12 hours the levels of these inflammatory mediators are

reduced in GRK5-L mice but not in GRK5+/+ mice suggesting regulation by GRK5 at

the transcriptional levels. This could be very important in terms of disease
pathogenesis since cytokines and chemokines are required during early phases of
infection. An increased or continued production, however, leads to tissue injury and
chronic inflammation. Our studies, thus suggest that GRK5 could be a potential drug
target because inhibition of GRK5 might result in lowered but not complete

suppression of cytokine levels. This might then subsequently lead to a resolution of

256

inflammation. The next logical steps here are to establish the role of GRK5 in human
sepsis and other human inflammatory diseases.

Unlike GRK5, GRK2 was found to negatively regulate LPS induced signaling
under in viva conditions. Previous studies showed that the expression levels of both
GRK2 and GRK5 increase in the neutrophils fi'om sepsis patients suggesting that
GRKs are involved in sepsis, our studies define a protective role for myeloid cell
GRK2 in sepsis. Interestingly, our previous findings in peritoneal macrophages
showed that LPS stimulation increases GRK2 but decreases GRK5 levels. Intriguingly,
GRK2 appears to be a negative regulator and GRK5 a positive regulator of
endotoxemia. Taken together, this suggests that endotoxemia is regulated by GRK2
and GRK5 by a feedback mechanism in an effort to maintain homeostasis. That is, the
negative regulator (GRK2) is upregulated whereas the positive regulator (GRK5) is
downregulated to prevent further TLR4 signaling and damage.

Because inhibition of NF KB and MAPK pathways have not been successful in
limiting all inflammatory diseases, investigation of novel regulators of NFKB and
MAPK pathways is an extensive area of research to treat inflammatory diseases. In
our studies we have discovered that GRK5 and GRK2 are two novel regulators of
NFKB and ERK signaling pathway respectively. Based on our studies, we propose that

GRK2 and GRK5 may be targets for therapeutic drug development in sepsis.
6.4 Future experiments

Some of the future experiments to further characterize the roles of GRK2 and 5 in

inflammatory signaling should focus onto investigating the regulation of other

257

 

inflammatory diseases by GRKs. In particular, double gene deletions of GRKs with
inflammatory disease models such as LDL receptor knockout (mouse model of
atherosclerosis) or 1L—10 knockout (mouse model of colitis) would be useful models
to define the roles of GRKs in these inflammatory diseases. The regulation of
inflammatory signaling by GRK2 and 5 in human cells should be studied. If the results
are similar in human cells, drug development can be considered whereby inhibitors for
GRK5 would be very useful in reducing the tissue injury seen in sepsis. A microarray
approach to investigate the genes that are specifically up or downregulated in LPS
stimulated GRK2 and 5 deleted macrophages would be useful to determine the
downstream effects of GRKs on inflammatory signaling. Investigating whether
signaling by other TLRs such as TLR2, TLR3 and TLR9 is similarly regulated by
GRK2 and 5 would add to our understanding of the role of GRKs in inflammatory
signaling. Peptidoglycans from gram positive bacteria serve as ligands for TLR2
which then leads to gram positive bacterial sepsis. TLR3 and TLR9 on the other hand
are activated by viruses. The results from such studies will help us determine
treatment strategies as inflammatory diseases such as sepsis is a complex syndrome
involving different species and strains of pathogens.

One of the other factors to be considered is the regulation of signaling by
GRK2 and 5 in cells other than macrophages. In our studies, although we found a
similar response in terms of production of several inflammatory mediators, both in
primary macrophage cell culture supematants as well as in the plasma (up or down
regulation), some of the mediators found to be regulated in macrophages were found

to be unaffected in plasma and vice versa. One possible explanation for these results is

258

the fact that other cell types such as T cells as well as cells such as fibroblasts and
endothelial cells are also involved in the production and release of inflammatory
mediators. Hence, it is also essential to determine the cell type specific regulation of
the inflammatory signaling by GRK2 and 5. The results from such studies would help
us determine treatment strategies as a cell type specific drug approach or a global

targeting of GRKs.

259

 

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