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INVESTIGATION OF REACTIVE OXYGEN SPECIES (ROS) AS
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Kyoungmun Lee

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6/01 cJCIRC/DatoDuopGS-sz

INVESTIGATION OF REACTIVE OXYGEN SPECIES (ROS) AS THE
SIGNALING MEDIATORS OF IMMUNOSTIMULATION

By

Kyoungmun Lee

A THESIS

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

MASTER OF SCIENCE
Cell and Molecular Biology Program

2003

ABSTRACT

INVESTIGATION OF REACTIVE OXYGEN SPECIES (ROS) AS THE
SIGNALING MEDIATORS OF IMMUNOSTIMULATION

BY

Kyoungmun Lee

Reactive oxygen species (ROS) are known to influence various functions
of cells including proliferation and apoptosis. When Jurkat T cells were
stimulated by H2O2, activation-associated phosphorylation of mitogen-activated
protein kinases (MAPKs) was induced. H202 inhibited activities of protein
tyrosine phosphatases (PTPs) that were immunoprecipitated from cells. Matrix-
assisted laser desorption ionization (MALDI) analysis showed that H2O2
treatment of recombinant CD45 modified active site cysteine residue. Ectopic
expression of different PTPs inhibited H2O2-induced phosphorylation of specific
members of MAPKs. N-aoetylcysteine inhibited 1-ch|oro-2,4-dinitrobenzene
(DNCB)-induced ROS production, CD45 inhibition, Lck activation, and c—Jun
NH2-tenninal kinase (JNK) and p38 phosphorylation. Curcumin, but not
resveratrol, inhibited phosphorylation of signal transducer and activator of
transcription 1 (STAT1) and STAT3 induced by interferon-a and Concanavalin A
(Con A). Both curcumin and resveratrol inhibited Con A-induced ROS
production, JNK phosphorylation and lL-2 mRNA expression. On the other hand,

curcumin, but not resveratrol, ablated Con A-induced c-Fos expression.

This dissertation is dedicated to my parents and the many educators who have

helped me attain this goal.

ACKNOWLEDGEMENTS

I greatly appreciate Dr. Walter J. Esselman for his guidance and support through
my years in his research group. I wish to express my gratitude to Dr. Richard
Schwartz and Dr. Kathleen Gallo for their guidance. I am also grateful to Dr.

Louis King and Dr. Ronda Hussain for their invaluable assistance.

TABLE OF CONTENTS

List of Figures ................................................................................... vn

Chapter One: Literature Review ............................................................ 1
Introduction ............................................................................. 2
Generation of ROS .................................................................. 2
NO and ROS .......................................................................... 5
Physiological Functions of ROS ................................................... 6
ROS in Inflammation .................................................................. 8
ROS in Diseases ...................................................................... 11
ROS in T Cells ........................................................................ 15
ROS-induced Signaling Pathways ................................................ 16
Conclusion .............................................................................. 17
References ............................................................................. 18

Chapter Two: cAMP potentiates H2O2-induced ERK1/2 phosphorylation without

the requirement for MEK1/2 phosphorylation ................................. 33
Abstract ................................................................................. 34
Introduction ............................................................................ 35
Materials and Methods ............................................................ 39
Results ................................................................................. 41
Discussion .............................................................................. 51
Acknowledgements .................................................................. 57
References ............................................................................. 58

Chapter Three: Inhibition of PTPs by H2O2 regulates the activation of distinct

MAPK pathways ...................................................................... 62
Abstract ................................................................................. 63
Introduction ............................................................................. 64
Materials and Methods .............................................................. 69
Results ................................................................................... 72
Discussion .............................................................................. 83
References .............................................................................. 92

Chapter Four: Inhibition of CD45 by reactive oxygen species (ROS) mediates
1-chloro-2,4-dinitrobenzene (DNCB)-induced immunostimulation of T cells

.............................................................................................. 99
Footnotes .............................................................................. 100
Abstract ................................................................................ 101

Introduction ........................................................................... 103
Materials and Methods ............................................................. 106

Results ................................................................................. 1 10
Discussion ............................................................................. 124
References ............................................................................ 1 30

Chapter Five: Curcumin inhibits STAT1 and STAT3 phosphorylation and c-Fos

expression in Jurkat T lymphocytes ........................................... 135
Abstract ................................................................................ 1 36
Introduction ........................................................................... 1 38
Materials and Methods ............................................................. 141
Results ................................................................................. 144
Discussion ............................................................................. 160
Acknowledgements ................................................................. 167
References ........................................................................... 168

vi

LIST OF FIGURES

Chapter Two
H202 potentiates TCR-induced ERK phosphorylation. ..................... 42
H202 induces MEK1/2 and ERK1/2 phosphorylation and Lck mobility shift.
.............................................................................................. 43
U73122 and EGTA inhibit MEK1I2 and ERK1I2 phosphorylation by H202.
............................................................................................... 47
Effect of cAMP on H202-induced ERK1/2 and MEK112 phosphorylation.
............................................................................................... 50
A model of cooperation between H202 and cAMP in ERK1/2
phosphorylation. ....................................................................... 54

Chapter Three
H202 induces MAPK phosphorylation. ........................................... 73
Treatment of cells with H202 inhibits the activity of PTPs. .................. 75
PTP overexpression affects H202-induced MAPK phosphorylation. ..... 78

H202-induced MAPK phosphorylation is regulated by differential signaling
pathways. ............................................................................... 82

PTP inhibition by H202 regulates the activation of distinct MAPK pathways.
............................................................................................... 91

Chapter Four
DNCB enhances TCR- and H202-induced MAPK phosphorylation. ...111

NAC inhibits DNCB-induced intracellular ROS production. ............... 112
PP2, the Src-family kinase inhibitor, and NAC inhibit DNCB-induced JNK

and p38 phosphorylation. .......................................................... 114
NAC inhibits DNCB-induced Lck activation. ................................... 116
DNCB reversibly inhibits CD45 activity in Jurkat cells. ..................... 117

vii

Inhibition of CD45 by ROS regulates DNCB-induced activation of distinct

MAPK pathways. ..................................................................... 120

H202 inhibits the modification of the active site cysteine residue of

recombinant CD45 by C13-iodoacetic acid. ................................... 121
Chapter Five

Curcumin and resveratrol inhibit Con A-induced ROS production. ..... 145

Curcumin, but not resveratrol, inhibits STAT1 and STAT3 phosphorylation

induced by lFN-a and Con A. .................................................... 147
Curcumin and resveratrol inhibit lL-2 mRNA induction by Con A. ...... 150
Curcumin and resveratrol inhibit Con A—induced JNK phosphorylation. 152
Curcumin and resveratrol comparably inhibit Con A-induced c-jun

promoter activation but distinctly inhibit AP-1 activation. .................. 153
Curcumin but not resveratrol inhibits Con A-induced c-Fos expression. 155

Curcumin and resveratrol do not inhibit Con A-induced Elk-1 activity or
CREB phosphorylation. ............................................................ 157

Anti-inflammatory effects mediated by antioxidant and non-antioxidant
properties of curcumin and resveratrol. ........................................ 166

viii

CHAPTER ONE

LITERATURE REVIEW

Introduction

Reactive Oxygen Species (ROS) are generated by the incomplete
reduction of oxygen during various biological processes. It has been known that
ROS mediate diverse effects on the function of the cells. Because ROS can be
generated rapidly in response to extracellular stimuli and can be degraded
efficiently, they have been regarded as potential second messengers.
Supporting this concept, various growth factor receptors, cytokine receptors, and
GPCRs (G-protein coupled receptors) have been shown to produce ROS

including H202 when cognate ligand binds the receptor (1).

Generation of ROS

The superoxide anion (02") is formed by the univalent reduction of oxygen
molecule (02). In a cellular system, superoxide anion is produced enzymatically
by NADPH oxidase or xanthine oxidase or non-enzymatically by semi-ubiquione
in the repiratory chain of mitochondria. Superoxide is converted to H202
spontaneously or by superoxide dismutase. In the presence of reduced
transition metals, H202 can be converted to the highly reactive hydroxyl radical
(OH').
Mithochondn’al respiratory chain

ROS are continually produced from the mitochondrial respiratory chain in
living cells. The major sites of ROS production in the respiratory chain lay within
complexes l (NADH dehydrogenase) and Ill (ubiquinone-cytochrome c

reductase) (2). Ceramide and arachidonic acid produce ROS formation from

mitochondria by direct interaction with the components of respiratory chain (3, 4).
ROS production from mitochondria has been linked to the propagation of
apoptotic signals and JNK activation. The importance of regulating ROS
production from mitochondria was observed in the development of oxidative
stress in mice deficient in mitochondrial SOD (Mn-SODISOD2) (5) or in
mitochondrial glutathione-peroxidase-1 (GPx1) (6).
NADPH oxidase

Professional phagocytes such as neutrophils, macrophages and
eosinohils produce high amount of ROS in the context of pathogen killing.
Although originally termed respiratory burst because the process accompanies
the consumption of 02, it has been identified that NADPH oxidase, not
mitochodrial respiratory burst, is responsible for the process. The components of
NADPH oxidase complex are separated between the cytosol and the plama
membrane in nonstimulated cells. The cytosolic complex composed of
p47p”°"/p67""°"/p40"”°" (phox for phagocyte oxidase) and the small GTPase Rac
move to the plasma membrane on stimulation and combine with the membrane-
bound flavocytochrome b558 (a heterodimer containing gp91""°" and p22"”°") to
form the active oxidase. Chronic granulomatous disease (CGD) is caused by a
defect in any of the genes encoding gp91””°", p47p”°", p67"”°" or p22p”°". The
genetic disorder results in the lack of ROS production and poor clearance of
many bacterial and fungal pathogens (7). However, it has been questioned that
ROS produced by oxidative burst are the major device for directly killing of

bacteria. Proteases may be more important than ROS as bactericidal agents

because macrophages from mice deficient in cathepsin G protease can no longer
kill bacteria, although their oxidative burst is not impaired (8). The
immunodeficiency caused by CGD may not be primarily due to a defect in
bacterial killing but rather due to a defect in macrophage and lymphocyte
activation (9). Mice deficient in p47””°" expression showed impaired NF-xB
activation in response to lipopolysaccharide (10). ERK1I2 activation was
inhibited by catalase when the respiratory burst in rat alveolar macrophage was
stimulated by zymosan-activated serum (11).

Albeit a smaller amount compared to phagocytes, generation of ROS by
NADPH oxidase has been also observed in lymphocytes, fibroblasts (12),
vascular smooth muscle cells (13), endothelial cells (14), carotid body (15), lung
(16) and kidney (17). In parallel to the demonstration, a number of cDNAs
encoding homologues of gp91phox have been cloned and named Nox (for
NADPH oxidase) or Duox (for Dual oxidase). Nox1 is highly expressed in colon
epithelial cells (18). Nox2 designates the classical gp91phox in phagocytic cells.
Nox3 was cloned from fetal kidney (19). Nox4 was found in kidney (20) and in
osteoclasts (21). NOX5 is highly expressed in lymphocytes in spleen and lymph
nodes (22), suggesting that the enzyme may have a role in lymphocyte function.
Duox1 and 2 contain peroxidase domain in the N-terminal region and are
expressed in thyroid gland (23).

Xanthine oxidase
Xanthine oxidase has been suggested to have a major role for ROS

production during ischemic/reperfusion. In addition, levels of circulating xantine

oxidase have been shown to be increased in inflammatory diseases such as
arthritis (24), artherosclerosis (25) and septoc shock (26). Xanthine oxidase is
able to bind to glycosaminoglycans on the surface of vascular endothelial cells
(27). Endothelium-bound xanthine oxidase is increased as a result of tissue

inflammation (28). Pro-inflammatory cytokines such as TNF-oc, lL-1j3, and lFN-y

increased the activity of xanthine oxidase in mammary epithelial cells (29). A
major role of ROS produced by xanthine oxidase in inflammation may be the
increase of the adhesion of neutrophils to endothelial cells (30, 31). Interestingly,
xanthine oxidase is capable of producing NO (32) and the activity of the enzyme
is inhibited by NO (33).
Myeloperoxidase

Although microbicidal functions of MPO are well established in vitro,
humans deficient in MPO expression generally do not have a higher risk of
infection (34). Using an antibody detecting HOCI-modified protein, the
generation of HOCI in vivo was demonstrated in human atherosclerotic plaques
(35), which myeloperoxidase co-Iocalized (36). HOCI activates MMP-7 in

atherosclerotic plaques (37). MP0 also inhibits endothelium-dependent vascular

relaxation by consuming NO (38).

N0 and ROS
Nitric oxide (NO) has been recognized as an important signaling molecule
in vessel and neurons (39), although it may function as the agents damaging

pathogens in pathological situations (40). Nitric Oxide (NO) activates guanylate

cyclase by binding to the heme moiety of the enzyme. The production of cGMP
by guanylate cyclase has been shown to regulate the relaxation of smooth
muscle cells and the inhibition of platelet adhesion (41).

By analogy with NO, ROS have both signaling and bactericidal functions
depending on the circumstances. The important aspect is that ROS and NO
influence the production and function of the other species. H202 may participate
in the activation of guanylate cyclase activation by stimulating NOS (42) or by
interaction of the cyclase with oxidized form of catalase called compound I (43).
The interaction of NO with ROS is regarded to be responsible for the generation
of RNS such as nitrosonium cation (NO+), nitroxyl anion (NO-), and peroxynitrite
(ONOO-) (44). . It has been shown that NOS is capable of producing ROS when
the substrate is limited (45). ROS production from NOS was observed in diabetic
rats (46). NOS expressed in mitochondria was identified (47). NO production in
mitochondria can contribute to the formation of ROS from respiratory chain by
building up semi-ubiquinone (48), by enhancing oxygen consumption (49), or by
inhibiting cytochrome oxidase (50). NO-induced cell death in human

osteoarthritic synoviocytes is mediated by tyrosine kinase activation and ROS

formation (51 ).

Physiological Functions of ROS
Various physiological functions involving ROS have been described as

follows (reviewed in (52)): 1) regulation of vascular tone, 2) sensing of oxygen

tension, 3) enhancement of signal transduction, and 4) the maintenance of redox
homeostasis
Regulation of vascular tone

H202 induced a relaxation of the aorta (53). Endothelial cells produce
H202 that acts as an EDHF (endothelium-derived hyperpolarizing factor) and
mediates the relaxation of smooth muscle cells in response to shear stress (54)
or bradykinin (55). H202 may promote vascular relaxation by stimulating guanyl
cyclase activity (56). Interestingly, without involvement of endothelial cells, H202
mediates the contraction of smooth muscle cells induced by angiotensin II (57).
ROS mediate stretch-induced contraction of bovine coronary artery via activation
of EGFR (58). Relaxation of the carotid artery in response to acetylcholine was
impaired in CuZnSOD-deficient mice (59).
Sensing of oxygen tension

When arterial oxygen tension decreases, chemoreceptor cells of the
carotid body release neurotransmitters that activate the sensory nerve endings of
the carotid sinus nerve. Integration of the carotid sinus nerve input in the
respiratory control centers of the brain stem results in an increased activity of the
respiratory muscle with increased ventilation (60). EPO-producing cells, located
mainly in the kidney in adults, release EPO in response to hypoxia. EPO
reaches the bone marrow and activates erythropoiesis. Production of
prostaglandins and other eicosanoid mediators is also influenced by oxygen

tension because 02 is the substrate for the oxygenation of arachidonic acid and

the hydroperoxy radical is required for the activation of cyclooxygenase and
lipoxygenase (61 ).
Enhancement of signal transduction

It has been shown that various growth factors: (eg. PDGF, EGF, VEGF),
cytokines (TNF-a, lL-1j3), and hormones (insulin, leptin) produce ROS upon
cognate receptor activation. The attenuation of ROS production by antioxidants
or ROS-scavenging enzymes inhibits signaling pathways activated by those
stimuli. In addition, transactivation of EGF and PDGF receptors by angiotensin II
is mediated by ROS (62, 63).
Maintenance of redox homeostasis

The effects of ROS in signaling have often been attributed to the shift in
the redox potential of the cells. The ratio of glutathione disulfide (GSSG)!
glutathione (GSH) is a good indicator of the cellular redox state (64). Cells
contain systems to control the amount of ROS. The system is regulated by the
redox potential of the cells. The importance of the system is manifested by the

dysregulation of the inflammatory process when the system was altered.

ROS in Inflammation
Inflammation is a complex set of interactions among soluble factors and
cells that can arise in response to traumatic, infectious, or autoimmune injury
(65). Immune cells infiltrate to the inflammatory sites and are activated to protect
wounds from invading pathogens. However, excessive and/or sustained

activation of immune cells may inhibit the healing procedure of wound (66) and

become a source for the inflammatory diseases (see section 6). ROS have been
involved in the various stages of inflammation from initiation to resolution (67).
H202 is present in exhaled air and the amount is increased in patients with
inflammatory lung diseases (68, 69).
Chemotaxis

ROS induce chemotactic migration of various types of cells to the
inflammatory sites by increasing chemokine expression in macrophages (70).
Lysyl oxidase plays a critical role in the formation and repair of extracellular
matrix by oxidizing lysine residues in elastin and collagen (71). It has been
shown that H202 produced by the activity of lysyl oxidase mediates Chemotaxis
for smooth muscle cells (72).
Adhesion

ROS induce the adhesion of leukocytes to endothelial cells (73). H202
enhances eosionphil adhesion to endothelial cells by inducing the expression of
[32 integrin (74). Firm adhesion of leukocytes, which is mediated by 82-integrins
(CD11/CD18), is initiated by engagement of activated complement, LTB4 and
PAF with their receptors on rolling leukocytes. The enhanced generation of
oxidants results in the activation and deposition of complement, and
phospholipase A2-mediated production of LTB4 and PAF in endothelial cells.
Oxidants also mediate the initial expression of P-selectin by mobilizing the
leukocyte rolling receptor from its preformed pool (Weibel-Palade bodies) in
endothelial cells. Sustained rolling and adhesion of leukocytes on endothelial

cells are ensured by an oxidant-dependent synthesis of endothelial cell adhesion

molecules, such as E-selectin and lCAM-1 (30). Oxidants, derived from either
endothelial cells or leukocytes, elicit those responses by activating specific
nuclear transcription factors (e.g., NF-KB).
Cytokine expression and activation

H202 induces gene expression of TGF-j31 (75) and connective tissue
growth factor (76). ROS also activate latent form of TGF-B1 (77). In turn, TGF-
81 induces IL-6 expression in fibroblasts by ROS production (78, 79). Therefore,
a feed-fonrvard mechanism is established in which ROS induce the generation of
ROS (80). This mechanism may contribute to the amplification of inflammatory
signals.
Angiogenesis

Exposure of endothelial cells to H202 induces angiogenesis (81). NF-kB
antisense nucleotide inhibited H202-induced angiogenesis in endothelial cells
(82). H202 induces VEGF expression in endothelial cells (83) and in
keratinocytes (84). ROS scavengers inhibit the angiogenesis induced by
lymphocytes (85) and macrophages (86).
Remodeling of Extracellular Matrix

Matrix metalloproteinases (MMPs) are proteases important for cellular
migration and tissue remodeling (87). Oxidants increase the activity of
metalloproteinases (MMPs) (37, 88) and mediate the induction of the MMP gene
expression (89). Lipid peroxidation products induce the expression of fibrogenic
cytokines and collagen (90). ROS degrade hyaluronic acid in the extracellular

matrix, which amplifies pro-inflammatory signals (91). ROS mediate the collagen

1O

expression induced by TGF-B1 (92). ROS are involved in collagen-induced

platelet aggregation and activation (93).
Generation of Autoimmunity

Increased expression of heat shock protein 65 coincides with a population
of infiltrating T lymphocytes in atherosclerotic lesions of rabbits specifically
responding to heat shock protein 65 (94). Lox-1 was identified as a receptor that
mediated the presentation of HSP as an antigen by dendritic cells (95). ROS
increase the level of Lox-1 mRNA in aortic endothelial cells (96).
Enhancement of Immune Response

Injury and inflammation lead to hypoxia and elevate lactate in wounds.
Lactate enhances angiogenic activity in macrophages (97). Lactate decreases
intracellular gluathione levels and enhances lL-2 production in T lymphocytes

(98).

ROS in Diseases

Atherosclerosis

Elevated levels of cholestrol were shown to promote leukocyte-endothelial
cell adhesion via ROS production from NADPH oxidase (99). A high level of
homocysteine that was associated with the high incidence of atherosclerosis was
shown to produce ROS in coronary artery via TNF-or production (100).
Homocysteine also induced the expression of lox-1, receptor for oxLDL, in
endothelium (96) and enhances T cell proliferation (101). SOD is expressed at

higher levels in endothelial cells that are exposed laminar flow shear stress and

11

show the lower susceptibility to develop atherogenesis (102). The level of
xanthine oxidase as well as anti-xanthine oxidase antibodies is elevated in
patients with coronary heart diseases (103). The interaction of advanced
glycation end products with corresponding cell surface receptor (RAGE)
produces ROS and decreases GSH levels (104). ROS are involved in AGE-
mediated vascular dysfunction through vascular endothelial growth factor (VEGF)
production (105).

Diabetes

ROS provide a multi-faceted role in the vascular and neuronal
complication caused by hyperglycemia in diabetes (reviewed in (106)).
Increased glycolysis generates high level of NADH that provides a source of
ROS by NADH oxidase and mitochondrial respiratory chain (107). Excess
glucose is consumed by polyol pathway that depletes NADPH and thereby
decreases cellular reducing capacity. High level of glucose may generate the
non-enzymatic glycation of proteins yielding AGEs that have an important role in
the complications of the inflammation. Gene transfer of SOD reverses
endothelial dysfuction in aorta of diabetic rats (108).

A high level of glucose was shown to induce the rearragement of actin
cytoskeleton in mesangial cells through ROS production (109). Hyperglycemia
activates MMP-9 in a ROS-dependent manner in bovine aortic endothelial cells
(110). Leptin, a hormone that controls body weight, induces production of ROS
from mitochondria and oxidation of fatty acids (111). Hyperglycemia-induced

mitochondrial ROS production induces plasminogen activator inhibitor-1 by

12

increasing Sp1 glycosylation (112). Interestingly, placental vessels from
gestational diabetic patients showed a loss of relaxation response to exogenous
and endogenous (lactate-derived) H202 (113). Lox-1 expression was
upregulated in vascular endothelial cells of streptozotocin-induced diabetic rats
(114).
Rheumatoid Arthritis

The rheumatoid synovial microenvironment is relatively ischemic and
hypoxic (115). Hypoxia/reoxygenation induces NF-KB activation and lCAM-1
expression in synovial fibroblasts with the enhanced adhesion of lymphocytes
(116). Xanthine oxidase mediates bone resorption induced by TNF—a and IL-18
(117). TNF-or is a major cytokine for inflammatory arthritis such as rheumatoid
arthritis where it stimulates synovial hyperplasia and cartilage destruction. It has
been shown that TNF-or induces c-fos gene expression in chondrocytes by
NADPH-dependent ROS production (118). Antioxidants inhibit TNF-a-induced
IL-8, MCP—1, and collagenase expression in synovial cells (119). lntegrin
induced MMP-1 gene expression by ROS-dependent IL-10L induction in rabbit
synovial fibroblasts (120). IL-1j3 also induces c-fos and collagenase expression
in chondrocytes in ROS-dependent manner (121). The risk of rheumatoid
arthritis is increased in patients with low levels of serum antioxidants such as or-
tocopherol and B-carotene (122). The transfer of SOD and catalase genes

ameliorated antigen-induced arthritis in rats (123). N-acetylcystein also
alleviated collagen-induced arthritis in mice (124).

lschemia/Reperfusion

13

Xanthine oxidase and NADPH oxidase have been shown to produce high
amount of ROS during ischemia/reperfusion injury (125, 126). SOD gene
transfer ameliorated tissue damage caused by ischemialreperfusion (127, 128).
Conversely, glutathione peroxidase knockout mice are susceptible to myocardial
ischemia reperfusion (129).

Neurodegenerative Diseases

ROS generated by oxidation of dopamine has been implicated in the
destruction of dopaminerglc neurons in Parkinson’s disease (130). Amyloid [3-
protein activates NADPH oxidase in microglial cells, which contributes to ROS-
mediated inflammatory process in Alzheimer’s disease (131). It has been shown
that amyloid B activates cells by engaging RAGE (132). The intracellular redox
state modulates the balance between proliferation and differentiation in glial
precursor cells (133). The level of uric acid, a general antioxidant in the plasma,
was significantly reduced in the brains of patients with Parkinson’s (134) and
Alzheimer’s disease (135). The decrease of cytochrome oxidase activity by
mutations of mitochondrial DNA was associated with late-onset Alzheimer's
disease (136).

Cancer

Cancer cells produce high amount of R08 (137). When fibroblasts were
transfected with oncogenic form of Ras, the proliferation capacity of the cells was
correlated with the amount of ROS produced upon transfection (138). ROS
production by tumor cells has been suggested to induce proliferation and

angiogenesis. Humans with MPO deficiency show a high incidence of malignant

14

tumors as MPO-deficient leukocytes exhibit a poor lytic action against malignant
cells (34). Ectopic expression of Nox1, an isofon'n of nonphagoctyic NADPH
oxidase, in fibroblasts induced malignant transformation that was reversed by
catalase coexpression (139). Transfection of cancer cells with 12-LOX or 15-
LOX enhances their tumorgenic potential (140, 141).
HIV infection

A massive loss of glutathione in plasma as well as in pheripheral blood
lymphoctes is observed in HIV infection (142). NAC is regarded to be a useful

treatment for HIV infection by replenishing GSH pool (143, 144).

ROS in T Cells

Functional activation of T cells has been shown to be enhanced by the
exposure of ROS and by the shift of the intracellular redox status (145-147). The
enhancement of T cell function by ROS may have important consequences when
the ligand concentration for TCR or costimulatory receptors is suboptimal. ROS
in the inflammatory condition may lower the threshold for triggering T cell
activation. According to this model, simultaneous injection of glutathione was
found to inhibit in vivo immunization by small amounts of antigen (148). There is
evidence that phagocytes release ROS in a quantal manner in the immunological
synapse created by the interaction of T cell receptor with MHC-bound peptide
(149). Helper T cells are not properly activated in p47phox-deficient mice (150).

The profile of cytokine production by antigen-presenting cells is influenced by the

15

intracellular redox status (151), thereby regulating the balance between Th1 and

Th2 cell types (152).

ROS-induced Signaling pathways
Activation of Mitogen-Activated Protein Kinase (MAPK)

One well documented result of cell stimulation with H202 is MAPK
activation. The MAPK family members considered in this report include ERK,
p38, and JNK. ERK activation has been mainly implicated in proliferation in
response to growth factors, whereas p38 and JNK activation are more important
to stress responses, such as in inflammation. It has been suggested that the
combination of the magnitude and kinetics of activation of each member of
MAPK family determines the appropriate response of the cell according to the
specific stimulus (153). In the case of T cells, the distinct activation profile of
three members of the MAPK family has been shown to influence the specific
stages of thymocyte development as well as the precise effector function of
mature T cells (154).

Inhibition of Protein Tyrosine Phosphatase (PTP)

One of the potential molecules directly affected by ROS are the protein
tyrosine phosphatases (PTPs). All PTP contain a conserved cysteine residue
that is located in the motif HC(X5)RSfl’ (155), and the mutation of the cys
resulted in the loss of activity. The cysteine acts as a nucleophile, forming a
thiophosphoenzyme intermediate during hydrolysis. The positively charged

electrostatic field provided by nearby amino acid residues such as Arg and His

16

maintains the cysteine residue in the form of thiolate anion (-S-) that was more
susceptible to the oxidation by ROS compared to other cysteine residues.
Supporting this concept, treatment of purified PTPs with H202 inhibits the activity
of the enzyme (156) and exposure of cells to H202 also leads to the
downregulation of PTP activity (157, 158). Hyperglycemia has been reported to
decrease PTP activity in platelets in a ROS-dependent manner (159).
Stimulation of cells with insulin or EGF inhibits PTP1B activity with endogenous
production of ROS (160, 161). PDGF increases intracellular ROS and
inactivates LMW PTP and SHP-2 by oxidation (162, 163). These results suggest

that PTP inhibition by ROS has physiological significance during cell stimulation.

Conclusion

Cells have the ability to adapt to the change in the redox status occurred
inside the cells as well as outside the cells. Generation of ROS from
mitochondrial respiratory chain intrinsically suggests that cells need to cope with
ROS. Generation of ROS at the inflammatory sites indicates that cells need to
fulfill their functions during exposure to ROS. It is conceived that cellular
adaptation to the oxidative environment occurs through the, activation of signaling
pathways. Moreover, ROS production itself is a regulated process, as evidenced
by the modulation of the activity of ROS—producing enzymes by cytokines and
growth factors. Therefore, it is important to understand how cellular signaling

pathways are activated by ROS.

17

10.

11.

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32

CHAPTER TWO

CAMP POTENTIATES H202-INDUCED ERK112 PHOSPHORYLATION
WITHOUT THE REQUIREMENT FOR MEK112 PHOSPHORYLATION

This work was published in Cellular Signaling (2001), Vol. 13, p. 645 - 652 l

33

ABSTRACT
In Jurkat T lymphocytes, hydrogen peroxide (H202) potentiates the
phosphorylation level of extracellular signal regulated kinase 1 and 2 (ERK1/2)
caused by T cell receptor (T CR) stimulation with anti-CD3 and anti-CD28 or anti-
CD3 alone. Submillimolar concentrations of H202 induced phosphorylation of
ERK1I2 and MAP/ERK kinase 1 and 2 (MEK1I2) without antigenic stimulation.
H202 also induced the electrophoretic mobility shift of Lck from 56 kDa to 60 kDa.
The MEK inhibitor, P098059 attenuated ERK1/2 and MEK1/2 phosphorylation as
well as the migration shift of Lck induced by H202. The phospholipase C (PLC)
inhibitor, U73122, and EGTA reduced the phosphorylation of both ERK1/2 and
MEK1/2 induced by H202. Interestingly, an increase of intracellular cAMP level
with forskolin or 8-(4-chIorophenylthio)-cAMP augmented ERK1I2
phosphorylation by H202, while inhibiting MEK1I2 phosphorylation by H202.
These results demonstrate an alternative pathway that results in augmentation of

ERK1I2 phosphorylation without concomitant MEK1I2 phosphorylation in T cells.

34

INTRODUCTION
Both exogenous treatment and endogenously produced hydrogen peroxide
(H202) has been shown to affect a number of cellular functions including gene
activation, proliferation, and apoptosis [1]. H202 is produced from mitochondria
in the reduction of O2 to H2O during respiration [2]. Substantial amount of H202

is produced and secreted by activated phagocytes through NADPH oxidase
system at sites of inflammation [3]. In both cases, superoxide (02") may be

converted to H202 spontaneously or by the action of superoxide dismutase [4].
There is increasing evidence to suggest that H202 generated at low levels during
normal cell signaling may act as a second messenger [5]. Various growth factor
receptors, cytokine receptors, and G-protein coupled receptors have been shown
to generate H202 following ligand activation (reviewed in [6]). It is not clear
whether H202 production by these receptors is linked to the activation of NADPH
oxidase, although several examples of NADPH oxidase system similar to that in
phagocytes have been found in nonphagocytic cells [7,8].

H202 is capable of affecting the function of proteins by oxidizing thiol
groups in Cys amino acid residues or in cofactor molecules. Transcription
factors such as AP-1 and NF-ch respond to redox change caused by H202 [9].
In addition the Cys118 residue of Ras was shown to be sensitive to oxidants
such as H202 and nitric oxide (NO) [10]. The most well documented effect of
H202 on signaling is due to the fact that H202 functions as a general inhibitor of
protein tyrosine phosphatases (PTPs) by oxidizing the essential catalytic Cys

residue to a sulfenic acid (Cys-SOH) [11]. The catalytic Cys residue of PTPs is

35

vulnerable to H202 because the pKa of the residue is about 5.5 as compared to a
pKa of about 8 for most other protein Cys residues [6]. This low pKa is
maintained by basic amino acid residues in the active site of the enzyme [12]. It
has been proposed that, because H202 is a mild oxidant compared to other
reactive oxygen species, the inhibition of PTPs would be the predominant cellular
effect at low concentrations of H202 [13]. This is supported by the rapid increase
of tyrosine phosphorylation in cells treated with H202. Inhibition of PTPs by H202
leads to unopposed action of protein tyrosine kinases which activate
phosphorylation cascades leading to downstream signals such as
phosphorylation of Mitogen-Activated Protein Kinase (MAPK) [14].

Certain lymphocytes, including Jurkat cells, produce reactive oxygen
species (ROS) responding to anti-Fas antibody (Ab) [15], and activation of the T
cell receptor (TCR) by anti-CD3 Ab has been proposed to produce ROS
including H202 [16]. C028 costimulation produces ROS by a lipoxygenase,
resulting IL-2 gene expression through NF-xB activation [17]. Moreover, H202
may be supplied to T cells at the site of inflammation where activated
granulocytes release large amounts of H202 leading to relatively large local
concentrations (estimated at 10 to 100 uM) [18-20]. Treatment of cells with
micromolar concentrations of H202 was previously shown to enhance lL-2 gene
expression after TCR activation as well as mitogenically induced T cell
proliferation [20,21].

P44/42 MAPK (ERK1/2) is activated by a variety of extracellular signals

including growth factors, cytokines, T cell antigens, phorbol esters, and

36

hormones [22]. The enzyme is activated by phosphorylation at a tyrosine and a
threonine residue located in the “activation loop" [23]. These residues are
phosphorylated by the dual-specificity kinases, MEK1 and 2 [24]. The activated
form of ERK1/2 not only phosphorylates cytosolic substrates such as RSK, but
also translocates to the nucleus, phosphorylating and thereby activating
transcription factors such as Elk-1 [25]. Activation of MEK1/2 is also controlled
by phosphorylation of serine residues [26]. MAPK kinase kinases (MAPKKK)
such as Raf-1 are responsible for phosphorylating MEK1/2 [27].

There is a complex interaction between cAMP and the ERK signaling
pathway [28]. cAMP activation of protein kinase A (PKA) is known to disturb the
Raf-19 MEK1/2 -) ERK1I2 cascade by phosphorylating Raf-1, an event which
inhibits its activation by the Ras G-protein [29,30]. Besides inhibiting Raf-1,
cAMP can also lead to ERK activation, changing the components in ERK
cascade by activating Rap1 in B-raf (a member of MAPKKK family) expressing
PC12 cells [31]. Interestingly, ERK activation by PKA can also be achieved by
phosphorylating and inhibiting PTPs that dephosphorylate tyrosine residue in
ERK [32,33]. The inhibition of these PTPs by PKA has a positive role in the
maintenance of ERK activation. In a feedback loop, ERK can affect the level of
cAMP by phosphorylating a family of cAMP-specific phosphodiesterases
(PDE4D) [34]. For instance, a short isoforrn of PDE4D, PDE4D1, is activated by
ERK phosphorylation, whereas the long isoforrns are inhibited by ERK
phosphorylation. In the case of long PDE4D isoforms, the inhibitory effect of

ERK phosphorylation is ablated when PKA phosphorylates the

37

phosphodiesterases [35,36]. These complex cross talk mechanisms are
proposed to control the amplitude and duration of ERK1/2 phosphorylation,
thereby modulating the specific outcome depending on the stimuli [37].

We have addressed one aspect of the cross talk affecting ERK1I2 by
treating cells with H202 and appropriate inhibitors and activators. Treatment of T
cells with H202 in the micromolar range reduced the threshold for effective T cell
signaling as assessed by phosphorylation of ERK1/2. H202 induced the
migration shift of Lck from p56 to p60, an event relevant to ERK1I2 activation and
T cell activation. We found that cAMP had an unanticipated role in H202-induced
ERK1I2 phosphorylation in T cells. When intracellular cAMP levels were
increased, the H202-induced ERK1I2 MAPK phosphorylation was potentiated,
whereas upstream MAPK Kinase (MAPKK), MEK1/2, phosphorylation was
inhibited. Thus we conclude that H202 can affect the cross talk between ERK
MAPK cascade and cAMP signaling in T cells. These results illustrate an
alternative pathway that results in augmentation of ERK1/2 phosphorylation

without concomitant MEK1/2 phosphorylation in T cells.

38

MATERIALS AND METHODS
Reagents and Antibodies
H202, PD98059, ethylene glycol-bis[beta-aminoethyl ether]-N,N,N’,N’-tetraacetic
acid (EGTA), forskolin, 8—(4-chlorophenylthio)adenosine-3‘:5‘-cyclic
monophosphate and 8-(4-chlorophenylthio)guanosine-3‘:5‘—cyclic
monophosphate were purchased from Sigma (St. Louis, MO). U73122 was
purchased from Calbiochem (La Jolla, CA). Monoclonal anti-CD3 clone 235 (lgM
type) and anti-CD28 clone NE51 (lgG type) were generously provided by Dr. Shu
Man Fu (University of Virginia, Charlottesville, VA). Phospho-p44/42 (ERK1/2),
phospho-MEK1/2, and p44/42 (ERK1/2) antibody were purchased from New
England Biolabs (Beverly, MA). Lck and ERK2 antibody were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-
conjugated goat anti-mouse and goat anti-rabbit antibody were purchased from
Bio—Rad (Hercules, CA). Horseradish peroxidase-conjugated rabbit anti-goat
antibody was purchased from Calbiochem.
Cell culture
Jurkat cell lines were cultured in RPMI 1640 medium (Life Technologies, Inc.
Gaithersburg, MD) supplemented with 10% heat-inactivated fetal bovine serum
(Life Technologies), streptomycin/penicillin (100 units/ml; Life Technologies), and
50 11M beta-mercaptoethanol (Sigma). Cells were maintained in an exponential

growth state (1 .650 x 105 cells/ml).

39

Stimulation and activation of cells

Cells were reconstituted in the concentration of 1 X 106 cells/ml media before
stimulation. Because PD98059, forskolin and U73122 were dissolved in dimethyl
sulfoxide (DMSO), the same volume of DMSO was included in the control
sample whenever these reagents were used. All antibody stimulation (anti-CD3
& anti-CD28) and H202 stimulation were performed for 10 min.

Cell lysis

Cells were washed twice with ice-cold phosphate-buffered saline (137 mM NaCl,
3 mM KCI, 8 mM Na2HPO4, 1 mM KH2P04, pH 7.4) and then lysed in an
appropriate volume (3 x 107 cells/ml) of lysis buffer (1% Nonidet P-40 (Pierce),
20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol, 5 mM EDTA, 2 mM
phenylmethylsulfonyl fluoride, 0.23 units/ml aprotinin, 0.5 pg/ml leupeptin, 10
ug/ml DNase I, and 0.2 mM sodium orthovanadate) for 30 min on ice. Cell nuclei
were pelleted by centrifugation at 10,000 rpm at 4°C for 10 min.

Immunoblotting

Lysates (30 pg) were separated with 10% SDS—PAGE gel and proteins were
transferred to nitrocellulose (Bio-Rad). The membrane was blocked in 5% nonfat
dry milk in TBS/T (80 mM Tris, pH 8.0, 3.6% (wlv) NaCl, 0.1% Tween-20 (Bio-
Rad)) for 1 hr and incubated in primary antibody at 4°C overnight, followed by
incubation in secondary antibody for 1 hr. Between treatments, the membrane
was washed five times with TBS/T solution. Bands were visualized with

chemiluminescence (Amersham, Arlington Heights, IL).

40

RESULTS
H202 enhances TCR-induced ERK1/2 phosphorylation.

We determined whether treatment of Jurkat T cells with H202 and with
anti-CD3/CD28 antibodies would reduce the threshold of T cell activation at the
level of ERK1/2 phosphorylation. As shown in Fig. 1, 50 pM of H202 enhanced
ERK1/2 phosphorylation by TCR stimulation with anti-CD3/CD28 and anti-CD3.
Anti-CD28 stimulation alone or together with 50 11M H202 was not able to induce
ERK1I2 phosphorylation. Treatment with H202 alone at this concentration did not
increase ERK1/2 phosphorylation. Anti-CD3ICD28 was more potent than anti-
CD3 alone in the induction of ERK1/2 phosphorylation, and anti-CD3 with H202
increased ERK1/2 phosphorylation up to the level induced by anti-CD3ICD28.
Phosphorylated ERK1/2 was detected using a phospho-p42l44 MAPK (ERK1/2)
antibody which binds preferentially to the doubly phosphorylated form of ERK1/2
(phosphorylated at both tyrosine and threonine residues).

H202 treatment increases ERK1/2 and MEK1/2 phosphorylation.

To define the pathway leading to the enhancement of ERK1/2
phosphorylation by H202, we first determined the concentration of H202 required
to induce phosphorylation ERK1/2. As shown in Fig. 2A (middle panel), 0.1 mM
H202 was the minimum amount able to increase ERK1/2 phosphorylation without
other TCR stimulation. Treatment with increasing amount of H202 showed that
ERK1 (p44) was phosphorylated at low H202 concentrations (~0.1 mM), followed

by the phosphorylation of ERK2 (p42) at higher concentrations (~0.5 mM). This

41

H202 (50 MM) ' + ' + " + " +

Anti-CD3 ' ' + + + + ' lB: phospho-

AntI-CDZ8 - - + , + - - + + ERK1I£§K1
w&,~- , 2:12; 2. :meam

mflfigihflgm ERK2
1 2 3 4 5 6 7

 

FIGURE 1. H202 potentiates TCR-induced ERK phosphorylation. Anti-CD3
(clone 235 1/500) and anti-CD28 (clone NE51, 1l500) were treated for 10 min
with or without 50 pM H202, as indicated. After blotting with phospho—ERK1/2
Ab, the membrane was reprobed with ERK2 Ab to show that similar amounts of

Iysates were present in each lane. Result is representative of triplicate

experiments.

42

FIGURE 2. H202 induces MEK1l2 and ERK1I2 phosphorylation and Lck
mobility shift. (A) H202 induces ERK1/2 and MEK1/2 phosphorylation. Cells
were treated with the Indicated amount of H202 for 10 min. After blotting with
phospho—MEK1/2 and phospho-ERK1I2 Ab, the membrane was reprobed with
ERK2 Ab as a loading control. (B) PD98059 reduces H202-induced ERK1/2 and
MEK1/2 phosphorylation. Cells were pretreated with PD98059 for 30 min at the
indicated concentration, followed by stimulation with 0.5 mM H202 for 10 min.
(C) H202 induces the migration shift of Lck, which is blocked by PD98059.
PD98059 was pretreated for 30 min at the indicated concentration, and cells
were stimulated by 0.5 mM H202 for 10 min subsequently. The ratio of band

intensity of p60 over that of p56 is plotted under the blot. Results are

representative of triplicate experiments (:I:SD).

43

A
H202(mM) o 0.1 0.5 0.71.0
tmww IB: phospho-MEK112

u 14541-3 M w mafi—p‘“ ERK1 I8. phospho-
Mfmz ERK2 ERK1/2

 

IB: ERK2

B

H202 (0.5 mM) - + + +

P098059 (11M) _ _ 30 90
mg > . f Iszhospho-MEK1l2
11* +1144 ERK1 lB:phospho—
“*«4 . _._,;<-p42ERK2 ERK1I2

C

H202(0.5mM) - + + +
PD98059 (11M) - - 30 90

 

 

 

IB: Lck
1.5
Ratio 1 ,
p60/p56 o 5
O _

 

44

suggests that ERK1 is more responsive to H202 than ERK2 at low H202
concentrations.

An increase in the phosphorylation of MEK1/2 was also detected after
treatment with 0.1 mM H202 (Fig. 2A, upper panel). MEK1/2 phosphorylation
was detected using a phospho-MEK1I2 antibody specific for the doubly
phosphorylated form of MEK1/2 (two serine residues). Because the molecular
weights (~46 KDa) of MEK1 and 2 isoforrns are very close, only one band was
detected. The increase of MEK1/2 phosphorylation indicates that, under these
conditions, H202 acts by activating upstream kinases such as Raf-1. Ser/T hr
phosphatases, such as those which dephosphorylate MEK1/2, are likely to be
resistant to the inhibitory effect of H202 at submillimolar concentrations [11].
MEK inhibitor suppresses H202-induced phosphorylation of ERK1/2 and
MEK1/2.

To determine whether MEK1/2 mediates ERK1I2 phosphorylation by
H202, we treated the cells with the MEK inhibitor, P098059. P098059 inhibited
both ERK1/2 and MEK1/2 phosphorylation by H202 (Fig 2B), suggesting that
MEK1/2 is involved in the signaling pathway leading to ERK1I2 phosphorylation.
H202 induced-migration shift of Lek from p56 to p60 is blocked by MEK
inhibitor.

The MAPK-mediated phosphorylation of Ser-59 in Lck correlates with the
electrophoretic mobility shift of Lck from 56 kDa to 60 kDa [38]. The shift is
relevant to T cell activation, because anti-C03 [39], phorbol 12-myristate 13-

acetate (PMA) [40], and ionomycin [41] were shown to induce this migration shift.

45

ERK binding to Lck has been proposed to have a positive role in T cell activation
by excluding SHP-1 recruitment to Lck [42]. We found that H202 induced the
migration shift of Lck, and the MEK inhibitor P098059 in a dose dependent
fashion blocked the Lck shift by H202 (Fig. 20), suggesting that ERK is
responsible for the H202-induced Lck shift.
U73122 and EGTA reduce H202-induced ERK1/2 and MEK1/2
phosphorylation.

Phospholipase C (PLC) hydrolyzes phosphatidylinositol 4,5-bisphosphate
(PIP2) to diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3), which activate
protein kinase C (PKC) and increase intracellular Ca” level, respectively [43].
Treatment of Jurkat cells with the PKC activator, PMA [44] and the calcium
ionophore, ionomycin [45] were independently shown to result in phosphorylation
of ERK1/2. H202 and pervanadate were shown to induce tyrosine
phosphorylation of PLCy and production of inositol phosphates [46]. We used
the PLC inhibitor, U73122, to determine whether phospholipase C was involved
in ERK1I2 phosphorylation by H202. As shown in Fig 3A, U73122 reduced the
level of phosphorylation in both ERK1/2 and MEK1/2 by H202 in a dose-
dependent manner. This suggests that part of the H202 signaling pathway
leading to ERK1/2 phosphorylation involves phosphoinositide turnover caused by
PLC.

H202 is known to activate calcium channels in the plasma membrane of
several types of plant cells [47,48], and H202 produced by EGF receptor has an

important role in calcium influx from eXtracellular space [49]. Exogenously added

46

A
H202 (0.5 mM) - +
U73122 (uM) ' '
flew- .- Iszhospho-MEK1/2
. ERK1 IB: hos ho
”3 m tmfim ergo/2p
:2; Wm WWW .1::133122 IB: ERK1/2

H202 (0.5mM) - + +
EGTA (0.5 mM),..- .

N+
O)

 

B

   
  
   

IB: phospho-MEK1/2

1 wt" 9 p44 ERK1 132 phospho-
*‘ P42 ERK2 ERK1I2

IB: ERK2

FIGURE 3. U73122 and EGTA inhibit MEK1l2 and ERK1I2 phosphorylation
by H202. (A) U73122 reduces H202—induced MEK1/2 and ERK1/2
phosphorylation. Cells were pretreated with U73122 for 10 min at the indicated
concentration, followed by stimulation with 0.5 mM H202 for 10 min. After blotting
with phospho-MEK1/2 and phospho-ERK1/2 Ab, the membrane was reprobed
with ERK1/2 Ab as a loading control. (B) EGTA attenuates H202-induced
ERK1/2 and MEK1/2 phosphorylation. Cells were incubated with 0.5 mM EGTA
for 30 min, followed by stimulation with 0.5 mM H202 for 10 min. After blotting
with phospho-MEK1/2 and phospho—ERK1/2 Ab, the membrane was reprobed
with ERK2 Ab as a loading control. Results are representative of three

independent experiments.

47

H202 has been shown to increase the intracellular calcium level in lymphocytes
including Jurkat T cells [50]. We determined whether calcium influx participated
in H202-induced ERK1/2 phosphorylation by incubating cells with EGTA before
H202 stimulation. As shown in Fig. 3B, extracelluar calcium depletion by 0.5 mM
EGTA reduced H2O2- induced ERK1I2 and MEK1/2 phosphorylation. These
results (Fig. 3) suggest that the H202-induced signaling pathways leading to
ERK1/2 phosphorylation include PLC activation and calcium influx. MEK1/2
appeared to mediate the signals from phosphoinositide hydrolysis and calcium
influx, because the level of phosphorylation in ERK1I2 was modulated by
U73122 and EGTA in parallel with that in MEK1/2. We conclude that U73122
and EGTA inhibit H202- induced MEK1/2 phosphorylation, and reduced activity of
MEK1/2 subsequently resulted in decreased phosphorylation level of ERK1/2.
Cyclic AMP potentiates H202-induced ERK1/2 phosphorylation while
inhibiting MEK1/2 phosphorylation.

An elevated intracellular cAMP inhibits calcium influx and
phosphatidylinositol turnover caused by TCR activation in some T cells [51,52].
Upregulation of intracellular cAMP level inhibits PLCy-1 tyrosine phosphorylation
caused by CD3 stimulation [53]. Because our results suggested that PLC
inhibitor and EGTA reduced ERK1I2 phosphorylation by H202 (Fig. 3A), inhibition
of phosphatidylinositol hydrolysis and calcium influx by cAMP would be expected
to negatively affect H202-induced ERK1/2 phosphorylation. In addition, PKA has
been shown to interfere with ERK MAPK pathway by phosphorylating Raf-1 in

Jurkat T lymphocytes [54]. In contrast, ERK1I2 shows much less sensitivity than

48

JNK, another member of MAPK family, to cAMP-mediated inhibition in T cells
[55]. In addition, a high concentration (0.5 mM) of the cell-permeable cAMP
analogue was shown to induce ERK1/2 phosphorylation without antigenic
stimulation in Jurkat T lymphocytes [33]. When we determined the role of cAMP
in ERK1/2 phosphorylation by H202, unexpected results were observed. As
shown in Fig. 4A, the level of H202-induced phosphorylation in ERK1I2 was
upregulated by forskolin (an adenlyate cyclase activator), whereas H202-induced
MEK1/2 phosphorylation was downregulated by the same reagent. The
concentration of forskolin used in our study (30 11M) did not cause ERK1/2
phosphorylation or MEK1/2 phosphorylation without H202 (Fig. 4A, lane 2).
Interestingly, ERK2 (p42) phosphorylation was highly upregulated by forskolin as
compared to ERK1 (p44) phosphorylation. To confirm that the effect of forskolin
was mediated by an increase in intracellular cAMP levels, we treated cells with 8-
(4-chlorophenylthio)-cAMP, a membrane permeable cAMP analogue, before
H202 stimulation. As shown in Fig. 4B, treatment with 50 11M 8-(4-
chlorophenylthio)-cAMP also potentiated ERK1I2 phosphorylation, while
inhibiting MEK1/2 phosphorylation by H202. This concentration of the cAMP
analogue did not increase either ERK1/2 phosphorylation or MEK1/2
phosphorylation without H202 stimulation. Like forskolin, ERK2 (p42) was more
sensitive to the amplifying effect by the cAMP analogue than the ERK1 (p44)
isoforrn. The same concentration (50 11M) of 8-(4-chlorophenylthio)-cGMP had
no effect on H202-induced MEK and ERK phosphorylation, further indicating the

specificity of the action of cAMP (data not shown).

49

A
H202 (0.5 mM) - - + +
Forskolin (30 11M) - + - +

 
 
 
  

IB: phospho—MEK1/2

W “ "71.214 ERK1 IB: phospho-
. ”+942 ERK2 ERK1/2

m m WW+M4 ERK1IB. ERK1/2

mm y W G— p42 ERK2
B
H202 (0.5 mM) - - + +
cAMP (50 11M) - + ' +
were -' - IB: phospho—MEK1/2

master; Emit/2 "

“flu.“ IB: ERK2

FIGURE 4. Effect of cAMP on H202-induced ERK1I2 and MEK1l2
phosphorylation. (A) Forskolin potentiates H202—induced ERK1/2
phosphorylation while inhibiting H202—induced MEK1/2 phosphorylation. Cells
were treated with 30 11M forskolin for 30 min and stimulated by H202 for 10 min.
After blotting with phospho-ERK1/2 and phospho-MEK1/2 Ab, the membrane
was reprobed with ERK1/2 Ab as a loading control. (B) 8-(4-chlorophenylthio)
cAMP enhances H202—induced ERK1/2 phosphorylation while inhibiting H202-
induced MEK1/2 phosphorylation. Cells were pretreated with 50 11M 8-(4-
chlorophenylthio) cAMP for 20 min, and subsequently stimulated by 0.5 mM H202
for 10 min. After blotting with phospho—ERK1/2 and phospho-MEK1/2 Ab, the
membrane was reprobed with ERK2 Ab as a loading control. Results are

representative of triplicate experiments.

50

DISCUSSION

The complex relationship and cross talk between signaling molecules has
represented one of the greatest challenges to the understanding of signal
transduction pathways. To contribute to the understanding of the complex
relationships between PTPs and PTKs we have studied the effect of the PTP
inhibitor, H202, on T cell activation. In this report we demonstrate that treatment
with 50 pM H202 potentiated ERK1/2 phosphorylation caused by TCR stimulation
with anti-CD3 and anti-0028 or anti-CD3 alone. Submillimolar concentrations of
H202 increased the phosphorylation levels of ERK1/2 and MEK1/2 in Jurkat T
cells. H202 treatment also induced the electrophoretic mobility shift of Lck from
56 kDa to 60 kDa. This shift of Lck was blocked by P098059 (MEK inhibitor),
supporting the notion that ERK1/2 phosphorylation by H202 is responsible for the
migration shift of Lck.

Because the MEK inhibitor (P098059), the PLC inhibitor (U73122) and
EGTA decreased the 0.5 mM H202 induced-phosphorylation level of both
ERK1I2 and MEK1/2, we conclude, that at this concentration, H202 probably
resulted in phosphorylation of ERK1/2 via upstream signaling pathways which
activate MEK1/2. The activation of upstream signaling pathway is believed to
involve H202-induced inhibition of proximal PTPs such as CD45 and SHP-1,
which regulate members of Src and Syk tyrosine kinase families. In support of
this view, millimolar concentration of H202 did not increase the general tyrosine

phosphorylation level in a Lck deficient Jurkat cell variant (J.CaM1.6) [56] and did

51

not cause ERK1/2 phosphorylation in ZAP70 deficient (p116) Jurkat cell line
variant [57].

In contrast, the effect of cAMP on H202-induced MEK1/2 and ERK1/2
phosphorylation suggests that H202 may activate other signaling pathways,
which increase ERK1/2 phosphorylation without activating MEK1/2. It has
previously been suggested that sustained activation of ERK1/2 may be
independent of MEK activation [58]. In our experiments, involving only 10
minutes of stimulation, cAMP may have promoted transient MEK1/2
phosphorylation while promoting sustained ERK1/2 phosphorylation. Although
this difference of kinetics may explain why MEK1/2 phosphorylation seems to be
suppressed by cAMP, it does not completely explain the effect of cAMP on
ERK1/2 phosphorylation by H202. Because cAMP actually amplifies the level of
H202-induced ERK1I2 phosphorylation, rather than maintaining ERK1/2
phosphorylation at the same level, it seems to be reasonable to assume that
another pathway leading to increased ERK1/2 phosphorylation is being activated
by H202 and that this pathway is potentiated by cAMP. It is known that cAMP
can activate Rap1 in a PKA-dependent manner [31] or via cAMP-activated
GDP/GTP exchange factor [59,60]. In the presence of B-Raf MAPKKK, activated
Rap1 can lead to ERK activation [31]. However, if cAMP enhances H202-
induced ERK1/2 phosphorylation via B-raf, cAMP should also upregulate H202-
induced MEK1/2 MAPKK phosphorylation concomitant with ERK1/2
phosphorylation, which was not the case in our experiments. Another

consideration is that cAMP and H202 may activate other MAPK kinases (MAPKK)

52

that can phosphorylate ERK1/2. However, this seems unlikely since dual-
specificity kinases that phosphorylate ERK1I2 other than MEK1/2 have not been
reported and the specificity of the MAPK signaling cascade is believed to be
tightly controlled between MAPKK and MAPK [61].

A more attractive possibility is that the increased phosphorylation of
ERK1/2 is due to the inhibition of a phosphatase, thus allowing upstream kinases
to act unopposed. Evidence for this has come from experiments as follows.
PKA was shown to phosphorylate serine residues in He-PTP [33], a
hematopoietic-specific PTP [62]. The serine residue is located in the KIM
domain of the phosphatase, which interacts with ERK1/2 [63]. When this residue
was phosphorylated by PKA, the interaction between He-PTP and ERK1/2 was
inhibited, thereby leading to the loss of He-PTP activity towards the tyrosine
residue in the activating loop of ERK1/2 [33]. It is possible that He-PTP becomes
more sensitive to H202-induced inhibition when the complex of the phosphatase
and ERK1/2 is dissociated by cAMP (Fig. 5). Maintenance of phosphorylated Tyr
residue in ERK1/2 may make it a better substrate for subsequent Thr
phosphorylation by MEK1/2. The low level of MEK1/2 activity is probably
sufficient to increase the phosphorylation level of both Tyr and Thr residues in
ERK1/2.

Although ERK1 and 2 seem to be functionally equivalent, it is not clear
why two isoforrns of ERK exist and whether there are signaling pathways
differentially regulating ERK1 and ERK2 isoforrns [64]. Scaffolding proteins such

as MP1 may have such a role, favoring the activation of ERK1 over ERK2 by

53

TCR

 

V .
H202 tyrosine kinases AC <" FOFSKO'm
. . . \V /,',\(e.g., Lck, Zap70)

   

adaptors ,
(e. 9., LAT SLP76) cAMP

EGTA
Gm; 805
PlP2 IP3 + DAG
Ca2+ Ras
\ PKC _> R a“ P098059

  

(H202

cytoplasm

 

 

cell membrane

 

 

Figure 5. A model of cooperation between H202 and cAMP in ERK1I2
phosphorylation. Cyclic AMP inhibits upstream signaling pathways activated by
H202, resulting in decrease of H202-induced MEK1/2 phosphorylation. H202 also
inhibits ERK-dephosphorylating PTPs (e.g. He-PTP) that may be dissociated
from ERK1/2 by PKA phosphorylation, leading to overall enhancement of ERK1/2
phosphorylation by cAMP. Arrows indicate activation pathways, whereas the
blunt arrows indicate downregulation. Broken lines indicate that the components

of the pathway are not clearly defined.

54

linking MEK1 with ERK1 [65]. Our observation that cAMP amplifies the effect of
H202 on phosphorylation of ERK2 more than phosphorylation of ERK1 indicates
that ERK phosphatases affected by both H202 and cAMP may take part in
differentially tuning the phosphorylation level of ERK isoforrns. This is in
agreement with a report showing that tyrosine-phosphorylated ERK2 binds to He-
PTP with higher affinity than tyrosine-phosphorylated ERK1 [66].

ERK1/2 phosphorylation by epidermal growth factor (EGF) is potentiated
by forskolin in a prostate cancer cell line [67] and a choriocarcinoma cell line [68].
Because EGF receptor stimulation generates H202 [69], the synergistic activation
of ERK1/2 may be attributed to cooperation between H202 and cAMP, as shown
in our study. However, the effect of cAMP on EGF-induced and H202-induced
ERK1I2 phosphorylation shows high cell-type specificity. cAMP or forskolin are
known to attenuate EGF-induced ERK activation in hepatocytes [70], fibroblasts
[29], and adenocarcinoma cells [71]. cAMP weakens H202-induced ERK1/2
phosphorylation in smooth muscle cells [72] and myofibroblasts [73]. The effect
combined effect of H202 and cAMP may be also applied to the cross talk
between other receptors generating cAMP and producing H202.

The results in this study suggest that there are at least two signaling
pathways regulating ERK1/2 phosphorylation in T cells. One is the classical
MAPK pathway involving the sequential activation through MAPKKK -)MAPKK
->MAPK by phosphorylation. The other is the pathway mediated by ERK1/2
phosphatases such as He-PTP. H202 seems to utilize both pathways to induce

ERK1/2 phosphorylation. When the cAMP level was increased, the inhibition of

55

the H202—induced ERK MAPK cascade resulted in the decreased level of
MEK1/2 phosphorylation, whereas the synergistic inhibition of ERK
phosphatases led to the enhancement of ERK1/2 phosphorylation (Fig. 5).
Although the ERK MAPK cascade is necessary for inducing ERK1/2
phosphorylation in T cells, the amplitude of ERK1/2 phosphorylation may be
modulated by a different pathway, which is regulated by H202 and cAMP. When
the environment provides these signaling molecules by other cell types or
cytokines, this pathway may affect the amplitude and duration of ERK1/2

phosphorylation, which may lead to different outcomes of TCR stimulation.

56

ACKNOWLEDGEMENTS
This study was supported by National Institutes of Health Grant number
Al42794 and by the Jean P. Schultz Endowed Oncology Research Fund. The
authors wish to acknowledge the generous gift of anti-CD3 and C028 antibodies

from Drs. Shu Man Fu and S.-S. J. Sung (University of Virginia, Charlottesville,
VA)

57

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61

CHAPTER THREE

INHIBITION OF PTPS BY H202 REGULATES THE ACTIVATION OF DISTINCT
MAPK PATHWAYS

This work was published in Free Radical in Biology and Medicine (2002), Vol.

33, p. 1121 — 1132.

62

ABSTRACT
It has been shown that endogenous production of reactive oxygen species (ROS)
during T cell activation regulates signaling events including MAPK activation.
Protein tyrosine phosphatases (PTPs) have been regarded as targets of ROS
including which modify the catalytic cysteine residues of the enzymes. We have
analyzed the interplay between the inhibition of PTPs and the activation of MAPK
by H202. Stimulation of Jurkat T cells with H202 induces the phosphorylation of
ERK, p38, and JNK members of MAPK family. H202 stimulation of T cells was
found to inhibit the PTP activity of CD45, SHP-1, and HePTP. Transfection of
cells with thHP-1 decreased H202-induced ERK and JNK phosphorylation
without affecting p38 phosphorylation. Transfection with thePTP inhibited
H202-induced ERK and p38 phosphorylation without inhibiting JNK
phosphorylation. The Src-family kinase inhibitor, PP2, inhibited the H202-
induced phosphorylation of ERK, p38 and JNK. The phospholipase C (PLC)
inhibitor, U73122, or the protein kinase C (PKC) inhibitor, Ro-31-8425, blocked
H202-induced ERK phosphorylation, whereas the same treatment did not inhibit
p38 or JNK phosphorylation. Taken together, these results suggest that
inhibition of PTPs by H202 contributes to the induction of distinct MAPK activation

profiles via differential signaling pathways.

63

INTRODUCTION

Reactive Oxygen Species (ROS) are generated by the incomplete
reduction of oxygen during various biological processes [1]. It has been known
that ROS mediate diverse effects on the function of the cells [2]. Because ROS
can be generated rapidly in response to extracellular stimuli and can be
degraded efficiently, they have been regarded as potential second messengers
[3]. Supporting this concept, various growth factor receptors, cytokine receptors,
and GPCRs (G-protein coupled receptors) have been shown to produce ROS
including H202 when cognate ligand binds the receptor [4].

It has been shown that T cells produce endogenous ROS in response to
various physiological stimuli. ROS production in thymocytes stimulated with
Concanavalin A has been suggested to modulate JNK activation [5]. CD28
costimulation has been shown to produce ROS by a lipoxygenase, resulting in IL-
2 gene transcription through NF-xB activation [6]. Recently, it has been
demonstrated that endogenously produced ROS during T cell receptor (T CR)
activation regulate ERK activation and Fas ligand expression [7]. In addition to
the endogenous production of ROS, exogenously provided ROS have been
shown to affect T cell function at inflammatory sites, where activated phagocytes
release high amount of ROS through the NADPH oxidase system [8]. Amine
oxidase in the endothelial cells has also been proposed as a source of H202
during T cell migration [9]. Abnormal regulation of T cell function in complex
diseases such as rheumatoid arthritis [10], atherosclerosis [11], AIDS [12], and

cancer [13] has been attributed to the oxidative environment in pathological

situations. Although it is clear that endogenously or exogenously produced ROS
affect T cell signaling, the target molecules of ROS and the mechanism of ROS
regulation have not been precisely defined.

Compared to the other members of ROS, H202 is more stable and
membrane permeable leading to the proposal that H202 can function as a second
messenger. Both exogenous treatment and endogenous production of H202 has
been suggested to contribute cellular signaling by inhibiting protein tyrosine
phosphatases (PTPs) [14]. PTPs contain an essential catalytic cysteine residue
in their active sites with a pKa (~5.5) as compared to the pKa (~8) of other
cysteine residues in most proteins [15]. The low pKa makes the thiolate anion
especially susceptible to the inhibitory action of H202. H202 has been shown to
inhibit PTPs in vitro [16] as well as inside cells [17]. For example, it has been
shown that H202 treatment substantially reduces total cellular PTP activity in the
MO7e [18] and HER14 cells [19]. Specifically, H202 was found to inhibit PTP
activity of CD45 in Jurkat T lymphocytes [20] and neutrophils [21]. PTP activity
of SHP-1 was also found to be inhibited by H202 in SHP-1 transfected HELA
cells [22]. Recent reports have shown that endogenously produced ROS
inactivate PTP1B when insulin receptor [23] or EGF receptor [24] were triggered,
suggesting that inhibition of PTPs by H202 may be a physiologically important
event during cell signaling.

CD45, SHP-1, and HePTP are PTPs predominantly expressed in T cells
and it has been known that the activities of these PTPs are important in T cell

activation. CD45 is the most abundant PTP in T cell, accounting for about 75%

65

PTP activity in T cell membrane [25]. CD45 activity is critical for T cell activation
and T cells deficient in CD45 expression failed to generate signals responding to
TCR-engaging antibodies [26-28]. One requirement for CD45 in T cell activation
has been understood to be the dephosphorylation of the inhibitory tyrosine
residue of Src-family kinases such as Lck and Fyn [29]. However, it has been
reported that CD45 may also dephosphorylate the activation loop tyrosine
residue of Src-family tyrosine kinases [30,31], suggesting that the role of CD45 in
T cell signaling may depend on the types of stimuli and the accessibility of the
substrates to the phosphatase. SHP-1 is a PTP which is predominantly
expressed in hematopoietic cells [32]. Thymocytes from SHP-1 deficient mice
(designated motheaten) showed the enhancement of constitutive as well as
induced tyrosine phosphorylation of the TCR complex [33], suggesting a negative
role of SHP-1 in TCR signaling. Supporting this concept, SHP-1 has been shown
to dephosphorylate SLP76 [34] and Zap70 [35], which are important signaling
molecules in T cell activation. HePTP is expressed exclusively in hematopoietic
cells [36]. Overexpression of HePTP in T cells resulted in down-regulation of
ERK activation and lL-2 promoter activation, suggesting that HePTP has a
negative role in T cell activation [37]. It has been shown that HePTP
dephosphorylates the activating tyrosine residue of ERK [38], and PMA- and
TCR-induced ERK activation is increased in spleen cells from HePTP knockout
mice [39], indicating that ERK is an authentic substrate of HePTP.

One well documented result of cell stimulation with H202 is MAPK

activation [40]. The MAPK family members considered in this report include

66

ERK, p38, and JNK. ERK activation has been mainly implicated in proliferation
in response to growth factors, whereas p38 and JNK activation are more
important to stress responses, such as in inflammation [41]. It has been
suggested that the combination of the magnitude and kinetics of activation of
each member of MAPK family determines the appropriate response of the cell
according to the specific stimulus [42]. In the case of T cells, the distinct
activation profile of three members of the MAPK family has been shown to
influence the specific stages of thymocyte development as well as the precise
effector function of mature T cells [43].

In this report, the role of PTPs on H202-induced MAPK activation was
analyzed by measurement of PTP activity immunoprecipitated from intact cells
and by transfection of PTP vectors. Treatment of T cells with H202 inhibited PTP
activity of CD45, SHP-1, and l-lePTP, suggesting that PTPs are targets of H202.
Ectopic expression of thHP-1 inhibited H202-induced ERK and JNK
phosphorylation without inhibiting p38 phosphorylation. On the other hand,
ectopic expression of HePTP specifically affected H202-induced ERK and p38
phosphorylation without affecting JNK phosphorylation. The differential effect of
PTP transfection suggests that each PTP controls the distinct signaling pathway
leading to MAPK phosphorylation. The activity of Src-family tyrosine kinase was
necessary for H202-induced ERK, p38, and JNK phosphorylation, whereas PLC
and PKC activity was dispensable in the case of p38 and JNK phosphorylation

induced by H202. Taken together, results in this study suggest that inhibition of

67

PTPs by H202 contributes to the distinct activation profile of three members of

MAPK family.

68

MATERIALS AND METHODS

Reagents and antibodies. H202 was purchased from Sigma (St. Louis,
MO). PP2, U73122 and Ro-31-8425 were obtained from Calbiochem (La Jolla,
CA). Antibodies used in this study were obtained as follows: phospho-ERK (New
England Biolabs, Beverly, MA); phospho-p38 and phospho-JNK (Promega,
Madison, WI); ERK2, JNK, normal lgG, and CD45 for immunoprecipitation
(Santa Cruz Biotechnology, Santa Cruz, CA); CD45 for western blotting and
SHP-1 (Transduction Laboratories, Lexington, KY); haemagglutinin (HA)
antibody, clone 16812 (Babco, Richmond, CA); horseradish peroxidase-
conjugated donkey anti-rabbit antibody and sheep anti-mouse antibodies
(Amersham, Arlington Heights, IL); and horseradish peroxidase-conjugated rabbit
anti-goat antibody (Calbiochem). cDNAs for thHP-1, ClS-SHP-1, thePTP
and C/S-HePTP containing in the pEFlHA vector were generously provided by
Dr. T. Mustelin [37,44].

Cell culture and stimulation. Jurkat T cells and Jurkat TAg cells were
cultured in RPMI 1640 medium with 25 mM HEPES buffer (Biofluids: Rockville,
MD) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Life
Technologies: Rockville, MD), streptomycin/penicillin (100 units/ml) and 2 mM
glutamine (Life Technologies). Cells were maintained in an exponential growth
phase (0.2-1.0 x 106 cells/ml). Cells were reconstituted in the concentration of 1
x 106 cells/ml media described above before stimulation. Because PP2, U73122,

and Ro-31-8425 were dissolved in dimethyl sulfoxide (DMSO), the same volume

69

of DMSO was included in the control sample whenever these reagents were
used.

Western Blotting. Cells were washed twice with ice-cold phosphate-
buffered saline and then lysed in lysis buffer (Cell Signaling: Beverly, MA) for 30
min on ice. Cell nuclei were pelleted by centrifugation at 8,000 x g at 4°C for 10
min. Lysates were separated on a 10% SDS-PAGE gel and proteins were
transferred to nitrocellulose membrane. Immunoblotting was performed as
previously described [45]. Bands were visualized by chemiluminescence
(Amersham).

Transfection. Transient transfection of Jurkat TAg cells was performed
using DMRlE-C (Life Technologies). Typically, 6 pl of DMRlE-C was mixed with
24 pg of SHP-1, HePTP vector or pEFlneo control vector in 250 pl of OPTl-MEM
media (Life Technologies) at room temperature to form lipid-DNA complexes.
After 40 min, 0.5 x 10‘5 cells in 50 pl of OPTI-MEM were added to the mixture.
After 5 hrs of incubation at 37°C, 1 ml RPMI medium containing 10% FBS was
added. After two days, cells were stimulated and subjected to lysis.

Immunoprecipitation and PTP assay. Cells were stimulated by H202
and, after washing with PBS, subject to lysis with immunoprecipitation buffer (15
mM KCI, 10 mM HEPES (pH 7.6), 2 mM MgCl2, 0.1% NP40, 1 pg/ml aprotinin, 1
pg/ml leupeptin, 1 pg/ml pepstatin A, 1 mM PMSF) for 10 min in the ice. Cell
supematants were incubated with antibody at 4°C for the indicated times
followed by addition of 30 pl Gammabind-Sepharose beads (Amersham) and

incubation for an additional two hours at 4°C. The beads were washed and then

70

mixed with 100 pl PTP buffer (25 mM imidazole (pH 7.2), 45 mM NaCl, 1 mM

EDTA) containing 20 mM or 40 mM pNPP followed by incubation at 37°C for 5
min or 30 min. Absorbance of the supernatant was determined at 410 nm. For
each PTP activity calculation the absorbance of pNPP hydrolyzed from beads
with control lgG (or anti-HA lgG) alone was subtracted from the absorbance of
lysate immunoprecipitates. The absorbance obtained from PTP immune
complexes was then normalized to the absorbance obtained with control lgG
immunoprecipitates from Iysates to determine fold change. In the case of HePTP
immunoprecipitation, the absorbance was normalized to that of anti-HA immune
complexes from cells transfected with control vector. In all cases, average
values with standard deviation were obtained after three independent
experiments. The statistical analysis was done using student’s t test and p value
was determined. After measurement of PTP activity, the beads were recovered
and subjected to SDS-PAGE gel for the analysis for western blotting to detect the

presence of PTPs.

71

RESULTS

H202 induces activation-related phosphorylation of ERK, p38, and
JNK MAPKs. The goal of this study is to understand the mechanism of H202-
induced signaling leading to MAPK phosphorylation and activation. The
phosphorylation level of MAPKs was measured by western blotting with
antibodies that specifically recognize both phospho-Tyr and phospho-Thr
residues that are regarded to be necessary and sufficient for the activation of
MAPKs. As shown in Fig. 1, stimulation of Jurkat T cells with H202 induced
specific phosphorylation of all three members of MAPK family, ERK, p38, and
JNK. ERK phosphorylation rapidly increased by 10 min (lane 2) of H202
stimulation and decreased after 30 min. p38 phosphorylation also rapidly
increased, but the phosphorylation was more sustained compared to ERK. In
contrast, JNK phosphorylation was delayed when compared to ERK and p38.
The peak of JNK phosphorylation was at about 30 min (lane 4), and this
phosphorylation level was still detected up to 2 hrs after stimulation. Accordingly,
in subsequent analysis in this report, the level of ERK and p38 phosphorylation
was measured at 10 min and JNK phosphorylation was measured at 30 min after
H202 stimulation.

Treatment of cells with H202 inhibits the activity of PTPs. In this
report we wished to test the hypothesis that the observed specific activation of
MAPKs was, at least in part, due to the inhibition of PTPs which influence both
upstream signaling pathways leading to MAPK phosphorylation, and to the direct

dephosphorylation of the MAPKs themselves.

72

H202 - + + + + + +

mIn - 1o 20 30 45 60 120
-u *. 1:13:23 IB: phospho-ERK
...- a... W IB: phospho-p38
m an m ~ lB:phospho-JNK
M... w W m “7'5“" «nu-b“ lB: ERK2

1 2 3 4 5 6 7

Figure 1. H202 induces MAPK phosphorylation. Jurkat T cells were
stimulated by 400 pM H202 for the indicated times. Phosphorylation levels were
measured by immunoblotting the whole cell lysate with antibodies specific for the
phosphorylated forms of the enzymes. The blot was reprobed with ERK2
antibody to show that equivalent amounts of protein were in each lane. The

representative blots of three independent experiments are shown.

73

CD45 is the most predominant phosphatase in T cell membrane and the
enzyme is critical for T cell activation through regulation of Src—family kinase
(Lck, Fyn in T cell) activity [46,47]. Treatment of Jurkat T cells with H202 for 10
min inhibited PTP activity of endogenous, immunoprecipitated CD45 by 60%
(Fig. 2A, lanes 2 and 3; N = 3, P < 0.005). Western blotting with CD45 antibody
confirmed the presence of CD45 in the immune complex (Fig. 2A, anti-CD45
immunoblot).

Because we hypothesized that SHP-1 is also a target of H202, we
measured the PTP activity of SHP-1 after H202 treatment. lmmunoprecipitates of
endogenous SHP-1 showed ~18 fold higher activity compared to the activity of
control lgG immunoprecipitates (Fig. 23, lanes 1 and 2). H202 treatment of the
cells for 10 min inhibited the PTP activity of SHP-1 immunoprecipitates by about
50% (Fig. 28, lanes 2 and 3; N = 3, P = 0.027). Western blotting the immune
complex with SHP-1 antibody showed that endogenous SHP-1 was successfully
immunoprecipitated with the antibody (Fig. 2B, anti-SHP-1 blot).

We next wished to examine the activity of the MAPK tyrosine
phosphatase, HePTP, which is expressed in T cells. Because a precipitating
antibody for endogenous HePTP was not available, Jurkat TAg cells ectopically
expressing haemagglutin (HA) tagged HePTP [37] were used and HA-HePTP
was immunoprecipitated using anti-HA antibody. Cells transfected with vectors
were treated with H202 for 10 min followed by anti-HA immunoprecipitation and
measurement of PTP activity. Anti-HA immunoprecipitates of thePTP

transfectants exhibited about a 50 fold greater PTP activity compared to

74

Figure 2. Treatment of cells with H202 inhibits the activity of PTPs. (A) After
Jurkat T cells were stimulated with 400 pM H202 for 10 min, CD45 was
immunoprecipitated with anti-CD45 Ab. The immune complex was incubated with
20 mM pNPP for 5 min and the activity was calculated as described in the
Methods. *, p < 0.005 versus the activity of CD45 immune complex from cells
without H202 stimulation. The bottom blot shows the presence of endogenous
CD45 in the anti-CD45 immune complex. (B) After Jurkat T cells were stimulated
by 400 pM H202 for 10 min, SHP-1 was immunoprecipitated with SHP-1 antibody
for two hours and the washed immune complex was incubated with 40 mM pNPP
for 30 min. The activity was calculated as described in the Methods. *, p = 0.027
versus the activity of SHP-1 immune complex from cells without H202 stimulation.
The bottom blot shows endogenous SHP-1 immunoprecipitated by the antibody.
(C) Jurkat TAg cells were transfected with 12 pg of HePTP vector or pEFlneo
vector. After two days, cells were stimulated by 400 pM H202 for 10 min.
Ectopically expressed HePTP with HA tag was immunoprecipitated using HA
antibody for two hours and the immune complex was incubated with 20 mM
pNPP for 5 min. The activity was calculated as described in the Methods. *, p <
0.01 versus the activity of anti-HA immune complex from thePTP transfected
cells without H202 stimulation. The bottom blot shows the presence of HA-
tagged HePTP in anti-HA immune complex. In each case, the value in the first
lane was set as 1 and the values in the other lanes were represented as fold

change with standard deviation after three independent experiments.

75

H202

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anti-CD45

IP: control

 

 

 

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33:8 En.
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endogenous
“T“. ""'""" *SHP-i
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wt

 

vector wt

transfection

 

 

H202

 

 

 

 

 

60‘

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76

immunoprecipitates from cells transfected with empty vector (Fig. 2C, lanes 1
and 2). The activity of HePTP immunoprecipitated from H202-treated cells was
inhibited by about 60% (Fig. ZC, lanes 2 and 3; N = 3, P < 0.01). As a control,
immunoprecipitates from cells expressing the catalytically inactive Cys to Ser
mutant (CIS) form of HePTP had no phosphatase activity (Fig. 2C, lanes 1 and
4). The presence of similar amounts of HePTP in each immune complex was
confirmed by western blotting with anti-HA antibody (Fig. 2C, anti-HA blot).
These experiments demonstrate that, under the conditions of H202 stimulation
which lead to MAPK activation, the PTP activity of several key PTPs was indeed
inhibited.

thHP-1 overexpression inhibits H202-induced ERK and JNK
phosphorylation without affecting p38 phosphorylation. The effect of PTP
overexpression on H202-induced MAPK phosphorylation was analyzed in order
to address the question of whether the inhibition of PTPs by H202 was relevant to
MAPK activation. SHP-1 was chosen as a representative enzyme that regulates
the upstream signaling pathways leading to MAPK phosphorylation. SHP-1 has
been suggested to dephosphorylate proximal tyrosine kinases such as Lck and
Zap70. Overexpression of thHP-1 moderately reduced H202-induced ERK
phosphorylation and substantially inhibited JNK phosphorylation (Fig. 3A, lane 3).
In contrast, overexpression of SHP-1 did not affect H202-induced p38
phosphorylation. The effect of SHP-1 overexpression was dependent on the
catalytic activity of SHP-1, because overexpression of catalytically inactive SHP-

1 containing a mutation of the catalytic Cys to Ser (CIS) did not suppress the

77

Figure 3. PTP overexpression affects H202-induced MAPK
phosphorylation. (A) Jurkat TAg cells were transfected with pEFlneo vector,
thHP-1, or CIS-SHP-1 vector; and (B) with pEF/neo vector, thePTP, or CIS-

HePTP vector. After two days of transfection, cells were stimulated with 200 pM

H202 and the ERK and p38 phosphorylation levels were measured after 10 min
and the JNK phosphorylation level was analyzed after 30 min. After blotting with
phospho-specific antibodies, the blots were reprobed with ERK2 or JNK antibody
to show that equivalent amount of protein were in each lane. Anti-HA blot
confirmed the expression of HA-tagged SHP-1 and HA-tagged HePTP in the
transfected cells. The representative blots from three independent experiments

are shown.

78

A. HA-SHP-1

 

 

vector wt CIS
H202 - + + +
10min :1; , f: :.'....;:.: 332$; IB: phospho-ERK
m .......... -- IB: phosphc-p38

1"" -— IB: HA (SHP-1)
* - fl 4. IB: ERK2
~~--.... .. , .. IB: phospho-JNK
' - ' lB:HA(SHP-1)

 

30 min

 

gun-u a... I... can. leJNK
1 2 3 4

 

    

 

 

B.
HA-HePTP
vector wt C/S
H202 - + + +
10min I. , If. $213322; IB: phospho-ERK
"' “- """""""" -I. IB: phospho-l338
“ ” IB: HA (HePTP)
mm ' i“ ‘ " " ' IB: ERK2
30 min .............. *W“ a» - IB: phospho-JNK

"— IB: HA (HePTP)

w m *0 m IBZJNK
1 2 3 4

79

H202-induced phosphorylation of the MAPKs (Fig 3A, lane 4). Because
ectopically expressed SHP-1 contains HA tag in the N terminus [44], the
expression of transfected SHP-1 vectors was verified by anti-HA antibody (Fig
3A, anti-HA blot).

wtl-lePTP overexpression suppresses H202-induced ERK and p38
phosphorylation, but not JNK phosphorylation. HePTP was chosen as an
example of a PTP which directly dephosphorylates MAPK at the activation loop
Tyr residue. Overexpression of thePTP substantially inhibited H202-induced
ERK and p38 phosphorylation (Fig. SB, lane 3). Conversely, overexpression of
CIS-HePTP potentiated ERK and p38 phosphorylation induced by H202,
suggesting a dominant-negative effect (Fig. 3B, lane 4). The enhancement of
H202—induced p38 phosphorylation by CIS-HePTP overexpression was less
dramatic than the potentiation of ERK phosphorylation. In contrast with ERK and
p38, JNK phosphorylation was not affected by either wt or CIS-HePTP
overexpression. These results suggest that SHP-1 and HePTP may differentially
regulate each member of MAPK phosphorylation when H202 stimulates T cells.

The Src-family kinase inhibitor, PP2, inhibits H202-induced MAPK
phosphorylation. To better understand the signaling pathway leading to MAPK
activation by H202, reagents that specifically inhibit signaling enzymes were
applied during H202 stimulation. Src family tyrosine kinases such as Lck and Fyn
are associated with cytoplasmic portions of TCR components and their activation
is the first step in TCR activation [48]. We treated cells with the Src family

kinase-specific inhibitor, PP2, to assess the involvement of these kinases in

80

H202-induced MAPK phosphorylation. PP2 treatment inhibited the H202-induced
phosphorylation of all three MAPKs (Fig. 4A, lane 3), suggesting that the activity
of the proximal tyrosine kinases for T cell activation (such as Lck and Fyn) was
required for H202-induced MAPK phosphorylation. The possibility that PP2
treatment made the cells nonfunctional was negated by the observation that
PMA-induced ERK phosphorylation was not inhibited by the same condition of
PP2 treatment (data not shown), as previously reported [49].

U73122 (PLC inhibitor) and Ro-31-8425 (PKC inhibitor) inhibit H202-
induced ERK phosphorylation, whereas p38 and JNK phosphorylation are
not affected. PLC and PKC are important enzymes that mediate the signaling
response in T cell activation. PLCy is activated by tyrosine phosphorylation [50]
during TCR stimulation, and it hydrolyzes PIP; to form IP3 and diacylglycerol
(DAG). It is believed that PKC contributes to ERK phosphorylation by promoting
Raf-1 activation when T cell receptor is triggered [51]. We used reagents which
specifically inhibit PLC and PKC to assess their involvement in H202-induced
MAPK phosphorylation. Treatment of cells with either U73122 or Ro-31-8425
abrogated H202-induced ERK phosphorylation (Fig. 4B, lanes 3 and 4).
Surprisingly, neither inhibitor at the same concentration affected H202-induced
p38 and JNK phosphorylation (Fig. 4B, lanes 3 and 4). These results suggest
that the activity of PLC and PKC is especially required for H202-induced ERK

phosphorylation.

81

 

 

 

 

 

PP2 - - +
10 min 7 .'.'....-.,. ‘ 1:1; Egg IB: phospho-ERK
~ **"‘* ' " IB: phospho—p38
. _ '31 ERK2
30 min . m - IB: phospho-JNK
. - fl' IB: JNK
1 2 3
B- H202 - + + +
U73122 - - + -
Ro31-8425 - - - +
10 min “a '_: 2:352:38: phospho-ERK
”'5. an... sell- '33 phospho-p38
30 min ' -. ‘- '1'!!! IB: phospho-JNK

""_'"" “A“ “a. IBzJNK
1 2 3 4

Figure 4. H202-induced MAPK phosphorylation is regulated by differential
signaling pathways. (A) Cells were incubated with 20 pM PP2 for 2 hrs and
stimulated by 400 pM H202. (B) Cells were incubated with 2 pM U73122 or 1
pM Ro-31-8425 for 10 min and stimulated by 400 pM H202. In both (A) and (B),
ERK and p38 phosphorylation levels were measured after 10 min stimulation with
H202 and the JNK phosphorylation level was analyzed after 30 min stimulation.
After blotting with phospho-specific antibodies, the blots were reprobed with
ERK2 or JNK antibody to show that equivalent amounts of protein were in each

lane. The representative blots from three independent experiments are shown.

82

DISCUSSION

The level of tyrosine phosphorylation inside the cell is determined by the
balance between the activity of protein tyrosine kinases (PTKs) and of protein
tyrosine phosphatases [52]. Although various potential schemes for the
regulation of PTKs have been proposed, rigorously verified regulatory
mechanisms for PTPs are poorly understood [53]. Inhibition of PTP activity by
ROS is regarded as one physiological means of regulation of PTPs. It has been
documented that ROS are produced endogenously and inhibit PTP activity when
receptors are stimulated, and blockade of the receptor-triggered ROS production
restores PTP activity [23]. In addition, there are various situations involving
inflammatory responses and associated diseases in which T cells are exposed to
exogenous ROS. Because of the potential importance of ROS modulation of T
cells by inhibition of PTPs, we designed experiments to use Jurkat cells treated
with H202 as a model system to understand the dynamic relationship between
several PTPs and signaling events leading to MAPK activation.

The current experiments were designed to evaluate the role of several
interacting PTPs important for T cell activation and for MAPK activation. We
chose to study CD45, SHP-1 and HePTP because of their important involvement
in T cell activation, as well as for their respective roles in the stages of the
signaling events leading to MAPK activation. Our strategy was to verify the effect
of H202 on PTP activity in intact cells followed by verification of the role of PTPs

in MAPK phosphorylation by ectopic expression of individual PTPs.

83

When Jurkat T cells were stimulated by H202, all three members of MAPK
family, ERK, p38, and JNK, become phosphorylated at the activating Tyr and Thr
residues as detected by phopho—specific antibodies (Fig. 1). The peak of H202-
induced JNK phosphorylation was delayed (about 30 min) compared to that of
ERK and p38 phosphorylation (about 10 min). The fact that this kinetic pattern of
MAPK phosphorylation was similar to that described for TCR-induced MAPK
phosphorylation [54] suggests that phosphorylation induced by H202 treatment
and TCR triggering share similar signaling pathways. The lag in JNK
phosphorylation could be due to mechanical events involving cytoskeletal
rearrangement required to fully activate JNK. GTPases leading to the activation
of JNK, such as Rac and cdc42, are also effectors of actin filament organization
[55]. Recently, it has been shown that agents that disrupt the cytoskeleton inhibit
both ROS production and JNK activation induced by TNFa in ECV-304 cells [56].

To test whether H202 treatment of Jurkat T cells inhibited PTP activity,
CD45, SHP-1, and HePTP were immunoprecipitated from cells and the PTP
activity of the immunoprecipitates were measured. Stimulation of Jurkat T cells
with H202 substantially inhibited the activity of all three tested PTPs (Fig. 2),
indicating that PTPs are potential targets of exogenously produced H202. All the
tested PTPs seemed to have a similar sensitivity to H202. This result supports
the idea that PTP inhibition by H202 is an intrinsic property of PTPs which

universally contain a highly reactive, negatively charged cysteine residue in the

catalytic site [57].

As a way of addressing the hypothesis that PTP inhibition by H202 is
relevant to H202-induced MAPK activation, the effect of SHP-1 and HePTP
overexpression on MAPK phosphorylation by H202 was analyzed. If PTPs are
targets of H202, overexpression of PTP would be expected to overcome the
effect of H202. An example of this approach is seen in a study in which the
overexpression of SHP-1 inhibited the activation of NF-AT induced by
peroxyvanadium, an inhibitor of PTPs [58]. SHP-1 overexpression also reduced
the activation of UV-induced MAPKAP kinase 2, which is thought to occur by
generation of ROS [59]. Our results also show that overexpression of thHP-1
inhibited H202-induced ERK and JNK phosphorylation (Fig. 3A). The catalytic
activity of SHP-1 was necessary for the effect, because the overexpression of the
catalytically inactivated ClS-SHP-1 did not reduce H202-induced ERK and JNK
phosphorylation. In contrast, SHP-1 overexpression did not affect H202-induced
p38 phosphorylation, suggesting that SHP-1 is primarily involved in the ERK and
JNK activation pathways (summarized in Fig. 5). The participation of SHP-1
activity on ERK activation has been previously suggested by the reports showing
that the Ras-ERK MAPK pathway was enhanced in motheaten thymocytes [33];
and SHP-1 overexpression reduced TCR-induced ERK activation [44]. The
possibility that SHP-1 negatively regulates JNK activation comes from the report
that SHP-1 associates with Vav [60], a GEF (GTP-exchanging factor) of Rac1
that has been implicated in JNK activation [61].

HePTP expression inhibited H202-induced ERK and p38 phosphorylation

without affecting JNK phosphorylation (Fig. 3B). This result suggests that ERK

85

and p38 are substrates of HePTP. Analysis of HePTP knockout mice conflrrned
that ERK is the authentic substrate of HePTP, because TCR- and PMA-mediated
ERK phosphorylation was enhanced in the absence of HePTP [39]. Sorbitol-
induced p38 phosphorylation was notenhanced in the same study, raising doubt
that p38 is a substrate of HePTP. The study of K562 cells stably expressing
HePTP also provides the evidence that ERK rather than p38 is a substrate of
HePTP, because CIS-HePTP bound to Tyr-phosphorylated ERK2 more
efficiently than p38 [62]. HePTP expression in our study also suggests the
highest affinity between ERK2 and CIS-HePTP, because the H202-induced
phosphorylation level of ERK2 was enhanced by CIS-HePTP overexpression
(Fig. 3B, lane 4). However, the ability of thePTP expression to inhibit H202-
induced p38 phosphorylation was as effective as for ERK phosphorylation (Fig.
3B, lane 3), raising the possibility that the mutation of Cys to Ser in CIS-HePTP
inadvertently changed the affinity of ERK and p38 for HePTP. The possibility
that p38 is a substrate of HePTP should still be considered because thePTP
overexpression inhibits TCR-induced p38 phosphorylation as well as ERK
phosphorylation; and p38 MAPK is detected in the endogenous HePTP
immunoprecipitation complex [38]. 1
To better understand the signaling pathway leading to H202-induced
MAPK phosphorylation, the effect of reagents inhibiting specific signaling
enzymes was analyzed. When the H202-induced signaling pathway was
investigated using the Src-family kinase inhibitor, PP2, it was found that Src-

family tyrosine kinases (most likely Lck and Fyn in T cells) are required for

86

phosphorylation of all three MAPK forms (Fig. 4A). This finding correlates with
reports showing that H202 treatment of Jurkat T cell leads to the increase of Lck
activity [63]. This result suggests that the proximal tyrosine kinases responsible
for T cell activation may be subject to ROS regulation. Because it has not been
demonstrated that the direct addition of H202 to the purified Src-family kinases
increases the tyrosine kinase activity in vitro [1], it is probable that the activation
of Src-family tyrosine kinases in intact cells treated by H202 occurs by the
inhibition of CD45 and/or SHP PTPs, which regulate these tyrosine kinases. It is
interesting that the activity of Src-family kinases is required for H202-induced
MAPK phosphorylation in a situation where H202 substantially inhibits the activity
of CD45 (Fig. 2A), because CD45 is the primary phosphatase implicated in the
dephosphorylation of the inhibitory Tyr residue of Src-family kinases in T cells. If
CD45 activity is inhibited by H202, unchecked phosphorylation of the inhibitory
Tyr residue may contribute to the inactivation of Src-family kinases. However,
the inhibition of CD45 activity by H202 may also lead to the hyperphosphorylation
of activating Tyr residues of Src-family kinases. It has been reported that Lck is
phosphorylated at both activating Tyr394 and inhibitory Tyr505 residue in CD45
negative cell line [30]. H202 treatment of T cells also leads to the
phosphorylation of both the activating Tyr394 and the inhibitory Tyr505 residues
of Lck [63]. lmportantly, the activity of Lck (which has phosphates on both Tyr
residues after H202 treatment [64] or in the absence of CD45 [31]) was actually
increased, suggesting that activation of Lck by phosphorylation of Tyr394 is

dominant over inhibition induced by Tyr505 phosphorylation. Therefore, it is

87

probable that H202 treatment leads to the activation of Src-family kinases by
inhibiting CD45 activity.

We found that thHP-1 overexpression did not inhibit H202-induced p38
phosphorylation (Fig. 3A) while the Src-family kinase inhibitor, PP2, effectively
inhibited p38 phosphorylation (Fig. 4A). This results suggest that the activity of
Src-family kinases (Lck and Fyn) was not completely controlled by SHP-1. There
is controversy surrounding the idea that Lck is the direct substrate of SHP-1.
While it has been shown that co-expression of Lck and SHP-1 resulted in the
dephosphorylation of activating Tyr394 residue of Lck in 293 cells [65], co-
expression of Lck and SHP-1 did not change the activity of Lck in COS-7 cells
[66]. The discrepancy may be caused by the fact that SH2-domain deleted form
of SHP-1 was used for expression in the former study. The two tandem SH2
domains in the N terminus of SHP-1 inhibit PTP activity by sterically blocking the
catalytic domain [67]. Because the removal of SH2 domains increases the
catalytic activity of SHP-1, the form of SHP-1 without SH2 domains has often
been used for transfection to increase the effect of overexpression. However,
SH2 domains of SHP-1 are important for localization of SHP-1 because the SH2
domains have been implicated in the binding of tyrosine-phosphorylated motifs in
inhibitory receptors such as Kir [68] and CD5 [69]. The localization of SHP-1
may be important for the phosphatase to access specific substrates. Because
the overexpression of thHP-1 with intact SH2 domains effectively inhibited
H202-induced JNK phosphorylation (Fig. 3A) and PI3K activity in COS-7 cells

[66], the overexpression of thHP-1 may have the enough potential to affect the

88

signaling pathway in which endogenous SHP-1 participates. Therefore, caution
is needed for the identification of the substrates of SHP-1 by overexpression of
SH2 domain-deleted SHP-1.

Exogenous treatment of H202 has been shown to induce tyrosine
phosphorylation of PLCy and to initiate PLC-mediated hydrolysis of inositol
phospholipids [70,71]. We found that PLC activity was required for H202-induced
ERK phosphorylation because the PLC inhibitor U73122 inhibited H202-induced
ERK phosphorylation (Fig. 4B). It has been known that PKC activation by DAG
is required for PKC phosphorylation and activation of Raf-1 [72,73]. PKC activity
was also necessary for H202-induced ERK phosphorylation because the PKC
inhibitor Ro-31-8425 inhibited H202-induced ERK phosphorylation (Fig. 4B). The
sequential activation pathway (PLC-)PKC-)Raf-1) may explain why PLC activity
is needed when H202 induces ERK phosphorylation.

In contrast to H202-induced ERK phosphorylation, the PLC inhibitor and
PKC inhibitor had no effect on H202-induced p38 and JNK phosphorylation (Fig.
4B). This result suggests that PKC activation by DAG production from PLC
activity may not be involved in the effect of H202 on p38 and JNK
phosphorylation. ASK1 is a characterized redox-regulated MAPKKK which
associates with Trx (thioredoxin). The dissociation of ASK1-Trx complex occurs
after the oxidation of Trx and ASK1 becomes activated and mediates the
activation of p38 and JNK MAPK pathway [74]. It will be interesting to check
whether PKC activity is dispensable for the activation of ASK1 by H202 in Jurkat

T cells.

89

Taken together, the differential effect of PTP overexpression and of
enzyme inhibitors on the induction of phosphorylation among MAPK members by
H202 suggests that each member of MAPK is activated by H202 through distinct
signaling pathways (Fig. 5). The observation that PTP overexpression specifically
affects H202-induced MAPK phosphorylation and that PTP activity is decreased
by H202 treatment indicates that the inhibition of PTPs by H202 is one important
mechanism for H202 to modulate the signaling pathways leading to differential

MAPK pathway activation.

90

/

 

 

p38

 

 

TCR

 

Lck, FynE

 

 

 

 

/

PLC

PKC I

ERK

 

 

 

 

\W

 

 

JNK

 

 

PTPs

H202

H202

w.

Figure 5. PTP inhibition by H202 regulates the activation of distinct MAPK

pathways.

The solid arrows indicate MAPK pathways of TCR activation,

whereas the blocked arrows indicate predicted sites of inhibition by PTPs. The

dashed line indicates a partial effect.

91

[1]

[2]

[3]

[4]

[5]

[6]

[7]

[3]

[9]

[10]

[11]

[12]

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98

CHAPTER FOUR

INHIBITION OF CD45 BY REACTIVE OXYGEN SPECIES (ROS) MEDIATES 1-
CHLORO-2,4-DINITROBENZENE (DNCBHNDUCED IMMUNOSTIMULATION
OF T CELLS

99

FOOTNOTES

1 This study was supported by National Institutes of Health Grant number

Al42794 and by the Jean P. Schultz Endowed Oncology Research Fund.

2 Address correspondence to: Dr. W. J. Esselman, Department of Microbiology
and Molecular Genetics, Michigan State University, East Lansing, MI 48824,
USA. Phone: 517-353-9752 x 1532. Fax: 517-353-9667. E-mail:

esselman@msu.edu

3 Abbreviations used in this paper: GSH, gluathione; Trx, thioredoxin; DNCB, 1-
chloro-2,4-dinitrobenzene; PTP, protein tyrosine phosphatase; ROS, reactive
oxygen species; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terrninal
kinase; ERK, extracellular-signal regulated kinase; p-JNK, phospho—JNK; p-ERK,
phospho-ERK; p-p38, phospho-p38; Ter, thioredoxin reductase; LMW, low
molecular weight; CM-HzDCF-DA, 5-(and-6)-chloromethyl-2',7'-
dichlorodihydrofluorescein diacetate acetyl ester; PLC, phospholipase C; PKC,
protein kinase C; EGF, epidermal growth factor; PDGF, platelet-derived growth

factor

100

ABSTRACT

DNCB has been utilized to modulate T cell activation in contact
hypersensitivity and in the treatment of melanoma. Because It has been
suggested that DNCB treatment of cells leads to the production of reactive
oxygen species (ROS) by inhibiting thioredoxin reductase and by depleting
glutathione, the relevance of ROS production on DNCB-induced
immunostimulation of T cells was investigated. The specific immunostimulatory
effect of DNCB was shown by the observation that DNCB strongly potentiated
TCR-induced p38 and JNK phosphorylation, while having no effect on ERK
phosphorylation. DNCB treatment of Jurkat cells increased intracellular ROS
level, which was prevented by an antioxidant, N-acetylcysteine (NAC). The
inhibition of Lck activity by PP2 or NAC corresponded with the decrease of
DNCB-induced JNK and p38 phosphorylation. The activity of CD45, a protein
tyrosine phosphatase (PTP) that regulates Lck, was inhibited when cells were
stimulated by DNCB or H202. Inclusion of DTT in the lysate or pretreatment of
the cells with NAC restored CD45 activity inhibited by DNCB, suggesting that
ROS were involved in the process. To assess the mechanism of CD45 inhibition
by ROS, the sequential modification of recombinant CD45 with differential
isotypes of iodoacetic acid was applied. H202 inhibited the incorporation of the
first modifying agent to the active site cysteine residue (Cys817) of CD45,
indicating that the modification of the cysteine residue was responsible for the
inhibition of CD45 activity by H202. Taken together, these results suggest that

ROS-induced inhibition of CD45 activity is responsible for the subsequent

101

activation of Lck and the differential regulation of MAPKs in DNCB-induced

immunostimulation of T cells.

102

INTRODUCTION

The topical application of DNCB to the skin has been known to provoke a
delayed-type hypersensitivity (DTH) reaction (1). In addition to the experimental
use of the compound to study DTH, DNCB has been tried to improve the
conditions of derrnatoses, HIV infection and malignant melanoma (2-4).
Recently, it has been shown that chemotherapy for melanoma using DNCB is
dependent on T cell-elicited immune response (5). Therefore, it is important to
understand the mechanism of DNCB-induced immunostimulation of T cells.
Although DNCB has been thought to induce DTH by functioning as a hapten (6),
it has been reported that immunostimulatory properties of DNCB cannot be solely
explained by the hapten model (7). An additional mechanism of
immunostimulation by DNCB involves the inhibition of mammalian thioredoxin
reductase (T er) activity by alkylating the catalytic selenocysteine and cysteine
residues (8). With the inhibition of Ter activity, thioredoxin (T rx) and other
substrates of Ter such as lipid hydroperoxides will remain in the oxidized state
and the reducing capacity of the cell will be impaired (9). Another interesting
feature is that Ter alkylated by DNCB produces superoxide with the
consumption of NADPH, like NADPH oxidase (10). In addition, DNCB depletes
GSH by forming a conjugate with GSH, in a reaction catalyzed by glutathione S-
transferase (GST) (11). All these effects change the intracellular milieu to a pro-
oxidative state which contributes to the profound immunostimulatory effect of
DNCB (12, 13). The relevance of DNCB-induced inhibition of Ter for the

immunostimulatory effect of the compound is supported by the reports that Ter

103

activity is decreased in vitiliginous skin that shows impaired DTH reactions by
DNCB (14, 15).

Cells maintain specific redox gradient in their intracellular compartments
with various regulatory machinery including glutathione (GSH) and thioredoxin
(Trx) (16-18). The change in the intracellular redox status affects diverse
aspects of the cell including proliferation and apoptosis (19, 20). Recent reports
have shown that the change in the redox status is used to modulate the
appropriate responses of the cells to extracellular stimuli. The most well
documented response in this regard is the production of endogenous ROS upon
exposure to extracellular stimuli (21). Phagocytic cells contain NADPH oxidase
in the plasma membrane, which produces high amount of ROS which has a role
in bacterial killing (22). Nonphagocytic cells also harbor a family of NADPH
oxidase-like enzymes, the homologues of which are being continuously cloned
and characterized (23, 24).

One of the potential molecules directly affected by ROS are the protein
tyrosine phosphatases (PTPs). Because the catalytic cysteine residue of PTP
exhibits a low pKa as compared to other cysteine residues (25), it has been
suggested that ROS inhibit PTP activity by modifying catalytic cysteine residue.
Supporting this concept, treatment of purified PTPs with H202 inhibits the activity
of the enzyme (26) and exposure of cells to H202 also leads to the
downregulation of PTP activity (27, 28). Stimulation of cells with insulin or EGF
inhibits PTP1B activity with endogenous production of ROS (29, 30). PDGF

increases intracellular ROS and inactivates LMW PTP and SHP-2 by oxidation

104

(31, 32). These results suggest that PTP inhibition by ROS has physiological
significance during cell stimulation. However, it has been difficult to
experimentally elucidate the mechanism of PTP inhibition by ROS because few
methods have been developed to examine the status of the active site cysteine
residue.

In this study, the relevance of ROS production to immunostimulation
induced by DNCB was investigated in Jurkat T lymphocytes. The
immunostimulatory effect of DNCB was manifested by the selective
enhancement of TCR-induced JNK and p38 phosphorylation. Stimulation of
Jurkat cells with DNCB was found to produce endogenous ROS. The
importance of ROS production for DNCB-induced immunostimulation was shown
by the observations that an antioxidant, N-acetylcysteine, inhibited DNCB-
induced ROS production, Lck activation, and MAPK phosphorylation. The PTP
activity of CD45 was downregulated by DNCB or H202 treatment of the cell,
which was reversed by the addition of DTT to the lysates. H202 inhibited the
incorporation of C‘3-iodoacetic acid to the active site cysteine residue of CD45,
indicating that the modification of the cysteine residue is responsible for the
inhibition of CD45. Taken together, these results suggest that ROS production
by DNCB results in the reversible inhibition of CD45 activity in T cells and
subsequent activation of Lck mediates the immunostimulatory effect of DNCB by

promoting JNK and p38 phosphorylation.

105

MATERIALS AND METHODS
Reagents and Antibodies
H202 and DNCB were purchased from Sigma (St. Louis, MO). PP2, U73122,
and Ro31-8425 were obtained from Calbiochem (San Diego, CA). Monoclonal
anti-CD3 clone 235 (lgM type) and anti-0028 clone NE51 (lgG type) Abs were
provided by Dr. Shu Man Fu (University of Virginia, Charlottesville, VA). p-ERK
Ab was purchased from New England Biolabs (Beverly, MA). p-p38 and p-JNK
Abs were obtained from Promega (Madison, WI). JNK1, ERK2, normal lgG, Lck
and CD45 Abs were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). Horseradish peroxidase-conjugated rabbit Ab was purchased from
Amersham (Arlington Heights, IL). Horseradish peroxidase-conjugated goat Ab
was obtained from Calbiochem.
Cell culture and stimulation
Jurkat cells, clone E6-1, were grown in RPMI 1640 with 25 mM HEPES buffer
(Biofluids: Rockville, MD) supplemented with 10% heat-inactivated FBS (Life
Technologies: Rockville, MD), 100 units/ml streptomycin/penicillin (Life
Technologies) and 2 mM glutamine (Life Technologies). Cells were reconstituted
in the concentration of 106 cells/ml in serum-free RPMI before stimulation.
Because DNCB, PP2, U73122, and R031-8425 were dissolved in DMSO, same
volume of DMSO was included in control sample. Western blotting procedure
was done as previously described (33).

Measurement of R08

106

After 5 min of stimulation with DNCB, 5 pM CM-HZDCF-DA (Molecular Probes:

Eugene, OR) was added. After 1 hr incubation with the fluorochrome, cells were
analyzed by FACS at an excitation wavelength of 488 nm and emission at 520
nm.

Lck kinase assay

Lck was immunoprecipitated from cells by incubation of Iysates with Lck Ab for 2
hrs followed by incubation with protein A agarose for the additional 2 hrs. The
beads were washed three times with immunoprecipitation buffer and two times
with kinase buffer (20 mM PIPES (pH 7.2), 10 mM MnClz). The immune
complexes were incubated in kinase buffer containing 5 pCi [y-azP] ATP and 20
pM ATP for 30 min at 30 °C. After the addition of Lammelie sample buffer and
boiling, the supematants were separated by SDS-PAGE gel. The gel was dried
and autoradiographed using a phosphoimager.

lmmunoprecipitation and PTP assay

After stimulation by DNCB, cells were washed with PBS and subject to lysis with
lysis buffer (10 mM HEPES (pH 7.6), 2 mM MgCIz, 15 mM KCI, 0.1% NP40, 1
pg/ml aprotinin, 1 pg/ml leupeptin, 1 pg/ml pepstatin A, 1 mM PMSF) for 30 min
in the ice. Where indicated, 5 mM DTT was included in the lysis buffer. After
centrifugation for 30 s at room temperature, supematants were incubated with
CD45 Ab at 4°C for two hours followed by addition of 30 pl Gammabind-
Sepharose beads (Amersham) and incubation for the additional two hours at
4°C. The beads were mixed with 100 pl PTP buffer (25 mM imidazole (pH 7.2),

45 mM NaCl, 1 mM EDTA) containing 20 mM pNPP followed by incubation for 90

107

s at 37°C. Absorbance of the supernatant was determined at 410 nm. The
value obtained from CD45 immunoprecipitates was subtracted by the value
obtained from control lgG immunoprecipitates.

PTP assay of recombinant CD45

The bacterial expression vector pET3D-His6CD45, which expresses the
cytoplasmic domain of murine CD45 with a His6 tag in the amino terminus, was
kindly provided by Dr. Pauline Johnson (University of British Columbia,
Vancouver, Canada). The purification of CD45 from bacteria was done as
described (34). 1 pg of protein in 100 pl PTP buffer was incubated with 100 pM
H202 or with 100 pM DNCB for 1 h at room temperature. PTP activity assay was
initiated by adding 20 mM pNPP. After 5 min of incubation at room temperature,
absorbance was determined at 410 nm.

Carboxymethylation with iodoacetic acid, in-gel digestion, and MALDl-MS

analysis of CD45

10 pg of recombinant CD45 in 100 pl of 50 mM MES (pH 6.6) buffer was
incubated with and without 1 mM H202 for 30 min at room temperature.
Subsequently, 250 pM of C‘3-iodoacetic acid was added and the mixture was
incubated for 30 min in the darkness. After the incubation, the solution was
incubated with 10 mM DTT for 30 min. After adding Lamealli sample buffer, the
solution was boiled and subject to SDS-PAGE electrophoresis. After staining of
the gel with Coomassie reagent (Bio-rad), CD45 band was cut out and chopped
into 1 mm-size pieces. After washing the gel pieces with ammonium

bicarbonate, the gel pieces were dehydrated by acetonitrile and vaccum-dry.

108

After reducing the gel pieces by incubation with DTT at 56°C for 30 min, the gel
pieces were subject to alkylation by C‘z-iodoacetic acid for 20 min at room
temperature in the darkness. The gel pieces were washed with ammonium
bicarbonate, dehydrated by acetonitrile and dried under vacuum. The gel pieces
were incubated by trypsin (Promega) in the ice for 45 min and subsequently at
37°C overnight. After harvesting the supernatant, the gel pieces were
suspended in the solution containing 60 % acetonitrile and 3 % trifluoroacetic
acid and were sonicated for 30 min. After mixing the supernatant from sonication

with the previous harvest, the solution was dried down to 20 pl under vacuum. 6
pl of 3 % trifluoroacetic acid was added to the solution and the mixture was

sonicated for 10 min. The solution was analyzed by MALDI-MS.

109

Results

DNCB potentiates TCR- and H202-induced MAPK phosphorylation.
To determine whether DNCB treatment affects T cell activation process, the
effect of DNCB on the phosphorylation in the activation domain of MAPKs was
analyzed. DNCB treatment induced JNK and p38 phosphorylation and
potentiated TCR-induced JNK and p38 phosphorylation (Fig. 1A, p-JNK and p-
p38 immunoblots). Interestingly, DNCB was a poor stimulator of ERK
phosphorylation and did not enhance TCR-induced ERK phosphorylation (Fig.
1A, p-ERK blot). To compare the sensitivity of MAPK phosphorylation induced
by TCR activation and by direct oxidant challenge, cells were stimulated by H202
with and without DNCB. In contrast to TCR activation, H202-induced ERK
phosphorylation was potentiated by DNCB as effectively as JNK and p38
phosphorylation (Fig. 1B). This result indicated that TCR-induced JNK and p38
activation is specifically affected by cellular redox status.

DNCB induces endogenous ROS production in T cells. To determine
whether DNCB treatment influences the oxidative potential of the T cell, the
intracellular ROS level was measured by the cell-permeable and ROS-sensitive
fluorochrome, CM-HzDCF-DA. Stimulation of T cell with DNCB increased the
fluorescence of the intracellular fluorochrome (Fig. 2, lanes 1 and 2). This result
confirmed the previous report that DNCB treatment produced endogenous ROS
in Jurkat T cells (35). Pretreatment of the cells with NAC inhibited basal level of
fluorescence (lanes 1 and 3) and DNCB-induced increase of fluorescence (lanes

2 and 4), indicating that NAC functioned as antioxidants.

110

A B

 

 

 

DNCB - - + + DNCB - - +
Anti-CD3/CDZB - + + - H202 + , +
I p-JNK
"T" ‘1 .. w +JNK1
7". 7'" """’ " ~— JNK1
.. 7". M p-p38 if“ p-p38
“* " ‘"ERK1
Cup-— ERK2
" '” “" " ‘ ERK2 1 2 3

Figure 1. DNCB enhances TCR- and H202-induced MAPK phosphorylation.
(A) Cells were incubated with 30 mM DNCB for 1 hr and were subsequently

stimulated with anti-CD3 (1:100) and anti-CD28 (1:100) for 30 min. (B) Cells

were incubated with 30 mM DNCB for 1 hr and were subsequently stimulated
with 200 mM H202 for 30 min. After blotting with phospho-MAPK antibodies, the
membranes were reprobed with ERK2 antibody to show that equivalent amount
of protein was present in each lane. The blots were the representative from

three independent experiments.

111

NAC - - + +
DNCB - + - +

 

 

 

Mean fluorescence
(fold increase)
0 _. re w a a:

Figure 2. NAC inhibits DNCB-induced intracellular ROS production. Cells
were stimulated with 30 mM DNCB and incubated with 5 mM CMHzDCF-DA for 1
hr. The fluorescence of the unstimulated cells was set 1 and the fluorescence
from DNCB-treated cells was represented as fold increase with standard
deviation after four independent experiments. *, p < 0.05 versus value obtained

from cells stimulated by DNCB alone.

112

NAC inhibits DNCB-induced JNK and p38 phosphorylation.
Because we observed that NAC inhibited DNCB-induced ROS production, we
checked the effect of ROS on DNCB-induced MAPK phosphorylation by
pretreatment of cells with NAC. As shown in Fig. 3A, NAC inhibited DNCB-
induced JNK and p38 phosphorylation. NAC pretreatment did not inhibit PMA-
induced ERK phosphorylation (data not shown), indicating that application of
NAC specifically affected JNK and p38. This result suggested that ROS
production is required for DNCB-induced JNK and p38 phosphorylation.

Src-family kinase inhibitor, PP2, inhibits DNCB-induced JNK and p38
phosphorylation. We analyzed the signaling pathways leading to MAPK
phosphorylation by DNCB with the application of the reagents which specifically
inhibit signaling enzymes. Src family tyrosine kinases such as Lck and Fyn are
associated with cytoplasmic portions of TCR components and their activation is
the first step in TCR activation [48]. To determine whether the activity of the
kinases is required for DNCB-induced JNK and p38 phosphorylation, the Src-
family kinase inhibitor, PP2, was pretreated before DNCB stimulation. PP2
inhibited DNCB-induced JNK and p38 phosphorylation (Fig. 3B). PP2 treatment
did not make cells nonfunctional because the same concentration of PP2 did not
inhibit PMA-induced ERK phosphorylation (data not shown), as previously
reported (36). This result suggested that Lck (and Fyn) activation is required for
DNCB-induced JNK and p38 phosphorylation.

The PLC inhibitor (U73122) and the PKC inhibitor (Ro-31-8425) do not

affect DNCB-induced JNK and p38 phosphorylation. Activation of PLCy by

113

 

C

U73122 - - + -

Ro31-8425 - - - +

DNCB - + + +
m h h " p_JNK

. . ”a“ m.+JNK1
«II- ” I". ”I! N“

MN m p38
1 2 3 4

 

Figure 3. PP2, the Src-family kinase inhibitor, and NAC inhibit DNCB-
induced JNK and p38 phosphorylation. (A) Cells were pretreated with 20 mM
PP2 for 2 hrs and subsequently incubated with 30 mM DNCB for 1 hr. (B) Cells
were pretreated with 20 mM NAC for 30 min and subsequently stimulated with 30
mM DNCB. (C) Cells were pretreated with 4 mM U73122 or with 1 mM Ro31-
8425 for 10 min and stimulated with 30 mM DNCB for 1 hr. After blotting with
phospho-MAPK antibodies, the membranes were reprobed with ERK2 antibody
to show that equivalent amount of protein was present in each lane. The blots

are the representative of three independent experiments.

114

tyrosine phosphorylation and subsequent activation of PKC by diacylglycerol is
an important event in T cell activation. To determine the involvement of these
enzymes in DNCB-induced JNK and p38 phosphorylation, cells were treated with
the PLC inhibitor, U73122, or with the PKC inhibitor, Ro—31-8425, before
stimulation with DNCB. Both inhibitors did not inhibit DNCB-induced JNK and
p38 phosphorylation (Fig. 3C).

NAC inhibits DNCB-induced Lck kinase activity. Because we
observed that PP2 inhibited DNCB-induced JNK and p38 phosphorylation, we
checked whether Lck is activated when cells are stimulated with DNCB. DNCB
treatment increased Lck kinase activity, judged by autophosphorylation of the
enzyme (Fig. 4, lanes 1 and 2). NAC pretreatment inhibited the induction of Lck
kinase activity by DNCB (Fig. 4, lanes 2 and 3), suggesting that ROS production
was involved in the activation of Lck.

DNCB or H202 treatment of Jurkat T cells reversibly inhibits PTP
activity of CD45. Because it has been reported that the direct addition of
oxidants did not increase Lck activity (37), we measured the PTP activity of
CD45 that regulated Lck activation. CD45 PTP activity, measured in specific
immunoprecipitates after treatment of Jurkat cells with DNCB or H202, was found
to decrease by about 40% (Fig. 5A, lanes 2 and 3). The addition of 5 mM DTT in
lysis buffer did not significantly affect the basal CD45 activity (Fig. 5A, lanes 1
and 4). However, a considerable portion of CD45 activity inhibited by DNCB or
by H202 was reversed by 5 mM DTT (Fig. 5A, lanes 2 and 5 and lanes 3 and 6).

This result suggested that inhibition of cellular CD45 activity by DNCB was

115

NAC - - + +
DNCB - . +7 . + . .. -
1 2 3 4

Figure 4. NAC inhibits DNCB-induced Lck activation. Cells were stimulated
with 30 mM DNCB for 1 hr. Lck was immunoprecipitated and
autophosphorylation activity in the immune complex was assayed by

incorporation of [g-32P] ATP. The radiograph is the representative of three

independent experiments.

116

Figure 5. DNCB reversibly inhibits CD45 activity in Jurkat cells. (A) Cells
were stimulated with 30 mM DNCB or with 300 mM H202 for 1 hr. 5 mM DTT
was included in lysis buffer at the indicated. PTP activity of CD45
immunoprecipitates was measured and average value was represented with
standard deviation after three independent experiments. *, p < 0.05 versus CD45
activity without treatment (lane 1). (B) Cells were incubated with 20 mM NAC for
30 min and subsequently stimulated with 30 mM DNCB for 1hr. PTP activity of
CD45 immunoprecipitates was measured and average value was represented
with standard deviation after three independent experiments. *, p < 0.05 versus
CD45 activity without treatment (lane 1). (C) Recombinant CD45 (1 mg) was
incubated with 100 mM DNCB or with 100 mM H202 for 1 hr. The PTP activity
without treatment was set 100% and the activity with DNCB or H202 was
represented as % activity with standard deviation after four independent

experiments. **, p < 0.05 versus CD45 activity without treatment (lane 1).

117

A DNCB

H202

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+ I
. *
. *
I'IJI.JIIJ|¢J.|I.
anlv 8 6 4. 2 0
m xanoo ocsEE_
..w 3.8 s 3258 at
D

 

 

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B NAC

B
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xanoo ocaEE
300 s 23:8 at".

118

mediated by reversible modification of a thiol group by ROS. NAC pretreatment
prevented the decrease of CD45 activity by DNCB (Fig. 53), further supporting
the notion that DNCB-induced inhibition of cellular CD45 activity was mediated
by ROS. The possibility that DNCB inhibited CD45 activity by direct alkylation
was excluded by the observation that the addition of DNCB to recombinant CD45
did not affect PTP activity (Fig. 5C, lanes 1 and 3). H202 effectively inhibited
recombinant CD45 activity (Fig. 5C, lanes 1 and 2), indicating that ROS directly
inhibited CD45 activity. These results suggest that the inhibition of CD45 activity
by DNCB-induced ROS production mediates the immuno-stimulatory effect of
DNCB (Fig. 6).

H202 inhibits the modification of the active site cysteine residue of
recombinant CD45 by C‘a-iodoacetic acid. Because we observed that H202
directly inhibited CD45 activity (Fig. 50), we tried to examine the mechanism how
ROS inhibit CD45 activity. Since it has been suggested that the active site
cysteine of PTPs is a peculiar target of ROS because of the low pKa, we
determined to look at the status of the active site cysteine residue (CysB17) of
CD45. lodoacetic acid reacts with the reduced sulfhydryl group (-SH), but not
with the oxidized forms such as sulfenic (-SOH), sulfinic (-SOzH), or sulfonic (-
SOaH) acids. Therefore, we used the reactivity with iodoacetic acid as the
indicator of oxidation. After incubation of CD45 with and without H202, the
protein was subject to modification by C‘3-iodoacetic acid. The solution was
subsequently incubated with D'l'l' to reverse the oxidized residues. The protein

was purified by SOS-PAGE electrophoresis and the gel piece containing CD45

119

CD45 DNCB»

TLck R\‘/
/Ter
/1 /R08
/ RfS targets \/

ERK
JNK p38

favors Th1 differentiation

Figure 6. Inhibition of CD45 by ROS regulates DNCB-induced activation of
distinct MAPK pathways. Arrows indicate activation pathways and blocked

arrows indicate inhibition.

120

Figure 7. H202 inhibits the modification of the active site cysteine residue
of recombinant CD45 by C13-iodoacetic acid. Recombinant CD45 (10 pg)
was incubated with or without 1 mM H202 for 30 min. After incubation with C13-
iodoacetic acid, the protein was reduced by DTT and was subsequently purified
by SOS-PAGE electrophoresis. The protein in the gel was subject to alkylation
with C12-iodoacetic acid and was digested by trypsin. The peptide fragment was
analyzed by MALDl-MS. The results shown are representative of two

independent experiments.

121

 

 

PTP active site tryptic peptide
VNAFSNFFSGPIWHCSAGVGR 2325.9

2325 9

283.8

2299.9

2225.8

2316.9

 

 

 

 

+ H202

2284.7
2300.8

$23234

2225.7

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2316.8
2323

 

122

underwent the second modification by C‘Z-iodoacetic acid. CD45 was digested
by trypsin and the masses of the resultant peptide fragments were analyzed by
MALDl-MS. The mass of the peptide fragment containing Cys817 from H202-
treated sample was about 2 Da lower than the mass of the peptide fragment from
untreated sample (Fig. 7B). This mass difference corresponded to the mass
difference between C‘3-iodoacetic acid and C‘z-iodoacetic acid. The result
indicated that the first modification by c‘3-icdcacetic acid was inhibited by H202.
After treatment of DTI', the unmodified cysteine residues from H202-treated
sample became more available to the second modification by C‘z-iodoacetic
acid. The relative intensity of the peak of the peptide containing Cy3817 from
H202-treated sample was lower than that from untreated sample. This result
indicated that some of the cysteine residues oxidized by H202 underwent the

irreversible oxidation into sulfinic or sulfonic acids, which were not reduced by

DTT.

123

DISCUSSION

It has been reported that DNCB-induced contact sensitivity is correlated
with NADPH—dependent oxygen consumption and elevated glutathione disulfide
(GSSG) level in mouse skin (7). Because it has been demonstrated that DNCB
inhibits Ter and depletes GSH inside the cell, the DTH reaction invoked by
DNCB may linked to its pro-oxidant properties. As T cells are important
mediators of DNCB-induced hypersensitivity and chemotherapy (5), the
relevance between the cellular redox alteration induced by DNCB and the
immunostimulation of T cells was investigated.

DNCB treatment of Jurkat cells induced JNK and p38 phosphorylation and
potentiated TCR-induced JNK and p38 phosphorylation (Fig 1A). In accordance
with the report that a mild oxidative shift enhances T cell signaling events
including JNK and p38 activation (38), this result suggests that the redox change
induced by DNCB may be involved in the modulation of T cell activation process.
The report that JNK2 and p38 activation stimulates CD4+ T cell differentiation
into Th1 cells (39) is consistent with the observations that DNCB treatment
promotes Th1-type cytokine expression (5, 9). Therefore, our result suggests
that direct stimulation of T cells by DNCB contributes to the differentiation into
Th1 type by selectively augmenting TCR-induced JNK and p38 activation.
Interestingly, DNCB was a poor stimulator of ERK phosphorylation and did not
enhance TCR-induced ERK phosphorylation. The enhancement of JNK and p38
phosphorylation without affecting ERK phosphorylation is a characteristic of

CD28 costimulation and lL-7 treatment (40-42). It will be interesting to check

124

whether the alteration of redox potential is involved in the activation of MAPKs
by those stimuli. It has been previously reported that 0028 activates NF-KB by
ROS-dependent signaling pathway involving 5-lipoxygenase activation (43).

The Src—family kinase inhibitor, PP2, decreased DNCB-induced JNK and
p38 phosphorylation (Fig. 3). This result suggests that DNCB-induced Lck (and
Fyn) activation is required for the downstream MAPK activation. In contrast with
PP2, the PLC inhibitor (U73122) or the PKC inhibitor (Ro-31-8425) did not affect
DNCB-induced JNK and p38 phosphorylation. The functionality of these
inhibitors was confirmed by the observation that the same concentration of
inhibitors abrogated H202-induced ERK phosphorylation (28). H202-induced JNK
and p38 phosphorylation, like DNCB, was not affected by PLC and PKC
inhibitors (28). In addition, it has been reported that chelerythrine-induced JNK
and p38 activation was inhibited by antioxidants but not by PKC inhibitors (44).
These observations suggest that PLC and PKC activity are not required for ROS-
induced JNK and p38 activation and support the specificity of ROS-induced
signaling pathway.

Stimulation of Jurkat cells with DNCB resulted in the activation of Lck (Fig.
4). NAC pretreatment inhibited DNCB-induced Lck activation, suggesting that
ROS production was involved. It is not likely that ROS produced by DNCB
directly activate Lck because the addition of oxidants to purified Lck did not
increase kinase activity (37). Therefore, ROS may indirectly regulate Lck activity
by affecting PTPs that control the phosphorylation of Lck. It has been known that

phosphorylation of Tyr394 in the activation loop of Lck induces kinase activity

125

(45), whereas phosphorylation of Tyr505 in the C—terminal domain inhibits the
activity (46). It has been reported that CD45 regultes Lck activity by
dephosphorylating both residues of Lck. Although the model that CD45 activates
Lck by dephosphorylating Tyr505 in T cell activation has been upheld, it has
been reported that Lck may be hyperphosphorylated at Tyr 394 in some CD45
deficient T cells (47, 48). H202 treatment of Jurkat cells inhibited CD45 activity
and led to the hyperphosphorylation of both residues of Lck (49). lmportantly,
Lck kinase activity is actually increased in cells stimulated by H202 or in cells
deficient in CD45 (47). These results suggest that the activating effect of Tyr394
phosphorylation is dominant over the inhibitory effect of Tyr505 phosphorylation
when both residues are phosphorylated. Therefore, the net effect of CD45
inhibition can lead to the activation of Lck (50). The importance of the inhibition
of Lck activity by CD45 has been shown in the report that mice expressing
constitutively active Lck by mutating Tyr505 into Phe develop thymic lymphoma
on a CD45-l- background (51).

CD45 is the prototypical PTP that is most abundantly expressed in T
cells. Because it has been suggested that the catalytic cysteine residues of
PTPs are susceptible to oxidation, the effect of DNCB-induced ROS production
on PTP activity of CD45 was investigated. As shown in Fig. 5A, the exposure of
Jurkat cells to DNCB or to H202 resulted in the substantial inhibition of CD45
activity immunoprecipitated from the cells. When 5 mM 011' was included in the
lysis buffer, significant portion of CD45 activity that was inhibited by DNCB and

H202 was recovered. This result suggests that both the extracellular addition of

126

ROS and the intracellular ROS production by DNCB reversibly inhibit CD45
activity. Pretreatment of cells with NAC before DNCB stimulation also prevented
the inhibition of CD45 activity, further indicating the involvement of ROS for the
process. In contrast with H202, DNCB did not directly inhibit recombinant CD45
activity. The result excluded the possibility that DNCB inhibited CD45 activity by
direct modification. If intracellular environment somehow induces the
modification of CD45 by DNCB without involving ROS, the inhibited portion of
CD45 activity as such may not be restored by DTT because Ter inhibition by
DNCB-mediated alkylation is irreversible (8).

Although it has been suggested that ROS inhibit PTP activity by oxidizing
the catalytic cysteine residue of the active site, only a few PTPases that are small
in size have been shown to contain redox-sensitive catalytic cysteine residues. It
has not been amenable to demonstrate the principle in the case of receptor-like
PTPs such as CD45 which contain the tandem PTPase domains. As a way to
elucidate the mechanism of PTP inhibition by ROS, we developed the method to
detect the status of the active site cysteine residue of CD45 by sequential
modification with differential iodoacetic acids by mass. H202 treatment inhibited
the first modification of the active site cysteine residue by C‘3-iodoacetic acid
because the oxidized cysteine residue was resistant to the alkylation. However,
the active site cysteine residue from CD45 sample treated with H202 became
modified by the second modification with C‘z-iodoacetic acid because the
oxidized cysteine residue was reduced by DTT before C‘z-iodoacetic acid

labeling. The lower peak intensity of the peptide fragment containing Cys817

127

from H202-treated sample compared to that from untreated sample suggested
that not all the oxidized cysteines were reduced by DTT. This result indicated
that the catalytic cysteine residue of CD45 was indeed oxidized by H202 into both
reversible and irreversible state. .

NAC inhibited DNCB-induced ROS production, CD45 inhibition, Lck
activation, and JNK and p38 phosphorylation. The correlative sensitivity of those
signaling events to NAC indicates that they are intimately connected processes.
NAC is a precursor of GSH that is the most abundant intracellular thiols and has
the capacity to directly scavenge ROS. It has been known that NAC and other
antioxidants inhibit JNK and p38 activation induced by other alkylating agents
and chemotherapeutic compounds. Therefore, JNK and p38 activation induced
by the alteration of the redox status is the important process for those reagents
including DNCB to induce appropriate cellular signaling.

Taken together, these results show that CD45 activity is reversibly
regulated by ROS in T cells and suggests a role for ROS for the subsequent
activation of Lck and differential regulation of MAPKs in DNCB-induced
immunostimulation (Fig. 6). The change in redox state by DNCB affected T cell
activation by specific activation of the JNK and p38 MAPK signaling pathways.
Since JNK and p38 activation has been associated with induction of Th1 cells
(39), our results provide the potential molecular mechanism how DNCB promotes
Th1 differentiation. With the use of differential iodoacetic acid, the catalytic
cysteine residue of CD45 was probed as the target of ROS. Since it has been

documented that TCR triggering produces endogenous ROS (54), it will be

128

interesting to determine whether PTP activity is regulated by redox status during

T cell activation.

129

10.

11.

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134

CHAPTER FIVE

CURCUMIN INHIBITS STAT1 AND STAT3 PHOSPHORYLATION AND C-FOS

EXPRESSION IN JURKAT T LYMPHOCYTES

135

ABSTRACT
Curcumin and resveratrol are polyphenolic compounds present in dietary
products that show anti-inflammatory, anti-neoplastic and antioxidant properties.
However, the association of antioxidant properties of the polyphenols with their
clinical effects has not been convincingly established. To understand the
relationship between anti-inflammatory effects and antioxidant properties of
curcumin and resveratrol, we compared the effect of the compounds on signaling
events in Jurkat T cells stimulated by interferon-or (IFN-or) and Concanavalin A
(Con A). The antioxidant properties of curcumin and resveratrol were manifested
by the inhibition of Con A-induced Reactive Oxygen Species (ROS) production.
Curcumin, but not resveratrol, inhibited phosphorylation of signal transducer and
activator of transcription 1 (STAT1) and STAT3 induced by lFN-a and Con A.
This result suggests that the effect of curcumin on STATs is mediated by its non-
antioxidant properties. Both curcumin and resveratrol inhibited Con A-induced
IL-2 mRNA expression, indicating that antioxidant properties of the compounds
also contribute to their anti-inflammatory effects. Although curcumin and
resveratrol comparably inhibited Con A-induced c-Jun NHz-terminal kinase (JNK)
phosphorylation and c-jun promoter activation, curcumin displayed more
profound inhibition of AP-1 activity. The differential sensitivity of AP-1 was
attributed to the finding that curcumin, but not resveratrol, ablated Con A-induced
c-Fos expression. Con A-induced Elk-1 activity and CREB phosphorylation were
not affected by either curcumin or resveratrol, suggesting that non-antioxidant

properties of curcumin inhibited c-Fos expression by blocking STAT1 and STAT3

136

activation. Taken together, these results demonstrate that non-antioxidant and
antioxidant properties of curcumin and resveratrol contribute to their overlapping

but distinct anti-inflammatory effects.

137

INTRODUCTION

Recently there has been a renewed level of interest in the anti-
inflammatory properties of naturally occurring polyphenols such as curcumin and
resveratrol [1]. The anti-inflammatory activity of this class of compounds has
been known for many years and recent studies support their beneficial effects in
clinical models of inflammation and cancer [2, 3]. In accordance with their clinical
effects, polyphenolic compounds have been reported to modulate a broad
spectrum of signaling events in a variety of types of cells [1, 4]. Many of the
polyphenols, including curcumin and resveratrol, have been demonstrated to
have antioxidant properties [5, 6]. Accordingly, it has been suggested that the
effect of the compounds is mediated by the regulation of redox-sensitive
signaling pathways [4]. However, the association of antioxidant properties of
polyphenols with their clinical effects has not been convincingly established [3].
Therefore, it is important to address the question whether all the effects of the
polyphenolic compounds are due to their antioxidant properties.

Curcumin is a dietary yellow pigment from the rhizome of Curcuma longa
L. . Curcumin displays anti-tumor and anti-inflammatory properties in models of
tumorigenesis. atherosclerosis, arthritis, Alzheimer’s disease and multiple
sclerosis [7-11]. The antioxidant properties of curcumin have been described in
the reports showing that curcumin inhibits the activities of lipoxygenase,
cyclooxygenase and xanthine oxidase [12-14]. With regard to immune cells,
curcumin inhibited superoxide production of macrophages stimulated with

phorbol myristate acetate (PMA) [15] and inhibited the response of blood

138

mononuclear cells to phytohemagglutinin (PHA) and mixed lymphocyte reactions
[16]. Recently it has been reported that curcumin inhibits the activation of signal
transducers and activators of transcription (STATs) in cells stimulated by
oncostatin M and interleukin (lL)-12 [11, 17].

Resveratrol is a polyphenolic compound naturally present in high amounts
in the skin of red grapes. Resveratrol has been regarded as a factor involved in
the “French Paradox”, in which it has been observed that moderate consumption
of red wine is associated with the low incidence of coronary heart disease [18].
The antioxidant properties of resveratrol have been mechanistically related to its
beneficial effect on atherosclerosis by research showing that resveratrol inhibits
the oxidation of low-density lipoprotein (LDL) particles, vascular reduced
nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, lipoxygenase
and cyclooxygenase [19-21]. The anti-neoplastic effect of resveratrol has also
been associated with its antioxidant properties [22]. Incubation of U937
lymphoma cells with resveratrol decreased tumor necrosis factor (T NF)-a-
induced ROS production and nuclear factor (NF)-KB activation [23]. Recently, it
has been shown that resveratrol inhibits Con A-induced lL-2 gene transcription
and NF-KB activation in splenic T cells [24].

In order to understand the relationship between anti-inflammatory effects
and antioxidant properties of curcumin and resveratrol, we compared the effect of
the compounds on signaling events in Jurkat T cells stimulated by IFN-a and Con

A. Examination of the signaling effects of the two related antioxidant polyphenols

identified the signaling pathways sensitive to both compounds as well as the

139

pathways that are differentially regulated. The comparison of the cellular effects
between curcumin and resveratrol led us to the conclusion that curcumin acts in
a non-antioxidant manner in suppressing STAT1 and STAT3 activation and

subsequent c-Fos expression.

140

MATERIALS AND METHODS

Reagents and Antibodies Curcumin (1 ,7-bis(4-hydroxy-3-
methoxyphenyl)-1,6-heptadiene-3,5-dione) and resveratrol (5-[(1E)-2-(4-
hydroxyphenyl)ethenyl]-1,3-benzenediol 3,4',5-trihydroxy-trans-stilbene) were
purchased from Sigma (St. Louis, MO). Curcumin and resveratrol were
dissolved in dimethyl sulfoxide (DMSO) freshly prior to use. Human recombinant
lFN-aA was obtained from Calbiochem (San Diego, CA). Con A was obtained
from Amersham (Piscataway, NJ). Antibodies used in this study were obtained
as follows: p-STAT1 (T yr701), p-STAT3 (T yr705), p-ERK1/2 (Thr202 and
Tyr204), STAT1, STAT3 and p38 (Cell Signaling, Beverly, MA); p-p38 (Thr180
and Tyr182) and p-JNK1I2 (Thr183 and Tyr185) (Promega, Madison, WI); 1p-
CREB (Ser133) (Upstate, Charlottesville, VA). ERK2 and JNK1 (Santa Cruz
Biotechnology, Santa Cruz, CA); horseradish peroxidase-conjugated donkey
anti-rabbit antibody and sheep anti-mouse antibodies (Amersham); and
horseradish peroxidase-conjugated rabbit anti-goat antibody (Calbiochem).

Plasmids Luciferase reporter plasmids containing c-jun and c—fos
promoters were kindly provided by Dr. Y. Kawakami (La Jolla Institute, CA).
Luciferase reporter containing five Gal4 binding sites with E1B minimal promoter
and the expression vector for Gal4-Elk1 transactivation domain fusion were the
generous gifts from Dr. R. A. Maurer (Oregon health science Univ., OR). Dr. M.
Karin (University of California at Davis, CA) generously provided luciferase
reporter containing two AP-1 binding sites from collagenase-ct enhancer with rat

prolactin minimal promoter.

141

Cell culture Jurkat cells, clone E6-1, were grown in RPMI 1640 media
with 25 mM HEPES buffer (Biofluids, Rockville, MD) supplemented with 10%
heat-inactivated FBS (Life Technologies, Rockville, MD), 100 units/ml
streptomycin/penicillin and 2 mM glutamine (Life Technologies). Cells were
maintained in an exponential growth phase (0.2 — 1.0 x 106 cells/ml).

Measurement of R08 1 x 10‘5 cells were reconstituted in 1 ml serum-free
RPMI medium. After stimulation, cells were incubated with 5 tiM of 5-(and-6)-
chloromethyl-Z',7'-dichlorodihydro-fluorescein diacetate acetyl ester (CM-HzDCF-
DA) (Molecular Probes, Eugene, OR) was added. After 1 hr incubation with the
fluorochrome, fluorescence-activated cell sorter (FACS) analysis was conducted
at an excitation wavelength of 488 nm and emission at 520 nm.

Immunoblotting 1.5 x 106 cells were reconstituted in 1.5 ml serum-free
RPMI medium. After stimulation, cells were washed twice with ice-cold
phosphate-buffered saline (PBS) and then lysed in lysis buffer (Cell Signaling) for
30 min on ice. Lysates were pelleted by centrifugation at 8,000 x g at 4°C for 10
min and supematants were recovered. Proteins were separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were
transferred to nitrocellulose membrane. Blotting of the membrane was
performed as previously described [25].

Measurement of IL-2 mRNA 4 x 106 cells were reconstituted in 4 ml
RPMI medium containing 10% FBS. After stimulation of cells, RNA was
extracted using Trizol reagent (Life Technologies) and the amount of RNA was

quantified by spectrophotometer. The amount of lL-2 mRNA was measured by

142

Quantikine mRNA kit (R&D systems, Minneapolis, MN) according to
manufacturer’s instruction.

Transfection and luciferase assay The luciferase reporter vectors and
B-gal reporter plasmids were mixed with DMRIE-C reagent (Life Technologies) in
OPTl-MEM media (Life Technologies) at room temperature. After 40 min, 0.5 x
106 cells were added to the mixture. After 5 hrs of incubation at 37°C, 1 ml RPMI
medium containing 10% F BS was added. After 16 hrs, cells were stimulated and
lysed with lysis buffer (Promega). The luciferase activity was determined by
luciferase reagent (Promega) and was normalized by B-galactosidase activity
determined by the galactosidase reagent (BD Biosciences, Palo Alto, CA).

Statistical analysis Data are expressed as means with standard

deviation. Data were analyzed by Student’s t-test to determine the level of

significance (P < 0.05)

143

RESULTS

Curcumin and resveratrol inhibit Con A-induced ROS production.
We compared the antioxidant properties of curcumin and resveratrol to determine
whether they exhibit comparable antioxidant capacity. Intracellular ROS levels in
Jurkat T cells were measured by the use of the cell-permeable and ROS-
sensitive fluorochrome, CM-HZDCF-DA. lFN-a stimulation did not induce a
significant change in fluorescence (Fig. 1, lanes 1 and 2). In contrast, Con A
stimulation led to about a 3 fold increase in fluorescence (Fig. 1, lanes 1 and 3).
This result suggests that endogenous ROS were produced in T cells stimulated
by Con A but not lFN-or. The increase of fluorescence by Con A was significantly
inhibited by curcumin (lanes 3 and 4) and by resveratrol (lanes 3 and 5). This
result confirmed that curcumin and resveratrol have antioxidant properties. The
degree of inhibition exhibited by curcumin and resveratrol was not statistically
different (lanes 4 and 5), indicating that curcumin and resveratrol manifest the
comparable antioxidant capacity in the system examined in this study.

Curcumin but not resveratrol blocks lFN-a-induced STA T1 and
STAT3 phosphorylation. The activation of STATs is the primary signaling
event activated by interferon [26]. Binding of interferons to their cognate
receptors leads to the activation of Janus kinases (JAKs) associated with the
receptors. When STATs become phosphorylated at the conserved tyrosine
residue (Tyr701 in STAT1 and Tyr 705 in STAT3) by JAKs, they undergo
dimerization and subsequent translocation to the nucleus to bind cognate DNA

sequences. As a way to examine the effect of curcumin and resveratrol on the

144

res - - - - + - *-
cur - - - + - -
Con A - - + + + - -
IFth - - - - - -

 

Mean fluorescence
(fold increase)
to

 

 

 

Figure 1. Curcumin and resveratrol inhibit Con A-induced ROS production.
Cells were preincubated with 20 tiM curcumin (cur) or with 50 pM resveratrol
(res) for 30 min and were subsequently stimulated with 50 jig/ml Con A or with
105 units/ml lFN-ot. After 5 min of stimulation, 5 pM CM-HzDCF-DA was added.
After 1 hr, cells were analyzed by FACS at an excitation wavelength of 488 nm
and emission at 520 nm. The mean fluorescence of the sample in the first lane
was set as 1 and the fluorescence of the other samples were presented as fold
increase. The average value with standard deviation is presented (resveratrol: n
= 4, curcumin: n = 3, lFN-oc: n = 3). *, p < 0.05 vs. the value obtained from cells

from Con A stimulation alone.

145

IF N-ot-induced signaling pathway, we compared the effect of the compounds on
lFN-a-induced STAT1 and STAT3 phosphorylation. Treatment of Jurkat cells
with IF N-ci resulted in the robust phosphorylation of both STAT1 and STAT3 (Fig.
2A, lanes 1 and 2). Both isoforrns of STAT1 (STAT1a and STAT1B) and STAT3
(STAT3a and STATSB) were phosphorylated in proportion to their cellular
abundance. Curcumin blocked lFN-a-induced STAT1 and STAT3 phos-
phorylation (Fig. 2A, lanes 2 and 3). In contrast, resveratrol did not affect either
STAT1 or STAT3 phosphorylation (Fig. 2A, lanes 2 and 4). This result suggests
that the observed inhibitory effect of curcumin on STAT1 and STAT3

phosphorylation was not due to its antioxidant properties because a related

antioxidant, resveratrol, had no effect.

Curcumin but not resveratrol blocks Con A-induced STA not and
STA T38 phosphorylation. It has been known that STATs are activated not only
by interferons but also by cytokines and growth factors [27]. To determine
whether the inhibitory effect of curcumin on STAT1 and STAT3 phosphorylation
is stimulus-specific, Con A-induced STAT phosphorylation was examined. Con A
stimulation induced phosphorylation of STAT1or (91 kDa) without detectable
STAT1B phosphorylation (84 kDa) (Fig. 2B, p-STAT1 blot, lanes 1 and 2).
Because blotting with STAT1 antibody (Ab) showed that the amount of STAT1a
was much higher than STAT18, preferential phopshorylation of STAT1a may
have resulted from the low signal strength from Con A stimulation. Interestingly,
Con A-induced STAT3 phosphorylation involved only the STAT3B isofon'n (82

kDa) (Fig. 23, p-STAT3 blot, lanes 1 and 2), even though STAT3or (93 kDa) was

146

Figure 2. Curcumin, but not resveratrol, inhibits STAT1 and STAT3
phosphorylation induced by IFN-a and Con A. Cells were pretreated with 20
pM curcumin (cur) or with 50 pM resveratrol (res) for 30 min and subsequently
stimulated (A) with 5 x 104 units/ml lFN-a or (B) with 50 jig/ml Con A for 10 min.
After blotting with p-STAT1 and p-STAT3 Abs, the membranes were reprobed
with STAT1 and STAT3 Abs to show that equivalent amount of protein was

present in each lane. Blots are representative of three independent experiments.

(IB: immunoblot)

147

cur res re

p-STAT1
(Tyr701)

STAT1

 

+stat3a p—STAT3
{3131313 (T yr705)

“’7 ‘7; """'¢stat3a
p... a ””4 statab STAT3
1 2 3 4

   
  

res .3

i, . v7: ,i+stat1a p-STAT1
at» mi+fi811fi (Tyr701)

 
 

STAT1

4818131 p-STAT3
{-th38 (Tyr705)

~' ~ I I I +stat3a
.2...” ”awards STAT3
1 2 3 4

148

far more abundant as determined by a STAT3 Ab blot. Curcumin completely
inhibited Con A-induced STAT1a and STAT3B phosphorylation (Fig. 2B, lanes 2
and 3). However, a significant level of STAT1a and STATSB phosphorylation
was still detectable in the presence of resveratrol (Fig. 2B, lanes 2 and 4). The
selective effect of curcumin on Con A-induced STAT1ot and STAT3I3
phosphorylation further confirmed that the inhibition of STAT phosphorylation by
curcumin was mediated by its unique non-antioxidant properties.

Curcumin and resveratrol inhibit Con A-induced lL-2 mRNA
expression. lL-2 is the pivotal cytokine produced when T cells are activated. To
determine the effect of curcumin and resveratrol on the outcome of T cell
activation, the level of lL-2 mRNA was assessed. Stimulation of Jurkat T cells by
Con A increased lL-2 mRNA level (Fig. 3, lanes 1 and 3). IFN-or did not increase
lL-2 mRNA level (Fig. 3, lanes 1 and 2). Curcumin effectively blocked the
increase of IL-2 mRNA induced by Con A (Fig. 3, lanes 3 and 4). Resveratrol
also significantly decreased the amount of lL-2 mRNA induced by Con A (Fig. 3,
lanes 3 and 5). The inhibition of IL-2 mRNA expression by both curcumin and
resveratrol strongly supports the idea that endogenous ROS production has an
important role in Con A-induced IL-2 gene expression.

Curcumin and resveratrol selectively inhibit Con A-induced JNK
phosphorylation. To understand the mechanism how curcumin and resveratrol
inhibit Con A-induced IL-2 mRNA expression, the effect of curcumin and
resveratrol on Con A-induced signaling pathways was further compared. As

mitogen-activated protein kinases (MAPKs) are important mediators of the T cell

149

 

 

 

 

 

 

 

 

   

 

Con A - - + + +

IFth 3 - - - -
z (I
‘E 3’»
‘7‘ 9 1 -
=1 2
a

1 2 3 4 5

Figure 3. Curcumin and resveratrol inhibit lL-2 mRNA induction by Con A.
Cells were incubated with 20 pM curcumin (cur) or with 50 pM resveratrol (res)
for 30 min, followed by 25 jig/ml Con A stimulation for 4 hrs. The amount of lL-2
mRNA was measured by Quantikine mRNA kit. The value of the amount of lL-2
mRNA (attomole) divided by the amount of RNA extracted (pg) is presented with
standard deviation after three independent experiments. *; p < 0.05 vs. value

from cells stimulated with Con A alone.

150

activation process [28], the status of activation-associated phosphorylation of c-
Jun NHz-termianl kinase (JNK), p38 and extracellular signal-regulated kinase
(ERK) MAPKs was examined. Con A stimulation of Jurkat cells induced
phosphorylation of all three MAPK members, judged by western blotting with
phospho—specific antibodies (Fig. 4A and 4B, lanes 1 and 2). Pretreatment of
cells with curcumin or resveratrol partially inhibited JNK phosphorylation (Fig. 4A
and 4B, p-JNK blot, lanes 2 and 3). In contrast, the level of p38 and ERK
phosphorylation was not affected by either curcumin or resveratrol (Fig. 4A and
4B, p-p38 and p-ERK blot, lanes 2 and 3). The selective effect of both
antioxidants on JNK phosphorylation suggests that the signaling pathway leading
to JNK phosphorylation harbors a component regulated by endogenous ROS.
Curcumin and resveratrol comparably inhibit c-jun promoter
activation but difl'erentially inhibit AP-1 activation. The activation of JNK
leads to the phosphorylation and the activation of c-Jun, and one of the target
genes activated by c-Jun is the c-jun promoter itself [29]. Accordingly, c-jun
promoter activity was measured as the readout of JNK activation. Con A
stimulation increased the activity of luciferase reporters containing c-jun promoter
(c-jun-luc) (Fig. 5A, lanes 1 and 2). Pretreatment of the cells with either curcumin
or resveratrol significantly inhibited Con A-induced c-jun-luc activation (Fig. 5A,
lanes 2, 4 and 6). This result suggests that the inhibition of JNK activation by
curcumin and resveratrol leads to the decrease of c-jun promoter activation.
Since AP-1 is composed of Jun and Fos dimers, we next measured AP-1 activity

as the consequence of c-Jun expression. Con A stimulation increased the

151

Cur - - + + Res
CO“ A - + + - '3 CO” A
u. g, _, _ {-JNKZ
NK
* wing 4-JNK1
it.--” JNK1
...uu --m~i'r RK
m... m tERK; p-ERK
1 2 3 4
Figure 4. Curcumin and resveratrol

- - + +
_ + + _ IB
III-aut- J. ”- ““‘ ‘PJNKZ

p-JNK
---o -~- +JNK1
«It Hm m ............ JNK1
“a. p-p38
“ deli w 91"“ p38

an. ”H n~ ERK2
3

inhibit Con A-induced JNK

phosphorylation. Cells were pretreated (A) with 20 pM curcumin (cur) or (B)

with 50 pM resveratrol (res) for 30 min and subsequently stimulated with 25

jig/ml Con A for 1 hr. After blotting with phospho-specific MAPK Abs, blotting

with JNK1, p38, and ERK2 Abs showed that equivalent amount of protein was

present in each lane.

experiments. (IB: immunoblot)

152

The blots are representative of three independent

A CUI’ res

 

 

 

 

 

 

 

 

 

 

 

   

 

Con A 8 - + - + - +
2:2
:5 3 8.
g (u 3..
m g 4 " *
5 .s "
é '2
6 V
B 5 6
OUT res
Con A - + + +

8

 

MN
00!

AP-1-luc actiVIty

(fold increase)

 

 

 

 

000

Figure 5. Curcumin and resveratrol comparably inhibit Con A-induced c-
jun promoter activation but distinctly inhibit AP-1 activation. Cells were
transfected with (A) 0.5 pg luciferase vector containing c-jun promoter (c-jun-luc)
or (B) with 1 pg luciferase reporter containing two AP-1 binding sites (AP-1-luc).
After 16 hrs, cells were preincubated with 20 pM curcumin (cur) or with 50 pM
resveratrol (res) for 30 min and subsequently stimulated by 25 jig/ml Con A for 4
hrs. The normalized luciferase activity of the sample in lane 1 was set as 1 and
the activities of the other samples were represented as fold increase. The
average value with standard deviation is from three independent experiments. *;

p < 0.05 vs. activity from cells stimulated with Con A alone.

153

activity of luciferase reporters containing AP—1 binding sites (AP-1-luc) (Fig. 5B,
lanes 1 and 2). Surprisingly, there was a dramatic difference in the capacity of
curcumin and resveratrol in the inhibition of AP-1-luc. Curcumin almost
completely inhibited AP-1-luc (~ 80%) (Fig. 5B, lanes 2 and 3), while resveratrol
exhibited only modest inhibition (~ 20%) (lanes 2 and 4).

Curcumin but not resveratrol inhibits Con A-induced c-Fos
expression. To understand the differential sensitivity of AP-1-luc to curcumin
and resveratrol, we examined another member of AP-1 family, c-Fos. Con A
stimulation of Jurkat cells induced c-Fos protein expression (Fig. 6A, lanes 1 and
2). Of the two bands detected with anti-c-Fos Ab, the upper band most likely
represents the hyperphosphorylated form of the protein [30]. Curcumin
completely abrogated the Con A-induced c-Fos expression (Fig. 6A, lanes 2 and
3). In contrast, resveratrol did not affect Con A-induced c-Fos expression (Fig.
6A, lanes 2 and 4). The observation that curcumin but not resveratrol inhibited
Con A-induced c-Fos expression indicates that the inhibitiory effect of curcumin
on c-Fos expression is mediated by its non-antioxidant properties.

To determine whether the inhibitory effect of curcumin on c-Fos
expression is exerted at the level of c-fos gene expression, we measured the
activity of luciferase reporters containing c-fos promoter (c-fos-luc). Curcumin
inhibited Con A-induced c-fos-luc activation by 50 % (Fig. 6B, lanes 2 and 3),
while resveratrol showed no effect (lanes 2 and 4). This result indicates that c-
fos promoter inhibition by curcumin is responsible for the decrease in c-Fos

expression.

154

 

 

 

 

104

. fl .

 

c-fos-luc activity
(fold increase)
I-

 

 

 

 

 

 

 

 

 

Figure 6. Curcumin but not resveratrol inhibits Con A-induced c-Fos
expression. (A) Cells were pretreated with 20 pM curcumin (cur) or with 50 pM
resveratrol (res) for 30 min and subsequently stimulated with 25 jig/ml Con A for
1 hr. The blot is the representative of three independent experiments. (IB:
immunoblot) (B) Cells were transfected with 0.5 pg luciferase vector containing c-
fos promoter (c-fos-luc). After 16 hrs, cells were preincubated with 20 pM
curcumin or with 50 pM resveratrol for 30 min and subsequently stimulated by 25
pg/ml Con A for 2 hrs. The normalized luciferase activity of the sample in lane 1
was set as 1 and the activities of the other samples were represented as fold
increase. The average value with standard deviation is from three independent

experiments. *, p < 0.05 vs. activity from cells stimulated with Con A alone.

155

Curcumin and resveratrol do not inhibit Con A-induced Elk-1 activity
and CREB phosphorylation. The transcriptional induction of c-fos is known to
be regulated by the following factors [reviewed in [31]]: 1) serum response
element (SRE) recognized by serum response factor (SRF) and a ternary
complex factor such as Elk-1; 2) cyclic adenosine monophsophate response
element (CRE) recognized by CRE-binding protein (CREB); and 3) sis-inducible
element (SIE) recognized by STAT proteins. Accordingly, we determined
whether the effect of curcumin on c-Fos expression could be attributed to the
activation status of transcription factors other than STAT1 and 3. The
transcriptional activity of Elk-1 is potentiated when MAPKs phosphorylate Elk-1 in
the transactivation domain [32]. The level of Elk-1 phosphorylation was
assessed by an assay in which a GaI4-Elk1 transactivation domain fusion protein
was co-expressed with a Gal4-dependent luciferase reporter (designated as
Gal4-luc). In this system, GaI4-luc activity increases when the Gal4-Elk1 fusion
protein is activated by phosphorylation of the Elk-1 transactivation domain [33].
As shown in Fig. 7A, Con A stimulation led to about 20 fold increase of Gal4-luc
activity (lanes 1 and 2). Pretreatment of the cells with either curcumin or
resveratrol did not inhibit Con A-induced Gal4-luc activity (Fig. 7A, lanes 2, 3 and
4). This result suggests that Elk-1 phosphorylation was intact in the presence of
the compounds.

It has been known that CREB phosphorylation at Ser133 residue by
protein kinase A (PKA), calcium/calmodulin-dependent protein kinase (CaMK) or

MAPK—dependent kinases induces transcriptional activity of the protein [34]. The

156

Figure 7. Curcumin and resveratrol do not inhibit Con A-induced Elk-1
activity or CREB phosphorylation. (A) Cells were transfected with 0.25 pg
luciferase reporter containing Gal4 binding sites (Gal4-luc) plus 0.25 pg Gal4-Elk-
1 expression vector. After 16 hrs, cells were preincubated with 20 pM curcumin
(cur) or with 50 pM resveratrol (res) for 30 min and subsequently stimulated by
25 pg/ml Con A for 2 hrs. The normalized luciferase activity of the sample in lane
1 was set as 1 and the activities of the other samples were represented as fold
increase. The average value with standard deviation is presented from three
independent experiments. (B) Cells were pretreated with 20 pM curcumin or with
50 pM resveratrol for 30 min and subsequently stimulated with 25 pg/ml Con A
for 1 hr. After blotting with p-CREB Ab, the membrane was reprobed with CREB
Ab to show the equal amount of the protein in each lane. Blots are

representative of three independent experiments. (IB: immunoblot)

157

>

O
O
3

Elk-1 activity
(Gal4-Iuc:

foldincrease)
o a. a a: s s 83>

I
+

CUT res
+ +

 

 

 

  

 

 

   

3 4

CUT res
+ + + _____|B

m M W p-CREB (Ser133)

 

158

level of CREB phosphorylation was determined by blotting with phospho—CREB
antibodies. As shown in Fig. 73, Con A stimulation induced CREB
phosphorylation (lanes 1 and 2). Treatment of cells with either curcumin or
resveratrol did not affect Con A-induoed CREB phosphorylation (Fig. 7B, lanes 2,
3, and 4). This result suggests that the CRE site in the c-fos promoter is not the
mediator of the inhibitory effect of curcumin. The observation that curcumin did
not inhibit Elk-1 and CREB phosphorylation strongly suggests that curcumin
inhibited Con A-induced c-Fos expression (Fig. 6) by inhibiting STAT1 and
STAT3 activation (Fig. 23).

159

DISCUSSION

In this report, we have studied the effect of curcumin and resveratrol on IL-
2 and c-Fos promoters to understand how these agents affect complex
inflammatory signal integration. It has been known that the signaling pathways
leading to the activation of AP-1 and NF-KB transcription factors contain redox-
sensitive components [35]. Accordingly, antioxidant polyphenols including
curcumin and resveratrol have been reported to inhibit AP-1 and NF-xB
activation in a wide range of stimuli [23, 36, 37]. However, the degree and the
mechanism of the effect exerted by these compounds show considerable
variation depending on the types of stimuli and on the cellular systems examined
(reviewed in [1]). More importantly, it has not been clearly demonstrated that all
the effects displayed by the polyphenols are due to their antioxidant properties.
As an approach to disentangle the complexity of their effect on the inflammatory
signaling, we have compared two related but distinct polyphenols (curcumin and
resveratrol) in Jurkat cells stimulated by lFN-a and Con A.

Stimulation of Jurkat cells with Con A, but not lFN-a, induced endogenous

ROS production (Fig. 1). This result demonstrates that ROS production is
regulated by specific stimulus. Both curcumin and resveratrol inhibited Con A-
induced ROS production (Fig. 1), suggesting that they have comparable
antioxidant capacities in the system examined in this study. Based on inhibitor
studies, NADPH oxidase and lipoxygenase have been implicated in ROS
production during T cell stimulation [38, 39]. Further support comes from a report

showing that 5-lipoxygenase was activated following T cell receptor (T CR)

160

stimulation [40] and from a report describing the abundant expression of Nox5, a
homologue of gp91p”°" NADPH oxidase component, in B and T lymphocytes [41].
Curcumin and resveratrol may inhibit the activity of these ROS-producing cellular
enzymes and/or may directly scavenge ROS (a known in vitro effect).

Curcumin inhibited lFN-a- and Con A-induced STAT1 and STAT3
phosphorylation (Fig. 2). The related antioxidant, resveratrol, had no effect on
either STAT1 or STAT3 phosphorylation. In addition, curcumin inhibited STAT1
and STAT3 phosphorylation induced both by Con A that produced ROS, and by
lFN-a that did not produce ROS (Fig. 1). These observations led us to the
conclusion that the inhibitory effect of curcumin on STATs is mediated by its non-
antioxidant properties. The mechanism of the inhibition of STAT phosphorylation
by curcumin is not clear. Curcumin may affect the activities of tyrosine kinases
that phosphorylate STATs [11] or the activities of protein tyrosine phosphatases
(PTPs) that dephosphorylate STATs. Alternatively, curcumin may affect protein-
protein interaction that is important for STAT phosphorylation [42]. An example
of a non-antioxidant effect of curcumin is the inhibition of sarco endoplasmic
reticulum Ca2*-ATPases (SERCA) [43]. Whether this and other non-antioxidant
properties of curcumin are linked to the inhibition of STAT phosphorylation will be
' investigated further.

Alternative splicing of STAT1 and STAT3 gene results in the shorter

isoforrns (STAT1B and STAT3B) that do not contain C-terrninal region of the
longer isoforrns (STAT1a and STAT3a) [44]. Although it has been suggested

that shorter isoforms of STAT1 and STAT3 may inhibit the transcriptional

161

activation of their respective longer isoforms, it has been shown that STAT1B and
STAT3|3 act as distinctive genetic regulators [45, 46]. Con A stimulation of Jurkat
cells led to the phosphorylation of STAT1a without STAT1B phosphorylation (Fig.
23). This result may be caused by the weak signal strength from Con A
stimulation, because the intensity of Con A-induced STAT phosphorylation was
much lower compared to that of IFN-a. In contrast, Con A stimulation induced
STATSB phosphorylation without STAT3a phosphorylation. This result is not

explained by the weak signal strength from Con A stimulation, because the

cellular abundance of STAT3a is much higher than STAT3B. Therefore, this
result suggests that there is a mechanism that distinctively regulates STAT3a
and STAT3B phosphorylation. It has been previously shown that expression and
phosphorylation of STAT3a and STAT3B is differentially regulated during
granulocyte differentiation [47]. Because STAT3B can be transcriptionally active
under conditions where STAT3a is not [46], phosphorylation of STAT3B without
STAT3a phosphorylation may lead to the activation of STATSB. The importance
of the transcriptional activity of STAT3B is supported by a report showing that
specific ablation of STATBB resulted in the impairment of c-fos promoter
activation without affecting STAT3a expression and phosphorylation [48].
Reactive oxygen species (ROS) not only participate in killing bacteria but
also play an active role in pro-inflammatory signaling [49]. The exposure of

immune cells to exogenous ROS at the inflammatory sites as well as

endogenous ROS produced by cellular activation contributes to the etiology of

162

various inflammatory conditions [50]. The inhibition of Con A-induced lL-2 mRNA
expression by curcumin and resveratrol (Fig. 3) is consistent with the previous
reports showing that the attenuation of the oxidative environment by antioxidants
inhibits T cell proliferation as well as the expression of lL-2 [51-53].
Consequently, the results in this study and others indicate that endogenous ROS
production as exemplified by Con A stimulation has an important role in cellular
activation.

It has been suggested that the distinct activation profile of MAPK
members contributes to the precise outcome of T cell activation [28]. While it has
been demonstrated that exogenous oxidants such as H202 activated JNK, p38,
and ERK MAPKs in Jurkat cells [54], it is not clear to what extent these kinases
are regulated by endogenous ROS production. In the context of Con A
stimulation, JNK may be the most sensitive target of endogenous ROS because
both curcumin and resveratrol inhibited JNK phosphorylation without affecting
p38 and ERK (Fig. 4). There are several candidate mediators for redox-
dependent activation of JNK. Apoptosis signal-regulating kinase 1 (ASK1) is a
MAPK kinase kinase (MAPKKK) that is activated by the dissociation of
thioredoxin (Trx) from the kinase. The dissociation is stimulated when Trx is
oxidized by exogenous oxidants as well as by endogenous ROS-producing
stimuli such as TNF-or [55]. Glutathione S-transferase (GST) binds to JNK to
inhibit its activation. The interaction of JNK with GST is disrupted by ultraviolet
(UV) irradiation or H202 treatment, conferring JNK activation [56]. In addition,

phosphatases which directly dephosphorylate JNK such as M3/6 [57] may be

163

regulated by oxidants because the phosphatases contain reactive cysteine
residues in their active sites.

The correlation of the inhibition of JNK phosphorylation (Fig. 4) and the
reduction of c-jun promoter activation and AP-1 activity (Fig. 5) by curcumin and
resveratrol suggests that the inhibition of transcriptional activity may be mediated
by the decrease of JNK activation. Because AP-1 is one of the critical
transcriptional regulators for lL-2 promoter activation [58], the inhibition of AP-1
activity by curcumin and resveratrol may be responsible for the inhibition of lL-2
mRNA expression (Fig. 3). Interestingly, the degree of inhibition of lL-2 mRNA
expression by resveratrol (~70 %) (Fig. 3) was more profound than the degree of
inhibition of AP-1 activity (~20 %) (Fig. 5B). This result supports the idea that
AP-1 has a cooperative effect with other transcription factors in lL-2 promoter
activation [58]. In addition, resveratrol has been shown to inhibit other
transcription factors important for lL-2 expression such as NF-xB [24]. The other
consideration is that inhibition of JNK pathway by resveratrol may cause a
negative effect on lL-2 mRNA stabilization [59].

The inhibition of c-Fos expression by curcumin (Fig. 6A) explains its more
robust inhibition of AP-1 activity compared to resveratrol (Fig. 5B). Inhibition of c-
Fos expression by curcumin was attributed to the reduction of c-fos promoter
activation (Fig. 68). It was interesting that inhibition of c-Fos expression by
curcumin was almost complete (Fig. 6A), although c-fos promoter activation was
downregulated by 50% (Fig. 68). This result suggests that posttranscriptional

and posttranslational regulation of c-Fos expression may also be affected by

164

curcumin [30, 60]. When we examined the activity of transcription factors
regulating c-fos promoter, it was observed that curcumin did not inhibit Elk-1 or
CREB phosphorylation (Fig. 7). Because it has been shown that the complex of
STAT1 and STAT3 binds to the SIE region of c-fos promoter [61, 62], the
inhibitory effect of curcumin on c-fos promoter activation may be mediated by the
impairment of STAT1 and STAT3 activation at the SIE region (although effects at
other promoter sites cannot be completely eliminated).

Taken together, the results in this study show that both antioxidant
properties and non-antioxidant properties of curcumin and resveratrol mediate
their overlapping but distinct effects in Jurkat T cells (Fig. 8). The concomitant
inhibition of ROS production, JNK activation, and lL-2 mRNA expression by both
curcumin and resveratrol provides the evidence that antioxidant properties of the
compounds contribute to their anti-inflammatory effects. On the other hand, the
observation that curcumin but not resveratrol inhibited STAT1 and STAT3
phosphorylation induced by lFN-a and Con A indicates that the inhibitory effect of
curcumin on STATs is mediated by its non-antioxidant properties. The inhibition
of Con A-induced STAT1 and STAT3 activation by curcumin resulted in the
downregulation of c-Fos expression, illustrating that non-antioxidant properties of
curcumin participate in its anti-inflammatory effect. Because constitutive
activation of STATs and c-Fos potentially leads to cellular transformation [63, 64],
the use of curcumin for the inhibition of STAT activation and c-Fos expression
may provide a promising modality for the amelioration of inflammatory and

neoplastic diseases.

165

curcumin

resveratrol
curcumin (antioxidant)
(non-antioxidant)
ROS
i- ‘JNigA
STAT1&3 ERK p38 c-Jun

l I \ \AP-1
P-STAT1&3 p-Elk-1 p-CREB H / l
I SIE SRE CRE | c-Fos lL-Z

 

Figure 8. Anti-inflammatory effects mediated by antioxidant and non-

antioxidant properties of curcumin and resveratrol. Arrows indicate

activation pathways, whereas blunted arrows indicate inhibition.

166

ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grant number Al42794

and by the Jean P. Schultz Endowed Oncology Research Fund.

167

10.

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