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dissertation entitled

REGULATION OF RECEPTOR ACTIVITY MODIFYING PROTEIN
EXPRESSION AS A NOVEL MECHANISM FOR MODULATION
OF ADRENOMEDULLIN ACTIVITY IN RAT MESANGIAL CELLS

presented by

WOJCIECH NOWAK

has been accepted towards fulfillment
of the requirements for

P . D
h degree in Physiology

 

 

 

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6/01 cJClRC/DatoDuopGS-DJ 5

REGULATION OF RECEPTOR ACTIVITY MODIFYING PROTEIN EXPRESSION
AS A NOVEL MECHANISM FOR MODULATION OF ADRENOMEDULLIN
ACTIVITY IN RAT MESANGIAL CELLS
By

Wojciech Nowak

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Department of Physiology

2003

ABSTRACT
REGULATION OF RECEPTOR ACTIVITY MODIFYING PROTEIN EXPRESSION
As A NOVEL MECHANISM FOR MODULATION OF ADRENOMEDULLIN
ACTIVITY IN RAT MESANGIAL CELLS
By

Wojciech Nowak

Adrenomedullin (AM), a recently discovered potent vasodilatory peptide, exhibits
myriad of physiologic effects by binding to a calcitonin-like receptor (CL receptor). CL
receptor is a member of a well-characterized group of G protein-coupled receptors
(GPCRs) and exhibits a remarkable capability of ligand specificity switching contingent
upon its association with specific member of a novel group of receptor activity modifying
proteins (RAMPS). When co-expressed and dimerized with RAMP-1, CL receptor
functions as a calcitonin gene-related peptide receptor (CGRPI). Conversely, interaction
with RAMP-2 or RAMP-3 renders it a fully functional AM receptor (AM; or AMz,
respectively). While the model of ligand-GPCR interaction is generally regarded as well
established, the discovery of RAMPs prompted a new look at the paradigm for receptor
phenotype determination. It also provided for a plethora of opportunities to recognize and
potentially exploit new mechanisms responsible for modulation of receptor activity.

This thesis focuses on the functional interplay between AM and its receptor
complex (CL receptor+RAMP-2/3). In particular, in the effort to identify the molecular
pathway(s) involved in influencing actions of AM, the herein described experiments
investigate the mechanisms and effects of extracellular signals on RAMP gene and
protein expression as well as the consequent alteration of AM activity. The experimental

model utilized is that of rat glomerular mesangial cells (MC) in culture. MC constitute a

Vital structural and functional part of the renal glomerulus and as such, aberration of their
normal biology has been shown to directly contribute to the genesis and progression of
several glomerulopathies. In addition, MC are the site of production and/or the target for
numerous cytokines, hormones, and other extracellular signals of which AM is one of the
more recently characterized. AM exhibits potent anti-proliferative and anti-migratory
effects on rat MC. These biological responses to AM are receptor mediated, hence
amenable to alterations involving any component of the AM receptor complex.

Data from this investigation show that upregulation of RAMP—3 gene and protein
expression results in the increase of AM-mediated cyclic AMP production and a
concomitant decrease in mesangial cell proliferation. Furthermore, the current study
identifies platelet-derived growth factor (PDGF) and lipopolysaccharide (LPS) as
extracellular factors increasing RAMP-3 mRNA expression and cell-membrane
associated RAMP-3 protein abundance. Both, PDGF and LPS, attain their effects Via
activation of mitogen-activated protein kinase signal transduction pathway(s). Moreover,
the effect of PDGF and LPS on RAMP-3 expression results from stabilization of the
RAMP-3 transcript, as observed by an increase in RAMP-3 mRNA half-life, and is
independent of new RNA synthesis.

In summary, this study demonstrates that differential expression of RAMP-3
represents a novel mechanism for regulation of mesangial cell’s responsiveness to AM. It
also identifies the process of RAMP-3 expression as a molecular target for cytokines
(PDGF) and other extracellular signals (LPS) known to be involved in the maintenance of

normal mesangial cell biology and/or its alteration in disease.

To my parents, who fostered my curiosity
and

my wife whose encouragement allows its growth.

iv

ACKNOWLEDGEMENTS

The work presented in this dissertation combines efforts of many individuals
whose help and support made it all possible.

I would like to extend my Sincere appreciation to the committee members: Drs.
Nambi Aiyar, Gregory Fink, Laura McCabe, Harvey Sparks, and William Spielman.
Their guidance and assistance continued to be Vital throughout all of the stages of this
project and my graduate education. To that extent, I would like to especially thank my
major advisor, Dr. William Spielman, for the mentoring and encouragement offered to
me at all times. I am also greatly appreciative of the hours spent by Drs. McCabe and
Fink sharing their invaluable thoughts on the project. I also would like to thank Dr.
Sparks for tremendous coaching he endowed me with during my graduate as well as
medical studies. I am equally deeply indebted to Dr. Aiyar for kindly arranging for my
Visit to SmithKline Beecham and training me in the methodology for assessment of
adenylate cyclase activity.

My day-to-day laboratory experiences were greatly enriched by the friendly
interactions with my colleagues: Carol Hall, Jennifer Bomberger, Dr. Kwan-Ki Huang
and Dr. Narayanan Parameswaran. I am particularly thankfiil for the immeasurable
technical support and knowledge base offered to me by Carol as well as the wealth of
ideas and scientific guidance provided by Nara.

I also extend a special thanks to the office and computer support staff of the

Physiology Department at Michigan State University for their helping hand.

TABLE OF CONTENTS

List of Tables ..................................................................................... vii
List of Figures .................................................................................... Vii
1. Introduction .................................................................................... 1
2. Literature review .............................................................................. 3
2.1. Mesangial cell ...................................................................... 3
2.1.1. General characteristics and cell functions ........................... 3
2.1.2. Mesagial cell in disease ................................................ 9
2.2. Adrenomedullin .................................................................... 10
2.2.1. Adrenomedullin gene ................................................... 10
2.2.2. Adrenomedullin protein structure ..................................... 11
2.2.3. Sites of Adrenomedullin gene and protein expression ............. 12
2.2.4. Signals responsible for Adrenomedullin secretion .................. 13
2.2.5. Biological actions of Adrenomedullin ................................ 17
2.2.6. Actions of Adrenomedullin in mesangial cells ...................... 22
2.2.7. Effects of Adrenomedullin gene alteration and somatic
Adrenomedullin gene delivery ......................................... 24
2.2.8. Actions of other preproadrenomedullin gene products ............. 29
2.3. Adrenomedullin receptor complex ................................................ 30
2.3.1 Adrenomedullin receptor system discovery ........................... 30
2.3.2. Other receptors that interact with RAMPS ............................ 35
2.3.3. Characteristics of RAMP gene and protein structure ................ 38
2.3.4. Tissue and cell specific RAMP expression and RAMP
subtype-CL receptor selectivity ........................................ 40
2.3.5. Mechanism of RAMP-receptor interaction ........................... 42
2.3.6. Regulation of RAMP gene expression ................................. 50

3. RAMP mRNA expression profile and the effects of RAMP over-expression on
AM—induced adenylate cyclase activity and [3H]thymidime incorporation in rat

mesangial cells ................................................................................... 53
3.1. Introduction ............................................................................ 53

3.2. Materials and methods ............................................................... 55
3.2.1. Materials .................................................................... 55

3.2.2. Cell Culture ................................................................. 55

3.2.3. RT-PCR analysis ........................................................... 56

3.2.4. RAMP 1, 2, 3, and CRLR cloning and expression .................... 57

3.2.5. Membrane preparation and adenylate cyclase assay .................. 58

3.2.6. [3H]thymidine incorporation ............................................. 59

3.2.7. Statistical analysis ......................................................... 59

vi

3.3. Results ............................................................................... 60
3.3.1. AM receptor components in rat mesangial cells .................... 60
3.3.2. Effects of RAMP over-expression on AM-induced adenylate

cyclase activity and [3H]thymidine incorporation in RMC ....... 60
3.4. Discussion ........................................................................... 65

4. Effects of platelet-derived growth factor and lipopolysaccharide on RAMP

expression in rat mesangial cells ............................................................ 69
4.1 . Introduction ......................................................................... 69
4.2. Materials and methods ............................................................ 72
4.2. 1. Materials .................................................................. 72
4.2.2. Cell Culture ............................................................... 73
4.2.3. RAMP 1, 2, 3, and CRLR cloning and expression .................. 73
4.2.4. Membrane preparation and adenylate cyclase assay ................ 74
4.2.5. [3H]thymidine incorporation ............................................ 75
4.2.6. Northern blot analysis .................................................... 75
4.2.7. Western blot analysis .................................................... 76
4.2.8. Statistical analysis ....................................................... 76
4.3. Results ................................................................................ 77
4.3.1. Effect of PDGF on RAMP mRNA expression ....................... 77
4.3.2. Effect of PDGF on RAMP protein expression and
AM-mediated adenylate cyclase activity .............................. 82
4.3.3. Effect ofLPS on RAMP mRNA expression .......................... 87
4.3.4. Effect of LPS on RAMP protein expression and AM-mediated
adenylate cyclase activity ................................................ 91
4.4. Discussion ............................................................................. 95
5. Mechanism of PDGF and LPS-dependent RAMP-3 up-regulation ..................... 100
5. 1. Introduction ........................................................................... 100
5.2. Materials and methods .............................................................. 103
5.2.1. Materials .................................................................. 103
5.2.2. Cell Culture ............................................................... 103
5.2.3. Northern blot analysis ................................................... 104
5.2.4. Analysis of RNA stability ............................................... 105
5.3. Results ................................................................................. 106
5.3.1. Involvement of signal transduction pathway(s) in PDGF
and LPS-dependent RAMP-3 up-regulation ......................... 106
5.3.2. Mechanism of PDGF and LPS-stimulated
RAMP-3 expression ..................................................... 111
5.4. Discussion ............................................................................ l 18
6. Summary and conclusions .................................................................... 122
6.1. Major hypothesis and results of the study ....................................... 122
6.4.Limitations of this study ............................................................. 126
6.5.Positive outcomes of this study .................................................... 128
References .......................................................................................... l3 1

LIST OF TABLES

Table 1. Biological Actions of Adrenomedullin ................................................ 21

Table 2. Pathophysiological states associated with elevated plasma
adrenomedullin levels .................................................................... 28

Table 3. Effect of AM22.52 on AM-mediated AC activity in RAMP-2 or RAMP-3
transfected RMC .......................................................................... 63

Table 4. Effect of a-amanitin pretreatment on PDGF and LPS- induced RAMP-3
mRNA expression ....................................................................... 1 14

viii

LIST OF FIGURES

Figure 1. Longitudinal section through a renal glomerulus .................................... 8

Figure 2. Receptor activity modifying protein (RAMP) and calcitonin
receptor-like receptor (CRLR) expression profile in quiescent rat
mesangial cells (RMC) ................................................................ 62

Figure 3. Effect of RAMP-1, RAMP-2, and RAMP-3 overexpression on
AM-stimulated adenylate cyclase activity in RMC ................................ 63

Figure 4. Effect of RAMP-2 and 3 over-expression on [3H]thymidine
incorporation in RMC .................................................................. 64

Figure 5. A-a. Effect of platelet-derived growth factor (PDGF) on RAMP-3 mRN A
expression in RMC (graph) ............................................................ 78

Figure 5. A-b and A-c. Effect of platelet-derived growth factor (PDGF) on
RAMP-3 mRNA expression in RMC (representative Northern blots) .......... 79

Figure 5. B. Effect of platelet-derived growth factor (PDGF) on RAMP-2 mRN A
expression in RMC ..................................................................... 80

Figure 6. Effect of platelet-derived growth factor (PDGF) on RAMP-3 mRN A
expression in RMC ..................................................................... 81

Figure 7. Effect of PDGF on membrane-associated RAMP-3 protein expression
in RMC ................................................................................... 83

Figure 8. A. Effect of PDGF on AM-mediated adenylate cyclase activity in RMC.
AC activity increased with increasing doses of AM in PDGF treated and
controls ................................................................................... 84

Figure 8. B. Effect of PDGF on AM-mediated adenylate cyclase activity in RMC.
PDGF caused a concentration-dependent increase in AM-stimulated
AC activity ............................................................................... 85

Figure 8. C. Effect of PDGF on AM-mediated adenylate cyclase activity in RMC.
Effect of AM-(22-52), the AM receptor antagonist, on AM-mediated AC
activity in rat mesangial cells exposed to PDGF .................................... 86

Figure 9. Effect of lipopolysaccharide (LPS) on RAMP-3 mRN A expression
in RMC ................................................................................... 88

ix

Figure 10.

Figure 11.

Figure 12.

Figure 13.

Figure 14.

Figure 15.

Figure 16.

Figure 17.

Figure 18.

Figure 18.

Figure 19.

Figure 20.

Figure 21.

Effect of lipopolysaccharide (LPS) on RAMP-3 mRNA expression
in RMC; temporal effect ............................................................. 89

Effect of lipopolysaccharide (LPS) on RAMP-2 mRN A expression
in RMC ................................................................................. 90

Effect of lipopolysaccharide (LPS) on membrane-associated RAMP-3
protein expression in RMC ........................................................... 92

Effect of lipopolysaccharide (LPS) on AM-mediated adenylate cyclase
activity in RMC; response to pre-treatment with AM22.52 ........................ 93

Effect of lipopolysaccharide (LPS) on AM-mediated adenylate cyclase
activity in RMC; response to varied concentrations of AM ..................... 94

Effects of AG1296, PD15303 5, and PD168393 on PDGF-stimulated
RAMP-3 mRNA expression in rat mesangial cells .............................. 108

Effects of SBZO3580 (10uM) and PD98059 (louM) on PDGF-induced
RAMP—3 expression in RMC ....................................................... 109

Effects of SB203580, PD98059, and Wortmannin (Wort) on LPS-induced
RAMP-3 mRN A expression in RMC ............................................. 110

A. Effect of Actinomycin D (ActD) on PDGF and LPS-stimulated
RAMP-3 expression in RMC ....................................................... 113

B. Effect of Actinomycin D (ActD) on PDGF and LPS-stimulated
RAMP-3 expression in RMC. Control experiment verifying that
PDGF and LPS-increased transcription of MEK-l is inhibited by ActD.....114

Effect of Cycloheximide (CI-IX) on PDGF and LPS-induced RAMP-3

mRNA expression in RMC ......................................................... l 15
Effect of PDGF on RAMP-3 mRNA rate of decay in RMC .................... 116
Effect of LPS on RAMP-3 mRNA rate of decay in RMC ...................... 117

KEY TO ABBREVIATIONS

Adrenomedullin

Adrenomedullin receptor (molecular constituents: CL receptor+RAMP2)‘
Adrenomedullin receptor (molecular constituents: CL receptor+RAMP3)‘
Adrenomedullin receptor (generic)

Amylin

Amylin receptor (molecular constituents: CTR+RAMP1)'
Amylin receptor (molecular constituents: CTR+RAMP2)'
Amylin receptor (molecular constituents: CTR+RAMP3).
Angiotensin-II

Arginine-vasopressin

Adenosine 3’-5’ cyclic monophosphate

Calcitonin gene-related peptide

CGRP receptor (molecular constituents: CL receptor+RAMPl)'
Calcitonin-like (receptor/protein); formerly CRLR.
Calcitonin receptor-like receptor; currently CL receptor.
Calcitonin

Calcitonin receptor

Endothelial cells

Extracellular signal-regulated kinase

End-stage renal disease

Endothelin-l

Glomerular filtration rate

G protein-coupled receptor

Human aortic endothelial cells

Human umbilical vein cells

Interferon

Interleukin

c-Jun N-terminal kinase

Nco-nitro-L-arginine methyl ester

Lipopolysaccharide

Mean arterial blood pressure

Mitogen-activated protein kinase

MAPK/ERK kinase

Nitric oxide

 

° Nomenclature recommended by International Union of Pharmacology (IUPHAR). XXXII (as described
by Poyner et a1. Pharmacol Rev 54: 233-246., 2002.). This naming system extends to all of the established
receptors for the calcitonin family of peptides. It assigns the receptor name afier the endogenous ligand to
which it exhibits the highest binding affinity, following the guidelines of IUPHAR (as described by Ruffolo
et al. in The 1UPHAR Compendium of Receptor Characterization and Classification, 2nd ed, pp7-8,
IUPHAR Media, London, UK, 2000.).

p38 MAPK
PAMP
PDGF
PDGFR
PI3K

PKA

PKC

PP2A

RBF
RCP
RMC
RT-PCR
SAPKS
TGF
TLR

UTRs
VEGF
VSMC

p38 mitogen-activated protein kinase
Proadrenomedullin N-terminal 20 peptide
Platelet-derived growth factor
Platelet-derived growth factor receptor
Phosphotidyl inositol 3-kinase

Protein kinase A

Protein kinase C

Protein phosphatase 2A

Receptor activity modifying protein
Renal blood flow

Receptor component protein

Rat mesangial cells

Reverse transcription-polymerase chain reaction
Stress activated protein kinases
Transforming growth factor

Toll-like receptors

Tumor necrosis factor

3'-Untranslated regions

Vascular endothelial growth factor
Vascular smooth muscle cells

xii

1. INTRODUCTION

The recent discovery of receptor activity modifying proteins (RAMPs) has
significantly altered our understanding of mechanisms involved in the regulation of G
protein-coupled receptors. RAMP-1, 2, and 3 have been shown to differentially couple to
calcitonin receptor (CTR) and calcitonin-like receptor (CL receptor) giving rise to
distinct receptor phenotype characteristics (199), (41). Receptor trafficking,
glycosylation, and a direct RAMP-receptor interaction have all been proposed as possible
mechanisms responsible for the critical role of RAMPs in CTR and CL receptor ligand
specificity determination (for reviews see Foord et al. (82) and Sexton et al. (294)).
Accumulating data suggests that the dynamic alteration of RAMP expression levels may
provide for yet another mode by which these proteins regulate the function of CL
receptor hence changing the cellular responses initiated by adrenomedullin (AM)
exposure. Given that AM plays an important role in normal as well as pathological
processes in mesangial cells, elucidation of mechanisms responsible for changes in AM-
receptor interactions becomes of critical importance. The current study was undertaken to
investigate the effects of altered RAMP gene expression on the responsiveness of
mesangial cells to exogenous AM. In addition, this thesis characterizes platelet-derived
growth factor and lipopolysaccharide as agents regulating AM-mediated cellular
responses by their ability to alter RAMP-3 mRN A abundance.

This brief introductory chapter is followed by literature review in chapter two.
Chapter two offers an in-depth discussion of the scientific literature describing the
biology of mesangial cell, characterization of adrenomedullin and its receptor complex,

and our current understanding of the adrenomedullin-receptor-RAMP interaction.

Chapters three through five are organized according to the specific aims of this study and
independently consist of brief introduction, experimental data, and specific discussion
relevant to the particular aim. Thus, chapter three explores the gene expression profile of
RAMP-1, 2, 3, and CL receptor in mesangial cells cultured under basal conditions. It also
identifies the effects of RAMP-1, 2, or 3 overexpression on AM-mediated mesangial cell
proliferation and adenylate cyclase activity. Chapter four incorporates the major aim of
examining PDGF and LPS effects on RAMP-3 mRN A and membrane-associated protein
expression. It also describes the effects of PDGF and LPS on AM-induced adenylate
cyclase activity in mesangial cells. Chapter five investigates fiirther the effects of PDGF
and LPS on RAMP-3 mRNA expression with an aim to characterize the molecular
mechanisms responsible for the observed changes. By utilizing specific pharmacological
inhibitors, the experiments described in this chapter scrutinize the involvement of PDGF
receptor and MAPK pathways in the case of PDGF-induced effects as well as PI3K and
MAPK pathways in regards to LPS. Lastly, experiments described in chapter five were
also designed to examine the effect of PDGF and LPS on the stability of RAMP-3 mRN A
as measured by the mRNA half-life. The closing chapter of this thesis, chapter six, offers
a list of major hypothesis tested and the experimental results obtained in the course of
this study. It also enumerates the limitations of this study and discusses its positive
outcomes focusing on the possible implications of the current findings and anticipated

future perspectives.

2. LITERATURE REVIEW

2.1.Mesangial cell.

2.1.1. General characteristics and cell functions.

Since the initial identification of mesangial cells by Zimmermann in 1933 (374),
much has been learned about the characteristics of this independent cell type localized
mainly to the trigonum of the glomerular tuft (Figure 1). Similarities of mesangial cells to
smooth muscle cells (presence of contractile elements and angiotensin II receptors (165),
(115), (307), (50)) as well as their architectural arrangement in the glomerulus suggest
several important functions.

As specialized contractile smooth muscle cells with a direct contact to the
glomerular endothelium, mesangial cells play a pivotal role in the regulation of
glomerular hemodynamics. By contracting, mesangial cells reduce blood flow to select
capillary loops thus effectively reducing the intraglomerular filtration area. This
observation was first supported by the fact that substances capable of reducing the
ultrafiltration coefficient would also cause robust contraction of isolated glomeruli and
cultured mesangial cells (59).

Another important aspect of mesangial-endothelial cell interaction is underscored
by the nature of their intimate apposition. Unlike other renal cell-cell interactions,
mesangial cells communicate directly with the endothelial lining of glomerular capillaries
without the interference of the glomerular basement membrane. Consequently,
endothelial fenestrations allow for free contact of plasma and various macromolecules

with mesangial cells. Macromolecular uptake by mesangial cells has been reported by

number of investigators and led to the current understanding of yet another function of
these highly specialized renal cells: handling of macromolecules, immune complexes,
and advanced glycation end products (72), (306), (333), (87). It, has been estimated that a
subpopulation of 3 to 7 percent of all glomerular mesangial cells in the rat express F c and
C3b receptors as well as the common leukocyte antigen and la determinants (289). These
cells, as initially hypothesized by Schreiner et al. (289) and subsequently confirmed by
Lovett and Sterzel (187), are involved in recognizing and presenting foreign antigens and
stimulating lymphocyte proliferation. Thus, through their phagocytic abilities and the
expression of above-mentioned surface receptors, mesangial cells aid glomerular resident
macrophages in modulation of glomerular inflammatory processes (187). Currently this
notion is well established and grossly evident upon considering the pathophysiology of
IgA nephropathy and related diseases, where complement activation is thought to be of
primary pathological importance (vide infra). In addition, several investigators reported
pro—inflammatory like responses of mesangial cells while challenged with sub-maximal
doses of C5b. Namely, immune complex formation stimulated mesangial cell production
of TNF, IL-1, IL-6, various eicosanoids and reactive oxygen species. It also enhanced
elaboration of extracellular matrix leading to pan-glomerular expansion of the
mesangium (6), (186), (287), (101), (5).

As alluded to previously, mesangial cells constitute a dynamic cell type, which is
a site of production and a target for a variety of cytokines and growth hormones (for
review see (285), (225), (183)). Of particular interest to this thesis is the production and
mesangial cell response to platelet-derived growth factor (PDGF). PDGF, a major

cytokine secreted by mesangial cells acts in turn as their most potent mitogen as

established for mouse, rat, and human cell lines (200), (79), (3). By interacting with a
specific tyrosine kinase receptor (PDGF receptor, PDGFR), widely expressed by
mesangial cells, PDGF has been shown to significantly increase mesangial cell thymidine
incorporation rate (by 10 to 15-fold) as well as augment the synthesis of extracellular
matrix components (197), (360). In addition, PDGF also induces directed mesangial cell
migration (17), (161). Thus, studies suggest that PDGF is not only a causal factor in
development of mesangial hypercellularity and excessive matrix accumulation (the
fundamental changes observed in several glomerular disease states) but also participates
in mesangial cell repopulation following sub-total glomerular damage characteristic to
several forms of glomerulonephritis (1). Despite the clear relationship of PDGF effects
on mesangial cells and the etiology of a number of glomerular diseases, PDGF plays a
vital role in the pr0per development and function of normal mesangial cells. Genetic
knock-out mice for PDGF or PDGFR exhibit failure of mesangial cell growth. In
addition, both mutant strains reveal a lack of glomerular tuft architecture due to the
absence of mesangial cells resulting in formation of poorly defined, greatly distended
capillary sacks (182), (309). Accordingly, these observations establish the need for PDGF
in normal mesangial cell growth and development. They also emphasize the importance
of another mesangial cell function: the assistance in appropriate generation and
maintenance of glomerular capillary loop structure.

Perhaps the most obvious function of mesangial cells is that of structural support
for the entire glomerular unit. Spanning the majority of the central portion of the
glomerulus, mesangial architecture provides for the skeletal structure defining the

glomerular capillary loop arrangement (see figure 1). The importance of this function is

clearly illustrated by yet another experimental model, the antithymocyte antibody, Thy
1.1-dependent mesangiolysis model. An intravenous administration of rabbit anti—rat
thymocyte serum reactive with Thy-l-like antigens present on rat mesangial cells induces
an abrupt mesangial cell injury with sequential mesangiolytic and mesangial-
proliferative/infiltrative lesions. Concomitant with mesangial injury and lysis, a rapid loss
of characteristic glomerular capillary architecture occurs. Furthermore, restoration of the
typical capillary pattern follows the progressive mesangial cell recovery (128), (353),
(363), (362). Interestingly, neutralization of PDGF or PDGFR directly by injection of
antibodies attenuates the pathological findings following anti-Thy 1.1 administration,
thus once again providing strong evidence for involvement of PDGF in the etiology of
glomerulonephritis (135), (134).

The structural support by mesangial cells is mainly afforded through liberation of
extracellular matrix components forming an organized mesangial matrix. The major
constituents of the mesangial matrix are type IV and V collagen, the glycoproteins
laminin and fibronectin, the basement membrane heparan sulphate proteoglycan
(perlecan), as well as chondroitin/dermatan sulphate proteoglycans: biglycan, decorin,
and versican (325). In addition, metalloproteinases and their specific inhibitors, also
synthesized by mesangial cells, form a part of the extracellular matrix. AS initially
suggested by Davies et al. (49), these metalloproteinases, while regulated by a variety of
grth factors and cytokines, appear to be largely involved in modulation of mesangial
matrix turnover via proteolytic as well as cell-signaling capabilities. Mesangial cells,
embedded in the mesangial matrix, do not merely synthesize this supporting scaffold but

also participate in an extensive "cross-talk" with the extracellular matrix molecules and

other soluble mediator substances. These mesangial cell-extracellular matrix interactions
exert major effects on such phenotypic features as cell growth, apoptosis, and
differentiation. As a result, they have been causally implicated in influencing mesangial
cell biology during embryonic development, tissue repair and glomerular disease (48,

264,286,310,343)

 

AA-afferent arteriole PE-parietal epithelium US-urinary space
EA- efferent arteriole M—mesangial cell P-prox. tubule
MD- macula densa E-fenestrated endothelium

GC- granular cell GBM-glom.basement memb.

EGM- extraglomerular mesangium PO-podocyte

Figure 1. Longitudinal section through a renal glomerulus. A: diagram. B: Light
microscopic view (x s 490). Adapted from Kriz et al.,1988 (166).

2.1.2. Mesangial cell in disease.

The aforementioned functions of mesangial cells are tightly regulated in a healthy _
state. On the other hand, changes in mesangial cell biology are well-documented events
participating in the development and progress of diseases like glomerulosclerosis, IgA
nephropathy, diabetic nephropathy, hypertensive nephrosclerosis, mesangioproliferative
nephropathy, and others. Excessive mesangial cell proliferation and matrix deposition are
considered to be pathognomonic for several renal diseases (160), (77), (64). These
alterations instigate the maladaptive process of glomerular hypertrophy and capillary
obliteration resulting in glomerulosclerosis, often leading to end-stage renal disease
(ESRD) (322), (150), (81), (344). The 1999 Annual Data Report of the US Renal Data
System (U SRDS) described a dramatic increase in ESRD over the past decade reaching a
stunning 287 new cases per one million population in the United States in 1997. In View
of the current epidemiological data, it becomes apparent that better understanding of the
etiology and pathophysiology of renal disease is crucial in order to prevent fiirther rise in
the incidence of this ailment. Considering that mesangial cell proliferation and mesangial
matrix accumulation are the hallmark of glomerulosclerosis (and related kidney
pathologies) and the fact that AM along with other substances regulates mesangial cell
biology, it becomes imperative to further investigatethe mechanism(s) responsible for
AM actions in mesangial cells. While the exact mechanisms responsible for AM-
mesangial cell interactions are still not entirely clear, their nature (in particular at the
receptor level) may be at least partially revealed by examining the dynamic interplay of
AM receptor components in physiogical and pathophysiological states. To that extend,

current experiments were designed to characterize the AM receptor complex in mesangial

cells. Furthermore, this study examined the effect(s) of PDGF and lipopolysaccharide
(substances of great importance in the development of glomerular injury) on the

components of AMR and the associated changes of AM activity in mesangial cells.

2.2. Adrenomedullin.

2.2.1 Adrenomedullin gene.

The first report describing the isolation and initial characterization of
adrenomedullin (AM) was published in April 1993. Kitamura and colleagues described a
polypeptide obtained from extracts of human pheochromocytoma tissue capable of
raising intracellular cyclic AMP levels in rat platelets. It also appeared to have a powerful
hypotensive activity through its vasodilatory effect on the resistance vessels (154).
Furthermore, Kitamura er al. utilized an AM-specific radioimmunoassay (RIA) to
demonstrate measurable amounts of this peptide in circulation. A few months later, the
same research group presented the human AM gene sequence, thus introducing a new
circulating hormone to the expending repertoire of known bioactive molecules inscribed
in the human genome (157). Since the initial discovery, approximately one thousand
publications and two international symposia have been dedicated to the characterization
and better understanding of AM, making it one of the most vigorously studied novel
hormones.

Human AM (hAM) gene is found in a single locus of chromosome 11, it
encompasses 4 exons and 3 introns, and it’s flanked at the 5’-end by RNA polymerase II

responsive TATA, CAAT, and GC boxes. It also encodes several binding sites for

10

activator protein-2 (AP-2) (123), a cyclic AMP-regulated enhancer (75), nuclear factor-
KB (125), hypoxia-inducible factor-1 (HIP-l) (44), hypoxia response elements (HRES)
(88), and a binding site for steroidogenic factor-l (SF-1) (210). The presence of such a
variety of functional elements on the hAM gene suggests a complex involvement of
numerous factors in the regulation of the gene which in its entirety codes for a 185 amino
acid long precursor protein: preproadrenomedullin (preproAM). Enzymatic processing of
this precursor leads to formation of proadrenomedullin (proAM) and subsequently yields
a production of AM as well as another biologically active polypeptide,
“proadrenomedullin N-terminal 20 peptide” (PAMP) (155). Interestingly, more recent
investigations suggested several other biologically active and structurally stable AM
fragments to be present in the circulation. Notably, Gumusel and colleagues proposed
that differential endopeptidase-induced cleavage of the proAM gives rise to AM153.135,
termed adrenotensin; while yet another form of precursor processing results in AM11-26
peptide isOlated from bovine adrenal medulla by Kitamura et. a1 (93), (159). As described
below, the biological activity of these preproadrenomedullin-derived peptides varies
significantly and has been proposed to depend on the presence or absence of vital protein

structural components.

2.2.2. Adrenomedullin protein structure.

Recognition of the structural homology of AM to calcitonin gene-related peptide
(CGRP), a potent vasodilator and central nervous system as well as peripherally acting
neurotransmitter, secured AM’s membership in the CGRP family of peptides. Despite a

rather low overall amino sequence homology (estimated at 24% with CGRP), 52 amino

11

acid long AM shares common features of a ring structure formed by one intramolecular
disulfide bond (between residues 16 and 21) and an amidated carboxyl terminal end with
the other members of the family: calcitonin (CT), or- and B-CGRP, and amylin (AMY)
(114). The integrity of these structures is essential for the biological activity of AM, as it
is also true for the related peptides (63), (156). Conversely, the terminal carboxyl-end
fragments devoid of the proximal ring structure serve as selective peptidyl competitive
antagonists to their respective full-length counterparts. Accordingly, peptide fragment
AM22.52 serves as a receptor blocker for AM, while a-CGRPsm and B-CGRP3.37 are
utilized as competitive antagonists for the CGRP receptors (63), (3 7), (184). As expected,
the peptidic nature of these antagonists imposes inherent limitations on their use during
pharmacological manipulations. Thus, numerous efforts are directed towards the
discovery of selective, non-peptide receptor antagonists to the members of the CGRP
family. At present, three such compounds have been reported. _ BIBN4096BS,
WO98/11128 (Compound 1), and SB-273779 all have been characterized as highly
selective non-peptide antagonists for the subtypes of CGRP receptor (56), (62), (7).
Unfortunately, a non-peptide antagonist selective for AM receptors is not currently

available.

2.2.3. Sites of Adrenomedullin gene and protein expression.

At present, it has been established that AM exists ubiquitously in a great variety
of tissues. With the aid of highly sensitive RIA-s and/or by immunohistochemical studies,
AM was identified in the adrenal medulla, heart, aorta, kidney, lung, brain, pancreas,

skin, and other tissues (154), (122), (275), (284), (193), (196), (194). AM protein and/or

12

gene expression were confirmed in many cell types of which cardiac myocytes (44),
vascular smooth muscle cells and endothelial cells (311), (313), (312), renal mesangial
cells (174), (203), (242), renal distal and collecting tubular cells (14), (283), (137),
pulmonary cells (195), and a number of human tumor cell lines (223), (158), (88), (318)

are but a few examples suggesting a wide biological role for AM.

2.2.4. Signals responsible for Adrenomedullin secretion.

Complementing the wide cell type and tissue distribution of AM is an equally
generous collection of cytokines, hormones, and extracellular factors implicated in the
regulation of AM production and secretion. Since AM appears to be secreted
constitutively by cells and not stored by secretory granules, regulation of AM production
parallels precisely its secretion (317), (127), (146). An extensive collaborative work of
several Japanese researchers enumerated a multitude of substances having an effect on
AM secretion. Of these, inflammatory cytokines: interleukin-la and B (IL-l-a/B), tumor
necrosis factor or and B (TNF-or/B), and LPS appear to stimulate AM production in
human, bovine, porcine, and rat vascular smooth muscle cells (VSMC) (312), (313),
(329). Cultured fibroblasts exhibit essentially the same responses to IL-l-or/B, TNF-or/B,
and LPS as the VSMC (126), while some remarkable species variation was noted on
examination of the endothelial cells (EC). TNF-or and LPS elevated AM secretion from
porcine, rat, and bovine EC. IL-l stimulated only rat endothelial AM production, whereas
human derived EC responded negatively to TNF and IL-1 administration (311), (127),
(329). Conversely, LPS showed a potent stimulatory effect on AM secretion by

macrophages (168), (167), (370). In general, data from several laboratories suggest that

13

pro-inflammatory and endotoxic factors stimulate significant AM production and release
from vascular cell types allowing this potent vasodilatory peptide to contribute to the
hypotensive events and vascular collapse of sepsis as well as other inflammatory states.
Of the other cytokines tested, transforming growth factor-B (TGF-[3) inhibits AM
secretion with the greatest potency in VSMC and EC (314), (178). Interferon-y (IFN-y)
Shares the same effect as TGF-B on vascular cell lines (329), (127), (314), whilst
exhibiting significant stimulation of AM release from cultured astrocytes (316), (169).
Hofbauer et al. also suggested a permissive role for IFN-y, as administration of this
cytokine potentiated the additive effect of IL-lB and TNF-or on AM gene expression in
rat aortic VSMC (120). Other cytokines: fibroblast growth factor (FGF), epidermal
grth factor (EGF), and platelet-derived growth factor (PDGF) demonstrate negligible
effect on regulation of AM secretion (312). A broad panel of vasoactive peptides,
including arginine-vasopressin (AVP), endothelin-1 (ET-1), angiotensin-II (ANG II),
bradykinin, adrenaline, and substance P stimulate formation of AM (270), (269), (146),
(206), (336), (314). This effect was also demonstrated for thrombin, where use of
submaximal doses led to considerable rise of AM in EC and skin keratinocytes (127),
(146). Furthermore, an investigation of hormonal influences on AM production revealed
a complex interaction between several classes of steroid and non-steroid derived
hormones and a potential for a tissue-specific control of AM gene activation.
Dexamethasone, cortisol, aldosterone, thyroid hormone, and retinoic acid all exhibit
significant augmentation of AM gene expression and/or AM secretion from a variety of
cultured cell types (329), (124), (205). This effect is also correlated with the in viva

observations where, for example, raised lung mRNA and plasma levels of AM were

14

reported in hyperthyroid rats as well as adrenalectomized rats following dexamethasone
administration (215), (105). Tissue-specific regulation of AM production studied by Abe
et al. in ovarian granulosa cells revealed a gonadotropin-sensitive pattern, where presence
of follicle stimulating hormone (F SH) achieved a long term inhibition of AM secretion
(4). Additionally, progesterone and an anti-estrogen (tamoxifen) augment AM plasma

and gene expression levels, respectively (133), (3 72).

Stress factors, namely hypoxia and fluid shear also have been reported to
influence AM gene expression and protein production. Hypoxia as well as oxidative
stress induction (via hydrogen peroxide, cobalt chloride or desferrioxamine mesylate)
increase AM secretion and its gene expression unanimously in many cell types, including
cardiac myocytes (368), (44), coronary artery EC (224), VSMC, Madin-Darby canine
kidney (MDCK) cells, mesangial cells (218), retinal pigment epithelial (RPE) cells (338),
colorectal carcinoma cell line (DLD-l) (223), and neuroblastoma cell lines (IMR-32 and
NB69) (159). Accumulating evidence suggests that this hypoxia-induced production of
AM is regulated by the hypoxia-inducible factor-l (HIF-l) transcription factor present in
the promoter consensus sites of the AM gene (88), (226), (44). Regulation of AM
secretion by fluid shear stress, on the other hand appears to be somewhat controversial.
Chun et al. reported a time and shear stress intensity-dependent elevation of AM mRNA
in endothelial cells derived from the human umbilical vein (HUVEC) (42). In contrast,
Shinoki et a1. observed a marked decrease in AM gene expression and peptide secretion
from cultured human aortic endothelial cells (HAOEC) exposed to shear stress loading
apparatus protocol (304). Researchers agree that the effect of physical stress on AM

secretion is likely to be a consequence of AM gene regulation, presumably through

15

activation/deactivation of the shear stress responsive element (SSRE) located in the AM
gene promoter region (304); the issue of differential regulation reported for HUVEC and

HAOEC, however awaits further investigation.

Considering the diverse nature of factors involved in the regulation of AM
production and secretion, perhaps a more inclusive approach is to study the involvement
of various signal transduction pathways in the synthesis of this peptide. In general, it
appears that stimulation of cAMP-protein kinase A pathway results in a decrease of AM
secretion as observed in glomerular epithelial cells (174), VSMC (314), and granulosa
cells (4). However, exposure of cultured rat endothelial cells to forskolin, a direct
adenylate cyclase (AC) activator, and a cell-permeable analog of CAMP, 8-bromo-cAMP,
had no effect on AM production, once again pointing out the cell specific nature of AM
regulation (127). On the other hand, activation of phospholipase C-protein kinase C and
Ca++lcalmodulin pathways leads to AM gene expression and/or accumulation of
immunorective-AM (ir-AM) in the supernatants of cultured VSMC (104), cardiac
myocytes (336), and others. Since the inflammatory cytokines are known to enhance
nitric oxide (NO) formation and their profound effect on AM gene expression has been
well established by a number of investigators, Hofbauer and others attempted to
characterize the exact relationship between NO pathway and AM expression. As
expected, rat aortic VSMC, endothelial cells, hepatocytes, and mesangial cells exhibited
marked elevation in AM production induced by NO donation using S-nitroso-N-
acetylpenicillamine (SNAP). Furthermore, NO deprivation by Nm-nitro-L-arginine
methyl ester (L-NAME) attenuated TNF-or, IL-IB, and INF-y induced AM gene

expression. Interestingly, exposure of these cells to 8-bromo-cGMP, an analog of cGMP,

l6

however had no effect on AM secretion, while pretreatment with a guanylate cyclase
(GC) inhibitor, lH-oxodiazolo-quinoxalin-1 (ODQ), failed to attenuate the SNAP-
assisted increase in AM mRNA expression (120). Compatible with these findings are the
reports of Isumi et al. where exposure of rat EC to 8-bromo-cGMP had no effect on AM
synthesis (127) and Dotsch et al. who upon incubation of human umbilical vein
endothelial cells (HUVEC) with the NO donors (sodiumnitroprusside, SNP;
morpholinosydnonimine,SIN-1; and phospodiesterase V inhibitor, zaprinast) observed an
increase in both AM secretion and mRNA expression (57). Altogether, these results
support that NO directly enhances AM production, as observed in response to the pro-
inflammatory cytokine exposure, but the NO-dependent signaling involved appears to be

unrelated to the classical GC-cGMP-nitric oxide synthase pathway.

2.2.5. Biological actions of Adrenomedullin.

By far, the best-characterized actions of AM are those related to the
cardiovascular system. Initiated by the first report on AM, its potent and long-lasting
hypotensive effect has been subsequently studied by a number of laboratories. To date
however, it appears that virtually every organ system examined is affected to a significant
degree by the biological activity of this circulating hormone. Accordingly, a
comprehensive review of all of the actions of AM reaches beyond the scope of this work.
Thus, only a limited discussion regarding AM actions in the cardiovascular and renal

systems with a particular attention to AM effects in the renal glomerular mesangial cells

17

will follow. Please refer to table 1 for the generalized overview of the multi-fiinctional
properties of AM.

Studies of AM actions on vascular beds of rat, cat, dog, pig, sheep, and human
collectively show that AM elicits relaxation of the resistance vessels thereby achieving a
long-lasting drop in the mean arterial blood pressure (MAP) (90), (118), (30), (68), (255),
(222). In most systems examined, vasodilating effect of AM is attenuated by L-NAME
indicating at least partial involvement of the NO-dependent pathway, (73), (208), (116),
however regional as well as interspecies variations have been reported (231), (32), (16).
As demonstrated by Parkes et al., decrease in MAP induced by intravenous AM
administration to conscious sheep is accompanied by an increase in heart rate and a slight
drop in stroke volume (collectively resulting in an elevation of cardiac output) along with
a marked decrease in total peripheral resistance (253). In addition, a direct positive
inotropic effect on the heart has been reported and appears to be CAMP and NO
independent (315), (152).

Besides the direct vasomotor activity, AM appears to play an important role in the
cell growth regulation. Its effects on cell growth and apoptosis depend largely on the cell
type and the experimental conditions examined. Miller et al. characterized AM activity in
several human tumor cell lines, where AM acts (perhaps in an autocrine fashion) as a
potent growth factor promoting neoplastic proliferation (204). Similarly, AM stimulates
cell proliferation in Swiss 3T3 fibroblasts (356), human oral keratinocytes (143), rat
gastric epithelial cells (346), and human retinal pigment epithelial cells (337). On the
other hand, AM has a general anti-proliferative effect on select cardiovascular cell types:

it inhibits growth of rat VSMC (142) and hypertrophy of cultured myocytes and

18

fibroblasts (335), (334). Vascular endothelial cells however depend on the presence of
AM for proper growth and morphology as illustrated by the embryonic lethality of AM
gene knockout (KO) mice. Independent research laboratories reported marked endothelial
cell pathology and a poor development of vitelline vasculature in the KO mice (303),
(300). Also, AM was shown to have an anti-apoptotic effect on cultured rat endothelial
cells, reflecting its Vital role not only during vascular embryogenesis but also as a cell
survival factor following the differentiation of the vascular system. The exact mechanism
responsible for AM inhibition of apoptosis in EC remains illusive. Despite its parallel to
NO-induced anti-apoptotic effects in EC, AM appears to signal through a cGMP-
independent pathway (282). Likewise, its effect is not dependent on the ability to elevate
intracellular pools of cAMP, however it has been reported to regulate endothelial cell
death by activating a known anti-apoptotic gene, MAX (147), (297).

Beginning with the initial studies on renal artery AM administration in the
anesthetiZed dogs, it became clear that AM exerts profound effects on renal function.
Ebara and co-workers reported that intrarenal infusion of AM at concentrations sub-
optimal for heart rate and MAP alterations resulted in an increase of renal blood flow
(RBF), total urine output, and urinary sodium excretion. These findings, unaccompanied
by changes in glomerular filtration rate (GFR), suggest a direct glomerular effect of AM
(61). As expected, at higher concentrations, AM infusion affords marked depression of
MAP but concomitantly it increases GFR, vasodilates afferent and efferent arterioles, and
decreases distal tubular sodium reabsorption thus further augmenting fractional sodium
excretion (116), (137), (242). Additional studies confirmed these AM-induced renal

vasodilatory and natriuretic actions to be NO-dependent (208), (116), (341). Contrary to

19

these findings and paradoxical to renal effects of AM in general, Leclerc and Brunette
reported a cAMP-dependent sodium-sparing capacity of AM. By influencing the
sodium/hydrogen exchangers of the distal tubular system, AMeffect resembled the well-
established mechanism of aldosterone activity (179).

Analogous to the findings for the vascular cell types, AM is expressed by a
variety of renal cells where it has also been shown to elicit numerous biological effects
including those of growth-regulation. Using reverse transcription-polymerase chain
reaction (RT-PCR), Owada et al. examined the AM gene localization in microdissected
rat nephron segments. RT-PCR demonstrated the presence of AM mRNA in the
glomerulus, cortical collecting duct, outer medullary collecting duct, and inner medullary
collecting duct but not in proximal convoluted tubule or medullary thick ascending limb.
Further analysis by northern blotting revealed especially high AM expression in the
glomerular mesangial cells. Mesangial cells not only produced the peptide but responded
to its administration by robust generation of intracellular cAMP in the presence and

absence of fetal calf serum (242).

20

Generalized AM actions:
Vascular effects:

0 IV administration of AM results in sustained hypotension via NO, CAMP,
and/or PG generation depending on the vascular bed studied

0 direct effects on heart: positive ionotropic and chronotropic effects
coronary artery dilation

Renal effects:

0 intrarenal AM infusion increases RBF, GFR, Na+ excretion, and urine
flow

0 stimulates mesangial cell contraction and inhibits PDGF-induced ET-l
production
inhibits PDGF-induced MAPK-dependent mesangial cell proliferation
stimulates p38 and PI3-K dependent mesangial cell hyaluronic acid
release

0 stimulates intrarenal renin release

Endocrine effects:

0 inhibits ACTH release

0 inhibits aldosterone production/secretion
o inhibits insulin secretion

Effects in Bone:
o promotes osteoblast growth and protein synthesis; increases the relative
area of mineralized bone

Effects in Luna:

0 inhibits histamine-induced bronchoconstriction

- inhibits LPS-induced alveolar macrophage release of neutrophil
chemoattractants

Immunologic effects:
0 bacteriocidal/static against gram+/- bacteria (at supra-physiologic
concentrations)

CNS effects:

0 ICV AM administration attenuates AVP release

0 ICV AM administration inhibits thirst drive

0 ICV AM administration is pro-anorexic

o ICV AM administration results in hypertension and increased HR (in
contrast to peripheral vasodilitory effects)

Table 1. Biological Actions of Adrenomedullin.

21

References:

(90). (73). (63), (364)

(315),(107), (170)

(116), (65). (219)
(40)

(328), (292)
(251)

(132)

(278), (254)
(361)
(I96)

(46).. (45)

(364)

(140)

(11)

(367)
(216)
(323)
(279),(274)

2.2.6. Actions of Adrenomedullin in mesangial cells.

Of particular interest to this work are the actions of AM on the glomerular
mesangial cells. As mentioned previously, mesangial cells elaborate basal levels of AM
but they have also been reported to produce AM in response to various
cellular/exogenous factors (174). The basal rate of AM secretion from rat mesangial cells
was estimated at 0.26:0.05 fmol/ 105 cells/h and approximates one-third to one-fifth of
the rate deduced for myocytes and EC (203). In addition, mesangial cells have been
shown to express AM-sensitive receptors and are a prime target for AM autocrine and/or
paracrine activity (240). While the fact that AM modulates mesangial cell contraction is
unequivocal, its bearing on the direct regulation of RBF remains highly controversial
(40), (362). On the other hand, the effects of AM on mesangial cell growth, apoptosis,
cell migration, free radical generation, and extracellular matrix production have been
extensively studied and are well established. We have recently reviewed in detail the
current understanding of AM actions on mesangial cells (249). In general, the
autocrine/paracrine nature of AM actions affords its role as a local modulator of
mesangial function while the ability to stimulate production of intracellular cAMP,
initially described by Khono et a1 and Chini et al, serves as its modus operandi (40),
(163). Several research groups, including our laboratory, documented potent inhibitory
effect of AM on mesangial cell growth (40), (162), (292), (203), (247). This anti-
proliferative action is evident in the quiescent mesangial cell culture as well as in the
cells exposed to such mitogenic factors as PDGF, epidermal growth factor (EGF),
elevated transmural pressure, and to a lesser degree endothelin-l (38), (239). AM

achieves the anti-proliferative effect via inhibition of the extracellular signal-regulated

22

kinase (ERK) activity in a cAMP/PKA-dependent manner (40), (100), (247). This effect
on grth regulation is complemented by AM-induced apoptosis in mesangial cells,
' which is compatible with a general finding that elevation of intracellular cAMP leads to
an increase in programmed mesangial cell death (214), (229). Parameswaran et al.
demonstrated an AM-mediated increase in the activity of protein phosphatase 2A (PPZA)
that leads to a concerted inhibition of ERK, thus a decrease in cell proliferation, and a
concomitant AM-induced stimulation of apoptosis in mesangial cells (248). Further
characterization of the pro-apoptotic action of AM determined it to be p38 MAPK
dependent and to coincide with a significant caspase-3 and caspase-8 activation (250),
(247), (246). In addition to counteracting the effects of PDGF on mesangial cell
proliferation, AM also inhibits PDGF-induced mesangial cell migration and ET-l
production, which are both implicated in the pathogenesis and progression of glomerular
disease (161), (162). Other actions of AM in mesangial cells include inhibition of
reactive oxygen metabolite formation (38) and ANG—II stimulated mesangial cell
migration (161). In general, apart from its stimulatory effect on mesangial cell hyaluronic
acid (a major glycosaminoglycan component of the extracellular mesangial matrix)
release (251), AM appears to play an important protective role in pathogenesis and
progression of glomerular disease. In that respect, besides its antiproliferative and pro-
apoptotic functions, the antagonistic nature of functional interplay between AM and
PDGF (a prime cytokine implicated in renal disease) deserves firrther investigation. The
present study will, in part, characterize the effect of PDGF on the AM receptor system

and its consequence in regards to AM actions in mesangial cells (see chapter 3).

23

2.2.7. Effects of Adrenomedullin gene alteration and somatic Adrenomedullin gene
delivery.

Much has been learned from the recent advances in gene engineering regarding
the role of AM in normal development as well as its involvement in pathogenesis and
progress of various diseases.

Caron and Smithies first reported generation of the AM gene knockout (K0) in
mice. The complete lack of the preproAM gene resulted in a 100 percent mortality of
mice at midgestation and characteristic cardiac abnormalities with a presence of extreme
hydrops fetalis (28). Findings from another laboratory confirmed the need of AM gene
for survival past mid-embryonic age, however the selective disruption of AM gene with
salvage of the PAMP encoding region led only to a mild form of subcutaneous edema
without previously reported frank hydrops (300). Others suggested the presence of poor
placental circulation, namely compromised vitelline vascularity and abnormal umbilical
artery cOntraction, as a cause for lethality in a complete AM gene KO (86), (303).
Interestingly, AM gene KO mice were rescued by osmotic pump-assisted infusion Of AM
to embryos prior to the midgestational age (106). Altogether, these findings emphasize
the importance of AM in embryogenesis, in particular with regard to the cardiovascular
system. Since the heterozygote mice for the AM gene KO (AM’H) are viable and fertile,
further studies granted additional information on the role of AM in the maintenance of
the circulatory system. Using angiotensin II and salt loading as a model previously
described to cause severe end-organ damage in rodents (13), (190), Shimosawa et al.
suggested a cardioprotective role for AM against oxidative stress and ANG II-induced

coronary artery injury. They reported a marked increase in coronary artery lesions and

24

elevation in all oxidative stress indices studied in AM"+ mice as compared to controls. In
addition, ANG Illsalt loading failed to upregulate cardiac AM production in the KO but
not the Sham mice (300). The AM'” mice also Showed (a diminished nitric oxide
production and a Significant increase in the basal blood pressure as compared with wild-
type littermates (303). As expected, transgenic mice overexpressing the AM gene had a
significantly lower mean blood pressure, which was normalized by intravenous injection
of L-NMMA, a NO synthase inhibitor. In addition, mice overexpressing AM exhibited an
increase in the survival rate following induction of septic shock by lipopolysaccharide
(LPS) administration. The transgenic mice were less sensitive to LPS-induced
hemodynamic changes and showed a remarkable resistance to organ damage clearly
associated with septic shock and grossly evident in the wild-type animals (302). These
observations together with the finding that AM concentrations are greatly elevated during
septic shock (more so that in any other pathological state reported (117)) establish the
protective role for AM and possibly point towards a new therapeutic approach in the
treatment of sepsis.

Several laboratories described a successful adenovirus—assisted AM gene transfer.
In order to better characterize the role of AM in maintenance of a healthy cardiovascular
system, human AM (hAM) gene was administered intravenously to the following
hypertensive rat models: spontaneously hypertensive rats (34), Deoxyxorticosterone
acetate (DOCA)—salt hypertensive rats (55), Dahl salt sensitive rats (371), and Goldblatt
hypertensive rats (345). For all rat models, researchers reported expression of human AM
gene in the heart, aorta, kidney, lung, liver, and adrenal glands following the gene

delivery. Detectable levels of hAM were also present in plasma and estimated by Zhang

25

et al. at 6.4i1 .4 ng/ml three days post-injection (371). Introduction of hAM gene caused a
robust and long lasting reduction in the mean arterial blood pressure in all hypertensive
rat models. In addition, lefi ventricular hypertrophy, _ interstitial fibrosis and
cardiomyocyte diameter were significantly reduced as compared with non-manipulated
animals (55), (345), (371). A direct protection of vascular walls was also demonstrated by
local hAM gene delivery into the carotid artery following a balloon-injury procedure.
Compared to the control animals, carotids of rats exposed to hAM gene demonstrated
considerable decrease in neointimal formation and a significant reduction in intima/media
ratio, indices of healthy vascular wall regeneration (35). These findings underscore the
importance of AM in post-injury re-endothelialization and complement previous reports
suggesting its function as an endothelial cell survival factor (147). While this pro-
endothelial effect of AM, hypothetically also beneficial in the ischemia/reperfusion injury
has not been yet directly tested, Chao et al. investigated hAM gene delivery in the rat
model of myocardial infarction by coronary occlusion/reperfusion. Introduction of hAM
gene greatly reduced the infarct size, rate of sustained ventricular fibrillation, and the
extent of apoptosis of myocytes confined to the ischemic area (3 5).

The protective effects of AM gene delivery also extend to the kidney, where
elevation in renal hemodynamic parameters (GFR and RBF), reduction in glomerular
sclerosis, attenuation of tubular and interstitial damage, and urinary protein excretion
were all observed (55), (345). In addition, the Dahl salt sensitive rats showed marked
increase in tubular and interstitial cell proliferation that correlated with renal injury but

was significantly reduced by hAM gene delivery (371).

26

Several lines of evidence suggest strongly an involvement of AM in a variety of
disease states: plasma AM levels are elevated in a broad range of diseases (see table 2),
AM gene KO/overexpression studies emphasize its importance in proper embryogenesis
and normal cardiovascular/renal system function, somatic AM gene delivery as well as
intravenous AM administration (151), (227), (354), (148), (136) protect against
multifaceted nature of pathologies.

These findings, especially the evident protective cardio-renal role of AM,
prompted a recent sprout of interesting clinical trials examining the effects of exogenous
AM administration in healthy as well as disease-affected human subjects (198), (259),
(355). Intravenous (iv) infusion of AM in healthy volunteers, patients with essential
hypertension, and patients diagnosed with IgA nephropathy confirmed potent
hemodynamic and neurohumoral effects of this polypeptide. In all subjects AM
administration produced significant fall in arterial pressure with concomitant increase in
heart rate and cardiac output. Stimulation of the sympathetic nervous system and renin
release were also noted (332). Nagaya et al. investigated i.v. AM effects in patients with
congestive heart failure and pulmonary hypertension. AM treatment appeared to have
beneficial effects in both groups of patients. It significantly improved cardiac firnction by
eliciting an increase in cardiac stroke index, ejection fraction, left ventricular posterior
wall thickening and urinary sodium excretion (221). AM administration also improved
pulmonary parameters by causing a significant decrease in pulmonary vascular resistance

and an appreciable reduction in mean pulmonary arterial pressure (220).

27

 

Disease state:

 

0 Cardiovascular disorders:
Essential hypertension
Acute myocardial infarction
Cerebrovascular disease
Heart failure
Preeclampsia
Hemorrhagic shock
Pulmonary hypertension
Mitral stenosis
Subarachnoid hemorrhage
Raynauds disease

0 Renal disorders:
Chronic renal failure
Renal failure of mixed etiology
End-stage renal failure
IgA nephropathy
Glomerulonephritis

0 Respiratory disorders:
Chronic obstructive pulmonary disease
Asthma (acute only)

0 Endocrine disorders:
Type I diabetes
NIDDM
Primary adrenal insufficiency
Thyrotoxicosis (Grave’s disease)
Primary hyperaldosteronism

0 Other conditions:
Hepatic cirrhosis
Cancer of lung, GI tract, ACTH-secreting adenoma
Sepsis
Wegener’s granulomatosis

 

 

 

Table 2. Pathophysiological states associated with elevated plasma adrenomedullin

levels. Adapted from Hinson et a1. 2000 (114).

28

2.2.8. Actions of other preproadrenomedullin gene products.

Like AM, PAMP exhibits a potent hypotensive effect, albeit the exact mechanism
of its action is related to an inhibition of catecholamine secretion from sympathetic nerve
endings rather than a direct vasodilatory mechanism akin to AM (153), (299), (321),
(320). In the central nervous system PAMP appears to regulate blood glucose, food
intake and gastric empting. Ohinata et al. showed that a significant elevation in blood
glucose as well as inhibition of food intake and gastric empting occurred within 30
minutes or less following central administration of PAMP (233), (234). These effects
seem to be independent of the CL receptor system and have been shown to rely on
bombesin (BN) receptor activation in the case of blood glucose regulation and a BN-
independent, PAMP-specific receptor responsible for food intake and gastric empting
suppression (235). PAMP-specific receptors, while currently not cloned, are likely
extensively distributed as suggested by 12SI-PAMP binding studies (130). Their activation
by PAMP presumably leads to peripheral vasodilatation (235). PAMP has been also
reported to stimulate aldosterone secretion from the rat adrenal glomerulosa cells in a
cAMP-dependent manner (113), (326), inhibit ACTH secretion from cultured pituitary
cells (280), and regulate renin synthesis and secretion from the juxtaglomerular cells of
the kidney (185).

Another protein akin to AM, adrenotensin appears to have vascular effects
opposite to those of AM (93). In particular, cat pulmonary arterial rings and isolated rat
aortas showed a dose-dependent contractile response to adrenotensin (92), (373). In
addition, intravenous injection of adrenotensin significantly elevated the mean arterial

pressure in anesthetized rats and induced the proliferation of cultured vascular smooth

29

muscle cells. Interestingly, these effects were attenuated by the concomitant exposure to
AM. Also, there was a significant reciprocal inhibition noted in the endogenous release of
adrenotensin and AM from the rat aorta (3 73).

More recently another endogenous, biologically active peptide derived from
differential processing of AM or its pre—pro form has been characterized by Kitamura er
a1. AM 11.26 was isolated from bovine adrenal medulla and produced strong pressor effect
when administered to conscious, unanesthetized rats. Surprisingly concomitant with the
hypertensive effect, AM”.26 dose-dependently increased the heart rate presumably
overriding the baroreceptor response by a potent stimulation of catecholamine release
( 159).

The antagonistic nature of the biological effects observed in the peptides derived
from a differential enzymatic processing of the precursor protein suggests an interesting
system of intra-molecular regulation of preproadrenomedullin. Clearly, further studies are
necessary to better characterize the newly proposed products of preproAM and their

relationship to AM and PAMP.

2.3. Adrenomedullin receptor complex.

2.3.1 Adrenomedullin receptor system discovery.

The vast array of AM activities described in a number of tissues (for review
please see table 1) is thought to be mediated by AM interaction with a cell-surface
receptor. Until recently, the identity of this receptor has been elusive and particularly
confusing due to numerous inconsistent reports on the pharmacological inhibition of AM

activities. For example, the vasodilatory effects of AM were shown to be blocked by

30

CGRP8.37 in a number of tissues, thus thought to be signaled through the CGRP receptor
(232), (258), (94), (68). To the contrary, some reported that CGRP3.37 had no effect on
AM-mediated actions although, at the concentrations used, it potently inhibited responses
to CGRP administration thus arguably pointing to the existence of AM-specific receptors.
For example, unique AM receptors were implicated in AM-induced vasodilatation of the
guinea pig pulmonary artery, hypotensive effects in Long-Evans rats, control of
aldosterone production in rat adrenals, and many other AM-mediated actions (108), (31),
(260), (319). Yet others documented AM-induced responses that were sensitive to both
peptidal inhibitors: CGRP8-37 and AM22.52 (240).

The initial attempts at molecular cloning of AM receptor resulted in data
comparably confusing to that of the pharmacological studies described above. Kapas et
al. first reported cloning of AM receptor from rat lung that was capable of binding 1251_
AM and causing elevation in intracellular cAMP when transfected into COS-7 cells
(144). FUrther analysis of this receptor revealed its similarity to previously described
orphan receptor called L1 or G10d (69), (103). Several months later, Kapas and Clark
documented that a previously identified canine receptor, RDC-l also had a CGRP and
AM-like characteristics and with a considerable homology to the L1 receptor, it too was
capable of supporting CGRP and AM-induced increase in cAMP when transfected into
COS-7 cells (145). Later, a human counterpart to the rat AM receptor identified by Kapas
et al. was cloned and the characterization of the AM receptor biology seemed to be well
underway (102). Disappointingly, subsequent attempts by other laboratories to further
examine the nature of the proposed AM receptors failed to reproduce previously reported

observations (149), (29), (199).

31

A Viable alternative for an AM receptor emerged shortly afier the initial finding
by Aiyar and co-workers that an orphan receptor, calcitonin receptor-like receptor
(CRLR) exhibited a well-characterized CGRP1 receptor pharmacology with a weak AM
cross-reactivity. Expression of human CRLR in human embryonic kidney 293 (HEK 293)
cells afforded a robust increase in CGRP-induced cAMP accumulation. Both, specific
binding of 125I-CGRP and CGRP-stimulated cAMP production were dose-dependently
inhibited by pre-treatment with CGRP3.37 (9). Later, similar results were obtained for rat
CRLR (99) and porcine CRLR (66). Interestingly, not all cell lines examined supported
the findings reported by Aiyar et al. and the laboratories that originally documented
cloning the rat (33), (230) and human (80) CRLR sequences classified this receptor as an
orphan. They recognized that CRLR is a seven-transmembrane-domain G-protein-
coupled receptor (GPCR), however they failed to identify its native ligand. Together with
receptors for calcitonin, gastric inhibitory peptide, glucagon, pituitary adenylate cyclase
activating hormone, vasoactive intestinal peptide, secretin, growth hormone releasing
hormone and parathyroid hormone CRLR forms the family B of GPCR-s. It consists of
464 and 461 amino acids and has a remarkable 50% and 54% amino acid sequence
identity with rat and human calcitonin receptor, respectively (33), (80). Its identity to the
calcitonin receptor is even greater in the transmembrane regions where it is estimated at
almost 80%, further implying possible commonalities in structure and/or function
between these to receptors (41).

In 1998, publication by McLatchie and colleagues presented new evidence that
did not only explain the elusiveness of the AM and CGRP receptor but also introduced a

novel concept for the receptor phenotype determination (199). In the effort to clone the

32

gene encoding the human CGRP receptor, the group utilized a DNA library from human
neuroblastoma SK-N-MC cells previously reported to bind 12SI-CGRP and show CGRP-
mediated cAMP accumulation (375). Systematic analysis of complementary RNA-s
derived from the DNA library using a Xenopus oocyte system (expressing an endogenous
CGRP receptor and an exogenous cAMP—sensitive cystic fibrosis transmembrane
regulator, CFTR) yielded a 148 amino acid protein capable of significant induction of
CGRP-mediated cAMP production. This protein, named receptor-activity modifying
protein 1 (RAMP-l), however did not by itself constitute a functional CGRP receptor
since its expression in HEK 293T, COS-7, or Swiss3T3 cells did not result in
pharmacological responses characteristic of a CGRP receptor. Conversely, concomitant
expression of RAMP-1 and CRLR in HEK 293T cells and oocytes restored the
pharmacological findings of the CGRP-specific receptor. Since HEK 293T, COS-7, and
Swiss3T3 cells do not express endogenous CRLR, co-expression of both proteins
(CRLR+RAMP1) was necessary to reconstitute a functional CGRP receptor. Further
database search identified two more RAMP-l-like proteins: RAMP-2 and RAMP-3,
which as a group exhibit approximately 31 percent identity and 56 percent similarity to
each other. Evaluation of RAMP-2 and RAMP-3 in the similar fashion to that described
for RAMP-1 led McLatchie and her colleagues to an astounding conclusion. For the first
time a member of a well-characterized GPCR family, CRLR, was shown to change its
receptor phenotype and recognize a different ligand based on a specific association with
one of the RAMP proteins. Explicitly, co-expression of CRLR with RAMP-1
reconstitutes a firlly functional CGRP receptor, while concomitant transfection of

CRLR/RAMP-Z or CRLR/RAW-3 establishes an AM receptor (199). Other laboratories

33

extended the above findings to multiple cell lines reporting that RAMP-2/CRLR complex
functions as an AM receptor in human endothelial and vascular smooth muscle cells
(139), rat osteoblast-like UMR-106 and COS-7 cells (26), and Drosophila Schneider 2
cells (10) while RAMP-I/CRLR interaction leads to the formation of a CGRP receptor in
the same cell lines. Interestingly, although RAMP-2 and RAMP-3 have a relatively low
homology with an estimated 30% sequence identity, they generate essentially a
pharmacologically indistinguishable adrenomedullin receptor when co-expressed with
CRLR in HEK 293T cells (83). While other laboratories reported no significant
differences between RAMP-2/CRLR and RAMP-3/CRLR receptor phenotypes, it may be
speculated that variations in their regulatory system exist. Supportive of this notion are
the findings from RAMP expression studies in animal disease models where variable
RAMP-2 and RAMP-3 mRNA expression was observed (see section 2.3.6). In addition,
the current study offers convincing evidence that RAMP-2 and RAMP-3 expression in
differentially regulated by platelet-derived growth factor (PDGF) and lipopolysaccharide
(LPS) in the rat glomerular mesangial cells (see section 4.3).

In contrast to the effects of coupling with CRLR, notable differences between
RAMP-2 and RAMP-3 were found in their interaction with the calcitonin receptor
(CTR). As described in the following section, RAMP-2 and RAMP-3 co-expressed with
CTR generate receptors characterized by marked phenotype variation and receptor
specificity (41) (327) (376).

A recent revision of nomenclature for the receptors of the calcitonin family of
peptides has been recommended in order to reflect the current state of knowledge

regarding the molectual profile of these receptors as well as their name compliance with

34

the present guidelines of the International Union of Pharmacology (for details, please
refere to footnote on page XI). The revised nomeclature will be adopted for the remainder

of this work.

2.3.2. Other receptors that interact with RAMPS.

Following the discovery of RAMPS and their interaction with calcitonin-like
receptor, CL receptor (formerly known as calcitonin receptor—like receptor, CRLR), an
obvious question was posed: can RAMPS interact with other receptors? Considering the
remarkable molecular identity between CL receptor and calcitonin receptor (CTR), CTR
seemed to be a plausible receptor target for the novel family of RAMPS. Overwhelming
evidence suggests that RAMP expression is not required for the biological responses
induced by calcitonin binding to its receptor (41) (295). However, shortly after the
publication by McLatchie et al. several independent investigators reported that
coexpression of RAMPS with CTR results in generation of the amylin receptor.
Cotransfection of human CTR with RAMP-1 (AMY 1) or RAMP-3 (AMY 3) engendered
an amylin receptor phenotype in COS-7 and rabbit aortic endothelial cells. RAMP-2
appeared to have only minimal effect on amylin receptor generation (AMY 2) (41) (212).
Likewise, transfection of RAMP-1 and RAMP-3 into Chinese hamster ovary (CHO)-K1
cells, which endogenously express a CTR, produce an amylin receptor (41). Further
studies showed that RAMP-2 is also capable of conferring an amylin receptor phenotype
upon CTR however the cellular background and the subtype of CTR present greatly
influence this finding. Namely, the most commonly abundant isoform of the human CTR

(the insert negative hCTRn.), unlike the hCTRm isoform, is not capable of interacting

35

with RAMP-2 and consequently fails to establish an amylin receptor in COS-7 cells. On
the other hand, cotransfection of RAMP-2 with either isoform of CTR in CHO-P cells
generated a high affinity amylin receptor (AMYz) (327) (376). Tilakaratne et al.
theorized that since the endogenous RAMP expression is Similar in both cell lines,
cellular factors other than CTR and RAMPS might contribute to the final receptor
phenotype generation. It is worth noting that unlike their interaction with CL receptor,
RAMP-2 and RAMP-3 couple differentially to the CTR. The significance of these
differences is currently poorly understood.

While to date there is no direct evidence that RAMPS interact with receptors other
than CTR and CL receptor, several observations make this an attractive speculation. The
ubiquitous abundance of RAMPS in a great number of tissues far exceeds that of CTR
and CL receptor. For example, RAMP mRNA expression was reported in the brain
regions that are devoid of any CTR or CL receptor presence. Interestingly, these areas
(specifically the subfornical organ and area postrema) express RAMP-1 and RAMP-3
and have been previously shown to be under the control of both adrenomedullin and
amylin (266) (267) (273) (296) (339). Furthermore, the expression of RAMPS in brain
correlates to that of the other members of the group B family of GPCRs, notably the
pituitary adenylate cyclase-activating polypeptide receptor and the glucagon receptor
(342) (201). These findings suggest that RAMPS may couple with other, yet unidentified,
molecules to form functional receptors for adrenomedullin and amylin. They also hint at
the ability of RAMPS to interact with the other members of the B family of GPCRs.
Latest analysis of several GPCRs as potential partners for RAMPS however failed to

detect any concerted cell-surface expression or a direct physical interaction between

36

RAMPS and selected receptors. All receptors tested in this study belong to the family B
of GPCRs. While two of them exhibit marked degree of amino acid homology with CL
receptor, PTH/PTHrP-R and GluR (approx. 45% homology), the others, vasopressin
VlaR and V2R are only vaguely related to the RAMP-interacting receptors: CL receptor -
and CTR (76).

A comparison between RAMP tissue distribution and that of 150 known receptors
was performed in the effort to identify other potential targets for RAMP interaction. A
statistically significant correlation was found between RAMPS and two receptors: the
EP4 prostanoid receptor and an orphan neuropeptide-like receptor, nonetheless further
studies are needed to elucidate if RAMPS indeed interact with these and/or other
receptors (106).

Recently, another receptor accessory protein has been investigated for its ability
to interact with CL receptor. CGRP-receptor component protein (RCP), an intracellular
peripheral membrane protein that is not related to the RAMP protein family, was reported
to potentiate the CGRP-mediated responses in Xenopus laevis oocytes (189). While RCP
appears to physically interact with CL receptor affecting both CGRP- and AM-evoked
responses, it does not exhibit a chaperone-like activity nor does it affect the ligand-
binding pharmacology. Instead, it is proposed to couple CL receptor directly to the
downstream signaling molecules, in particular the subunits of G-proteins and/or
adenylate cyclase complexes (70) (271). Since the data characterizing RCP and its
receptor interactions are relatively scarce, it is especially difficult at present to define the

role of RCP as it may pertain to the receptor biology of CL protein.

37

2.3.3. Characteristics of RAMP gene and protein structure.

RAMP-l and RAMP-2 sequences were originally cloned from human
neuroblastoma SK—N-MC cell cDNA library, whereas RAMP-3 cDNA was isolated from
human spleen. Sequential comparison of RAMP cDNA-s, proposed by McLatchie and
co-workers, with the genomic map revealed their chromosomal location and gene
organization. RAMP-l gene resides on chromosome 2, the gene for RAMP-2 is on
chromosome 17, and that of RAMP-3 localizes to chromosome 7 (199). Since the scan of
approximately 95% of the human genome revealed no more RAMP-like sequences, it is
likely that RAMPI, 2, and 3 are the only unique members of this gene family (106). The
genes encoding RAMP-1 and RAMP-3 share several characteristics. Composed of three
exons and divided by large introns, they span an access of 24 kilobases (Kb). The
RAMP-2 gene on the other hand is relatively small (approx. 5 Kb) and consists of four
exons. Common to all of the genes is the localization of the 5’UTR and the signal peptide
sequences on the first exon and the C-terrninal and transmembrane domains on the last
exon of the corresponding RAMP genes (294).

The analysis of hydrophobicity plots suggest a substantial similarity of protein
topology among the RAMPS despite their relatively low amino acid sequence identity
which is estimated at 30%. The RAMP interspecies sequence similarity however is well
conserved with approximately 90% identity between rat and mouse. Likewise, the rodent
sequences show roughly 70%, 65%, and 85% identity with human RAMP-1, RAMP-2,
and RAMP-3, respectively (217) (121). RAMP-1 and RAMP-3, both 148 amino acid
proteins, have a 26 amino acid long N-terminal signal peptide followed by an

approximately 90 amino acid extracellular domain, a single 20 amino acid

38

transmembrane segment, and a Short 10 amino acid C-terminal tail (199). RAMP-2,
composed of 175 amino acids, has essentially the same protein structure organization. As
intrinsic membrane proteins, RAMPS are relatively small and have a predicted size of
approximately 14,000-17,000 molecular weight (M,). Several residues are uniformly
conserved in all RAMP-s across many species suggesting their importance in
preservation of a common secondary structure and/or function. These include four
cysteine residues localized to the extracellular domain and two amino acid sequences:
DPPXX and LVVWXSK present in the N-terminal and C-terminal parts of the
transmembrane domain, respectively. Two additional cysteine residues are found in the
human, rat, and mouse RAMP-1 and RAMP-3 (199). Furthermore, several consensus
sites for N-glycosylation have been identified on RAMP-2 and RAMP-3. Analysis of
human, rat, and mouse RAMP-3, for example consistently identify four N-glycosylation
sites of which all four are also present in mouse RAMP-2. Interestingly, RAMP-1 amino
acid seqUence is devoid of any consensus sites for N-glycosylation, perhaps implicating a
different mechanism for post-transcriptional modifications among the RAMP subtypes
(211). Other sequences possibly involved in the regulation of RAMP-s include protein
kinase C and protein kinase A phosphorylation sites present in the intracellular C-
terminus of RAMP-1 and -3 (but not RAMP-2) and the unique NHERF motif in the C-
terminus of RAMP-3, which previously has been implicated in the activation of the
sodium-hydrogen ion exchanger (96) (95) (217). Clearly, the functional importance of
these sequences requires further investigation for it may provide us with a better
understanding of RAMP regulation and their interaction as a functional part of the

adrenomedullin receptor complex.

39

2.3.4. Tissue and cell specific RAMP expression and RAMP subtype-CL receptor
selectivity.

The distribution of RAMP mRNA has been partially examined in the human, rat
and mouse tissues. While species differences have been shown to exist, in general,
RAMP-1 is abundantly present in the heart, brain, skeletal muscle, thymus, spleen, fat
and kidney, while RAMP-2 is in the heart, aorta, kidney, spleen, fat, and lung. RAMP-3
exhibits the most ubiquitous distribution with the greatest abundance in the kidney, heart,
brain, and lung (199) (217) (294) (121) (237) (339). Cell specific RAMP mRNA
expression has also been observed as originally presented in human HEK193T, Kelly,
and SK-N-MC cells (199) and subsequently extended to other cell lines studied (277)
(139) (339) (159) (8). In fact, the existence of RAMPS in certain cell types but not others
led to the initial classification of CL receptor as an orphan receptor following a
functionally unsuccessful expression of CL receptor in COS-7 cells which do not express
RAMPS in basal conditions (80). At present, it is well understood that the cellular
background of endogenously expressed RAMPS defines the functional character of the
observed receptor subtype; moreover, it may have a significant influence on the receptor
phenotype detected following transfection of RAMPS and/or CL receptor into various cell
types. For example, rabbit aortic endothelial cells (RAECs) which endogenously express
rabbit RAMP-2 (rRAMP-Z) but not rRAMP-l, exhibit selective responses to AM which
are blocked by AM22.52 but not CGRP8.37. Transfection of human RAMP-1 (hRAMP-l)
into the endogenous background of RAECs affords CGRP-mediated responses
selectively inhibited by CGRP3-37 but not AM22-52. Cotransfection of hRAMP-l and

hRAMP-3 into RAECS however significantly diminishes CGRP-evoked responses

40

suggesting greater affinity of hRAMP-3 for the endogenous CL receptor than that of
hRAMP-l. Conversely, transfection of hRAMPs into RACES does not alter their
responsiveness to AM possibly implying a stronger interaction between rRAMP-2 and
rCL receptor (constituting an endogenous rabbit AMz) than any interaction of rCL
receptor and hRAMPs (213). The notion of intra- and inter-species differences in
affinities for CL receptor among RAMP subtypes has also been documented in other cell
types. Buhlmann et al. studied the rat osteogenic sarcoma UMR 106-06 cells, which
endogenously express rCL receptor and rRAMP-Z but little rRAMP-l, as well as the
COS-7 cells which lack native CL receptor or RAMP expression. Transfection of
hRAMPs into these cell lines revealed a higher affinity interaction between RAMP-1 and
CL receptor than RAMP-2 and the receptor (26). As exemplified above, the concept of
variable RAMP subtype—CL receptor affinity affords additional possibilities for
modulation of receptor phenotype by the RAMP family of proteins. Importantly, it also
hints that a well-regulated mechanism for RAMP-RAMP interaction may exist and it too,
along with the RAMP-CL receptor interaction, could be responsible for the final receptor
type determination. To that extent, a competitive and/or an inhibitory relationship among
the RAMP subtypes could be theorized. Corroborative with this hypothesis are the
findings that RAMP-1 (199) and RAMP-3 (294) form stable homodimers. To date,
formation of RAMP heterodimers however has not been reported.

Of particular interest to this study is the renal expression of RAMP mRNA.
Abundant expression in the renal tissue was reported for RAMP-2 and RAMP-3 with
similar expression in both cortical and medullary parts of the rat kidney (369). Utilizing

competitive, quantitative RT-PCR Totsune et al. measured the mRNA expression levels

41

for RAMP-2 and RAMP-3 in a normal Munich-Wistar rat kidney. RAMP-2 mRNA was
detected at 26.5:19 mmol per mole of GAPDH and RAMP-3 at 77:04 mmol per mole
of GAPDH (meaniSE). CL receptor was also present in. the renal tissue, albeit at
significantly lower concentrations. (330). As previously described, RAMP-2 and RAMP-
3 can independently constitute a functional AM receptor when associated with CRLR.
Renal expression of both RAMP-2 and RAMP-3 affords a conceivably complex
mechanism for regulation of AM activity in the kidney during the basal as well as disease
states (for further discussion see section 2.3.6). A more precise cellular localization of
RAMP expression in the renal tissue could aid our understanding of the molecular
mechanisms involved. To date, however there is only one report on RAMP expression
profile in a non-embryonic kidney cell line. Robert-Nicoud and co-workers reported basal
RAMP-3 expression and its marked upregulation by vasopressin in mouse clonal cortical
collecting duct (CCD) principal cells (268). Since these cells participate in sodium and
water reabsorption under a direct influence of vasopressin and because adrenomedullin
has a known natriuretic activity, the effect of vasopressin on RAMP-3 expression may
represent a feedback mechanism by which CCD ensure the fluid balance. With an aim to
further elucidate the ambiguous kidney cell profile of RAMP expression, the present
study characterizes the mRNA expression of RAMPS in the renal mesangial cells and

their effect on AM activity.

2.3.5. Mechanism of RAMP-receptor interaction.

As initially postulated by McLatchie et al. and further theorized by Foord and

Marshall, the consequences of RAMP-CL receptor interaction are at least threefold:

42

1. RAMPS participate in the trafficking of CL receptor, thus regulating its appearance at
the cell surface. 2. RAMPS influence the state of CL receptor glycosylation. 3. RAMPS,
at least in part, determine the distinct pharmacology of CL receptor by their differential
association with the receptor. Furthermore, they hypothesized that the particular
pharmacological profile may be imposed upon CL receptor by RAMP-directed
glycosylation, induction of conformational change, and/or direct physical association
between CL receptor and a RAMP family member (199) (82).

To date, the most convincing evidence clearly defining the role of RAMPS in
directed trafficking of CL receptor to the cell-surface plasma membrane comes from the
confocal microscopic studies of the epitope-tagged CL receptors and RAMPS along with
the fluorescence-associated cell-sorting (FACS) analysis. Several independent
laboratories reported significant increase in the cell membrane presentation of CL
receptors following co-expression with all subtypes of RAMPS. RAMP-l, 2, and 3 induce
CL receptor translocation with a comparable efficacy resulting on average in the cell
membrane presentation of 25% of total CL receptor protein. Moreover, a strict
requirement for CL receptor presence was detected for RAMP cell membrane
translocation (199) (83) (41) (173). This reciprocal need for co-expression strongly
suggested a direct physical interaction between CL receptor and RAMPS, which was
shortly demonstrate by co-immunoprecipication studies (111). In contrast to CL receptor,
CTR localizes to cell membranes independent of RAMPS presence (41) (119) (36). Also,
as previously discussed, CTR does not interact with RAMPS in order to function as a
classical calcitonin receptor. Nonetheless, when co—expressed with CTR, RAMP-1 cell-

surface translocation is readily observed and it correlates with an increase in specific

43

binding of amylin to CTR as examined in COS-7 cells. Similar findings were reported for
RAMP-3/CTR interaction (41) (294). Accordingly, while RAMPS regulate CL receptor
protein trafficking as well as its receptor pharmacology, their interaction with CTR is
currently understood only at the level of receptor phenotype determination.

Initial findings pointing to a change in molecular mass of CL receptor when
cotransfected with RAMP-1 prompted a thorough investigation of the CL receptor
protein structure expressed at the cell surface. HEK 293T cells transfected with Myc-
tagged CL receptor presented with a single immunoreactive band of approximately 58K
relative M,. When transfected together with RAMP-1, CL receptor immunoreactivity
shifted to M,=66K and a concomitant disappearance of the original 58K band was
observed. Parallel to these findings, 125I-labelled CGRP cross-linking to the surface
receptors was found to exist only in the RAMP-I/CL receptor transfected HEK 293T
cells. Furthermore, endoglycosidase F and H aided analysis of the two CL receptor forms
determined that unlike the 58K species, the 66K form represents a terminally
glycosylated, hence endoglycosidase H resistant, CL receptor. Since the 66K form is the
species capable of 12SI-CGRP binding and present only when co-expressed with RAMP-
1, it was proposed that mature glycosylation of CL receptor is directed by RAMP-1 and
results in acquisition of CGRP binding specificity (199). Corroborative with this
hypothesis were the findings from coimmunoprecipitation studies, where the cytosolic
and membranous RAMP-I/CL receptor complexes were identified. While investigators
reported both immature (core-glycosylated) and terminally glycosylated CL receptor in
the cytosol, only the fully glycosylated, mature form of CL receptor appeared at the cell

membrane as a RAMP-UCL receptor complex. In contrast to RAMP-1, cotransfection of

44

RAMP-2 or RAMP-3 with CL receptor does not change the receptor’s glycosylation
status preserving its native core-glycosylation and the associated selective AM specificity
(199) (83). As documented by Fraser er al., the N-terminal extracellular domain of
RAMPS is responsible for RAMP-directed differential glycosylation of CL receptor.
Using RAMP-IIRAMP-Z chimeras, they showed that only RAMP-1 N-terminus
containing hybrids are capable of inducing terminal glycosylation of CL receptor thus
securing the CGRP-specific receptor phenotype (83). Altogether, these initial findings
suggested that RAMP-specific CL receptor glycosylation might provide a mechanism for
their ability to determine the receptor phenotype. Born and co-workers (91) offered a
closer look at the glycosylation of CL receptor by performing site-directed mutagenesis
of the three predicted N-glycosylation Sites for the human CL receptor. They reported
that N-glycosylation at residues Asn60 and Asn112 is required for proper transport of the
hCL receptor to the cell membrane. Surprisingly, while mutations at these residues
significantly decreased the frequency of receptor complex-ligand binding, they did not
preclude RAMP-l-CL receptor and 12"'I-CGRP interactions resulting in an adenylate
cyclase activity with an EC50 comparable to that of the controls (91) (25). Analysis of
asparagine at position 117 revealed that despite its predicted potential for N-
glycosylation, this modification does not occur in the wild type hCL receptor.

”7 position resulted in a close to complete

Nonetheless, site-directed mutations at Asn
ablation of CGRP-evoked adenylate cyclase activity. This functional receptor defect was
accompanied by an unaffected receptor cell surface expression and a slight instability of

117

the RAMP-l-CL receptor complex (91). Accordingly, it implicates Asn , and perhaps

the neighboring amino acid residues, in facilitating a direct protein-protein interaction

45

between RAMP and CL receptor in order to establish a stable and functional receptor
complex. As pointed out by Gujer et al., the amino acid sequence flanking Asn117
residue is highly conserved among several receptors belonging to the same receptor class
as CL receptor, possibly emphasizing the apparent importance of the sequence for the
proper receptor function. Unfortunately, there are no reports on the effects of the above-
mentioned mutations on the RAMP-2- or RAMP-3-CL receptor interaction and the AM
receptor complex function. Taken together, the site-directed mutagenesis studies suggest
a crucial role for CL receptor N-glycosylation in proper cell surface delivery and hint at
other, glycosylation independent, events regulating the direct RAMP-CL receptor
interaction and the resultant specific ligand binding ability.

Interestingly, more recent reports presented additional evidence undermining the
importance of CL receptor glycosylation in receptor phenotype regulation while focusing
on the significance of the direct RAMP-CL receptor interaction. Contrary to the initial
findings in the HEK 293T cells, cotransfection of CL receptor and RAMP-1 or RAMP-2
in Drosophila Schneider 2 (82) cells resulted in mature glycosylation of CL receptor
which was independent of the expressed RAMP subtype. In addition, radioligand binding
studies revealed that albeit uniformly glycosylated, CL receptor retains CGRP or AM
receptor characteristics, identical to those found in the previous studies, based on the
presence of RAMP-l or RAMP-2, respectively (10). Hilairet er a1. extended the analysis
of CL receptor glycosylation to mammalian cells by finding that indeed the direct
RAMP-CL receptor protein interaction and not the glycosylation state of CL receptor
determine the receptor ligand specificity. They found that while the core-glycosylated CL

receptor form predominates when cotransfected with RAMP-2 or RAMP-3, AM appears

46

to bind only to the mature (N-glycosylated) form of the receptor on the cell surface.
Furthermore, the cross-linking analysis showed that RAMP-2 and RAMP-3 form stable
heterodimers with CL receptor. Together, as a ternary protein receptor complex, RAMP-
CL receptor participate in the ligand binding as depicted by the consistent incorporation
of 12S'I-AM into the RAMP-2 and RAMP-3 protein structure. Analogous interaction was
also found for the RAMP-l-CL receptor heterodimer complexes and their selective
binding of 12SI-CGRP (112). To further analyze the nature of RAMP-CL receptor
interaction, Kuwasako et al. utilized RAMP deletion mutant constructs and identified the
key RAMP residues responsible for the generation of the functional receptor complex.
The deletion of human RAMP-2 residues 86-92 and RAMP-3 residues 59-65 resulted in a
Significant decrease in specific 1251-AM binding, hence an attenuated AM-directed cAMP
production in HEK 293 cells. Interestingly, the cell surface expression of human RAMP-
CL receptor complexes was not affected by the mutations, suggesting that the identified
RAMP residues directly interact with CL receptor to form a high affinity agonist-binding
site and/or evoke necessary conformational change of the receptor complex associated
with the acquisition of ligand specificity (171). The same group obtained similar findings
when they co-expressed rat CL receptor with rat RAMP deletion mutants in HEK 293T
cells. Here, the deletions of residues 93-99 and 58-64 from rRAMP-2 and rRAMP-3,
respectively, significantly inhibited high-affinity 11'SI-AM binding and AM-evoked cAMP
production despite full cell surface expression of the receptor heterodimers (172). It is
worth noting that the sequence identity between the key residues identified for RAMP-2
and RAMP-3 is minimal in both human and rat sequences. Also, single substitutions

within these sequences do not result in a significant loss of ligand recognition, thus

47

emphasizing again the importance of the entire sequence as a possible structural
determinant of the ligand-binding pocket (171). The direct involvement of RAMPS in
defining the ligand recognition site was also suggested by the observations of Mallee and
colleagues. Intrigued by two independent reports on marked species selectivity of non-
peptidal CGRP receptor antagonists, BIBN4096BS and Compound 1 ((56) (62)), they
postulated that precise species variations in RAMP-1 dictate the observed variability in
affinities reported for these antagonists of CL receptor-RAMP-l complex. Using
recombinant human/rat CL receptor-RAMP-l complexes they Showed that hCL receptor-
rRAMP-l heterodimers presented with the rat receptor pharmacology, while those
composed of rCL receptor-hRAMP-l retained human CGRP receptor characteristics.
Furthermore, they identified lysine at position 74 in the RAMP-1 sequence as the key
amino acid involved in modulation of antagonist affinity for CL receptor-RAMP-l. A
single lysine to tryptOphan substitution in rat RAMP-l at this position resulted in a
greater then 100 fold increase in antagonist affinities, resembling those native to the
human receptor complex (191). Considering the fact that both BIBN4096BS and
Compound 1 are competitive antagonists at the ligand binding site of the CL receptor-
RAMP-l receptor (56) (62), the above findings Strongly imply that RAMP-l forms an
integral structural part of the receptor binding site responsible for the recognition of both
CGRP and the small molecular antagonists.

Similar to other related GPCRs, CL receptor has been shown to undergo receptor
internalization following ligand exposure (173). Several laboratories scrutinized the role
of RAMPS in the ligand-induced CL receptor cycling phenomenon. Notably, Kuwasako

and colleagues visualized RAMP and CL receptor localization and trafficking by

48

fluorescent microscopy in HEK 293 cells. They confirmed the previous findings
suggesting a requirement of RAMPS for the appearance of CL receptors at the plasma
membrane. Green fluorescent protein (GFP) labeled CL receptor was not found in the
plasma membranes and it failed to generate CGRP or AM-evoked responses without
concomitant RAMPS expression. On the other hand, co-expression of RAMPS and CL
receptor resulted in ample cell surface appearance of both proteins restoring ligand-
induced intracellular cAMP production and calcium mobilization. Furthermore, AM-
mediated internalization of CL receptor-GFP was noted in association with all three
RAMP subtypes. This internalization process appears to be largely clathrin-coated pit-
mediated and involves specific targeting of both CL receptor and RAMPS to lysosomes.
Again, no CL receptor-GFP internalization occurred in the absence of RAMP expression
regardless of the ligand presence (173). Further examination of the sub-cellular
localization of CL receptor and RAMPS revealed that unlike CL receptor, which remains
in the endoplasmic reticulum (ER) when expressed alone, RAMP-1 is present both in the
ER and the Golgi predominantly in a disulfide—linked homodimer form. When co-
expressed with CL receptor, RAMP-l acquires several intramolecular disulfide bonds
and in association with CL receptor it co-localizes to cell membrane with a 1:1
heterodimer stoichiometry. Upon agonist exposure, CL receptor undergoes rapid
phosphorylation and the entire heterodimer complex associates with B-arrestin and
becomes endocytosed via clathrin-mediated process (111). Altogether these findings
establish unequivocally a direct association between CL receptor and RAMPS and
suggest that RAMPS may also play a role in regulation of ligand-induced CL receptor

internalization and/or a chaperone like activity during the receptor trafficking.

49

2.3.6. Regulation of RAMP gene expression.

A large number of recent publications demonstrated the dynamic nature of RAMP
mRNA expression. Indeed, RAMP gene expression is not constitutive and undergoes
dramatic changes in a number of pathophysiological states and as a consequence of
exogenous substance administration.

For example, a high expression of CL receptor and RAMP-2 mRNA is observed
in lungs of normal mice, but it is markedly decreased in endotoxemic mice treated with
LPS. In contrast, LPS treatment significantly increases RAMP-3 gene expression in
lungs, spleen, and thymus (237). Furthermore, Frayon et al. evaluated glucocorticoid
regulation of RAMPS and CL receptor by dexamethasone administration in human
coronary arteries vascular smooth muscle cells (VSMC). Untreated cells expressed
detectable levels of CL receptor, abundant amount of RAMP-2 and lesser amount of
RAMP-1. Dexamethasone treatment elevated RAMP-1 mRNA levels significantly while
those for RAMP-2 remained unchanged, resulting in a 5-fold increase in the ratio of
RAMP-1 to RAMP-2 expression. Slight variations in CL receptor expression were also
noted, albeit these appeared to be inconsistent with various dexamethasone
concentrations used. Altogether, these results suggest that glucocorticoids may be
involved in the regulation of RAMP expression and preferentially lead to the generation
of CGRP-responsive receptors in the human coronary VSMC (84). As previously
mentioned, vasopressin also has been shown to affect RAMP expression, leading to a 17-
fold increase in RAMP-3 mRN A levels in mouse kidney cortical collecting duct principal

cells (268). This observation, while intriguing, currently remains only poorly understood

50

due to the lack of any well-established interaction between vasopressin and the AM
receptor system.

Changes in RAMP mRNA expression have been also clearly demonstrated in a
variety of pathophysiological conditions. A concerted upregulation of AM, CL receptor,
and RAMP-2 mRNA expression was shown in the rat heart following myocardial
infarction (236), as well as in the state of congestive heart failure induced by coronary
ligation (331). In the chronic model of heart failure, RAMP-1 and RAMP-3 mRNA as
well as protein levels were significantly increased in both atria and ventricles of rats 6
months afier an aortic banding procedure (47). Furthermore, studies of the LPS-induced
mouse sepsis model documented an elevation of AM and RAMP-3 with a concomitant
decrease in CL receptor and RAMP-2 mRNA expression in the lung tissue (23 7).

The alterations in the AM-CL receptor-RAMP axis have also been noted in the
pathological states of the kidney where modified RAMP expression pattern was reported
following ureteral obstruction and a sub-total renal mass ablation. In the rat model of
nephropathy caused by unilateral ureteral obstruction (UUO), RAMP-1, RAMP-2, and
CL receptor gene expression were significantly upregulated, whereas RAMP-3
expression remained unaltered (217). Contrary to these findings, Totsune et al. (330)
demonstrated a 50% and 70% decrease in RAMP-3 and 30% to 43% decrease in CL
receptor rnRN A expression on the fourth and fourteenth day following 5/6 nephrectomy
in rats, respectively. No significant changes in RAMP-2 and AM mRNA levels were
noted in the nephrectomized animals as compared to the sham-operated controls. An
interesting side observation was the attenuation of RAMP-2 expression by sodium intake

restriction, which occurred irrespective of the nephrectomy procedure. The varied

51

expression of the AM receptor complex components reported by the two laboratories is
likely to represent the principal differences of the pathophysiological changes occurring
in these two models of nephropathy. Specifically, while UUO leads to primary renal
interstitial fibrosis perpetuated by elevated concentrations of TGF-[3, the sub-total renal
mass ablation results in renal failure with a compensatory renal hypertrophy and elevated
sodium excretion in the remnant kidney (21), (141). Despite these differences however
both studies Show dynamic changes within the AM receptor system (with a particularly
impressive quantitative changes in the RAMP expression profile). Taken together with
the previously discussed actions of AM in the kidney (see section 2.2.5-6), these
observations strongly suggest an important role for AM in the pathophysiology of renal

disease as it may be regulated at the level of RAMP mRN A expression.

52

3. RAMP mRNA expression profile and the effects of RAMP over-
expression on AM-induced adenylate cyclase activity and [3H]thymidine

incorporation in rat mesangial cells.

3.1. Introduction.

The recent discovery of receptor activity modifying proteins (RAMPS) by
McClatchie et al. (199) has significantly altered our understanding of mechanisms
involved in the regulation of G protein-coupled receptors (GPCRS). RAMP-1, 2, and 3
are distinct gene products and have been characterized as single-transmembrane domain
proteins capable of direct interaction with two related members of GPCR: calcitonin
receptor (CTR) and calcitonin-like receptor (CL receptor) (41), (199), (26), (139). While
the exact nature of RAMP-CTR and RAMP-CL receptor interactions remains elusive, it
has been clearly documented that RAMPS facilitate trafficking and determine the
phenotype of these receptors (199), (41), (173). In particular, with regard to CL receptor,
RAMP-l and CL receptor co-expression renders the receptor a firlly functional calcitonin
gene-related peptide receptor (CGRPI). Co-transfection of CL receptor with RAMP-2 or
RAMP-3, on the other hand, confers upon CL receptor adrenomedullin receptor
characteristics (AMI and AM;, respectively) (199). Consequently, differential expression
of RAMPS can potentially function as a regulatory step for CL receptor activity and its
ligand specificity towards CGRP and/or AM.

AM, a 52 amino acid peptide recently isolated from a pheochromocytoma (154),
has been shown to activate CL receptor causing an elevation in intracellular cAMP in

several systems including rat mesangial cells (40), (163), (240). In particular, by

53

activation of cAMP-PKA pathway, AM exerts anti-proliferative, pro-apoptotic, and anti-
migratory effects on rat mesangial cells (38), (247). Since disproportionate mesangial
proliferation and matrix deposition are a hallmark pathological change accompanying
several glomerular diseases (64), (160), the anti-proliferative effects of AM suggest an
attractive, reno-protective role for this hormone.

As thoroughly discussed in section 2.3.4, abundant expression of AM receptor
complex components was reported in the rat kidney tissue, where expression of both
RAMP-2 and RAMP-3 predominates (369), (330). To date however there is no report on
the specific non-embryonic renal cell type RAMP and CL receptor expression. Here we
have investigated the mRNA expression profile of AM receptor complex and the
functional consequence of RAMP overexpression on the AM responsiveness in rat

mesangial cells.

54

3.2. Materials and methods.
3.2.1. Materials.

Adrenomedullin and adrenomedullin(22-52) fragment were purchased from
Sigma‘9 RBI” (St. Louis, MO). RPMI-1640, fetal bovine serum, penicillin/streptomycin,
trypsin-EDTA were from GibcoBRL® (Grand Island, NY).

All other reagents were of highest quality available.

3.2.2. Cell Culture.

Rat mesangial cell (RMC) cultures were established from glomeruli obtained
from kidney cortex of 55 to 70 g male rats (Sprague-Dawley, Charles River, MA).
Glomeruli were isolated by sequential sieving which removes tubules (300 to 150 m
sieves), then retains glomeruli on the 63 m sieve, as described by Wolthuis et al. (358).
Isolated glomeruli were incubated for 10 min at 37 °C in collagenase(750 U/ml), then
plated in flasks in RPMI 1640 medium supplemented with 0.6 U/ml of insulin, 100 U/ml
of penicillin, 100 (1ng of streptomycin and 15% fetal bovine serum. Cells were grown
at 37 C in 5% carbon dioxide with medium changed twice a week. At confluency, cells
were sub-cultured by rinsing with calcium and magnesium free phosphate buffered saline
and then incubating with 0.05% trypsin supplemented with 20 mM EDTA. The correct
cell type was confirmed by the following criteriazl. a stellate morphology using phase
contrast microscopy; 2. microfilaments and sub-plasmalemmal cytoplasmic densities
using transmission electron microscopy; 3. insensitive to puromycin aminonucleoside; 4.
positive immunofluorescence staining for actin and desmin but negative for keratin and

factor VIII antigens and 5. positive contraction reaction with angiotensin II (15). For the

55

experiments, passages 15-24 were used. Specific confluency for different experiments

was determined by preliminary studies.

3.2.3. RT-PCR analysis.

Total RNA was isolated from rat mesangial cells using TRIzol® reagent
(GibcoBRLQ, Grand Island, NY ). Following sodium acetate/ethanol precipitation and
several ethanol washes, RNA was used as a template in RT-PCR amplification procedure.
RT-PCR reaction was carried out using SUPERSCRIPTTM One-Step RT-PCR with
Platinum® T aq. (GibcoBRL‘D, Grand Island, NY) in accordance with manufacturer’s
specifications. Specific primers used were as follows:

RAMP-1: sense: 5’-GGG GAG ACG CTG TGG TG-3’
antisense: 5’-ATG CCC TCA GTG CGC TT-3’
RAMP-2: sense: 5’-GCA ACT GGA CTT TGA TTA GCA G-3’
antisense: 5’-GGC CAG AAG CAC ATC CTC T-3’
RAMP-3: sense: 5’-ACC TGT CGG AGT TCA TCG TG-3’
antisense: 5’-CTT CAT CCG GGG GGT CTT C-3’
CRLR/CL receptor: sense: 5’-GCA GCA GAG TCG GAA GAA GG-3’
antisense: 5’-GCC ACT GCC GTG AGG TGA-3’
GAPDH: sense: 5’-AGA CAG CCG CAT CTT CTT GTG C-3’
antisense: 5’-CTC CTG GAA GAT GGT GAT GG-3’
Reactions were carried out, using Perkin-Elmer Model 9600 thermal cycler, in 50 ul total

reaction volumes subjected to the following conditions: 1) 50°C for 30 min (1 cycle), 2)

94°C for 5 min (1 cycle), 3) 94°C for 30 s; 50-55°C for 30 s; 72°C for 30 s (40 cycles), 4)

56

72°C for 15 min (1 cycle). Products were separated by gel electrophoresis and
subsequently visualized by ethidium bromide staining and UV illumination. Photographs
of the gels were taken and digitalized with UMAX Astra2000P flat-bed scanner. To
assure the identity of the products, cDNA was extracted from the gel and sequenced by
standard dye-termination DNA sequencing procedure. Sequences of cDNAs obtained in
that fashion were analyzed for Similarity to published full-length sequences of RAMPI,
2, 3, and CL receptor via two-sequence comparison method from BLAST database
(http://www.ncbi.nlm.nih.gov/BLAST). These analyses revealed identity of RT-PCR
products to the corresponding full-length cDNA sequences. Several cDNAs acquired by

this method were used as probes in northern blot hybridizations.

3.2.4. RAMP l, 2, 3, and CRLR cloning and expression.

Full-length cDNA of human RAMP-1, 2, 3, and bovine CL receptor were cloned
into Myc-tagged and pCDN vectors obtained from Clonetech (Palo Alto, CA). Multiple
DNA preparation batches were used. Expression vectors were transfected into RMC
using LipofectAMINE PLUSTM Reagent (GibcoBRL®, Grand Island, NY) following the
suggested protocol. In brief, RMC were plated on P-100 tissue culture plates a day prior
to the transfection to achieve an approximate 60-70% confluency. On the following day,
cells were transfected with a total of 2ug of each vector-cDNA in a serum-free
transfection medium for 4 hrs at 37°C/5% C02. The total amount of transfected cDNA
was kept constant by adding empty Myc or pCDN vector. Afterwards, 4ml of serum-
containing medium was added and cells were incubated for an additional 24-36 hrs. This

transient transfection protocol resulted in an approximate 70% transfection efficiency as

57

assessed by green fluorescent protein (GFP). Successful introduction of RAMP-1, 2, and
3 cDNA was further verified by western blot analysis revealing a significant increase in
appropriate membrane-associated RAMP protein expression as compared to vector-

transfected cells.

3.2.5. Membrane preparation and adenylate cyclase assay.

Cells were harvested from P150 plates and homogenized in Tris HCl, pH 7.4,
(10mM)/EDTA (10mM) buffer. Membranes were prepared by homogenization in a
Dounce ground glass homogenizer, centrifuged for 20 min at 12,000g at 4°C and washed
in Tris HCl, pH 7.4, (50mM)/MgCl (10mM) buffer. Final concentration of 40ug of
protein/assay tube was obtained and the membranes were immediately subjected to
adenylate cyclase assay, as follows. Membrane associated adenylate cyclase activity was
measured as the rate of conversion of [a32P]ATP to [32P]cAMP as described by
Elshourbagy et al. (67). Accordingly, membranes were incubated for 20 min at 30°C with
appropriate drugs and assay mix containing ATP regeneration system (SOmM Tris-HCl,
pH 7.4, IOmM MgClz, 1.2 mM ATP, 0.1 mM cAMP, 2.8 mM phosphoenolpyruvate, and
5.2 )4ng myokinase) and 1.0 uCi of [a32Pj-ATP. Total reaction volume was IOOuL,
drug as well as AM concentrations were as described for particular experiments.
Reactions were stopped with 1 ml of stop solution containing 0.28mM “cold” cAMP,
0.33mMATP, and 22,000dpm of 3H-cAMP. Contents of the assay tubes were washed
through a Dowex and subsequently alumina columns to separate the degradation products
of ATP as previously described by Salmon et al. (276). Elution profiles were performed

prior to experiments to determine the amount of water (for Dowex columns) and

58

imidazole (for alumina columns) needed to wash and elute the products. Products eluted
from alumina column were counted for the presence of 3H-cAMP and a32P-cAMP. Each
experiment was done in triplicates, repeated at least 3 times, and expressed as percentage

of AM-mediated adenylate cyclase activity compared to basal.

3.2.6. [3H]thymidine incorporation.

Cells were plated in 24 well plates (30000 cells/well) and grown for 2 days with
subsequent serum starving for 48 hrs. Then, they were treated with the compounds for a
period of 16 hrs and pulsed with [3H]thymidine for 4 hrs. The radioactivity was counted
in Beckman LS counter, after washing the cells and stopping the reaction with 5%
trichloro acetic acid and solubilizing the cells in 0.5 ml of 0.25 N sodium hydroxide.

Each experiment was performed in quadruplicates and repeated at least three times.

3.2.7. Statistical analysis.
Data are presented as meaniS.E.M. Multiple group comparisons were made using
a two-way analysis of variance (ANOVA). Single group comparisons exercised Student’s

t-test method. Statistical significance was set at P<0.05

59

3.3. Results.

3.3.1. AM receptor components in rat mesangial cells.

We first characterized the expression of AM receptor components in quiescent
RMC by RT-PCR as delineated in materials and methods. Under basal conditions, RMC
express CL receptor (formerly known as CRLR) as well as all three subtypes of RAMP-s:

RAMP-1, RAMP-2, and RAMP-3 (Fig. 2).

3.3.2. Effects of RAMP over-expression on AM-induced adenylate cyclase activity
and [3H]thymidine incorporation in RMC.

To investigate the effect of RAMP over-expression on mesangial cell
responsiveness to AM, we transiently transfected RMC with RAMP-1, RAMP-2, and
RAMP-3 and subsequently examined AM-mediated adenylate cyclase activity and
[3H]thymidine incorporation. .

Transfection of RAMP-2 or RAMP-3 resulted in 85.4 e302 and 54.5 21:2.63
percent increase in AM-induced adenylate cyclase activity, respectively (Fig. 3). These
responses were further potentiated by co-transfection of CL receptor with RAMP-2 or
RAMP-3 (136.6zt7.9 and 90.71i3.8 percent increase, respectively). AM-induced
adenylate cyclase activity in RAMP-2 and RAMP-3 transfected cells was also inhibited
by at least 50% following pretreatment of the membranes with 10 LtM AM22.52, AM
receptor antagonist, indicating that this effect is AM receptor mediated (table 3).
Transfection of RAMP-1, on the other hand, had no effect on AM-mediated responses
(Fig. 3) despite successful introduction of RAMP-1 cDNA and subsequent elevation in

RAMP-1 protein expression as verified by western blotting.

60

We further hypothesized that in addition to increasing adenylate cyclase activity,
RAMP overexpression will also potentiate an AM-mediated decrease in [3H]thymidine
incorporation. As predicted, transfection of RAMP-2 or RAMP-3 significantly
potentiated AM-evoked inhibition of [3H]thymidine incorporation (Fig. 4). Again,
RAMP-1 transfection had no significant effect on mesangial cell responsiveness to AM
as assessed by [3H]thymidine incorporation assay (—41.6i4.9 and -29.8:i:5.4 inhibition of

[3H]thymidine incorporation for RAMP-1 and vector alone, respectively; p>0.1).

61

 

Figure 2. Receptor activity modifying protein (RAMP) and calcitonin receptor-like
receptor (CRLR or CL receptor) expression profile in quiescent rat mesangial cells
(RMC). RT-PCR was performed on total RNA isolated from RMC using primer
sequences designed from published sequences as described in materials and methods. The
identity of PCR products resolved by electrophoresis was confirmed by DNA sequencing.
The product of complete reaction devoid of RNA was loaded in lane A (negative control).
Lane B contains the product of reaction carried in the absence of reverse transcriptase
(no-RT control). RMC express detectable levels of CL receptor as well as all 3 subtypes

of RAMPS.

62

 

 

 

 

 

 

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RAMP-3

Vector

Figure 3. Effect of RAMP-l, RAMP-2, and RAMP-3 overexpression on AM-stimulated
adenylate cyclase activity in RMC. RMC were transfected with vector or vectorrRAMP-
l, RAMP-2, or RAMP-3 at z60% confluence. Cells were allowed to grow for another 24-
36 hours. Adenylate cyclase (AC) assay in response to 100 nM AM was performed as
described in materials and methods. Overexpression of RAMP-2 and RAMP-3
significantly enhanced AM-stimulated AC activity in RMC. Overexpression of RAMP-1
had no significant effect on AC activity. *p50.01, n.s.-statistically not significant,

experiments performed in triplicates, n23.

 

 

 

 

 

 

 

 

Vector RAMP-2 RAMP-2 RAMPJ RAMP-3
over- over- over- over-
expressed expressed expressed expressed

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nM AM 20.64 13.02 $4.44 £2.63 $4.60
(percent from basal tSE)

 

 

Table 3. Effect of AMnsz on AM-mediated AC activity in RAMP-2 or RAMP-3 transfected RMC. Prior to

performing the AC activity assay membranes were pretreated with 10 W of AM;;.,;, an AM receptor
antagonist. AM22.52 significantly inhibited AM-mediated AC activity in RAMP-2 and RAMP-3
overexpressed RMC. pS0.01, experiments performed in triplicates, n=3.

63

 

 

 

 

   

 

 

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Figure 4. Effect of RAMP-2 and 3 over-expression on [3H]thymidine incorporation in
RMC. Cells were transiently transfected with RAMP-2 or RAMP-3 and allowed to grow
for additional 24-36 hours. Subsequently, labeled thymidine incorporation in response to
100 nM AM was followed as per protocol described in materials and methods. RAMP-2
and RAMP-3 over-expression significantly potentiated AM-mediated decrease of
[3H]thymidine incorporation in RMC; *p<0.05 compared with vector; experiments

performed in quadruplicates; n=4.

64

3.4. Discussion.

Several laboratories reported the RAMP gene expression profiles for a number of
cell lines and tissues (see section 2.3.4). It is now clear that RAMP mRNA expression
varies among cell lines and can be dramatically altered during different physiological
states. Since RAMPS constitute a structural part of the AM receptor complex and are
capable of modifying its ligand Specificity, it becomes crucial to identify the expression
patterns of these receptor-associated proteins in order to fully understand the biology of
the ligands involved. Here, we have identified for the first time the expression profile of
RAMPS in the rat mesangial cells (RMC). Cultured RMC express in basal conditions all
subtypes of RAMPS as well as CL receptor. This theoretically allows for generation of
both CGRP (CGRP): CL receptor+RAMP-1) and AM (AM1: CL receptor+RAMP-2 and
AM2: CL receptor+RAMP-3) specific receptors and correlates well with previously
reported findings on the responsiveness of cultured RMC to the exogenous administration
of the CGRP family of peptides. Osajima et al. (240) observed a 7-fold increase in AM-
mediated cAMP generation as compared to only 2-fold rise in cAMP levels following
CGRP administration in RMC. In addition, the ICso calculated for the inhibition of
peptide-induced cAMP formation was approximately 10'8 M for AM22.52 as compared to
106M for CGRP3.37. Accordingly, these data point out that both CGRP and AM-specific
receptors are present in RMC but the AM-sensitive receptors are preferentially expressed,
suggesting the predominant role for AM in regulating mesangial cell biology. Of note is
the finding that amylin had no effect on cAMP generation in RMC even at high
concentrations (240). Considering the current understanding of the amylin receptor

biology, this finding together with our RAMP profile data implies the lack of functional

65

calcitonin receptor expression in RMC; this hypothesis however awaits future
confirmation.

The expression of all three subtypes of RAMPS offers an additional theoretical
possibility for AM receptor modulation in RMC. As previously described, compelling
evidence exists to suggest a competitive interaction and homodimer formation among
different RAMP subtypes. This leads to a variable ligand specificity of the membrane-
associated receptors, as it may depend on the quantitative as well as the qualitative nature
of the RAMPS available for the receptor complex formation. For example, transient
expression of CL recepors in UMR-106 cells revealed that AM receptor expression and
AM-specific receptor binding were enhanced with RAMP-2 co-expression but
precipitously reduced by 50 percent with concomitant introduction of RAMP-1 cDNA
(26). Similarly, in rabbit aortic endothelial cells co-expression of RAMP-3 along with
RAMP-1 decreased the functional response to CGRP by 50 percent (212). These findings
may be explained by the direct competition of RAMP interaction with CL receptor, or
alternatively they may reflect a process of RAMP-RAMP hetero- and/or homo-
dimerization, with the latter recently reported by Sexton et al. (294).

The AM-dependent cAMP formation and the related antiproliferative effect of
AM on mesangial cells have been previously demonstrated by our laboratory as well as
others (40), (247), (292). Activation of PKA has been shown to induce an antimitogenic
effect on mesangial cells. Because AM increases adenylate cyclase activity and hence
PKA, we hypothesized that overexpression of RAMP-2 and RAMP-3, in addition to
increasing adenylate cyclase activity, will also potentiate an AM-mediated decrease in

[3Hjthymidine incorporation. As predicted, transfection of RAMP-2 or RAMP-3

66

significantly potentiated AM-evoked inhibition of [3H]thymidine incorporation (Fig.4).
Since the excessive mesangial cell proliferation (as indexed by [3H]thymidine
incorporation) is a hallmark pathological change present in several renal diseases (160),
(77), (64), these findings suggest that the postulated reno-protective effects of AM may at
least in part be modulated at the receptor level. In particular, the upregulation of RAMP-2
and RAMP-3, the integral components of AM receptor complex, increases the cell’s
responsiveness to AM-mediated antiproliferative effects.

The overexpression of RAMP-1 had no significant effect on mesangial cell
responsiveness to AM as assessed by adenylate cyclase activity and [3H]thymidine
incorporation assays. These findings are consistent with previously reported data
describing CL receptor-RAMP-l as a receptor complex with low AM binding specificity
(26), (83). Also, it is worth noting that the overexpression of RAMP-1 had no significant
effect on the basal adenylate cyclase activity as the vector transfected and the RAMP-1
transfected cells exhibited a comparable response to AM stimulation (Fig. 3). This
observation comes as a contrast to the proposed hypothesis of competitive interaction
among the RAMP subtypes, perhaps once again underscoring the importance of cell-
specific differences in RAMP behavior, which has been previously discussed in section
2.3.4.

In summary, experimental results presented herein demonstrate that RMC express
all three subtypes of RAMPS in basal conditions. Overexpression of RAMP-2 or RAMP-
3 (but not RAMP-1) leads to a predicted change in functional responsiveness of RMC to
AM. Namely, RMC transfected with exogenous RAMP-2 or RAMP-3 cDNA exhibit a

significant decrease in AM-mediated cell proliferation as assessed by [3H]thymidine

67

incorporation. These results suggest that the effects of AM on RMC proliferation may at
least in part be dependent on the profile of RAMP expression and/or cellular availability
for intermolecular interactions. Moreover, the membranous adenylate cyclase activity in
response to AM is also significantly increased in RAMP-2 or RAMP-3 transfecter RMC.
This response appears to be AM receptor specific as pretreatment with AM22-52, an AM
receptor antagonist, abolishes it (Table 3).

Since alteration in RAMP-1 gene expression does not appear to affect the actions
of AM in mesangial cells, we will refrain from further consideration of RAMP-l gene

regulation.

68

4. Effects of platelet-derived growth factor and lipopolysaccharide on

RAMP expression in rat mesangial cells.

4.1. Introduction.

Platelet-derived growth factor (PDGF) is a well-characterized cytokine
secreted by many cell types including the glomerular mesangial cells. In mesangial cells
PDGF acts in an autocrine and paracrine fashion as their most potent mitogen (2), (200),
(79), (3). By interacting with a specific tyrosine kinase receptor (PDGF receptor,
PDGFR), widely expressed by mesangial cells, PDGF has been shown to significantly
increase mesangial cell thymidine incorporation rate as well as augment the synthesis of
extracellular matrix components (197), (360). In addition, PDGF also potently induces
directed mesangial cell migration (17), (161). A number of studies suggest that PDGF is
not only a causal factor in development of mesangial hypercellularity and excessive
matrix accumulation (the fundamental changes observed in several glomerular disease
states) but also participates in mesangial cell repopulation following sub-total glomerular
damage characteristic to several forms of glomerulonephritis (1). Consequently, PDGF
has been recognized as a prime cytokine responsible for mediating both proliferative and
migratory responses in the mesangium during glomerular injury (1), (241), (188). The
clearly opposing effects of AM and PDGF on cell growth were examined during the
initial characterization of then newly discovered AM polypeptide. AM was found to
potently decrease PDGF-induced mesangial cell proliferation (39), (292). Our current
understanding of AM biology, especially at the receptor level, offers a new angle on

investigation of the regulation of proliferative/anti-proliferative events in mesangial cells.

69

In particular, the mechanisms responsible for the functional interplay between PDGF and
AM receptor deserve firrther examination. Hence, the present study was undertaken to
examine the possible effect(s) of PDGF on AM receptor complex expression in rat
mesangial cells.

The endotoxin, lipopolysaccharide (LPS), is a known component of the outer
membrane of Gram-negative organisms that plays a crucial role in mediating sepsis-
evoked circulatory failure. Recently it has been argued that in addition to inducing
generalized nitric oxide formation (256), (51), (209) and downregulating the angiotensin-
aldosterone system (24), LPS achieves its effects by directly influencing AM activity. In
fact, LPS has been proposed as a leading mediator responsible for stimulation of AM
production during sepsis in viva (366), (305), (301), (308) and in a cell culture setting
(312). The dramatic rise in intravascular levels of AM during sepsis has been reported in
humans as well as animals and is estimated to be greater than in any other pathological
condition studied (117), (228), (351). Careful examination of the circulating levels of AM
revealed its robust increase during the early, hyperdynamic phase of sepsis characterized
by an increase in cardiac output, decrease in total peripheral resistance, hyperglycemia,
and hyperinsulinemia (349), (365), (348). The above-mentioned physiological changes
have also been observed in healthy humans exposed to intravenous AM (175). Wang et
al. demonstrated the causative effects of AM in sepsis by Studying rats after intravenous
synthetic AM administration. Inducing circulating concentrations of AM that resemble
those during clinical sepsis resulted in a hyperdynamic response characteristic of early
sepsis. Moreover, administration of anti-AM neutralizing antibodies to animals after

induction of sepsis by cecal ligation and puncture procedure prevented the development

70

of the hyperdynamic response (348). In addition, the AM-induced hyperdynamic state
has been reported to maintain renal blood flow during the early endotoxic Shock thereby
protecting against kidney failure (228). Interestingly, it has (been documented that despite
the sustained elevation in AM levels throughout sepsis, a marked decrease in vascular
responsiveness to AM takes place during the later stages characterized by multi-organ
failure and circulatory collapse. This presumably facilitates the transition from the
hyperdynamic to the hypodynamic state where marked increase in vascular resistance,
decrease in cardiac output and peripheral oxygen delivery are concomitantly observed
(350). It is hypothesized that the initial responsiveness to elevated AM levels prolongs
the hyperdynamic state thus protecting against the cardiovascular failure while the later
loss of AM reactivity culminates with multiple organ failure and impending mortality.
The mechanism responsible for these changes is not currently well understood. However
the process of AM receptor desensitization, which has been documented to occur in
vascular smooth muscle (129) as well as mesangial cells (245), may potentially explain
the observed shift in AM sensitivity during the progression of sepsis. Alternatively,
changes in AM receptor affinity and/or receptor-adenylate cyclase uncoupling as well as
the direct effect of LPS on AM receptor complex gene expression could also provide for
the observed temporal changes in AM receptor activity. Despite the ambiguity regarding
the exact mechanisms involved, researchers agree on the beneficial effects of AM activity
during sepsis. The protective role of AM has been elegantly demonstrated by Shido el al.
(302), who reported that transgenic mice overexpressing AM exhibited a remarkable
resistance to the induction and progression of septic shock. Mice overexpressing AM in

their vasculature showed greater vasomotor stability, diminution in the end-organ

71

damage, and consequently greater survival rates following LPS challenge equated to
bacterial endovascular burden encountered in septicemia.

The production and cellular responsiveness of AM- are clearly affected by both
PDGF and LPS. In View of the wide spectrum of pathologies with PDGF and/or LPS
involvement as well as the fact that AM appears to play an important protective role
during the development and progress of these pathological states, further investigation of
the interactions between AM and these regulatory molecules needs to be considered. In
particular little is currently known about the effects of PDGF and LPS at the AM receptor
level. PDGF and/or LPS can conceivably regulate the gene expression of the AM
receptor components, thus leading to the observed alterations in AM responsiveness.
Accordingly, this chapter will describe the experiments designed to inspect the possible
effect(s) of PDGF and LPS on the mRNA and protein expression of the AM receptor

complex (AM; and AM;) present in mesangial cells.

4.2. Materials and methods.
4.2.1. Materials.

Adrenomedullin, adrenomedullin(22-52) fragment and PDGF-BB were purchased
from Sigma” RBI® (St. Louis, MO). Lipopolysacccharide, Ecoli 055:B5 was from
Calbiochem (La Jolla, CA). RPMI-1640, fetal bovine serum, penicillin/streptomycin,
trypsin-EDTA were from GibcoBRL0 (Grand Island, NY). RAMP-2 and RAMP-3

polyclonal primary antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz,

72

CA). Horseradish peroxidase-conjugated secondary Anti-rabbit IgG antibodies were from
Sigma” R131® (St. Louis, MO).

All other reagents were of highest quality available.

4.2.2. Cell Culture.

Rat mesangial cell (RMC) cultures were established from glomeruli as described
in section 3.2.2. RMC were maintained in RPMI-1640 with 15% fetal bovine serum
unless otherwise stated for particular experiments. Passages 15-24 were used for

subsequent experiments with Specific confluency determined by preliminary studies.

4.2.3. RAMP l, 2, 3, and CRLR cloning and expression.

Full-length cDNA of human RAMP-1, 2, 3, and bovine CL receptor were cloned
into Myc-tagged and pCDN vectors obtained from Clonetech (Palo Alto, CA). Multiple
DNA preparation batches were used. Expression vectors were transfected into RMC
using LipofectAMINE PLUSWI Reagent (GibcoBRL®, Grand Island, NY) following the
suggested protocol. In brief, RMC were plated on P-100 tissue culture plates a day prior
to the transfection to achieve an approximate 60-70% confluency. On the following day,
cells were transfected with a total of 211g of each vector-cDNA in a serum-free
transfection medium for 4 hrs at 37°C/5% C02. The total amount of transfected cDNA
was kept constant by adding empty Myc or pCDN vector. Afterwards, 4ml of serum-
containing medium was added and cells were incubated for an additional 24-36 hrs. This
transient transfection protocol resulted in an approximate 70% transfection efficiency as

assessed by green fluorescent protein (GFP).

73

4.2.4. Membrane preparation and adenylate cyclase assay.

Cells were harvested from P150 plates and homogenized in Tris HCl, pH 7.4,
(10mM)/EDTA (10mM) buffer. Membranes were prepared by homogenization in a
Dounce ground glass homogenizer, centrifuged for 20 min at 12,000g at 4°C and washed
in Tris HCl, pH 7.4, (50mM)/MgCl (10mM) buffer. Final concentration of 40ug of
protein/assay tube was obtained and the membranes were immediately subjected to
adenylate cyclase assay, as follows. Membrane associated adenylate cyclase activity was
measured as the rate of conversion of [a32P]ATP to [32P]cAMP as described by
Elshourbagy et a1. (67). Accordingly, membranes were incubated for 20 min at 30°C with
appropriate drugs and assay mix containing ATP regeneration system (SOmM Tris-HCl,
pH 7.4, IOmM MgClz, 1.2 mM ATP, 0.1 mM cAMP, 2.8 mM phosphoenolpyruvate, and
5.2 jig/ml myokinase) and 1.0 uCi of [0132P]ATP. Total reaction volume was IOOuL, drug
as well as AM concentrations were as described for particular experiments. Reactions
were stopped with 1 ml of stop solution containing 0.28mM “cold” cAMP, 0.33mM
ATP, and 22,000dpm of 3H-cAMP. Contents of the assay tubes were washed through a
Dowex and subsequently alumina columns to separate the degradation products of ATP
as previously described by Salmon et al. (276). Elution profiles were performed prior to
experiments to determine the amount of water (for Dowex columns) and imidazole (for
alumina columns) needed to wash and elute the products. Products eluted from alumina
column were counted for the presence of 3H-cAMP and a32P-cANIP. Each experiment
was done in triplicates, repeated at least 3 times, and expressed as percentage of AM-

mediated adenylate cyclase activity compared to basal.

74

4.2.5. [3H]thymidine incorporation.

Cells were plated in 24 well plates (30000 cells/well) and grown for 2 days with
subsequent serum starving for 48 hrs. Then, they were treated with the compounds for a
period of 16 hrs and pulsed with [3H]thymidine for 4 hrs. The radioactivity was counted
in Beckman LS counter, after washing the cells and stopping the reaction with 5%
trichloro acetic acid and solubilizing the cells in 0.5 ml of 0.25 N sodium hydroxide.

Each experiment was performed in quadruplicates and repeated at least three times.

4.2.6. Northern blot analysis.

Immediately after cell culture medium was aspirated from the tissue culture
plates, 2-4 ml of TRIzol” was added, dispersed uniformly, and plates were stored at -
80°C until firrther use. Following a quick thaw, cells were scraped with cell lifters and
transferred to 15 ml centrifuge tubes. Total cellular RNA was isolated with TRIzol®
according to manufacturer’s specifications. RNA was precipitated by adding 3 M sodium
acetate and absolute ethanol, washed with 75% ethanol, pelleted in microcentrifuge tubes,
and dried prior to resuspension in RNAse-free water. Its purity was checked by
measuring the ratio of absorbance@260m/absorbance@2go..m. All of the RNA used had the
ratio equal or greater to 1.8. A standardized aliquot of RNA (30 ug) was separated by
electrophoresis on a formaldehyde agarose denaturing gel and transferred to an Optitran®
membrane (Schleicher & Schuell, Keene, NH) by capillary transfer. Subsequently, RNA
samples were immobilized to the membrane by ultraviolet cross-linking. Membranes
were successively hybridized at 42°C for 16-24 hrs with four parts (15 ml of Fonnamide,

0.6 ml Denhardt's solution, 1.5 ml of 1 M phosphate buffer, 7.5 ml of 20x SSC, 1.5 ml of

75

SDS, 2.4 ml of diethylpyrocarbonate water, and 1.5 ml of salmon sperm DNA)/blot and
one part 32P-dCTP-labeled cDNA probes (specific for RAMP-l, 2, 3 or 188 ribosomal
subunit; RAMP probes were obtained as RT-PCR products. using RAMP-specific primers
designed from published sequences). The cDNA was radiolabeled using a random prime
labeling kit. Following hybridization for 16-24 hrs, the membranes were washed and
placed in an x-ray cassette for the requisite exposure time. Signals were quantitated by

phosphoimager analyses and expressed relative to 183 levels.

4.2.7. Western blot analysis.

Western blot analysis was done as described before (247). Briefly, equal
concentrations of protein samples were subjected to sodium dodecyl sulfate
polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The
membranes were subsequently blocked with 5% non-fat dry milk in Tris buffered saline
containing 0.05% Tween-20 (TBS-T) and incubated with RAMP-2 or RAMP-3
polyclonal primary antibodies at final concentration of 400 ng/ml followed by horse
radish peroxidase-conjugated secondary Anti-rabbit IgG antibodies at final dilution of
1:10,000, according to manufacturer’s instructions. Enhanced chemiluminescence (ECL)

kit (Pierce, Rockford, IL) was used to Visualize the blots.

4.2.8. Statistical analysis.
Data are presented as meaniS.E.M. Multiple group comparisons were made using
a two-way analysis of variance (ANOVA). Single group comparisons exercised Student’s

t-test method. Statistical significance was set at P<0.05

76

4.3 Results.

4.3.1. Effect of PDGF on RAMP mRNA expression.

In concordance with findings from other cell systems, over-expression of RAMP-
2 and RAMP-3 in mesangial cells also led to potentiated AM responsiveness (fig. 3).
Considering the fact that altered RAMP expression has been reported in several disease
states and that PDGF is a prime factor responsible for pathOphysiological changes in
glomerular biology, we hypothesized that PDGF may also modify RAMP expression. To
test this hypothesis, we investigated the effects of PDGF-BB on mesangial cell RAMP
mRNA abundance. Exposure of mesangial cells to exogenous PDGF (0.1-100 ng/ml)
increased RAMP-3 mRNA expression in a concentration-dependent manner (Fig. 5A)
while it had no effect on RAMP-2 mRNA abundance (Fig. SB).

In order to evaluate the temporal character of the PDGF-induced RAMP-3 mRN A
expression, we quantified the RAMP-3 mRNA abundance during the 48 hour period
following introduction of 50 ng/ml PDGF into the mesangial cell culture medium.
Significant increase in RAMP-3 mRNA levels was observed within 6 hours post PDGF
exposure with a peak elevation noted at 24 hours (Fig. 6). Thus RAMP-3 gene
upregulation in response to 50 ng/ml PDGF is time-dependent within the exposure period

studied.

77

i?
3

 

 

 

 

= 9
e
.5 g 100
m N 90
o .9
’5. E 80
5 g 70
«g ‘3 60
en
E g 50
E a 33
Z a 20
2 8 10
I-
E a. .
V 0.1 1 10 50 100
PDGF (ng/ml)

Figure 5A. Effect of platelet-derived grth factor (PDGF) on RAMP-3 mRNA
expression in RMC. Cells were treated with vehicle (basal) or doses of PDGF, as
indicated, for 24 hours. RNA was extracted and northern blot analysis performed as
described in materials and methods. Blots were first probed for RAMP-3 and then
stripped and reprobed for 183 RNA. Raw values were converted to ratios of RAMP-3
mRNA to 18s and then expressed as percent change from basal. A-a: PDGF increased
RAMP-3 mRNA expression in a dose-dependent manner; *p<0.01, **p<0.001 as

compared with 0.1ng/ml PDGF dose, n=5.

78

A-b A-c _

PDGF ng/ml 0 0.1 1 I0 50

V
ridg‘;a ’\

'-

288 ’3' ""5 :21“ V1 i‘i‘f

 

Figure 5A. Effect of platelet-derived growth factor (PDGF) on RAMP-3 mRNA
expression in RMC. A-b and A-c: representative Northern blots of RAMP-3 expression

in response to PDGF in RMC.

79

A
ns 5‘3
3‘3

8

 

m
U
G
a.

 

 

 

 

 

RAMP-2 mRNA expression
(percent basal) _
S

PDGF
BASAL SOng/ml

Figure 5B. Effect of platelet-derived growth factor (PDGF) on RAMP-2 mRN A
expression in RMC. Cells were treated with vehicle (basal) or doses of PDGF, as
indicated, for 24 hours. RNA was extracted and northern blot analysis performed as
described in materials and methods. Blots were first probed for RAMP-2 and then
stripped and reprobed for 18s RNA. Raw values were converted to ratios of RAMP-2
mRN A to 18s and then expressed as percent change from basal. B: PDGF had no
significant effect on RAMP-2 expression as compared to basal; n.s.-statistically not
significant, n=4. Insert shows a representative northern blot of RAMP-2 expression in

response to PDGF in RMC.

80

\D
O
1

\l
O
1

Us
6
1

s—s
O
I

   

 

I
H
O

p

t—

0.5 2 6 12 24 48

Time (hrs)

RAMP-3 mRNA expression
(percent change from basal)
U
6

Figure 6. Effect of platelet-derived growth factor (PDGF) on RAMP-3 mRN A expression
in RMC. Cells were treated with vehicle (basal) or 50 ng/ml PDGF for time periods as
indicated. RNA was extracted and northern blot analysis performed as described in
materials and methods. Blots were first probed for RAMP-3 and then stripped and
reprobed for 183 RNA. Raw values were converted to ratios of RAMP-3 mRNA to 18s
and then expressed as percent change from basal. PDGF increased RAMP-3 mRNA

expression in a time-dependent manner.

81

4.3.2. Effect of PDGF on RAMP protein expression and AM-mediated adenylate
cyclase activity.

To determine whether the PDGF-induced increase in RAMP-3 mRNA
corresponds to an elevated expression of RAMP-3 protein, we examined RAMP-3
protein levels in the membrane-associated fraction of cells cultured in the presence or
absence of PDGF. Western blot data revealed a 3.3 $0.28 fold increase in the amount of
RAMP-3 protein in cells exposed to PDGF as compared to controls (Fig. 7). As
predicted, PDGF-induced elevation of RAMP-3 correlated to a functional increase in cell
responsiveness to various concentrations of AM as measured by AM-mediated adenylate
cyclase activity (Fig. 8A). Also, membranes of cells treated with various concentrations
of PDGF (1-100 ng/ml) exhibited a concentration-dependent increase in AM-stimulated
AC activity (Fig. 8B). This effect was inhibited by pre-treatment of membranes with

AM(22-52), suggesting a direct involvement of AM-specific receptors (Fig. 8C).

82

h
I

U
L

fl
1

 

 

 

 

 

G

 

Membrane-associated RAMP-3
protein expression (fold basal)
N

BASAL PDGF

Figure 7. Effect of PDGF on membrane-associated RAMP-3 protein expression in RMC.
Cells were incubated with PDGF (50 ng/ml) for 24 hours. Subsequently membranes were
extracted as per membrane preparation protocol for AC assay (see materials and
methods) and equal concentrations of protein were loaded onto a gradient polyacrylamide
gel. Western blotting was performed as described in the materials and methods. Raw
values were converted to ratios of RAMP-3 protein to actin and expressed as fold of basal
(with basal expression arbitrarily set at 1). Right: insert represents an exemplary Western
blot obtained in this set of experiments. PDGF significantly increased RAMP-3 protein

expression in the membrane-associated fraction of RMC. *p<0.001; n=4.

83

 

 

 

 

 

 

80‘ *
’5 G 70.. ClControl
«g a 60- IPDGF(50ng/m1)
a in 50-
a E
2.? 4"
o E. 30‘
*3 g 20~
">1 Lt
g a 10-
i v 0‘—=;=fi I I

 

401 1 10 100
AM (nM)

 

Figure 8. Effect of PDGF on AM-mediated adenylate cyclase activity in RMC. Cells
were incubated in the presence or absence of PDGF (50 ng/ml) for 24 hours. Membranes
were then extracted and AC activity assay in response to indicated concentrations of AM
was performed. A: AC activity increased with increasing doses of AM in PDGF treated
and controls. PDGF treated cells exhibited significantly higher AC activity as compared
to corresponding basals (*p<0.01) and control treated cells (“#p<0.01). Experiments were

performed in triplicates, n=5.

84

8

8

 

Adenylate cyclase activity
(percent from basal)
8 8

 

   

 

 

G

 

     

10 so 100 PDGF (ng/ml)

AM 100 nM

Figure 8. Effect of PDGF on AM-mediated adenylate cyclase activity in RMC. B: Cells
were treated with different concentrations of PDGF, as indicated, for 24 hours.
Membranes were then extracted and AC activity assay in response to 100 nM AM was
performed. PDGF caused a concentration-dependent increase in AM-stimulated AC
activity, which closely correlated with PDGF-stimulated RAMP-3 mRNA expression

(Fig. 5A); experiments were done in triplicates, n=3.

85

O

1

H

83388

I

l
t...

 

1
+1.

 

 

 

 

Adenylate cyclase activity
(percent from basal)
6
i

 

 

Control
+ ++ + ++ AM-(22-52)

Figure 8. Effect of PDGF on AM-mediated adenylate cyclase activity in RMC. C: Effect
of AM-(22-52), the AM receptor antagonist, on AM-mediated AC activity in rat
mesangial cells exposed to PDGF. Cells were grown in the presence of PDGF (50 ng/ml)
or its absence (control) for 24 hours. Next, membranes were extracted and pre-treated
with AM-(22-52) at 100 nM (+) or 111M (H) for 10 minutes prior to AC activity assay in
response to 100 nM AM. PDGF-dependent increase in AM-mediated AC activity was
significantly inhibited by AM receptor antagonist, AM-(22-52); *p<0.01 (as compared
with PDGF treatment), *p<0.01 (as compared with control); experiments performed in

triplicates; n=3.

86

4.3.3. Effect of LPS on RAMP mRNA expression.

Similar to the PDGF findings, LPS treatment of mesangial cells also induced the
mRNA expression of RAMP-3. The LPS-evoked increase‘in RAMP-3 mRNA was dose-
dependent with the LPS concentrations of 1-30 ug/ml (Fig. 9). LPS concentrations above
30 ug/ml appeared to be highly toxic to mesangial cells, resulting in gross cell structure
changes and loss of vital cell adhesion properties. RAMP-3 mRNA expression was
significantly increased by 30 minutes following the introduction of 10 ug/ml LPS into the
cell culture medium and remained elevated throughout the 24-hour period studied (Fig.
10).

Next, we investigated the effect of LPS on RAMP-2 gene expression by RT-PCR
and northern blot analysis. Exposure of mesangial cells to various concentrations of LPS
(1-30 rig/ml) did not affect the basal levels of RAMP-2 expression as assessed by RT-

PCR (data not shown) and northern blot analysis (see Fig 11. for representative data).

87

N
UI
G

N
G
©

t—1
UI
O
.t____-r#¥-r_-fiu_‘ l

   

U!
G

RAMP-3 mRNA expression
(percent from basal)
e E

 

1 10
LPS (pg/m1)

Figure 9. Effect of lipopolysaccharide (LPS) on RAMP-3 mRN A expression in RMC.
Cells were incubated with vehicle (basal) or doses of LPS, as indicated, for 24 hours.
RNA was extracted and northern blot analysis performed as described in materials and
methods. Blots were first probed for RAMP-3 and then stripped and reprobed for 185
RNA. Raw values were converted to ratios of RAMP-3 mRNA to 18s and then expressed
as percent change from basal. LPS increased RAMP-3 mRN A expression in a dose-

#fl
1.

dependent manner; *p<0.001 as compared with basa p<0.001 as compared with l

ug/ml LPS dose; n=3.

88

50“

 

RAMP-3 mRNA expression
(percent change from basal)
:-

 

G
t

0.5 4

Time (hrs)

 

Figure 10. Effect of lipopolysaccharide (LPS) on RAMP-3 mRNA expression in RMC;
temporal effect. Cells were treated with vehicle (basal) or 10 11ng LPS for time periods
as indicated. RNA was extracted and northern blot analysis performed as described in
materials and methods. Blots were first probed for RAMP-3 and then stripped and
reprobed for 185 RNA. Raw values were converted to ratios of RAMP-3 mRNA to 185
and then expressed as percent change from basal. LPS significantly increased RAMP-3
mRNA expression by half an hour of exposure. Peak elevation of RAMP-3 mRN A
expression is observed within one hour of LPS exposure and it is sustained during the 24-

hour period; *p<0.001 as compared with basal; n=3.

89

1 10 30 LPS (pg/ml)

 

 

Figure 11. Effect of lipopolysaccharide (LPS) on RAMP-2 mRNA expression in RMC.
Cells were treated with vehicle (basal) or doses of LPS, as indicated, for 24 hours. RNA
was extracted and northern blot analysis performed as described in materials and
methods. Blots were first probed for RAMP-2 and then stripped and reprobed for 18s
RNA. Depicted above is a northern blot of RAMP-2 expression representative of three
independent experiments. LPS, at concentrations examined, has no significant effect on

RAMP-2 mRN A expression as compared to basal.

90

4.3.4. Effect of LPS on RAMP protein expression and AM-mediated adenylate
cyclase activity.

Parallel to the LPS-mediated increase in RAMP-3 mRNA, we also noted an
elevation in RAMP-3 protein expression in the cell membrane fraction of mesangial cells
treated with 10 11ng LPS for 24 hours. Specifically, a 2.942t0.23 fold increase in
RAMP-3 membrane-associated protein was observed (Fig. 12).

Since previously observed elevation in RAMP-3 protein expression resulted in a
concomitant increase in mesangial cell responsiveness to AM, we tested if this was also
true for the LPS-induced increase in RAMP-3 protein. Our data indicate, that exposure of
mesangial cells to LPS also increases AM-mediated adenylate cyclase activity. As
predicted, this effect is inhibited by AM22.52 in a concentration dependent manner, hence
it appears to be AM receptor specific (Fig. 13). Furthermore, the significant increase in
AM responsiveness following LPS treatment is observed in membranes stimulated with
varied AM concentrations, thus augmenting a basal, AM dose-dependent trend in

adenylate cyclase activity (Fig. 14).

91

 

 

 

 

 

 

”Arte BASAL LPS
|— '10-'29,
2a ‘ -. 9:
“gt:

:1
.E‘c
Oo-
¢%2-
m
“2
ta-
:0
3.51-
as
Ee
or...
EQ-

0

 

 

 

 

 

BASAL LPS

Figure 12. Effect of lipopolysaccharide (LPS) on membrane-associated RAMP-3 protein
expression in RMC. Cells were incubated with LPS (10 ug/ml) for 24 hours.
Subsequently membranes were extracted as per membrane preparation protocol for AC
assay (see materials and methods) and equal concentrations of protein were loaded onto a
gradient polyacrylamide gel. Western blotting was performed as described in the
materials and methods. Raw values were converted to ratios of RAMP-3 protein to actin
and expressed as fold of basal (with basal expression arbitrarily set at 1). Insert represents
an exemplary Western blot obtained in this set of experiments. LPS significantly
increased RAMP-3 protein expression in the membrane-associated fraction of RMC.

*p<0.01; n=4.

92

 

 

 

 

 

 

 

Q 100 -

.2 '7:

H

g a so -

3'2

.3 E 60 —

ash-t

8 E 40 — r

'2 a 20

>1 9 -

5 E,- #

3 0 W
Control LPS + ++ AM

2252

Figure 13. Effect of lipopolysaccharide (LPS) on AM-mediated adenylate cyclase activity
in RMC; response to pre-treatment with AM22.52. Cells were incubated in the presence of
LPS (10 ug/ml) or its absence (control) for 24 hours. Membranes were then extracted and
AC activity assay in response to 100 nM AM was performed. LPS treated cells exhibited
significantly higher AC activity as compared to control treated cells; *p<0.01.
Membranes pre-treated with AM22-52 (AM receptor antagonist) at 100 nM (+) or 111M
(++) for 10 minutes prior to AC activity assay exhibited significantly attenuated response
to 100 nM AM. #p<0.01 (as compared with LPS treatment; experiments performed in

triplicates; n=3.

93

$8-

70 - DControl
I LPS (10ug/ml)

 

(percent from basal)

 

Adenylate cyclase activity

-10 J 1 10 100
AM (nM)

Figure 14. Effect of lipopolysaccharide (LPS) on AM-mediated adenylate cyclase activity
in RMC; response to varied concentrations of AM. Cells were incubated in the presence
or absence of LPS (10 ug/ml) for 24 hours. Membranes were then extracted and AC
activity assay in response to indicated concentrations of AM was performed. AC activity
increased with increasing doses of AM in LPS treated cells and controls. LPS treated
cells exhibited significantly higher AC activity as compared to corresponding basals
(*p<0.01) and control treated cells (“p<0.01). Experiments were performed in triplicates,

n=5 .

94

4.4. Discussion.

Mesangial cells express a functional AM receptor complex (AM1 and AM;) in
culture. As observed by several investigations of various cell lines, over-expression of
RAMP-2 or RAMP-3 also increases RMC responsiveness to AM as measured by AM-
induced adenylate cyclase activity ((83), (121) and Fig. 3). Fraser et a1. (83) and
Husmann et al. (121) have reported that co-transfection of HEK-293 and COS-7 cells
with CL receptor and RAMP2/3 resulted in an increased 12’I-AM binding affinity and an
augmented cAMP accumulation in response to various concentrations of AM. Clearly,
modification of RAMP expression can serve as a major mechanism for effectively
altering cell’s responsiveness to AM not only in other cell types, but also in mesangial
cells. Here, we propose that two factors that are known to influence the adrenomedullin
signaling system in mesangial cells: PDGF and LPS, may modulate this system through
their action(s) on RAMP expression.

PDGF is a well-known, important autocrine/paracrine growth factor in mesangial
cells and has been shown to interact with adrenomedullin anti-proliferative pathway.
Specifically, PDGF-induced mitogenesis is blocked by adrenomedullin in rat mesangial
cells in culture (39), (292). Indeed, if PDGF enhances adrenomedullin signaling in
mesangial cells, it may serve as a negative feedback mechanism in regulating normal
turnover of mesangial cells. Thus, we investigated the effects of PDGF on
adrenomedullin receptor system, including its effects on CL receptor, RAMP2 and
RAMP3 in rat mesangial cells. We report here that PDGF regulates adrenomedullin
receptor signaling through modulation of RAMP3 mRN A expression. PDGF did not have

any effect on CL receptor (data not shown) or RAMP2 mRNA expression. Others have

95

found similar results in both animal and cell culture models. For example, Totsune et a1.
presented findings from a rat model of 5/6 nephrectomy where RAMP-2 mRNA levels
were unchanged while a significant decrease in RAMP-3 mRNA was found in the
remnant kidney (330). In general, ample evidence exists for differential expression of
RAMP mRNA in tissues from animal disease models, including those with renal
pathology (217), (237), (331), (236), (47), (263), (369). Frayon et al. have reported that
glucocorticoid treatment in VSMC in Culture causes a transient increase in RAMP-1
mRNA expression without any change in RAMP-2 expression (84). This glucocorticoid-
directed RAMP expression results in a CGRP responsive receptor (CGRP1: CL
receptor+RAMP-1). These results also suggest that in vivo, glucocorticoid regulation of
RAMPI mRN A expression may lead to a subsequent consequence on ligand recognition.
Kitamuro and colleagues (159) observed suppression of RAMP-2 (and no change in
RAMP-1) mRNA expression in two neuroblastoma cell lines following a
pharmacological induction of hypoxic state with cobalt chloride and desferrioxamine
mesylate. They hypothesized that the differential expression of RAMP-2 may in fact
constitute a viable mechanism for cellular adaptation to hypoxic stress by modifying AM
receptor complex expression. Similarly, Robert-Nicoud et al. reported vasopressin-
induced RAMP-3 mRNA levels in mouse clonal cortical collecting duct (CCD) principal
cell line (268). To date, it is not known if the well-established role for RAMP-3 in AM
receptor pharmacology can be extended to other, unrelated systems such as that of
vasopressin. However, ample evidence from related studies supports the hypothesis that
PDGF-mediated changes in RAMP-3 expression in mesangial cells may have functional

consequence in terms of adrenomedullin signaling. In fact, our results suggest that

96

PDGF-induced RAMP-3 expression leads to an increase in AM-stimulated adenylate
cyclase activity. This effect, if present in vivo, may augment adrenomedullin signaling
and hence increase the anti-proliferative effect of AM, opposing the proliferative effect of
PDGF. This negative feedback mechanism may be present to keep the mesangial cell
grth in check. Alternatively, this system of PDGF-induced RAMP-3 expression may
be altered or absent under disease conditions thus resulting in an aberrant negative
feedback and consequently leading to uncontrolled mesangial growth. While this
hypothesis is attractive, clearly further studies are essential. It is worth noting that PDGF
had no significant effect on RAMP-2 expression suggesting a differential regulation
between closely related RAMP-2 and RAMP-3.

As alluded to previously, LPS exerts direct effect on AM secretion resulting in a
large increase in the circulating levels of this polypeptide during sepsis. In turn, the high
levels of AM during sepsis bear a great importance on the overall outcomes following the
septic insult. Studies using transgenic mice overexpressing AM } in their vasculature
showed a marked protective role of AM during LPS induced sepsis. Transgenic mice
responded to LPS administration with a smaller decrease in blood pressure, less severe
end-organ damage, and overall significantly greater 24-hour survival rate as compared to
the control animals (302). Recent studies suggest that LPS may also influence the AM
receptor system. Ono er al. reported that the high basal expression of CL receptor and
RAMP-2 mRNA in lungs of normal mice was markedly decreased in endotoxemic mice
treated with LPS. Furthermore, they noted that under these conditions RAMP-3 gene
expression increased significantly in lungs, spleen, and thymus (237). Similarly, research

from another laboratory also reported a marked upregulation in pulmonary RAMP-3 gene

97

expression following an induction of polymicrobial sepsis by cecal ligation and puncture
(CLP) in male rats (238). Interestingly, this effect was seen only during the early stage of
sepsis and was absent 20 hours following CLP procedureAlso, in contrast to the findings
reported by Ono et al., they found no alterations in the expression of CL receptor or
RAMP-2 in lung tissues at 5 or 20 hours post CLP. Altogether, these reports present a
strong evidence for LPS-directed AM receptor complex regulation. In addition, they
document a differential response of the individual AM receptor components during the
state of sepsis.

The current investigation identifies LPS as an extracellular agent capable of
potent induction of RAMP-3 gene expression and RAMP-3 protein production in
cultured rat mesangial cells. This effect may provide at least a partial mechanism
responsible for LPS interactions with the AM system discussed in the previous sections.
To that extent, our experimental findings clearly demonstrate that the LPS-mediated
elevation in RAMP-3 protein levels is accompanied by a significant increase in mesangial
responsiveness to AM. Specifically, cells exposed to LPS concentrations similar to those
observed in fulminant septicemia showed a greater than 90 percent increase in cAMP
production in response to AM stimulation (Fig. 13). Considering that the decline in
vascular reactivity of the end-organs to AM and the consequent transition to the
hypodynamic phase of sepsis hallmark the impending morbidity and mortality of
endotoxic shock ((347), (302)), the effect of LPS on the AM activity in RMC observed
here may indeed provide for a protective mechanism.

As already noted for PDGF, LPS also did not exhibit any effects on RAMP-2

gene expression in RMC. This finding correlates with the data from the animal sepsis

98

model, where rats subjected to the CLP procedure showed no evidence of alteration in
pulmonary tissue RAMP-2 gene expression (23 8).

This part of the study identifies PDGF, a pleotrophic cytokine well established to
influence mesangial cell biology, and LPS, a potent causative agent of sepsis as novel
factors capable of regulating RAMP-3 mRNA and membrane-associated RAMP-3
protein expression in RMC. Both PDGF and LPS selectively increase RAMP-3 (but not
RAMP-2) mRNA expression in a dose-dependent manner (Fig. 5A and Fig. 9). These
effects correlate with an elevation in cellular responsiveness to AM since membranes of
RMC exposed to PDGF or LPS exhibit a significant increase in AM-stimulated adenylate

cyclase activity (Fig. 8 and Fig. 13).

99

5. Mechanism of PDGF and LPS-dependent RAMP-3 up-regulation.

5.1. Introduction.

The present section of the study was designed to evaluate possible signal
trasduction pathways responsible for PDGF and LPS effects on RAMP-3 gene and
protein expression. Current literature has extensively characterized the molecular
pathways affected by both of these agents (138), (71), (78), (109), (359), (357), thus
clearly identifying possible target mechanisms involved. Considering the fact that both
PDGF and LPS cause similar upregulation of RAMP-3 gene and protein expression in
mesangial cells, it is reasonable to suggest that both exercise the same signal transduction
pathway(s) to achieve their molecular effects. Accordingly, we have investigated the role
of mitogen-activated protein kinase (MAPK) pathways as these are well-established
primary intracellular enzymatic cascades responsible for the majority of cellular actions
for PDGF and LPS alike. In general, mitogen-activated protein kinases belong to a group
of intracellular proteins forming discrete signaling cascades, which in turn serve as a
focal point for a multitude of diverse extracellular stimuli capable of regulating
fundamental cellular processes.

PDGF activates its receptor, PDGFR, by binding it with a one-to-one
stoichiometry and initiating receptor autodimerization followed by intrinsic tyrosine
kinase activitation. This in turn results in phosphorylation of several cytoplasmic 8H2
domains-containing proteins. These proteins (phospholipase C, GTPase activating
protein, and tyrosine phosphatase SH-PTP), now in active form, proceed with specific

intracellular enzymatic reactions interacting with a variety of other downstream proteins,

100

including those of the MAPK cascade (2), (192). Alternatively, activated PDGFR can
directly stimulate Ras protein by recruiting Grb-2/ SOS complex to the plasma membrane
location of Ras. This also initiates an enzymatic cascade leading to stimulation of MAPK,
translocation to the nucleus, and phosphorylation of specific transcription factors
responsible for regulation of gene expression originally shown to be induced by PDGF
(192), (176).

The intracellular signaling for LPS has only recently been actively studied with an
appreciable success in identifying the precise molecular machinery involved. Unlike the
well-established cell surface events for PDGF, the precise nature of the LPS receptor and
its function are still under active investigation. Current data indicate that LPS present in
plasma binds to LPS-binding protein and is delivered to the cell membrane where it is
capable of interacting with cell surface receptors, including CD14 and CD1 1b/ 18 (74).
Since CD14 lacks a transmembrane domain it is believed that LPS/CD14 interaction with
surface Toll-like receptors (TLR), in particular TLR-4 and its accessory protein MD-2,
confers upon the LPS receptor complex the ability to activate the intracellular enzymatic
machinery (20), (12), (22), (181), (298), (54). Several signal transduction pathways have
been reported to respond to LPS exposure (53), (52). However, activation of MAPK
cascade, especially the extracellular signal-regulated kinases ERK-l/ERK-2 (p42/p44
MAPK) and p38 MAPK, is regarded as a hallmark of LPS-induced intracellular Signaling
in variety of cell types including mesangial cells (281), (291), (60), (352), (58), (290),
(262), (265). Interestingly, p38 MAPK was initially isolated as 38-kDa protein which was

rapidly tyrosine phOSphorylated in response to exogenous LPS stimulation (98), (97).

101

Activation of signaling pathways, like the MAPK cascade, represents a common
mechanism by which variety of extracellular effector molecules regulate gene
transcription. However, post-transcriptional events also play a significant role in the
overall control of gene expression. Our current knowledge emphasizes the importance of
mRNA molecules not only as coding for appropriate proteins but also as dynamic self-
regulatory molecules capable of controlling their own processing, transport, localization,
proper translation, and life span (340), (89), (202). Through the cis-acting signals,
directly encoded by the mRNA, a host of protein factors (“end-effectors” of various
signaling cascades) are capable of interacting with the message thus regulating its
function (207). Accordingly, in order to elucidate the effects of extracellular agents on
gene regulation, one must consider their potential influence on these post-translational
events. In particular, understanding the cellular events that control the half-life of an
mRNA is critical during an investigation of gene expression regulation. The half-lives of
different mRNAs vary considerably from as short as 10 minutes to greater than 24 hours
and are largely dependent on the cell type, mRNA sequence characteristics, and a
Significant influence of numerous Signal transduction events (257), (272). Modulation of
mRN A stability is governed by at least two independent processes: the unique decay rates
characteristic of different mRNA species and the apperent stability of mRNA
dynamically regulated by a host of extracellular signals. Since the stability of an mRNA
is a key determinant of its steady state level and subsequently the translated protein level
(272), (27), (131), we have also investigated the effects of PDGF and LPS on the half-life

of RAMP-3 message.

102

5.2. Materials and methods.
5.2.1. Materials.

Adrenomedullin, adrenomedullin(22-52) fi'agment and PDGF-BB, Actimonycin
D, ct-amanitin were purchased from Sigma” RBI” (St. Louis, MO). Lipopolysacccharide,
Ecoli 0552B5, AG 1296, PD 98059, PD 153035, PD168393, SB203580, and
cycloheximide (CI-IX) were from Calbiochem (La Jolla, CA). RPMI-1640, fetal bovine
serum, penicillin/streptomycin, trypsin-EDTA were from GibcoBRL” (Grand Island,
NY). RAMP-2 and RAMP-3 polyclonal primary antibodies were from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Horseradish peroxidase-conjugated secondary
Anti-rabbit IgG antibodies were from Sigma” R131O (St. Louis, MO).

All other reagents were of highest quality available.

5.2.2. Cell Culture.

Rat mesangial cell (RMC) cultures were established from glomeruli as described
in section 3.2.2. RMC were maintained in RPMI-1640 with 15% fetal bovine serum
unless otherwise stated for particular experiments. Passages 15-24 were used for

subsequent experiments with specific confluency determined by preliminary studies.

103

5.2.3. Northern blot analysis.

Immediately after cell culture medium was aspirated from the tissue culture plates, 2-4 ml
of TRIzol’D was added, dispersed uniformly, and plates were stored at -80°C until further
use. Following a quick thaw, cells were scraped with cell lifters and transferred to 15 ml
centrifirge tubes. Total cellular RNA was isolated with TRIzol%ccording to
manufacturer’s specifications. RNA was precipitated by adding 3 M sodium acetate and
absolute ethanol, washed with 75% ethanol, pelleted in microcentrifuge tubes, and dried
prior to resuspension in RNAse-free water. Its purity was checked by measuring the ratio
of absorbance@250nm/absorbance@230nm. All of the RNA used had the ratio equal or greater
to 1.8. A standardized aliquot of RNA (30 pg) was separated by electrophoresis on a
formaldehyde agarose denaturing gel and transferred to an Optitran® membrane
(Schleicher & Schuell, Keene, NH) by capillary transfer. Subsequently, RNA samples
were immobilized to the membrane by ultraviolet cross-linking. Membranes were
successively hybridized at 42°C for 16-24 hrs with four parts (15 ml of Formamide, 0.6
ml Denhardt's solution, 1.5 ml of l M phosphate buffer, 7.5 ml of 20x SSC, 1.5 ml of
SDS, 2.4 ml of diethylpyrocarbonate water, and 1.5 ml of salmon sperm DNA)/blot and
one part 32P-dCTP-labeled cDNA probes (specific for RAMP-1, 2, 3 or 18S ribosomal
subunit; RAMP probes were obtained as RT-PCR products using RAMP-specific primers
designed from published sequences). The cDNA was radiolabeled using a random prime
labeling kit. Following hybridization for 16-24 hrs, the membranes were washed and
placed in an x-ray cassette for the requisite exposure time. Signals were quantitated by

phosphoimager analyses and expressed relative to 18$ levels.

104

5.2.4. Analysis of RNA stability.

Rat mesangial cells were grown on P-150 tissue culture plates to approximately
80% confluency (as described above) and subsequently rendered quiescent by serum-
starving for 24 hours. Next, cells were incubated with PDGF-BB (50 ng/ml) or LPS

(lOug/ml) for 16 hours, washed and placed in serum-fiee medium containing

Actinomycin D, an inhibitor of gene transcription, at final concentration of lOttg/ml and
in the presence or absence ofPDGF-BB (50 ng/ml) for O, 1, 2, 4, and 8 hours. Total RNA
was isolated and analyzed by Northern blotting as described above. RNA degradation
curves were obtained by setting the 100% value to the amount of RAMP mRNA present
immediately prior to Actinomycin D exposure [maximum value at time 0 (10)]. mRNA
levels remaining at indicated times following to were compared as per-cent of the
maximum value. A one-phase exponential decay curve was fitted including the maximum
value at to and decay rate constant, K, calculated for each nonlinear regression curve. The

half-life of RAMP message was calculated as equal to In (2/K).

105

5.3.Results.

5.3.1. Involvement of signal transduction pathway(s) in PDGF and LPS-dependent
RAMP-3 up-regulation.

PDGF has been Show to exert a myriad of biological effects principally by acting
through its receptor, PDGFR, known to have an intrinsic tyrosine kinase activity (110).
To investigate whether PDGF-induced RAMP-3 mRNA expression is PDGFR specific,
we utilized several pharmacological tyrosine kinase inhibitors. AG 1296, a specific and
selective inhibitor of PDGFR tyrosine kinase activity (164), abrogated PDGF-induced
response (Fig. 15). PD153035 and PD168393, selective inhibitors of epidermal grth
factor receptor-associated tyrosine kinase (23), (85), did not, however, have any effect on
PDGF- stimulated RAMP-3 mRNA expression (Fig. 15). Accordingly, the observed
increase in RAMP-3 mRN A abundance after exposure to PDGF is PDGFR mediated and
receptor-associated tyrosine kinase dependent. V

Because initiation of intracellular tyrosine kinase activity often triggers a cascade
of events mediated via mitogen-activated protein kinases (MAPK-s), we investigated the
effects of PD 98059 (IOuM) and SB 203580 (1011M) (selective inhibitors of MAP kinase
kinase (MEK) and p38 MAP kinase, respectively) on PDGF-induced RAMP-3
expression. Both inhibitors significantly attenuated the PDGF-induced response (Fig. 16),
indicating that RAMP-3 expression in RMC is, at least in part, regulated by MAPK
pathway.

Similarly, considering the fact that MAPK-s (especially MEK, p38 kinase, and

PI3-kinase) often serve as an enzymatic relay for LPS mediated intracellular responses,

106

we have examined the effects of SB 203580, PD 98059, and Wortmannin on LPS-
induced RAMP-3 mRNA expression. Pretreatment of RMC with pharmacological doses
of SB 203580 and PD 98059 ablated the LPS-evoked elevation in RAMP-3 message by
over 75 and 90 percent, respectively. Conversely, inhibition of PI3-K with Wortmannin
had no statistically significant outcome. None of the blockers showed any appreciable

effect on RAMP-3 expression when used alone (Fig. 17).

107

PDGF(50ng/ml)
+ AG1296 (10 pg/rni)
+ PD153035(0.1ug/ml)
' .' i .PDl68393(0.1ug/ml)

 

Control

   

Figure 15. Effects of AG1296, PD153035, and PD168393 on PDGF-stimulated RAMP-3
mRNA expression in rat mesangial cells. Cells were pre-treated with inhibitors and
subsequently exposed to PDGF (50 ng/ml) for 24 hours. Representative northern blot
showing the selective dependence of PDGF-induced response on the activation of PDGF

receptor but not EGF receptor-associated tyrosine kinase activity.

108

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RAMP-3 mRNA expression
(percent change from basal)
A
Q

PDGF + + + - -

SB203580 - + - + -
PD98059 - - + - +

Figure 16. Effects of SB203 580 (IOuM) and PD98059 (IOuM) on PDGF-induced
RAMP-3 expression in RMC. Cells were pre-treated with inhibitors and subsequently
exposed to PDGF (50 ng/ml) for 24 hours. RNA was collected as in previous experiments
and analyzed by Northern blot hybridization with RAMP-3 probes. To correct for loading
variability, blots were stripped and reprobed for 18s RNA. Raw values were converted to
ratios of RAMP-3 mRN A to 185 and then expressed as percent change from basal. Insert:
representative Northern blot of RAMP-3 expression in response to pre-treatment of RMC
with SB203580 and PD98059 followed by PDGF exposure. Both SBZOSSSO and

PD98059 significantly attenuated PDGF-induced RAMP-3 expression; *p<0.001, n=5.

109

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RAMP-3 mRNA expression
(percent change from basal)
on
Q

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0 _ T L
LPS + + + + - — -
SB203580 - + — - + - -
PD98059 - - + - - + -
Wort - - - + - - +

Figure 17. Effects of SB203580, PD98059, and Wortmannin (Wort) on LPS-induced
RAMP-3 mRNA expression in RMC. Cells were pre-treated with inhibitors and
subsequently exposed to LPS (10 ug/ml) for 24 hours. RNA was collected as in previous
experiments and analyzed by Northern blot hybridization with RAMP-3 probes. To
correct for loading variability, blots were stripped and reprobed for 18s RNA. Raw values
were converted to ratios of RAMP-3 mRN A to 185 and then expressed as percent change
from basal. SBZO3580 (10 nM) and PD98059 (10 nM) significantly attenuated LPS-
induced RAMP-3 expression, *p<0.001 as compared to LPS treated cells (n23); while
Wortmannin (0.1 11M) had no effect (n=5), n.s.- no statistical significance as compared to

LPS treated cells.

110

5.3.2. Mechanism of PDGF and LPS-stimulated RAMP-3 expression.

To determine whether PDGF and LPS affect transcriptional events leading to
increased abundance of RAMP-3, we tested Actinomycin D and a-amanitin (inhibitors of
DNA-dependent RNA synthesis) for their ability to influence the PDGF and LPS-
dependent effect. Preincubation of mesangial cells with Actinomycin D (5 rig/ml) did not
alter PDGF or LPS-induced RAMP-3 expression (Fig. 18A) suggesting the lack of
regulation at a transcriptional level. To verify the effectiveness of transcriptional
inhibition by Actinomycin D, Northern blots were re-probed with mitogen-activated
protein kinase (MAPK) kinase, MEK-l, cDNA and quantification of MEK-l RNA was
performed. As previously reported by Schramek et al. (288), PDGF induced MEK-l
mRNA and this effect was inhibited by Actinomycin D, indicating a good transcriptional
inhibition by Actinomycin D. Interestingly, LPS also elevated MEK-l mRN A expression
in an Actinomycin D-sensitive manner. To our knowledge, this incidental finding albeit
previously reported for other cell types, appears to be novel for RMC (Fig. 188).
Similarly, pre-incubation of the cells with ct-amanitin (l rig/ml) for 5-6 hrs did not have
any effect on PDGF-induced or LPS-induced RAMP-3 mRN A elevation (Table 4).

Next, a requirement for new protein synthesis was examined by pretreatment of
PDGF or LPS exposed cells to cycloheximide (CHX), a potent eukaryotic translational
inhibitor. At 10 ug/ml, CHX significantly inhibited PDGF and LPS-mediated RAMP-3
mRNA expression, identifying a requisite step Ofde novo protein synthesis (Fig. 19).

Given that neither PDGF nor LPS-induced increase in RAMP-3 mRNA
expression were mediated through transcriptional events, we firrther hypothesized that

these agents enhance RAMP-3 mRNA stability, thus leading to increased abundance of

111

RAMP-3 message. Accordingly, we analyzed the effect of PDGF and LPS on the half-
life ofRAMP-3 mRNA as described in materials and methods (5.2.4.). The baseline half-
life of RAMP-3 mRNA in mesangial cells is approximately 73 minutes. PDGF (50
ng/ml) treatment raised the apparent RAMP-3 mRNA half-life to 331.6 minutes, resulting
in a 4.99 fold increase (Fig. 20) while exposure to LPS (lOug/ml) attained a 2.59 fold
increase (Fig. 21). This indicates that the mechanism of PDGF-induced and LPS-induced
RAMP-3 expression is mediated by a posttranscriptional event of mRNA stability

enhancement.

112

 

 

 

 

 

 

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Figure 18. Effect of Actinomycin D (ActD) on PDGF and LPS-stimulated RAMP-3

expression in RMC. RMC were serum starved overnight and treated with PDGF (50

ng/ml) or LPS (10 ug/ml) or vehicle (basal) in the absence or presence of 5 jig/ml ActD

for 24 hours, as indicated. Northern blot analysis was performed as described in materials

and methods and the blots were probed for RAMP-3. A: ActD did not inhibit PDGF or

LPS-induced RAMP-3 mRN A expression suggesting that RAMP-3 expression in RMC

may not be regulated by increase in transcription. Insert: representative northern blot for

this experiment; n23.

113

a PDGF LPS
3 + + ActD

MEK-l —* ”311.....- .... -~ “.31 .,

Figure 18. Effect of Actinomycin D (ActD) on PDGF and LPS-stimulated RAMP-3
expression in RMC. B: control experiment Verifying that PDGF and LPS-increased
transcription of MEK-l is inhibited by ActD. This experiment is shown to eliminate the
possibility that ActD is inactive. The representative northern blot shown here for MEK-l

expression is the same one that was used for probing RAMP-3.

 

 

 

 

 

 

 

 

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Table 4. Efl’ect of or-amanitin pretreahnent on PDGF and LPS- induced RAMP-3 mRNA expression. RMC
were incunated for 5 to 6 hrs in preueatrnent medium contating 1 rig/ml ct-amanitin. Cells were
subsequently washed and exposed to PDGF or LPS for an additional 24 hrs. RAMP-3 mRNA was
examined in the usual manner, as described previously. a-Amanitin did not inhibit PDGF or LPS-induced
RAMP-3 mRNA expression. This correlates with findings from the ActD pretreatment experiments and

suggests an involvement of a transcriptionally independent mechanism for RAMP-3 expression in RMC.

114

 

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Figure 19. Effect of Cycloheximide (CHX) on PDGF and LPS-induced RAMP-3 mRNA
expression in RMC. RMC were serum starved overnight and treated with vehicle (basal),
PDGF (50 ng/ml), or LPS (10ug/ml) in the presence or absence of CHX (lOttg/ml) for 24
hours. RNA was extracted and northern blot analysis performed as indicated in materials
and methods. Blots were first probed for RAMP-3 and then stripped and reprobed for 18s
RNA. Raw values were converted to ratios ofRAMP-3 rnRN A to 185 and then expressed
as percent change from basal. CHX significantly attenuated PDGF-induced and LPS-

induced RAMP-3 mRNA expression; 'pS0.001 as compared to PDGF treated cells;

”p50.001 as compared to LPS treated cells; n=3.

115

 

 

time (hrs) '6— 1 2 4 8

 

RAMP-3 mRNA expression

(percent of max.

 

 

time (hrs)

Figure 20. Effect of PDGF on RAMP-3 mRNA, rate of decay in RMC. RNA stability
assay was performed on RMC as described in materials and methods. RNA from each
time point was analyzed by Northern hybridization and relative RAMP-3 mRN A
abundance expressed as percent of that present at time=0 (percent of maximum value).
RAMP-3 mRNA half-lives were calculated using the decay constants (K) obtained from
one-phase exponential decay curves. Insert: representative Northern blot obtained
following the RNA stability assay protocol. PDGF significantly increased RAMP-3
mRNA half-life from 1.1 to 5.5 hours. Regression lines (half-lives) for RNA decay from
PDGF (SOng/ml) treated (a) and no PDGF (A) RMC were compared by analysis of

covariance. Regressions were regarded Significantly different with a p300]; n=3.

116

 

 

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75‘ O -LPS

 

RAMP-3 mRNA expression
(percent of max. value at b0)
0|
?

 

 

25- .
T
T
G I I l I
0 2 4 6 8
time (hrs)

Figure 21. Effect of LPS on RAMP-3 mRN A rate of decay in RMC. RNA stability assay
was performed on RMC as described in materials and methods. RNA from each time
point was analyzed by Northern hybridization and relative RAMP-3 mRN A abundance
expressed as percent of that present at time=0 (percent of maximum value). RAMP-3
mRN A half-lives were calculated using the decay constants (K) obtained from one-phase
exponential decay curves. Insert: representative Northern blot obtained following the
RNA stability assay protocol LPS significantly increased RAMP-3 mRN A half-life
from 1.3 to 3.5 hours. Regression lines (half-lives) for RNA decay from LPS (10 ug/ml)

treated (0) and no LPS (0) RMC were compared by analysis of covariance. Regressions

were regarded significantly different with a p30.01; n=3.

117

5.4. Discussion.

One of the aims in this Study was to investigate the mechanism of PDGF and
LPS-dependent induction of RAMP-3 expression at the signaling level. In particular, we
examined the role of mitogen-activated protein kinases (MAPKS), a highly conserved
family of protein serine/threonine kinases that are fundamental to multiple signaling
cascades relaying extracellular signals to the nucleus. The MAPK family of proteins
includes the extracellular signal-regulated kinases (ERKS) and the stress activated protein
kinases (SAPKS): c-Jun N-terrninal kinase (JNK) and p38 kinase (293), (43). Upon
phosphorylation, activated MAPKS translocate into nucleus where they activate
transcription factors thus regulating expression of a wide variety of target genes (244).
MAPKS are also implicated in direct regulation of target mRNA stability, thus
influencing the relative mRNA abundance, which is independent of their effects on the
transcriptional machinery. This action is presumably mediated by specific recruitment of
proteins, like AUFl proteins, to various parts of the 3'-untranslated regions (UTRs) of
mRNA molecules. A number of instability elements (for example the AU-rich element,
ARE) have been identified in the UTRs and are thought to constitute the target for the
regulatory proteins like AUFl. Recently, several laboratories reported that the binding of
AUFl and similar proteins to UTRs is dependent on protein kinase directed
phosphorylation. For example, INK and p38 MAPK signaling pathways have been shown
to directly stabilize mRNAs of VEGF, interleukin-2, -3, -6, and —8 (207), (257), 272).

Cellular actions of PDGF have been shown to involve both its receptor tyrosine

kinase activity and MAPK pathways (110), (192), (261). MAPKS are also activated in

118

many cell types upon LPS exposure (52), (60), (352). To examine the role of MAPKS, we
utilized specific pharmacological inhibitors to block these signaling molecules.

Our observation that AG1296 (PDGF receptor tyrosine kinase blocker) but not
PD153035 and PD168393 (selective inhibitors of epidermal growth factor receptor-
associated tyrosine kinase) blocked the PDGF-stimulated RAMP-3 mRNA expression
suggests that PDGF actions in mesangial cells are indeed mediated through PDGF
receptor tyrosine kinase activity. Furthermore, we showed that pharmacological
inhibition of MEK (kinase upstream of ERK) and p38 MAPK, reversed the PDGF-
dependent elevation of RAMP-3 mRNA abundance, indicating that activation of these
kinases by PDGF may be important in the regulation of RAMP-3 expression.

Similarly, we showed that LPS-induced RAMP-3 gene expression is almost
completely abolished by inhibition of MAPK kinase (MEK) with PD 98059. This finding
also implicates MAPK signaling in LPS-directed regulation of RAMP-3 gene in
mesangial cells. Since LPS-dependent regulation of gene expression is known to involve
a series of positive and negative signal transduction pathways, we also investigated the
role of phosphatidyl inositol 3-kinasei(PI3K) in LPS-induced RAMP-3 gene expression.
LPS activates PI3K consequently leading to a potent inhibition of MAPK pathways
(ERKl/2, p38, and JNK), thus imposing a firnctional “braking mechanism” on the LPS-
induced gene activation (180), (243), (252). For example, inhibition of PI3K enhanced
LPS-dependent nitric oxide (NO) production in murine macrophages (252), while LPS-
dependent induction of NO synthase in astrocytes and lipoprotein lipase expression in
macrophages were inhibited by direct activation of PI3K (243), (324). In contrast to the

above studies showing the negative involvement of PI3K pathway in LPS signaling, our

119

findings suggest that PI3K is not engaged in LPS-dependent RAMP-3 gene regulation.
Explicitly, we have observed that mesangial cells pretreated with pharmacological doses
of Wortmannin, a specific inhibitor of PI3K, exhibited no significant change in RAMP-3
mRNA expression following LPS exposure as compared to non-pretreated cells.

To our knowledge this is the first report of an involvement of MAPK pathways in
regulating RAMP-3 expression in mesangial cells. Considering that MAPK pathways
serve as a common denominator for a variety of divergent intracellular signals, this
finding may bear an important implication for the complexity of RAMP gene expression
regulation.

While both Frayon el al. and Robert-Nicoud et al. demonstrated that exogenous
addition of factors can alter RAMP expression, they did not address the underlying
mechanisms, which may be responsible for these effects. Here, we present evidence that
PDGF and LPS-induced RAMP-3 expression is transcriptionally independent, as it was
not inhibited by Actinomycin D or ct-amanitin. Furthermore, our data indicate that PDGF
and LPS act Via stabilizing RAMP-3 mRN A, consequently increasing the apparent half-
life of the message by nearly 5 and 2.6 fold, respectively. While we established the
requirement of de nova protein synthesis for this effect, future studies are necessary to
firrther characterize the exact mechanism(s) by which PDGF and LPS influence the
stability of RAMP-3 message. Recently, Kitamuro et al. (159) also proposed that the total
abundance of RAMP message may be regulated specifically by changes in RAMP
mRNA stability. They estimated the basal half-life of RAMP-2 mRNA for two human
neuroblastoma cell lines, IMR-32 and NB69, to exceed 6 hours. In addition, they reported

an increase in RAMP-2 mRNA stability in IMR-32 cells under hypoxic conditions.

120

Interestingly, exposure of IMR-32 cells to a hypoxia-mimetic agent, CoClz (cobalt
chloride), decreased RAMP-2 mRNA half-life while NB69 cells did not exhibit any
change in response to hypoxia or CoClz. It is worth noting that neither PDGF nor LPS
had a significant effect on RAMP-2 expression in mesangial cells, suggesting a
differential gene regulation between closely related RAMP-2 and RAMP-3.

In conclusion, this chapter presents evidence for the mechanism(s) which may be
involved in the observed PDGF and LPS-induced increase in RAMP-3 mRNA and
RAMP-3 membrane protein expression. Our results indicate that PDGF-induced RAMP-
3 mRNA elevation is MEK and p38 MAPK dependent (Fig. 16). Moreover, as predicted,
this effect relies on the activation of the PDGF receptor and its inherent tyrosine kinase
activity (Fig. 15). LPS also mediates RAMP-3 expression via MEK/p38 MAPK pathway
but appears to be independent of PI3K signaling (Fig. 17). In addition, both PDGF and
LPS regulate RAMP-3 gene expression in a post-transcriptional manner as
pharmacological inhibition of transcription by actinomycin D and ct-amanitin appears to
have no effect on the original observations (Fig. 18 and Table 4). The dependence on de
nova protein synthesis was however evidenced by significant attenuation of PDGF and
LPS-induced RAMP-3 mRN A expression following preexposure to cycloheximide (Fig.
19). Furthermore, our results demonstrate for the first time that PDGF and LPS augment
the abundance of RAMP-3 mRNA via stabilization of the message and a consequent
increase in RAMP-3 mRNA half-life (Fig. 20 and Fig. 21). As alluded to previously, this
effect may result from the activation of MEK/p38 MAPK pathway(s) leading to
subsequent direct targeting of cis-acting signals encoded by RAMP-3 mRNA and/or

regulation of mRN A interacting proteins. This hypothesis however awaits further testing.

121

6. Summary and conclusions.

6.1. Major hypothesis and results of the study.

In general, we hypothesized that the regulation of RAMPS expression provides a
mechanism for modulation of AM activity in mesangial cells.

The major aims of this study were to investigate the effects of two extracellular
signaling agents, PDGF and LPS, on the regulation of RAMP expression and the
consequent changes in mesangial cell responsiveness to AM. After documenting that both
PDGF and LPS induce RAMP-3 mRNA and protein expression, we also scrutinized the
molecular mechanisms responsible for this observation. Accordingly, as summarized
below, the following major hypothesis were tested and the corresponding experimental
results obtained:

Hypothesis #1:
Over-expression of RAMP-1, 2, and 3 results in an increase in firnctional
responsiveness of RMC to AM as measured by AM-induced cAMP production
and [3H]thymidine incorporation.
Results:
1. At basal cell culture conditions RMC express all of the essential components
of the AM receptor system, namely CL receptor (formerly CRLR), RAMP-1,
2, and 3, as examined by RT-PCR method.

2. Over-expression of RAMP-2 and RAMP-3 cDNA in RMC results in a

significant increase of AM-induced adenylate cyclase activity as measured by

AM-induced cAMP production.

122

3. Over-expression of RAMP-1 in RMC does not effect AM-induced cAMP
production. .

4. Over-expression of RAMP-2 and RAMP-3 cDNA in RMC results in marked
enhancement of AM-mediated decrease in mesangial cell proliferation as
measured by [3H]thymidine incorporation.

5. Over-expression of RAMP-1 cDNA in RMC does not have any significant
effect on basal level of AM-mediated decrease in [3H]thymidine
incorporation.

Conclusion:
Accept hypothesis #1 for RAMP-2 and RAMP-3.

Reject hypothesis #1 for RAMP-1.

Hypothesis #2:
PDGF and LPS up-regulate RAMP-2 and RAMP-3 expression and increase

functional responsiveness of RMC to AM.
Results:
1. Exposure of cultured RMC to PDGF increases RAMP-3 mRN A expression
and RAMP-3 protein content in the cell membrane-associated fraction of
RMC cell lysate.
2. RMC treated with PDGF exhibit a significant increase in AM-mediated

adenylate cyclase activity as measured by AM-induced cAMP production.

123

3. Treatment of RMC with LPS in culture results in a significant increase of
RAMP-3 mRNA expression and a concordant elevation of RAMP-3 protein
levels in the cell membrane-associated fraction of RMC lysate.

4. RMC treated with LPS show significant increase in AM-induced cAMP
production.

5. Exposure of cultured RMC to PDGF or LPS does not affect the basal level of
RAMP-2 expression.

Conclusion:
Accept hypothesis #2 for RAMP-3.

Reject hypothesis #2 for RAMP-2.

Hypothesis #3:
A. PDGF and LPS-mediated elevation in RAMP-3 mRNA expression is MAPK-
dependent
B. PDGF-mediated change in RAMP-3 mRNA expression requires PDGF
receptor activation.
C. LPS-mediated elevation in RAMP-3 mRNA expression is independent of
PI3K.
Results:
1. Pharmacological inhibition of MAPK kinase (MEK) and p38 MAPK
abrogated the PDGF-induced RAMP-3 mRNA elevation. LPS effect
on RAMP-3 expression was also arrested by MEK and p38 MAPK

inhibition.

124

2. Pre-treatment of cultured RMC with AG 1296, a specific and selective

Conclusion:

inhibitor of PDGF receptor tyrosine kinase activity, abrogated PDGF-
induced RAMP-3 mRN A elevation. Pre-treatment with PD153035 and
P0168393, selective inhibitors of epidermal growth factor receptor-

associated tyrosine kinase, exhibited no effect.

. Pre-treatrnent of cultured mesangial cells with wortmannin, a potent

PI3K inhibitor, did not have any significant effect on LPS—induced

RAMP-3 mRNA expression.

Accept Hypothesis #3, A-C.

Hypothesis #4:

PDGF and LPS increase RAMP-3 mRNA abundance by stabilizing the message

thus increasing the apparent half-life of RAMP-3 mRNA.

Results:

1.

PDGF and LPS-induced elevation of RAMP-3 mRNA abundance is
transcriptionally independent in RMC, since inhibition of transcription
by Actinomycin D or ct-amanitin did not alter PDGF and LPS-
mediated effects.

PDGF and LPS-stimulated RAMP-3 expression requires de nova
protein synthesis as it was inhibited by pre—treatment of RMC with

cycloheximide.

125

3. Treatment of RMC with PDGF and LPS results in a significant
increase of RAMP-3 mRNA half-life and an apparent abundance of

the RAMP-3 message.

6.2. Limitations of this study.

The main limitation of this study stems from the inherent constraints of a cell
culture system. All of the experimental data obtained in this work are based on biological
responses observed in rat mesangial cells cultured in a homogeneous, controlled milieu.
Clearly, mesangial cells in viva experience a much more dynamic environment, where
interactions with other cell types, paracrine and endocrine influences, as well as
hemodynamic alterations within the glomerular apparatus unquestionably contribute to
the final cellular response. In addition, as previously discussed, cellular responses are
greatly varied among cells derived from even closely related species; hence one should
avoid a mere extrapolation of the observations from the current study to those expected
for human cell lines. For these reasons, the experimental data presented and conclusions
drawn from this work should be interpreted with caution, bearing in mind their innate
limitations. While our choice of rat mesangial cell culture model presents the above-
discussed limitations, it nevertheless lends itself nicely to experimental manipulations. It
is well characterized in the scientific literature, relatively inexpensive, non-laborious to
maintain, and easily reproducible.

Another limitation of this work is the use of pharmacological inhibitors to
investigate the involvement of various signaling pathways and post-transcriptional

mechanisms. Since the specificity and effectiveness of inhibitory activity of these agents

126

cannot be unequivocally determined, one must always consider their use carefully. In the
present study, only well characterized and widely used pharmacological inhibitors were
utilized. In addition, previously published inhibitory concentrations of these agents were
strictly observed.

Methodology for the analysis of mRNA turnover and mRNA half-life
determination is currently under active investigation: The existence of numerous
technical problems and shortcomings is perhaps the single most reason why so many
different methods have been developed over the years (272), (19). In this study we used a
relatively convenient way of analyzing mRNA kinetics. We utilized a pharmacological
inhibition of transcription followed by serial determination of remaining mRNA of
interest. While this is the most frequently used method in the scientific literature, it too
has some limitations. As an indirect study of mRN A stability it examines the steady-state
level of mRN A assuming that changes in mRN A abundance affect directly the stability of
a given message. In addition, the mathematical derivation of the half-life presumes that
mRNA decays according to the first-order kinetics. Recent data suggests however, that
such assumptions while true for prokaryotic mRN A processing events do not always hold
in case of mammalian transcriptional machinery (18). On the other hand, this approach
allows for minimal investigational error due to an uncomplicated, easily reproducible
experimental setup. It is compatible with analysis of relatively scarce and unstable
mRNAs and bypasses often-troublesome genetic manipulations of the message required
by alternative approaches. Thus, for practical reasons this method is widely used and has

been elected in the current investigation.

127

6.3. Positive outcomes of this study.

The original publication of McLatchie et al. implicated RAMPS in regulation of
CL receptor at the level of receptor trafficking, glycosylation, and a direct RAMP-
receptor interaction (199). Accumulating data suggests that the dynamic alteration of
RAMP expression levels may provide for yet another mode by which these proteins
regulate the function of CL receptor and possibly other receptors. A number of previously
published observations reported significant changes in RAMP gene expression in
response to pathophysiological states or direct administration of an exogenous substance;
they however do not propose a mechanism underlining these changes. The experimental
outcomes of this work identify a novel mechanism by which AM receptor complex is
regulated in response to exogenous agents. Here, we have demonstrated for the first time
that regulation of RAMP-3 message stability by PDGF and LPS leads to an alteration of
cell’s functional responsiveness to an endogenous AM. We have also shown the
involvement of MEK and p38 MAPK pathway in the regulation of RAMP-3 mRNA
expression in mesangial cells. Considering the reno-protective effects of AM
(specifically, the AM-mediated decrease in mesangial cell proliferation and increase in
cell apoptosis), the regulation of RAMP-3, a well established component of AM receptor
system, by PDGF and LPS may constitute one mechanism of cellular adaptation during
the development and progress of kidney disease. In addition, the molecular mechanisms
identified in this thesis may provide for plausible pharmacological targets aiming at
alleviation of such pathophysiological processes as glomerulonephritis and renal

consequences of septic shock.

128

Overall, the current study identified glomerular mesangial cells as yet another cell
type in which RAMPS play an important role for AM receptor phenotype determination.
Presumably by the mechanisms originally described by McLatchie et al. (199) and the
direct regulation of RAMP gene stability outlined in the current work, RAMPS modulate
AM activity in mesangial cells. In general, RAMPS unique ability to change receptor
ligand specificity as described for RAMP-CL receptor and RAMP-CTR interactions as
well as RAMPS potential to act as possible partners for hundreds of GPCRS identified in
the Human Genome Project (177) hint at their distinct role with an evolutionary
importance. Namely, RAMP-GPCRS interactions afford a combinatorial power for large
phenotypic receptor diversity within a relatively small gene pool. Future research will
unquestionably pursue the identification of novel RAMP-receptor interactions as well as
the precise nature of their regulation.

In summary, we report that mesangial cells express CL receptor, RAMP-1, 2, and
3 at basal conditions. PDGF, a pleotrophic cytokine well established to influence
mesangial cell biology, and LPS, a potent causative agent of sepsis derived from the outer
membrane of Gram-negative bacteria, increase RAMP-3 mRNA and membrane-
associated RAMP-3 protein expression. This effect correlates with an elevation in cellular
responsiveness to AM as measured by AM-stimulated adenylate cyclase activity.
Moreover, our data show the PDGF-induced RAMP-3 mRN A elevation to be MEK and
p38 MAPK dependent. LPS also mediates RAMP-3 expression via MEK pathway but
appears to be independent of PI3K signaling. Both agents, PDGF and LPS, regulate
RAMP-3 gene expression in a post-transcriptional manner. They augment the abundance

of RAMP-3 mRN A Via stabilization of the message and a consequent increase in RAMP-

129

3 mRN A half-life. Taken together, these data suggest an important and novel mechanism

of regulation of RAMP-3 in rat mesangial cells.

130

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