Novel regulation of adrenomedullin receptor by PDGF: role of
receptor activity modifying protein-3
Wojciech
Nowak,
Narayanan
Parameswaran,
Carolyn S.
Hall,
Nambi
Aiyar,
Harvey V.
Sparks, and
William
S.
Spielman
Department of Physiology, Michigan State University, East
Lansing, Michigan 48824
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ABSTRACT |
Receptor activity modifying protein-3
(RAMP-3) has been shown to complex with the calcitonin receptor-like
receptor, establishing a functional receptor for adrenomedullin (AM).
AM exhibits potent antiproliferative and antimigratory effects on rat
mesangial cells (RMCs). In this study we investigated the effect of
platelet-derived growth factor (PDGF) on RAMP-3 expression in RMCs. We
show here that PDGF-BB stimulates RAMP-3 mRNA expression in a
concentration-dependent manner. Pretreatment with actinomycin-D and
-amanitin demonstrates that this effect is independent of new RNA
synthesis. Furthermore, PDGF increased the half-life of RAMP-3 mRNA
from 66.5 to 331.6 min. Using selective inhibitors, our results also
indicate that the increase in RAMP-3 mRNA is mitogen-activated protein
kinase (MAPK) kinase (MEK)/MAPK and p38 MAPK dependent. PDGF also
caused a corresponding elevation in membrane-associated RAMP-3 protein. Associated with this increase, PDGF pretreatment led to a significantly higher AM-mediated adenylate cyclase activity, suggesting a functional consequence for the PDGF-induced increase in RAMP-3 expression. Taken
together, these data identify PDGF-dependent regulation of RAMP-3
expression as a possible mechanism for modulating the responsiveness of
the mesangial cell to AM.
G protein coupled receptors; receptor signaling; calcitonin
receptor-like receptor
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INTRODUCTION |
THE RECENT DISCOVERY
OF receptor activity modifying proteins (RAMPs) by McClatchie et
al. (31) has significantly altered our understanding of
mechanisms involved in the regulation of G protein-coupled receptors
(GPCR). RAMP-1, RAMP-2, and RAMP-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 (CR) and CR-like receptor (CRLR) (4, 7, 20, 31).
Although the exact nature of RAMP-CR and RAMP-CRLR interactions remains
elusive, it has been clearly documented that RAMPs facilitate
trafficking and determine the phenotype of these receptors (7,
28, 31). In particular, with regard to CRLR, RAMP-1 and CRLR
coexpression renders the receptor a fully functional calcitonin
gene-related peptide (CGRP) receptor. Cotransfection of CRLR with
RAMP-2 or RAMP-3, on the other hand, confers on CRLR adrenomedullin
(AM) receptor characteristics (31). Consequently,
differential expression of RAMPs may prove to function as a regulatory
step for CRLR activity and its ligand specificity toward CGRP and/or AM
in physiological and pathophysiological states alike. In fact, RAMP
gene expression has been observed to be nonconstitutive and reported to
undergo dynamic alterations in several disease states including renal
diseases (32, 33, 47, 48).
AM, a 52-amino acid peptide recently isolated from a pheochromocytoma
(23), has been shown to activate CRLR causing an elevation in intracellular cAMP in several systems including rat mesangial cells
(RMCs) (6, 26, 35). In particular, by activation of the
cAMP-protein kinase A (PKA) pathway, AM exerts antiproliferative, proapoptotic, and antimigratory effects on RMCs (5,
37). Because disproportionate mesangial proliferation and matrix
deposition are hallmark pathological changes accompanying several
glomerular diseases (10, 25), the antiproliferative
effects of AM suggest an attractive, renoprotective role for this
hormone. AM has also been shown to decrease platelet-derived growth
factor (PDGF)-induced mesangial cell proliferation (6,
44). PDGF is a prime cytokine responsible for mediating both
proliferative and migratory responses in the mesangium during
glomerular injury (1, 29, 36). Elucidating the mechanisms
of functional interplay between PDGF and AM receptor will provide novel
information on regulation of proliferative/antiproliferative events in
mesangial cells. Hence the present study was undertaken to examine the
possible effect(s) of PDGF on AM receptor complex (CRLR-RAMP-3)
expression in RMCs. Our results suggest that PDGF causes a significant
increase in RAMP-3 mRNA and protein expression, with a corresponding
elevation in AM-stimulated adenylate cyclase activity. Furthermore, the PDGF-mediated increase in RAMP-3 mRNA abundance is dependent on mRNA
stability but not on transcription.
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MATERIALS AND METHODS |
Materials.
AM, AM-(22-52) fragment, actinomycin D,
-amanitin, and PDGF-BB were purchased from Sigma RBI (St.
Louis, MO). AG-1296, PD-98059, PD-153035, PD-168393, and SB-203580 were
from Calbiochem (La Jolla, CA). RPMI 1640, fetal bovine serum,
penicillin/streptomycin, and trypsin-EDTA were from GIBCO BRL (Grand
Island, NY). All other reagents were of highest quality available.
Cell culture.
Rat mesangial cell (RMC) cultures were established from glomeruli
obtained from kidney cortex of 55-70 g male rats (Sprague-Dawley; Charles River). Glomeruli were isolated by sequential sieving, which
removes tubules (300- to 150-µm sieves) and then retains glomeruli on
the 63-µm sieve, as described by Wolthuis et al. (51). Isolated glomeruli were incubated for 10 min
at 37°C in collagenase (750 U/ml) and then plated in flasks in RPMI
1640 medium supplemented with 0.6 U/ml insulin, 100 U/ml penicillin, 100 µg/ml streptomycin, and 15% fetal bovine serum. Cells were grown
at 37°C in 5% carbon dioxide with medium changed twice a week. At
confluence, cells were subcultured by rinsing with calcium- and
magnesium-free PBS and then incubating with 0.05% trypsin supplemented
with 20 mM EDTA. The correct cell type was confirmed by the following
criteria: 1) a stellate morphology using phase-contrast microscopy; 2) microfilaments and subplasmalemmal
cytoplasmic densities using transmission electron microscopy;
3) insensitivity 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 ANG II (2). For the experiments,
passages 15-24 were used. Specific confluence for
different experiments was determined by preliminary studies.
RT-PCR analysis.
Total RNA was isolated from RMCs using Trizol reagent (GIBCO BRL).
After sodium acetate-ethanol precipitation and several ethanol washes,
RNA was used as a template in an RT-PCR amplification procedure. The
RT-PCR reaction was carried out using Superscript One-Step RT-PCR with
Platinum Taq (GIBCO BRL), in accordance with the
manufacturer's specifications. The 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 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, with a Perkin-Elmer model 9600 thermal cycler, in 50 µl of 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); and 4) 72°C for 15 min
(1 cycle). Products were separated by gel electrophoresis and
subsequently visualized by ethidium bromide staining and ultraviolet
illumination. Photographs of the gels were taken and digitalized with a
UMAX Astra 2000P flat-bed scanner. To ensure the identity of the
products, cDNA was extracted from the gel and sequenced by a standard
dye-termination DNA sequencing procedure. Sequences of cDNAs obtained
in that fashion were analyzed for similarity to published full-length sequences of RAMP-1, -2, -3, and CRLR via a two-sequence comparison method from the BLAST database (http://www.ncbi.nlm.nih.gov/BLAST). These analyses revealed the identity of RT-PCR products for the corresponding full-length cDNA sequences. Several cDNAs
acquired by this method were used as probes in Northern blot hybridizations.
RAMP-1, -2, -3, and CRLR cloning and expression.
Full-length cDNA of human RAMP-1, -2, -3, and bovine CRLR 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 Plus Reagent (GIBCO BRL)
following the suggested protocol. In brief, RMC were plated on P-100
tissue culture plates a day before the transfection to achieve an
~60-70% confluence. On the following day, cells were
transfected with a total of 2 µg of each vector-cDNA in a serum-free
transfection medium for 4 h at 37°C and 5% CO2. The total amount of transfected cDNA was kept constant by adding empty Myc
or pCDN vector. Afterward, 4 ml of serum-containing medium were added
and cells were incubated for an additional 24-36 h. This transient
transfection protocol resulted in an ~70% transfection efficiency as
assessed by green fluorescent protein.
Membrane preparation and adenylate cyclase assay.
Cells were harvested from P-150 plates and homogenized in 10 mM
Tris · HCl (pH 7.4)-10 mM EDTA buffer. Membranes were prepared by homogenization in a Dounce ground glass homogenizer, centrifuged for
20 min at 12,000 g at 4°C, and washed in 50 mM
Tris · HCl (pH 7.4)-10 mM MgCl2 buffer. A final
concentration of 40 µg 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 [
-32P]ATP to
[32P]cAMP as described by Elshourbagy et al.
(11). Accordingly, membranes were incubated for 20 min at
30°C with appropriate drugs and an assay mix containing an ATP
regeneration system (50 mM Tris · HCl, pH 7.4, 10 mM
MgCl2, 1.2 mM ATP, 0.1 mM cAMP, 2.8 mM
phosphoenolpyruvate, and 5.2 µg/ml myokinase) and 1.0 µCi of [
-32P]ATP. Total reaction volume was 100 µl, and drug as well as AM concentrations were as described for
particular experiments. Reactions were stopped with 1 ml of stop
solution containing 0.28 mM "cold" cAMP, 0.33 mM ATP, and 22,000 dpm [3H]cAMP. The contents of the assay tubes were washed
through a Dowex column, and subsequently through alumina columns, to
separate the degradation products of ATP as previously described by
Salmon et al. (41). Elution profiles were
performed before 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 the alumina column were
counted for the presence of [3H]cAMP and
[
-32P]cAMP. Each experiment was done in triplicate,
repeated at least three times, and expressed as a percentage of
AM-mediated adenylate cyclase activity compared with basal.
[3H]thymidine incorporation.
Cells were plated in 24-well plates (30,000 cells/well) and grown for 2 days with subsequent serum starving for 48 h. They were then
treated with the compounds for a period of 16 h and pulsed with
[3H]thymidine for 4 h. The radioactivity was counted
in a Beckman LS counter after the cells were washed, the reaction was
stopped with 5% TCA, and the cells were solubilized in 0.5 ml of 0.25 N sodium hydroxide. Each experiment was performed in quadruplicates and
repeated at least three times.
Northern blot analysis.
Immediately after the cell culture medium was aspirated from the tissue
culture plates, 2-4 ml of Trizol were added and dispersed uniformly, and plates were stored at
80°C until further use. After
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 the manufacturer's specifications. RNA was precipitated
by adding 3 M sodium acetate and absolute ethanol and then washed with
75% ethanol, pelleted in microcentrifuge tubes, and dried before
resuspension in RNase-free water. The purity of the RNA was checked by
measuring the ratio of absorbance at 260 nm to absorbance at 280 nm.
All of the RNA used had a ratio
1.8. A standardized aliquot of
RNA (30 µg) was separated by electrophoresis on a formaldehyde
agarose denaturing gel and transferred to an Optitran membrane
(Schleicher and 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 h with four parts of a solution
containing 15 ml of formamide, 0.6 ml of Denhardt's solution,
1.5 ml of 1 M phosphate buffer, 7.5 ml of 20× SSC, 1.5 ml of SDS, 2.4 ml of diethylpyrocarbonate water, and 1.5 ml of salmon sperm DNA per blot and one part of [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. After hybridization for 16-24 h, the membranes were
washed and placed in an X-ray cassette for the requisite exposure time.
Signals were quantitated by phosphorimager analyses and expressed
relative to 18S levels.
Analysis of RNA stability.
Rat mesangial cells were grown on P-150 tissue culture plates to
~80% confluence (as described above) and subsequently rendered quiescent by serum starving for 24 h. Next, cells were incubated with PDGF-BB (50 ng/ml) for 16 h, washed, and placed in serum-free medium containing actinomycin D, an inhibitor of gene
transcription, at a final concentration of 10 µg/ml and in
the presence or absence of PDGF-BB (50 ng/ml) for 0, 1, 2, 4, and
8 h. 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 before
actinomycin D exposure [maximum value at time 0 (t0)]. mRNA levels remaining at
indicated times following t0 were compared as a
percent of the maximum value. A one-phase exponential decay curve was
fitted including the maximum value at t0 and
decay rate constant K, calculated for each nonlinear regression curve. The half-life of the RAMP message was calculated as
equal to ln (2/K).
Western blot analysis.
Western blot analysis was done as described before (37).
Briefly, equal concentrations of protein samples were subjected to
SDS-PAGE 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-3 polyclonal primary antibodies at a final concentration of 400 ng/ml
(Santa Cruz Biotechnology, Santa Cruz, CA) followed by horseradish peroxidase-conjugated secondary anti-rabbit IgG antibodies at a final
dilution of 1:10,000 (Sigma), according to the manufacturer's instructions. An enhanced chemiluminescence (ECL) kit (Pierce, Rockford, IL) was used to visualize the blots.
Statistical analysis.
Data are presented as means ± SE. Multiple group comparisons were
made using a two-way ANOVA. Single group comparisons exercised the
Student's t-test method. Statistical significance was set at P < 0.05.
 |
RESULTS |
AM receptor components in RMCs.
We first characterized the expression of AM receptor components in
quiescent RMCs by RT-PCR as described in MATERIALS AND METHODS. Under basal conditions, RMCs express CRLR as well as all three subtypes of RAMP (RAMP-1, -2, and -3; Fig.
1).

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Fig. 1.
Receptor activity modifying protein (RAMP) and calcitonin
receptor-like receptor (CRLR) expression profile in quiescent rat
mesangial cells (RMCs). RT-PCR was performed on total RNA isolated from
RMCs 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 RT (no-RT control). RMCs express
detectable levels of CRLR as well as all 3 subtypes of RAMPs.
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Effects of RAMP overexpression on AM-induced adenylate cyclase
activity and [3H]thymidine incorporation in RMCs.
To investigate the effect of RAMP overexpression on mesangial cell
responsiveness to AM, we transiently transfected RMCs with RAMP-1, -2, and -3 and subsequently examined AM-mediated adenylate cyclase activity
and [3H]thymidine incorporation. Western blot analysis
revealed significantly higher levels of RAMP-1, -2, and -3 protein
expression in the membrane fraction compared with vector transfected
cells (data not shown). Transfection of RAMP-2 or RAMP-3 resulted in a
85.4 ± 3.02 and 54.5 ± 2.63% increase in AM-induced
adenylate cyclase activity, respectively (Fig.
2). These responses were further potentiated by cotransfection of CRLR with RAMP-2 or RAMP-3 (136.6 ± 7.9 and 90.71 ± 3.8% increase, respectively). AM-induced
adenylate cyclase activity in RAMP-2/RAMP-3-transfected cells was also
inhibited by pretreatment with AM-(22-52), AM
receptor antagonist, indicating that this effect is AM receptor
mediated (results not shown). Transfection of RAMP-1, on the other
hand, had no effect on AM-mediated responses (Fig. 2), which is
consistent with previously reported data describing RAMP-1-CRLR as a
receptor complex with low AM binding specificity (4, 15).

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Fig. 2.
Effect of RAMP-1, RAMP-2, and RAMP-3 overexpression on
adrenomedullin (AM)-stimulated adenylate cyclase activity in RMCs. RMCs
were transfected with vector or vector+RAMP-1, RAMP-2, or RAMP-3 at
~60% confluence. Cells were allowed to grow for another 24-36
h. 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
RMCs. Overexpression of RAMP-1 had no significant effect on AC
activity. * P 0.01; n.s., statistically not
significant; experiments performed in triplicates; n 3.
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The antiproliferative effect of AM on mesangial cells has been
previously demonstrated by our laboratory as well as others (6,
37, 44). 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
[3H]thymidine incorporation. As predicted, transfection
of RAMP-2 or RAMP-3 significantly potentiated AM-mediated inhibition of [3H]thymidine incorporation (Fig.
3). Again, RAMP-1 transfection had no
significant effect on mesangial cell responsiveness to AM as assessed
by [3H]thymidine incorporation assay (
41.6 ± 4.9 and
29.8 ± 5.4% inhibition of
[3H]thymidine incorporation for RAMP-1 and vector alone,
respectively; P > 0.1).

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Fig. 3.
Effect of RAMP-2 and RAMP-3 overexpression on
[3H]thymidine incorporation in RMCs. Cells were
transiently transfected with RAMP-2 or RAMP-3 and allowed to grow for
additional 24-36 h. Subsequently, labeled thymidine incorporation
in response to 100 nM AM was followed per protocol described in
MATERIALS AND METHODS. RAMP-2 and RAMP-3 overexpression
significantly potentiated AM-mediated decrease of
[3H]thymidine incorporation in RMCs;
* P < 0.05 compared with vector; experiments
performed in quadruplicates; n = 4.
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Effect of PDGF on RAMP mRNA expression.
In concordance with findings from other cell systems, overexpression of
RAMP-2 and -3 in mesangial cells also led to potentiated AM
responsiveness. 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.
4A), whereas it had no effect
on the RAMP-2 message (Fig. 4B).

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Fig. 4.
Effect of platelet-derived growth factor (PDGF) on RAMP-3 and
RAMP-2 mRNA expression in RMCs. Cells were treated with vehicle (basal)
or doses of PDGF, as indicated, for 24 h. RNA was extracted, and
Northern blot analysis was performed as described in MATERIALS
AND METHODS. Blots were first probed for RAMP-2 or RAMP-3 and
then stripped and reprobed for 18S RNA. Raw values were converted to
ratios of RAMP-3 mRNA to 18S and then expressed as percent change from
basal. Aa: PDGF increased RAMP-3 mRNA expression in a
dose-dependent manner. * P < 0.01, ** P < 0.001 compared with 0.1 ng/ml PDGF dose;
n = 5. Ab and Ac: representative
Northern blots of RAMP-3 expression in response to PDGF in RMCs.
B: PDGF had no significant effect on RAMP-2 expression
compared with basal; n.s., statistically not significant;
n = 4. Right: a representative Northern blot
of RAMP-2 expression in response to PDGF in RMCs.
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Mechanism of PDGF-stimulated RAMP-3 expression.
To determine whether PDGF affects transcriptional events leading to
increased abundance of RAMP-3, we tested actinomycin D and
-amanitin
(inhibitors of DNA-dependent RNA synthesis) for their ability to
influence the PDGF-dependent effect. Preincubation of mesangial cells
with actinomycin D (5 µg/ml) did not alter PDGF-induced RAMP-3
expression (Fig. 5A),
suggesting the lack of regulation at a transcriptional level. To verify
the effectiveness of transcriptional inhibition by actinomycin D,
Northern blots were reprobed with mitogen-activated protein kinase
(MAPK) kinase (MEK)-1 cDNA, and quantification of MEK-1 RNA
was performed. As previously reported by Schramek et al.
(43), PDGF induced MEK-1 mRNA, and this effect was
inhibited by actinomycin D (Fig. 5B), indicating a good
transcriptional inhibition by actinomycin D. Similarly, preincubation
of the cells with
-amanitin (1 µg/ml) for 5-6 h did not have
any effect on PDGF-induced RAMP-3 mRNA elevation. Coexposure of cells
to
-amanitin and PDGF resulted in a 58.49 ± 2.56% increase in
RAMP-3 mRNA expression above basal compared with a 55.46 ± 3.47%
increase in cells treated with 50 ng/ml PDGF alone (statistical
difference between these results is not significant, with
P = 0.665, n = 3). Next, a requirement for new protein synthesis was examined by pretreatment of PDGF- exposed
cells to cycloheximide (CHX), a potent eukaryotic translational inhibitor. At 10 µg/ml, CHX significantly inhibited PDGF-induced RAMP-3 mRNA expression, identifying a requisite step of de novo protein synthesis (Fig. 6).

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Fig. 5.
Effect of actinomycin D (ActD; 5 µg/ml) on
PDGF-stimulated RAMP-3 expression in RMCs. RMCs were serum starved
overnight and treated with or without PDGF (50 ng/ml) in the presence
of ActD for 24 h. Northern blot analysis was performed as
described in MATERIALS AND METHODS, and the blot was probed
for RAMP-3 or mitogen-activated protein kinase kinase (MEK)-1 mRNA
expression. A: ActD did not inhibit PDGF-induced RAMP-3
expression, suggesting that RAMP-3 expression in mesangial cells may
not be regulated by increase in transcription. Inset:
representative Northern blot for this experiment; n 3. B: control experiment verifying that PDGF-increased
transcription of MEK-1 is inhibited by ActD. This experiment is shown
to eliminate the possibility that ActD is inactive. The representative
Northern blot shown here for MEK-1 expression is the same one that was
used for probing RAMP-3.
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Fig. 6.
Effect of cycloheximide (CHX; 10 µg/ml) on PDGF-induced
RAMP-3 expression in RMCs. RMCs were serum starved overnight and
treated with or without PDGF (50 ng/ml) in the presence of CHX for
24 h. RNA was extracted, and Northern blot analysis was 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 of RAMP-3 mRNA to 18S and then
expressed as percent change from basal. Inset:
representative Northern blot of RAMP-3 expression in response to
PDGF ± CHX. CHX significantly attenuated PDGF-induced RAMP-3 mRNA
expression; ** P 0.001; n = 3.
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Because a PDGF-induced increase in RAMP-3 mRNA expression
was not mediated through transcriptional events, we further
hypothesized that PDGF enhances RAMP-3 mRNA stability, thus leading to
increased abundance of the RAMP-3 message. Accordingly, we analyzed the effect of PDGF on the half-life of RAMP-3 mRNA as described in MATERIALS AND METHODS. PDGF (50 ng/ml) treatment increased
the apparent half-life of RAMP-3 mRNA from 66.5 to 331.6 min (Fig. 7). This indicates that the mechanism of
PDGF-induced RAMP-3 expression is mediated by a posttranscriptional
event of mRNA stability enhancement.

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Fig. 7.
Effect of PDGF on RAMP-3 mRNA rate of decay in RMCs. RNA
stability assay was performed on RMCs as described in MATERIALS
AND METHODS. RNA from each time point was analyzed by Northern
blot hybridization, and relative RAMP-3 mRNA abundance was expressed as
a percentage of that present at time 0 (percentage of
maximum value). RAMP-3 mRNA half-lives were calculated by using the
decay constants (K) obtained from one-phase exponential
decay curves. Inset: representative Northern blot obtained
following the RNA stability assay protocol. PDGF significantly
increased RAMP-3 mRNA half-life from 1.1 to 5.5 h. Regression
lines (half-lives) for RNA decay from PDGF-treated ( )
and no PDGF ( ) RMCs were compared by analysis of
covariance. Regressions were regarded significantly different with a
value of P 0.01; n 3.
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Involvement of signal transduction pathway(s) in PDGF-dependent
RAMP-3 upregulation.
PDGF has been shown to exert myriad biological effects principally by
acting through its receptor, PDGFR, known to have an intrinsic tyrosine
kinase activity (18). 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 (27), abrogated the PDGF-induced response (Fig.
8). The effect was dose dependent for
AG-1296 concentrations of 0.1-30 µM (data not shown). PD-153035
and PD-168393, selective inhibitors of epidermal growth factor
receptor-associated tyrosine kinase (3, 17), did not,
however, have any effect on PDGF-stimulated RAMP-3 mRNA expression
(Fig. 8). Accordingly, the observed increase in RAMP-3 mRNA abundance
after exposure to PDGF is PDGFR mediated and receptor-associated tyrosine kinase dependent.

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Fig. 8.
Effects of AG-1296, PD-153035, and PD-168393 on
PDGF-stimulated RAMP-3 mRNA expression in RMCs. Cells were pretreated
with inhibitors and subsequently exposed to PDGF (50 ng/ml) for 24 h. 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.
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Because initiation of intracellular tyrosine kinase activity often
triggers a cascade of events mediated via MAPKs, we investigated the
effects of PD-98059 (10 µM) and SB-203580 (10 µM) (selective inhibitors of MEK and p38 MAPK, respectively) on PDGF-induced RAMP-3
expression. Both inhibitors significantly attenuated the PDGF-induced
response (Fig. 9), indicating that RAMP-3
expression in RMCs is, at least in part, regulated by the MAPK pathway.

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Fig. 9.
Effects of SB-203580 (10 µM) and PD-98059 (10 µM) on
PDGF-induced RAMP-3 expression in RMCs. Cells were pretreated with
inhibitors and subsequently exposed to PDGF (50 ng/ml) for 24 h.
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 mRNA to 18S and then expressed as
percent change from basal. Inset: representative Northern
blot of RAMP-3 expression in response to pretreatment of RMC with
SB-203580 and PD-98059 followed by PDGF exposure. Both SB-203580 and
PD-98059 significantly attenuated PDGF-induced RAMP-3 expression;
* P < 0.001; n = 5.
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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 compared with controls (Fig.
10). 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. 11A). Also, membranes of
cells treated with various concentrations of PDGF (1-100 ng/ml)
exhibited a concentration-dependent increase in AM-stimulated adenylate
cyclase activity (Fig. 11B). This effect was inhibited by
pretreatment of membranes with AM-(22-52), suggesting a direct involvement of AM-specific receptors (Fig. 11C).

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|
Fig. 10.
Effect of PDGF on membrane-associated RAMP-3 protein
expression in RMCs. Cells were incubated with PDGF (50 ng/ml) for
24 h. 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 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:
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.
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|

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Fig. 11.
Effect of PDGF on AM-mediated AC activity in RMCs.
Cells were incubated in the presence or absence of PDGF (50 ng/ml) for
24 h. 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
control cells. PDGF-treated cells exhibited significantly higher AC
activity compared with corresponding basals (* P < 0.01) and control-treated cells (## P < 0.01). Experiments were performed in triplicates; n = 5. B: cells were treated with different concentrations of
PDGF, as indicated, for 24 h. 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.
4A); experiments were done in triplicates; n = 3. C: effect of AM-(22-52), the AM
receptor antagonist, on AM-mediated AC activity in RMCs exposed to
PDGF. Cells were grown in the presence of PDGF (50 ng/ml) or its
absence (control) for 24 h. Next, membranes were extracted and
pretreated with AM-(22-52) at 100 nM (+) or 1 µM
(++) for 10 min before AC activity assay in response to 100 nM AM.
PDGF-dependent increase in AM-mediated AC activity was significantly
inhibited by AM-(22-52). * P < 0.01 (compared with PDGF treatment),
P < 0.01 (compared with control);
experiments were performed in triplicates; n = 3.
|
|
 |
DISCUSSION |
Initial reports characterizing glomerular mesangial cells
emphasized their importance in providing structural integrity to the
glomerulus as well as their likely involvement in glomerular filtration
rate regulation (for reviews see Refs. 8 and 42). Recent
discoveries focus on the contribution of the mesangial cell to the
genesis and progression of several glomerulopathies. Excessive
mesangial cell proliferation and matrix deposition are considered to be
the hallmark for several renal diseases (10, 12, 25).
These alterations in mesangial cell biology instigate the maladaptive
process of glomerular hypertrophy resulting in glomerulosclerosis,
often leading to end-stage renal failure (13, 21, 46, 49,
52). Several agents have been proposed to influence the course
and progression of renal disease (24). Of these, one of
the most recently identified peptides, AM, has been shown to have
several actions of glomerular mesangial cells, suggesting that it may
serve to be an important autocrine/paracrine factor that regulates
mesangial cell turnover. We have recently reviewed the current
understanding of the actions of AM on glomerular mesangial cells
(38). Restoration of normal kidney function and histology
by AM has been suggested in several in vivo studies of kidney disease
models. Dobrzynski et al. (9) reported that renal damage
in deoxycorticosterone acetate salt hypertensive rat models was
significantly attenuated by AM gene delivery. Others also confirmed the
beneficial effects of AM administration in humans as well as animal
models of renal disease (22, 30, 50). Although the exact
mechanism for the renoprotective effect of AM is unclear at this time,
it is hypothesized that the antiproliferative (and proapoptotic)
effects of AM on mesangial cells may in part serve to protect the
kidney during development of glomerular disease. Accordingly,
identification of mechanisms responsible for modulation of the activity
of AM in mesangial cells will benefit our understanding of mesangial
growth regulation and the associated progress of several renal pathologies.
Mesangial cells express a functional AM receptor complex
(CRLR+RAMP-2/RAMP-3) in culture. In addition, as observed by others in
different cell lines, overexpression of RAMP-2 or RAMP-3 also increased
RMC responsiveness to AM as measured by AM-induced adenylate cyclase
activity (15, 19). Fraser et al. (15) and
Husmann et al. (19) have reported that
cotransfection of HEK-293 and COS-7 cells with CRLR and RAMP-2/RAMP-3
exhibit increased 125I-labeled 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 the responsiveness of cells to AM
not only in other cell types, but also in mesangial cells. Here, we
propose that factor(s) that influence the AM signaling system in
mesangial cells 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 the AM
antiproliferative pathway. Specifically, PDGF-induced mitogenesis is blocked by AM in RMCs in culture. Indeed, if PDGF enhances AM
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 the AM receptor system, including its effects on CRLR, RAMP-2, and RAMP-3 in RMCs. We report
here that PDGF regulates AM receptor signaling through modulation of
RAMP-3 mRNA expression. PDGF did not have any effect on CRLR or RAMP-2
mRNA expression. Others have found similar results in both animal and
cell culture models. For example, Totsune et al. (47)
presented findings from a rat model of 5/6 nephrectomy where RAMP-2
mRNA levels were unchanged whereas a significant decrease in RAMP-3
mRNA was found in the remnant kidney. In general, ample evidence
exists for differential expression of RAMP mRNA in tissues from animal
disease models, including those with renal pathology (32-34,
39, 48, 53). Frayon et al. (16) have reported that
glucocorticoid treatment in vascular smooth muscle cells in
culture causes a transient increase in RAMP-1 mRNA expression without
any change in RAMP-2 expression. This glucocorticoid-directed RAMP
expression results in a CGRP-responsive receptor (CRLR+RAMP-1). These
results also suggest that, in vivo, glucocorticoid regulation of RAMP-1
mRNA expression may lead to a subsequent consequence on ligand
recognition. Similarly, Robert-Nicoud et al. (40) reported
vasopressin-induced RAMP-3 mRNA levels in the mouse clonal cortical
collecting duct principal cell line. 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 AM signaling. In fact, our
results suggest that PDGF-stimulated RAMP-3 expression leads to
AM-stimulated adenylate cyclase activity. This effect, if present in
vivo, may augment AM signaling and hence increase the antiproliferative effect of AM, opposing the proliferative effect of PDGF. This negative
feedback mechanism may be present to keep the mesangial cell growth 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. Though this hypothesis is attractive, clearly further
studies are essential.
Although both Frayon et al. (16) and Robert-Nicoud et al.
(40) 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 for PDGF-induced RAMP-3 expression to be transcriptionally independent, because it was not inhibited by actinomycin D or
-amanitin.
Furthermore, our data indicate that PDGF acts via stabilizing RAMP-3
mRNA, consequently increasing the apparent half-life of the message by
nearly fivefold. Though we established the requirement of de novo
protein synthesis for this effect, future studies are necessary to
further characterize the exact mechanism(s) by which PDGF influences the stability of the RAMP-3 message. It is worth noting that PDGF had
no significant effect on RAMP-2 expression, suggesting a differential regulation between the closely related RAMP-2 and RAMP-3.
One of the aims of this study was to investigate the mechanism of
PDGF-induced RAMP-3 expression at the signaling level (particularly the
role of MAPKs). Cellular actions of PDGF have been shown to involve
both its receptor tyrosine kinase activity and MAPK pathways. To
examine the role of MAPKs, we utilized specific inhibitors to block
these signaling molecules. The effect of AG-1296 (PDGFR tyrosine kinase
blocker) suggests that the effects of PDGF on RAMP-3 expression are
indeed mediated through PDGFR tyrosine kinase activity. Furthermore,
inhibition of MEK (kinase upstream of extracellular signal-related
kinase) by PD-98059, and p38 MAPK by SB-203580, suggests that
activation of these kinases by PDGF may be important in the regulation
of RAMP-3 expression. 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.
The discovery of the GPCR-associated family of RAMP proteins prompted a
new look at the paradigm for receptor phenotype determination. It also
provided a plethora of opportunities to recognize novel mechanisms
responsible for modulation of receptor activity. RAMP-1, -2, and -3 have been shown to differentially couple to CR and CRLR, giving rise to
distinct receptor phenotype characteristics (7, 31).
Receptor trafficking, glycosylation, and a direct RAMP-receptor
interaction have all been proposed as possible mechanisms responsible
for the critical role of RAMPs in CR and CRLR ligand specificity
determination [for reviews see Foord et al. (14) and
Sexton et al. (45)]. Accumulating data suggest that the dynamic alteration of RAMP expression levels may provide for yet another mode by which these proteins regulate the function of CRLR and
possibly other receptors. The current study identified a model for RAMP
mRNA regulation by a growth factor as a means for alteration of the
functional responsiveness of a cell to a ligand.
In summary, we report that mesangial cells express CRLR, RAMP-1,
RAMP-2, and RAMP-3 at basal conditions. PDGF, a pleotrophic cytokine
well established to influence mesangial cell biology, increases 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 mRNA elevation to be MEK and p38 MAPK
dependent and posttranscriptionally regulated. PDGF augments the
abundance of RAMP-3 mRNA via stabilization of the message and a
consequent increase in RAMP-3 mRNA half-life. Taken together, these
data suggest an important and novel mechanism for regulation of RAMP-3
in RMCs.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the expert assistance of Dr. Kazuhito
Totsune (Second Department of Internal Medicine, Tohoku University School of Medicine, Seiryo-machi, Aoba-ku, Sendai, Miyagi, Japan) in
the selection of appropriate primer sequences for RT-PCR analysis of
rat mesangial cell CRLR and RAMP expression.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
W. S. Spielman, Michigan State Univ., Dept. of Physiology,
103A Giltner Hall, East Lansing, MI 48824 (E-mail:
spielman{at}msu.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 23, 2002;10.1152/ajpcell.00561.2001
Received 26 November 2001; accepted in final form 22 January 2002.
 |
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