Coexpression of Calcitonin Receptor-Like Receptor and Receptor Activity-Modifying Protein 2 or 3 Mediates the Antimigratory Effect of Adrenomedullin
Abstractq/#r*, 百拇医药
Three isoforms of the receptor activity-modifying protein (RAMP) are thought to transport the calcitonin receptor-like receptor (CRLR) to the plasma membrane to function as calcitonin gene-related peptide or adrenomedullin receptors, but their role remains largely unknown. We investigated whether coexpression of RAMP and CRLR are involved in the regulation of cell migration using a monolayer-wounding protocol. Quantification of gene transcripts revealed expression of all RAMP isoforms and CRLR in cultured rat vascular smooth muscle cells (VSMCs), RAMP2 and RAMP3 in rat endothelial cells, and RAMP1 in rat fibroblasts. CRLR expression was minimal in endothelial cells and fibroblasts. Adrenomedullin potently suppressed the migration of VSMCs, whereas calcitonin gene-related peptide did not suppress migration in any cell type. The antimigratory effect of adrenomedullin on VSMCs was potentiated by transfecting CRLR cDNA. Cotransfection of RAMP2 or RAMP3 with CRLR into VSMCs resulted in a slower migratory rate, and this effect was enhanced by adrenomedullin. Migration of fibroblasts was also suppressed after cotransfection of RAMP2 or RAMP3 with CRLR. cAMP agonists had no effect on VSMC migration, and a cAMP antagonist failed to abrogate the antimigratory effect of adrenomedullin. Thus, coexpression of CRLR and RAMP2 or RAMP3 mediates the inhibitory effect of adrenomedullin on cell migration, independent of cAMP-dependent signaling pathways.
Introductionl4!1h, 百拇医药
ADRENOMEDULLIN IS A POTENT vasodilator peptide (1) and belongs to the calcitonin family of peptides that include calcitonin gene-related peptide (CGRP). Adrenomedullin is synthesized and secreted from endothelial cells, vascular smooth muscle cells (VSMCs), and fibroblasts (2, 3, 4, 5) and has been shown to regulate cell proliferation (6, 7)and apoptosis (8, 9). Adrenomedullin stimulates the proliferation of rat VSMCs via activation of ERK/MAPK (6, 7), whereas it antagonizes c-Myc-induced apoptosis in endothelial cells (9). It has been shown that adrenomedullin suppresses the migration of VSMCs stimulated by a variety of chemoattractants, including serum, platelet-derived growth factor, angiotensin II, and lysophosphatidylcholine (10, 11). cAMP has been implicated as an intracellular second messenger in the inhibition of VSMC migration (10, 12). Thus, adrenomedullin may modify the process of atherosclerosis by antagonizing VSMC migration in addition to its mitogenic and antiapoptotic actions.
Calcitonin receptor-like receptor (CRLR) was originally identified as a seven-transmembrane orphan receptor (13, 14). It was later shown that human CRLR transfected into human embryo kidney 293 cells exhibited the pharmacology of a CGRP1 receptor. Recently three isoforms of receptor activity-modifying protein (RAMP) were isolated (15). CRLR can function as either a CGRP receptor or an adrenomedullin receptor, depending on which RAMP isoforms are expressed. The RAMP isoforms have varied tissue expression and facilitate expression of CRLR. Coexpression of RAMP1 and CRLR conferred CGRP1 and adrenomedullin receptor activities (16), whereas RAMP2 or RAMP3 coexpressed with CRLR resulted in normal adrenomedullin receptor pharmacology (17). The expression of adrenomedullin and CGRP receptors determined by RAMP and CGRP were evaluated by the ability to activate cAMP, but other second-messenger pathways may be involved in mediating their activity. These findings led us to examine whether the RAMP isoforms and CRLR are involved in the migration of VSMCs.
Materials and Methods(9z3a5, http://www.100md.com
Materials(9z3a5, http://www.100md.com
Rat adrenomedullin, adrenomedullin (22–52), adrenomedullin (1–25), proadrenomedullin NH2-terminal 20 peptide, and CGRP (8–37) were purchased from the Peptide Institute (Osaka, Japan), Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Rp-cAMPS) from Sigma (St. Louis, MO), forskolin and prostaglandin I2 from Wako Pure Chemical Industries (Osaka, Japan), DMEM from Flow Laboratories (Irvine, Scotland, UK), and fetal bovine serum (FBS) and calf serum (CS) from Cell Culture Laboratories (Cleveland, OH). RAMP1-pcDNA, RAMP2-pcDNA, RAMP3-pcDNA, and CRLR-pcDNA were gifts from Dr. Steven M. Foord. PCR primers were synthesized by JbioS Inc. (Saitama, Japan).(9z3a5, http://www.100md.com
Cell culture and transfection protocol(9z3a5, http://www.100md.com
Primary rat aortic VSMCs and endothelial cells were prepared and maintained as described (9, 18, 19, 20). COS7 cells were cultured in DMEM supplemented with 10% FBS. Diploid rat fibroblast cell line originally derived from Rat-1 were cultured in DMEM supplemented with glutamine and 10% CS (21, 22). Transient transfections were performed using a modified transferrin receptor-operated protocol using Transfast (Promega Corp., Madison, WI) as described (9, 20, 22). Salmon sperm DNA was used as a carrier to adjust the total DNA content of each transfection.
cAMP and ERK/MAPK assays46itn, 百拇医药
The cAMP and ERK/MAPK-dependent reporter systems (PathDetect trans-reporting system, Stratagene, La Jolla, CA) were used to measure cAMP and ERK/MAPK activity (20). The systems include the fusion activator plasmids consisting of the DNA-binding domain of the yeast GAL4 fusion activator (pFR-Luc) and the activation domain of the CREB or Elk1 transcription factor (pFC2-CREB or pFC2-Elk1). Cells plated in 96-well plates were cotransfected with pFC2-CREB or pFC2-Elk1 (50 ng each/well), pFR-Luc reporter plasmid (1 µg each/well), pRL-TK vector (1 µg each/well; Promega Corp.) expressing renilla luciferase as an internal control with or without RAMP2-pcDNA/RAMP3-pcDNA and CRLR. Cells were incubated for 24 h after transfection in DMEM containing 10% FBS, replaced with media containing 1% FBS and incubated for an additional 24 h, followed by the addition of adrenomedullin for the indicated time, after which firefly and renilla luciferase activities were measured using the dual luciferase reporter assay system (Promega Corp.) in a single-tube assay format using MicroLumatPlus (EG&G Berthold, Wildbad, Germany). The firefly luciferase activity of each sample was normalized to an internal reference standard of renilla luciferase activity.
Quantification of mRNA]mg3%, 百拇医药
Rat RAMP and CRLR transcripts were quantified with a LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) PCR protocol in which fluorescence of SYBR green dye bound to amplified products is measured (18). Total RNA was extracted, first-strand cDNA synthesized, and each amplification reaction performed as described (9, 20). Primers used for amplification were: RAMP1, forward 5'-ctttcattgtgctccccatt-3' and reverse 5'-ggtctgtccctgatgctgtt-3'; RAMP2, forward 5'-tcaaaagggaagatggagga-3' and reverse 5'-gtctgcctcgtactccaagc-3'; RAMP3, forward 5'-aaggtggacgtctggaagtg-3' and reverse 5'-tgcctatggatacccgtgat-3'; CRLR, forward 5'-gcaaggtgtcccagttcatt-3' and reverse 5'-cccccagccaagaaaataat-3', and the reaction produced 151-bp, 152-bp, 148-bp, and 148-bp products, respectively. Conventional RT-PCR employed the same set of primers, and the amplification reactions were examined by 1.5% agarose gel electrophoresis.]mg3%, 百拇医药
Cell migration assay
Confluent monolayers in 6-well dishes (Corning, Inc., Corning, NY) were denuded with a 300-µm-wide surgical blade (Feather Safety Razor, Osaka, Japan), and phase-contrast 100x photomicroscopy was performed after 0, 6, 12, and 24 h at two distinct sites with the center of the wound (marked with a scratch) in the middle of each frame (18). The mean distance between the wound edge and the nuclei of the 10 farthest-migrating cells along a perpendicular line was measured with Adobe (San Jose, CA) Photoshop 5.0 software after the incorporation of photomicrographs using GT8000 (Epson, Tokyo, Japan), and normalization to a standard field width. Migration distance was calculated from the mean distances in successive frames of a particular field. Cell proliferation assays demonstrated that DNA synthesis in confluent cultures was negligible during the monolayer wounding experiments, and repopulation within 24 h is almost entirely due to cell migration (18). Subconfluent VSMCs were transfected with RAMP/CRLR, CRLR alone, or salmon sperm DNA, and rendered confluent after incubation for 48 h. Cells were incubated for 24 h with or without test reagents before the cell migration assay. Migration assays were performed with 10% FBS supplementation for VSMCs and endothelial cells and 10% CS with glutamine for fibroblasts.
Statistical analysis;*cul2, 百拇医药
Data were expressed as mean ± SEM. Differences between groups were examined for statistical significance using the unpaired t test. P values less than 0.05 were considered statistically significant.;*cul2, 百拇医药
Results;*cul2, 百拇医药
Expression of RAMP and CRLR;*cul2, 百拇医药
Quantification of mRNA transcripts using the real-time quantitative PCR method revealed that RAMP1 is expressed in fibroblasts and VSMCs, whereas RAMP2 and RAMP3 are expressed in endothelial cells and VSMCs (Fig. 1). CRLR mRNA expression in endothelial cells and fibroblasts was only 5% and 0.8%, respectively, of that in VSMCs (Fig. 1).;*cul2, 百拇医药
fig.ommitteedfig.ommitteed;*cul2, 百拇医药
Figure 1. Expression of RAMP isoforms and CRLR in rat endothelial cells, fibroblasts, and VSMCs. Quantification of RAMP1, RAMP2, RAMP3, and CRLR gene transcripts. Each gene transcript is expressed as percent ± SEM of transcript in rat VSMCs.
Antimigratory effect of adrenomedullin^, 百拇医药
Cell migratory rate was evaluated using a monolayer-wounding protocol in which cells migrated from the confluent to the denuded area. A wounded confluent monolayer of VSMCs cultured with 10% FBS and nutrients migrated rapidly at approximately 4 µm/h until the denuded area was repopulated (Fig. 2, A and B). Adrenomedullin (10-6 M) inhibited the migration of VSMCs but not that of endothelial cells or fibroblasts, whereas CGRP (10-6 M) did not have an antimigratory effect on any cell type tested (Fig. 3). Proadrenomedullin NH2-terminal 20 peptide, adrenomedullin (1–25), adrenomedullin (22–52), and CGRP (8–37) did not inhibit cell migration either.^, 百拇医药
fig.ommitteedfig.ommitteed^, 百拇医药
Figure 2. Migration of VSMCs and the effect of coexpression of RAMP2 and CRLR. A and B, Cell migration was initiated by wounding a confluent monolayer of nontransfected VSMCs. The leading edges immediately (A) and 24 h after wounding (B). C and D, The leading edges of a confluent monolayer cotransfected with RAMP and CRLR for 72 h and pretreated with 10-6 M adrenomedullin immediately (C) and 24 h after wounding (D).
fig.ommitteedfig.ommitteed#., 百拇医药
Figure 3. Effects of adrenomedullin on cell migration. Confluent endothelial cells (A), fibroblasts (B), and VSMCs (C) pretreated with or without adrenomedullin (10-6 M) or CGRP (10-6 M) for 24 h were denuded, and cell migration rates were determined. The graphs represent the average migration rate ± SEM during 12 h after denudement. *, P < 0.05 vs. untreated culture (n = 10).#., 百拇医药
Cotransfection of RAMP and CRLR#., 百拇医药
RAMP- and CRLR-expressing vectors were cotransfected into COS7 cells, and cAMP and ERK/MAPK activity was measured using the PathDetect system (Stratagene). RAMP isoforms cotransfected with CRLR increased cAMP activity in response to adrenomedullin, whereas transfection of CRLR alone did not (Fig. 4). Adrenomedullin failed to induce ERK/MAPK activity in RAMP/CRLR-transfected COS7 cells (data not shown). These results confirmed the expression of the adrenomedullin receptor by cotransfecting RAMP isoform with CRLR functionally coupled to a cAMP pathway.
fig.ommitteedfig.ommitteedlk:u\y$, http://www.100md.com
Figure 4. Adrenomedullin stimulates cAMP activity in COS7 cells expressing RAMP/CRLR. COS7 cells were cotransfected with pFR-Luc (firefly luciferase), pFA-CREB, pRL-TK (renilla luciferase), and CRLR-pcDNA with or without RAMP1-pcDNA, RAMP2-pcDNA, or RAMP3-pcDNA. Transcriptional activity of GAL4-CREB (pFA-CREB) after incubation with (closed column) or without (open column) adrenomedullin (10-6 M) was analyzed using firefly luciferase as a reporter gene normalized with renilla luciferase as an internal control. Each column represents mean ± SEM (n = 8).lk:u\y$, http://www.100md.com
Quantification of RAMP2 mRNA 48 h after cotransfection of RAMP2 and CRLR into endothelial cells and VSMCs were 1299 ± 182% and 272 ± 144%, respectively, of untransfected cells, whereas RAMP1 mRNA level after cotransfection of RAMP1 and CRLR into fibroblasts was 636 ± 151% of untransfected cells, indicating satisfactory transfection efficiency.lk:u\y$, http://www.100md.com
Mediation of RAMP/CRLR receptorslk:u\y$, http://www.100md.com
To determine whether the antimigratory effect of adrenomedullin is mediated by receptors consisting of RAMP and CRLR, we examined the migration rate of VSMCs and fibroblasts cotransfected with RAMP and CRLR. Transfection of CRLR into VSMCs markedly potentiated the antimigratory effect of adrenomedullin in a concentration-dependent manner (10-9–10-6 M; Fig. 5). Cotransfection of RAMP2 or RAMP3 with CRLR into VSMCs reduced the migratory rate to a greater extent than transfection of CRLR alone, and this effect was enhanced by adrenomedullin (Fig. 6A). The effect of adrenomedullin to suppress VSMC migration was blocked by an adrenomedullin receptor antagonist, CGRP (8–37) (Fig. 6B). However, CGRP did not affect the migration of VSMCs cotransfected with RAMP1 and CRLR (data not shown). In fibroblasts that express limited CRLR and no RAMP2 or RAMP3, the migration rate was not reduced after CRLR transfection alone, but cotransfection of RAMP2/RAMP3 with CRLR reduced the migratory rate by 49% to 58% (Fig. 7A). In cotransfected fibroblasts, adrenomedullin did not potentiate the antimigratory effect, but antiadrenomedullin antiserum significantly increased the cell migration rate (Fig. 7B).
fig.ommitteedfig.ommitteed)62t.ne, http://www.100md.com
Figure 5. Migration inhibition by adrenomedullin of VSMCs transfected with CRLR. Each column represents mean ± SEM (n = 10). *, P < 0.05 vs. control.)62t.ne, http://www.100md.com
fig.ommitteedfig.ommitteed)62t.ne, http://www.100md.com
Figure 6. Effects of RAMP and CRLR on VSMC migration. A, After cotransfection with RAMP2 or RAMP3 with CRLR or transfection with CRLR alone into rat VSMCs and fibroblasts, the migration rate was determined in the presence (closed column) or absence (open column) of adrenomedullin (10-6 M). B, The effect of adrenomedullin to inhibit migration of VSMCs cotransfected with RAMP2 with CRLR was abrogated by an adrenomedullin receptor antagonist, CGRP (8–37). Each column represents mean ± SEM (n = 10). *, P < 0.05 vs. control; **, P < 0.05 vs. AM(-) (n = 10).)62t.ne, http://www.100md.com
fig.ommitteedfig.ommitteed)62t.ne, http://www.100md.com
Figure 7. Effects of RAMP and CRLR on fibroblast migration. A, After cotransfection with RAMP2 or RAMP3 with CRLR or transfection with CRLR alone into rat fibroblasts, the migration rate was determined in the presence (closed column) or absence (open column) of adrenomedullin (10-6 M). B, The migration rate of fibroblasts cotransfected with RAMP2 with CRLR increased by addition of antiadrenomedullin antiserum to the media. Each column represents mean ± SEM (n = 10). NS, Not significant. *, P < 0.05 vs. control (n = 10).
To elucidate whether the inhibitory effect of adrenomedullin on VSMC migration via RAMP/CRLR is linked to cAMP generation, we examined the effects of forskolin and prostaglandin I2 on VSMC migration. Neither forskolin (10-3 M) nor prostaglandin I2 (10-5 M) reduced VSMC migration rate (Fig. 8A). Furthermore, the cAMP antagonist Rp-cAMPS did not affect adrenomedullin-induced inhibition of VSMC migration (Fig. 8B). To determine which signaling pathway other than cAMP is involved, we tested the following pharmacological inhibitors: phosphatidylinositol 3-kinase inhibitors (wortmannin, LY294002), tyrosine kinase inhibitors (genistein, herbimycin A, ST638), protein kinase C inhibitors (GF109203X, U73122), nuclear factor-B inhibitors (MG132), ras farnesyltransferase inhibitors (manumycin), p38 MAPK inhibitor (SB203580), methyl ethyl ketone inhibitor (PD98059), JAK-2 inhibitor (AG490), cGMP-dependent protein kinase inhibitor (KT5823), and soluble guanylate cyclase inhibitor (ODQ). None of the inhibitors antagonized the effect of adrenomedullin (data not shown).
fig.ommitteedfig.ommitteed'jp, 百拇医药
Figure 8. cAMP is not involved in antimigratory effect by adrenomedullin in rat VSMCs. A, Cells were treated with or without adrenomedullin (10-6 M), forskolin (10-3 M), or prostaglandin I2 (10-5 M), and the migration rate was determined. B, Cells, pretreated with or without Rp-cAMPS, were incubated with adrenomedullin (10-6 M), and cell migration rate was determined. Each column represents mean ± SEM (n = 10). *, P < 0.05 vs. control (n = 10).'jp, 百拇医药
Discussion'jp, 百拇医药
In the present study, we demonstrated using a monolayer-wounding assay that adrenomedullin is a potent antimigratory factor for VSMCs, although adrenomedullin failed to alter migration of fibroblasts or endothelial cells cultured under the same conditions. Using LightCycler technology (Roche Molecular Biochemicals) that allows reliable quantification results of gene expression, we have demonstrated that rat VSMCs abundantly expressed RAMP2, RAMP3, and CRLR, whereas rat endothelial cells and fibroblasts, which did not show any antimigratory effect by adrenomedullin, did not coexpress CRLR and RAMP2 or RAMP3. The migration of VSMCs that coexpress RAMP1 and CRLR was not suppressed by CGRP. These data suggest that adrenomedullin receptors consisting of the RAMP2 or RAMP3 isoforms coupled with CRLR may be involved in the antimigratory effect, whereas CGRP receptors consisting of RAMP1 and CRLR do not exert a similar effect.
Using transferrin receptor-operated gene transfer, we have shown that coexpression of either of the two isoforms, RAMP2 or RAMP3, with CRLR in COS7 cells resulted in increased cAMP activity by adrenomedullin. Coexpression of RAMP1 and CRLR also enhanced cAMP response to adrenomedullin as previously described (16). These results suggest that the functional expression of the RAMP/CRLR adrenomedullin receptor is coupled to adenylate cyclase. We have also demonstrated high transfection efficiency of our gene transfer protocol, not only in endothelial cells and fibroblasts but in VSMCs as well, by quantifying RAMP mRNA after cotransfection of RAMP and CRLR.997e%|{, 百拇医药
We reported previously that adrenomedullin stimulated intracellular cAMP in our VSMCs and endothelial cells (8, 23). However, quantification of RAMPs and CRLR revealed limited expression of CRLR transcripts in endothelial cells, suggesting the possibility that adrenomedullin receptors other than RAMP/CRLR may also mediate cAMP response in endothelial cells. Cotransfection of RAMP2 or RAMP3 with CRLR did not result in further marked cAMP elevation in response to adrenomedullin in endothelial cells and VSMCs (data not shown). These results suggest that cell type specificity of adrenomedullin antimigratory actions can be explained by cell type-specific expression of RAMP isoforms and CRLR, rather than by cAMP response.
Transfection of CRLR alone into VSMCs potentiated the antimigratory effect of adrenomedullin in a dose-dependent fashion. Furthermore, cotransfection of CRLR with RAMP2 or RAMP3 markedly reduced the migration of VSMCs, an effect that was potentiated by adrenomedullin. Because VSMCs synthesize and secrete less adrenomedullin than fibroblasts (3), it is possible that the antimigratory response to exogenous adrenomedullin may be more profound in VSMCs than fibroblasts. Our study suggests that coexpression of CRLR with RAMP2 or RAMP3 mediates the adrenomedullin-induced antimigratory effect in VSMCs.48c2v|, 百拇医药
It has been shown that fibroblasts synthesize and secrete abundant adrenomedullin-like immunoreactivity and endogenous adrenomedullin has autocrine/paracrine roles in fibroblasts (3, 24). Our fibroblasts also secrete 3–5 ng/107 cells per 6 h of adrenomedullin molecules into serum-free cultured media as detected by our specific RIA and more than 10 times of that when cultured in serum-supplemented media. Both antiadrenomedullin antiserum (Fig. 7B) and adrenomedullin receptor antagonist (data not shown) increased the cell-migratory rate of fibroblasts cotransfected with RAMP2/CRLR but not that of VSMCs, suggesting a differential role for endogenous adrenomedullin in cell migration.
It has been reported that cAMP suppresses migration of VSMCs stimulated by FBS and platelet-derived growth factor-BB as chemoattractants using transwell cell culture chambers (10). However, in our study of monolayer denuding, cAMP agonists did not reduce the migratory rate and a cAMP antagonist did not abrogate the antimigratory effect of adrenomedullin. Additionally, CGRP had no antimigratory effect on VSMCs that coexpress RAMP1 and CRLR and induce cAMP. These data suggest that cAMP is not involved in inhibiting VSMC migration under full-growth stimulation with FBS. In an attempt to identify signals for antimigratory effect mediated by RAMP/CRLR, we used various pharmacological inhibitors to antagonize adrenomedullin, but none were effective. The molecular mechanisms mediated by RAMP/CRLR remain to be elucidated. The reasons for the discrepancies between our results and the previous study remain unknown, but they are not necessarily contradictory. The migration assays between the two studies are different. In the studies using the Boyden chamber, VSMCs suspended in media without platelet-derived growth factor, lysophosphatidylcholine or FBS may migrate toward chemoattractants in the lower chemotaxis chamber. However, in the monolayer-denuding assay, confluent cells that had been contact inhibited migrate after denudement and being exposed to 10% FBS and nutrients. Monolayer-wounding assays may more closely reflect the initial stages of vascular remodeling than chemotaxis chambers. Adrenomedullin may have two mechanisms in inhibiting migration, one mediated via CRLR and RAMP2/RAMP3 independent of cAMP and a second chemotactic mechanism dependent on cAMP.
Accumulating lines of evidence supports the role for adrenomedullin as a growth factor in both nontumor (3, 6, 7, 25, 26, 27, 28) and tumor cells (29, 30, 31, 32) probably via activation of ERK/MAPK (6, 7). In contrast, adrenomedullin antagonizes mitogenesis in certain cell types (24, 33, 34, 35). Differential growth responses to adrenomedullin in each cell type may be dependent on intracellular signaling pathway downstream to adrenomedullin receptor. For example, in cells lacking the ERK/MAPK activation signaling pathway, such as fibroblasts, stimulation of adrenomedullin receptor and subsequent activation of cAMP may cause inhibition of cell growth, whereas in the majority of cell types, including our VSMCs, adrenomedullin stimulates ERK/MAPK and promotes mitogenesis. Our present results show that RAMP/CRLR receptor is not coupled to ERK/MAPK but mediates inhibition of cell migration via cAMP-independent mechanism.m, http://www.100md.com
In conclusion, we have demonstrated that receptors consisting of CRLR and RAMP2 or RAMP3 mediate adrenomedullin-induced inhibition of migration. Molecular mechanisms downstream to the receptors remain to be determined.
Acknowledgments&&, http://www.100md.com
The authors gratefully acknowledge Steven M. Foord, Ph.D. (GlaxoSmithKline) for RAMP1-pcDNA, RAMP2-pcDNA, RAMP3-pcDNA, and CRLR-pcDNA plasmids.&&, http://www.100md.com
Received May 1, 2002.&&, http://www.100md.com
Accepted for publication October 21, 2002.&&, http://www.100md.com
References&&, http://www.100md.com
Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, Eto T 1993 Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 192:553–560&&, http://www.100md.com
Hinson JP, Kapas S, Smith DM 2000 Adrenomedullin, a multifunctional regulatory peptide. Endocr Rev 21:138–167&&, http://www.100md.com
Isumi Y, Minamino N, Katafuchi T, Yoshioka M, Tsuji T, Kangawa K, Matsuo H 1998 Adrenomedullin production in fibroblasts: its possible function as a growth regulator of Swiss 3T3 cells. Endocrinology 139:2552–2563&&, http://www.100md.com
Sugo S, Minamino N, Kangawa K, Miyamoto K, Kitamura K, Sakata J, Eto T, Matsuo H 1994 Endothelial cells actively synthesize and secrete adrenomedullin. Biochem Biophys Res Commun 201:1160–1166
Sugo S, Minamino N, Shoji H, Kangawa K, Kitamura K, Eto T, Matsuo H 1994 Production and secretion of adrenomedullin from vascular smooth muscle cells: augmented production by tumor necrosis factor-{alpha} . Biochem Biophys Res Commun 203:719–726ax9@}, 百拇医药
Iwasaki H, Eguchi S, Shichiri M, Marumo F, Hirata Y 1998 Adrenomedullin as a novel growth-promoting factor for cultured vascular smooth muscle cells: role of tyrosine kinase-mediated mitogen-activated protein kinase activation. Endocrinology 139:3432–3441ax9@}, 百拇医药
Iwasaki H, Shichiri M, Marumo F, Hirata Y 2001 Adrenomedullin stimulates proline-rich tyrosine kinase 2 in vascular smooth muscle cells. Endocrinology 142:564–572ax9@}, 百拇医药
Kato H, Shichiri M, Marumo F, Hirata Y 1997 Adrenomedullin as an autocrine/paracrine apoptosis survival factor for rat endothelial cells. Endocrinology 138:2615–2620ax9@}, 百拇医药
Shichiri M, Kato H, Doi M, Marumo F, Hirata Y 1999 Induction of Max by adrenomedullin and calcitonin gene-related peptide antagonizes endothelial apoptosis. Mol Endocrinol 13:1353–1363
Horio T, Kohno M, Kano H, Ikeda M, Yasunari K, Yokokawa K, Minami M, Takeda T 1995 Adrenomedullin as a novel antimigration factor of vascular smooth muscle cells. Circ Res 77:660–6644p, http://www.100md.com
Kohno M, Yokokawa K, Yasunari K, Minami M, Kano H, Hanehira T, Yoshikawa J 1998 Induction by lysophosphatidylcholine, a major phospholipid component of atherogenic lipoproteins, of human coronary artery smooth muscle cell migration. Circulation 98:353–3594p, http://www.100md.com
Kohno M, Yokokawa K, Kano H, Yasunari K, Minami M, Hanehira T, Yoshikawa J 1997 Adrenomedullin is a potent inhibitor of angiotensin II-induced migration of human coronary artery smooth muscle cells. Hypertension 29:1309–13134p, http://www.100md.com
Chang CP, Pearse 2nd RV, O’Connell S, Rosenfeld MG 1993 Identification of a seven transmembrane helix receptor for corticotropin-releasing factor and sauvagine in mammalian brain. Neuron 11:1187–11954p, http://www.100md.com
Njuki F, Nicholl CG, Howard A, Mak JC, Barnes PJ, Girgis SI, Legon S 1993 A new calcitonin-receptor-like sequence in rat pulmonary blood vessels. Clin Sci 85:385–388
McLatchie LM, Fraser NJ, Main MJ, Wise A, Brown J, Thompson N, Solari R, Lee MG, Foord SM 1998 RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 393:333–339qx, 百拇医药
Buhlmann N, Leuthauser K, Muff R, Fischer JA, Born W 1999 A receptor activity modifying protein (RAMP)2-dependent adrenomedullin receptor is a calcitonin gene-related peptide receptor when coexpressed with human RAMP1. Endocrinology 140:2883–2890qx, 百拇医药
Aiyar N, Rand K, Elshourbagy NA, Zeng Z, Adamou JE, Bergsma DJ, Li Y 1996 A cDNA encoding the calcitonin gene-related peptide type 1 receptor. J Biol Chem 271:11325–11329qx, 百拇医药
Shichiri M, Hirata Y 2001 Antiangiogenesis signals by endostatin. FASEB J 15:1044–1053qx, 百拇医药
Shichiri M, Kato H, Marumo F, Hirata Y 1997 Endothelin-1 as an autocrine/paracrine apoptosis survival factor for endothelial cells. Hypertension 30:1198–1203qx, 百拇医药
Shichiri M, Yokokura M, Marumo F, Hirata Y 2000 Endothelin-1 inhibits apoptosis of vascular smooth muscle cells induced by nitric oxide and serum deprivation via MAP kinase pathway. Arterioscler Thromb Vasc Biol 20:989–997
Shichiri M, Hanson KD, Sedivy JM 1993 Effects of c-myc expression on proliferation, quiescence, and the G0 to G1 transition in nontransformed cells. Cell Growth Differ 4:93–104i, 百拇医药
Shichiri M, Sedivy JM, Marumo F, Hirata Y 1998 Endothelin-1 is a potent survival factor for c-Myc-dependent apoptosis. Mol Endocrinol 12:172–180i, 百拇医药
Iwasaki H, Eguchi S, Shichiri M, Marumo F, Hirata Y 1998 Down-regulation of adenylate cyclase coupled to adrenomedullin receptor in vascular smooth muscle cells. Eur J Pharmacol 352:131–134i, 百拇医药
Tsuruda T, Kato J, Kitamura K, Kawamoto M, Kuwasako K, Imamura T, Koiwaya Y, Tsuji T, Kangawa K, Eto T 1999 An autocrine or a paracrine role of adrenomedullin in modulating cardiac fibroblast growth. Cardiovasc Res 43:958–967i, 百拇医药
Cornish J, Callon KE, Coy DH, Jiang NY, Xiao L, Cooper GJ, Reid IR 1997 Adrenomedullin is a potent stimulator of osteoblastic activity in vitro and in vivo. Am J Physiol 273:E1113–E1120i, 百拇医药
Semplicini A, Ceolotto G, Baritono E, Malendowicz LK, Andreis PG, Sartori M, Rossi GP, Nussdorfer GG 2001 Adrenomedullin stimulates DNA synthesis of rat adrenal zona glomerulosa cells through activation of the mitogen-activated protein kinase-dependent cascade. J Hypertens 19:599–602
Andreis PG, Markowska A, Champion HC, Mazzocchi G, Malendowicz LK, Nussdorfer GG 2000 Adrenomedullin enhances cell proliferation and deoxyribonucleic acid synthesis in rat adrenal zona glomerulosa: receptor subtype involved and signaling mechanism. Endocrinology 141:2098–210460z@f, 百拇医药
Withers DJ, Coppock HA, Seufferlein T, Smith DM, Bloom SR, Rozengurt E 1996 Adrenomedullin stimulates DNA synthesis and cell proliferation via elevation of cAMP in Swiss 3T3 cells. FEBS Lett 378:83–8760z@f, 百拇医药
Forneris M, Gottardo L, Albertin G, Malendowicz LK, Nussdorfer GG 2001 Expression and function of adrenomedullin and its receptors in Conn’s adenoma cells. Inter J Mol Med 8:675–67960z@f, 百拇医药
Martinez A, Miller MJ, Unsworth EJ, Siegfried JM, Cuttitta F 1995 Expression of adrenomedullin in normal human lung and in pulmonary tumors. Endocrinology 136:4099–410560z@f, 百拇医药
Moody TW, Coy D, Cuttitta F, Montuenga LM 2000 Proadrenomedullin NH(2)-terminal 20 peptide (PAMP) and adrenomedullin bind to teratocarcinoma cells. Peptides 21:101–10760z@f, 百拇医药
Rocchi P, Boudouresque F, Zamora AJ, Muracciole X, Lechevallier E, Martin PM, Ouafik L 2001 Expression of adrenomedullin and peptide amidation activity in human prostate cancer and in human prostate cancer cell lines. Cancer Res 61:1196–120660z@f, 百拇医药
Chini EN, Choi E, Grande JP, Burnett JC, Dousa TP 1995 Adrenomedullin suppresses mitogenesis in rat mesangial cells via cAMP pathway. Biochem Biophys Res Commun 215:868–87360z@f, 百拇医药
Segawa K, Minami K, Sata T, Kuroiwa A, Shigematsu A 1996 Inhibitory effect of adrenomedullin on rat mesangial cell mitogenesis. Nephron 74:577–57960z@f, 百拇医药
Tsuruda T, Kato J, Kitamura K, Kuwasako K, Imamura T, Koiwaya Y, Tsuji T, Kangawa K, Eto T 1998 Adrenomedullin: a possible autocrine or paracrine inhibitor of hypertrophy of cardiomyocytes. Hypertension 31:505–510(Nozomi Fukai Masayoshi Shichiri Naoko Ozawa Mika Matsushita and Yukio Hirata)
Three isoforms of the receptor activity-modifying protein (RAMP) are thought to transport the calcitonin receptor-like receptor (CRLR) to the plasma membrane to function as calcitonin gene-related peptide or adrenomedullin receptors, but their role remains largely unknown. We investigated whether coexpression of RAMP and CRLR are involved in the regulation of cell migration using a monolayer-wounding protocol. Quantification of gene transcripts revealed expression of all RAMP isoforms and CRLR in cultured rat vascular smooth muscle cells (VSMCs), RAMP2 and RAMP3 in rat endothelial cells, and RAMP1 in rat fibroblasts. CRLR expression was minimal in endothelial cells and fibroblasts. Adrenomedullin potently suppressed the migration of VSMCs, whereas calcitonin gene-related peptide did not suppress migration in any cell type. The antimigratory effect of adrenomedullin on VSMCs was potentiated by transfecting CRLR cDNA. Cotransfection of RAMP2 or RAMP3 with CRLR into VSMCs resulted in a slower migratory rate, and this effect was enhanced by adrenomedullin. Migration of fibroblasts was also suppressed after cotransfection of RAMP2 or RAMP3 with CRLR. cAMP agonists had no effect on VSMC migration, and a cAMP antagonist failed to abrogate the antimigratory effect of adrenomedullin. Thus, coexpression of CRLR and RAMP2 or RAMP3 mediates the inhibitory effect of adrenomedullin on cell migration, independent of cAMP-dependent signaling pathways.
Introductionl4!1h, 百拇医药
ADRENOMEDULLIN IS A POTENT vasodilator peptide (1) and belongs to the calcitonin family of peptides that include calcitonin gene-related peptide (CGRP). Adrenomedullin is synthesized and secreted from endothelial cells, vascular smooth muscle cells (VSMCs), and fibroblasts (2, 3, 4, 5) and has been shown to regulate cell proliferation (6, 7)and apoptosis (8, 9). Adrenomedullin stimulates the proliferation of rat VSMCs via activation of ERK/MAPK (6, 7), whereas it antagonizes c-Myc-induced apoptosis in endothelial cells (9). It has been shown that adrenomedullin suppresses the migration of VSMCs stimulated by a variety of chemoattractants, including serum, platelet-derived growth factor, angiotensin II, and lysophosphatidylcholine (10, 11). cAMP has been implicated as an intracellular second messenger in the inhibition of VSMC migration (10, 12). Thus, adrenomedullin may modify the process of atherosclerosis by antagonizing VSMC migration in addition to its mitogenic and antiapoptotic actions.
Calcitonin receptor-like receptor (CRLR) was originally identified as a seven-transmembrane orphan receptor (13, 14). It was later shown that human CRLR transfected into human embryo kidney 293 cells exhibited the pharmacology of a CGRP1 receptor. Recently three isoforms of receptor activity-modifying protein (RAMP) were isolated (15). CRLR can function as either a CGRP receptor or an adrenomedullin receptor, depending on which RAMP isoforms are expressed. The RAMP isoforms have varied tissue expression and facilitate expression of CRLR. Coexpression of RAMP1 and CRLR conferred CGRP1 and adrenomedullin receptor activities (16), whereas RAMP2 or RAMP3 coexpressed with CRLR resulted in normal adrenomedullin receptor pharmacology (17). The expression of adrenomedullin and CGRP receptors determined by RAMP and CGRP were evaluated by the ability to activate cAMP, but other second-messenger pathways may be involved in mediating their activity. These findings led us to examine whether the RAMP isoforms and CRLR are involved in the migration of VSMCs.
Materials and Methods(9z3a5, http://www.100md.com
Materials(9z3a5, http://www.100md.com
Rat adrenomedullin, adrenomedullin (22–52), adrenomedullin (1–25), proadrenomedullin NH2-terminal 20 peptide, and CGRP (8–37) were purchased from the Peptide Institute (Osaka, Japan), Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Rp-cAMPS) from Sigma (St. Louis, MO), forskolin and prostaglandin I2 from Wako Pure Chemical Industries (Osaka, Japan), DMEM from Flow Laboratories (Irvine, Scotland, UK), and fetal bovine serum (FBS) and calf serum (CS) from Cell Culture Laboratories (Cleveland, OH). RAMP1-pcDNA, RAMP2-pcDNA, RAMP3-pcDNA, and CRLR-pcDNA were gifts from Dr. Steven M. Foord. PCR primers were synthesized by JbioS Inc. (Saitama, Japan).(9z3a5, http://www.100md.com
Cell culture and transfection protocol(9z3a5, http://www.100md.com
Primary rat aortic VSMCs and endothelial cells were prepared and maintained as described (9, 18, 19, 20). COS7 cells were cultured in DMEM supplemented with 10% FBS. Diploid rat fibroblast cell line originally derived from Rat-1 were cultured in DMEM supplemented with glutamine and 10% CS (21, 22). Transient transfections were performed using a modified transferrin receptor-operated protocol using Transfast (Promega Corp., Madison, WI) as described (9, 20, 22). Salmon sperm DNA was used as a carrier to adjust the total DNA content of each transfection.
cAMP and ERK/MAPK assays46itn, 百拇医药
The cAMP and ERK/MAPK-dependent reporter systems (PathDetect trans-reporting system, Stratagene, La Jolla, CA) were used to measure cAMP and ERK/MAPK activity (20). The systems include the fusion activator plasmids consisting of the DNA-binding domain of the yeast GAL4 fusion activator (pFR-Luc) and the activation domain of the CREB or Elk1 transcription factor (pFC2-CREB or pFC2-Elk1). Cells plated in 96-well plates were cotransfected with pFC2-CREB or pFC2-Elk1 (50 ng each/well), pFR-Luc reporter plasmid (1 µg each/well), pRL-TK vector (1 µg each/well; Promega Corp.) expressing renilla luciferase as an internal control with or without RAMP2-pcDNA/RAMP3-pcDNA and CRLR. Cells were incubated for 24 h after transfection in DMEM containing 10% FBS, replaced with media containing 1% FBS and incubated for an additional 24 h, followed by the addition of adrenomedullin for the indicated time, after which firefly and renilla luciferase activities were measured using the dual luciferase reporter assay system (Promega Corp.) in a single-tube assay format using MicroLumatPlus (EG&G Berthold, Wildbad, Germany). The firefly luciferase activity of each sample was normalized to an internal reference standard of renilla luciferase activity.
Quantification of mRNA]mg3%, 百拇医药
Rat RAMP and CRLR transcripts were quantified with a LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) PCR protocol in which fluorescence of SYBR green dye bound to amplified products is measured (18). Total RNA was extracted, first-strand cDNA synthesized, and each amplification reaction performed as described (9, 20). Primers used for amplification were: RAMP1, forward 5'-ctttcattgtgctccccatt-3' and reverse 5'-ggtctgtccctgatgctgtt-3'; RAMP2, forward 5'-tcaaaagggaagatggagga-3' and reverse 5'-gtctgcctcgtactccaagc-3'; RAMP3, forward 5'-aaggtggacgtctggaagtg-3' and reverse 5'-tgcctatggatacccgtgat-3'; CRLR, forward 5'-gcaaggtgtcccagttcatt-3' and reverse 5'-cccccagccaagaaaataat-3', and the reaction produced 151-bp, 152-bp, 148-bp, and 148-bp products, respectively. Conventional RT-PCR employed the same set of primers, and the amplification reactions were examined by 1.5% agarose gel electrophoresis.]mg3%, 百拇医药
Cell migration assay
Confluent monolayers in 6-well dishes (Corning, Inc., Corning, NY) were denuded with a 300-µm-wide surgical blade (Feather Safety Razor, Osaka, Japan), and phase-contrast 100x photomicroscopy was performed after 0, 6, 12, and 24 h at two distinct sites with the center of the wound (marked with a scratch) in the middle of each frame (18). The mean distance between the wound edge and the nuclei of the 10 farthest-migrating cells along a perpendicular line was measured with Adobe (San Jose, CA) Photoshop 5.0 software after the incorporation of photomicrographs using GT8000 (Epson, Tokyo, Japan), and normalization to a standard field width. Migration distance was calculated from the mean distances in successive frames of a particular field. Cell proliferation assays demonstrated that DNA synthesis in confluent cultures was negligible during the monolayer wounding experiments, and repopulation within 24 h is almost entirely due to cell migration (18). Subconfluent VSMCs were transfected with RAMP/CRLR, CRLR alone, or salmon sperm DNA, and rendered confluent after incubation for 48 h. Cells were incubated for 24 h with or without test reagents before the cell migration assay. Migration assays were performed with 10% FBS supplementation for VSMCs and endothelial cells and 10% CS with glutamine for fibroblasts.
Statistical analysis;*cul2, 百拇医药
Data were expressed as mean ± SEM. Differences between groups were examined for statistical significance using the unpaired t test. P values less than 0.05 were considered statistically significant.;*cul2, 百拇医药
Results;*cul2, 百拇医药
Expression of RAMP and CRLR;*cul2, 百拇医药
Quantification of mRNA transcripts using the real-time quantitative PCR method revealed that RAMP1 is expressed in fibroblasts and VSMCs, whereas RAMP2 and RAMP3 are expressed in endothelial cells and VSMCs (Fig. 1). CRLR mRNA expression in endothelial cells and fibroblasts was only 5% and 0.8%, respectively, of that in VSMCs (Fig. 1).;*cul2, 百拇医药
fig.ommitteedfig.ommitteed;*cul2, 百拇医药
Figure 1. Expression of RAMP isoforms and CRLR in rat endothelial cells, fibroblasts, and VSMCs. Quantification of RAMP1, RAMP2, RAMP3, and CRLR gene transcripts. Each gene transcript is expressed as percent ± SEM of transcript in rat VSMCs.
Antimigratory effect of adrenomedullin^, 百拇医药
Cell migratory rate was evaluated using a monolayer-wounding protocol in which cells migrated from the confluent to the denuded area. A wounded confluent monolayer of VSMCs cultured with 10% FBS and nutrients migrated rapidly at approximately 4 µm/h until the denuded area was repopulated (Fig. 2, A and B). Adrenomedullin (10-6 M) inhibited the migration of VSMCs but not that of endothelial cells or fibroblasts, whereas CGRP (10-6 M) did not have an antimigratory effect on any cell type tested (Fig. 3). Proadrenomedullin NH2-terminal 20 peptide, adrenomedullin (1–25), adrenomedullin (22–52), and CGRP (8–37) did not inhibit cell migration either.^, 百拇医药
fig.ommitteedfig.ommitteed^, 百拇医药
Figure 2. Migration of VSMCs and the effect of coexpression of RAMP2 and CRLR. A and B, Cell migration was initiated by wounding a confluent monolayer of nontransfected VSMCs. The leading edges immediately (A) and 24 h after wounding (B). C and D, The leading edges of a confluent monolayer cotransfected with RAMP and CRLR for 72 h and pretreated with 10-6 M adrenomedullin immediately (C) and 24 h after wounding (D).
fig.ommitteedfig.ommitteed#., 百拇医药
Figure 3. Effects of adrenomedullin on cell migration. Confluent endothelial cells (A), fibroblasts (B), and VSMCs (C) pretreated with or without adrenomedullin (10-6 M) or CGRP (10-6 M) for 24 h were denuded, and cell migration rates were determined. The graphs represent the average migration rate ± SEM during 12 h after denudement. *, P < 0.05 vs. untreated culture (n = 10).#., 百拇医药
Cotransfection of RAMP and CRLR#., 百拇医药
RAMP- and CRLR-expressing vectors were cotransfected into COS7 cells, and cAMP and ERK/MAPK activity was measured using the PathDetect system (Stratagene). RAMP isoforms cotransfected with CRLR increased cAMP activity in response to adrenomedullin, whereas transfection of CRLR alone did not (Fig. 4). Adrenomedullin failed to induce ERK/MAPK activity in RAMP/CRLR-transfected COS7 cells (data not shown). These results confirmed the expression of the adrenomedullin receptor by cotransfecting RAMP isoform with CRLR functionally coupled to a cAMP pathway.
fig.ommitteedfig.ommitteedlk:u\y$, http://www.100md.com
Figure 4. Adrenomedullin stimulates cAMP activity in COS7 cells expressing RAMP/CRLR. COS7 cells were cotransfected with pFR-Luc (firefly luciferase), pFA-CREB, pRL-TK (renilla luciferase), and CRLR-pcDNA with or without RAMP1-pcDNA, RAMP2-pcDNA, or RAMP3-pcDNA. Transcriptional activity of GAL4-CREB (pFA-CREB) after incubation with (closed column) or without (open column) adrenomedullin (10-6 M) was analyzed using firefly luciferase as a reporter gene normalized with renilla luciferase as an internal control. Each column represents mean ± SEM (n = 8).lk:u\y$, http://www.100md.com
Quantification of RAMP2 mRNA 48 h after cotransfection of RAMP2 and CRLR into endothelial cells and VSMCs were 1299 ± 182% and 272 ± 144%, respectively, of untransfected cells, whereas RAMP1 mRNA level after cotransfection of RAMP1 and CRLR into fibroblasts was 636 ± 151% of untransfected cells, indicating satisfactory transfection efficiency.lk:u\y$, http://www.100md.com
Mediation of RAMP/CRLR receptorslk:u\y$, http://www.100md.com
To determine whether the antimigratory effect of adrenomedullin is mediated by receptors consisting of RAMP and CRLR, we examined the migration rate of VSMCs and fibroblasts cotransfected with RAMP and CRLR. Transfection of CRLR into VSMCs markedly potentiated the antimigratory effect of adrenomedullin in a concentration-dependent manner (10-9–10-6 M; Fig. 5). Cotransfection of RAMP2 or RAMP3 with CRLR into VSMCs reduced the migratory rate to a greater extent than transfection of CRLR alone, and this effect was enhanced by adrenomedullin (Fig. 6A). The effect of adrenomedullin to suppress VSMC migration was blocked by an adrenomedullin receptor antagonist, CGRP (8–37) (Fig. 6B). However, CGRP did not affect the migration of VSMCs cotransfected with RAMP1 and CRLR (data not shown). In fibroblasts that express limited CRLR and no RAMP2 or RAMP3, the migration rate was not reduced after CRLR transfection alone, but cotransfection of RAMP2/RAMP3 with CRLR reduced the migratory rate by 49% to 58% (Fig. 7A). In cotransfected fibroblasts, adrenomedullin did not potentiate the antimigratory effect, but antiadrenomedullin antiserum significantly increased the cell migration rate (Fig. 7B).
fig.ommitteedfig.ommitteed)62t.ne, http://www.100md.com
Figure 5. Migration inhibition by adrenomedullin of VSMCs transfected with CRLR. Each column represents mean ± SEM (n = 10). *, P < 0.05 vs. control.)62t.ne, http://www.100md.com
fig.ommitteedfig.ommitteed)62t.ne, http://www.100md.com
Figure 6. Effects of RAMP and CRLR on VSMC migration. A, After cotransfection with RAMP2 or RAMP3 with CRLR or transfection with CRLR alone into rat VSMCs and fibroblasts, the migration rate was determined in the presence (closed column) or absence (open column) of adrenomedullin (10-6 M). B, The effect of adrenomedullin to inhibit migration of VSMCs cotransfected with RAMP2 with CRLR was abrogated by an adrenomedullin receptor antagonist, CGRP (8–37). Each column represents mean ± SEM (n = 10). *, P < 0.05 vs. control; **, P < 0.05 vs. AM(-) (n = 10).)62t.ne, http://www.100md.com
fig.ommitteedfig.ommitteed)62t.ne, http://www.100md.com
Figure 7. Effects of RAMP and CRLR on fibroblast migration. A, After cotransfection with RAMP2 or RAMP3 with CRLR or transfection with CRLR alone into rat fibroblasts, the migration rate was determined in the presence (closed column) or absence (open column) of adrenomedullin (10-6 M). B, The migration rate of fibroblasts cotransfected with RAMP2 with CRLR increased by addition of antiadrenomedullin antiserum to the media. Each column represents mean ± SEM (n = 10). NS, Not significant. *, P < 0.05 vs. control (n = 10).
To elucidate whether the inhibitory effect of adrenomedullin on VSMC migration via RAMP/CRLR is linked to cAMP generation, we examined the effects of forskolin and prostaglandin I2 on VSMC migration. Neither forskolin (10-3 M) nor prostaglandin I2 (10-5 M) reduced VSMC migration rate (Fig. 8A). Furthermore, the cAMP antagonist Rp-cAMPS did not affect adrenomedullin-induced inhibition of VSMC migration (Fig. 8B). To determine which signaling pathway other than cAMP is involved, we tested the following pharmacological inhibitors: phosphatidylinositol 3-kinase inhibitors (wortmannin, LY294002), tyrosine kinase inhibitors (genistein, herbimycin A, ST638), protein kinase C inhibitors (GF109203X, U73122), nuclear factor-B inhibitors (MG132), ras farnesyltransferase inhibitors (manumycin), p38 MAPK inhibitor (SB203580), methyl ethyl ketone inhibitor (PD98059), JAK-2 inhibitor (AG490), cGMP-dependent protein kinase inhibitor (KT5823), and soluble guanylate cyclase inhibitor (ODQ). None of the inhibitors antagonized the effect of adrenomedullin (data not shown).
fig.ommitteedfig.ommitteed'jp, 百拇医药
Figure 8. cAMP is not involved in antimigratory effect by adrenomedullin in rat VSMCs. A, Cells were treated with or without adrenomedullin (10-6 M), forskolin (10-3 M), or prostaglandin I2 (10-5 M), and the migration rate was determined. B, Cells, pretreated with or without Rp-cAMPS, were incubated with adrenomedullin (10-6 M), and cell migration rate was determined. Each column represents mean ± SEM (n = 10). *, P < 0.05 vs. control (n = 10).'jp, 百拇医药
Discussion'jp, 百拇医药
In the present study, we demonstrated using a monolayer-wounding assay that adrenomedullin is a potent antimigratory factor for VSMCs, although adrenomedullin failed to alter migration of fibroblasts or endothelial cells cultured under the same conditions. Using LightCycler technology (Roche Molecular Biochemicals) that allows reliable quantification results of gene expression, we have demonstrated that rat VSMCs abundantly expressed RAMP2, RAMP3, and CRLR, whereas rat endothelial cells and fibroblasts, which did not show any antimigratory effect by adrenomedullin, did not coexpress CRLR and RAMP2 or RAMP3. The migration of VSMCs that coexpress RAMP1 and CRLR was not suppressed by CGRP. These data suggest that adrenomedullin receptors consisting of the RAMP2 or RAMP3 isoforms coupled with CRLR may be involved in the antimigratory effect, whereas CGRP receptors consisting of RAMP1 and CRLR do not exert a similar effect.
Using transferrin receptor-operated gene transfer, we have shown that coexpression of either of the two isoforms, RAMP2 or RAMP3, with CRLR in COS7 cells resulted in increased cAMP activity by adrenomedullin. Coexpression of RAMP1 and CRLR also enhanced cAMP response to adrenomedullin as previously described (16). These results suggest that the functional expression of the RAMP/CRLR adrenomedullin receptor is coupled to adenylate cyclase. We have also demonstrated high transfection efficiency of our gene transfer protocol, not only in endothelial cells and fibroblasts but in VSMCs as well, by quantifying RAMP mRNA after cotransfection of RAMP and CRLR.997e%|{, 百拇医药
We reported previously that adrenomedullin stimulated intracellular cAMP in our VSMCs and endothelial cells (8, 23). However, quantification of RAMPs and CRLR revealed limited expression of CRLR transcripts in endothelial cells, suggesting the possibility that adrenomedullin receptors other than RAMP/CRLR may also mediate cAMP response in endothelial cells. Cotransfection of RAMP2 or RAMP3 with CRLR did not result in further marked cAMP elevation in response to adrenomedullin in endothelial cells and VSMCs (data not shown). These results suggest that cell type specificity of adrenomedullin antimigratory actions can be explained by cell type-specific expression of RAMP isoforms and CRLR, rather than by cAMP response.
Transfection of CRLR alone into VSMCs potentiated the antimigratory effect of adrenomedullin in a dose-dependent fashion. Furthermore, cotransfection of CRLR with RAMP2 or RAMP3 markedly reduced the migration of VSMCs, an effect that was potentiated by adrenomedullin. Because VSMCs synthesize and secrete less adrenomedullin than fibroblasts (3), it is possible that the antimigratory response to exogenous adrenomedullin may be more profound in VSMCs than fibroblasts. Our study suggests that coexpression of CRLR with RAMP2 or RAMP3 mediates the adrenomedullin-induced antimigratory effect in VSMCs.48c2v|, 百拇医药
It has been shown that fibroblasts synthesize and secrete abundant adrenomedullin-like immunoreactivity and endogenous adrenomedullin has autocrine/paracrine roles in fibroblasts (3, 24). Our fibroblasts also secrete 3–5 ng/107 cells per 6 h of adrenomedullin molecules into serum-free cultured media as detected by our specific RIA and more than 10 times of that when cultured in serum-supplemented media. Both antiadrenomedullin antiserum (Fig. 7B) and adrenomedullin receptor antagonist (data not shown) increased the cell-migratory rate of fibroblasts cotransfected with RAMP2/CRLR but not that of VSMCs, suggesting a differential role for endogenous adrenomedullin in cell migration.
It has been reported that cAMP suppresses migration of VSMCs stimulated by FBS and platelet-derived growth factor-BB as chemoattractants using transwell cell culture chambers (10). However, in our study of monolayer denuding, cAMP agonists did not reduce the migratory rate and a cAMP antagonist did not abrogate the antimigratory effect of adrenomedullin. Additionally, CGRP had no antimigratory effect on VSMCs that coexpress RAMP1 and CRLR and induce cAMP. These data suggest that cAMP is not involved in inhibiting VSMC migration under full-growth stimulation with FBS. In an attempt to identify signals for antimigratory effect mediated by RAMP/CRLR, we used various pharmacological inhibitors to antagonize adrenomedullin, but none were effective. The molecular mechanisms mediated by RAMP/CRLR remain to be elucidated. The reasons for the discrepancies between our results and the previous study remain unknown, but they are not necessarily contradictory. The migration assays between the two studies are different. In the studies using the Boyden chamber, VSMCs suspended in media without platelet-derived growth factor, lysophosphatidylcholine or FBS may migrate toward chemoattractants in the lower chemotaxis chamber. However, in the monolayer-denuding assay, confluent cells that had been contact inhibited migrate after denudement and being exposed to 10% FBS and nutrients. Monolayer-wounding assays may more closely reflect the initial stages of vascular remodeling than chemotaxis chambers. Adrenomedullin may have two mechanisms in inhibiting migration, one mediated via CRLR and RAMP2/RAMP3 independent of cAMP and a second chemotactic mechanism dependent on cAMP.
Accumulating lines of evidence supports the role for adrenomedullin as a growth factor in both nontumor (3, 6, 7, 25, 26, 27, 28) and tumor cells (29, 30, 31, 32) probably via activation of ERK/MAPK (6, 7). In contrast, adrenomedullin antagonizes mitogenesis in certain cell types (24, 33, 34, 35). Differential growth responses to adrenomedullin in each cell type may be dependent on intracellular signaling pathway downstream to adrenomedullin receptor. For example, in cells lacking the ERK/MAPK activation signaling pathway, such as fibroblasts, stimulation of adrenomedullin receptor and subsequent activation of cAMP may cause inhibition of cell growth, whereas in the majority of cell types, including our VSMCs, adrenomedullin stimulates ERK/MAPK and promotes mitogenesis. Our present results show that RAMP/CRLR receptor is not coupled to ERK/MAPK but mediates inhibition of cell migration via cAMP-independent mechanism.m, http://www.100md.com
In conclusion, we have demonstrated that receptors consisting of CRLR and RAMP2 or RAMP3 mediate adrenomedullin-induced inhibition of migration. Molecular mechanisms downstream to the receptors remain to be determined.
Acknowledgments&&, http://www.100md.com
The authors gratefully acknowledge Steven M. Foord, Ph.D. (GlaxoSmithKline) for RAMP1-pcDNA, RAMP2-pcDNA, RAMP3-pcDNA, and CRLR-pcDNA plasmids.&&, http://www.100md.com
Received May 1, 2002.&&, http://www.100md.com
Accepted for publication October 21, 2002.&&, http://www.100md.com
References&&, http://www.100md.com
Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, Eto T 1993 Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 192:553–560&&, http://www.100md.com
Hinson JP, Kapas S, Smith DM 2000 Adrenomedullin, a multifunctional regulatory peptide. Endocr Rev 21:138–167&&, http://www.100md.com
Isumi Y, Minamino N, Katafuchi T, Yoshioka M, Tsuji T, Kangawa K, Matsuo H 1998 Adrenomedullin production in fibroblasts: its possible function as a growth regulator of Swiss 3T3 cells. Endocrinology 139:2552–2563&&, http://www.100md.com
Sugo S, Minamino N, Kangawa K, Miyamoto K, Kitamura K, Sakata J, Eto T, Matsuo H 1994 Endothelial cells actively synthesize and secrete adrenomedullin. Biochem Biophys Res Commun 201:1160–1166
Sugo S, Minamino N, Shoji H, Kangawa K, Kitamura K, Eto T, Matsuo H 1994 Production and secretion of adrenomedullin from vascular smooth muscle cells: augmented production by tumor necrosis factor-{alpha} . Biochem Biophys Res Commun 203:719–726ax9@}, 百拇医药
Iwasaki H, Eguchi S, Shichiri M, Marumo F, Hirata Y 1998 Adrenomedullin as a novel growth-promoting factor for cultured vascular smooth muscle cells: role of tyrosine kinase-mediated mitogen-activated protein kinase activation. Endocrinology 139:3432–3441ax9@}, 百拇医药
Iwasaki H, Shichiri M, Marumo F, Hirata Y 2001 Adrenomedullin stimulates proline-rich tyrosine kinase 2 in vascular smooth muscle cells. Endocrinology 142:564–572ax9@}, 百拇医药
Kato H, Shichiri M, Marumo F, Hirata Y 1997 Adrenomedullin as an autocrine/paracrine apoptosis survival factor for rat endothelial cells. Endocrinology 138:2615–2620ax9@}, 百拇医药
Shichiri M, Kato H, Doi M, Marumo F, Hirata Y 1999 Induction of Max by adrenomedullin and calcitonin gene-related peptide antagonizes endothelial apoptosis. Mol Endocrinol 13:1353–1363
Horio T, Kohno M, Kano H, Ikeda M, Yasunari K, Yokokawa K, Minami M, Takeda T 1995 Adrenomedullin as a novel antimigration factor of vascular smooth muscle cells. Circ Res 77:660–6644p, http://www.100md.com
Kohno M, Yokokawa K, Yasunari K, Minami M, Kano H, Hanehira T, Yoshikawa J 1998 Induction by lysophosphatidylcholine, a major phospholipid component of atherogenic lipoproteins, of human coronary artery smooth muscle cell migration. Circulation 98:353–3594p, http://www.100md.com
Kohno M, Yokokawa K, Kano H, Yasunari K, Minami M, Hanehira T, Yoshikawa J 1997 Adrenomedullin is a potent inhibitor of angiotensin II-induced migration of human coronary artery smooth muscle cells. Hypertension 29:1309–13134p, http://www.100md.com
Chang CP, Pearse 2nd RV, O’Connell S, Rosenfeld MG 1993 Identification of a seven transmembrane helix receptor for corticotropin-releasing factor and sauvagine in mammalian brain. Neuron 11:1187–11954p, http://www.100md.com
Njuki F, Nicholl CG, Howard A, Mak JC, Barnes PJ, Girgis SI, Legon S 1993 A new calcitonin-receptor-like sequence in rat pulmonary blood vessels. Clin Sci 85:385–388
McLatchie LM, Fraser NJ, Main MJ, Wise A, Brown J, Thompson N, Solari R, Lee MG, Foord SM 1998 RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 393:333–339qx, 百拇医药
Buhlmann N, Leuthauser K, Muff R, Fischer JA, Born W 1999 A receptor activity modifying protein (RAMP)2-dependent adrenomedullin receptor is a calcitonin gene-related peptide receptor when coexpressed with human RAMP1. Endocrinology 140:2883–2890qx, 百拇医药
Aiyar N, Rand K, Elshourbagy NA, Zeng Z, Adamou JE, Bergsma DJ, Li Y 1996 A cDNA encoding the calcitonin gene-related peptide type 1 receptor. J Biol Chem 271:11325–11329qx, 百拇医药
Shichiri M, Hirata Y 2001 Antiangiogenesis signals by endostatin. FASEB J 15:1044–1053qx, 百拇医药
Shichiri M, Kato H, Marumo F, Hirata Y 1997 Endothelin-1 as an autocrine/paracrine apoptosis survival factor for endothelial cells. Hypertension 30:1198–1203qx, 百拇医药
Shichiri M, Yokokura M, Marumo F, Hirata Y 2000 Endothelin-1 inhibits apoptosis of vascular smooth muscle cells induced by nitric oxide and serum deprivation via MAP kinase pathway. Arterioscler Thromb Vasc Biol 20:989–997
Shichiri M, Hanson KD, Sedivy JM 1993 Effects of c-myc expression on proliferation, quiescence, and the G0 to G1 transition in nontransformed cells. Cell Growth Differ 4:93–104i, 百拇医药
Shichiri M, Sedivy JM, Marumo F, Hirata Y 1998 Endothelin-1 is a potent survival factor for c-Myc-dependent apoptosis. Mol Endocrinol 12:172–180i, 百拇医药
Iwasaki H, Eguchi S, Shichiri M, Marumo F, Hirata Y 1998 Down-regulation of adenylate cyclase coupled to adrenomedullin receptor in vascular smooth muscle cells. Eur J Pharmacol 352:131–134i, 百拇医药
Tsuruda T, Kato J, Kitamura K, Kawamoto M, Kuwasako K, Imamura T, Koiwaya Y, Tsuji T, Kangawa K, Eto T 1999 An autocrine or a paracrine role of adrenomedullin in modulating cardiac fibroblast growth. Cardiovasc Res 43:958–967i, 百拇医药
Cornish J, Callon KE, Coy DH, Jiang NY, Xiao L, Cooper GJ, Reid IR 1997 Adrenomedullin is a potent stimulator of osteoblastic activity in vitro and in vivo. Am J Physiol 273:E1113–E1120i, 百拇医药
Semplicini A, Ceolotto G, Baritono E, Malendowicz LK, Andreis PG, Sartori M, Rossi GP, Nussdorfer GG 2001 Adrenomedullin stimulates DNA synthesis of rat adrenal zona glomerulosa cells through activation of the mitogen-activated protein kinase-dependent cascade. J Hypertens 19:599–602
Andreis PG, Markowska A, Champion HC, Mazzocchi G, Malendowicz LK, Nussdorfer GG 2000 Adrenomedullin enhances cell proliferation and deoxyribonucleic acid synthesis in rat adrenal zona glomerulosa: receptor subtype involved and signaling mechanism. Endocrinology 141:2098–210460z@f, 百拇医药
Withers DJ, Coppock HA, Seufferlein T, Smith DM, Bloom SR, Rozengurt E 1996 Adrenomedullin stimulates DNA synthesis and cell proliferation via elevation of cAMP in Swiss 3T3 cells. FEBS Lett 378:83–8760z@f, 百拇医药
Forneris M, Gottardo L, Albertin G, Malendowicz LK, Nussdorfer GG 2001 Expression and function of adrenomedullin and its receptors in Conn’s adenoma cells. Inter J Mol Med 8:675–67960z@f, 百拇医药
Martinez A, Miller MJ, Unsworth EJ, Siegfried JM, Cuttitta F 1995 Expression of adrenomedullin in normal human lung and in pulmonary tumors. Endocrinology 136:4099–410560z@f, 百拇医药
Moody TW, Coy D, Cuttitta F, Montuenga LM 2000 Proadrenomedullin NH(2)-terminal 20 peptide (PAMP) and adrenomedullin bind to teratocarcinoma cells. Peptides 21:101–10760z@f, 百拇医药
Rocchi P, Boudouresque F, Zamora AJ, Muracciole X, Lechevallier E, Martin PM, Ouafik L 2001 Expression of adrenomedullin and peptide amidation activity in human prostate cancer and in human prostate cancer cell lines. Cancer Res 61:1196–120660z@f, 百拇医药
Chini EN, Choi E, Grande JP, Burnett JC, Dousa TP 1995 Adrenomedullin suppresses mitogenesis in rat mesangial cells via cAMP pathway. Biochem Biophys Res Commun 215:868–87360z@f, 百拇医药
Segawa K, Minami K, Sata T, Kuroiwa A, Shigematsu A 1996 Inhibitory effect of adrenomedullin on rat mesangial cell mitogenesis. Nephron 74:577–57960z@f, 百拇医药
Tsuruda T, Kato J, Kitamura K, Kuwasako K, Imamura T, Koiwaya Y, Tsuji T, Kangawa K, Eto T 1998 Adrenomedullin: a possible autocrine or paracrine inhibitor of hypertrophy of cardiomyocytes. Hypertension 31:505–510(Nozomi Fukai Masayoshi Shichiri Naoko Ozawa Mika Matsushita and Yukio Hirata)