Syndecan-4 Is Required for Thrombin-induced Migration and Proliferation in Human Vascular Smooth Muscle Cells

Thrombin is a mitogen and chemoattractant for vascular smooth muscle cells (SMCs) and may contribute to vascular lesion formation. We have previously shown that human SMCs, when stimulated with thrombin, release basic fibroblast growth factor (bFGF), causing phosphorylation of FGF receptor-1 (FGFR-1). Treatment with bFGF-neutralizing antibodies (anti-bFGF) or heparin inhibits thrombin-induced DNA synthesis. We concluded that thrombin may stimulate entry into the cell cycle via bFGF release and FGFR-1 activation. In the present study, we demonstrate a requirement for not only FGFR-1 but also syndecan-4, a transmembrane heparan-sulfate proteoglycan. Inhibition of syndecan-4 expression using small interfering RNA (siRNA) resulted in reduced DNA synthesis by human SMCs after stimulation with thrombin (10 nmol/liter). Anti-bFGF antibody, which inhibits DNA synthesis in control cells, had no inhibitory effect when syndecan-4 expression was reduced by siRNA. Thrombin- or bFGF-induced SMC migration, determined in Boyden chamber assays, was reduced in cells treated with syndecan-4 or FGFR-1 siRNA or by anti-bFGF. Thrombin induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in a biphasic pattern. Although thrombin-mediated ERK phosphorylation at 5 min was not affected by syndecan-4 or FGFR-1 siRNA, ERK phosphorylation at later time points was reduced. We conclude that thrombin-released bFGF binds to syndecan-4 and FGFR-1, which is required for thrombin-induced mitogenesis and migration. Thrombin is a mitogen and chemoattractant for vascular smooth muscle cells (SMCs) and may contribute to vascular lesion formation. We have previously shown that human SMCs, when stimulated with thrombin, release basic fibroblast growth factor (bFGF), causing phosphorylation of FGF receptor-1 (FGFR-1). Treatment with bFGF-neutralizing antibodies (anti-bFGF) or heparin inhibits thrombin-induced DNA synthesis. We concluded that thrombin may stimulate entry into the cell cycle via bFGF release and FGFR-1 activation. In the present study, we demonstrate a requirement for not only FGFR-1 but also syndecan-4, a transmembrane heparan-sulfate proteoglycan. Inhibition of syndecan-4 expression using small interfering RNA (siRNA) resulted in reduced DNA synthesis by human SMCs after stimulation with thrombin (10 nmol/liter). Anti-bFGF antibody, which inhibits DNA synthesis in control cells, had no inhibitory effect when syndecan-4 expression was reduced by siRNA. Thrombin- or bFGF-induced SMC migration, determined in Boyden chamber assays, was reduced in cells treated with syndecan-4 or FGFR-1 siRNA or by anti-bFGF. Thrombin induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in a biphasic pattern. Although thrombin-mediated ERK phosphorylation at 5 min was not affected by syndecan-4 or FGFR-1 siRNA, ERK phosphorylation at later time points was reduced. We conclude that thrombin-released bFGF binds to syndecan-4 and FGFR-1, which is required for thrombin-induced mitogenesis and migration. Vascular smooth muscle cell (SMC) 1The abbreviations used are: SMC, vascular smooth muscle cell; FGF, fibroblast growth factor; bFGF, basic FGF; anti-bFGF, bFGF-neutralizing antibody; FGFR-1, FGF receptor-1; EGF, epidermal growth factor; HSPG, heparan sulfate proteoglycan; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PKCα, protein kinase C α; Ab, antibody; siRNA, small interfering RNA; RT, reverse transcription; ANOVA, analysis of variance; HS, heparan sulfate. proliferation and migration are key events in atherosclerosis and restenosis after vascular injury (1Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9988) Google Scholar, 2Schwartz S.M. deBlois D. O'Brien E.R. Circ. Res. 1995; 77: 445-465Crossref PubMed Scopus (898) Google Scholar). The G-protein-coupled receptor agonist thrombin may contribute to disease progression through stimulation of mitogenic signaling. In a previous study with cultured human SMCs, we demonstrated that thrombin releases basic fibroblast growth factor (bFGF), which is bound to the pericellular matrix and causes phosphorylation of the FGF receptor-1 (FGFR-1) (3Rauch B.H. Millette E. Kenagy R.D. Daum G. Clowes A.W. Circ. Res. 2004; 94: 340-345Crossref PubMed Scopus (72) Google Scholar). Preventing FGFR-1 phosphorylation by bFGF-neutralizing antibodies or by heparin inhibits thrombin-induced mitogenesis. Since heparin binds bFGF and inhibits bFGF-FGFR-1 interactions, we hypothesized that an endogenous heparan sulfate proteoglycan (HSPG) may be involved in mediating the effects of thrombin-released bFGF and thus be required for thrombin-induced mitogenesis. Syndecan-4 is a transmembrane HSPG with a core protein of 35 kDa carrying mainly heparan sulfate side chains (4Bellin R. Capila I. Lincecum J. Park P.W. Reizes O. Bernfield M.R. Glycoconj. J. 2002; 19: 295-304Crossref PubMed Scopus (34) Google Scholar). Syndecans and the HSPG glypican have been shown to bind bFGF and increase bFGF-FGFR-1 interactions (5Steinfeld R. Van Den Berghe H. David G. J. Cell Biol. 1996; 133: 405-416Crossref PubMed Scopus (230) Google Scholar). Of the family of syndecans (syndecan-1 to -4), syndecan-4 is the only member with an intracellular domain that is capable of binding phosphatidylinositol 4,5-bisphosphate and protein kinase C α (PKCα), which allows it to support PKC-mediated signaling (6Oh E.S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 7Oh E.S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 11805-11811Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 8Horowitz A. Simons M. J. Biol. Chem. 1998; 273: 25548-25551Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Recently, several unique functions have been described for syndecan-4. It clusters in focal contacts and affects integrin function, thereby modulating cell adhesion, migration, and morphology (9Longley R.L. Woods A. Fleetwood A. Cowling G.J. Gallagher J.T. Couchman J.R. J. Cell Sci. 1999; 112: 3421-3431Crossref PubMed Google Scholar, 10Mostafavi-Pour Z. Askari J.A. Parkinson S.J. Parker P.J. Ng T.T. Humphries M.J. J. Cell Biol. 2003; 161: 155-167Crossref PubMed Scopus (183) Google Scholar, 11Simons M. Horowitz A. Cell. Signal. 2001; 13: 855-862Crossref PubMed Scopus (122) Google Scholar, 12Woods A. Couchman J.R. Curr. Opin. Cell Biol. 2001; 13: 578-583Crossref PubMed Scopus (186) Google Scholar, 13Bass M.D. Humphries M.J. Biochem. J. 2002; 368: 1-15Crossref PubMed Scopus (122) Google Scholar). Since thrombin activates FGFR-1 by releasing bFGF into the pericellular matrix and syndecan-4 functions as a mediator for bFGF signaling and a cofactor for FGFR-1 (for a review, see Ref. 13Bass M.D. Humphries M.J. Biochem. J. 2002; 368: 1-15Crossref PubMed Scopus (122) Google Scholar), we tested the possibility that thrombin-induced mitogenesis and migration require syndecan-4 for binding thrombin-released bFGF. Materials—Antibodies (Abs) against syndecan-4 and FGFR-1 were from Santa Cruz Biotechnology (Santa Cruz, CA). Ab against phosphorylated ERK1/2 was from Cell Signaling Technology (Beverly, MA). Ab to an HSPG neo-epitope, exposed after heparitinase III digestion (anti-ΔHS, clone 3G10), was from Seikagaku Corp. (Tokyo, Japan). Protein A-agarose was from Roche Diagnostics. Neutralizing Ab against human bFGF was a generous gift from Dr. Michael A. Reidy (University of Washington) (14Lindner V. Reidy M.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3739-3743Crossref PubMed Scopus (598) Google Scholar). ERK1/2 antiserum was a generous gift from Dr. Karen Bornfeld (University of Washington). Human α-thrombin was from American Diagnostica (Greenwich, CT). Recombinant bFGF, heparin (porcine intestinal mucosa), and heparitinase I, II, and III were from Sigma. All cell culture solutions were from Invitrogen. Cell Culture—Human aortic SMCs were prepared by the explant technique as described previously (15Kalmes A. Vesti B.R. Daum G. Abraham J.A. Clowes A.W. Circ. Res. 2000; 87: 92-98Crossref PubMed Scopus (166) Google Scholar). Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum, 200 units/ml penicillin, and 200 μg/ml streptomycin. Cells at passage 5–12 were used for experiments. Gene Silencing with Small Interfering RNA (siRNA)—The following sequences were chosen to generate siRNA: for syndecan-4, 5′-AAGGCCGATACTTCTCCGGAG-3′ (sense), 5′-AAGGCTCTCCGGAG-CGATACT-3′ (scrambled); for FGFR-1, 5′-AAGTCGGACGCAACAGAGAAA-3′ (sense), 5′-AACAGAGAAAGTCGGACGCAA-3′ (scrambled). SiRNAs were generated in vitro using a siRNA construction kit (Ambion, Austin, TX) according to the manufacturer's instructions. Cells were transfected with siRNAs (final concentration 10 nmol/liter) by calcium phosphate precipitation for 15 h, as described (16Deroanne C. Vouret-Craviari V. Wang B. Pouyssegur J. J. Cell Sci. 2003; 116: 1367-1376Crossref PubMed Scopus (107) Google Scholar). Cells were washed three times with phosphate-buffered saline, once with medium containing 15% fetal bovine serum, and allowed to recover for at least 9 h. Cells were detached with trypsin, counted, and reseeded for experiments in 15% serum. Immunoprecipitation and Western Blotting—Immunoprecipitation of FGFR-1 was carried out as described previously (3Rauch B.H. Millette E. Kenagy R.D. Daum G. Clowes A.W. Circ. Res. 2004; 94: 340-345Crossref PubMed Scopus (72) Google Scholar). For detection of syndecan-4 or HSPG neo-epitope (ΔHS), cells were lysed with 8 m urea buffer (25 mmol/liter Tris-HCl, 8 m urea, pH 7.5, 2 mmol/liter phenylmethylsulfonyl fluoride). Cell lysates were frozen at –80 °C to disrupt syndecan-4 dimers (13Bass M.D. Humphries M.J. Biochem. J. 2002; 368: 1-15Crossref PubMed Scopus (122) Google Scholar). Cell extracts were dialyzed against 0.1 mol/liter sodium acetate, pH 7.0, 0.1 mmol/liter calcium acetate. Samples were concentrated using centrifugal filter tubes (Amicon®, cut-off 10,000, Millipore, MA) and resuspended in 100 μl of digestion buffer (10 mmol/liter calcium acetate, 18 mmol/liter sodium acetate, 0.1 m Tris-HCl, pH 7.4, 2 mmol/liter phenylmethylsulfonyl fluoride). Samples were incubated with heparitinase I–III (10 units/ml heparitinase I and II, 2.5 units/ml heparitinase III) for 4 h at 37 °C to digest HS side chains. Protein concentrations were determined to ensure equal protein loading. To determine ERK phosphorylation, cells were seeded into 6-well plates (80,000–100,000 cells/well) and incubated in serum-free medium for 48 h with a change of medium 24 h before stimulation. For Western blot analysis, samples were boiled in Laemmli sample buffer and subjected to SDS-PAGE followed by transfer to nitrocellulose. Primary antibodies used were monoclonal Ab against syndecan-4 (clone 5G9, Santa Cruz Biotechnology), anti-ΔHS (clone 3G10, Seikagaku Corp.), anti-ERK1/2 (Cell Signaling), anti-actin (Sigma), and anti-FGFR1 (Santa Cruz Biotechnology). Protein was visualized by enhanced chemiluminescence (kit from Amersham Biosciences) according to the manufacturer's protocol. Results were quantified by densitometry of films using ImageQuant software (Amersham Biosciences). Semiquantitative RT-PCR—Untreated SMCs or those transfected with 10 nmol/liter siRNA were seeded into 100-mm dishes (5–6 × 105 cells/dish) and incubated in serum-free medium for 48 h. RNA was prepared using RNeasy® spin columns (Qiagen, Valencia, CA). Semiquantitative RT-PCR was performed with Ready-To-Go™ RT-RCR Beads (Amersham Biosciences) using 1 μg of total RNA and 15 pmol of primer according to the manufacturer's instructions. Primer pairs, product size, and annealing temperature were as follows: FGFR-1, sense, 5′-CGGTGTGCCTGTGGAGGAACTT-3′, antisense, 5′-GTTACAGCTGACGGTGGAGTCT-3′ (408-bp fragment, 65 °C) (17Jayson G.C. Vives C. Paraskeva C. Schofield K. Coutts J. Fleetwood A. Gallagher J.T. Int. J. Cancer. 1999; 82: 298-304Crossref PubMed Scopus (48) Google Scholar); syndecan-2, sense, 5′-GGGAGCTGATGAGGATGTAG-3′, antisense, 5′-CACTGGATGGTT-TGCGTTCT-3′ (394-bp fragment, 60 °C); syndecan-4, sense, 5′-CTCCTAGAAGGCCGATACTTCT-3′, antisense, 5′-GGACCTCCGTTCTCT-CAAAGAT-3′ (345-bp fragment, 60 °C); glypican, sense, 5′-ATCACCGACAAGTTCTGGGGTA-3′, antisense, 5′-CATCTTCTCACTGCACA-GTGTC-3′ (317-bp fragment, 60 °C) (18Dobra K. Andang M. Syrokou A. Karamanos N.K. Hjerpe A. Exp. Cell Res. 2000; 258: 12-22Crossref PubMed Scopus (65) Google Scholar). For semiquantitative analysis, glyceraldehyde-3-phosphate dehydrogenase was co-amplified: sense, 5′-TGATGACATCAAGAAGGTGGTGAA-3′, antisense, 5′-TCCTTGGAGGCCATGTAGGCCAT-3′ (238-bp fragment, 60–65 °C) (19Debey S. Meyer-Kirchrath J. Schror K. Biochem. Pharmacol. 2003; 65: 979-988Crossref PubMed Scopus (25) Google Scholar). Conditions for PCR after reverse transcription were: 95 °C for 1 min, specific annealing temperature for 1 min, 72 °C for 2.5 min, and a final elongation step at 72 °C for 15 min. DNA Synthesis—SMCs (7,500–8,750 cells/cm2) were incubated in serum-free medium for 48 h with a change of medium 24 h before stimulation. Cells were stimulated by the addition of thrombin (10 nmol/liter) or bFGF (1 ng/ml). [3H]Thymidine (1 μCi/ml) was added 16–18 h after stimulation. After 26–28 h, cells were washed three times with ice-cold phosphate-buffered saline followed by incubation in 10% trichloroacetic acid overnight at 4 °C. Cells were washed in trichloroacetic acid, and DNA was solubilized in 0.1 n NaOH. Radioactivity was measured by liquid scintillation counting. Boyden Chamber Migration Assay and Cell Spreading—Modified Boyden chamber assays were performed for 5 h at 37 °C with 48-well microchemotaxis chambers (Neuro Probe) and polycarbonate filters (10-μm pores; Nucleopore Corp.) coated with monomeric collagen (100 μg/ml Vitrogen 100 in 0.1 mol/liter acetic acid, Collagen Corp., Palo Alto, CA), as described (15Kalmes A. Vesti B.R. Daum G. Abraham J.A. Clowes A.W. Circ. Res. 2000; 87: 92-98Crossref PubMed Scopus (166) Google Scholar). Cells (20,000/well) were added to the upper chamber, and chemoattractants or serum-free medium were added to the lower chamber. Migration was measured as the number of cells/high power field (×100) that had migrated across the membrane. Assays were performed in quadruplicate. For cell spreading experiments, cells were seeded on glass slides coated with monomeric collagen (100 μg/ml Vitrogen 100 in 0.1 mol/liter acetic acid) and allowed to spread for up to 6 h in serum-free medium. Cells were fixed and stained with Diff-Quick staining solution (Baxter, Detroit, MI) according to the manufacturer's instructions. Statistics—All experiments were performed at least three times in duplicate, triplicate, or quadruplicate. Statistical analysis was performed using one-way ANOVA followed by Bonferroni's multiple comparison test or by a paired two-tailed t test as indicated. Values of p < 0.05 were considered significant. Inhibition of Syndecan-4 and FGFR-1 Expression by siRNA in Human Vascular SMCs—Specific siRNA significantly reduced RNA levels for syndecan-4 and FGFR-1 in human SMCs, respectively, but had no effect on RNA of syndecan-2 or glypican, two proteoglycans that bind bFGF (5Steinfeld R. Van Den Berghe H. David G. J. Cell Biol. 1996; 133: 405-416Crossref PubMed Scopus (230) Google Scholar). RNA with a from syndecan-4 or FGFR-1 siRNA had no effect on RNA levels Syndecan-4 protein levels were determined in extracts from human aortic SMCs by Western Although the specific siRNA syndecan-4 protein expression by a RNA had no effect on syndecan-4 expression To determine in the cell were affected by syndecan-4 siRNA, an (anti-ΔHS, clone that an HSPG neo-epitope after heparitinase digestion was used on Western of SMC lysates prepared as described to those described by using J.A. M.A. Biochem. J. 2004; PubMed Scopus (72) Google Scholar), were of syndecan-4 core was reduced in cells treated with syndecan-4 siRNA. that are only syndecan-4 is by siRNA to syndecan-4. siRNA was also generated to FGFR-1, as determined in from SMC lysates Cell in SMCs with Syndecan-4 syndecan-4 is involved in the of the (9Longley R.L. Woods A. Fleetwood A. Cowling G.J. Gallagher J.T. Couchman J.R. J. Cell Sci. 1999; 112: 3421-3431Crossref PubMed Google Scholar, R. M. M. C. Couchman J.R. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), we syndecan-4 siRNA affects cell with syndecan-4 cells spread at 1 and h in serum-free medium. by 6 h, cells were spread on collagen Cell spreading in 15% to be and the number of cells that in 15% overnight was not in syndecan-4 siRNA cells when with control that the of syndecan-4 cell spreading on spreading was not in cells with FGFR-1 functions of syndecan-4 and Syndecan-4 and FGFR-1 for Thrombin- and bFGF-induced Boyden chamber assays, or bFGF-induced migration of SMCs transfected with syndecan-4 siRNA was when with the migration of control cells In migration was not affected by syndecan-4 siRNA. SMCs transfected with FGFR-1 siRNA also migrated thrombin control cells, and control cells, bFGF-neutralizing antibodies had no inhibitory effect on thrombin-induced migration The migration of SMCs was not from control cells treated with RNA for syndecan-4 or FGFR-1 siRNA not Syndecan-4 and FGFR-1 for DNA Synthesis—SMCs transfected with syndecan-4 or FGFR-1 siRNA or with RNA were stimulated with thrombin or bFGF. In cells treated with syndecan-4 siRNA, DNA synthesis in to thrombin or bFGF was reduced bFGF-neutralizing thrombin-induced DNA synthesis in control cells but had no inhibitory effect when syndecan-4 expression was reduced by siRNA. of of thrombin-induced DNA synthesis by heparin was only in control had no effect in The effects of reduced FGFR-1 expression were to those with reduced syndecan-4 DNA synthesis was in cells with reduced FGFR-1 and bFGF-neutralizing antibodies no inhibitory effect significantly DNA synthesis only in control In to control cells, stimulation with bFGF of cells treated with FGFR-1 siRNA not in DNA synthesis. of ERK1/2 Syndecan-4 and determine ERK 1/2 is involved in and intracellular that are by thrombin, SMCs and SMCs transfected with syndecan-4 siRNA, or FGFR-1 siRNA were stimulated with thrombin from 5 min to 4 h. The of ERK phosphorylation at 5 min in control cells and siRNA cells was the at later time it was reduced in SMCs transfected with syndecan-4 siRNA or FGFR-1 siRNA In previous we and have shown that thrombin-induced mitogenesis and migration require the of a (15Kalmes A. Vesti B.R. Daum G. Abraham J.A. Clowes A.W. Circ. Res. 2000; 87: 92-98Crossref PubMed Scopus (166) Google Scholar, E. H. M. Abraham R. C. A. Nature. 1999; PubMed Scopus Google Scholar). In SMCs, thrombin the receptor by releasing growth factor from the cell (15Kalmes A. Vesti B.R. Daum G. Abraham J.A. Clowes A.W. Circ. Res. 2000; 87: 92-98Crossref PubMed Scopus (166) Google Scholar). In we demonstrated that in human SMCs, thrombin not the thrombin causes release of bFGF into the pericellular matrix with FGFR-1 phosphorylation, which be by bFGF-neutralizing antibodies and by heparin (3Rauch B.H. Millette E. Kenagy R.D. Daum G. Clowes A.W. Circ. Res. 2004; 94: 340-345Crossref PubMed Scopus (72) Google Scholar). In the present study, we have demonstrated that the cell heparan sulfate proteoglycan syndecan-4 is required for thrombin-induced mitogenesis and migration by using syndecan-4 siRNA to syndecan-4 core protein We that human SMCs with reduced syndecan-4 or FGFR-1 and in to thrombin or bFGF 4 and The of effect is to the of of thrombin-mediated mitogenesis by cells with bFGF-neutralizing antibodies (3Rauch B.H. Millette E. Kenagy R.D. Daum G. Clowes A.W. Circ. Res. 2004; 94: 340-345Crossref PubMed Scopus (72) Google Scholar). In the bFGF-neutralizing not or migration and proliferation of FGFR-1 that the thrombin-induced is by syndecan-4 and as as and and bind bFGF and increase bFGF-FGFR-1 interactions (5Steinfeld R. Van Den Berghe H. David G. J. Cell Biol. 1996; 133: 405-416Crossref PubMed Scopus (230) Google Scholar). In syndecan-4 is to a unique in (9Longley R.L. Woods A. Fleetwood A. Cowling G.J. Gallagher J.T. Couchman J.R. J. Cell Sci. 1999; 112: 3421-3431Crossref PubMed Google Scholar, 13Bass M.D. Humphries M.J. Biochem. J. 2002; 368: 1-15Crossref PubMed Scopus (122) Google Scholar, R. J. R.D. Simons M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The of syndecan-4 a with phosphatidylinositol 4,5-bisphosphate and PKCα, which (6Oh E.S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 7Oh E.S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 11805-11811Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 8Horowitz A. Simons M. J. Biol. Chem. 1998; 273: 25548-25551Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). It been that a protein after bFGF binds to kinase the of syndecan-4, for phosphatidylinositol 4,5-bisphosphate and of the syndecan-4 in the binding and of A. E. Simons M. J. Cell Biol. 2002; PubMed Scopus Google Scholar). It is that thrombin-induced signaling of and of into focal C. S. C. G. K. M. H. J. 2000; PubMed Google Scholar). syndecan-4 activates (6Oh E.S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 7Oh E.S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 11805-11811Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 8Horowitz A. Simons M. J. Biol. Chem. 1998; 273: 25548-25551Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), it is that thrombin may effects on migration and in via bFGF and syndecan-4 activation. Thrombin also through the and ERK is required for cell cycle progression induced by thrombin V. Van E. Van E. Pouyssegur J. Biochem. J. 1993; PubMed Scopus Google Scholar). In SMCs, the receptor was required to ERK min (15Kalmes A. Vesti B.R. Daum G. Abraham J.A. Clowes A.W. Circ. Res. 2000; 87: 92-98Crossref PubMed Scopus (166) Google Scholar). In human SMCs, we that of ERK by thrombin required syndecan-4 and FGFR-1 in the of syndecan-4 and FGFR-1 in thrombin-mediated SMC proliferation It be of to the involved in syndecan-4 ERK activation. of spreading of syndecan-4 siRNA cells on slides is in with previous that syndecan-4 is involved in cell spreading through the of focal contacts (9Longley R.L. Woods A. Fleetwood A. Cowling G.J. Gallagher J.T. Couchman J.R. J. Cell Sci. 1999; 112: 3421-3431Crossref PubMed Google Scholar, 13Bass M.D. Humphries M.J. Biochem. J. 2002; 368: 1-15Crossref PubMed Scopus (122) Google Scholar). may the reduced migration of SMCs Although we not focal the that cells were spread by 6 h that human SMCs have to focal is with using from Although cells are to focal (13Bass M.D. Humphries M.J. Biochem. J. 2002; 368: 1-15Crossref PubMed Scopus (122) Google Scholar, K. K. H. S. E. K. H. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), focal is when the cells are cultured on the cell binding of in the of culture medium with the of K. K. H. S. E. K. H. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Of but when and in the M. S. S. M. J. 2001; PubMed Scopus Google Scholar, S. E. Glycoconj. J. 2002; 19: PubMed Scopus Google Scholar). the specific of syndecan-4 for focal in human cells, is and In we demonstrate a requirement for syndecan-4 and FGFR-1 for thrombin-induced migration, and ERK in human vascular We are to Dr. Michael A. Reidy (University of Washington) for the bFGF