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.
Rauch
Bernhard H
BH
University of Washington School of Medicine, Department of Surgery, Seattle, Washington 98195-6410, USA. rauchb@uni-duesseldorf.de
Millette
Esther
E
Kenagy
Richard D
RD
Daum
Guenter
G
Fischer
Jens W
JW
Clowes
Alexander W
AW
D001011:Aorta / Q000166:cytology; D015153:Blotting, Western; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D020868:Gene Silencing; D006493:Heparin / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008562:Membrane Glycoproteins / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology*; D010766:Phosphorylation; D011509:Proteoglycans / Q000502:physiology*; D034741:RNA, Small Interfering / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D051496:Receptor, Fibroblast Growth Factor, Type 1; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053671:Syndecan-4; D013917:Thrombin / Q000378:metabolism*; D013997:Time Factors
D008562:Membrane Glycoproteins; D011509:Proteoglycans; D034741:RNA, Small Interfering; D017468:Receptors, Fibroblast Growth Factor; C508304:SDC4 protein, human; D053671:Syndecan-4; D016222:Fibroblast Growth Factor 2; D006493:Heparin; D004247:DNA; C496348:FGFR1 protein, human; D020794:Receptor Protein-Tyrosine Kinases; D051496:Receptor, Fibroblast Growth Factor, Type 1; D048049:Extracellular Signal-Regulated MAP Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D013917:Thrombin