Platelet-derived growth factor-BB, insulin-like growth factor-I, and phorbol ester activate different signaling pathways for stimulation of vascular smooth muscle cell migration.
Vascular smooth muscle cell (VSMC) migration is an important process in the development of vascular occlusive disease. To investigate mitogen regulation of VSMC migration, a cell-layer-scrape assay was used to measure migration 20 h after stimulation of VSMC with platelet-derived growth factor-BB (PDGF-BB), insulin-like growth factor I (IGF-I), or phorbol 12-myristate 13-acetate (PMA). The contributions of cell proliferation were eliminated by treatment of VSMC with hydroxyurea, which suppressed DNA synthesis.PDGF-BB stimulated VSMC migration 2.5-fold, while PMA and IGF-I stimulated migration 1.7- and 1.5-fold, respectively. The importance of protein kinase C (PKC), ERK, and phosphoinositide-3' kinase (PI3 kinase) in mitogen-stimulated migration was investigated, using specific inhibitors of these signaling molecules. PDGF-BB-stimulated migration was inhibited by the general PKC inhibitor RO 31-8220 (40%), the MEK inhibitor PD98059 (31%), and the PI3 kinase inhibitor wortmannin (22%) but not by PMA-induced downregulation of conventional and novel PKC isoforms. IGF-I-stimulated migration was inhibited by RO 31-8220 (34%) and wortmannin (37%) but was much less affected by PD98059 (19%) or PKC downregulation (10%). PMA-stimulated migration was inhibited by RO 31-8220 (53%), PD98059 (50%), wortmannin (45%), and PKC downregulation (47%). Western analysis confirmed that ERK was strongly activated by PDGF-BB and PMA but not by IGF-I. To examine potential in vivo negative regulators of VSMC migration, we analyzed the ability of heparin, an analogue of heparan sulfate, and TGFbeta to attenuate mitogen-stimulated migration. Heparin but not TGFbeta inhibited VSMC migration stimulated by all three mitogens. Delayed-addition experiments showed that RO 31-8220 retained substantial inhibitory activity even if added 3 h after PMA or IGF-I stimulation and 5 h after PDGF-BB addition, suggesting that sustained PKC activation is important for migration. The MEK inhibitor retained some effectiveness for 5 h after PDGF-BB stimulation but only 1 h after PMA addition. Western analysis showed ERK activation was transient after PMA treatment but sustained for 6 h after PDGF-BB treatment. Heparin strongly inhibited migration even if added 5-7 h after mitogen stimulation, suggesting that heparin may inhibit both short- and long-term signals necessary for migration. The present studies indicate that PMA and IGF-I activate a limited number of second messengers resulting in moderate stimulation of migration; in contrast PDGF-BB stimulates multiple signaling pathways resulting in strong stimulation of migration and lessened sensitivity to inhibitory signals.
Department of Pathology, Harvard Medical School, Boston, Massachusetts, 02115-5701, USA. lapuk@warren.med.harvard.edu
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Pukac
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Huangpu
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Karnovsky
D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology*; D000077214:Becaplermin; D002465:Cell Movement / Q000187:drug effects* / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000187:drug effects; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020574:Proto-Oncogene Proteins c-sis; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D000925:Anticoagulants; D000077214:Becaplermin; D004791:Enzyme Inhibitors; D006133:Growth Substances; D006493:Heparin; D007334:Insulin-Like Growth Factor I; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010982:Platelet-Derived Growth Factor; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D020574:Proto-Oncogene Proteins c-sis; D013755:Tetradecanoylphorbol Acetate; D016212:Transforming Growth Factor beta