Thrombospondin-1 induces activation of focal adhesion kinase in vascular smooth muscle cells

Abstract

Purpose: Vascular smooth muscle cell (VSMC) proliferation and migration are important events in the development of intimal hyperplasia. Thrombospondin-1 (TSP-1), an extracellular matrix protein present in intimal hyperplastic lesions, has been shown to stimulate VSMC proliferation and migration. We hypothesized that the focal adhesion plaque, specifically the focal adhesion kinase (FAK) protein, may be important in the signal transduction pathway for TSP-1-induced VSMC migration. Methods: Growth- arrested bovine aortic VSMCs were treated with TSP-1 (20 μg/mL) for set intervals (15, 30, and 120 minutes) and compared with VSMCs grown in serum- free medium (negative control) or in the presence of a known mitogen and chemotactic factor, platelet-derived growth factor (10 ng/mL; positive control). Crude cell lysates and anti-FAK immunoprecipitates were assayed for phosphotyrosine activity by means of antiphosphotyrosine immunoblotting. The blots were quantified by means of densitometric analysis. Results: TSP-1 increased tyrosine phosphorylation of three protein bands of molecular weights 68, 125 (consistent with FAK), and 180 kDa. Immunoprecipitation with FAK antibody, followed by antiphosphotyrosine immunoblotting, indicated that there was an increase in tyrosine phosphorylation of FAK at 15, 30, and 120 minutes in the TSP-1-treated groups (P < .05). Conclusion: Tyrosine phosphorylation of FAK is induced by TSP-1 stimulated VSMCs. This suggests an outside-inside signaling pathway by which TSP-1 stimulates VSMC migration.