Tenascin-C Enhances Crosstalk Signaling of Integrin αvβ3/PDGFR-β Complex by SRC Recruitment Promoting PDGF-Induced Proliferation and Migration in Smooth Muscle Cells

Migration and proliferation of smooth muscle cells (SMCs) are key events during neointimal formation in pathological conditions of vessels. Tenascin-C (TNC) is upregulated in the developing neointima of lesions. We evaluated the effects of TNC on responses of SMCs against platelet-derived growth factor (PDGF) stimulation. TNC coated on substrate promoted PDGF-BB-induced proliferation and migration of rat SMC cell line A10 in BrdU incorporation and transwell assays, respectively. Immunoblotting showed that TNC substrate enhanced autophosphorylation of PDGFR-beta after PDGF-BB stimulation. Integrin alpha v beta 3 is known to be a receptor for TNC in SMCs. In immunofluorescence and immunoblot of integrin alpha v subunit, clustering of alpha v-positive focal adhesions and upregulated alpha v expression were observed in the cells on TNC substrate. Immunoprecipitation using anti-integrin alpha v beta 3 antibody demonstrated that PDGFR-beta and integrin alpha v beta 3 were co-precipitated and that the relative amount of PDGFR-beta after the stimulation was increased by TNC treatment. TNC also promoted phosphorylation of focal adhesion kinase (FAK) at tyrosine (Y) 397 and Y925. The phosphorylated FAK was localized at focal adhesions in immunofluorescence. Phosphorylated SRC at Y418 was also seen at focal adhesions. Immunoprecipitation with alpha v antibody showed increased SRC association with the integrin signaling complex in the cells on TNC after PDGF treatment. In the cells on TNC substrate, crosstalk signaling between integrin alpha v beta 3 and PDGFR-beta could be amplified by SRC and FAK recruited to focal adhesions, followed by enhanced proliferation and migration of A10 cells by PDGF-BB. J. Cell. Physiol. 226: 2617-2624, 2011. (C) 2010 Wiley-Liss, Inc.