Autocrine thrombospondin partially mediates TGF-beta 1-induced proliferation of vascular smooth muscle cells

Transforming growth factor (TGF)-beta1 has been implicated in vascular heal ing responses after mechanical injury. Using cultured rat aortic smooth mus cle cells (RASMC), we examined the hypothesis that production and secretion of thrombospondin (TSP) contributes to TGF-beta1-induced proliferation. We found that TGF-beta1 enhanced production and secretion of TSP, with peak l evels of secreted TSP observed 24 h after treatment. RASMC treated with TGF -beta1 secreted a mitogenic activity that was transferable in conditioned m edia and partially inhibited by C6.7, a monoclonal anti-TSP antibody. Exoge nous TSP stimulated a proliferative response, with maximal [H-3]thymidine i ncorporation occurring 24 h earlier than maximal [H-3]thymidine incorporati on in response to TGF-beta1-treatment. Pretreatment with C6.7 or polyclonal anti-TSP neutralizing antibodies inhibited TGF-beta1-induced proliferation of RASMC. Proliferative responses to TGF-beta1 were also inhibited by pret reatment with an anti-beta (3) integrin monoclonal blocking antibody (F11), RGD peptides, and the anti-alpha (v)beta (3) disintegrin echistatin. Treat ment with TSP and TGF-beta1 increased c-Jun NH2-terminal kinase (JNK)1 acti vity, with peak effects observed at 15 min and 4 h, respectively. Treatment with C6.7 or F11 inhibited TGF-beta -induced activation of JNK1. In summar y, these studies support the hypothesis that TGF-beta -induced JNK1 activat ion and proliferation of RASMC require secretion of TSP and ligation of alp ha (v)beta (3)-integrins.