Cross Talk Among Smad, MAPK, and Integrin Signaling Pathways Enhances Adventitial Fibroblast Functions Activated by Transforming Growth Factor–β1 and Inhibited by Gax

OBJECTIVE: We investigated whether Smad, mitogen-activated protein kinase (MAPK), and integrin signaling pathways cross-talk to enhance adventitial fibroblast (AF) bioactivity, which was activated by transforming growth factor (TGF)-beta1 and inhibited by Gax. METHODS AND RESULTS: Cultured AFs were stimulated with Ad-Gax, TGF-beta1, and siRNA-Gax. Assays for AFs viabilities demonstrated that TGF-beta1 and siRNA-Gax enhanced AFs proliferative, migratory, and adherent abilities, whereas Gax counteracted TGF-beta1-activated actions. Flow cytometry revealed that TGF-beta1 and siRNA-Gax increased S phase cells; however, Gax decreased AFs in the S phase and increased those in the G0-G1 and apoptotic phases. RT-PCR, Western blotting, and immunocytochemistry showed that TGF-beta1 and siRNA-Gax upregulated the expression of cytokines in Smad, MAPK, and integrin signaling pathways, and downregulated that of p15, p16, and p21. Conversely, Gax induced downregulation of these cytokines and upregulation of p15, p16, and p21. Thus, these signaling pathways cross-talk to enhance AF bioactivity; Gax effectively counteracts TGF-beta1 effects, blocks the cross-talk of these pathways, inhibits AF functions, and increases AF apoptosis. CONCLUSIONS: Our findings indicate that cross-talk among Smad, MAPK, and integrin signaling pathways may account mainly for the mechanism of AF functions. Gax is a promising therapeutic gene for dissecting the signaling pathways controlling AF bioactivities.