Thrombin Activates Latent TGFβ1 via Integrin αvβ1 in Gingival Fibroblasts

Transforming growth factor beta (TGF beta) regulates cell proliferation, differentiation, migration, apoptosis, and extracellular matrix production. It also plays a pivotal role in the pathogenesis of gingival overgrowth. Thrombin is a key player in tissue repair, remodeling, and fibrosis after an injury, and it exerts profibrotic effects by activating protease-activated receptors. Connective tissue growth factor (CTGF or CCN2) modulates cell adhesion, migration, proliferation, matrix production, and wound healing. It is overexpressed in many fibrotic disorders, including gingival overgrowth, and it is positively associated with the degree of fibrosis in gingival overgrowth. In human gingival fibroblasts, we previously found that TGF beta 1 induced CCN2 protein synthesis through c-jun N-terminal kinase and Smad3 activation. Thrombin stimulates CCN2 synthesis through protease-activated receptor 1 and c-jun N-terminal kinase signaling. Curcumin inhibited TGF beta 1- and thrombin-induced CCN2 synthesis. In this study, we demonstrated that thrombin and protease-activated receptor 1 agonist SFLLRN induced latent TGF beta 1 activation and Smad3 phosphorylation in human gingival fibroblasts. Pretreatment with a TGF beta-neutralizing antibody, TGF beta type I receptor inhibitor SB431542, and Smad3 inhibitor SIS3 inhibited approximately 86%, 94%, and 100% of thrombin-induced CCN2 synthesis, respectively. Furthermore, blocking integrin subunits alpha v and beta 1 with antibodies effectively inhibited SFLLRN-induced Smad3 phosphorylation and CCN2 synthesis and increased activated TGF beta 1 levels; however, similar effects were not observed for integrins alpha v beta 3 and alpha v beta 5. These results suggest that protease-activated receptor 1-induced CCN2 synthesis in human gingival fibroblasts is mediated through integrin alpha v beta 1-induced latent TGF beta 1 activation and subsequent TGF beta 1 signaling. Moreover, curcumin dose dependently decreased thrombin-induced activated TGF beta 1 levels. Curcumin-inhibited thrombin-induced CCN2 synthesis in human gingival fibroblasts is caused by the suppression of latent TGF beta 1 activation.