Thrombin Activates Latent TGFβ1 via Integrin αvβ1 in Gingival Fibroblasts

Transforming growth factor β (TGFβ) 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 TGFβ1 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 TGFβ1- and thrombin-induced CCN2 synthesis. In this study, we demonstrated that thrombin and protease-activated receptor 1 agonist SFLLRN induced latent TGFβ1 activation and Smad3 phosphorylation in human gingival fibroblasts. Pretreatment with a TGFβ-neutralizing antibody, TGFβ 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 αv and β1 with antibodies effectively inhibited SFLLRN-induced Smad3 phosphorylation and CCN2 synthesis and increased activated TGFβ1 levels; however, similar effects were not observed for integrins αvβ3 and αvβ5. These results suggest that protease-activated receptor 1-induced CCN2 synthesis in human gingival fibroblasts is mediated through integrin αvβ1-induced latent TGFβ1 activation and subsequent TGFβ1 signaling. Moreover, curcumin dose dependently decreased thrombin-induced activated TGFβ1 levels. Curcumin-inhibited thrombin-induced CCN2 synthesis in human gingival fibroblasts is caused by the suppression of latent TGFβ1 activation.