TGF-B1 regulation of alpha-smooth muscle actin expression in fibroblasts is dependent on the deformability of the substrate

Abstract

Wound contraction is a fundamental process in wound repair and is required for closure, but the interactions between the inflammatory cytokines that regulate this process and the cytoskeletal elements in fibroblasts that provide the contractile forces are poorly understood. I examined the effect of TGF-$\beta$1 on the ability of human gingival fibroblasts to contract collagen gels in vitro and on the expression of the putative fibroblast contractile marker, $\alpha$-smooth muscle actin ($\alpha$-SMA). TGF-$\beta$1 (10 ng/ml; 3 days) increased $\alpha$-SMA protein and mRNA 2-3 fold, as determined by western and northern blots and normalised for $\beta$-actin content. Western blots of cells in anchored collagen gels also showed a TGF-$\beta$1-induced increase of $\alpha$-SMA content but the effect was reduced compared to collagen-coated plastic. In floating collagen gels, TGF-$\beta$1 exerted no significant effect on $\alpha$-SMA protein. Notably, TGF-$\beta$1 increased the rate of contraction in anchored and floating collagen gels in serum-free cultures by 50% and 30% respectively but the difference was not evident in serum-containing cultures. Using an ELISA, active and latent forms of endogenous TGF-$\beta$1 were detected in all 3 models. Addition of exogenous TGF-$\beta$1 stimulated its own production. The highest levels of endogenous TGF-$\beta$1 were detected in monolayer cultures and the lowest levels in floating gels. Blocking $\alpha$2 and $\beta$1 subunits with monoclonal antibodies abolished the effect of TGF-$\beta$1 on $\alpha$-SMA expression. Collectively these data indicate that TGF-$\beta$1 may regulate wound contraction by affecting the cellular content of the contractile protein, $\alpha$-SMA and that generation of intracellular tension may regulate expression of specific cytoskeletal genes involved in cell contraction.