SMAD3 is essential for transforming growth factor-beta 1-induced urokinase type plasminogen activator expression and migration in transformed keratinocytes

Abstract

Transforming growth factor-beta 1 (TGF-beta 1) stimulates the extracellular matrix degrading proteases expression and cell migration in order to enhance cancer cells malignancy. In the present study, we analysed the role of TGF-beta 1-induced Smad3 activation in the urokinase type plasminogen activator (uPA) production, as well as in cell migration and E-cadherin downregulation in transformed PDV keratinocyte cell line. TGF-beta 1 signalling was interfered by the chemical inhibitor of the TGF-beta 1-receptor 1 (ALK5), SB505124, and the specific Smad3 inhibitor, SiS3. Our results showed that TGF-beta 1 stimulates uPA expression directly through ALK5 activation. The inhibition of Smad3 strongly reduced the capacity of TGF-beta 1 to stimulate uPA expression, in parallel decreasing the uPA inhibitor plasminogen activator inhibitor type 1 (PAI-1) expression. In addition, the transient expression of dominant negative Smad3 mutant inhibited the TGF-beta 1-induced uPA promoter transactivation. Moreover, Smad3-/- mouse embryonic fibroblasts were refractory to the induction of uPA by TGF-beta 1. The inhibition of both ALK5 and Smad3 dramatically blocked the TGF-beta 1-stimulated E-cadherin downregulation, F-actin reorganisation and migration of PDV cells. Taken together, our results suggest that the TGF-beta 1-induced activation of Smad3 is the critical step for the uPA upregulation and E-cadherin downregulation, which are the key events preceding the induction of cell migration by TGF-beta 1 in transformed cells.