Role of Ras and Mapks in TGFβ signaling

Normal signaling by TGFβ, in the absence of serum or exogenous factors, involves a rapid activation of Ras, Erks, and Sapks in proliferating cultures of TGFβ-sensitive untransformed epithelial cells and human carcinoma cells. Expression of either RasN17 or dominant-negative (DN) MKK4, or addition of the MEK1 inhibitor PD98059, can block the ability of TGFβ to induce AP-1 complex formation at the TGFβ₁ promoter and to autoinduce its own production. The primary components present in this TGFβ-stimulated AP-1 complex are JunD and Fra-2, although c-Jun, and possibly Fos B, may also be present. While there are two potential Smad binding elements (SBE's) in the TGFβ₁ promoter, supershift assays suggest that at least one of these does not bind Smad4, and the other is unable to bind factors activated by TGFβ. In contrast, TGFβ autoinduction is Smad3-dependent, as DN Smad3 inhibits the ability of TGFβ to stimulate TGFβ₁ promoter activity. Our results indicate that TGFβ can activate both the MKK4/Sapk and MEK/Erk pathways, through Ras and TGFβ R₁ and R₁₁, to induce TGFβ₁ production; Smad4 does not appear to be involved, and Smad3 appears to function independently of this Smad4. We also demonstrate that activation of the Ras/Mapk pathway by TGFβ positively modulates Smad1-signaling-pathway activation by TGFβ. In addition, Smad1 could enhance TGFβ activation of the SBE reporter SBE-luc and this effect could be blocked by co-expression of a DN TGFβ R₁ receptor or by the MEK1 inhibitor PD98059. This cross-talk between the MEK/Erk and Smad1 pathways was mediated through the four Erk consensus phosphorylation sites in the linker region of Smad1. Mutation of these sites resulted in a loss of the ligand- dependence of both Smad1-Smad4 interactions and nuclear accumulation of Smad1, as well as a loss of the ability of Smad1 to enhance TGFβ-mediated SBE activation. Our results provide evidence that Erk-mediated phosphorylation of Smad1 in response to TGFβ is critical for regulating Smad1 subcellular localization; this may be a key determinant in maintaining TGFβ-dependent transcriptional activation. (C) 2000 Elsevier Science Ltd.