Role of Ras and Mapks in TGFβ signaling

Normal signaling by TGF beta, in the absence of serum or exogenous factors, involves a rapid activation of Ras, Erks, and Sapks in proliferating cultures of TGF beta-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 TGF beta to induce AP-1 complex formation at the TGF beta(1) promoter and to autoinduce its own production. The primary components present in this TGF beta-stimulated AP-I 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 TGF beta(1) promoter, supershift assays suggest that at least one of these does not bind Smad4, and the other is unable to bind factors activated by TGF beta. In contrast, TGF beta autoinduction is Smad3-dependent, as DN Smad3 inhibits the ability of TGF beta to stimulate TGF beta(1) promoter activity. Our results indicate that TGF beta can activate both the MKK4/Sapk and MEK/Erk pathways, through Ras and TGF beta RI and R-11. to induce TGF beta(1) 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 TGF beta positively modulates Smad1-signaling-pathway activation by TGF beta. In addition, Smad1 could enhance TGF beta activation of the SEE reporter SBE-luc and this effect could be blocked by co-expression of a DN TGF beta R-I 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 TGF beta-mediated SEE activation. Our results provide evidence that Erk-mediated phosphorylation of Smad1 in response to TGF beta is critical for regulating Smad1 subcellular localization; this may be a key determinant in maintaining TGF beta-dependent transcriptional activation. (C) 2000 Elsevier Science Ltd. All rights reserved.