Requirement of Ras/MAPK pathway activation by transforming growth factor ß for transforming growth factor ß1 production in a Smad-dependent pathway

Our previous results have shown that transforming growth factor beta (TGF beta) rapidly activates Ras, as well as both ERKs and SAPKs, In order to address the biological significance of the activation of these pathways by TGF beta, here we examined the role of the Ras/MAPK pathways and the Smads in TGF beta(3) induction of TGF beta(1) expression in untransformed lung and intestinal epithelial cells. Expression of either a dominant-negative mutant of Ras (RasN17) or a dominant-negative mutant of MKK4 (DN MKK4), or addition of the MEK1 inhibitor PD98059, inhibited the ability of TGF beta(3) to induce AP-1 complex formation at the TGF beta(1) promoter, and the subsequent induction of TGF beta(1) mRNA, The primary components present in this TGF beta(3)-inducible AP-1 complex at the TGF beta(1) promoter were JunD and Fra-2, although c-Jun and FosB were also involved. Furthermore, deletion of the AP-1 site in the TGF beta(1) promoter or addition of PD98059 inhibited the ability of TGF beta(3) to stimulate TGF beta(1) promoter activity. Collectively, our data demonstrate that TGF beta(3) induction of TGF beta(1) is mediated through a signaling cascade consisting of Ras, the MAPKKs MKK4 and MEK1, the MAPKs SAPKs and ERKs, and the specific AP-1 proteins Fra-2 and JunD. Although Smads and Smad4 were not detectable in TGF beta(3)-inducible AP-1 complexes at the TGF beta(1) promoter, stable expression of dominant-negative Smad3 could significantly inhibit the ability of TGF beta(3) to stimulate TGF beta(1) promoter activity. Transient expression of dominant-negative Smad4 also inhibited the ability of TGF beta(3) to transactivate the TGF beta(1) promoter. Thus, although the Ras/MAPK pathways are essential for TGF beta(3) induction of TGF beta(1), Smads may only contribute to this biological response in an indirect manner.