Requirement of Ras/MAPK pathway activation by transforming growth factor β for transforming growth factor β₁ production in a Smad-dependent pathway

Our previous results have shown that transforming growth factor β (TGFβ) rapidly activates Ras, as well as both ERKs and SAPKs. In order to address the biological significance of the activation of these pathways by TGFβ, here we examined the role of the Ras/MAPK pathways and the Smads in TGFβ₃ induction of TGFβ₁ 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 TGFβ₃ to induce AP-1 complex formation at the TGFβ₁ promoter, and the subsequent induction of TGFβ₁ mRNA. The primary components present in this TGFβ₃-inducible AP-1 complex at the TGFβ₁ promoter were JunD and Fra-2, although c-Jun and FosB were also involved. Furthermore, deletion of the AP-1 site in the TGFβ₁ promoter or addition of PD98059 inhibited the ability of TGFβ₃ to stimulate TGFβ₁ promoter activity. Collectively, our data demonstrate that TGFβ₃ induction of TGFβ₁ 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 Smad3 and Smad4 were not detectable in TGFβ₃-inducible AP-1 complexes at the TGFβ₁ promoter, stable expression of dominant-negative Smad3 could significantly inhibit the ability of TGFβ₃ to stimulate TGFβ₁ promoter activity. Transient expression of dominant-negative Smad4 also inhibited the ability of TGFβ₃ to transactivate the TGFβ₁ promoter. Thus, although the Ras/MAPK pathways are essential for TGFβ₃ induction of TGFβ₁, Smads may only contribute to this biological response in an indirect manner.