Requirement of Ras/MAPK pathway activation by transforming growth factor ss for transforming growth factor ss(1) production in a Smad-dependent pathway

Our previous results have shown that transforming growth factor beta (TGF b eta) rapidly activates Ras, as well as both ERKs and SAPKs, In order to add ress 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 i ntestinal epithelial cells. Expression of either a dominant-negative mutant of Ras (RasN17) or a dominant-negative mutant of MKK4 (DN MKK4), or additi on of the MEK1 inhibitor PD98059, inhibited the ability of TGF beta(3) to i nduce AP-1 complex formation at the TGF beta(1) promoter, and the subsequen t 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 F ra-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. Coll ectively, our data demonstrate that TGF beta(3) induction of TGF beta(1) is mediated through a signaling cascade consisting of Ras, the MAPKKs MKK4 an d 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 st imulate TGF beta(1) promoter activity. Transient expression of dominant-neg ative Smad4 also inhibited the ability of TGF beta(3) to transactivate the TGF beta(1) promoter. Thus, although the Ras/MAPK pathways are essential fo r TGF beta(3) induction of TGF beta(1), Smads may only contribute to this b iological response in an indirect manner.