Requirement of Ras/MAPK pathway activation by transforming growth factor beta for transforming growth factor beta 1 production in a Smad-dependent pathway.
Our previous results have shown that transforming growth factor beta (TGFbeta) rapidly activates Ras, as well as both ERKs and SAPKs. In order to address the biological significance of the activation of these pathways by TGFbeta, here we examined the role of the Ras/MAPK pathways and the Smads in TGFbeta(3) induction of TGFbeta(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 TGFbeta(3) to induce AP-1 complex formation at the TGFbeta(1) promoter, and the subsequent induction of TGFbeta(1) mRNA. The primary components present in this TGFbeta(3)-inducible AP-1 complex at the TGFbeta(1) promoter were JunD and Fra-2, although c-Jun and FosB were also involved. Furthermore, deletion of the AP-1 site in the TGFbeta(1) promoter or addition of PD98059 inhibited the ability of TGFbeta(3) to stimulate TGFbeta(1) promoter activity. Collectively, our data demonstrate that TGFbeta(3) induction of TGFbeta(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 Smad3 and Smad4 were not detectable in TGFbeta(3)-inducible AP-1 complexes at the TGFbeta(1) promoter, stable expression of dominant-negative Smad3 could significantly inhibit the ability of TGFbeta(3) to stimulate TGFbeta(1) promoter activity. Transient expression of dominant-negative Smad4 also inhibited the ability of TGFbeta(3) to transactivate the TGFbeta(1) promoter. Thus, although the Ras/MAPK pathways are essential for TGFbeta(3) induction of TGFbeta(1), Smads may only contribute to this biological response in an indirect manner.
Yue
J
J
Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
Mulder
K M
KM
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D016895:Culture Media, Serum-Free; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D051921:Fos-Related Antigen-2; D005799:Genes, Dominant; D011905:Genes, ras / Q000235:genetics; D007413:Intestinal Mucosa / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D048670:MAP Kinase Kinase 4; D020935:MAP Kinase Signaling System; D008907:Mink; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D012260:Ribonucleases / Q000378:metabolism; D015398:Signal Transduction; D051900:Smad3 Protein; D051901:Smad4 Protein; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000096:biosynthesis* / Q000378:metabolism*; D053773:Transforming Growth Factor beta1; D053782:Transforming Growth Factor beta3; D018631:ras Proteins / Q000502:physiology*
D001426:Bacterial Proteins; D016895:Culture Media, Serum-Free; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D051921:Fos-Related Antigen-2; C483820:Fosb protein, rat; C497874:Fosl2 protein, rat; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D051900:Smad3 Protein; C497787:Smad3 protein, rat; D051901:Smad4 Protein; C490811:Smad4 protein, rat; C508960:TGFB1 protein, human; C508962:Tgfb1 protein, rat; C509046:Tgfb3 protein, rat; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D053782:Transforming Growth Factor beta3; D008156:Luciferases; D048670:MAP Kinase Kinase 4; C482559:MAP2K4 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; D012260:Ribonucleases; D018631:ras Proteins; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one