Requirement of Smad3 and CREB-1 in mediating transforming growth factor-beta (TGF beta) induction of TGF beta 3 secretion.
Because increased transforming growth factor-beta (TGFbeta) production by tumor cells contributes to cancer progression through paracrine mechanisms, identification of critical points that can be targeted to block TGFbeta production is important. Previous studies have identified the precise signaling components and promoter elements required for TGFbeta induction of TGFbeta1 expression in epithelial cells (Yue, J., and Mulder, K. M. (2000) J. Biol. Chem. 275, 30765-30773). To determine how regulation of TGFbeta3 expression differs from that of TGFbeta1, we identified the precise signaling pathways and transcription factor-binding sites that are required for TGFbeta3 gene expression. By using mutational analysis in electrophoresis mobility shift assays (EMSAs), we demonstrated that the c-AMP-responsive element (CRE) site in the TGFbeta3 promoter was required for TGFbeta-inducible TGFbeta3 expression. Electrophoresis mobility supershift assays indicated that CRE-binding protein 1 (CREB1) and Smad3 were the major components present in this TGFbeta-inducible complex. Furthermore, by using chromatin immunoprecipitation assays, we demonstrated that CREB-1, ATF-2, and c-Jun bound constitutively at the TGFbeta3 promoter (-100 to +1), whereas Smad3 bound at this site only after TGFbeta stimulation. In addition, inhibition of JNK and p38 suppressed TGFbeta induction of TGFbeta3 transactivation, whereas inhibition of ERK and protein kinase A had no effect. Small interfering RNA-CREB1 and small interfering RNA-Smad3 significantly inhibited TGFbeta stimulation of TGFbeta3 promoter reporter activity and TGFbeta3 production. Our results indicate that TGFbeta activation of the TGFbeta3 promoter CRE site, which leads to TGFbeta3 production, is required for TGFbetaRII, JNK, p38, and Smad3 but was independent of protein kinase A, ERK, and Smad4.
Liu
Guangming
G
Department of Pharmacology, Penn State College of Medicine, Hershey, Pennsylvania 17033, USA.
Ding
Wei
W
Neiman
Jill
J
Mulder
Kathleen M
KM
D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology*; D006801:Humans; D011401:Promoter Regions, Genetic; D051381:Rats; D051900:Smad3 Protein / Q000502:physiology*; D053782:Transforming Growth Factor beta3 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
C508137:Creb1 protein, rat; D017362:Cyclic AMP Response Element-Binding Protein; D051900:Smad3 Protein; C497787:Smad3 protein, rat; D053782:Transforming Growth Factor beta3