Requirement of Smad3 and CREB-1 in mediating transforming growth factor-β (TGFβ) induction of TGFβ3 secretion

Guangming Liu, Wei Ding, Jill Neiman, Kathleen Mulder

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Abstract

Because increased transforming growth factor-β (TGFβ) production by tumor cells contributes to cancer progression through paracrine mechanisms, identification of critical points that can be targeted to block TGFβ production is important. Previous studies have identified the precise signaling components and promoter elements required for TGFβ induction of TGFβ1 expression in epithelial cells (Yue, J., and Mulder, K. M. (2000) J. Biol. Chem. 275, 30765-30773). To determine how regulation of TGFβ3 expression differs from that of TGFβ1, we identified the precise signaling pathways and transcription factor-binding sites that are required for TGFβ3 gene expression. By using mutational analysis in electrophoresis mobility shift assays (EMSAs), we demonstrated that the c-AMP-responsive element (CRE) site in the TGFβ3 promoter was required for TGFβ-inducible TGFβ3 expression. Electrophoresis mobility supershift assays indicated that CRE-binding protein 1 (CREB1) and Smad3 were the major components present in this TGFβ-inducible complex. Furthermore, by using chromatin immunoprecipitation assays, we demonstrated that CREB-1, ATF-2, and c-Jun bound constitutively at the TGFβ3 promoter (-100 to +1), whereas Smad3 bound at this site only after TGFβ stimulation. In addition, inhibition of JNK and p38 suppressed TGFβ induction of TGFβ3 transactivation, whereas inhibition of ERK and protein kinase A had no effect. Small interfering RNA-CREB1 and small interfering RNA-Smad3 significantly inhibited TGFβ stimulation of TGFβ3 promoter reporter activity and TGFβ3 production. Our results indicate that TGFβ activation of the TGFβ3 promoter CRE site, which leads to TGFβ3 production, is required for TGFβRII, JNK, p38, and Smad3 but was independent of protein kinase A, ERK, and Smad4.

Original languageEnglish (US)
Pages (from-to)29479-29490
Number of pages12
JournalJournal of Biological Chemistry
Volume281
Issue number40
DOIs
StatePublished - Oct 6 2006

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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