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Originally published In Press as doi:10.1074/jbc.M600579200 on August 4, 2006
J. Biol. Chem., Vol. 281, Issue 40, 29479-29490, October 6, 2006
Requirement of Smad3 and CREB-1 in Mediating Transforming Growth Factor- (TGF ) Induction of TGF 3 Secretion*
Guangming Liu,
Wei Ding,
Jill Neiman, and
Kathleen M. Mulder1
From the
Department of Pharmacology, Penn State College of Medicine, Hershey, Pennsylvania 17033
Because increased transforming growth factor- (TGF ) production by tumor cells contributes to cancer progression through paracrine mechanisms, identification of critical points that can be targeted to block TGF production is important. Previous studies have identified the precise signaling components and promoter elements required for TGF induction of TGF 1 expression in epithelial cells (Yue, J., and Mulder, K. M. (2000) J. Biol. Chem. 275, 3076530773). To determine how regulation of TGF 3 expression differs from that of TGF 1, we identified the precise signaling pathways and transcription factor-binding sites that are required for TGF 3 gene expression. By using mutational analysis in electrophoresis mobility shift assays (EMSAs), we demonstrated that the c-AMP-responsive element (CRE) site in the TGF 3 promoter was required for TGF -inducible TGF 3 expression. Electrophoresis mobility supershift assays indicated that CRE-binding protein 1 (CREB1) and Smad3 were the major components present in this TGF -inducible complex. Furthermore, by using chromatin immunoprecipitation assays, we demonstrated that CREB-1, ATF-2, and c-Jun bound constitutively at the TGF 3 promoter (100 to +1), whereas Smad3 bound at this site only after TGF stimulation. In addition, inhibition of JNK and p38 suppressed TGF induction of TGF 3 transactivation, whereas inhibition of ERK and protein kinase A had no effect. Small interfering RNA-CREB1 and small interfering RNA-Smad3 significantly inhibited TGF stimulation of TGF 3 promoter reporter activity and TGF 3 production. Our results indicate that TGF activation of the TGF 3 promoter CRE site, which leads to TGF 3 production, is required for TGF RII, JNK, p38, and Smad3 but was independent of protein kinase A, ERK, and Smad4.
Received for publication, January 19, 2006
, and in revised form, August 2, 2006.
* This work was supported by National Institutes of Health Grants CA90765, CA92889, and CA100239 and Department of Defense Award DAMD17-03-1-0287 (to K. M. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 To whom correspondence should be addressed: Dept. of Pharmacology, Penn State College of Medicine, 500 University Dr., Hershey, PA 17033. Tel.: 717-531-6789; Fax: 717-531-5013; E-mail: kmm15{at}psu.edu.

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Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
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