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Mullerian-Inhibiting Substance Induces Gro-ß Expression in Breast Cancer Cells through a Nuclear Factor-B–Dependent and Smad1-Dependent Mechanism

¹ Department of Surgical Oncology, Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, Massachusetts; ² Department of Radiation Medicine, University of Kentucky, Lexington, Kentucky; and ³ Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Boston, Massachusetts

Requests for reprints: Shyamala Maheswaran, Massachusetts General Hospital Cancer Center, Building 149, 13th Street, Charlestown, MA 02129. Phone: 617-724-6552; Fax: 617-724-9648; E-mail: maheswaran{at}helix.mgh.harvard.edu.

Mullerian-inhibiting substance (MIS), a transforming growth factor-ß family member, activates the nuclear factor-B (NF-B) pathway and induces the expression of B-cell translocation gene 2 (BTG2), IFN regulatory factor-1 (IRF-1), and the chemokine Gro-ß. Inhibiting NF-B activation with a phosphorylation-deficient IB mutant abrogated MIS-mediated induction of all three genes. Expression of dominant-negative Smad1, in which serines at the COOH-terminal SSVS motif are converted to alanines, suppressed MIS-induced Smad1 phosphorylation and impaired MIS-stimulated Gro-ß promoter-driven reporter expression and Gro-ß mRNA. Suppressing Smad1 expression using small interfering RNA also mitigated MIS-induced Gro-ß mRNA, suggesting that regulation of Gro-ß expression by MIS was dependent on activation of NF-B as well as Smad1. However, induction of IRF-1 and BTG2 mRNAs by MIS was independent of Smad1 activation. Characterization of B-binding sequences within Gro-ß, BTG2, and IRF-1 promoters showed that MIS stimulated binding of p50 and p65 subunits to all three sites, whereas phosphorylated Smad1 (phospho-Smad1) protein was detectable only in the NF-B complex bound to the B site of the Gro-ß promoter. Consistent with these observations, chromatin immunoprecipitation assays showed recruitment of both phospho-Smad1 and p65 to the Gro-ß promoter in vivo, whereas p65, but not phospho-Smad1, was recruited to the BTG2 promoter. These results show a novel interaction between MIS-stimulated Smad1 and NF-B signaling in which enhancement of NF-B DNA binding and gene expression by phospho-Smad1 is dependent on the sequence of the B consensus site within the promoter. [Cancer Res 2007;67(6):2747–56]