Mullerian-inhibiting substance induces Gro-beta expression in breast cancer cells through a nuclear factor-kappaB-dependent and Smad1-dependent mechanism.
Mullerian-inhibiting substance (MIS), a transforming growth factor-beta family member, activates the nuclear factor-kappaB (NF-kappaB) pathway and induces the expression of B-cell translocation gene 2 (BTG2), IFN regulatory factor-1 (IRF-1), and the chemokine Gro-beta. Inhibiting NF-kappaB activation with a phosphorylation-deficient IkappaBalpha 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-beta promoter-driven reporter expression and Gro-beta mRNA. Suppressing Smad1 expression using small interfering RNA also mitigated MIS-induced Gro-beta mRNA, suggesting that regulation of Gro-beta expression by MIS was dependent on activation of NF-kappaB as well as Smad1. However, induction of IRF-1 and BTG2 mRNAs by MIS was independent of Smad1 activation. Characterization of kappaB-binding sequences within Gro-beta, 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-kappaB complex bound to the kappaB site of the Gro-beta promoter. Consistent with these observations, chromatin immunoprecipitation assays showed recruitment of both phospho-Smad1 and p65 to the Gro-beta 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-kappaB signaling in which enhancement of NF-kappaB DNA binding and gene expression by phospho-Smad1 is dependent on the sequence of the kappaB consensus site within the promoter.
Gupta
Vandana
V
Department of Surgical Oncology, Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, Massachusetts 02129, USA.
Yeo
Giminna
G
Kawakubo
Hirofumi
H
Rangnekar
Vivek
V
Ramaswamy
Preethi
P
Hayashida
Tetsu
T
MacLaughlin
David T
DT
Donahoe
Patricia K
PK
Maheswaran
Shyamala
S
D054304:Anti-Mullerian Hormone; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D054426:Chemokine CXCL2; D019743:Chemokines, CXC / Q000096:biosynthesis* / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D016147:Genes, Tumor Suppressor; D006023:Glycoproteins / Q000494:pharmacology*; D006801:Humans; D017874:Immediate-Early Proteins / Q000096:biosynthesis / Q000235:genetics; D050856:Interferon Regulatory Factor-1 / Q000096:biosynthesis / Q000235:genetics; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D051898:Smad1 Protein / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D013735:Testicular Hormones / Q000494:pharmacology*; D051996:Transcription Factor RelA / Q000378:metabolism; D025521:Tumor Suppressor Proteins
C499706:CXCL2 protein, human; D054426:Chemokine CXCL2; D019743:Chemokines, CXC; D006023:Glycoproteins; D017874:Immediate-Early Proteins; D050856:Interferon Regulatory Factor-1; D016328:NF-kappa B; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C497399:SMAD1 protein, human; D051898:Smad1 Protein; D013735:Testicular Hormones; D051996:Transcription Factor RelA; D025521:Tumor Suppressor Proteins; C070114:BTG2 protein, human; D054304:Anti-Mullerian Hormone