BRCA1 Interacts with Smad3 and Regulates Smad3-Mediated TGF-β Signaling during Oxidative Stress Responses

BACKGROUND: BRCA1 is a key regulatory protein participating in cell cycle checkpoint and DNA damage repair networks. BRCA1 plays important roles in protecting numerous cellular processes in response to cell damaging signals. Transforming growth factor-beta (TGF-β) is a potent regulator of growth, apoptosis and invasiveness of tumor cells. TFG-β activates Smad signaling via its two cell surface receptors, the TbetaRII and ALK5/TbetaRI, leading to Smad-mediated transcriptional regulation. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report an important role of BRCA1 in modulating TGF-β signaling during oxidative stress responses. Wild-type (WT) BRCA1, but not mutated BRCA1 failed to activate TGF-β mediated transactivation of the TGF-β responsive reporter, p3TP-Lux. Further, WT-BRCA1, but not mutated BRCA1 increased the expression of Smad3 protein in a dose-dependent manner, while silencing of WT-BRCA1 by siRNA decreased Smad3 and Smad4 interaction induced by TGF-β in MCF-7 breast cancer cells. BRCA1 interacted with Smad3 upon TGF-β1 stimulation in MCF-7 cells and this interaction was mediated via the domain of 298–436aa of BRCA1 and Smad3 domain of 207–426aa. In addition, H(2)O(2) increased the colocalization and the interaction of Smad3 with WT-BRCA1. Interestingly, TGF-β1 induced Smad3 and Smad4 interaction was increased in the presence of H(2)O(2) in cells expressing WT-BRCA1, while the TGF-β1 induced interaction between Smad3 and Smad4 was decreased upon H(2)O(2) treatment in a dose-dependent manner in HCC1937 breast cancer cells, deficient for endogenous BRCA1. This interaction between Smad3 and Smad4 was increased in reconstituted HCC1937 cells expressing WT-BRCA1 (HCC1937/BRCA1). Further, loss of BRCA1 resulted in H(2)O(2) induced nuclear export of phosphor-Smad3 protein to the cytoplasm, resulting decreased of Smad3 and Smad4 interaction induced by TGF-β and in significant decrease in Smad3 and Smad4 transcriptional activities. CONCLUSIONS/SIGNIFICANCE: These results strongly suggest that loss or reduction of BRCA1 alters TGF-β growth inhibiting activity via Smad3 during oxidative stress responses.