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Prolactin Blocks Nuclear Translocation of VDR by Regulating Its Interaction with BRCA1 in Osteosarcoma Cells

Division of Biomedical Sciences (C.D., E.U., K.E.C., A.W.N., R.A.L., A.M.W.) and Department of Biochemistry (C.D., C.B., A.W.N., R.A.L.) University of California, Riverside, California 92521

Address all correspondence and requests for reprints to: Ameae M. Walker, Division of Biomedical Sciences, University of California, Riverside, California 92521. E-mail: Ameae.Walker{at}ucr.edu.

Based on their content of prolactin receptors, osteosarcoma cells were predicted to be responsive to prolactin (PRL), but whether PRL would be beneficial or contribute to pathogenesis was unclear. 1,25(OH)₂ vitamin D₃ [1,25(OH)₂D₃] has antiproliferative effects on osteosarcoma cells, and a complex interregulatory situation exists between PRL and 1,25(OH)₂D₃. Using osteosarcoma cells, Western blot, real time RT-PCR, and promoter-luciferase assays, we have examined the interaction between PRL and 1,25(OH)₂D₃ and demonstrated that physiological concentrations of PRL block increased osteocalcin and vitamin D receptor (VDR) expression in response to 1,25(OH)₂D_3. This blockade was shown to be the result of lack of nuclear accumulation of the VDR in response to 1,25(OH)₂D₃. Although inhibition of proteasomic degradation with MG132 had no effect on the VDR itself in a 30-min time frame, it relieved the blockade by PRL. Analysis of ubiquitinated proteins brought down by immunoprecipitation with anti-VDR showed PRL regulation of a 250-kDa protein-VDR complex. P250 was identified as the breast cancer tumor suppressor gene product, BRCA1, by Western blot of the VDR immunoprecipitate and confirmed by immunoprecipitation with anti-BRCA1 and blotting for the VDR in the absence and presence of PRL. Knockdown of BRCA1 inhibited nuclear translocation of the VDR and the ability of 1,25(OH)₂D₃ to induce the VDR. This, to our knowledge, is the first demonstration of a role for BRCA1 in nuclear accumulation of a steroid hormone and the first demonstration that PRL has the potential to affect the cell cycle through effects on BRCA1.

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Coregulators:   VDR