Steroid receptor co-activator-1 mediates 1,25-dihydroxyvitamin D3-stimulated alkaline phosphatase in human osteosarcoma cells

For steroid hormone function to occur, nuclear receptors interact with a se ries of coactivators including steroid receptor coactivator-1 (SRC-1). The SRC-1 binds the vitamin D receptor (VDR in the presence of ligand in an act ivation function 2 (AF-2)-dependent manner. in order to understand the role of this interaction in 1,25-dihydroxy-vitamin D-3 [1,25(OH)(2)D-3]-mediate d gene expression, the level of SRC-1 expression was altered in MG-63 cells . Previous studies had demonstrated that MG-63 cells express the VDR and th at 1,25(OH)(2)D-3 regulates expression of alkaline phosphatase (ALP). Analy sis of MG-63 cells demonstrated that SRC-1 is expressed. A full-length cDNA coding for SRC-1 was inserted in antisense orientation into an expression vector (anti-SRC-1). The MG-63 cells were transfected with anti-SRC-1 or mo ck vector and stable transformants were selected. Western blot analysis sho wed a 95% reduction in SRC-1 protein as compared with mock cells. We determ ined the effect of normal and reduced SRC-1 expression in MG-63 cells on 1, 25(OH)(2)D-3-mediated stimulation of ALP. Whereas 10(-8) M 1,25(OH)(2)D-3 p roduced a 3.6-fold stimulation in ALP in mock cells expressing normal level s of SRC-1, it did not alter ALP in cells expressing reduced levels of SRC- 1. Thus, SRC-1 is required for 1,25(OH)(2)D-3-mediated gene expression of A LP by human MG-63 cells.