Steroid Receptor Co-activator-1 Mediates 1,25-Dihydroxyvitamin D₃-Stimulated Alkaline Phosphatase in Human Osteosarcoma Cells

Abstract

For steroid hormone function to occur, nuclear receptors interact with a series 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 activation function 2 (AF-2)-dependent manner. In order to understand the role of this interaction in 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]-mediated 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 that 1,25(OH)₂D₃ regulates expression of alkaline phosphatase (ALP). Analysis 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 mock vector and stable transformants were selected. Western blot analysis showed a 95% reduction in SRC-1 protein as compared with mock cells. We determined the effect of normal and reduced SRC-1 expression in MG-63 cells on 1,25(OH)₂D₃-mediated stimulation of ALP. Whereas 10⁻⁸ M 1,25(OH)₂D₃ produced a 3.6-fold stimulation in ALP in mock cells expressing normal levels 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)₂D₃-mediated gene expression of ALP by human MG-63 cells.