Androgen Receptor Regulates Bone Sialoprotein Gene Expression

Objectives: Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. Androgens are steroid hormones that are essential for skeletal development. Androgen receptor (AR) is a transcription factor and a member of the steroid receptor superfamily that plays an important role in male sexual differentiation and prostate cell proliferation. Methods: To determine the molecular mechanism of androgen (dihydrotestosterone; DHT) and AR effects on BSP transcription, we conducted Western blot, Northern blot, real-time PCR, transient transfection analyses with chimeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene and gel shift assays. Results: AR protein levels were increased after AR overexpression in ROS 17/2.8 cells. BSP mRNA levels were increased by AR overexpression. However, the endogenous and overexpressed BSP mRNA levels were not change by DHT (10-8M, 24 h). Transient transcription analyses revealed that AR overexpression increased luciferase activity in the pLUC3 luciferase construct (nts -116 to +60), as well as in longer constructs (pLUC4; -425 to +60, pLUC5; -801 to +60, pLUC6; -938 to +60) transfected into ROS 17/2.8 cells. The effect of AR overexpression was abrogated by 2bp mutations in either the cAMP response element (CRE) or activator protein 1 (AP1)/glucocorticoid response element (GRE). Gel shift analyses showed that AR overexpression increased binding to the CRE and AP1/GRE elements. Notably, the CRE-protein complexes were supershifted by phospho-CREB antibody and CREB, c-Fos, c-Jun, and AR antibodies disrupted the complexes formation. The AP1/GRE-protein complexes were supershifted by c-Fos antibody, and c-Jun, and AR antibodies disrupted the complexes formation. Conclusions: These studies show that AR induces BSP gene transcription by targeting the CRE and AP1/GRE elements in the promoter of the rat BSP gene.