Bone sialoprotein (BSP) is a noncollagenous protein of the extracellular matrix in mineralized connective tissues that has been implicated in the nucleation of hydroxyapatite. Forskolin (FSK), an activator of adenylate cyclase, increased the intracellular cAMP level, which stimulates the proliferation and differentiation of osteoblasts. Fibroblast growth factor 2 (FGF2) is a potent mitogen in many cell types, including osteoblasts. In human prostate cancer DU145 cells, FSK (1 μM) and FGF2 (10 ng/ml) increased BSP and Runx2 mRNA and protein levels at 3 and 12h, respectively. Transient transfection analyses were performed using chimeric constructs of the human BSP gene promoter linked to a luciferase reporter gene. Treatment of DU145 cells with FSK (1 μM) and FGF2 (10 ng/ml) increased the luciferase activities of constructs between -60LUC to -927LUC and -108LUC to -927LUC, including the human BSP gene promoter. Effects of FSK and FGF2 abrogated in constructs included 2bp mutations in the two cAMP response elements (CRE1 and CRE2). Luciferase activities induced by FSK and FGF2 were blocked by protein kinase A and tyrosine kinase inhibitors. Gel mobility shift analyses showed that FSK and FGF2 increased the binding of CRE1 and CRE2. CRE1-protein complexes were supershifted by phospho-CREB1 and c-Fos antibodies, and disrupted by CREB1, c-Jun, JunD, Fra2, p300, Runx2, Dlx5 and Smad1 antibodies. CRE2-protein complexes were disrupted by CREB1, phospho-CREB1, c-Fos, c-Jun, JunD, Fra2, p300, Runx2, Dlx5 and Smad1 antibodies. These studies demonstrate that FSK and FGF2 stimulate BSP transcription in DU145 human prostate cancer cells by targeting the CRE1 and CRE2 elements in the human BSP gene promoter.
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