Forskolin Regulates Bone Sialoprotein Expression in Human Prostate Cancer Cells

Objectives: Bone sialoprotein (BSP) is a mineralized tissue-specific protein that appears to function in the initial mineralization of bone. BSP is also expressed in osteotropic cancers, such as breast and prostate cancers. We previously demonstrated that cAMP up-regulated BSP gene expression in osteoblasts. Forskolin (FSK) activates adenylate cyclase activities and elevates intracellular cAMP levels. In this study we used FSK to look at the effects of cAMP on the expression of BSP in DU145 human prostate cancer cells. Methods: To determine the molecular basis of the transcriptional regulation of BSP gene by FSK in prostate cancer cells, we conducted real-time PCR, transient transfection analyses with chimerical constructs of the human BSP gene promoter linked to a luciferase reporter gene, and gel mobility shift assays. Results: FSK (1 µM) increased BSP and Osterix mRNA levels at 6 and 12 h in DU145 cells. In transient transfection analyses, using various sized human BSP gene promoter luciferase constructs, FSK (1 µM, 6 h) stimulated luciferase activity of the construct (-184HumanBSPLUC; -184 to +60) transfected into DU145 cells. The effects of FSK were partially inhibited by protein kinase C (PKC) inhibitor H7, and almost complete inhibited by protein kinase A (PKA) inhibitor H89 and 2bp mutation in the CRE1 element. Gel mobility shift assays with two CREs (CRE1; -79~-72, and CRE2; -673~-666) and FGF response element (FRE) ds-oligonucleotides revealed increased binding of nuclear proteins from FSK (1 µM, 6 h) stimulated DU145 cells. The CRE1-protein complexes formation was inhibited by anti-CREB antibody and supershifted by anti-phospho CREB and anti-Smad1 antibodies. Conclusion: These studies indicated that FSK regulates BSP transcription in prostate cancer cells and that FSK effects were mediated through CRE1, CRE2 and FRE elements in the human BSP gene promoter.