cAMP Regulates BSP Expression in Human Breast Cancer Cells

Objectives: Bone sialoprotein (BSP) is a sulfated and phosphorylated glycoprotein, found almost exclusively in mineralized connective tissues. BSP is also expressed in osteotropic cancers, such as breast, lung and prostate cancers. Forskolin (FSK) elevates intracellular cAMP levels. We previously reported that FGF2 and cAMP synergistically regulate BSP gene expression in osteoblast-like cells. Methods: To determine the molecular basis of the transcriptional regulation of BSP gene by cAMP in breast cancer cells, we conducted RT-PCR, transient transfection analyses with chimeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene and gel mobility shift assays. Results: FSK (1 µM) increased BSP, Runx2 and Osterix mRNA levels at 12 h in MCF7 breast cancer cells by RT-PCR. In transient transfection analyses, FSK (1 µM, 12 h) increased luciferase activity of the construct, pLUC3 (-116 to +60), transfected into MCF7 cells. Included within the DNA sequence that is unique to this region (between –116 and -43) are an inverted CCAAT box (ATTGG; –50 ~ –46), a cAMP response element (CRE; –75 ~ –68), a FGF2 response element (FRE; –92 ~ –85) and a pituitary-specific transcription factor-1 motif (Pit-1; –111 ~ –105). To determine the regulatory elements utilized by FSK, a series of 5' deletion constructs were prepared between -116 and -43. –60, -84, -108 and -116BSPLUC activities were increased by FSK. Effects of FSK abrogated in constructs included 2 bp mutations in the inverted CCAAT, CRE and Pit-1 motif. Gel shift assays with radiolabeled ds-oligonucleotides revealed CRE-protein complex was increased at 3 h, and Pit-1-protein complex decreased at 6 h by FSK. Whereas the CCAAT-protein complex did not change after stimulation by FSK. Conclusions: These studies show that cAMP induces BSP transcription in MCF7 cells through inverted CCAAT, CRE and Pit-1 elements in the proximal promoter of rat BSP gene.