Prostaglandin E2 stimulates bone sialoprotein (BSP) expression through cAMP and fibroblast growth factor 2 response elements in the proximal promoter of the rat BSP gene

Bone sialoprotein (BSP), an early marker of osteoblast differentiation, has been implicated in the nucleation of hydroxyapatite during de novo bone formation. Prostaglandin E-2 (PGE(2)) has anabolic effects on proliferation and differentiation of osteoblasts via diverse signal transduction systems. Because PGE(2) increases the proportion of functional osteoblasts in fetal rat calvarial cell cultures, we investigated the regulation of BSP, as an osteoblastic marker, by PGE(2). Treatment of rat osteosarcoma UMR 106 cells with 3 muM, 300 nM, and 30 nM PGE(2) increased the steady state levels of BSP mRNA about 2.7-, 2.5-, and 2.4-fold after 12 h. From transient transfection assays, the constructs including the promoter sequence of nucleotides (nt) -116 to +60 (pLUC3) were found to enhance transcriptional activity 3.8- and 2.2-fold treated with 3 muM and 30 nM PGE(2) for 12 h. 2-bp mutations were made in an inverted CCAAT box (between nt -50 and -46), a cAMP response element (CRE; between nt -75 and -68), a fibroblast growth factor 2 response element (FRE; nt -92 to -85), and a pituitary-specific transcription factor-1 motif (between nt -111 and -105) within pLUC3 and pLUC7 constructs. Transcriptional stimulation by PGE(2) was almost completed abrogated in constructs that included 2-bp mutations in either the CRE and FRE. In gel shift analyses an increased binding of nuclear extract components to double-stranded oligonucleotide probes containing CRE and FRE was observed following treatment with PGE(2). These studies show that PGE(2) induces BSP transcription in UMR 106 cells through juxtaposed CRE and FRE elements in the proximal promoter of the BSP gene.