Identification of a vitamin D-3-response element that overlaps a unique inverted TATA box in the rat bone sialoprotein gene

Bone sialoprotein (BSP), an early marker of osteoblast differentiation, has been implicated in the nucleation of hydroxyapatite during bone formation de novo. Our studies, using the osteoblastic cell line ROS 17/2.8, have revealed that rat BSP gene expression is suppressed by 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3], which is a powerful regulator of bone formation and resorption. To determine the molecular basis of the transcriptional suppression of BSP gene transcription by 1,25(OH)(2)D-3, we have conducted transient transfection analyses with chimaeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene. 1,25(OH)(2)D-3 suppressed expression in all constructs, including construct (pLUC 3; nt -116 to +60) that contained a putative vitamin D-3-response element (VDRE; AGGGTTTATAGGTCA; nt -28 to -14) that overlaps a unique inverted TATA (TTTATA) box. Mobility shift assays demonstrated strong binding of recombinant human vitamin D-3 receptor protein (hVDR) to the VDRE. Point mutations introduced into each half-site and analysed for 1,25(OH)(2)D-2-mediated suppression of-transcription and for hVDR binding either decreased or increased both transcriptional suppression and binding. In comparison with activating VDREs, the rat BSP VDRE bound VDR-VDR homodimers more avidly than VDR-RXR alpha heterodimers (where RXR is retinoid X receptor). These studies have therefore identified a novel 1,25(OH)(2)D-3 suppressor element that overlaps the inverted TATA box in the rat BSP gene and indicate that transcriptional suppression of the rat BSP gene by 1,25(OH)(2)D-3 might involve competition between the VDR and the TATA binding protein (TBP).