Calcium hydroxide regulates transcription of the bone sialoprotein gene via a calcium-sensing receptor in osteoblast-like ROS 17/2.8 cells

Bone sialoprotein (BSP) is a glycoprotein associated with mineralized tissues. In this study, we investigated the regulation of Bsp transcription by calcium hydroxide [Ca(OH)₂] in rat osteosarcoma-derived osteoblast-like ROS 17/2.8 cells and stromal bone marrow cells. Application of Ca(OH)₂ (0.4 mM) increased the levels of runt-related transcription factor 2 (Runx2) and BspmRNAs at 3 and 6 h and the level of BSP protein at 12 h. Transient transfection analyses were performed using chimeric constructs encompassing different regions of the rat Bsp gene promoter ligated to a luciferase reporter gene. It was found that Ca(OH)₂ increased the luciferase activities of the pLUC3 and pLUC4 constructs. Introduction of 2-bp mutations to the luciferase construct showed that the effects of Ca(OH)₂ were mediated by cAMP response-element (CRE) and fibroblast growth factor 2 (FGF2) response element (FRE). Luciferase activities induced by Ca(OH)₂ were blocked by protein kinase C (PKC), protein kinase A (PKA), phosphoinositide-3-kinase (PI3-K), and extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitors and by calcium-sensing receptor (CASR) antagonists. Gel-shift analyses showed that Ca(OH)₂ increased binding of nuclear protein to CRE and FRE. Dexamethasone-induced mineralization in stromal bone marrow cells was abrogated by CASR antagonists. These studies demonstrate that Ca(OH)₂ regulates Bsp transcription via the CASR by targeting CRE and FRE in the rat Bsp gene promoter.

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