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

Abstract

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 Bsp mRNA s 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 pLUC 3 and pLUC 4 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 ( FGF 2) response element ( FRE ). Luciferase activities induced by Ca( OH ) 2 were blocked by protein kinase C ( PKC ), protein kinase A ( PKA ), phosphoinositide‐3‐kinase ( PI 3‐K), and extracellular signal‐regulated kinase 1/2 ( ERK 1/2) 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.