Calcium hydroxide regulates bone sialoprotein gene transcription in human osteoblast-like Saos2 cells

Bone sialoprotein (BSP) is a mineralized tissue-specific protein expressed in differentiated osteoblasts that appears to function in the initial mineralization of bone. Calcium hydroxide (Ca(OH)(2)) is a basic salt that has been widely used for a variety of applications in dentistry, due to its antimicrobial effects and its capability of inducing hard tissue formation. However, details of the mechanism involved in the mineralization induced by Ca(OH)(2) are still unclear. In the present study, Ca(OH)(2) (0.4 mM) was found to increase the levels of BSP and Runx2 mRNA at 3 h in human osteoblast-like Saos2 cells. Transient transfection assays were performed using chimeric constructs of the human BSP gene promoter linked to a luciferase reporter gene. Treatment of Saos2 cells with Ca(OH)(2) (0.4 mM) increased the luciferase activities of the constructs between -60LUC and -927LUC at 12 h. Gel shift analysis showed that Ca(OH)(2) (0.4 mM) increased the binding of nuclear protein to CRE1, CRE2 and FRE. Antibodies against CREB1, c-Fos, c-Jun, JunD, Fra2 and P300 disrupted the formation of the CRE1- and CRE2-protein complexes, and antibodies against Dlx5, Msx2, Runx2 and Smad1 disrupted the formation of the FRE-protein complex. These findings demonstrate that Ca(OH)(2) stimulates BSP transcription by targeting the CRE1, CRE2 and FRE elements in the human BSP gene promoter.