Regulation of cementoblast gene expression by inorganic phosphate in vitro

Examination of mutant and knockout phenotypes with altered phosphate/pyrophosphate distribution has demonstrated that cementum, the mineralized tissue that sheathes the tooth root, is very sensitive to local levels of phosphate and pyrophosphate. The aim of this study was to examine the potential regulation of cementoblast cell behavior by inorganic phosphate (P(i)). Immortalized murine cementoblasts were treated with P(i) in vitro, and effects on gene expression (by quantitative real-time reverse-transcriptase polymerase chain reaction [RT-PCR]) and cell proliferation (by hemacytometer count) were observed. Dose-response (0.1-10 mM) and time-course (1-48 hours) assays were performed, as well as studies including the Na-P(i) uptake inhibitor phosphonoformic acid. Real-time RT-PCR indicated regulation by phosphate of several genes associated with differentiation/mineralization. A dose of 5 mM P(i) upregulated genes including the SIBLING family genes osteopontin (Opn, >300% of control) and dentin matrix protein-1 (Dmp-1, >3,000% of control). Another SIBLING family member, bone sialoprotein (Bsp), was downregulated, as were osteocalcin (Ocn) and type I collagen (Col1). Time-course experiments indicated that these genes responded within 6-24 hours. Time-course experiments also indicated rapid regulation (by 6 hours) of genes concerned with phosphate/pyrophosphate homeostasis, including the mouse progressive ankylosis gene (Ank), plasma cell membrane glycoprotein-1 (Pc-1), tissue nonspecific alkaline phosphatase (Tnap), and the Pit1 Na-P(i) cotransporter. Phosphate effects on cementoblasts were further shown to be uptake-dependent and proliferation-independent. These data suggest regulation by phosphate of multiple genes in cementoblasts in vitro. During formation, phosphate and pyrophosphate may be important regulators of cementoblast functions including maturation and regulation of matrix mineralization.