Inorganic phosphate (Pi) acts as a signaling molecule in bone-forming cells, affecting cell functions and gene expression. Particularly, Pi stimulates the expression of mineralization-associated genes such as matrix gla protein (MGP) and osteopontin (OPN) through the ERK1/2 pathway. With respect to the presence of elevated extracellular calcium and Pi levels during bone remodeling, we questioned whether calcium might play a role in the Pi-dependent effects in osteoblasts. We first showed by Western blot and real-time PCR that the concomitant presence of 10 mM Pi and 1.8 mM calcium is required to stimulate ERK1/2 phosphorylation and MGP/OPN genes expression. The mechanisms involved in the cellular effects of calcium in the presence of Pi were subsequently examined. Firstly, the use of the calcium-sensing receptor (CaSR) agonist gadolinium and the G-protein inhibitor pertussis toxin enabled us to determine that a CaSR mechanism is not involved in the Pi and calcium mediated cellular effects. By transmission electron microscopy, we next demonstrated that adding 10mM Pi to the culture medium containing 1.8mM calcium led to the formation calcium phosphate precipitates (CaPp). Moreover, treatment of osteoblasts with exogenous pre-synthesized CaPp stimulated ERK1/2 phosphorylation and MGP/OPN genes expression. In spite of high extracellular calcium and Pi concentrations, this stimulation was blunted in the presence of phosphocitrate, an inhibitor of crystal formation. Finally, we showed that despite that CaPp are not endocytosed, their effect on ERK1/2 phosphorylation and MGP/OPN genes expression were dependent on lipid rafts integrity. In summary, we showed that calcium is required for Pi-dependent ERK1/2 phosphorylation and regulation of mineralization-associated genes in osteoblasts and that its effect could originate from extracellular-related effects of CaPp that are dependent on the integrity of lipid rafts.
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