Regulation of the napi-cotransporter in osteoclasts by bone matrix through recruitment to the plasma membrane

Osteoclasts (OCs) are exposed to high ambient concentrations of phosphate (Pi) during the ATP dependent process of bone résorption. We have previously reported the existence of a Na-dependent Pi cotransporter in the avian OCs, which are a model system for the OC phenotype capable of bone résorption in vitro. Upon exposure of these OCs to bone particles in culture, there was an immediate stimulation of Pi transport, independent öl de novo protein synthesis. The stimulatory effect of bone particles was inhibited by peptides with the Arg-Giy-Asp (RGD) motif which are ligands for the ocvp3 integrin in osteoclasts. We used a polyclonal antibody to the N-terminal of NaPi-2 to detect a single band of -100 kDa in western blots on both OC lysates and membrane fractions. Immunofluorescence studies detected the protein in discrete vesicles in unpolarized OCs, and a redistribution of NaPi-cotransporters to the plasma membrane of OCs cultured on bone. The NaPi-cotransporter protein was induced in membrane fractions isolated from osteoclasts cultured in the presence of bone particles. In the rabbit OC, the nucleotide sequence of the Na-Pi cotransporter is identical to that in the renal proximal tubule (NaPi-6). The Na-Pi cotransporter is localized exclusively on the basolateral surface of the polarized OC and colocalizes with both the H-ATPase and NHE-1 on that membrane alone. In conclusion, our preliminary studies indicate that a Na-Pi cotransporter exists in the OC, immunologically related to the NaPi-2 family, and which may be regulated through integrin-mediated pathways in the presence of hone.