ATP-mediated mineralization of MC3T3-E1 osteoblast cultures

Abstract While bone is hypomineralized in hypophosphatemia patients and in tissue-nonspecific alkaline phosphatase (Tnsalp)-deficient mice, the extensive mineralization that nevertheless occurs suggests involvement of other phosphatases in providing phosphate ions for mineral deposition. Although the source of phosphate liberated by these phosphatases is unknown, pyrophosphate, ATP, pyridoxal-5′-phosphate (PLP) and phoshoethanolamine (PEA) are likely candidates. In this study, we have induced mineralization of MC3T3-E1 osteoblast cultures using ATP, and have investigated potential phosphatases involved in this mineralization process. MC3T3-E1 osteoblasts were cultured for 12 days and treated either with β-glycerophosphate (βGP) or ATP. Matrix and mineral deposition was examined by biochemical, cytochemical, ultrastructural and X-ray microanalytical methods. ATP added at levels of 4–5 mM resulted in mineral deposition similar to that following conventional treatment with βGP. Collagen levels were similarly normal in ATP-mineralized cultures and transmission electron microscopy and X-ray microanalysis confirmed hydroxyapatite mineral deposition along the collagen fibrils in the ECM. Phosphate release from 4 mM ATP into the medium was rapid and resulted in approximately twice the phosphate levels than after release from 10 mM βGP. ATP treatment did not affect mineralization by altering the expression of mineral-regulating genes such as Enpp1 , Ank , and Mgp , nor phosphatase genes indicating that ATP induces mineralization by serving as a phosphate source for mineral deposition. Levamisole, an inhibitor of TNSALP, completely blocked mineralization in βGP-treated cultures, but had minor effects on ATP-mediated mineralization, indicating that other phosphatases such as plasma membrane Ca2+ transport ATPase 1 (PMCA1) and transglutaminase 2 (TG2) are contributing to ATP hydrolysis. To examine their involvement in ATP-mediated mineralization, the inhibitors cystamine (TG2 inhibitor) and ortho-vanadate (PMCA inhibitor) were added to the cultures — both inhibitors significantly reduced mineralization whereas suppression of the phosphate release by ortho-vanadate was minor comparing to other two inhibitors. The contribution of PMCA1 to mineralization may occur through pumping of calcium towards calcification sites and TG2 can likely act as an ATPase in the ECM. Unlike the GTPase activity of TG2, its ATPase function was resistant to calcium, demonstrating the potentia