The role of tissue-nonspecific alkaline phosphatase in the phosphate-induced activation of alkaline phosphatase and mineralization in SaOS-2 human osteoblast-like cells

Tissue-nonspecific alkaline phosphatase (TNAP) plays a key role in mineralization by degrading inorganic pyrophosphate and providing free inorganic phosphate. We have previously reported that TNAP is induced by β-glycerophosphate and NaH2PO4 in short-term cultures of SaOS-2 human osteoblast-like cells and that PHEX (phosphate-regulating gene with homologies to endopeptidase on the X chromosome) mRNA is also induced after TNAP induction. In the present study, we have investigated the effects of levamisole, a TNAP inhibitor, and phosphonoformic acid (PFA), a type III sodium-phosphate cotransporter inhibitor, on the phosphate-induced expression of TNAP and mineralization. Levamisole inhibited β-glycerophosphate-induced mineralization, TNAP and PHEX expression, and the increase in enzymatic activity of NPP1 (5′-nucleotide pyrophosphatase phosphodiesterase 1), but did not inhibit NaH2PO4-induced mineralization. PFA completely inhibited NaH2PO4-induced mineralization and NPP1 enzymatic activation, and partly inhibited β-glycerophosphate-induced mineralization, but did not affect the increase in TNAP activity. These results suggest that phosphate derived from TNAP-induced hydrolysis of β-glycerophosphate yields signals that induce TNAP expression and mineralization, and that PHEX expression may be linked to TNAP expression. However, luciferase assays failed to detect any transcriptional activation of the promoter region of the human TNAP gene by β-glycerophosphate or NaH2PO4, suggesting that the effects of these phosphates may be indirect.