Osteoblast tissue-nonspecific alkaline phosphatase antagonizes and regulates PC-1

Tissue-nonspecific alkaline phosphatase (TNAP) is essential for bone matrix mineralization, but the central mechanism for TNAP action remains undefined. We observed that ATP-dependent Ca-45 precipitation was decreased in calvarial osteoblast matrix vesicle (MV) fractions from TNAP(-/-) mice, a model of infantile hypophosphatasia. Because TNAP hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PPi), we assessed phosphodiesterase nucleotide pyrophosphatase (PDNP/NTPPPH) activity, which hydrolyzes ATP to generate PPi. Plasma cell membrane glycoprotein-1 (PC-1), but not the isozyme B10 (also called PDNP3) colocalized with TNAP in osteoblast MV fractions and pericellular matrix. PC-1 but not B10 increased MV fraction PPi and inhibited Ca-45 precipitation by MVs. TNAP directly antagonized inhibition by PC-1 of MV-mediated Ca-45 precipitation. Furthermore, the PPi content of MV fractions was greater in cultured TNAP(-/-) than TNAP(+/+) calvarial osteoblasts. Paradoxically, transfection with wild-type TNAP significantly increased osteoblast MV fraction NTPPPH. Specific activity of NTPPPH also was twofold greater in MV fractions of osteoblasts from TNAP(+/+) mice relative to TNAP(-/-) mice. Thus TNAP attenuates PC-1/NTPPPH-induced PPi generation that would otherwise inhibit MV-mediated mineralization. TNAP also paradoxically regulates PC-1 expression and NTPPPH activity in osteoblasts.