I n the process of bone formation, osteoblasts mineralize the matrix by promoting the seeding of basic calcium phosphate crystals of hydroxyapatite in the sheltered interior of shed membrane-limited matrix vesicles (MVs) and by propagating hydroxyapatite mineral onto the collagenous extracellular matrix (osteoid; refs. 1 and 2). Tissue-nonspecific alkaline phosphatase (TNAP), an isozyme of a family of four homologous human alkaline phosphatase genes (3), plays a role in bone matrix mineralization. Deactivating mutations in the TNAP gene causes the inborn error of metabolism known as hypophosphatasia (4), characterized by poorly mineralized cartilage (rickets) and bones (osteomalacia), spontaneous bone fractures, and elevated extracellular inorganic pyrophosphate (PP i ) concentrations (5). The severity and expressivity of hypophosphatasia depends on the nature of the TNAP mutation (6). TNAP is present in MVs (7), and it has been proposed that the inorganic phosphate (P i )-generating activity of TNAP is required to generate the P i needed for hydroxyapatite crystallization (8-10). However, the ability of TNAP to hydrolyze PP i also has been hypothesized to be important to promote osteoblastic mineralization (11, 12), because PP i suppresses the formation and growth of hydroxyapatite crystals (13). In fact, heritable extracellular PP i deficiencies are models of ectopic calcification such as ankylosing spinal hyperostosis and pathologic soft-tissue ossification (14-16). PP i is produced by the nucleoside triphosphate pyrophosphohydrolase (NTPPPH) activity of a family of isozymes that include PC-1, B10͞PDNP3, and autotaxin (17-19). However, PC-1 seems to be the only NTPPPH present in MVs (20). TNAP knockout (KO) mice (21-23) recapitulate the heritable metabolic disease hypophosphatasia (5), whereas PC-1-null mice display hypermineralization abnormalities similar to cartilage calcification in osteoarthritis (14) and ossification of the posterior longitudinal ligament of the spine (15).We previously identified PC-1 as the likely NTPPPH isozyme to act on the same pathway with TNAP as antagonistic regulators of extracellular PP i concentrations (20). In this paper, we have tested the hypothesis that bone abnormalities caused by the lack of TNAP could be counterbalanced by the removal of PC-1 and vice versa. We show that bone mineralization in double-KO mice lacking both TNAP and PC-1 is essentially normal, providing evidence that TNAP and PC-1 are key regulators of bone mineralization by determining the normal steady-state levels of PP i . Our work suggests that TNAP and PC-1 may be useful therapeutic targets for the treatment of bone mineralization abnormalities. Materials and MethodsAkp2 and Enpp1 KO Mice. The generation and characterization of the Akp2 KO mice has been reported (22,23). These Akp2 KO mice were hybrids of C57BL͞6 ϫ 129͞J mouse strains. The generation of the Enpp1 KO mice has been reported briefly (24). These Enpp1 KO mice were hybrids of C57BL͞6 ϫ 129͞SvTerJ mouse strains. Akp2͞Enpp1 double-heterozy...

Tissue-nonspecific alkaline phosphatase (TNAP) hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PP(i)). Deletion of the TNAP gene (Akp2) in mice results in hypophosphatasia characterized by elevated levels of PP(i) and poorly mineralized bones, which are rescued by deletion of nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) that generates PP(i). Mice deficient in NPP1 (Enpp1(-/-)), or defective in the PP(i) channeling function of ANK (ank/ank), have decreased levels of extracellular PP(i) and are hypermineralized. Given the similarity in function between ANK and NPP1 we crossbred Akp2(-/-) mice to ank/ank mice and found a partial normalization of the mineralization phenotypes and PP(i) levels. Examination of Enpp1(-/-) and ank/ank mice revealed that Enpp1(-/-) mice have a more severe hypermineralized phenotype than ank/ank mice and that NPP1 but not ANK localizes to matrix vesicles, suggesting that failure of ANK deficiency to correct hypomineralization in Akp2(-/-) mice reflects the lack of ANK activity in the matrix vesicle compartment. We also found that the mineralization inhibitor osteopontin (OPN) was increased in Akp2(-/-), and decreased in ank/ank mice. PP(i) and OPN levels were normalized in [Akp2(-/-); Enpp1(-/-)] and [Akp2(-/-); ank/ank] mice, at both the mRNA level and in serum. Wild-type osteoblasts treated with PP(i) showed an increase in OPN, and a decrease in Enpp1 and Ank expression. Thus TNAP, NPP1, and ANK coordinately regulate PP(i) and OPN levels. The hypomineralization observed in Akp2(-/-) mice arises from the combined inhibitory effects of PP(i) and OPN. In contrast, NPP1 or ANK deficiencies cause a decrease in the PP(i) and OPN pools that leads to hypermineralization.

Endochondral ossification is a carefully orchestrated process mediated by promoters and inhibitors of mineralization. Phosphatases are implicated, but their identities and functions remain unclear. Alkaline phosphatase (TNAP) plays a crucial role promoting mineralization of the extracellular matrix by restricting the concentration of the calcification inhibitor inorganic pyrophosphate (PPi). Mutations in the TNAP gene cause hypophosphatasia, a heritable form of rickets and osteomalacia. Here we show that PHOSPHO1, a phosphatase with specificity for phosphoethanolamine and phosphocholine, plays a functional role in the initiation of calcification and that ablation of PHOSPHO1 and TNAP function prevents skeletal mineralization. Phospho1−/− mice display growth plate abnormalities, spontaneous fractures, bowed long bones, osteomalacia, and scoliosis in early life. Primary cultures of Phospho1−/− tibial growth plate chondrocytes and chondrocyte-derived matrix vesicles (MVs) show reduced mineralizing ability, and plasma samples from Phospho1−/− mice show reduced levels of TNAP and elevated plasma PPi concentrations. However, transgenic overexpression of TNAP does not correct the bone phenotype in Phospho1−/− mice despite normalization of their plasma PPi levels. In contrast, double ablation of PHOSPHO1 and TNAP function leads to the complete absence of skeletal mineralization and perinatal lethality. We conclude that PHOSPHO1 has a nonredundant functional role during endochondral ossification, and based on these data and a review of the current literature, we propose an inclusive model of skeletal calcification that involves intravesicular PHOSPHO1 function and Pi influx into MVs in the initiation of mineralization and the functions of TNAP, nucleotide pyrophosphatase phosphodiesterase-1, and collagen in the extravesicular progression of mineralization. © 2011 American Society for Bone and Mineral Research.