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...

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The ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) multigene family contains five members. NPP1-3 are type II transmembrane metalloenzymes characterized by a similar modular structure composed of a short intracellular domain, a single transmembrane domain and an extracellular domain containing a conserved catalytic site. The short intracellular domain of NPP1 has a basolateral membrane-targeting signal while NPP3 is targeted to the apical surface of polarized cells. NPP4-5 detected by database searches have a predicted type I membrane orientation but have not yet been functionally characterized. E-NPPs have been detected in almost all tissues often confined to specific substructures or cell types. In some cell types, NPP1 expression is constitutive or can be induced by TGF-beta and glucocorticoids, but the signal transduction pathways that control expression are poorly documented. NPP1-3 have a broad substrate specificity which may reflect their role in a host of physiological and biochemical processes including bone mineralization, calcification of ligaments and joint capsules, modulation of purinergic receptor signalling, nucleotide recycling, and cell motility. Abnormal NPP expression is involved in pathological mineralization, crystal depositions in joints, invasion and metastasis of cancer cells, and type 2 diabetes. In this review we summarize the present knowledge on the structure and the physiological and biochemical functions of E-NPP and their contribution to the pathogenesis of diseases.