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.

Extracellular matrix (ECM) mineralization is a physiological process in bone and a pathological one in soft tissues. The mechanisms determining the spatial restriction of ECM mineralization to bone physiologically are poorly understood. Here we show that a normal extracellular phosphate concentration is required for bone mineralization, while lowering this concentration prevents mineralization of any ECM. However, simply raising extracellular phosphate concentration is not sufficient to induce pathological mineralization, this is because of the presence in all ECMs of pyrophosphate, an inhibitor of mineralization. ECM mineralization occurs only in bone because of the exclusive coexpression in osteoblasts of Type I collagen and Tnap, an enzyme that cleaves pyrophosphate. This dual requirement explains why Tnap ectopic expression in cells producing fibrillar collagen is sufficient to induce pathological mineralization. This study reveals that coexpression in osteoblasts of otherwise broadly expressed genes is necessary and sufficient to induce bone mineralization and provides evidence that pathological mineralization can be prevented by modulating extracellular phosphate concentration.[Keywords: ECM; mineralization; TNAP; pyrophosphate; collagen] Supplemental material is available at http://www.genesdev.org.

We report three novel inhibitors of the physiological pyrophosphatase activity of alkaline phosphatase and show that these compounds are capable of reducing calcification in two models of vascular calcification (i.e., they suppress in vitro calcification by cultured Enpp1 −/− VSMCs and they inhibit the increased pyrophosphatase activity in a rat aortic model).Introduction: Genetic ablation of tissue-nonspecific alkaline phosphatase (TNALP) leads to accumulation of the calcification inhibitor inorganic pyrophosphate (PP i ). TNALP deficiency ameliorates the hypermineralization phenotype in Enpp1 −/− and ank/ank mice, two models of osteoarthritis and soft tissue calcification. We surmised that the pharmacological inhibition of TNALP pyrophosphatase activity could be used to prevent/ suppress vascular calcification. Materials and Methods: Comprehensive chemical libraries were screened to identify novel drug-like compounds that could inhibit TNALP pyrophosphatase function at physiological pH. We used these novel compounds to block calcification by cultured vascular smooth muscle cells (VSMCs) and to inhibit the upregulated pyrophosphatase activity in a rat aortic calcification model. Results: Using VSMC cultures, we determined that Enpp1−/− and ank/ank VSMCs express higher TNALP levels and enhanced in vitro calcification compared with wildtype cells. By high-throughput screening, three novel compounds, 5361418, 5923412, and 5804079, were identified that inhibit TNALP pyrophosphatase function through an uncompetitive mechanism, with high affinity and specificity when measured at both pH 9.8 and 7.5. These compounds were shown to reduce the calcification by Enpp1 −/− VSMCs. Furthermore, using an ex vivo rat whole aorta PP i hydrolysis assay, we showed that pyrophosphatase activity was inhibited by all three lead compounds, with compound 5804079 being the most potent at pH 7.5. Conclusions: We conclude that TNALP is a druggable target for the treatment and/or prevention of ectopic calcification. The lead compounds identified in this study will serve as scaffolds for medicinal chemistry efforts to develop drugs for the treatment of soft tissue calcification.

PHOSPHO1 is a phosphatase highly expressed in bone. We studied its functional involvement in mineralization through the use of novel small molecule inhibitors. PHOSPHO1 expression was present within matrix vesicles, and inhibition of enzyme action caused a decrease in the ability of matrix vesicles to calcify. Introduction:The novel phosphatase, PHOSPHO1, belongs to the haloacid dehalogenase superfamily of hydrolases and is capable of cleaving phosphoethanolamine (PEA) and phosphocholine to generate inorganic phosphate. Our aims in this study were to examine the expression of PHOSPHO1 in murine mineralizing cells and matrix vesicles (MV) and to screen a series of small-molecule PHOSPHO1-specific inhibitors for their ability to pharmacologically inhibit the first step of MV-mediated mineralization. Materials and Methods: q-PCR and immunohistochemistry were used to study the expression and localization profiles of PHOSPHO1. Inhibitors of PHOSPHO1's PEA hydrolase activity were discovered using highthroughput screening of commercially available chemical libraries. To asses the efficacy of these inhibitors to inhibit MV mineralization, MVs were isolated from TNAP-deficient (Akp2

Increased levels of ePP i in mice deficient in TNALP (i.e., Akp2 −/− ) lead to elevated OPN concentrations. We examined the skeletal phenotype of mice lacking both OPN and TNALP and concluded that the increased OPN levels contribute to the hypophosphatasia phenotype characteristic of Akp2 −/− mice. We also found that extracellular OPN regulates the PP i output by osteoblasts. Introduction: Akp2−/− display mineralization deficiencies characterized by rickets/osteomalacia. This defect has been attributed to the increased levels of extracellular inorganic pyrophosphate (ePP i ), a substrate of tissue-nonspecific alkaline phosphatase (TNALP) and a potent inhibitor of mineral deposition. Because elevated levels of ePP i induce Opn gene expression, the Akp2 −/− mice also display increased levels of osteopontin (OPN), another inhibitor of mineralization. Materials and Methods: Akp2−/− mice were bred into the Opn −/− line. The resulting double knockout mice were analyzed for skeletal abnormalities by histology and CT. Calvarial osteoblasts were assayed for their ability to mineralize in vitro and were probed for changes in gene expression. Results: Mice lacking both Akp2 and Opn showed partial normalization at the histological level with regard to mineral deposition and BMD. However, high ePP i levels remained in Akp2 −/− mice. We found that Opn