Elevated Skeletal Osteopontin Levels Contribute to the Hypophosphatasia Phenotype in Akp2−/− Mice

Abstract

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. 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. Akp2(-/-) mice were bred into the Opn(-/-) line. The resulting double knockout mice were analyzed for skeletal abnormalities by histology and muCT. Calvarial osteoblasts were assayed for their ability to mineralize in vitro and were probed for changes in gene expression. 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(-/-) mice have themselves elevated levels of ePP(i) attributable to an increase in Enpp1 and Ank expression and a concomitant downregulation of Akp2 expression in Opn(-/-) osteoblasts, but that Opn(-/-) mice have more mineralized osteoid than wildtype (WT) controls despite their elevated ePP(i) levels. Addition of exogenous OPN to Opn(-/-) osteoblasts results in downregulation of Enpp1 and Ank gene expression and a reduction of the PP(i) output by these cells. Deletion of both Akp2 and Opn can partially rescue the hypomineralized phenotype of Akp2(-/-) mice. However, these double knockout mice do not display corrected ePP(i) levels, and we conclude that regulation of hydroxyapatite deposition requires the coordinated actions of both PP(i) and OPN and that the hypophosphatasia phenotype in Akp2(-/-) mice results from the combined inhibitory action of increased levels of both ePP(i) and OPN. Our data also suggest that the ePP(i)-mediated regulation of OPN and the OPN-mediated regulation of ePP(i) are linked counterregulatory mechanisms that control the concentrations of these two important mineralization inhibitors, OPN and ePP(i).