• hypomineralization;
  • hypermineralization;
  • alkaline phosphatase;
  • inorganic pyrophosphate;
  • osteomalacia;
  • mouse models


Increased levels of ePPi 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 PPi 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 (ePPi), a substrate of tissue-nonspecific alkaline phosphatase (TNALP) and a potent inhibitor of mineral deposition. Because elevated levels of ePPi 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 μCT. 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 ePPi levels remained in Akp2−/− mice. We found that Opn−/− mice have themselves elevated levels of ePPi 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 ePPi levels. Addition of exogenous OPN to Opn−/− osteoblasts results in downregulation of Enpp1 and Ank gene expression and a reduction of the PPi output by these cells.

Conclusions: Deletion of both Akp2 and Opn can partially rescue the hypomineralized phenotype of Akp2−/− mice. However, these double knockout mice do not display corrected ePPi levels, and we conclude that regulation of hydroxyapatite deposition requires the coordinated actions of both PPi and OPN and that the hypophosphatasia phenotype in Akp2−/− mice results from the combined inhibitory action of increased levels of both ePPi and OPN. Our data also suggest that the ePPi-mediated regulation of OPN and the OPN-mediated regulation of ePPi are linked counterregulatory mechanisms that control the concentrations of these two important mineralization inhibitors, OPN and ePPi.