Methods: The expression of osteopontin (Opn), an inhibitor of bone mineralization, in Fgf23-/- mice was evaluated by in situ hybridization, immunohistochemical staining, qPCR analyses of cortical bone and ELISA measurement of serum Opn. To investigate whether Opn is contributing to the bone mineralization defect of Fgf23-/- mice, we generated Fgf23-/-/Opn-/- double knockout mice (DKO), and compared their phenotype to the one of wild-type (WT), Fgf23-/- and Opn-/- knockout mice.
Results: We found that expression of Opn was markedly increased in Fgf23-/- bones, as well as in the serum levels. Biochemical analyses showed that the hypercalcemia and hyperphophatemia observed in Fgf23-/- mice remained unchanged in DKO mice. However, additional ablation of Opn in Fgf23-/- mice resulted in larger, heavier, and more active DKO mice when compared to Fgf23-/- littermates. The length and radiopacity of the femora in the DKO mice were significantly increased. MicroCT and histomorphometrical analyses of femurs from DKO mice showed that cancellous bone volume and cortical thickness was restored to the values found in Opn-/-. More interestingly, osteoid volume was significantly decreased. We further reversed the hyperphophatemia of Fgf23-/- mice to hypophosphatemia by generating Fgf23-/-/NaPi2a-/- mice or by feeding low phosphate diet, and found serum Opn levels were significantly decreased when phosphate levels diminished, although still higher than the ones of WT.
Conclusions: Our results suggest that increased osteoblastic Opn expression is an important pathogenetic factor mediating the mineralization defect and the alterations in bone metabolism observed in Fgf23-/- bones. Moreover, hyperphophatemia mainly contributes to the markedly increased Opn levels in Fgf23-/- mice.