Lack of Klotho, the co-receptor for the bone-derived hormone fibroblast growth factor-23 (FGF23), leads to a severe impairment in bone mineralization in mice. However, the mechanisms underlying the Klotho deficiency-associated defects in bone mineralization are still poorly understood. Here, we sought to elucidate the vitamin D independent role of Klotho in bone mineralization. To ablate increased vitamin D signaling in Klotho knockout mice (Kl−/−), we crossed Kl−/− mice with mice expressing a non-functioning vitamin D receptor (VDRΔ/Δ). As expected, Kl−/− mice were characterized by increased serum 1,25-dihydroxyvitamin D3 (1,25 (OH)2D3) and Fgf23, and impaired bone mineralization as evidenced by μCT, quantitative backscattered electron imaging, and histomorphometric analysis. The mineralization defect in Kl−/− mice was associated with increased bone mRNA expression of ANK (progressive ankylosis), ENPP1 (ectonucleotide pyrophosphatase phosphodiesterase 1), ENPP3, and osteopontin as compared to WT and VDRΔ/Δ mice. In addition, we found increased pyrophosphate levels and osteopontin protein expression in bones of Kl−/− mice. However, ablation of vitamin D signaling in Kl−/−/VDRΔ/Δ compound mutants normalized serum Fgf23 levels, bone mineralization, bone pyrophosphate levels, and bony expression of ANK, ENPP1, ENPP3, and osteopontin. Treatment of differentiated primary osteoblasts isolated from WT mice with 1,25(OH)2D3, but not with recombinant FGF23, increased ANK, ENPP1, ENPP3 and osteopontin mRNA expression. Moreover, primary differentiated osteoblasts isolated from Kl−/− did not show cell autonomous changes in mRNA expression of ANK, ENPP1, ENPP3, or osteopontin as compared to WT cells. Our data suggest that Klotho lacks a vitamin D independent role in bone mineralization, and that the mineralization defect observed in Klotho deficient mice is entirely due to 1,25(OH)2D3-driven upregulation of the mineralization inhibitor osteopontin, and of the pyrophosphate-regulating factors ANK, ENPP1, ENPP3.
17 May 2014 - 20 May 2014