Deletion of PTH Rescues Skeletal Abnormalities and High Osteopontin Levels in Klotho ^−/− Mice

Abstract

Maintenance of normal mineral ion homeostasis is crucial for many biological activities, including proper mineralization of the skeleton. Parathyroid hormone (PTH), Klotho, and FGF23 have been shown to act as key regulators of serum calcium and phosphate homeostasis through a complex feedback mechanism. The phenotypes of Fgf23 ^−/− and Klotho ^−/− ( Kl ^−/− ) mice are very similar and include hypercalcemia, hyperphosphatemia, hypervitaminosis D, suppressed PTH levels, and severe osteomalacia/osteoidosis. We recently reported that complete ablation of PTH from Fgf23 ^−/− mice ameliorated the phenotype in Fgf23 ^−/−/PTH ^−/− mice by suppressing serum vitamin D and calcium levels. The severe osteomalacia in Fgf23 ^−/− mice, however, persisted, suggesting that a different mechanism is responsible for this mineralization defect. In the current study, we demonstrate that deletion of PTH from Kl ^−/− ( Kl ^−/−/PTH ^−/− or DKO) mice corrects the abnormal skeletal phenotype. Bone turnover markers are restored to wild-type levels; and, more importantly, the skeletal mineralization defect is completely rescued in Kl ^−/−/PTH ^−/− mice. Interestingly, the correction of the osteomalacia is accompanied by a reduction in the high levels of osteopontin (Opn) in bone and serum. Such a reduction in Opn levels could not be observed in Fgf23 ^−/−/PTH ^−/− mice, and these mice showed sustained osteomalacia. This significant in vivo finding is corroborated by in vitro studies using calvarial osteoblast cultures that show normalized Opn expression and rescued mineralization in Kl ^−/−/PTH ^−/− mice. Moreover, continuous PTH infusion of Kl ^−/− mice significantly increased Opn levels and osteoid volume, and decreased trabecular bone volume. In summary, our results demonstrate for the first time that PTH directly impacts the mineralization disorders and skeletal deformities of Kl ^−/− , but not of Fgf23 ^−/− mice, possibly by regulating Opn expression. These are significant new perceptions into the role of PTH in skeletal and disease processes and suggest FGF23-independent interactions of PTH with Klotho.

Author Summary

Maintenance of normal mineral ion homeostasis is crucial for many biological activities, including proper mineralization of the skeleton. PTH, Klotho, and FGF23 are the key regulators of blood mineral ion homeostasis. Klotho is a type-I membrane protein and has been identified as cofactor required for FGF23 to bind and activate its receptor. Loss of either Klotho or Fgf23 activity results in a similar abnormal phenotype, including severe defects in skeletal mineralization and alterations in mineral ion balance. Here we describe a new mouse model in which we eliminated PTH from Kl ^−/− mice, and we can show that the skeletal mineralization defect was completely rescued in Kl ^−/− / PTH ^−/− mice and that this phenomenon was accompanied by a reduction in the high levels of osteopontin in bone and serum. We also present additional data showing that continuous infusion of Kl ^−/− mice with PTH results in an elevation in Opn levels and subsequently increased osteoid volume. Interestingly, this result differs from our previous report in which we describe that the osteomalacia and the high Opn levels in Fgf23 ^−/− / PTH ^−/− mice persisted. Our finding suggests that PTH, possibly by regulating osteopontin, is responsible for the skeletal mineralization defect in Kl ^−/− mice, but not in Fgf23 ^−/− mice.