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.

Figure 1. Macroscopic phenotype of 6-wk-old mice.

(A) Overall phenotype of littermates. Complete deletion of PTH from Kl^−/− mice resulted in larger, heavier, and more active DKO mice compared to Kl^−/− littermates. The body weight of DKO mice is significantly higher than that of Kl^−/− mice (B), and the lifespan is slightly improved (C). ###: p<0.001 vs Kl^−/−.

Figure 2. Serum biochemical measurement.

Abnormal serum calcium levels observed in Kl^−/− and PTH^−/− mice were normalized in DKO mice (A). As for phosphate, DKO mice showed a further increase over already high levels in Kl^−/− and PTH^−/− mice (B). Kl^−/− and DKO mice showed similarly high CaPi product (C). The serum levels of intact FGF23 (D) and 1,25(OH)₂D (E) in DKO mice were partially reduced compared to that of Kl^−/− mice. *: p<0.05, **: p<0.01, ***: p<0.001vs WT; ###: p<0.001vs Kl^−/−; and $$: p<0.01, $$$: p<0.001 vs DKO.

Figure 3. Rescued skeletal phenotype.

Bone Mineral Density (BMD) of distal ends of femurs from 6-week-old mice (A). Radiographs of the tibiae from all genotypes at 6 wk of age indicate that length and radiopacity of the tibiae were restored in DKO mice (B). Alizarin red S and Alcian blue staining shows that the abnormally wide ribs in Kl^−/− mice (indicated by black arrow) were not observed in the DKO mice (C). Representative microCT images of distal femoral metaphyses (D) and midshaft cortical bone (E) and quantitative analysis (F, G). The midshaft cortical thickness (C.Th) of the DKO mice was restored to a volume comparable to that of WT and PTH^−/− mice. ***: p<0.001vs WT; ##: p<0.01, ###: p<0.001 vs Kl^−/−.

Figure 4. Histological and histomorphometric analyses.

Undecalcified sections of distal ends of femurs from 6wk-old littermates were stained with von Kossa and McNeal (A, B). High magnification of the secondary spongiosa shows heavily unmineralized osteoid in Kl^−/− mice but not in DKO mice (B). Histomorphometric analysis (C–N) confirmed that the skeletal architecture and mineralization defect of Kl^−/− mice were rescued in DKO mice. BV/TV: bone volumes; OV/TV: osteoid volume; OS/BS: osteoid surface/bone surface; OTh; osteoid thickness; Tb.Th: trabecular thickness; Tb.N: trabecular number; Tb.Sp: trabecular separation; Oc.S/BS: osteoclast surface/bone surface; N.Oc/B.Pm: osteoclast number/bone perimeter; MS/BS: mineral surface/bone surface; BFR: bone formation rate, and MAR: mineral apposition rate. *: p<0.05, **: p<0.01, ***: p<0.001vs WT; and #: p<0.05, ##: p<0.01, ###: p<0.001 vs Kl^−/−.

Figure 5. Measurement of serum CTX and PINP.

Measurement of serum CTX (A) and PINP (B), indicating normalized bone turnover in DKO mice. *: p<0.05, **: p<0.01vs WT; ##: p<0.01vs Kl^−/−.

Figure 6. Normalized expression of Opn in DKO mice.

In situ hybridization (A) and immunohistochemical staining (B) on paraffin sections prepared from tibiae of animals at 6 weeks of age. The elevated Opn expression in Kl^−/− mice was normalized in DKO mice. (C) Measurements of the serum Opn levels. Quantitive PCR confirmed the normal expression of Opn in the osteoblasts isolated from DKO mice (D). DKO osteoblasts also exhibited normal mineralization as evaluated by Alizarin red staining (E). *: p<0.05, ***: p<0.001vs WT; and #: p<0.05, ###: p<0.001 vs Kl^−/−.

Figure 7. Infusion of PTH increases Opn levels and leads to a more severe mineralization defect.

PTH infusion increased serum Opn levels (A). Undecalcified sections of distal ends of femurs were stained with von Kossa and McNeal (B, C). Histomorphometric analysis (D–M) showed that PTH infusion decreased the bone volume (BV/TV) and increased osteoid volume (OV/BV) in Kl^−/− mice. Serum CTX (N) and PINP (O) levels were significantly increased after PTH infusion. MdV/TV: mineralized bone volume/total volume. Ob.S/BS: osteoblast surface/bone surface. *: p<0.05, **: p<0.01, ***: p<0.001vs vehicle controls.