The metabolic bone disease associated with the Hyp mutation is independent of osteoblastic HIF1α expression

Abstract

Fibroblast growth factor-23 (FGF23) controls key responses to systemic phosphate increases through its phosphaturic actions on the kidney. In addition to stimulation by phosphate, FGF23 positively responds to iron deficiency anemia and hypoxia in rodent models and in humans. The disorder X-linked hypophosphatemia (XLH) is characterized by elevated FGF23 in concert with an intrinsic bone mineralization defect. Indeed, the Hyp mouse XLH model has disturbed osteoblast to osteocyte differentiation with altered expression of a wide variety of genes, including FGF23. The transcription factor Hypoxia inducible factor-1α (HIF1α) has been implicated in regulating FGF23 production and plays a key role in proper bone cell differentiation. Thus the goals of this study were to determine whether HIF1α activation could influence FGF23, and to test osteoblastic HIF1α production on the Hyp endocrine and skeletal phenotypes in vivo. Treatment of primary cultures of osteoblasts/osteocytes and UMR-106 cells with the HIF activator AG490 resulted in rapid HIF1α stabilization and increased Fgf23 mRNA (50-100 fold; p < 0.01-0.001) in a time- and dose-dependent manner. Next, the Phex gene deletion in the Hyp mouse was bred onto mice with a HIF1α/Osteocalcin (OCN)-Cre background. Although HIF1α effects on bone could be detected, FGF23-related phenotypes due to the Hyp mutation were independent of HIF1α in vivo. In summary, FGF23 can be driven by ectopic HIF1α activation under normal iron conditions in vitro, but factors independent of HIF1α activity after mature osteoblast formation are responsible for the disease phenotypes in Hyp mice in vivo.

Keywords: FGF23; Hypoxia inducible factor-1α; Phosphate; X-linked hypophosphatemia.

Fig. 1

AG490 activity on Fgf23 mRNA expression in vitro. A. AG490 (50 μM) stimulated FGF23 mRNA expression in primary cultures of differentiated osteoblasts/osteocytes (**p < 0.001) and B. UMR-106 cells (**p < 0.001). Inset: Hif1α protein was stabilized by both AG490 (50 μM) as assessed by immunoblot. (C.) Time- and (D.) dose-dependent effects on Fgf23 mRNA expression in UMR-106 cells versus vehicle (DMSO) after 2 h (*p < 0.05; **p < 0.01). E. AG490 (50 μM) activity on FGF23 mRNA expression is affected by pre-treatment with U0126 (*p < 0.05 vs untreated and U0126; ^@p < 0.05 vs AG490 treated).

Fig. 2

Effects of Hif1α(Cre⁺) on Hyp phenotypes, A&B. Body weights (g) of male (left) and female (right) WT, WT/Hif1α(Cre⁺), Hyp, and Hyp/Hif1α(Cre⁺) mice were recorded every other week from weeks 4 through 12. (n ≥ 6 mice per group; *p < 0.01, ^#p < 0.05). C. Serum intact FGF23 was elevated in Hyp versus WT in the presence or absence of the Hif1α(Cre) allele. Female Hyp/Hif1α(Cre⁺) mice had slightly higher serum FGF23 compared with female Hyp. Bone structure in control and Hyp genetic crosses. Representative μCT images of distal femurs from 12 week old WT, WT/Hif1α(Cre⁺), Hyp, and Hyp/Hif1α(Cre⁺); D. male (left panel), E. female (right panel). Compared to genotype controls, Hyp mice had markedly widened distal femur (white arrows) consistent with their known ricketic phenotype, as well as far less trabecular bone.