Controlling hypoxia-inducible factor-2α is critical for maintaining bone homeostasis in mice

Abstract

Pathological bone loss is caused by an imbalance between bone formation and resorption. The bone microenvironments are hypoxic, and hypoxia-inducible factor (HIF) is known to play notable roles in bone remodeling. However, the relevant functions of HIF-2α are not well understood. Here, we have shown that HIF-2α deficiency in mice enhances bone mass through its effects on the differentiation of osteoblasts and osteoclasts. In vitro analyses revealed that HIF-2α inhibits osteoblast differentiation by targeting Twist2 and stimulates RANKL-induced osteoclastogenesis via regulation of Traf6. In addition, HIF-2α appears to contribute to the crosstalk between osteoblasts and osteoclasts by directly targeting RANKL in osteoprogenitor cells. Experiments performed with osteoblast- and osteoclast-specific conditional knockout mice supported a role of HIF-2α in this crosstalk. HIF-2α deficiency alleviated ovariectomy-induced bone loss in mice, and specific inhibition of HIF-2α with ZINC04179524 significantly blocked RANKL-mediated osteoclastogenesis. Collectively, our results suggest that HIF-2α functions as a catabolic regulator in bone remodeling, which is critical for the maintenance of bone homeostasis.

Keywords: Bone; Diseases.

Fig. 1

Heterozygous Hif-2α KO mice show increased bone mass. a–e Analysis of femoral trabecular or calvarial bones from 4-month-old Hif-2α^+/− and WT mice. mRNA levels of Hif-2α in femoral bone from WT (Hif-2α^+/+) and Hif-2α^+/− mice (n = 4; a). Representative images of µCT reconstructions of femoral trabecular and cortical bones (b) and H&E and TRAP staining of trabecular bones (scale bar: 100 μm; c). Bone volume per tissue volume (BV/TV), trabecular bone thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N) were analyzed based on the µCT measurements (n = 8; b). BV/TV, the number of osteoblastic cells per bone perimeter (N.Ob/B.Pm), the osteoblast surface normalized by bone surface (Ob.S/BS), the number of osteoclastic cells per bone perimeter (N.Oc/B.Pm), and the osteoclast surface normalized by bone surface (Oc.S/BS) were assessed by bone histomorphometric analyses of the metaphyseal regions of femurs (n = 8; c). ELISA-based measurement of the serum concentrations of OCN (n = 5) and CTX-1 (n = 7) (d). The bone forming rate (BFR) and mineral apposition rate (MAR) were analyzed from measurements obtained using calcein double labeling of femurs (n = 4; e). f Representative µCT images and measurements of bone volume of calvarial defect models generated in Hif-2α^+/− and WT mice and in C57BL/6 mice infected with Ad-Hif-2α or Ad-control (Ad-C) (n = 8). g Representative images of µCT reconstructions of femoral trabecular bones are shown, and quantitative µCT analysis was used to measure the BV/TV, Tb.Th, Tb.Sp, and Tb.N of the femoral bones from OVX- or sham-operated mice (n = 8). Values are presented as the mean ± SEM (*P < 0.05, **P < 0.01, and ***P < 0.005). The effects of OVX and genetic deletion of Hif-2α as well as their interaction in mice were analyzed by two-way ANOVA (g BV/TV: interaction = 0.048 7, OVX < 0.000 1, genetic deletion of Hif-2α = 0.024 5)

Fig. 2

HIF-2α blocks osteoblast differentiation by inhibiting osteocalcin expression. a Primary calvarial preosteoblasts from WT mice were cultured in osteogenic differentiation medium containing 50 μg·mL⁻¹ L-AA and 5 μmol·L⁻¹ β-GP for 24 days. The transcript and protein levels of HIF-2α on the indicated culture days were determined by qRT-PCR and western blotting, respectively. The expression levels of Hif-2α, Ocn, Runx2, Rankl, and Hif-1α were analyzed by qRT-PCR (n = 3). b Western blotting and quantification of protein levels of HIF-1α and HIF-2α in undifferentiated or differentiated osteoblasts under normoxia or hypoxia (n = 4). DM, differentiation media. c Alkaline phosphatase (ALP) and alizarin red S (ARS) staining in primary calvarial preosteoblasts cultured in control media (CM) or differentiation media (DM). Calvarial preosteoblasts were obtained from WT or Hif-2α^+/− mice (n = 3). d Transcript levels of Hif-2α, Ocn, and Runx2 were detected by qRT-PCR in primary cultured calvarial preosteoblasts infected with 400 multiplicity of infection (MOI) of Ad-C or the indicated MOI of Ad-Hif-2α (n > 3). e Detection of the indicated mRNAs by qRT-PCR in osteoblasts transfected with control siRNA (si-C) or the indicated amounts (nM) of Hif-2α-siRNA (n = 6). f RUNX2-responsive luciferase reporters (6xOSE-luc or OG2-luc) were transfected into primary calvarial preosteoblasts infected with Ad-Hif-2α or Ad-C. Luciferase assays were performed, and the data are presented as fold changes relative to each CM group (n = 3). g qRT-PCR analysis of Twist1 and Twist2 (n = 4). h TWIST2 immunostaining in osteoblasts of bone tissue from WT and Hif-2α^+/− mice. Dotted lines indicate osteoblasts (scale bar: 10 μm). Quantification of TWIST2-positive osteoblasts is shown (n = 7). i Detection of the mRNA levels of Hif-2α, Twist2, Runx2, and Ocn following the siRNA-mediated silencing of Twist2 (Twist2-siR) in HIF-2α-overexpressing cells (n = 3). j ChIP assays were performed using primer pairs (1 and 2) designed to span the putative HIF-2α binding sites within the Twist2 promoter, along with an anti-HIF-2α antibody. k Representative µCT images and measurements of bone volume of calvarial defect models infected with Ad-C or Ad-Hif-2α and coinjected with adenovirus encoding Twist2 shRNA (Ad-shTwist2) (n = 3). Values are presented as the mean ± SEM (*P < 0.05, **P < 0.01, and ***P < 0.005)

Fig. 3

HIF-2α increases osteoblast-mediated osteoclastogenesis. a–c Primary calvarial preosteoblasts were infected with Ad-C or Ad-Hif-2α. qRT-PCR analysis of Rankl, Opg (left panel), and the Rankl/Opg ratio (right panel) (n = 3; a). ELISA analysis of secreted RANKL in culture media (n = 4; b) and detection of RANKL by immunofluorescence microscopy (scale bar: 100 μm; c). d Determination of Hif-2α, Rankl, and Opg expression following siRNA-mediated knockdown of HIF-2α in L-AA/β-GP-induced osteoblasts (n = 3). e RANKL immunostaining in bone obtained from Hif-2α^+/+ and Hif-2α^+/− mice. Scale bar: 100 μm. Rankl-positive osteoblasts were counted in the epiphyseal bone compartment. f Primary calvarial preosteoblasts infected with Ad-Hif-2α and BMMs were mixed and cocultured on glass covers, and TRAP staining was performed. g Osteoclasts derived from WT mice and osteoblasts from HIF-2α^+/+ or HIF-2α^+/− mice in the absence or presence of BMP-2 (100 ng/ml) were cocultured, and TRAP staining was performed (scale bar: 100 μm). h A ChIP assay of Ad-Hif-2α-infected osteoblasts was performed with anti-HIF-2α antibody and a primer pair designed to span the putative HIF-2α binding regions within the promoter of Rankl. Values are presented as the mean ± SEM (*P < 0.05, **P < 0.01, and ***P < 0.005; NS not significant)

Fig. 4

Osteoblast-specific depletion of HIF-2α increases bone mass. a Osteoblast-specific depletion of HIF-2α in Hif-2α^fl/fl and Hif-2α^fl/fl;Col1a1-Cre mice was determined by immunohistochemistry with anti-HIF-2α antibody. Scale bar: 10 μm b, c Analyses of femoral trabecular bones from 4-month-old Hif-2α^fl/fl and Hif-2α^fl/fl;Col1a1-Cre mice. Representative images of µCT reconstructions of trabecular bones (b) and H&E and TRAP staining (c). BV/TV, Tb.Th, Tb.Sp, and Tb.N were assessed based on the µCT measurements (n = 8; b), and BV/TV, N.Ob/B.Pm, Ob.S/BS, N.Oc/B.Pm, and Oc.S/BS were determined from the bone histomorphometric analysis of the metaphyseal regions of femurs (n = 8; c). Scale bar: 100 μm. d, e Quantitative µCT analysis of femoral trabecular bones (n = 8; d) and ELISA-based measurement of the serum concentrations of OCN (n = 5; e) and CTX-1 (n = 9; e) in OVX- or sham-operated 3-month-old Hif-2α^fl/fl and Hif-2α^fl/fl;Col1a1-Cre mice. f, g Osteoblast differentiation was validated in primary calvarial preosteoblasts from Hif-2α^fl/fl mice infected with Ad-C or Ad-Cre in the presence of differentiation medium. Osteoblast differentiation was examined by ALP and ARS staining (f), and its corresponding gene expression was determined by qRT-PCR (n = 4; g). Values are presented as the mean ± SEM (*P < 0.05; **P < 0.01, and ***P < 0.005). The effects of OVX and osteoblast-specific deletion of Hif-2α (cΚΟ) as well as their interaction in mice were analyzed by two-way ANOVA (d BV/TV: interaction = 0.034 1, OVX < 0.000 1, cΚΟ < 0.000 1; e OCN: interaction < 0.000 1, OVX < 0.000 1, cΚΟ < 0.000 1; e CTX-1: interaction = 0.041 4, OVX < 0.000 1, cΚΟ = 0.046 1)

Fig. 5

HIF-2α upregulation stimulates osteoclast differentiation. a The mRNA levels of osteoclast-related genes during the M-CSF/RANKL-induced osteoclastogenesis of BMMs (n = 4). b Protein levels of HIF-1α and HIF-2α in BMMs cultured with or without 100 ng·mL⁻¹ RANKL for 4 days under normoxia or hypoxia were examined by western blot analysis (left panel) and quantified by ImageJ (right panel) (n = 4). c TRAP staining and immunohistochemical staining of HIF-2α in serial paraffin sections of femoral trabecular bones from OVX-operated mice. Scale bar: 10 μm d–g BMMs were infected with Ad-C or Ad-Hif-2α and then cultured with M-CSF and RANKL for 5 days. TRAP staining and quantitative analysis of multinucleated cells are shown (n = 6) (d); mineral resorption pits in differentiated osteoclasts, as quantified by fluorescence intensity (n = 4) (e); F-actin ring formation (n = 3; f); and western blotting of HIF-2α and qRT-PCR analysis of Hif-2α and the osteoclast-related genes Trap, Ctsk, Nfatc1, Dcstamp, and Ocstamp (n ≥ 4; g). Scale bar: 100 μm. Values are presented as the mean ± SEM (*P < 0.05, **P < 0.01, and ***P < 0.005)

Fig. 6

HIF-2α promotes osteoclast function by upregulating TRAF6 expression. a–d BMMs isolated from 8-week-old Hif-2α^+/+ and Hif-2α^+/− mice were subjected to M-CSF/RANKL-induced differentiation in vitro. TRAP staining and quantitative analysis of multinucleated cells are shown (n = 4; a); mineral resorption pits (n = 4; b); F-actin ring formation (n = 4; c); and western blotting of HIF-2α / qRT-PCR analysis of Hif-2α and osteoclast-related genes (Trap, Ctsk, Nfatc1, Dcstamp, and Ocstamp) (n ≥ 4; d). e, f TRAP staining and quantification of multinucleated cells were performed in BMMs treated with 2.5 or 5 μmol·L⁻¹ ZINC04179524, a potent inhibitor of HIF-2α, during RANKL-mediated osteoclast differentiation (n = 4; e), and the RNA expression levels of osteoclast-related genes were analyzed by qRT-PCR (n = 4; f). g Traf6 expression was determined by qRT-PCR during the RANKL-mediated osteoclastogenesis of BMMs in Ad-Hif-2α-infected BMMs isolated from WT mice, in BMMs isolated from Hif-2α^+/− mice, and in BMMs treated with ZINC04179524 (n = 4). h BMMs infected with Ad-C or Ad-Hif-2α with or without 30 μmol·L⁻¹ T6DP were analyzed by TRAP staining and quantification of multinucleated cell numbers (n = 3). i A ChIP assay of Ad-Hif-2α-infected osteoclasts was performed with anti-HIF-2α antibody and a primer pair designed to span the putative HIF-2α binding regions within the promoter of Traf6. j TRAF6 immunostaining in osteoclasts from the bone tissue of Hif-2α^+/− and WT mice. k Mice calvaria were injected with RANKL (5 µg) and coinjected with 100 μmol·L⁻¹ ZINC04179524 or 100 μmol·L⁻¹ T6DP in the presence of Ad-Hif-2α (1 × 10⁹ CFU). Calvaria bone resorption was detected by μCT analysis and TRAP staining and quantified by bone volume measurement. Values are presented as the mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.005). Scale bar: 100 μm

Fig. 7

Osteoclast-specific depletion of HIF-2α increases bone mass. a Osteoclast-specific depletion of HIF-2α in Hif-2α^fl/fl and Hif-2α^fl/fl;Ctsk-Cre mice was determined by immunohistochemistry with anti-HIF-2α antibody (n = 3; scale bar: 10 μm). b, c Analysis of femoral trabecular bones from 4-month-old Hif-2α^fl/fl and Hif-2α^fl/fl;Ctsk-Cre mice. Quantitative µCT analysis of trabecular bones (n = 8; b), H&E and TRAP staining and bone histomorphometric analysis (n = 8; c). Scale bar: 100 μm. d, e Quantitative µCT analysis (n = 8; d) and measurement of serum OCN and CTX-1 concentrations (n = 6; e) in OVX or sham-operated Hif-2α^fl/fl and Hif-2α^fl/fl;Ctsk-Cre mice. f Schematic diagram depicting HIF-2α regulation of bone remodeling. Values are presented as the mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.005; ‘NS’ not significant). Scale bar: 100 μm. The effects of OVX and osteoclast-specific depletion of Hif-2α (cKO) as well as their interaction in mice were analyzed by two-way ANOVA (d BV/TV: interaction = 0.001 8, OVX < 0.000 1, cKO < 0.000 1; e OCN: interaction = 0.743 6, OVX < 0.000 1, cKO = 0.557 7; e CTX-1: interaction = 0.045 0, OVX < 0.000 1, cKO = 0.002 5)