Oxygen-sensing PHDs regulate bone homeostasis through the modulation of osteoprotegerin

Abstract

The bone microenvironment is composed of niches that house cells across variable oxygen tensions. However, the contribution of oxygen gradients in regulating bone and blood homeostasis remains unknown. Here, we generated mice with either single or combined genetic inactivation of the critical oxygen-sensing prolyl hydroxylase (PHD) enzymes (PHD1-3) in osteoprogenitors. Hypoxia-inducible factor (HIF) activation associated with Phd2 and Phd3 inactivation drove bone accumulation by modulating osteoblastic/osteoclastic cross-talk through the direct regulation of osteoprotegerin (OPG). In contrast, combined inactivation of Phd1, Phd2, and Phd3 resulted in extreme HIF signaling, leading to polycythemia and excessive bone accumulation by overstimulating angiogenic-osteogenic coupling. We also demonstrate that genetic ablation of Phd2 and Phd3 was sufficient to protect ovariectomized mice against bone loss without disrupting hematopoietic homeostasis. Importantly, we identify OPG as a HIF target gene capable of directing osteoblast-mediated osteoclastogenesis to regulate bone homeostasis. Here, we show that coordinated activation of specific PHD isoforms fine-tunes the osteoblastic response to hypoxia, thereby directing two important aspects of bone physiology: cross-talk between osteoblasts and osteoclasts and angiogenic-osteogenic coupling.

Keywords: HIF signaling; bone homeostasis; hypoxia; osteoprotegerin; oxygen sensing; prolyl hydroxylase.

Figure 1.

Differential regulation of osteoblastic HIF signaling through selective inactivation of PHDs. (A) Representative PIMO staining of tibial sections of mice at 8 wk of age. Arrowheads point to osteoblasts at the endosteal bone surface and osteocytes. Positively stained cells are marked with black arrowheads, and negative cells are marked with open arrowheads. (B) PIMO staining of a mouse kidney. (C) Quantification of PIMO-stained osteocytes and endosteal osteoblasts form tibia isolated from a wild-type mouse. (D) Real-time PCR analysis of runx2 (RUNX), osterix (OSX), collagen1α (Col1a), and osteocalcin (OCN) mRNA expression in calvarial osteoblasts isolated from mice at postnatal days 0 and 7 and at 4 wk of age. (E) Western blot analysis of whole-cell lysates isolated from calvarial osteoblasts at postnatal days 0 and 7 and at 4 wk of age. (F) Real-time PCR analysis of EPO, VEGF, and PGK mRNA expression in bone tissues isolated from littermate control mice (C) and OSX-Cre mutant mice. For each group analyzed, n = 4. (UD) Undetectable levels. (G,H) Immunohistochemical analysis of HIF-1α (G, top) and HIF-2α (G, bottom) expression in osteoblasts lining the endosteal surface and in osteocytes (H) in OSX-Cre mutant tibias. Arrowheads point to osteoblasts and osteocytes expressing HIF-1α or HIF-2α.

Figure 2.

Selective inactivation of specific PHD isoform combinations increases trabecular bone volume without disrupting blood homeostasis. (A) Representative μCT images of distal femurs isolated from 12-wk-old control (CNTRL) and OSX-Cre mutant mice (axial view of metaphyseal region). (B) Representative images of H&E-stained proximal tibias isolated from 12-wk-old control (CNTRL) and OSX-Cre mutant mice. (C) μCT analysis of trabecular bone volume (BV/TV) and trabecular number (Tb.N) from 12-wk-old control (C) and OSX-Cre mutant mice. For each group analyzed, OSX-Cre; Phd1^fl/fl; Phd2^fl/fl; Phd3^fl/fl (PH1/2/3), n = 6; OSX-Cre; Phd2^fl/fl; Phd3^fl/fl (PH2/3), n = 8; OSX-Cre; Phd1^fl/fl; Phd2^fl/fl (PH1/2), n = 7; and OSX-Cre; Phd1^fl/fl; Phd3^fl/fl (PH1/3), n = 7. (D) μCT analysis of cortical bone thickness (Ct.Th) of 12-wk-old control (C) and OSX-Cre mutant mice. For each group analyzed, OSX-Cre; Phd1^fl/fl; Phd2^fl/fl; Phd3^fl/fl(PH1/2/3), n = 6; OSX-Cre; Phd2^fl/fl; Phd3^fl/fl(PH2/3), n = 8; OSX-Cre; Phd1^fl/fl; Phd2^fl/fl (PH1/2), n = 7; and OSX-Cre; Phd1^fl/fl; Phd3^fl/fl (PH1/3), n = 4. (E) Percentage of hematocrit (HCT) in control (C) and OSX-Cre mutant mice at 12 wk of age. For each group analyzed, OSX-Cre; Phd1^fl/fl; Phd2^fl/fl; Phd3^fl/fl(PH1/2/3), n = 4; OSX-Cre; Phd2^fl/fl; Phd3^fl/fl(PH2/3), n = 5; OSX-Cre; Phd1^fl/fl; Phd2^fl/fl (PH1/2), n = 5; and OSX-Cre; Phd1^fl/fl; Phd3^fl/fl (PH1/3), n = 5. All data are represented as mean ± standard error of the mean (SEM). (*) P < 0.05; (**) P < 0.01; (***) P < 0.001, determined by Student's t-test.

Figure 3.

Osteoblastic inactivation of PHD2 and PHD3 inhibits osteoclastogenesis. (A) Representative images of CD31 immunohistochemistry to visualize vascularization in control (CNTRL) and OSX-Cre PHD mutant mice. Blood vessels are indicated with black arrowheads. (B, top) Quantification of blood vessels in bones in 12-wk-old control and OSX-Cre PHD mutant mice. (Bottom) Analysis of VEGF protein levels in the serum of 12-wk-old control (C) and OSX-Cre PHD mutant mice. (C, left) Representative images of serial sections stained for CD31 and endomucin. Colocalized staining of blood vessels is indicated with black arrowheads. (Right) Quantification of CD31^high/endomucin^high blood vessels in 8-wk-old control and mutant mice. (D) Representative images of Alcian blue-stained control (CNTRL) and OSX-Cre; Phd2^fl/fl; Phd3^fl/fl (PH2/3) mutant proximal tibias isolated from 12-wk-old mice. The arrowheads point to unremodeled cartilage. (E) Representative images of tartrate-resistant acid phosphatase (TRAP)-stained proximal tibias isolated from 12-wk-old control and mutant mice. The arrowheads point to TRAP-positive osteoclasts. (F) Quantification of TRAP-positive multinucleated osteoclasts in proximal tibias. The number of trabecular and endosteal osteoclasts per bone surface (trabecular N.Oc./BS, n = 6; endosteal N.Oc/BS, n = 4) is shown. (G) Real-time PCR analysis of OPG and RANKL mRNA in bone tissues isolated from 12-wk-old mice. For each group analyzed, n = 4. (H) Real-time PCR analysis of OPG and RANKL mRNA isolated from primary calvarial cultures. For each group analyzed, n = 4. (I) Analysis of OPG and RANKL protein levels in the serum of 12-wk-old mice as determined by ELISA. For each group analyzed, n = 4. (J, left) Representative image of dual calcein-labeled femurs from 12-wk-old mice. (Right) Assessment of the mineral apposition rate (MAR) and bone formation rate per bone surface (BFR/BS) by quantification of dual calcein-labeled sections of distal femur from 12-wk-old mice. For each group analyzed, n = 6. (K) Quantification of trabecular osteoblast numbers per bone surface (n = 3). (L) Quantification of TRAP-positive colonies from cocultures of wild-type bone marrow stromal cells and primary osteoblast cultures isolated from PHD2^fl/fl; PHD 3^fl/fl treated with either control or adeno-CRE virus (n = 3). (M) Analysis of secreted OPG and RANKL protein levels from coculture experiments as determined by ELISA (n = 3). All data are represented as mean ± standard error of the mean (SEM). (*) P < 0.05; (**) P < 0.01; (***) P < 0.001, determined by Student's t-test.

Figure 4.

HIF regulates osteoblastic OPG to modulate osteoclastogenesis. (A) Real-time PCR analysis of OPG and VEGF expression in MC3T3 E1 cells exposed to 21% oxygen (Nx) or 0.5% oxygen (Hx). (B) Real-time PCR analysis of OPG expression of MC3T3 E1 cells transfected with constitutively active variants of HIF-1α (HIF-1dPA) or HIF-2α (HIF-2dPA). (C, top) Schematic representation of the mouse Tnfrsf11b (OPG) promoter upstream of the transcriptional start site (TSS). Four potential HIF-binding sites (hypoxia-responsive element [HREs]) are indicated with white boxes. (Bottom) Chromatin immunoprecipitation (ChIP) assay analysis of HIF-2 binding to HREs located within the OPG promoter in MC3T3 E1 cells transfected with a nondegradable HA-tagged variant of HIF-2α. HIF-2α occupancy on the VEGF promoter HRE was used as the positive control. (D) Real-time PCR analysis of OPG mRNA from whole-bone homogenates of 12-wk-old mice. For each group analyzed, n = 3. (E) Analysis of OPG protein levels in the serum of 12-wk-old mice as determined by ELISA. For each group analyzed, n = 4. (F) Representative images of TRAP-stained proximal tibias isolated from 12-wk-old mice. (G,H) Quantification of TRAP-positive multinucleated trabecular (G; n = 5) and endosteal (H; n = 3) osteoclasts in proximal tibias by analyzing the number of osteoclasts per bone surface (N.Oc./BS). (I, top) Representative μCT images of distal femurs (axial view of metaphyseal region) isolated from 12-wk-old mice. (Bottom) Representative images from H&E staining of proximal tibias isolated from 12-wk-old mice. (J) μCT analysis of distal femurs of trabecular bone volume (BV/TV) and trabecular number (Tb.N) from 12-wk-old mice. (CNTRL) Control; (HIF1dPA) OSX-Cre; Hif1dPA; (HIF2dPA) OSX-Cre; HIf2dPA. (K, top) Representative μCT images of distal femurs (axial view of metaphyseal region) isolated from 12-wk-old mice. (Bottom) Representative H&E images of proximal tibias isolated from control (CNTRL) and OSX-Cre; Phd2^fl/fl; Phd3^fl/fl; HIF2α^fl/fl (PHD2/3/H2) mice. (L) μCT analysis of trabecular bone volume (BV/TV) and trabecular number (Tb.N) from 12-wk-old mice isolated from control (C) and OSX-Cre; Phd2^fl/fl; Phd3^fl/fl; HIF2α^fl/fl (PHD2/3/H2) mice. For each group analyzed, n = 5. All data are represented as mean ± standard error of the mean (SEM). (*) P < 0.05; (**) P < 0.01; (***) P < 0.001, determined by Student's t-test. Note: Data shown from CNTRL and PHD2/3 mice are taken from Figure 2.

Figure 5.

Loss of both HIF-1α and HIF-2α is necessary to alter osteoclastogenesis. (A,B) Real-time PCR analysis of PGK (left) and OPG (right) mRNA expression in primary osteoblast cultures isolated from Hif-1α^fl/fl (HIF-1fl/fl) or HIF-2α^fl/fl (HIF-2fl/fl) mice exposed to hypoxia (0.5% oxygen) and infected with adenovirus to drive expression of Cre-recombinase. (C,D) Real-time PCR analysis of OPG mRNA from whole-bone homogenates (C) and quantification of TRAP-positive multinucleated trabecular and endosteal osteoclasts (D) in proximal tibias of 12-wk-old OSX-Cre; Hif-1α^fl/fl (n = 3). mice (E,F) Real-time PCR analysis of OPG mRNA from whole-bone homogenates (E) and quantification of TRAP-positive multinucleated trabecular and endosteal osteoclasts (F) in proximal tibias of 12-wk-old OSX-Cre; Hif-2α^fl/fl mice (n = 3). (G) Representative images of TRAP-stained proximal tibias. TRAP-positive cells are stained in purple. (H) Real-time PCR analysis of OPG mRNA in bone tissues isolated from 12-wk-old mice (n = 3). (I) Quantification of TRAP-positive trabecular and endosteal multinucleated osteoclasts in proximal tibias of 12-wk-old mice (n = 3). All data are represented as mean ± standard error of the mean (SEM). (**) P < 0.01; (*) P < 0.05, determined by Student's t-test.

Figure 6.

Osteoblastic inactivation of PHD2 and PHD3 modulates OPG to protect mice against bone loss. (A) Representative μCT images of distal femurs (axial view of metaphyseal region) isolated from sham-operated (SHAM) and ovariectomized (OVX) control (CNTRL) and OSX-Cre; Phd2^fl/fl; Phd3^fl/fl (PH2/3) mice. (B) Representative images of H&E-stained proximal tibias isolated from sham-operated (SHAM) and ovariectomized (OVX) control (CNTRL) and OSX-Cre; Phd2^fl/fl; Phd3^fl/fl (PH2/3) mice. (C) μCT analysis of trabecular bone volume (BV/TV) and trabecular number (Tb.N) for sham-operated (SHAM) and ovariectomized (OVX) control (CNTRL) and OSX-Cre; Phd2^fl/fl; Phd3^fl/fl (PH2/3) mice. For each group analyzed, n = 9. (D) Representative images of TRAP-stained proximal tibias. The arrowheads point to TRAP-positive osteoclasts. (E) Quantification of TRAP-positive multinucleated osteoclasts in proximal tibias of sham-operated (SHAM) and ovariectomized (OVX) control (CNTRL) and OSX-Cre; Phd2^fl/fl; Phd3^fl/fl (PHD2/3) mice. (N.Oc./BS) Number of osteoclasts per bone surface. (F) Analysis of OPG protein levels in the serum of sham-operated (SHAM) or ovariectomized (OVX) control (CNTRL) and mutant mice as determined by ELISA (n = 6). For all experiments, operations were performed at 12 wk of age, and 6 wk post-surgery bone parameters were assessed. Data are represented as mean ± standard error of the mean (SEM). (**) P < 0.01; (****) P < 0.0001, as determined by Student's t-test.

Figure 7.

Selective inactivation of PHDs regulates HIF signaling dosage to differentially modulate bone homeostasis. Selective inactivation of PHDs modulates the dosage of HIF signaling with functional consequences for bone and hematopoietic homeostasis. We show that, by acting as a molecular rheostat of HIF activation, genetic inhibition of specific PHD isoform combinations differentially regulates two important aspects of bone physiology: cross-talk between osteoblasts and osteoclasts and angiogenic/osteogenic coupling. In conditions where genetic inactivation of three PHD isoforms occurs, robust HIF activation ensues, leading to enhanced angiogenesis that results in extreme ossification of trabecular bones and aberrant hematopoiesis. In contrast, the loss of specific dual combinations of PHD isoforms does not alter microvascular density or hematopoietic homeostasis but does enhance trabecular bone volume. This enhancement is due to HIF-mediated suppression of osteoblast-mediated osteoclastogenesis through the modulation of OPG.