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Cited by 126Bone Metabolism and DiseasesBone health and osteoporosis researchAlkaline Phosphatase Research Studies

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Osteocytic oxygen sensing controls bone mass through epigenetic regulation of sclerostin

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Stegen, Steve, Stockmans, Ingrid, Moermans, Karen, Thienpont, Bernard, Maxwell, Patrick H., Carmeliet, Peter, Carmeliet, Geert·2018·Nature Communications
DOI 10.1038/s41467-018-04679-7PMC PMC6028485
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Abstract

Preservation of bone mass is crucial for healthy ageing and largely depends on adequate responses of matrix-embedded osteocytes. These cells control bone formation and resorption concurrently by secreting the WNT/β-catenin antagonist sclerostin (SOST). Osteocytes reside within a low oxygen microenvironment, but whether and how oxygen sensing regulates their function remains elusive. Here, we show that conditional deletion of the oxygen sensor prolyl hydroxylase (PHD) 2 in osteocytes results in a high bone mass phenotype, which is caused by increased bone formation and decreased resorption. Mechanistically, enhanced HIF-1α signalling increases Sirtuin 1-dependent deacetylation of the Sost promoter, resulting in decreased sclerostin expression and enhanced WNT/β-catenin signalling. Additionally, genetic ablation of PHD2 in osteocytes blunts osteoporotic bone loss induced by oestrogen deficiency or mechanical unloading. Thus, oxygen sensing by PHD2 in osteocytes negatively regulates bone mass through epigenetic regulation of sclerostin and targeting PHD2 elicits an osteo-anabolic response in osteoporotic models.

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Figures

Fig. 1 Deletion of PHD2 in osteocytes increases bone mass. a PHD2 and β-actin immunoblot on whole-cell extracts isolated from osteocyte-enriched bone fractions of 8-week-old mice. Results are representative of three experiments. b HIF-1α, HIF-2α and Lamin A/C immunoblot on nuclear cell extracts isolated from osteocyte-enriched bone fractions. Results are representative of three experiments. c, d 3D microCT models (c) of the tibial metaphysis and quantification (d) of trabecular bone volume (BV/TV) and cortical thickness (Ct.Th) in 8-week-old mice (n = 8 Phd2ot+–10 Phd2ot−). e, f Von Kossa staining (e) of tibiae with quantification (f) of trabecular bone volume (BV/TV) (n = 8 Phd2ot+–10 Phd2ot−). Scale bar is 500 µm. Data are means ± SEM. *p < 0.05 vs. Phd2ot+, **p < 0.01 vs. Phd2ot+ (Student′s t-test)
Fig. 1 Deletion of PHD2 in osteocytes increases bone mass. a PHD2 and β-actin immunoblot on whole-cell extracts isolated from osteocyte-enriched bone fractions of 8-week-old mice. Results are representative of three experiments. b HIF-1α, HIF-2α and Lamin A/C immunoblot on nuclear cell extracts isolated from osteocyte-enriched bone fractions. Results are representative of three experiments. c, d 3D microCT models (c) of the tibial metaphysis and quantification (d) of trabecular bone volume (BV/TV) and cortical thickness (Ct.Th) in 8-week-old mice (n = 8 Phd2ot+–10 Phd2ot−). e, f Von Kossa staining (e) of tibiae with quantification (f) of trabecular bone volume (BV/TV) (n = 8 Phd2ot+–10 Phd2ot−). Scale bar is 500 µm. Data are means ± SEM. *p < 0.05 vs. Phd2ot+, **p < 0.01 vs. Phd2ot+ (Student′s t-test)CC BY · PubMed Central
Fig. 2 Bone formation exceeds bone resorption in Phd2ot− mice. a, b H&E staining (a) of the tibial metaphysis with quantification (b) of osteoblast number per bone surface (N.Ob/B.S) and osteoblast surface per bone surface (Ob.S/B.S) in 8-week-old mice (n = 8 Phd2ot+–10 Phd2ot−). White arrowheads in a indicate osteoblasts. c, d H&E staining (c) of the cortical diaphysis of tibiae with quantification (d) of osteocyte number per bone area (N.Ot/B.Ar) and percentage of empty osteocyte lacunae (n = 8 Phd2ot+–10 Phd2ot−). e, f Calcein labelling of mineralizing surfaces on trabeculae (e) with quantification (f) of the bone formation rate (BFR) and mineral apposition rate (MAR) (n = 8 Phd2ot+–10 Phd2ot−). White arrowheads in e indicate calcein incorporation. g Serum osteocalcin levels (n = 8 Phd2ot+–10 Phd2ot−). h, i TRAP staining (h) of the tibial metaphysis with quantification (i) of osteoclast surface per bone surface (Oc.S/B.S) (n = 8 Phd2ot+–10 Phd2ot−). Black arrowheads in h indicate osteoclasts. j Serum CTx-I levels (n = 8 Phd2ot+ –10 Phd2ot-). Data are means ± SEM. *p < 0.05 vs. Phd2ot+, **p < 0.01 vs. Phd2ot+, N.S. is not significant (Student′s t-test). Scale bars in a, e and h are 50 µm, scale bar in c is 100 µm
Fig. 2 Bone formation exceeds bone resorption in Phd2ot− mice. a, b H&E staining (a) of the tibial metaphysis with quantification (b) of osteoblast number per bone surface (N.Ob/B.S) and osteoblast surface per bone surface (Ob.S/B.S) in 8-week-old mice (n = 8 Phd2ot+–10 Phd2ot−). White arrowheads in a indicate osteoblasts. c, d H&E staining (c) of the cortical diaphysis of tibiae with quantification (d) of osteocyte number per bone area (N.Ot/B.Ar) and percentage of empty osteocyte lacunae (n = 8 Phd2ot+–10 Phd2ot−). e, f Calcein labelling of mineralizing surfaces on trabeculae (e) with quantification (f) of the bone formation rate (BFR) and mineral apposition rate (MAR) (n = 8 Phd2ot+–10 Phd2ot−). White arrowheads in e indicate calcein incorporation. g Serum osteocalcin levels (n = 8 Phd2ot+–10 Phd2ot−). h, i TRAP staining (h) of the tibial metaphysis with quantification (i) of osteoclast surface per bone surface (Oc.S/B.S) (n = 8 Phd2ot+–10 Phd2ot−). Black arrowheads in h indicate osteoclasts. j Serum CTx-I levels (n = 8 Phd2ot+ –10 Phd2ot-). Data are means ± SEM. *p < 0.05 vs. Phd2ot+, **p < 0.01 vs. Phd2ot+, N.S. is not significant (Student′s t-test). Scale bars in a, e and h are 50 µm, scale bar in c is 100 µmCC BY · PubMed Central
Fig. 3 PHD2 deletion reduces sclerostin expression through SIRT1. a Phex, Dmp1 and Sost mRNA levels in osteocyte-enriched bone fractions of 8-week-old mice (n = 8 Phd2ot+–10 Phd2ot−). b Sclerostin and β-actin immunoblot on whole-cell extracts isolated from osteocyte-enriched bone fractions. c Non-phosphorylated (non-phospho) β-catenin and Lamin A/C immunoblot (nuclear cell extracts), and total β-catenin and β-actin immunoblot (whole-cell extracts). Protein extracts are isolated from osteocyte-enriched bone fractions. d Axin2, Dkk-4, Lef-1 and Tcf-1 mRNA levels in osteocyte-enriched bone fractions (n = 8 Phd2ot+– 10 Phd2ot−). e Sirtuin 1 and Lamin A/C immunoblot on nuclear cell extracts isolated from osteocyte-enriched bone fractions. f Sirtuin 1, non-phospho β-catenin, Lamin A/C, sclerostin and β-actin immunoblot isolated from vehicle (IDGVEH) or IOX2-treated IDG-SW3 (IDGIOX2) cells after 14 days of osteogenic differentiation. IDGVEH cells were treated with vehicle (−) or SRT1720 (SRT); IDGIOX2 cells were treated with vehicle (−) or EX527 (EX). g Sirtuin 1, non-phospho β-catenin and Lamin A/C immunoblot on nuclear cell extracts isolated from IDGVEH or IDGIOX2 cells after 14 days of osteogenic differentiation in the presence of recombinant sclerostin. Cells were treated as in f. Data are means ± SEM. Immunoblot images are representative of three experiments. *p < 0.05 vs. Phd2ot+, **p < 0.01 vs. Phd2ot+ (Student′s t-test)
Fig. 3 PHD2 deletion reduces sclerostin expression through SIRT1. a Phex, Dmp1 and Sost mRNA levels in osteocyte-enriched bone fractions of 8-week-old mice (n = 8 Phd2ot+–10 Phd2ot−). b Sclerostin and β-actin immunoblot on whole-cell extracts isolated from osteocyte-enriched bone fractions. c Non-phosphorylated (non-phospho) β-catenin and Lamin A/C immunoblot (nuclear cell extracts), and total β-catenin and β-actin immunoblot (whole-cell extracts). Protein extracts are isolated from osteocyte-enriched bone fractions. d Axin2, Dkk-4, Lef-1 and Tcf-1 mRNA levels in osteocyte-enriched bone fractions (n = 8 Phd2ot+– 10 Phd2ot−). e Sirtuin 1 and Lamin A/C immunoblot on nuclear cell extracts isolated from osteocyte-enriched bone fractions. f Sirtuin 1, non-phospho β-catenin, Lamin A/C, sclerostin and β-actin immunoblot isolated from vehicle (IDGVEH) or IOX2-treated IDG-SW3 (IDGIOX2) cells after 14 days of osteogenic differentiation. IDGVEH cells were treated with vehicle (−) or SRT1720 (SRT); IDGIOX2 cells were treated with vehicle (−) or EX527 (EX). g Sirtuin 1, non-phospho β-catenin and Lamin A/C immunoblot on nuclear cell extracts isolated from IDGVEH or IDGIOX2 cells after 14 days of osteogenic differentiation in the presence of recombinant sclerostin. Cells were treated as in f. Data are means ± SEM. Immunoblot images are representative of three experiments. *p < 0.05 vs. Phd2ot+, **p < 0.01 vs. Phd2ot+ (Student′s t-test)CC BY · PubMed Central
Fig. 4 Decreased sclerostin affects osteoblasts and osteoclasts. a Alizarin Red staining of IDGVEH or IDGIOX2 cells after 14 or 21 days of osteogenic differentiation (n = 3). IDGVEH cells were treated with vehicle (−) or SRT1720 (SRT); IDGIOX2 cells were treated with vehicle (−) or EX527 (EX). b Alizarin Red staining of IDGVEH or IDGIOX2 cells after 14 days of osteogenic differentiation in the presence of recombinant sclerostin (n = 3). Cells were treated as in a. c Rankl and Opg mRNA levels in osteocyte-enriched bone fractions of 8-week-old mice (n = 8 Phd2ot+–10 Phd2ot-). d RANKL, OPG and β-actin immunoblot on whole-cell extracts isolated from osteocyte-enriched bone fractions. e RANKL, OPG and β-actin immunoblot on whole-cell extracts isolated from IDGVEH or IDGIOX2 cells after 14 days of osteogenic differentiation. Cells were treated as in a. f Quantification of TRAP-positive multinuclear cells (MNC), formed after co-culturing wild-type hematopoietic cells with IDG-SW3 cells, in which Phd2 was deleted or not via CRISPR-Cas9 (n = 3). g Rankl/Opg mRNA ratio in IDGVEH or IDGIOX2 cells after 14 days of osteogenic differentiation in the presence of recombinant sclerostin (n = 3). Cells were treated as in a. Data are means ± SEM. Immunoblot images are representative of three experiments. *p < 0.05 vs. Phd2ot+ (Student′s t-test), #p < 0.05 (two-way ANOVA)
Fig. 4 Decreased sclerostin affects osteoblasts and osteoclasts. a Alizarin Red staining of IDGVEH or IDGIOX2 cells after 14 or 21 days of osteogenic differentiation (n = 3). IDGVEH cells were treated with vehicle (−) or SRT1720 (SRT); IDGIOX2 cells were treated with vehicle (−) or EX527 (EX). b Alizarin Red staining of IDGVEH or IDGIOX2 cells after 14 days of osteogenic differentiation in the presence of recombinant sclerostin (n = 3). Cells were treated as in a. c Rankl and Opg mRNA levels in osteocyte-enriched bone fractions of 8-week-old mice (n = 8 Phd2ot+–10 Phd2ot-). d RANKL, OPG and β-actin immunoblot on whole-cell extracts isolated from osteocyte-enriched bone fractions. e RANKL, OPG and β-actin immunoblot on whole-cell extracts isolated from IDGVEH or IDGIOX2 cells after 14 days of osteogenic differentiation. Cells were treated as in a. f Quantification of TRAP-positive multinuclear cells (MNC), formed after co-culturing wild-type hematopoietic cells with IDG-SW3 cells, in which Phd2 was deleted or not via CRISPR-Cas9 (n = 3). g Rankl/Opg mRNA ratio in IDGVEH or IDGIOX2 cells after 14 days of osteogenic differentiation in the presence of recombinant sclerostin (n = 3). Cells were treated as in a. Data are means ± SEM. Immunoblot images are representative of three experiments. *p < 0.05 vs. Phd2ot+ (Student′s t-test), #p < 0.05 (two-way ANOVA)CC BY · PubMed Central
Fig. 5 SIRT1 regulates bone mass in Phd2ot− mice. a–c 3D microCT models of the tibial metaphysis (a) and quantification of trabecular bone volume (BV/TV; b) and cortical thickness (Ct.Th; c) in 8-week-old mice (n = 5 Phd2ot+–8 Phd2ot−). Mice were treated with vehicle, SRT1720 or EX527 for 5 weeks. d, e Calcein labelling of trabecular mineralizing surfaces (d) with quantification (e) of the bone formation rate (BFR) (n = 5 Phd2ot+–8 Phd2ot−). White arrowheads in d indicate calcein incorporation. f Serum osteocalcin levels (n = 5 Phd2ot+–8 Phd2ot−). g, h TRAP staining (g) of the tibial metaphysis with quantification (h) of the osteoclast surface per bone surface (Oc.S/B.S) (n = 5 Phd2ot+–8 Phd2ot−). Black arrowheads in g indicate osteoclasts. i Rankl/Opg mRNA levels in osteocyte-enriched bone fractions (n = 5 Phd2ot+–8 Phd2ot−). j HIF-1α, Sirtuin 1, non-phosphorylated (non-phospho) β-catenin, Lamin A/C (nuclear cell extracts), and sclerostin and β-actin immunoblot (whole-cell extracts). Protein extracts are isolated from osteocyte-enriched bone fractions. Results are representative of three experiments. k–n Axin2 (k), Dkk-4 (l), Lef-1 (m) and Tcf-1 (n) mRNA levels in osteocyte-enriched bone fractions (n = 5 Phd2ot+–8 Phd2ot−). Data are means ± SEM. #p < 0.05 (two-way ANOVA). Scale bars in a and g are 50 µm
Fig. 5 SIRT1 regulates bone mass in Phd2ot− mice. a–c 3D microCT models of the tibial metaphysis (a) and quantification of trabecular bone volume (BV/TV; b) and cortical thickness (Ct.Th; c) in 8-week-old mice (n = 5 Phd2ot+–8 Phd2ot−). Mice were treated with vehicle, SRT1720 or EX527 for 5 weeks. d, e Calcein labelling of trabecular mineralizing surfaces (d) with quantification (e) of the bone formation rate (BFR) (n = 5 Phd2ot+–8 Phd2ot−). White arrowheads in d indicate calcein incorporation. f Serum osteocalcin levels (n = 5 Phd2ot+–8 Phd2ot−). g, h TRAP staining (g) of the tibial metaphysis with quantification (h) of the osteoclast surface per bone surface (Oc.S/B.S) (n = 5 Phd2ot+–8 Phd2ot−). Black arrowheads in g indicate osteoclasts. i Rankl/Opg mRNA levels in osteocyte-enriched bone fractions (n = 5 Phd2ot+–8 Phd2ot−). j HIF-1α, Sirtuin 1, non-phosphorylated (non-phospho) β-catenin, Lamin A/C (nuclear cell extracts), and sclerostin and β-actin immunoblot (whole-cell extracts). Protein extracts are isolated from osteocyte-enriched bone fractions. Results are representative of three experiments. k–n Axin2 (k), Dkk-4 (l), Lef-1 (m) and Tcf-1 (n) mRNA levels in osteocyte-enriched bone fractions (n = 5 Phd2ot+–8 Phd2ot−). Data are means ± SEM. #p < 0.05 (two-way ANOVA). Scale bars in a and g are 50 µmCC BY · PubMed Central
Fig. 6 HIF-1α controls SIRT1 expression. a HIF-1α, HIF-2α and Lamin A/C immunoblot on nuclear cell extracts derived from IDGVEH or IDGIOX2 cells after transduction with scrambled shRNA (shScr), shHIF-1α (shH1) or shHIF-2α (shH2). Results are representative of three experiments. b Sirtuin 1, non-phosphorylated (non-phospho) β-catenin, Lamin A/C (nuclear cell extracts), and sclerostin and β-actin immunoblot (whole-cell extracts). Protein extracts are derived from IDGVEH or IDGIOX2 cells after genetic silencing of HIF-1α (shH1) or HIF-2α (shH2). Results are representative of three experiments. c–f Axin2 (c), Dkk-4 (d), Lef-1 (e), Tcf-1 (f) mRNA levels in IDGVEH or IDGIOX2 cells after genetic silencing of HIF-1α (shH1) or HIF-2α (shH2) (n = 3). Data are means ± SEM. #p < 0.05 (one-way ANOVA)
Fig. 6 HIF-1α controls SIRT1 expression. a HIF-1α, HIF-2α and Lamin A/C immunoblot on nuclear cell extracts derived from IDGVEH or IDGIOX2 cells after transduction with scrambled shRNA (shScr), shHIF-1α (shH1) or shHIF-2α (shH2). Results are representative of three experiments. b Sirtuin 1, non-phosphorylated (non-phospho) β-catenin, Lamin A/C (nuclear cell extracts), and sclerostin and β-actin immunoblot (whole-cell extracts). Protein extracts are derived from IDGVEH or IDGIOX2 cells after genetic silencing of HIF-1α (shH1) or HIF-2α (shH2). Results are representative of three experiments. c–f Axin2 (c), Dkk-4 (d), Lef-1 (e), Tcf-1 (f) mRNA levels in IDGVEH or IDGIOX2 cells after genetic silencing of HIF-1α (shH1) or HIF-2α (shH2) (n = 3). Data are means ± SEM. #p < 0.05 (one-way ANOVA)CC BY · PubMed Central
Fig. 7 Deletion of PHD2 in osteocytes stimulates angiogenesis. a, b CD31 immunostaining (a) of the tibial metaphysis with quantification (b) of blood vessel number and size in 8-week-old mice (n = 8 Phd2ot+–10 Phd2ot−). c, d CD31 immunostaining (c) of the cortical diaphysis of tibiae with quantification (d) of blood vessel number and size (n = 8 Phd2ot+–10 Phd2ot−). e Vegf and Plgf mRNA levels in femora of 8-week-old mice (n = 8 Phd2ot+–10 Phd2ot−) and in vehicle (IDGVEH) or IOX2-treated IDG-SW3 (IDGIOX2) cells (n = 3). f Scheme of experimental design. HIF-1α (shH1) or HIF-2α (shH2) were silenced in IDGIOX2 cells and a scrambled shRNA (shScr) was used as control. Conditioned medium was subsequently used to culture HUVECs in monoculture or in co-culture with IDG-SW3 cells. g Proliferation of HUVECs in monoculture according to the scheme in f (n = 3). h, i Human CD31 (hCD31) immunostaining (h) and quantification (i) of hCD31-positive surface in HUVECs co-cultured with IDG-SW3 cells, according to the scheme in f. When indicated, cells were treated with anti-VEGF164 antibody or recombinant mouse soluble VEGFR-1 (sFlt) (n = 3). Data are means ± SEM. *p < 0.05 vs. Phd2ot+ or IDGVEH, **p < 0.01 vs. Phd2ot+ or IDGVEH, ***p < 0.01 vs. Phd2ot+ or IDGVEH (Student′s t-test), #p < 0.05 (ANOVA: one-way ANOVA in g; two-way ANOVA in i). Scale bars in a and c are 100 µm, scale bar in h is 500 µm
Fig. 7 Deletion of PHD2 in osteocytes stimulates angiogenesis. a, b CD31 immunostaining (a) of the tibial metaphysis with quantification (b) of blood vessel number and size in 8-week-old mice (n = 8 Phd2ot+–10 Phd2ot−). c, d CD31 immunostaining (c) of the cortical diaphysis of tibiae with quantification (d) of blood vessel number and size (n = 8 Phd2ot+–10 Phd2ot−). e Vegf and Plgf mRNA levels in femora of 8-week-old mice (n = 8 Phd2ot+–10 Phd2ot−) and in vehicle (IDGVEH) or IOX2-treated IDG-SW3 (IDGIOX2) cells (n = 3). f Scheme of experimental design. HIF-1α (shH1) or HIF-2α (shH2) were silenced in IDGIOX2 cells and a scrambled shRNA (shScr) was used as control. Conditioned medium was subsequently used to culture HUVECs in monoculture or in co-culture with IDG-SW3 cells. g Proliferation of HUVECs in monoculture according to the scheme in f (n = 3). h, i Human CD31 (hCD31) immunostaining (h) and quantification (i) of hCD31-positive surface in HUVECs co-cultured with IDG-SW3 cells, according to the scheme in f. When indicated, cells were treated with anti-VEGF164 antibody or recombinant mouse soluble VEGFR-1 (sFlt) (n = 3). Data are means ± SEM. *p < 0.05 vs. Phd2ot+ or IDGVEH, **p < 0.01 vs. Phd2ot+ or IDGVEH, ***p < 0.01 vs. Phd2ot+ or IDGVEH (Student′s t-test), #p < 0.05 (ANOVA: one-way ANOVA in g; two-way ANOVA in i). Scale bars in a and c are 100 µm, scale bar in h is 500 µmCC BY · PubMed Central
Fig. 8 Phd2ot− mice are protected from disuse-induced bone loss. a–c 3D microCT models of the tibial metaphysis (a) and quantification of trabecular bone volume (BV/TV) (b) and cortical thickness (Ct.Th) (c) 4 weeks after hindlimb unloading and expressed as a percentage relative to grounded mice (n = 7 Phd2ot+–8 Phd2ot−). d, e Calcein labelling of trabecular mineralizing surfaces (d) with quantification (e) of the bone formation rate (BFR) (n = 7 Phd2ot+–8 Phd2ot−). White arrowheads in d indicate calcein incorporation. f Serum osteocalcin levels (n = 7 Phd2ot+–8 Phd2ot−). g, h TRAP staining (g) of the tibial metaphysis with quantification (h) of the osteoclast surface per bone surface (Oc.S/B.S) (n = 7 Phd2ot+–8 Phd2ot−). Black arrowheads in g indicate osteoclasts. i Rankl/Opg mRNA levels in osteocyte-enriched bone fractions (n = 7 Phd2ot+–8 Phd2ot−). j HIF-1α, Sirtuin 1, non-phosphorylated (non-phospho) β-catenin, Lamin A/C (nuclear cell extracts), and sclerostin and β-actin immunoblot (whole-cell extracts). Protein extracts are isolated from osteocyte-enriched bone fractions. Results are representative of four experiments. HU is hindlimb unloading. k–n Axin2 (k), Dkk-4 (l), Lef-1 (m) and Tcf-1 (n) mRNA levels in osteocyte-enriched bone fractions (n = 7 Phd2ot+–8 Phd2ot−). Data are means ± SEM. #p < 0.05 (two-way ANOVA). Scale bars in d and g are 50 µm
Fig. 8 Phd2ot− mice are protected from disuse-induced bone loss. a–c 3D microCT models of the tibial metaphysis (a) and quantification of trabecular bone volume (BV/TV) (b) and cortical thickness (Ct.Th) (c) 4 weeks after hindlimb unloading and expressed as a percentage relative to grounded mice (n = 7 Phd2ot+–8 Phd2ot−). d, e Calcein labelling of trabecular mineralizing surfaces (d) with quantification (e) of the bone formation rate (BFR) (n = 7 Phd2ot+–8 Phd2ot−). White arrowheads in d indicate calcein incorporation. f Serum osteocalcin levels (n = 7 Phd2ot+–8 Phd2ot−). g, h TRAP staining (g) of the tibial metaphysis with quantification (h) of the osteoclast surface per bone surface (Oc.S/B.S) (n = 7 Phd2ot+–8 Phd2ot−). Black arrowheads in g indicate osteoclasts. i Rankl/Opg mRNA levels in osteocyte-enriched bone fractions (n = 7 Phd2ot+–8 Phd2ot−). j HIF-1α, Sirtuin 1, non-phosphorylated (non-phospho) β-catenin, Lamin A/C (nuclear cell extracts), and sclerostin and β-actin immunoblot (whole-cell extracts). Protein extracts are isolated from osteocyte-enriched bone fractions. Results are representative of four experiments. HU is hindlimb unloading. k–n Axin2 (k), Dkk-4 (l), Lef-1 (m) and Tcf-1 (n) mRNA levels in osteocyte-enriched bone fractions (n = 7 Phd2ot+–8 Phd2ot−). Data are means ± SEM. #p < 0.05 (two-way ANOVA). Scale bars in d and g are 50 µmCC BY · PubMed Central
Fig. 9 Phd2ot− mice are protected from OVX-induced bone loss. a–c 3D microCT models of the tibial metaphysis (a) and quantification of trabecular bone volume (BV/TV) (b) and cortical thickness (Ct.Th) (c) 4 weeks after OVX and expressed as a percentage relative to sham-operated mice (n = 7 Phd2ot+–8 Phd2ot−). d, e TRAP staining (d) of the tibial metaphysis with quantification (e) of the osteoclast surface per bone surface (Oc.S/B.S) (n = 7 Phd2ot+–8 Phd2ot−). Black arrowheads in e indicate osteoclasts. f Rankl/Opg mRNA levels in osteocyte-enriched bone fractions (n = 7 Phd2ot+–8 Phd2ot−). g, h Calcein labelling of trabecular mineralizing surfaces (g) with quantification (h) of the bone formation rate (BFR) (n = 7 Phd2ot+–8 Phd2ot−). White arrowheads in g indicate calcein incorporation. i Serum osteocalcin levels (n = 7 Phd2ot+–8 Phd2ot−). j HIF-1α, Sirtuin 1, non-phosphorylated (non-phospho) β-catenin, Lamin A/C (nuclear cell extracts) and sclerostin and β-actin immunoblot (whole-cell extracts). Protein extracts are derived from osteocyte-enriched bone fractions. Results are representative of four experiments. k–n Axin2 (k), Dkk-4 (l), Lef-1 (m) and Tcf-1 (n) mRNA levels in osteocyte-enriched bone fractions (n = 7 Phd2ot+–8 Phd2ot−). Data are means ± SEM. #p < 0.05 (two-way ANOVA). Scale bars in d and g are 50 µm
Fig. 9 Phd2ot− mice are protected from OVX-induced bone loss. a–c 3D microCT models of the tibial metaphysis (a) and quantification of trabecular bone volume (BV/TV) (b) and cortical thickness (Ct.Th) (c) 4 weeks after OVX and expressed as a percentage relative to sham-operated mice (n = 7 Phd2ot+–8 Phd2ot−). d, e TRAP staining (d) of the tibial metaphysis with quantification (e) of the osteoclast surface per bone surface (Oc.S/B.S) (n = 7 Phd2ot+–8 Phd2ot−). Black arrowheads in e indicate osteoclasts. f Rankl/Opg mRNA levels in osteocyte-enriched bone fractions (n = 7 Phd2ot+–8 Phd2ot−). g, h Calcein labelling of trabecular mineralizing surfaces (g) with quantification (h) of the bone formation rate (BFR) (n = 7 Phd2ot+–8 Phd2ot−). White arrowheads in g indicate calcein incorporation. i Serum osteocalcin levels (n = 7 Phd2ot+–8 Phd2ot−). j HIF-1α, Sirtuin 1, non-phosphorylated (non-phospho) β-catenin, Lamin A/C (nuclear cell extracts) and sclerostin and β-actin immunoblot (whole-cell extracts). Protein extracts are derived from osteocyte-enriched bone fractions. Results are representative of four experiments. k–n Axin2 (k), Dkk-4 (l), Lef-1 (m) and Tcf-1 (n) mRNA levels in osteocyte-enriched bone fractions (n = 7 Phd2ot+–8 Phd2ot−). Data are means ± SEM. #p < 0.05 (two-way ANOVA). Scale bars in d and g are 50 µmCC BY · PubMed Central

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Authors
Stegen, S., Stockmans, I., Moermans, K., Thienpont, B., Maxwell, P. H., Carmeliet, P., & Carmeliet, G.
Affiliations
  • KU Leuven (BE)
  • University of Cambridge (GB)
  • VIB-KU Leuven Center for Cancer Biology (BE)
Corresponding author
Carmeliet, Geert
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2018-07-02
Volume
9
Issue
1
Pages
2557-2557
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-018-04679-7
PMC
PMC6028485
OpenAlex
W2810241167
Cited by
126
References
64
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4.62
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journal article
Language
en
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  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Lirias (submittedVersion, other-oa)
  • Apollo (University of Cambridge) (submittedVersion, cc-by)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Apollo (cc-by)
Funders
  • Wellcome Trust (096956/Z/11/Z)
  • National Institute for Health and Care Research (NF-SI-0514-10122)
  • Fonds Wetenschappelijk Onderzoek (12H5917N, G0A4216N)
  • Vlaamse regering
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bone Metabolism and Diseases
  • Health Sciences › Medicine › Orthopedics and Sports Medicine › Bone health and osteoporosis research
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Alkaline Phosphatase Research Studies
Keywords
osteocytes, oxygen sensing, prolyl hydroxylase 2, sclerostin, HIF-1α, SIRT1, histone deacetylation, Wnt/β-catenin signaling, bone mass regulation, bone formation, bone resorption, osteoporosis, estrogen deficiency, mechanical unloading, epigenetic regulation
MeSH terms
Acetylation, Animals, Carbazoles, Cell Line, Disease Models, Animal, Female, Glycoproteins, Heterocyclic Compounds, 4 or More Rings, Humans, Male, Mice, Inbred C57BL, Mice, Transgenic, Osteocytes, Osteogenesis, Osteoporosis, Oxygen, Promoter Regions, Genetic, Bone Density, Coculture Techniques, Intercellular Signaling Peptides and Proteins, Epigenesis, Genetic, Adaptor Proteins, Signal Transducing, Hypoxia-Inducible Factor 1, alpha Subunit, Sirtuin 1, Wnt Signaling Pathway, Primary Cell Culture, Hypoxia-Inducible Factor-Proline Dioxygenases

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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