Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

HIF-1α metabolically controls collagen synthesis and modification in chondrocytes

Nature volume 565, pages 511–515 (2019)Cite this article

Subjects

Abstract

Endochondral ossification, an important process in vertebrate bone formation, is highly dependent on correct functioning of growth plate chondrocytes1. Proliferation of these cells determines longitudinal bone growth and the matrix deposited provides a scaffold for future bone formation. However, these two energy-dependent anabolic processes occur in an avascular environment1,2. In addition, the centre of the expanding growth plate becomes hypoxic, and local activation of the hypoxia-inducible transcription factor HIF-1α is necessary for chondrocyte survival by unidentified cell-intrinsic mechanisms3,4,5,6. It is unknown whether there is a requirement for restriction of HIF-1α signalling in the other regions of the growth plate and whether chondrocyte metabolism controls cell function. Here we show that prolonged HIF-1α signalling in chondrocytes leads to skeletal dysplasia by interfering with cellular bioenergetics and biosynthesis. Decreased glucose oxidation results in an energy deficit, which limits proliferation, activates the unfolded protein response and reduces collagen synthesis. However, enhanced glutamine flux increases α-ketoglutarate levels, which in turn increases proline and lysine hydroxylation on collagen. This metabolically regulated collagen modification renders the cartilaginous matrix more resistant to protease-mediated degradation and thereby increases bone mass. Thus, inappropriate HIF-1α signalling results in skeletal dysplasia caused by collagen overmodification, an effect that may also contribute to other diseases involving the extracellular matrix such as cancer and fibrosis.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Skeletal dysplasia in Phd2chon− mice.
Fig. 2: Altered collagen processing in Phd2chon− growth plates.
Fig. 3: Enhanced collagen hydroxylation relies on glutamine-dependent αKG production.
Fig. 4: Stimulating glucose oxidation in PHD2-deficient chondrocytes avoids energy deficit and restores collagen synthesis.

Similar content being viewed by others

Data availability

Source data are provided in the online version of the paper. Uncropped blots are provided in Supplementary Fig. 1. Any additional information required to interpret, replicate or build upon the findings of this study are available from the corresponding author upon reasonable request.

References

  1. Kronenberg, H. M. Developmental regulation of the growth plate. Nature 423, 332–336 (2003).

    Article  ADS  CAS  Google Scholar 

  2. Buttgereit, F. & Brand, M. D. A hierarchy of ATP-consuming processes in mammalian cells. Biochem. J. 312, 163–167 (1995).

    Article  CAS  Google Scholar 

  3. Maes, C. et al. VEGF-independent cell-autonomous functions of HIF-1α regulating oxygen consumption in fetal cartilage are critical for chondrocyte survival. J. Bone Miner. Res. 27, 596–609 (2012).

    Article  CAS  Google Scholar 

  4. Pfander, D., Cramer, T., Schipani, E. & Johnson, R. S. HIF-1α controls extracellular matrix synthesis by epiphyseal chondrocytes. J. Cell Sci. 116, 1819–1826 (2003).

    Article  CAS  Google Scholar 

  5. Aro, E. et al. Hypoxia-inducible factor-1 (HIF-1) but not HIF-2 is essential for hypoxic induction of collagen prolyl 4-hydroxylases in primary newborn mouse epiphyseal growth plate chondrocytes. J. Biol. Chem. 287, 37134–37144 (2012).

    Article  CAS  Google Scholar 

  6. Schipani, E. et al. Hypoxia in cartilage: HIF-1α is essential for chondrocyte growth arrest and survival. Genes Dev. 15, 2865–2876 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Aragones, J., Fraisl, P., Baes, M. & Carmeliet, P. Oxygen sensors at the crossroad of metabolism. Cell Metab. 9, 11–22 (2009).

    Article  CAS  Google Scholar 

  8. Nakazawa, M. S., Keith, B. & Simon, M. C. Oxygen availability and metabolic adaptations. Nat. Rev. Cancer 16, 663–673 (2016).

    Article  CAS  Google Scholar 

  9. Hochachka, P. W., Buck, L. T., Doll, C. J. & Land, S. C. Unifying theory of hypoxia tolerance: molecular/metabolic defense and rescue mechanisms for surviving oxygen lack. Proc. Natl Acad. Sci. USA 93, 9493–9498 (1996).

    Article  ADS  CAS  Google Scholar 

  10. Wheaton, W. W. & Chandel, N. S. Hypoxia. 2. Hypoxia regulates cellular metabolism. Am. J. Physiol. Cell Physiol. 300, C385–C393 (2011).

    Article  CAS  Google Scholar 

  11. Cheng, K. et al. Hypoxia-inducible factor-1α regulates β cell function in mouse and human islets. J. Clin. Invest. 120, 2171–2183 (2010).

    Article  CAS  Google Scholar 

  12. Myllyharju, J. & Kivirikko, K. I. Collagens, modifying enzymes and their mutations in humans, flies and worms. Trends Genet. 20, 33–43 (2004).

    Article  CAS  Google Scholar 

  13. Vater, C. A., Harris, E. D. Jr & Siegel, R. C. Native cross-links in collagen fibrils induce resistance to human synovial collagenase. Biochem. J. 181, 639–645 (1979).

    Article  CAS  Google Scholar 

  14. Jarman-Smith, M. L. et al. Porcine collagen crosslinking, degradation and its capability for fibroblast adhesion and proliferation. J. Mater. Sci. Mater. Med. 15, 925–932 (2004).

    Article  CAS  Google Scholar 

  15. Gerstenfeld, L. C., Riva, A., Hodgens, K., Eyre, D. R. & Landis, W. J. Post-translational control of collagen fibrillogenesis in mineralizing cultures of chick osteoblasts. J. Bone Miner. Res. 8, 1031–1043 (1993).

    Article  CAS  Google Scholar 

  16. van Gastel, N. et al. Expansion of murine periosteal progenitor cells with fibroblast growth factor 2 reveals an intrinsic endochondral ossification program mediated by bone morphogenetic protein 2. Stem Cells 32, 2407–2418 (2014).

    Article  Google Scholar 

  17. Myllyharju, J. Prolyl 4-hydroxylases, key enzymes in the synthesis of collagens and regulation of the response to hypoxia, and their roles as treatment targets. Ann. Med. 40, 402–417 (2008).

    Article  CAS  Google Scholar 

  18. Lorendeau, D., Christen, S., Rinaldi, G. & Fendt, S. M. Metabolic control of signalling pathways and metabolic auto-regulation. Biol. Cell 107, 251–272 (2015).

    Article  CAS  Google Scholar 

  19. Elia, I. et al. Breast cancer cells rely on environmental pyruvate to shape the metastatic niche. Nature (in the press).

  20. Altman, B. J., Stine, Z. E. & Dang, C. V. From Krebs to clinic: glutamine metabolism to cancer therapy. Nat. Rev. Cancer 16, 619–634 (2016).

    Article  CAS  Google Scholar 

  21. Semenza, G. L. Molecular mechanisms mediating metastasis of hypoxic breast cancer cells. Trends Mol. Med. 18, 534–543 (2012).

    Article  CAS  Google Scholar 

  22. Marini, J. C. et al. Osteogenesis imperfecta. Nat. Rev. Dis. Primers 3, 17052 (2017).

    Article  Google Scholar 

  23. Wynn, T. A. & Ramalingam, T. R. Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat. Med. 18, 1028–1040 (2012).

    Article  CAS  Google Scholar 

  24. Bonnans, C., Chou, J. & Werb, Z. Remodelling the extracellular matrix in development and disease. Nat. Rev. Mol. Cell Biol. 15, 786–801 (2014).

    Article  CAS  Google Scholar 

  25. Mazzone, M. et al. Heterozygous deficiency of PHD2 restores tumor oxygenation and inhibits metastasis via endothelial normalization. Cell 136, 839–851 (2009).

    Article  CAS  Google Scholar 

  26. Ovchinnikov, D. A., Deng, J. M., Ogunrinu, G. & Behringer, R. R. Col2a1-directed expression of Cre recombinase in differentiating chondrocytes in transgenic mice. Genesis 26, 145–146 (2000).

    Article  CAS  Google Scholar 

  27. Maes, C. et al. Soluble VEGF isoforms are essential for establishing epiphyseal vascularization and regulating chondrocyte development and survival. J. Clin. Invest. 113, 188–199 (2004).

    Article  CAS  Google Scholar 

  28. Stegen, S. et al. Osteocytic oxygen sensing controls bone mass through epigenetic regulation of sclerostin. Nat. Commun. 9, 2557 (2018).

    Article  ADS  Google Scholar 

  29. Stegen, S. et al. HIF-1α promotes glutamine-mediated redox homeostasis and glycogen-dependent bioenergetics to support postimplantation bone cell survival. Cell Metab. 23, 265–279 (2016).

    Article  CAS  Google Scholar 

  30. Stegen, S. et al. Adequate hypoxia inducible factor 1α signaling is indispensable for bone regeneration. Bone 87, 176–186 (2016).

    Article  CAS  Google Scholar 

  31. Christen, S. et al. Breast cancer-derived lung metastases show increased pyruvate carboxylase-dependent anaplerosis. Cell Rep. 17, 837–848 (2016).

    Article  CAS  Google Scholar 

  32. Lorendeau, D. et al. Dual loss of succinate dehydrogenase (SDH) and complex I activity is necessary to recapitulate the metabolic phenotype of SDH mutant tumors. Metab. Eng. 43, 187–197 (2017).

    Article  CAS  Google Scholar 

  33. Buescher, J. M. et al. A roadmap for interpreting 13C metabolite labeling patterns from cells. Curr. Opin. Biotechnol. 34, 189–201 (2015).

    Article  CAS  Google Scholar 

  34. Elia, I. et al. Proline metabolism supports metastasis formation and could be inhibited to selectively target metastasizing cancer cells. Nat. Commun. 8, 15267 (2017).

    Article  ADS  Google Scholar 

  35. Koopman, W. J., Visch, H. J., Smeitink, J. A. & Willems, P. H. Simultaneous quantitative measurement and automated analysis of mitochondrial morphology, mass, potential, and motility in living human skin fibroblasts. Cytometry A 69, 1–12 (2006).

    Article  Google Scholar 

  36. Sunic, D., Belford, D. A., McNeil, J. D. & Wiebkin, O. W. Insulin-like growth factor binding proteins (IGF-BPs) in bovine articular and ovine growth-plate chondrocyte cultures: their regulation by IGFs and modulation of proteoglycan synthesis. Biochim. Biophys. Acta 1245, 43–48 (1995).

    Article  Google Scholar 

  37. Carmeliet, G., Himpens, B. & Cassiman, J. J. Selective increase in the binding of the alpha 1 beta 1 integrin for collagen type IV during neurite outgrowth of human neuroblastoma TR 14 cells. J. Cell Sci. 107, 3379–3392 (1994).

    CAS  PubMed  Google Scholar 

  38. Weis, M. A. et al. Location of 3-hydroxyproline residues in collagen types I, II, III, and V/XI implies a role in fibril supramolecular assembly. J. Biol. Chem. 285, 2580–2590 (2010).

    Article  CAS  Google Scholar 

  39. Creemers, L. B., Jansen, D. C., van Veen-Reurings, A., van den Bos, T. & Everts, V. Microassay for the assessment of low levels of hydroxyproline. Biotechniques 22, 656–658 (1997).

    Article  CAS  Google Scholar 

  40. Daci, E., Verstuyf, A., Moermans, K., Bouillon, R. & Carmeliet, G. Mice lacking the plasminogen activator inhibitor 1 are protected from trabecular bone loss induced by estrogen deficiency. J. Bone Miner. Res. 15, 1510–1516 (2000).

    Article  CAS  Google Scholar 

  41. Decuypere, J. P. et al. STIM1, but not STIM2, is required for proper agonist-induced Ca2+ signaling. Cell Calcium 48, 161–167 (2010).

    Article  CAS  Google Scholar 

  42. Masuyama, R. et al. TRPV4-mediated calcium influx regulates terminal differentiation of osteoclasts. Cell Metab. 8, 257–265 (2008).

    Article  CAS  Google Scholar 

  43. Grynkiewicz, G., Poenie, M. & Tsien, R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450 (1985).

    CAS  PubMed  Google Scholar 

  44. Missiaen, L., Luyten, T., Bultynck, G., Parys, J. B. & De Smedt, H. Measurement of intracellular Ca2+ release in intact and permeabilized cells using 45Ca2+. Cold Spring Harb. Protoc. 2014, 263–270 (2014).

    Article  Google Scholar 

  45. Storkebaum, E. et al. Impaired autonomic regulation of resistance arteries in mice with low vascular endothelial growth factor or upon vascular endothelial growth factor trap delivery. Circulation 122, 273–281 (2010).

    Article  CAS  Google Scholar 

  46. Laperre, K. et al. Development of micro-CT protocols for in vivo follow-up of mouse bone architecture without major radiation side effects. Bone 49, 613–622 (2011).

    Article  CAS  Google Scholar 

  47. Bouxsein, M. L. et al. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J. Bone Miner. Res. 25, 1468–1486 (2010).

    Article  Google Scholar 

  48. Callewaert, F. et al. Differential regulation of bone and body composition in male mice with combined inactivation of androgen and estrogen receptor-α. FASEB J. 23, 232–240 (2009).

    Article  CAS  Google Scholar 

  49. Verhaeghe, J., Van Herck, E., Van Bree, R., Van Assche, F. A. & Bouillon, R. Osteocalcin during the reproductive cycle in normal and diabetic rats. J. Endocrinol. 120, 143–151 (1989).

    Article  CAS  Google Scholar 

  50. Dempster, D. W. et al. Standardized nomenclature, symbols, and units for bone histomorphometry: a 2012 update of the report of the ASBMR Histomorphometry Nomenclature Committee. J. Bone Miner. Res. 28, 2–17 (2013).

    Article  Google Scholar 

  51. Wilkinson, D. G. in In Situ Hybridisation: A Practical Approach (ed. Wilkinson. D. G.) 75–80 (IRC Press, Oxford, 1992).

Download references

Acknowledgements

We thank I. Stockmans, K. Moermans, T. Luyten, J.-P. Decuypere, E. Van Herck and I. Jans for technical assistance. G.C. acknowledges funding from the Research Foundation-Flanders (FWO: G.0A72.13, G.0964.14 and G.0A42.16); P.C. from long-term structural funding – Methusalem Funding by the Flemish Government; S.-M.F. from Marie Curie-CIG, FWO-Odysseus II, and FWO-Research Grants/Projects; D.R.E. from NIH grants AR037318 (NIAMS) and HD070394 (NICHD); and G.B. from KU Leuven OnderzoeksToelage. S.S. is a postdoctoral fellow from the FWO (12H5917N). G.R. is supported by consecutive PhD fellowships from the Emmanuel van der Schueren - Kom op tegen Kanker foundation and FWO. S.L. is supported by a FWO doctoral grant for strategic basic research.

Reviewer information

Nature thanks F. Long, S. Ramasamy and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: Steve Stegen, Kjell Laperre

Authors and Affiliations

  1. Laboratory of Clinical and Experimental Endocrinology, Department of Chronic Diseases, Metabolism and Ageing, KU Leuven, Leuven, Belgium

    Steve Stegen, Kjell Laperre, Sophie Torrekens, Riet Van Looveren, Shauni Loopmans & Geert Carmeliet

  2. Laboratory of Angiogenesis and Vascular Biology, VIB Center for Cancer Biology, VIB, Leuven, Belgium

    Guy Eelen, Peter Fraisl, Stefan Vinckier & Peter Carmeliet

  3. Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology and Leuven Cancer Institute (LKI), KU Leuven, Leuven, Belgium

    Guy Eelen, Peter Fraisl, Stefan Vinckier & Peter Carmeliet

  4. Laboratory of Cellular Metabolism and Metabolic Regulation, VIB Center for Cancer Biology, VIB, Leuven, Belgium

    Gianmarco Rinaldi & Sarah-Maria Fendt

  5. Laboratory of Cellular Metabolism and Metabolic Regulation, Department of Oncology and Leuven Cancer Institute (LKI), KU Leuven, Leuven, Belgium

    Gianmarco Rinaldi & Sarah-Maria Fendt

  6. Laboratory of Molecular and Cellular Signalling, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium

    Geert Bultynck

  7. Molecular and Nanomaterials, KU Leuven, Leuven, Belgium

    Filip Meersman

  8. Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK

    Patrick H. Maxwell

  9. Department of Orthopaedics, University of Washington, Seattle, WA, USA

    Jyoti Rai, MaryAnn Weis & David R. Eyre

  10. Metabolomics Expertise Center, Department of Oncology, KU Leuven/VIB Center for Cancer Biology Leuven, Leuven, Belgium

    Bart Ghesquière

  11. State Key Laboratory of Ophtalmology, Zhongshan Ophtalmic Center, Sun Yat-Sen University, Guangzhou, China

    Peter Carmeliet

Authors
  1. Steve Stegen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kjell Laperre
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Guy Eelen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gianmarco Rinaldi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peter Fraisl
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sophie Torrekens
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Riet Van Looveren
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Shauni Loopmans
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Geert Bultynck
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Stefan Vinckier
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Filip Meersman
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Patrick H. Maxwell
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Jyoti Rai
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. MaryAnn Weis
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. David R. Eyre
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Bart Ghesquière
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Sarah-Maria Fendt
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Peter Carmeliet
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Geert Carmeliet
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

G.C. conceived the concept of the study and provided supervision. S.S., K.L. and G.C. designed research. S.S., K.L., G.E., P.F., S.L., S.T. and R.V.L. carried out molecular biology and/or in vivo experiments. G.R. and B.G. analysed metabolism by mass spectrometry. G.B. performed calcium measurements. S.V. assisted in microscopic analysis. F.M. carried out infrared spectroscopy. J.R., M.W. and D.R.E. performed mass spectrometry-based analysis of collagen hydroxylation. P.H.M., S.-M.F. and P.C. provided necessary materials. S.S., K.L., G.E., G.R., P.F., S.L., G.B., S.V., F.M., M.W., D.R.E., B.G., S.-M.F. and G.C. analysed and interpreted data. S.S., K.L. and G.C. wrote the manuscript. All authors agreed on the final version of the manuscript.

Corresponding author

Correspondence to Geert Carmeliet.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Phenotype of Phd2chon− mice.

a, Southern blot analysis showing efficient and selective recombination (black arrowhead) of the Phd2 gene in neonatal (P2.5) growth plate tissue from Phd2chon− mice. Representative images of 3 independent experiments are shown. b, Phd1 (also known as Egln2), Phd2 and Phd3 (also known as Egln3) mRNA levels in neonatal growth plates. n = 11 and 10 independent sample from Phd2chon+ and Phd2chon− mice, respectively. c, d, Immunoblot of PHD2 and β-actin (c), and of HIF1α, HIF-2α and lamin A/C (d) levels in growth plate tissue (c) and cultured chondrocytes (d). Representative images of 4 independent experiments are shown. e, Tibia length (n = 11 Phd2chon+ mice, n = 8 Phd2chon− mice), body weight (n = 11 Phd2chon+ mice, n = 8 Phd2chon− mice), lean body mass (n = 4 mice) and fat mass (n = 4 mice) of adult (14-week-old) mice. f, Growth-related phenotype in adult Phd2chon− mice. g, Sox9, Col2, Col10, Pthrp, Ihh, Mmp9, Mmp13 and Opn (also known as Spp1) mRNA levels in neonatal growth plates. n = 11 and 10 independent samples from Phd2chon+ and Phd2chon− mice, respectively. h, In situ hybridization for Col2, Col10, Pthrp and Ihh on neonatal growth plates. n = 4 biologically independent samples. Scale bar, 250 µm. i, Trabecular number (Tb.N) for P2.5 (n = 10) and 14-week-old (n = 11 Phd2chon+, n = 8 Phd2chon−) mice, trabecular thickness (Tb.Th) for P2.5 (n = 10) and 14-week-old (n = 11 Phd2chon+, n = 8 Phd2chon−) mice, cortical thickness (Ct.Th) for P2.5 (n = 10) and 14-week-old (n = 11 Phd2chon+, n = 8 Phd2chon−) mice, and calvarial thickness (Calv.Th; n = 4 mice) and porosity (Calv.Po; n = 4 mice) in neonatal and adult mice. Nd, not determined. j, Osteoblast number (N.Ob/B.S; n = 4 Phd2chon+, n = 5 Phd2chon− mice), osteoblast surface (Ob.S/B.S; n = 4 Phd2chon+, n = 5 Phd2chon− mice) and osteoid surface per bone surface (O.S/B.S; n = 6 mice), bone formation rate (BFR; n = 4 mice), mineral apposition rate (MAR; n = 4 mice), osteoclast surface per bone surface (Oc.S/B.S; n = 11 Phd2chon+, n = 7 Phd2chon− mice), blood vessel number per tissue surface (N.BV/T.S; n = 9 Phd2chon+, n = 6 Phd2chon− mice), and serum osteocalcin (OCN; n = 16 biologically independent samples), CTx-I (n = 8 biologically independent samples) and CTx-II levels (n = 9 biologically independent samples) in adult mice. k, l, Representative images of TRAP-positive multinuclear cells formed after one week of culture (k) with quantification of the number of osteoclasts formed per well (l). n = 4 biologically independent samples. Scale bar, 50 µm). Quantification was based on the number of nuclei per osteoclast (see Methods). m, COL1 and COL2 immunostaining of the metaphysis of neonatal mice (n = 8 mice). Scale bar, 100 µm. n, Quantification of the cell/extracellular matrix (ECM) ratio in two zones of the growth plate (n = 8 mice). Data are mean ± s.e.m. (c, d, l), or mean ± s.d. (b, e, g, i, j, n). *P < 0.05 vs Phd2chon+, **P < 0.01 vs Phd2chon+, ***P < 0.001 vs Phd2chon+; two-sided Student’s t-test). Exact P values: 0.000000001 (Phd2; b), 0.00000002 (Phd3; b), 0.00003 (c), 0.0014 (HIF-1α; d), 0.00005 (tibia length; e), 0.019 (body weight; e), 0.00003 (Tb.N P2.5; i), 0.003 (Tb.N 14 weeks; i), 0.006 (Tb.Th P2.5; i), 0.037 (Tb.Th 14 weeks; i) or 0.02 (Serum CTx-II; j).

Source data

Extended Data Fig. 2 Metabolic alterations in PHD2-deficient chondrocytes.

a, Rhodamine labelling of mitochondria, with quantification of mitochondrial content. Samples from n = 3 Phd2chon+, n = 4 Phd2chon− mice. Yellow line denotes cell membrane. b, c, Immunoblot of Myc (b), LC3-II (c) and β-actin levels in cultured chondrocytes. Representative images of 4 independent experiments are shown. d, Oxygen consumption in cultured chondrocytes. n = 9 biologically independent samples. e, Pimonidazole immunostaining on neonatal (P2.5) growth plates. The boxed area is magnified to the right of each panel. Number of pimonidazole-positive cells within the growth plate is shown. n = 4 mice. f, Glucose oxidation (GO), fatty acid oxidation (FAO) and glutamine oxidation (QO) in cultured chondrocytes. n = 6 biologically independent samples. g, Glucose (Glc) uptake and lactate (Lac) secretion. n = 6 biologically independent samples. h, Glycolytic flux. n = 6 biologically independent samples. i, Fractional contribution of 13C6-glucose to lactate, citrate (Cit), αKG, succinate (Suc), fumarate (Fum) and malate (Mal). n = 6 biologically independent samples. j–l, ATP content (j), energy charge (([ATP] + ½[ADP])/([ATP] + [ADP] + [AMP]); k), and energy status (ratio of ATP to AMP levels; l). n = 6 biologically independent samples. m, Apoptosis rate of cultured chondrocytes. n = 4 independent experiments. n, TUNEL immunostaining of neonatal growth plates. n = 6 mice. o, ATP production resulting from glycolysis, GO, FAO and QO in cultured chondrocytes. n = 6 biologically independent samples. p, Proliferation rate of cultured chondrocytes. n = 4 independent experiments. q, Immunoblot of Na+/K+ ATPase and β-actin. Representative images of 3 independent experiments are shown. r, Normalized Ca2+ rise in the cytosol of cultured chondrocytes upon stimulation with thapsigargin (TG) in the presence of EGTA. n = 4 biologically independent samples. s, Quantification of the Ca2+ release from the endoplasmic reticulum upon stimulation with thapsigargin. n = 4 biologically independent samples. t, 45Ca2+-loading capacity of the endoplasmic reticulum of permeabilised chondrocytes in intracellular-like medium supplemented with 5 mM Mg-ATP and 45Ca2+. n = 4 biologically independent samples. u, v, Total protein (u) and proteoglycan synthesis (v). n = 8 biologically independent samples. Data are mean ± s.e.m. (a–d, f–m, o–v), or mean ± s.d. (e, n). *P < 0.05 vs Phd2chon+, **P < 0.01 vs Phd2chon+, ***P < 0.001 vs Phd2chon+ (two-sided Student’s t-test), §P < 0.05 vs Phd2chon+ + vehicle (ANOVA). Exact P values: 0.03 (a), 0.016 (b); Phd2chon+ + vehicle vs Phd2chon− + vehicle, 0.0004 (c); Phd2chon+ + vehicle vs Phd2chon− + chloroquine, 0.0002 (c); 0.000002 (d), 0.0005 (e), 0.00003 (GO; f), 0.000004 (FAO; f), 0.00001 (Glc; g), 0.000001 (Lac; g), 0.0007 (h), 0.00002 (Lac; i), 0.000002 (Cit; i), 0.000001 (αKG; i), 0.00009 (Suc; i), 0.011 (Fum; i), 0.004 (Mal; i), 0.000001 (j), 0.00005 (k), 0.00000001 (l), 0.0007 (p), 0.012 (q), 0.0003 (s), 0.0002 (u), 0.003 (v). Scale bars: 10 µm (a), 250 µm (e, n).

Source data

Extended Data Fig. 3 HIF-1α silencing in PHD2-deficient chondrocytes.

a, Expression of indicated genes in cultured chondrocytes, transduced with scrambled shRNA (shScr) or shRNA targeting HIF-1α (shHIF-1α). n = 3 biologically independent samples. Gene symbols are listed in parentheses: Glut1 (Slc2a1), Glut4 (Slc2a4), Ldh-a (Ldha), Mct1 (Slc16a1), Mct4 (Slc16a4), Cox4-1 (Cox4i1), Cox4-2 (Cox4i2), Pgc-1a (Ppargc1a), Pgc-1b (Ppargc1b), Gls1 (Gls). b, HIF-1α and lamin A/C immunoblot of cultured chondrocytes, transduced with shScr or shHIF-1α. Representative images of 3 independent experiments are shown. c–f, Oxygen consumption (c), glycolytic flux (d), energy charge (e) and energy status (f) of cultured chondrocytes transduced with shScr or shHIF-1α. n = 6 biologically independent samples. g, p-AMPKT172 and AMPK immunoblot with quantification of p-AMPKT172:AMPK ratio in cultured chondrocytes transduced with shScr or shHIF-1α. Representative images of 3 independent experiments are shown. h, i, Proliferation (h) and collagen synthesis (i) in cultured chondrocytes transduced with shScr or shHIF-1α. n = 6 biologically independent samples. j, k, BiP (j), cleaved ATF6 (cATF6) (k) and β-actin immunoblot of cultured chondrocytes transduced with shScr or shHIF-1α. Representative images of 3 independent experiments are shown. l, m, OH-Pro (l; n = 6 biologically independent samples) and αKG levels (m; n = 5 biologically independent samples) in cultured chondrocytes transduced with shScr or shHIF-1α. Data are mean ± s.e.m. #P < 0.05 (ANOVA), §P < 0.05 vs Phd2chon+ + shScr, °P < 0.05 vs Phd2chon− + shScr (ANOVA). Exact P values: Phd2chon+ + shScr vs Phd2chon+ + shHIF-1α 0.0003 (b) or 0.002 (c); Phd2chon+ + shScr vs Phd2chon− + shScr 0.050 (b), 0.0000002 (c), 0.00003 (d), 0.004 (e), 0.000002 (f), 0.050 (g), 0.00002 (h), 0.00005 (i), 0.004 (j), 0.03 (k), 0.00012 (l) or 0.00005 (m); Phd2chon− + shScr vs Phd2chon− + shHIF-1α 0.045 (b), 0.00010 (c), 0.00011 (d), 0.0010 (e), 0.000012 (f), 0.03 (g), 0.00005 (h), 0.00010 (i), 0.004 (j), 0.006 (k), 0.00001 (l) or 0.0008 (m).

Source data

Extended Data Fig. 4 mTOR signalling and the unfolded protein response in PHD2-deficient chondrocytes.

a, Immunoblot and quantification of phosphorylated (at Ser2448) mTOR (p-mTORS2448), mTOR, phosphorylated (at Thr389 and Ser371) p70 S6 kinase (p-p70 S6KT389 and p-p70 S6KS371), p70 S6K, phosphorylated (at Ser235 and Ser236) S6 (p-S6S235/236) and S6, phosphorylated (at Thr37 and Thr46) 4E-BP1 (p-4E-BP1T37/46), 4E-BP1 and β-actin in cultured chondrocytes. Representative images of 3 independent experiments are shown. b, c, p-S6S235/236 immunostaining on neonatal (P2.5) growth plates (b; boxed areas are magnified, right) and quantification (c) of the p-S6+ area. n = 6 mice. PS, primary spongiosa. d, Immunoblot of p-S6S235/236 and S6 in cultured chondrocytes. Cells were cultured in full medium or in nutrient-deprived conditions (PBS), and then switched to full medium for the indicated times. Representative images of 3 independent experiments are shown. These findings demonstrate a lack of enhanced mTOR signalling. e, Immunoblot and quantification of BiP, phosphorylated eIF2α, ATF4 and cATF6 protein levels. Representative images of 3 independent experiments are shown. f, Spliced Xbp1 (Xbp1s) mRNA levels in neonatal growth plates. n = 8 biologically independent samples. g, h, BiP and cATF6 immunostaining (g) of neonatal growth plates with quantification (h) of the percentage of positive cells. n = 6 mice. Data are mean ± s.e.m. (a, d, e) or mean ± s.d. (c, f, h). *P < 0.05 vs Phd2chon+, **P < 0.01 vs Phd2chon+, ***P < 0.001 vs Phd2chon+ (two-sided Student’s t-test), §P < 0.05 vs Phd2chon+ + full medium, °P < 0.05 vs Phd2chon+ + full medium 0.5 h (ANOVA). Exact P values: Phd2chon+ + full medium vs Phd2chon+ + PBS, 0.003 (d); Phd2chon+ + full medium vs Phd2chon− + PBS, 0.005 (d); Phd2chon+ + full vs Phd2chon− 0.5 h, 0.03 (d); Phd2chon+ 0.5 h vs Phd2chon− 0.5 h, 0.04 (d); 0.04 (BiP; e); 0.02 (p-eIF2a; e); 0.0005 (ATF4; e); 0.02 (cATF6; e); 0.002 (f); 0.00005 (BiP; h); 0.000009 (cATF6; h). Scale bars, 250 µm.

Source data

Extended Data Fig. 5 PHD2 regulates COL2 modifications.

a, COL2 protein levels in neonatal (P2.5) growth plates, visualized by COL2 western blot. Protein loading was normalized to growth plate weight. Representative images of 2 experiments, each with 2 biologically independent replicates, are shown. b, COL2 immunostaining of extracellular matrix produced by cultured chondrocytes and after removal of cells, with quantification of the COL2 positive area. n = 4 mice. c, Amide I peak, representing collagen, from FTIR spectra of neonatal growth plates. Samples from n = 8 Phd2chon+ and n = 6 Phd2chon− mice. AUC, area under the curve. d, Transmission electron microscopy images of the collagen network in neonatal growth plates. n = 3 mice. e, Increase in hydroxylation and/or glycosylation (as percentage) of Pro and Lys residues in type II collagen of Phd2chon− mice compared to Phd2chon+ mice. n = 4 biologically independent samples. Glcgal, glucosyl-galactosyl. f, Total, intracellular and extracellular OH-Pro content of cultured chondrocytes. n = 4 biologically independent samples. g, Von Kossa staining of neonatal growth plates, with quantification of the percentage mineralized matrix. Boxed areas are enlarged to the right of each panel. n = 8 mice. h, i, Micromass mineralization, as evidenced by alizarin red staining (h), with quantification of alizarin red intensity (i), showing that increased matrix mineralization is not caused by HIF-1α-induced changes in mineralization capacity. n = 5 biologically independent samples. j, Expression of genes involved in mineralization (Ank, Tnap (also known as Alpl), Enpp1 and Spp1) is not changed in neonatal growth plates. n = 6 biologically independent samples. k, Phd2 mRNA levels in cultured periosteum-derived cells (PDC), calvarial osteoblasts (calv. OB) and trabecular osteoblasts (trab. OB). n = 4 biologically independent samples. l, Change in hydroxylation and/or glycosylation (as percentage) of Pro and Lys residues in type I collagen of Phd2chon− mice compared to Phd2chon+ mice. n = 4 biologically independent samples. m, OH-Pro levels in cultured PDC, and calvarial and trabecular osteoblasts. n = 4 biologically independent samples. n, OH-Pro content in bone tissue and supernatant after incubation with MMP9 or MMP13. n = 4 biologically independent samples. o, Fractional contribution of 13C5-glutamine to Pro. n = 3 biologically independent samples. p, Intracellular proline levels in cultured Phd2chon+ and Phd2chon− chondrocytes. n = 3 biologically independent samples. Data are mean ± s.d. (a, c, g, j), or mean ± s.e.m. (b, f, i, k–p). *P < 0.05 vs Phd2chon+, **P < 0.01 vs Phd2chon+, ***P < 0.001 vs Phd2chon+ (two-sided Student’s t-test), #P < 0.05 (ANOVA). Exact P values: 0.0003 (a), 0.02 (b), 0.006 (c), 0.0018 (Pro459; e), 0.008 (Pro744; e), 0.014 (Pro795; e), 0.015 (Pro826; e), 0.016 (Pro945; e), 0.00015 (Pro966; e), 0.008 (Pro986; e), 0.019 (Lys87; e), 0.002 (total; f), 0.0014 (extra; f), 0.012 (g), vehicle vs Phd2chon+, 0.0002 (i); IOX2 vs Phd2chon−, 0.0001 (i); Phd2chon+ vs Phd2chon−, 0.004 (i); 0.015 (Pro1011; l); 0.012 (Lys174; l). Scale bars: 50 µm (b), 0.5 µm (d) and 100 µm (g).

Source data

Extended Data Fig. 6 Genetic confirmation of HIF-1α signalling and metabolic adaptations.

a–c, Immunoblot of HIF-1α (a), GLS1 (b), PDK1 (c), lamin A/C and β-actin in cultured control or PHD2-deficient (PHD2KD) periosteal cells transduced with scrambled shRNA (shScr) or gene-specific shRNAs. Representative images of 3 independent experiments are shown. d, Toluidine blue staining of bone ossicles. Arrowheads indicate cartilage remnants. n = 5 biologically independent samples. Scale bar, 100 µm. e, 3D microCT models of bone ossicles, with quantification of the mineralized tissue volume (MV) relative to tissue volume (TV). n = 5 biologically independent samples. f, g, Immunoblot of GLS1 (f), PDK1 (g) and β-actin in cultured chondrocytes transduced with shScr or gene-specific shRNAs. Representative images of 3 independent experiments are shown. h, p-AMPKT172 and AMPK immunoblot with quantification of p-AMPKT172:AMPK ratio in cultured chondrocytes transduced with shScr or gene-specific shRNAs. Representative images of 3 independent experiments are shown. i–k, Proliferation (i), αKG levels (j) and OH-Pro content (k) in cultured chondrocytes transduced with shScr or gene-specific shRNAs. n = 5 biologically independent samples. Data are mean ± s.e.m. (a–c, f–k), or mean ± s.d. (e). #P < 0.05 (ANOVA), §P < 0.05 vs control/Phd2chon+ + shScr, °P < 0.05 vs PHD2KD/Phd2chon− + shScr (ANOVA). Exact P values: control-shScr vs control gene-specific shRNA, 0.0005 (a), 0.0003 (b), 0.010 (c), 0.0004 (shHIF-1α; e) or 0.00012 (shGLS1; e); control-shScr vs PHD2KD + shScr, 0.02 (a), 0.04 (b), 0.046 (c) or 0.003 (e); PHD2KD + shScr vs PHD2KD + gene-specific shRNA, 0.03 (a), 0.03 (b), 0.03 (c), 0.002 (shHIF-1α; e), 0.0005 (shGLS1; e), 0.049 (shPDK1; e); Phd2chon+ + shScr vs Phd2chon+ + gene-specific shRNA, 0.000010 (shGLS1; f), 0.006 (shPDK1; g), 0.00011 (shGLS1; i), 0.002 (shGLS1;j) or 0.0006 (shGLS1; k); Phd2chon+ + shScr vs Phd2chon− + shScr, 0.0020 (f), 0.00010 (g), 0.006 (h), 0.00012 (i), 0.00011 (j) or 0.0002 (k); Phd2chon− + shScr vs Phd2chon− + gene-specific shRNA, 0.005 (shGLS1; f), 0.00015 (shPDK1; g), 0.003 (shPDK1; h), 0.0002 (shPDK1; i), 0.00008 (shGLS1; j), 0.00010 (shGLS1; k) or 0.0006 (shPDK1; k); Phd2chon+ + shScr vs Phd2chon− + gene-specific shRNA, 0.002 (shGLS1; h).

Source data

Extended Data Fig. 7 PHD2-deficient chondrocytes display enhanced glutamine metabolism.

a, Intracellular glutamate (Glu), αKG, succinate, fumarate, malate and citrate levels in cultured chondrocytes, with or without BPTES treatment. n = 3 biologically independent samples. b, Ratio of αKG/Suc and αKG/Fum. n = 3 biologically independent samples. c, GLS1 and β-actin immunoblot of cultured chondrocytes transduced with scrambled shRNA (shScr) or shRNA against HIF-1α (shHIF-1α). Representative images of 3 independent experiments are shown. d, Immunoblot of GLS1, GLS2 and β-actin in cultured chondrocytes, compared to HeLa cells. Representative images of 3 independent experiments are shown. e, Fractional contribution of 13C5-glutamine to glutamate, αKG, succinate, fumarate, malate and citrate in cultured chondrocytes, with or without BPTES treatment. n = 3 biologically independent samples. f, Citrate mass isotopomer distribution (MID) from 13C5-glutamine. n = 3 biologically independent samples. g, Relative abundance of reductive carboxylation-specific mass isotopomers of citrate, malate and fumarate. n = 3 biologically independent samples. h, COL2 immunostaining of the tibia of neonatal (P2.5) mice treated with BPTES and/or αKG with quantification of the percentage COL2-positive matrix (green) relative to bone volume (BV). n = 5 for Phd2chon+ + vehicle or Phd2chon− + vehicle mice; n = 7 for Phd2chon+ + BPTES, Phd2chon+ + BPTES + αKG, Phd2chon− + BPTES or Phd2chon− + BPTES + αKG mice. Scale bar, 250 µm. Arrowheads indicate COL2 cartilage remnants. i, 3D microCT models of the tibial metaphysis of mice treated with BPTES, with or without αKG, and quantification of trabecular bone volume (TBV). n = 6 for Phd2chon+ + vehicle, Phd2chon+ + BPTES + αKG or Phd2chon− + vehicle; n = 5 for Phd2chon+ + BPTES; n = 7 for Phd2chon− + BPTES or Phd2chon− + BPTES + αKG. j, Immunoblot of HIF-1α and lamin A/C in cultured chondrocytes treated with BPTES, with or without αKG. Representative images of 3 independent experiments are shown. k, Relative mRNA levels of indicated genes in growth plates derived from mice treated with BPTES, with or without αKG. n = 3 biologically independent samples. l, Immunoblot of p-AMPKT172 and AMPK in cultured chondrocytes treated with BPTES, with or without αKG. Representative images of 3 independent experiments are shown. m, Proliferation, as determined by BrdU incorporation, of cultured chondrocytes, treated with BPTES, with or without αKG. n = 3 biologically independent samples. n, Tibia length of mice treated with BPTES, with or without αKG. n = 5 for Phd2chon+ + vehicle or Phd2chon− + vehicle; n = 7 for Phd2chon+ + BPTES, Phd2chon+ + BPTES + αKG, Phd2chon− + BPTES or Phd2chon− + BPTES + αKG. o, BiP, cATF6 and β-actin immunoblot in cultured chondrocytes treated with BPTES, with or without αKG. Representative images of 3 independent experiments are shown. Data are mean ± s.e.m. (a–g, j, l, m, o), or mean ± s.d. (h, i, k, n). *P < 0.05 vs Phd2chon+, **P < 0.01 vs Phd2chon+, ***P < 0.001 vs Phd2chon+ (two-sided Student’s t-test), #P < 0.05 (ANOVA), §P < 0.05 vs Phd2chon+ + shScr or vehicle, °P < 0.05 vs Phd2chon− + shScr or vehicle (ANOVA). Exact P values: Phd2chon+ + vehicle vs Phd2chon− + vehicle, 0.045 (αKG/Suc; b), 0.010 (αKG/Fum; b), 0.00008 (Glu; e), 0.00004 (αKG; e), 0.02 (Suc; e), 0.0007 (Fum; e), 0.009 (Mal; e), 0.050 (Cit; e), 0.00001 (h), 0.00003 (i), 0.03 (j), 0.0001 (P4ha1; k), 0.0006 (P4ha2; k), 0.00002 (P3h1; k), 0.0001 (Plod1; k), 0.0002 (Plod2; k), 0.0003 (Pdi; k), 0.0004 (Lox; k), 0.004 (l), 0.006 (m), 0.000002 (n), 0.0005 (BiP; o) or 0.002 (cATF6; o); Phd2chon+ + vehicle vs Phd2chon+ + BPTES, 0.00001 (Glu; e), 0.000003 (αKG; e), 0.02 (Suc; e), 0.0006 (Fum; e), 0.00005 (Mal; e), 0.02 (Cit; e), 0.00001 (h), 0.0001 (i), 0.002 (m) or 0.0000001 (n); Phd2chon+ + BPTES vs Phd2chon+ + BPTES + αKG, 0.004 (m) or 0.0003 (n); Phd2chon+ + vehicle vs Phd2chon− + BPTES, 0.03 (αKG/Fum; b), 0.005 (j), 0.0003 (P4ha1; k), 0.001 (P4ha2; k), 0.0006 (P3h1; k), 0.0001 (Plod1; k), 0.0006 (Plod2; k), 0.002 (Pdi; k), 0.0006 (Lox; k), 0.012 (l), 0.007 (m), 0.000007 (n), 0.003 (BiP; o) or 0.004 (cATF6; o); Phd2chon+ + vehicle vs Phd2chon− + BPTES + αKG, 0.00006 (h), 0.00002 (i), 0.03 (j), 0.0003 (P4ha1; k), 0.001 (P4ha2; k), 0.0001 (P3h1; k), 0.00008 (Plod1; k), 0.0007 (Plod2; k), 0.00003 (Pdi; k), 0.0003 (Lox; k), 0.04 (l), 0.019 (m), 0.000005 (n), 0.02 (BiP; o) or 0.0010 (cATF6; o); Phd2chon− + vehicle vs Phd2chon− + BPTES, 0.025 (αKG/Suc; b), 0.049 (αKG/Fum; b), 0.00001 (Glu; e), 0.00001 (αKG; e), 0.02 (Suc; e), 0.006 (Fum; e), 0.002 (Mal; e), 0.02 (Cit; e), 0.000002 (h) or 0.000005 (i); Phd2chon+ + shScr vs Phd2chon− + shScr, 0.007 (c); Phd2chon+ + vehicle vs Phd2chon+ + shHIF-1α, 0.006 (c); Phd2chon− + vehicle vs Phd2chon− + shHIF-1α, 0.013 (c); Phd2chon+ vs Phd2chon−, 0.007 (GLS1; d), 0.0013 (m + 4; f), 0.0006 (m + 5; f), 0.0006 (Cit; g), 0.0002 (Mal; g) or 0.048 (Fum; g); Phd2chon+ vs HeLa, 0.04 (GLS1; d) or 0.0088 (GLS2; d).

Source data

Extended Data Fig. 8 Inhibition of pyruvate uptake does not affect collagen or bone properties.

a, Intracellular αKG levels in cultured chondrocytes, with or without treatment with an inhibitor of monocarboxylate transporter 2 (MCT2i). n = 3 biologically independent samples. b, p-AMPKT172 and AMPK immunoblot with quantification of p-AMPKT172/AMPK ratio in cultured chondrocytes treated with MCT2i. Representative images of 3 independent experiments are shown. c, Tibia length of mice treated with MCT2i. n = 5 mice. d, Collagen synthesis in cultured chondrocytes, with or without MCT2i treatment. n = 4 biologically independent samples. e, BiP, cATF6 and β-actin immunoblot in cultured chondrocytes treated with MCT2i. Representative images of 3 independent experiments are shown. f, OH-Pro content in neonatal growth plates of mice treated with MCT2i. n = 5 biologically independent samples. g, Safranin O staining of the tibia of mice treated with MCT2i, and quantification of the percentage safranin O-positive matrix relative to bone volume. n = 5 mice. h, COL2 immunostaining of the tibia of mice treated with MCT2i, with quantification of the percentage COL2-positive matrix (green) relative to bone volume. n = 5 mice. Arrowheads indicate COL2 cartilage remnants. i, 3D microCT models of the tibial metaphysis of mice treated with MCT2i, and quantification of trabecular bone volume. n = 5 mice. Data are mean ± s.e.m. (a, b, d, e), or mean ± s.d. (c, f–i). #P < 0.05 (ANOVA), §P < 0.05 vs Phd2chon+ + vehicle (ANOVA). Exact P values: Phd2chon+ + vehicle vs Phd2chon− + vehicle, 0.00002 (a), 0.015 (b), 0.00001 (c), 0.00011 (d), 0.012 (BiP; e), 0.008 (cATF6; e), 0.00012 (f), 0.00013 (g), 0.00001 (h) or 0.00001 (i); Phd2chon+ + MCT2i vs Phd2chon− + MCT2i, 0.0003 (a), 0.000002 (c), 0.005 (d) or 0.0003 (f); Phd2chon+ + vehicle vs Phd2chon− + MCT2i, 0.02 (b), 0.004 (BiP; e), 0.002 (cATF6; e), 0.00007 (g), 0.0001 (h) or 0.00002 (i). Scale bars, 250 µm (g, h).

Source data

Extended Data Fig. 9 Administration of αKG increases collagen hydroxylation and bone mass in wild-type mice.

a, Intracellular αKG in cultured chondrocytes, with or without supplementation of dimethyl-αKG (hereafter αKG). n = 4 biologically independent samples. b, p-AMPKT172 and AMPK immunoblot with quantification of p-AMPKT172/AMPK ratio in cultured chondrocytes, with or without αKG supplementation. Representative images of 3 independent experiments are shown. c, Tibia length of mice treated with αKG. n = 5 mice. d, Collagen synthesis in cultured chondrocytes, with or without αKG supplementation. n = 4 biologically independent samples. e, Immunoblot of BiP, cATF6 and β-actin in cultured chondrocytes, with or without αKG supplementation. Representative images of 3 independent experiments are shown. f, OH-Pro content in neonatal growth plates of mice treated with αKG. n = 5 biologically independent samples. g, Safranin O staining of the tibia of mice treated with αKG, and quantification of the percentage Safranin O-positive matrix relative to bone volume. n = 5 mice. h, COL2 immunostaining of the tibia of mice treated with αKG, with quantification of the percentage COL2-positive matrix (green) relative to bone volume. n = 5 mice. Arrowheads indicate COL2-cartilage remnants. i, 3D microCT models of the tibial metaphysis of mice treated with αKG, and quantification of trabecular bone volume, n = 5 mice. j, Immunoblot of HIF-1α and lamin A/C in cultured chondrocytes, with or without αKG supplementation. Representative images of 3 independent experiments are shown. k, Relative mRNA levels of indicated genes in growth plates derived from mice treated with αKG. n = 3 biologically independent samples. Data are mean ± s.e.m. (a, b, d, e, j), or mean ± s.d. (c, f–i, k). #P < 0.05 (ANOVA), §P < 0.05 vs Phd2chon+ + vehicle (ANOVA). Exact P values: Phd2chon+ + vehicle vs Phd2chon− + vehicle, 0.000001 (a), 0.04 (b), 0.00000001 (c), 0.002 (d), 0.046 (BiP; e), 0.011 (cATF6; e), 0.0003 (f), 0.0002 (g), 0.000004 (h), 0.00002 (i), 0.03 (j), 0.00008 (P4ha1; k), 0.0007 (P4ha2; k), 0.0003 (P3h1; k), 0.004 (Plod1; k), 0.0005 (Plod2; k), 0.0005 (Pdi; k) or 0.002 (Lox; k); Phd2chon+ + vehicle vs Phd2chon+ + αKG, 0.000007 (a), 0.02 (f), 0.006 (g), 0.00002 (h) or 0.0003 (i); Phd2chon+ + vehicle vs Phd2chon− + αKG, 0.02 (b), 0.008 (BiP; e), 0.02 (cATF6; e), 0.005 (f), 0.0003 (g), 0.000002 (h), 0.000003 (i), 0.010 (j), 0.0010 (P4ha1; k), 0.002 (P4ha2; k), 0.0011 (P3h1; k), 0.02 (Plod1; k), 0.00001 (Plod2; k), 0.007 (Pdi; k) or 0.004 (Lox; k); Phd2chon+ + αKG vs Phd2chon− + αKG, 0.0002 (a), 0.00000007 (c) or 0.002 (d); Phd2chon− + vehicle vs Phd2chon− + αKG, 0.003 (a). Scale bars, 250 µm (g, h).

Source data

Extended Data Fig. 10 Normalization of glucose oxidation corrects the energy deficit in PHD2-deficient chondrocytes.

a–e, Glucose oxidation (a), oxygen consumption (b), palmitate oxidation (c), glutamine oxidation (d) and glycolytic flux (e) in cultured chondrocytes, with or without DCA treatment. n = 6 biologically independent samples (a), n = 3 biologically independent samples (b–e). f, Proliferation, as determined by BrdU incorporation, of cultured chondrocytes, with or without DCA treatment. n = 3 biologically independent samples. g, Tibia length of mice treated with DCA n = 5 Phd2chon+ + vehicle, Phd2chon− + vehicle or Phd2chon− + DCA; n = 7 Phd2chon+ + DCA. h, i, BiP (h), cATF6 (i) and β-actin immunoblot in cultured DCA-treated chondrocytes. Representative images of 3 independent experiments are shown. j, OH-Pro content in neonatal growth plates of mice treated with DCA, with or without BPTES. n = 5 Phd2chon+ + vehicle or Phd2chon− + vehicle; n = 7 Phd2chon+ + DCA, Phd2chon+ + DCA + BPTES, Phd2chon− + DCA or Phd2chon− + DCA + BPTES. k, COL2 immunostaining of the tibia of mice treated with DCA, with or without BPTES, with quantification of the percentage COL2-positive matrix (green) relative to bone volume. n = 5 Phd2chon+ + vehicle; n = 7 Phd2chon− + vehicle, Phd2chon+ + DCA, Phd2chon+ + DCA + BPTES, Phd2chon− + DCA or Phd2chon− + DCA + BPTES. Scale bar, 250 µm. Arrowheads indicate COL2 cartilage remnants. l, 3D microCT models of the tibial metaphysis of mice treated with DCA, with or without BPTES, and quantification of trabecular bone volume. n = 5 Phd2chon+ + vehicle or Phd2chon− + vehicle; n = 7 Phd2chon+ + DCA, Phd2chon+ + DCA + BPTES, Phd2chon− + DCA or Phd2chon− + DCA + BPTES. m, Intracellular αKG levels in cultured chondrocytes treated with DCA, with or without BPTES. n = 4 biologically independent samples. n, Relative mRNA levels of indicated genes in growth plates derived from mice treated with DCA, with or without BPTES. n = 3 biologically independent samples. Data are mean ± s.e.m. (a–f, h, i, m), or mean ± s.d. (g, j–l, n). #P < 0.05 (ANOVA), §P < 0.05 vs Phd2chon+ + vehicle, °P < 0.05 vs Phd2chon− + vehicle (ANOVA). Exact P values: Phd2chon+ + vehicle vs Phd2chon− + vehicle, 0.00002 (a), 0.0006 (b), 0.0009 (c), 0.0096 (e), 0.003 (f), 0.00006 (g), 0.003 (h), 0.005 (i), 0.0003 (j), 0.0000004 (k), 0.0002 (l), 0.002 (m), 0.00008 (P4ha1; n), 0.00003 (P4ha2; n), 0.0004 (P3h1; n), 0.004 (Plod1; n), 0.005 (Plod2; n), 0.0002 (Pdi; n) or 0.002 (Lox; n); Phd2chon+ + vehicle vs Phd2chon+ + DCA, 0.003 (a); Phd2chon− + vehicle vs Phd2chon− + DCA, 0.0003 (a), 0.002 (b), 0.003 (e), 0.02 (f), 0.00012 (g), 0.004 (h), 0.005 (i), 0.000003 (k) or 0.0001 (l); Phd2chon+ + vehicle vs Phd2chon− + DCA, 0.005 (c), 0.000008 (j), 0.0000002 (k), 0.00005 (l), 0.0004 (m), 0.0002 (P4ha1; n), 0.00003 (P4ha2; n), 0.00001 (P3h1; n), 0.0004 (Plod1; n), 0.001 (Plod2; n), 0.001 (Pdi; n) or 0.00008 (Lox; n); Phd2chon+ + vehicle vs Phd2chon+ + DCA + BPTES, 0.0000002 (j), 0.0003 (k), 0.004 (l) or 0.005 (m); Phd2chon− + vehicle vs Phd2chon− + DCA + BPTES, 0.00002 (j), 0.000000002 (k), 0.000003 (l), 0.0014 (m), 0.0008 (P4ha1; n), 0.0002 (P4ha2; n), 0.0001 (P3h1; n), 0.0001 (Plod1; n), 0.001 (Plod2; n), 0.001 (Pdi; n) or 0.0002 (Lox; n).

Source data

Supplementary information

Supplementary Information

This file contains the uncropped gels

Reporting Summary

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Stegen, S., Laperre, K., Eelen, G. et al. HIF-1α metabolically controls collagen synthesis and modification in chondrocytes. Nature 565, 511–515 (2019). https://doi.org/10.1038/s41586-019-0874-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-019-0874-3

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing