De novo serine synthesis regulates chondrocyte proliferation during bone development and repair

Abstract

The majority of the mammalian skeleton is formed through endochondral ossification starting from a cartilaginous template. Cartilage cells, or chondrocytes, survive, proliferate and synthesize extracellular matrix in an avascular environment, but the metabolic requirements for these anabolic processes are not fully understood. Here, using metabolomics analysis and genetic in vivo models, we show that maintaining intracellular serine homeostasis is essential for chondrocyte function. De novo serine synthesis through phosphoglycerate dehydrogenase (PHGDH)-mediated glucose metabolism generates nucleotides that are necessary for chondrocyte proliferation and long bone growth. On the other hand, dietary serine is less crucial during endochondral bone formation, as serine-starved chondrocytes compensate by inducing PHGDH-mediated serine synthesis. Mechanistically, this metabolic flexibility requires ATF4, a transcriptional regulator of amino acid metabolism and stress responses. We demonstrate that both serine deprivation and PHGDH inactivation enhance ATF4 signaling to stimulate de novo serine synthesis and serine uptake, respectively, and thereby prevent intracellular serine depletion and chondrocyte dysfunction. A similar metabolic adaptability between serine uptake and de novo synthesis is observed in the cartilage callus during fracture repair. Together, the results of this study reveal a critical role for PHGDH-dependent serine synthesis in maintaining intracellular serine levels under physiological and serine-limited conditions, as adequate serine levels are necessary to support chondrocyte proliferation during endochondral ossification.

References

[1] Hallett SA, Ono W, Ono N. Growth plate chondrocytes: skeletal development, growth and beyond. Int. J. Mol. Sci., 2019, 20: 6009

[2] Tsang KY, Tsang SW, Chan D, Cheah KS. The chondrocytic journey in endochondral bone growth and skeletal dysplasia. Birth Defects Res. C. Embryo Today, 2014, 102: 52-73

[3] Krakow D, Rimoin DL. The skeletal dysplasias. Genet. Med., 2010, 12: 327-341

[4] Einhorn TA, Gerstenfeld LC. Fracture healing: mechanisms and interventions. Nat. Rev. Rheumatol., 2015, 11: 45-54

[5] Little DG, Ramachandran M, Schindeler A. The anabolic and catabolic responses in bone repair. J. Bone Jt. Surg. Br., 2007, 89: 425-433

[6] Kronenberg HM. Developmental regulation of the growth plate. Nature, 2003, 423: 332-336

[7] Long F, Ornitz DM. Development of the endochondral skeleton. Cold Spring Harb. Perspect. Biol., 2013, 5: a008334

[8] Stegen S, Carmeliet G. Hypoxia, hypoxia-inducible transcription factors and oxygen-sensing prolyl hydroxylases in bone development and homeostasis. Curr. Opin. Nephrol. Hypertens., 2019, 28: 328-335

[9] van Gastel N et al. Lipid availability determines fate of skeletal progenitor cells via SOX9. Nature, 2020, 579: 111-117

[10] Stegen S et al. Glutamine metabolism controls chondrocyte identity and function. Dev. Cell, 2020, 53: e538

[11] Lee SY, Abel ED, Long F. Glucose metabolism induced by Bmp signaling is essential for murine skeletal development. Nat. Commun., 2018, 9

[12] Stegen S et al. HIF-1alpha metabolically controls collagen synthesis and modification in chondrocytes. Nature, 2019, 565: 511-515

[13] Locasale JW. Serine, glycine and one-carbon units: cancer metabolism in full circle. Nat. Rev. Cancer, 2013, 13: 572-583

[14] Yang M, Vousden KH. Serine and one-carbon metabolism in cancer. Nat. Rev. Cancer, 2016, 16: 650-662

[15] Mattaini KR, Sullivan MR, Vander Heiden MG. The importance of serine metabolism in cancer. J. Cell Biol., 2016, 214: 249-257

[16] Newman AC, Maddocks ODK. Serine and functional metabolites in cancer. Trends Cell Biol., 2017, 27: 645-657

[17] Schibler L et al. New insight on FGFR3-related chondrodysplasias molecular physiopathology revealed by human chondrocyte gene expression profiling. PLoS One, 2009, 4: e7633

[18] De la Fuente, A. et al. Proteome analysis during chondrocyte differentiation in a new chondrogenesis model using human umbilical cord stroma mesenchymal stem cells. Mol Cell Proteom. 11, M111.010496 (2012).

[19] van Gastel N et al. Engineering vascularized bone: osteogenic and proangiogenic potential of murine periosteal cells. Stem Cells, 2012, 30: 2460-2471

[20] Lefebvre V, Angelozzi M, Haseeb A. SOX9 in cartilage development and disease. Curr. Opin. Cell Biol., 2019, 61: 39-47

[21] Locasale JW et al. Phosphoglycerate dehydrogenase diverts glycolytic flux and contributes to oncogenesis. Nat. Genet., 2011, 43: 869-874

[22] Possemato R et al. Functional genomics reveal that the serine synthesis pathway is essential in breast cancer. Nature, 2011, 476: 346-350

[23] Vandekeere S et al. Serine synthesis via PHGDH is essential for heme production in endothelial cells. Cell Metab., 2018, 28: 573-587.e513

[24] Zhu J, Thompson CB. Metabolic regulation of cell growth and proliferation. Nat. Rev. Mol. Cell Biol., 2019, 20: 436-450

[25] Keibler MA et al. Metabolic requirements for cancer cell proliferation. Cancer Metab., 2016, 4: 16

[26] Lane AN, Fan TW. Regulation of mammalian nucleotide metabolism and biosynthesis. Nucleic Acids Res., 2015, 43: 2466-2485

[27] Reid MA et al. Serine synthesis through PHGDH coordinates nucleotide levels by maintaining central carbon metabolism. Nat. Commun., 2018, 9

[28] 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, 2014, 32: 2407-2418

[29] Ye J et al. Pyruvate kinase M2 promotes de novo serine synthesis to sustain mTORC1 activity and cell proliferation. Proc. Natl. Acad. Sci. USA, 2012, 109: 6904-6909

[30] DeNicola GM et al. NRF2 regulates serine biosynthesis in non-small cell lung cancer. Nat. Genet., 2015, 47: 1475-1481

[31] Zhao E et al. KDM4C and ATF4 cooperate in transcriptional control of amino acid metabolism. Cell Rep., 2016, 14: 506-519

[32] Adams CM. Role of the transcription factor ATF4 in the anabolic actions of insulin and the anti-anabolic actions of glucocorticoids. J. Biol. Chem., 2007, 282: 16744-16753

[33] Nigdelioglu R et al. Transforming growth factor (TGF)-beta promotes de novo serine synthesis for collagen production. J. Biol. Chem., 2016, 291: 27239-27251

[34] Cooper KL et al. Multiple phases of chondrocyte enlargement underlie differences in skeletal proportions. Nature, 2013, 495: 375-378

[35] Maddocks OD et al. Serine starvation induces stress and p53-dependent metabolic remodelling in cancer cells. Nature, 2013, 493: 542-546

[36] Wang W et al. Atf4 regulates chondrocyte proliferation and differentiation during endochondral ossification by activating Ihh transcription. Development, 2009, 136: 4143-4153

[37] Stegen S, van Gastel N, Carmeliet G. Bringing new life to damaged bone: the importance of angiogenesis in bone repair and regeneration. Bone, 2015, 70: 19-27

[38] Yoshida K et al. Targeted disruption of the mouse 3-phosphoglycerate dehydrogenase gene causes severe neurodevelopmental defects and results in embryonic lethality. J. Biol. Chem., 2004, 279: 3573-3577

[39] Ovchinnikov DA, Deng JM, Ogunrinu G, Behringer RR. Col2a1-directed expression of Cre recombinase in differentiating chondrocytes in transgenic mice. Genesis, 2000, 26: 145-146

[40] Guo W et al. Slug and Sox9 cooperatively determine the mammary stem cell state. Cell, 2012, 148: 1015-1028

[41] Maes C et al. Placental growth factor mediates mesenchymal cell development, cartilage turnover, and bone remodeling during fracture repair. J. Clin. Invest., 2006, 116: 1230-1242

[42] Sullivan MR et al. Increased serine synthesis provides an advantage for tumors arising in tissues where serine levels are limiting. Cell Metab., 2019, 29: 1410-1421.e1414

[43] Stegen S et al. Glutamine metabolism in osteoprogenitors is required for bone mass accrual and PTH-induced bone anabolism in male mice. J. Bone Min. Res., 2021, 36: 604-616

[44] Whittier DE et al. Guidelines for the assessment of bone density and microarchitecture in vivo using high-resolution peripheral quantitative computed tomography. Osteoporos. Int., 2020, 31: 1607-1627

Funding

Fund for Scientific Research-Flanders: G.0A42.16 Fund for Scientific Research-Flanders: G.0B34.18 Fund for Scientific Research-Falnders: G.0C51.20

Fund for Scientific Research-Flanders: 12H5917N

Fund for Scientific Research-Flanders: 1S46318N

Fund for Scientific Research-Flanders: G.0B3418

ERC Advanced Resesarch Grant: EU-ERC743074