Osteocytes act within a hypoxic environment to control key steps in bone formation.FGF23,a critical phosphate-regulating hormone,is stimulated by low oxygen/iron in acute and chronic diseases,however the molecular mec...Osteocytes act within a hypoxic environment to control key steps in bone formation.FGF23,a critical phosphate-regulating hormone,is stimulated by low oxygen/iron in acute and chronic diseases,however the molecular mechanisms directing this process remain unclear.Our goal was to identify the osteocyte factors responsible for FGF23 production driven by changes in oxygen/iron utilization.Hypoxia-inducible factor-prolyl hydroxylase inhibitors(HIF-PHI)which stabilize HIF transcription factors,increased Fgf23 in normal mice,as well as in osteocyte-like cells;in mice with conditional osteocyte Fgf23 deletion,circulating i FGF23 was suppressed.An inducible MSC cell line(‘MPC2’)underwent FG-4592 treatment and ATACseq/RNAseq,and demonstrated that differentiated osteocytes significantly increased HIF genomic accessibility versus progenitor cells.Integrative genomics also revealed increased prolyl hydroxylase Egln1(Phd2)chromatin accessibility and expression,which was positively associated with osteocyte differentiation.In mice with chronic kidney disease(CKD),Phd1-3 enzymes were suppressed,consistent with FGF23 upregulation in this model.Conditional loss of Phd2 from osteocytes in vivo resulted in upregulated Fgf23,in line with our findings that the MPC2 cell line lacking Phd2(CRISPR Phd2-KO cells)constitutively activated Fgf23 that was abolished by HIF1αblockade.In vitro,Phd2-KO cells lost iron-mediated suppression of Fgf23 and this activity was not compensated for by Phd1 or-3.In sum,osteocytes become adapted to oxygen/iron sensing during differentiation and are directly sensitive to bioavailable iron.Further,Phd2 is a critical mediator of osteocyte FGF23 production,thus our collective studies may provide new therapeutic targets for skeletal diseases involving disturbed oxygen/iron sensing.展开更多
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 a...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.展开更多
基金NIH grants F31-DK122679 and T32-HL007910(MLN)a postdoctoral research grant from the Research Foundation–Flanders(FWO/12H5917N)(SS)+6 种基金R01-AR074473(WRT)R21-AR059278,R01-DK112958,and R01-HL145528(KEW)The David Weaver Professorship(KEW)The Indiana University Melvin and Bren Simon Comprehensive Cancer Center FCRF is funded in part by NIHNational Cancer Institute(NCI)grant P30 CA082709National Institute of Diabetes and Digestive and Kidney Diseases(NIDDK)grant U54DK106846supported in part by NIH instrumentation grant 1S10D012270。
文摘Osteocytes act within a hypoxic environment to control key steps in bone formation.FGF23,a critical phosphate-regulating hormone,is stimulated by low oxygen/iron in acute and chronic diseases,however the molecular mechanisms directing this process remain unclear.Our goal was to identify the osteocyte factors responsible for FGF23 production driven by changes in oxygen/iron utilization.Hypoxia-inducible factor-prolyl hydroxylase inhibitors(HIF-PHI)which stabilize HIF transcription factors,increased Fgf23 in normal mice,as well as in osteocyte-like cells;in mice with conditional osteocyte Fgf23 deletion,circulating i FGF23 was suppressed.An inducible MSC cell line(‘MPC2’)underwent FG-4592 treatment and ATACseq/RNAseq,and demonstrated that differentiated osteocytes significantly increased HIF genomic accessibility versus progenitor cells.Integrative genomics also revealed increased prolyl hydroxylase Egln1(Phd2)chromatin accessibility and expression,which was positively associated with osteocyte differentiation.In mice with chronic kidney disease(CKD),Phd1-3 enzymes were suppressed,consistent with FGF23 upregulation in this model.Conditional loss of Phd2 from osteocytes in vivo resulted in upregulated Fgf23,in line with our findings that the MPC2 cell line lacking Phd2(CRISPR Phd2-KO cells)constitutively activated Fgf23 that was abolished by HIF1αblockade.In vitro,Phd2-KO cells lost iron-mediated suppression of Fgf23 and this activity was not compensated for by Phd1 or-3.In sum,osteocytes become adapted to oxygen/iron sensing during differentiation and are directly sensitive to bioavailable iron.Further,Phd2 is a critical mediator of osteocyte FGF23 production,thus our collective studies may provide new therapeutic targets for skeletal diseases involving disturbed oxygen/iron sensing.
基金supported by funding from the Research Foundation-Flanders(FWO:G.0A42.16, G.0B3418 and G0C5120N)the KU Leuven (C24/17/077)supported by long-term structural funding-Methusalem Funding by the Flemish Government and the European Research Council (ERC Advanced Research Grant EU-ERC743074)
文摘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.
