FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3128816991> ?p ?o ?g. }

• W3128816991 endingPage "108723" @default.
• W3128816991 startingPage "108723" @default.
• W3128816991 abstract "•Glutamine-dependent OXPHOS drives early erythroid differentiation•OXPHOS-induced ROS inhibit erythroblast enucleation•IDH1 downregulation augments ROS, leading to pathological erythroid differentiation•Vitamin C rescues erythroid differentiation under conditions of oxidative stress The metabolic changes controlling the stepwise differentiation of hematopoietic stem and progenitor cells (HSPCs) to mature erythrocytes are poorly understood. Here, we show that HSPC development to an erythroid-committed proerythroblast results in augmented glutaminolysis, generating alpha-ketoglutarate (αKG) and driving mitochondrial oxidative phosphorylation (OXPHOS). However, sequential late-stage erythropoiesis is dependent on decreasing αKG-driven OXPHOS, and we find that isocitrate dehydrogenase 1 (IDH1) plays a central role in this process. IDH1 downregulation augments mitochondrial oxidation of αKG and inhibits reticulocyte generation. Furthermore, IDH1 knockdown results in the generation of multinucleated erythroblasts, a morphological abnormality characteristic of myelodysplastic syndrome and congenital dyserythropoietic anemia. We identify vitamin C homeostasis as a critical regulator of ineffective erythropoiesis; oxidized ascorbate increases mitochondrial superoxide and significantly exacerbates the abnormal erythroblast phenotype of IDH1-downregulated progenitors, whereas vitamin C, scavenging reactive oxygen species (ROS) and reprogramming mitochondrial metabolism, rescues erythropoiesis. Thus, an IDH1-vitamin C crosstalk controls terminal steps of human erythroid differentiation. The metabolic changes controlling the stepwise differentiation of hematopoietic stem and progenitor cells (HSPCs) to mature erythrocytes are poorly understood. Here, we show that HSPC development to an erythroid-committed proerythroblast results in augmented glutaminolysis, generating alpha-ketoglutarate (αKG) and driving mitochondrial oxidative phosphorylation (OXPHOS). However, sequential late-stage erythropoiesis is dependent on decreasing αKG-driven OXPHOS, and we find that isocitrate dehydrogenase 1 (IDH1) plays a central role in this process. IDH1 downregulation augments mitochondrial oxidation of αKG and inhibits reticulocyte generation. Furthermore, IDH1 knockdown results in the generation of multinucleated erythroblasts, a morphological abnormality characteristic of myelodysplastic syndrome and congenital dyserythropoietic anemia. We identify vitamin C homeostasis as a critical regulator of ineffective erythropoiesis; oxidized ascorbate increases mitochondrial superoxide and significantly exacerbates the abnormal erythroblast phenotype of IDH1-downregulated progenitors, whereas vitamin C, scavenging reactive oxygen species (ROS) and reprogramming mitochondrial metabolism, rescues erythropoiesis. Thus, an IDH1-vitamin C crosstalk controls terminal steps of human erythroid differentiation. Hematopoietic stem cell (HSC) numbers as well as their differentiation state are tightly regulated throughout the lifetime of an individual, allowing the sustained production of all mature blood lineages under physiological conditions. Circulating mature erythrocytes are a terminally differentiated product of HSCs that have undergone a series of lineage-fate engagements that gradually restrict their potential, resulting in a commitment to the erythroid lineage. Commitment defines the beginning of erythropoiesis; a three-stage process characterized by early erythropoiesis, terminal erythroid differentiation, and reticulocyte maturation. Early erythropoiesis is characterized by commitment of multilineage progenitor cells into erythroid progenitor cells, with proliferation and development into erythroid burst-forming unit cells, followed by erythroid colony-forming unit cells and proerythroblasts. Terminal erythroid differentiation begins with proerythroblasts differentiating in a stepwise manner to early then late basophilic erythroblasts, polychromatic erythroblasts, and orthochromatic erythroblasts that enucleate to become reticulocytes. Erythropoiesis is a metabolically daunting process when evaluated at the level of cell numbers. In healthy adults, committed erythroid progenitors support the production of 2.4 million erythrocytes per second via a synchronized regulation of iron, glucose, fatty acid, and amino acid metabolism. Iron is indispensable for heme biosynthesis in erythroblasts (Oburoglu et al., 2016Oburoglu L. Romano M. Taylor N. Kinet S. Metabolic regulation of hematopoietic stem cell commitment and erythroid differentiation.Curr. Opin. Hematol. 2016; 23: 198-205Crossref PubMed Scopus (36) Google Scholar). The utilization of both glutamine and glucose in de novo nucleotide biosynthesis is a sine qua non for erythroid differentiation (Oburoglu et al., 2014Oburoglu L. Tardito S. Fritz V. de Barros S.C. Merida P. Craveiro M. Mamede J. Cretenet G. Mongellaz C. An X. et al.Glucose and glutamine metabolism regulate human hematopoietic stem cell lineage specification.Cell Stem Cell. 2014; 15: 169-184Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), and glutamine-derived production of succinyl-coenzyme A (succinyl-CoA) is also required for the production of heme (Burch et al., 2018Burch J.S. Marcero J.R. Maschek J.A. Cox J.E. Jackson L.K. Medlock A.E. Phillips J.D. Dailey Jr., H.A. Glutamine via α-ketoglutarate dehydrogenase provides succinyl-CoA for heme synthesis during erythropoiesis.Blood. 2018; 132: 987-998Crossref PubMed Scopus (11) Google Scholar). Furthermore, amino acids regulate mTOR signaling (Chung et al., 2015Chung J. Bauer D.E. Ghamari A. Nizzi C.P. Deck K.M. Kingsley P.D. Yien Y.Y. Huston N.C. Chen C. Schultz I.J. et al.The mTORC1/4E-BP pathway coordinates hemoglobin production with L-leucine availability.Sci. Signal. 2015; 8: ra34Crossref PubMed Scopus (33) Google Scholar) as well as lipid metabolism (Huang et al., 2018Huang N.J. Lin Y.C. Lin C.Y. Pishesha N. Lewis C.A. Freinkman E. Farquharson C. Millán J.L. Lodish H. Enhanced phosphocholine metabolism is essential for terminal erythropoiesis.Blood. 2018; 131: 2955-2966Crossref PubMed Scopus (10) Google Scholar) during erythropoiesis. The critical nature of metabolism in erythroid differentiation is further highlighted by the recent identification of “metabolic regulators” as an erythropoietin (EPO)-induced phosphorylation target set (Held et al., 2020Held M.A. Greenfest-Allen E. Jachimowicz E. Stoeckert C.J. Stokes M.P. Wood A.W. Wojchowski D.M. Phospho-proteomic discovery of novel EPO signal transducers including thioredoxin-interacting protein as a mediator of EPO-dependent human erythropoiesis.Exp. Hematol. 2020; 84: 29-44Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). It is important though to note that terminal erythroid differentiation is a distinctive process; each mitosis results in the production of daughter cells that differ, morphologically and functionally, from their parent cells. This sequential maturation is tightly regulated at each stage of erythroid differentiation, associated with decreased cell size, enhanced chromatin condensation, progressive hemoglobinization, and changes in membrane organization and transcriptome specificities (An et al., 2014An X. Schulz V.P. Li J. Wu K. Liu J. Xue F. Hu J. Mohandas N. Gallagher P.G. Global transcriptome analyses of human and murine terminal erythroid differentiation.Blood. 2014; 123: 3466-3477Crossref PubMed Scopus (160) Google Scholar; Hu et al., 2013Hu J. Liu J. Xue F. Halverson G. Reid M. Guo A. Chen L. Raza A. Galili N. Jaffray J. et al.Isolation and functional characterization of human erythroblasts at distinct stages: implications for understanding of normal and disordered erythropoiesis in vivo.Blood. 2013; 121: 3246-3253Crossref PubMed Scopus (159) Google Scholar; Li et al., 2014Li J. Hale J. Bhagia P. Xue F. Chen L. Jaffray J. Yan H. Lane J. Gallagher P.G. Mohandas N. et al.Isolation and transcriptome analyses of human erythroid progenitors: BFU-E and CFU-E.Blood. 2014; 124: 3636-3645Crossref PubMed Scopus (81) Google Scholar; Ludwig et al., 2019Ludwig L.S. Lareau C.A. Bao E.L. Nandakumar S.K. Muus C. Ulirsch J.C. Chowdhary K. Buenrostro J.D. Mohandas N. An X. et al.Transcriptional states and chromatin accessibility underlying human erythropoiesis.Cell Rep. 2019; 27: 3228-3240.e7Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar; Schulz et al., 2019Schulz V.P. Yan H. Lezon-Geyda K. An X. Hale J. Hillyer C.D. Mohandas N. Gallagher P.G. A unique epigenomic landscape defines human erythropoiesis.Cell Rep. 2019; 28: 2996-3009.e7Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Moreover, during the late stages of mammalian terminal erythroid differentiation, erythroblasts expel their nuclei and lose all organelles, including mitochondria (Moras et al., 2017Moras M. Lefevre S.D. Ostuni M.A. From erythroblasts to mature red blood cells: organelle clearance in mammals.Front. Physiol. 2017; 8: 1076Crossref PubMed Scopus (60) Google Scholar). Thus, the constraints of a late-stage terminally differentiating erythroblast differ significantly from that of an erythroid-committed progenitor early in erythroid development. Indeed, in mice, early-stage erythroid progenitors have been found to require mTORC1-mediated mitochondrial biogenesis and reactive oxygen species (ROS) production (Liu et al., 2017Liu X. Zhang Y. Ni M. Cao H. Signer R.A.J. Li D. Li M. Gu Z. Hu Z. Dickerson K.E. et al.Regulation of mitochondrial biogenesis in erythropoiesis by mTORC1-mediated protein translation.Nat. Cell Biol. 2017; 19: 626-638Crossref PubMed Scopus (60) Google Scholar; Luo et al., 2017Luo S.T. Zhang D.M. Qin Q. Lu L. Luo M. Guo F.C. Shi H.S. Jiang L. Shao B. Li M. et al.The promotion of erythropoiesis via the regulation of reactive oxygen species by lactic acid.Sci. Rep. 2017; 7: 38105Crossref PubMed Scopus (15) Google Scholar; Zhao et al., 2016Zhao B. Mei Y. Yang J. Ji P. Erythropoietin-regulated oxidative stress negatively affects enucleation during terminal erythropoiesis.Exp. Hematol. 2016; 44: 975-981Abstract Full Text Full Text PDF PubMed Google Scholar), while terminal erythropoiesis requires that cells be protected from oxidative stress (Case et al., 2013Case A.J. Madsen J.M. Motto D.G. Meyerholz D.K. Domann F.E. Manganese superoxide dismutase depletion in murine hematopoietic stem cells perturbs iron homeostasis, globin switching, and epigenetic control in erythrocyte precursor cells.Free Radic. Biol. Med. 2013; 56: 17-27Crossref PubMed Scopus (24) Google Scholar; Filippi and Ghaffari, 2019Filippi M.D. Ghaffari S. Mitochondria in the maintenance of hematopoietic stem cells: new perspectives and opportunities.Blood. 2019; 133: 1943-1952Crossref PubMed Scopus (20) Google Scholar; Hyde et al., 2012Hyde B.B. Liesa M. Elorza A.A. Qiu W. Haigh S.E. Richey L. Mikkola H.K. Schlaeger T.M. Shirihai O.S. The mitochondrial transporter ABC-me (ABCB10), a downstream target of GATA-1, is essential for erythropoiesis in vivo.Cell Death Differ. 2012; 19: 1117-1126Crossref PubMed Scopus (0) Google Scholar; Xu et al., 2019Xu P. Palmer L.E. Lechauve C. Zhao G. Yao Y. Luan J. Vourekas A. Tan H. Peng J. Schuetz J.D. et al.Regulation of gene expression by miR-144/451 during mouse erythropoiesis.Blood. 2019; 133: 2518-2528Crossref PubMed Scopus (5) Google Scholar; Zhao et al., 2016Zhao B. Mei Y. Yang J. Ji P. Erythropoietin-regulated oxidative stress negatively affects enucleation during terminal erythropoiesis.Exp. Hematol. 2016; 44: 975-981Abstract Full Text Full Text PDF PubMed Google Scholar). Significant differences in murine versus human erythroid development have been documented, especially as relates to the diversity of erythroid progenitor subpopulations in human bone marrow (BM) (Gautier et al., 2020Gautier E.F. Leduc M. Ladli M. Schulz V.P. Lefèvre C. Boussaid I. Fontenay M. Lacombe C. Verdier F. Guillonneau F. et al.Comprehensive proteomic analysis of murine terminal erythroid differentiation.Blood Adv. 2020; 4: 1464-1477Crossref PubMed Scopus (8) Google Scholar; Schulz et al., 2019Schulz V.P. Yan H. Lezon-Geyda K. An X. Hale J. Hillyer C.D. Mohandas N. Gallagher P.G. A unique epigenomic landscape defines human erythropoiesis.Cell Rep. 2019; 28: 2996-3009.e7Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar; Yan et al., 2018Yan H. Hale J. Jaffray J. Li J. Wang Y. Huang Y. An X. Hillyer C. Wang N. Kinet S. et al.Developmental differences between neonatal and adult human erythropoiesis.Am. J. Hematol. 2018; 93: 494-503Crossref PubMed Scopus (13) Google Scholar). Furthermore, regarding oxidative stress, it is important to note that murine and human erythropoiesis differ dramatically as a function of vitamin C/ascorbate plasma concentrations and expression of the SLC2A1/GLUT1 transporter, shuttling dehydroascorbic acid (DHA) across the membrane and rapidly reducing it to ascorbate (reviewed by May et al., 2001May J.M. Qu Z. Morrow J.D. Mechanisms of ascorbic acid recycling in human erythrocytes.Biochim. Biophys. Acta. 2001; 1528: 159-166Crossref PubMed Scopus (65) Google Scholar). Indeed, humans exhibit a deficiency in vitamin C production, due to inactivation of L-gulono-γ-lactone oxidase (GLO), the enzyme that catalyzes the terminal step of L-ascorbic acid (AA) biosynthesis (Burns, 1957Burns J.J. Missing step in man, monkey and guinea pig required for the biosynthesis of L-ascorbic acid.Nature. 1957; 180: 553Crossref PubMed Scopus (0) Google Scholar). While human erythrocytes express the highest level of the GLUT1 transporter, harboring greater than 200,000 molecules per cell (Helgerson and Carruthers, 1987Helgerson A.L. Carruthers A. Equilibrium ligand binding to the human erythrocyte sugar transporter. Evidence for two sugar-binding sites per carrier.J. Biol. Chem. 1987; 262: 5464-5475Abstract Full Text PDF PubMed Google Scholar; Mueckler, 1994Mueckler M. Facilitative glucose transporters.Eur. J. Biochem. 1994; 219: 713-725Crossref PubMed Google Scholar), we found that erythroid GLUT1 expression is a specific feature of mammals that have lost the ability to synthesize AA from glucose (Montel-Hagen et al., 2008aMontel-Hagen A. Blanc L. Boyer-Clavel M. Jacquet C. Vidal M. Sitbon M. Taylor N. The Glut1 and Glut4 glucose transporters are differentially expressed during perinatal and postnatal erythropoiesis.Blood. 2008; 112: 4729-4738Crossref PubMed Scopus (50) Google Scholar, Montel-Hagen et al., 2008bMontel-Hagen A. Kinet S. Manel N. Mongellaz C. Prohaska R. Battini J.L. Delaunay J. Sitbon M. Taylor N. Erythrocyte Glut1 triggers dehydroascorbic acid uptake in mammals unable to synthesize vitamin C.Cell. 2008; 132: 1039-1048Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). This potential evolutionary compensation, and subsequent alterations in the ability of plasma vitamin C to scavenge ROS/reactive nitrogen species (RNS), make it critical to evaluate the stepwise changes that regulate the progression of human erythropoiesis. Here, we demonstrate that during the early stages of human red blood cell development, erythroid progenitors exhibit increased oxidative phosphorylation (OXPHOS) activity. This correlated with the increased generation of the alpha-ketoglutarate (αKG) TCA-cycle intermediate, generated by the anaplerotic utilization of glutamine, and a directly augmented OXPHOS. However, upon terminal differentiation of erythroblasts, mitochondrial biomass, OXPHOS, mitochondrial ROS, and superoxide production were all markedly decreased. Supraphysiological levels of αKG markedly attenuated terminal erythroid differentiation and enucleation. The impact of αKG was directly coupled to its role in mitochondrial metabolism, as enucleation was similarly inhibited by mitochondrial ROS and superoxide, induced by MitoParaquat (MitoPQ) or DHA. Moreover, enucleation was rescued by ROS scavengers, including vitamin C, glutathione (GSH), N-acetylcysteine (NAC), and vitamin E. We identified the cytoplasmic isocitrate dehydrogenase 1 (IDH1) enzyme, catalyzing the interconversion between αKG and isocitrate, as a critical enzyme in this process. Lentiviral-mediated downregulation of IDH1, by significantly increasing mitochondrial metabolism and mitochondrial superoxide production, dramatically inhibited enucleation and resulted in the generation of abnormal multinucleated erythroblasts. Notably, both αKG and DHA markedly exacerbated the negative impact of IDH1 downregulation, while the vitamin C antioxidant rescued erythroid differentiation. Indeed, the vitamin-C-mediated quenching of ROS in erythroblasts accelerated human erythroid differentiation. This effect was specific to an IDH1-vitamin C axis; inhibition of TET2 in late-stage erythroblasts did not attenuate enucleation. Thus, our data identify IDH1 as a critical regulator of redox homeostasis, promoting late-stage erythropoiesis. Furthermore, these results highlight the therapeutic potential of vitamin C in human erythropoiesis, especially under pathological conditions where ROS are increased and vitamin C levels are not sufficient to combat oxidative damage. As glutaminolysis is required for erythroid lineage specification (Oburoglu et al., 2014Oburoglu L. Tardito S. Fritz V. de Barros S.C. Merida P. Craveiro M. Mamede J. Cretenet G. Mongellaz C. An X. et al.Glucose and glutamine metabolism regulate human hematopoietic stem cell lineage specification.Cell Stem Cell. 2014; 15: 169-184Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), we evaluated the utilization of glutamine during early stages of human erythropoiesis at day 4 following recombinant erythropoietin (rEPO) stimulation, a time point at which the erythroid markers CD36, CD71 (transferrin receptor), and glycophorin A (GlyA) are upregulated (Figure S1A). The anaplerotic contribution of glutamine can be evaluated by tracing glutamine labeled with heavy stable carbons and nitrogens (13C515N2). As shown in Figure 1A, the portion of glutamate derived from 13C515N2 glutamine, a measure of glutaminase activity, increased significantly following erythropoietin-induced erythroid differentiation of human CD34+ progenitors (p < 0.05; Figure 1A). Additionally, the contribution of labeled glutamine to the pool of glutamate (13C515N1 glutamate/13C0 glutamate) as well as the portion of 13C5 labeled αKG increased significantly upon erythroid differentiation. This glutamate-derived generation of αKG was critical for OXPHOS in erythroid progenitors, as the oxygen consumption rate (OCR) was abrogated by the aminooxyacetic acid (AOA) transaminase inhibitor, blocking conversion of glutamate to αKG, and was completely restored by αKG supplementation (Figure 1B). These data show that glutamine supports the tricarboxylic acid (TCA) cycle and downstream OXPHOS via the cataplerotic utilization of αKG. As each stage of terminal erythroid differentiation is defined by a decreased cell size with the final stage of erythropoiesis resulting in enucleation and reticulocyte maturation, we assessed whether mitochondrial biomass and transmembrane polarization decrease following commitment of progenitors to an erythroid lineage fate. Indeed, both mitochondrial biomass and polarization, evaluated by MitoGreen and MitoRed staining, respectively, decreased massively upon erythroid differentiation, monitored as a function of day of differentiation (days 0–10) as well as on erythroblast subsets fluorescence-activated cell sorting (FACS) sorted on the basis of their CD49d/GLUT1 staining profiles (p < 0.05 to p < 0.0001; Figures 1C, 1D, and S1B). These changes in mitochondrial biomass and polarization had functional consequences on terminal erythroid differentiation (Figure 1D). Erythroblasts at distinct stages of differentiation exhibited significant differences in their levels of OXPHOS, as estimated by their OCRs; basal OCR decreased from 146.9 ± 15.5 to 17.5 ± 0.6 pmol O2/min between the proerythroblast and orthochromatic erythroblast stages of differentiation (p < 0.0001; Figure 1E). Notably, the respiratory capacities of both polychromatic and orthochromatic subsets were minimal; neither of these terminal subsets exhibited a spare respiratory capacity, as measured by the ability of the cell to increase its respiration in response to an Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP)-mediated disruption of mitochondrial membrane potential (Figure 1E). Similarly, these differences were also detected with increasing time after EPO induction (Figure S1C). Consistent with a critical role for glutamine in the oxidative potential of the erythroblast, glutamine uptake was significantly higher in proerythroblasts and basophilic erythroblasts than in polychromatic/orthochromatic erythroblasts (p < 0.01; Figure 1F). Thus, in marked contrast with the high-energy state of early erythroid progenitors, terminal maturation results in a significant attenuation of glutamine uptake and an associated decrease in OXPHOS. As decreased mitochondrial function of terminally differentiated erythroblasts was associated with decreased glutamine uptake, we evaluated whether αKG, the anaplerotic product of glutamine that enters into the TCA cycle, directly contributed to the erythroblast’s respiratory capacity. Importantly, we found that αKG directly regulates the metabolic state of progenitors, as the injection of a cell-permeable αKG immediately augmented the maximal respiratory capacity of erythroblasts at both day 3 and day 7 of EPO induction (p < 0.001 and p < 0.0001; Figure 2A). This increase in OXPHOS did not alter the generation of CD34−CD36+ committed erythroid colony-forming unit (CFU-E) progenitors or GlyA+ erythroblasts or the upregulation of CD36 or CD71 erythroid markers (Figures 2B and S2A). While αKG did not alter mitochondrial biomass or polarization at early stages of development (day 3; Figure S2B), it substantially affected these parameters when it was added only at later stages (from days 7 to 10). Mitochondrial biomass and polarization were significantly higher in αKG-supplemented late-stage erythroblasts than in control erythroblasts (p < 0.01 and p < 0.001; Figure 2C). Furthermore, progression from the CD49d+GLUT1+ basophilic erythroblast to the CD49d−Glut1+ orthochromatic stage was significantly inhibited by αKG (p < 0.05 and p < 0.01; Figure 2D). This phenomenon was also associated with a significantly attenuated level of enucleation, decreasing by 32% ± 7% and 52% ± 7% in the presence of αKG at days 10 and 14 of erythroid differentiation, respectively (p < 0.001 and p < 0.01; Figure 2D). Notably, this effect was specific to αKG and was not recapitulated by other TCA-cycle intermediates such as succinate or citrate (Figure S2C). Generation of ROS within mitochondria is associated with the oxidative function of these organelles (Adam-Vizi and Tretter, 2013Adam-Vizi V. Tretter L. The role of mitochondrial dehydrogenases in the generation of oxidative stress.Neurochem. Int. 2013; 62: 757-763Crossref PubMed Scopus (47) Google Scholar; Murphy, 2009Murphy M.P. How mitochondria produce reactive oxygen species.Biochem. J. 2009; 417: 1-13Crossref PubMed Scopus (4029) Google Scholar), and αKG can directly contribute to ROS generation through the catalytic activity of αKG dehydrogenase (αKGDH) (Starkov et al., 2004Starkov A.A. Fiskum G. Chinopoulos C. Lorenzo B.J. Browne S.E. Patel M.S. Beal M.F. Mitochondrial alpha-ketoglutarate dehydrogenase complex generates reactive oxygen species.J. Neurosci. 2004; 24: 7779-7788Crossref PubMed Scopus (495) Google Scholar; Tretter and Adam-Vizi, 2004Tretter L. Adam-Vizi V. Generation of reactive oxygen species in the reaction catalyzed by alpha-ketoglutarate dehydrogenase.J. Neurosci. 2004; 24: 7771-7778Crossref PubMed Scopus (312) Google Scholar). NADH produced by αKGDH also stimulates superoxide production by complex I of the electron transport chain (ETC; Figure 3A) (Adam-Vizi and Tretter, 2013Adam-Vizi V. Tretter L. The role of mitochondrial dehydrogenases in the generation of oxidative stress.Neurochem. Int. 2013; 62: 757-763Crossref PubMed Scopus (47) Google Scholar; Murphy, 2009Murphy M.P. How mitochondria produce reactive oxygen species.Biochem. J. 2009; 417: 1-13Crossref PubMed Scopus (4029) Google Scholar). We therefore evaluated whether addition of αKG, the substrate of αKGDH, alters the redox state of the cell. Direct measurement of mitochondrial superoxide with the fluorescent MitoSOX indicator showed that αKG induced a 1.3-fold increase in superoxide (p < 0.01) as well as an overall increase of 1.3-fold in all mitochondrial ROS (p < 0.001), monitored as a function of DHR123 fluorescence (MitoROS) (Figure 3B). These data suggested that the negative impact of αKG on erythroid maturation was linked to its role in altering the erythroblast redox state. We therefore assessed whether directly increasing mitochondrial superoxide would inhibit erythroid maturation. Erythroblasts were treated with MitoPQ, a newly synthesized paraquat compound that is targeted to the mitochondria by conjugation to the lipophilic tri-phenylphosphonium cation (Robb et al., 2015Robb E.L. Gawel J.M. Aksentijević D. Cochemé H.M. Stewart T.S. Shchepinova M.M. Qiang H. Prime T.A. Bright T.P. James A.M. et al.Selective superoxide generation within mitochondria by the targeted redox cycler MitoParaquat.Free Radic. Biol. Med. 2015; 89: 883-894Crossref PubMed Google Scholar). This compound selectively increases superoxide production within mitochondria at 1,000-fold higher efficiency than untargeted paraquat (PQ) (Robb et al., 2015Robb E.L. Gawel J.M. Aksentijević D. Cochemé H.M. Stewart T.S. Shchepinova M.M. Qiang H. Prime T.A. Bright T.P. James A.M. et al.Selective superoxide generation within mitochondria by the targeted redox cycler MitoParaquat.Free Radic. Biol. Med. 2015; 89: 883-894Crossref PubMed Google Scholar); within the mitochondria, mitoPQ2+ is reduced to the radical monocation at the flavin site of complex I and the monocation then reacts with O2 to generate superoxide (O2−) (Figure 3C). MitoPQ dramatically increased mitochondrial superoxide (MitoSOX), MitoROS, and mitochondrial biomass (MitoGreen) in differentiating erythroblasts (p < 0.01 and p < 0.05; Figure 3D). The impact of both MitoPQ and αKG was specific to mitochondrial ROS, as total intracellular ROS levels were not significantly changed (Figures S3A and S3B). Notably, short-term MitoPQ treatment significantly altered late erythropoiesis, with a 25% ± 5% decrease in erythroblast enucleation (p < 0.01; Figure 3E). To assess whether mitochondrial superoxide production has similar effects on erythroblasts at different stages of differentiation, we FACS-sorted early basophilic and late orthochromatic erythroblasts on the basis of their GLUT1/CD49d profiles. Representative cytospins are shown in Figure 3F. Notably, MitoPQ dramatically decreased the ability of basophilic erythroblasts to differentiate to reticulocytes, attenuating enucleation by 94% ± 3% (p < 0.001; Figure 3F). In contrast, the maturation of orthochromatic erythroblasts was not affected by MitoPQ treatment (Figure 3F). These data show that a cell type that exhibits only very minimal OXPHOS potential, such as an orthochromatic erythroblast (Figure 1E), is not sensitive to a drug whose impact is dependent on mitochondrial function. In contrast, selectively increasing superoxide production in the mitochondrial matrix of an early erythroblast significantly inhibits erythroid maturation, revealing the critical role of this pathway in the proper progression of erythroid differentiation. As we found that oxidative stress, monitored as a function of mitochondrial superoxide production and OXPHOS, was associated with attenuated late-stage erythroid differentiation and enucleation, it was of interest to determine whether these parameters were directly responsible for these observed effects. Vitamin C has the potential to promote a redox environment, but its activity is countered by the oxidized form of vitamin C, DHA. The rapid reduction of intracellular DHA to vitamin C (Figure 4A) results in concomitant increases in endogenous ROS in tumor cell lines (Kc et al., 2005Kc S. Cárcamo J.M. Golde D.W. Vitamin C enters mitochondria via facilitative glucose transporter 1 (Glut1) and confers mitochondrial protection against oxidative injury.FASEB J. 2005; 19: 1657-1667Crossref PubMed Scopus (0) Google Scholar; Yun et al., 2015Yun J. Mullarky E. Lu C. Bosch K.N. Kavalier A. Rivera K. Roper J. Chio I.I. Giannopoulou E.G. Rago C. et al.Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH.Science. 2015; 350: 1391-1396Crossref PubMed Scopus (390) Google Scholar), but its role in erythroid cells is not known. Of note, DHA is transported via the GLUT1 glucose transporter, the most highly expressed transporter on human erythrocytes (Bianchi and Rose, 1986Bianchi J. Rose R.C. Glucose-independent transport of dehydroascorbic acid in human erythrocytes.Proc. Soc. Exp. Biol. Med. 1986; 181: 333-337Crossref PubMed Google Scholar; Helgerson and Carruthers, 1987Helgerson A.L. Carruthers A. Equilibrium ligand binding to the human erythrocyte sugar transporter. Evidence for two sugar-binding sites per carrier.J. Biol. Chem. 1987; 262: 5464-5475Abstract Full Text PDF PubMed Google Scholar; May, 1998May J.M. Ascorbate function and metabolism in the human erythrocyte.Front. Biosci. 1998; 3: d1-d10Crossref PubMed Google Scholar; Montel-Hagen et al., 2008bMontel-Hagen A. Kinet S. Manel N. Mongellaz C. Prohaska R. Battini J.L. Delaunay J. Sitbon M. Taylor N. Erythrocyte Glut1 triggers dehydroascorbic acid uptake in mammals unable to synthesize vitamin C.Cell. 2008; 132: 1039-1048Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, Montel-Hagen et al., 2009Montel-Hagen A. Si" @default.
• W3128816991 created "2021-02-15" @default.
• W3128816991 creator A5000821531 @default.
• W3128816991 creator A5001014952 @default.
• W3128816991 creator A5003583446 @default.
• W3128816991 creator A5004749543 @default.
• W3128816991 creator A5005174262 @default.
• W3128816991 creator A5008043966 @default.
• W3128816991 creator A5024575675 @default.
• W3128816991 creator A5025903107 @default.
• W3128816991 creator A5027053225 @default.
• W3128816991 creator A5029112093 @default.
• W3128816991 creator A5031967403 @default.
• W3128816991 creator A5034746599 @default.
• W3128816991 creator A5034912242 @default.
• W3128816991 creator A5036219613 @default.
• W3128816991 creator A5042127314 @default.
• W3128816991 creator A5042844951 @default.
• W3128816991 creator A5045924865 @default.
• W3128816991 creator A5049012918 @default.
• W3128816991 creator A5062895477 @default.
• W3128816991 creator A5070298817 @default.
• W3128816991 creator A5070724314 @default.
• W3128816991 creator A5075558296 @default.
• W3128816991 creator A5081972452 @default.
• W3128816991 date "2021-02-01" @default.
• W3128816991 modified "2023-10-17" @default.
• W3128816991 title "An IDH1-vitamin C crosstalk drives human erythroid development by inhibiting pro-oxidant mitochondrial metabolism" @default.
• W3128816991 cites W123698486 @default.
• W3128816991 cites W1492408334 @default.
• W3128816991 cites W1517698775 @default.
• W3128816991 cites W1954131635 @default.
• W3128816991 cites W1964903272 @default.
• W3128816991 cites W1976052439 @default.
• W3128816991 cites W1978040580 @default.
• W3128816991 cites W1981160854 @default.
• W3128816991 cites W1983189827 @default.
• W3128816991 cites W1987455406 @default.
• W3128816991 cites W1987761030 @default.
• W3128816991 cites W1991563020 @default.
• W3128816991 cites W1993627126 @default.
• W3128816991 cites W1995091371 @default.
• W3128816991 cites W1997846723 @default.
• W3128816991 cites W1998293871 @default.
• W3128816991 cites W1998367819 @default.
• W3128816991 cites W2006938530 @default.
• W3128816991 cites W2008092267 @default.
• W3128816991 cites W2017203219 @default.
• W3128816991 cites W2018729297 @default.
• W3128816991 cites W2031959283 @default.
• W3128816991 cites W2034914128 @default.
• W3128816991 cites W2036297641 @default.
• W3128816991 cites W2037314139 @default.
• W3128816991 cites W2040716137 @default.
• W3128816991 cites W2043498565 @default.
• W3128816991 cites W2043729227 @default.
• W3128816991 cites W2044354693 @default.
• W3128816991 cites W2045651327 @default.
• W3128816991 cites W2050905343 @default.
• W3128816991 cites W2054424756 @default.
• W3128816991 cites W2058483875 @default.
• W3128816991 cites W2058618870 @default.
• W3128816991 cites W2059823229 @default.
• W3128816991 cites W2065401691 @default.
• W3128816991 cites W2070152906 @default.
• W3128816991 cites W2074516296 @default.
• W3128816991 cites W2075023533 @default.
• W3128816991 cites W2080452140 @default.
• W3128816991 cites W2084327512 @default.
• W3128816991 cites W2087814592 @default.
• W3128816991 cites W2091864583 @default.
• W3128816991 cites W2094703195 @default.
• W3128816991 cites W2095081648 @default.
• W3128816991 cites W2096686378 @default.
• W3128816991 cites W2097743943 @default.
• W3128816991 cites W2098264151 @default.
• W3128816991 cites W2098989605 @default.
• W3128816991 cites W2103974249 @default.
• W3128816991 cites W2104852749 @default.
• W3128816991 cites W2111086157 @default.
• W3128816991 cites W2115851778 @default.
• W3128816991 cites W2116516144 @default.
• W3128816991 cites W2122341467 @default.
• W3128816991 cites W2130202974 @default.
• W3128816991 cites W2132338390 @default.
• W3128816991 cites W2135470678 @default.
• W3128816991 cites W2141202000 @default.
• W3128816991 cites W2144542772 @default.
• W3128816991 cites W2147849026 @default.
• W3128816991 cites W2148847314 @default.
• W3128816991 cites W2155800445 @default.
• W3128816991 cites W2159177346 @default.
• W3128816991 cites W2159722630 @default.
• W3128816991 cites W2164059454 @default.
• W3128816991 cites W2169596495 @default.
• W3128816991 cites W2178620944 @default.
• W3128816991 cites W2185914603 @default.
• W3128816991 cites W2266362809 @default.

• next