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Enhanced Phosphocholine Metabolism Is Essential for Terminal Erythropoiesis

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Enhanced Phosphocholine Metabolism Is Essential for Terminal Erythropoiesis Enhanced phosphocholine metabolism is essential for terminal erythropoiesis The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Huang, Nai-Jia et al. “Enhanced phosphocholine metabolism is essential for terminal erythropoiesis.” Blood 131 (2018): 2955-2966 © 2018 The Author(s) As Published 10.1182/BLOOD-2018-03-838516 Publisher American Society of Hematology Version Author's final manuscript Citable link https://hdl.handle.net/1721.1/125205 Terms of Use Creative Commons Attribution-Noncommercial-Share Alike Detailed Terms http://creativecommons.org/licenses/by-nc-sa/4.0/ From www.bloodjournal.org by guest on May 1, 2018. For personal use only. Blood First Edition Paper, prepublished online April 30, 2018; DOI 10.1182/blood-2018-03-838516 Enhanced phosphocholine metabolism is essential for terminal erythropoiesis Nai-Jia Huang1, Ying-Cing Lin2, Chung-Yueh Lin1,4, Novalia Pishesha1,3, Caroline A. Lewis1, Elizaveta Freinkman1, Colin Farquharson5, José Luis Millán6, Harvey Lodish*,1,3,4 1 Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA. 2Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139, USA 3 Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 4Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 5The Roslin Institute, University of Edinburgh, Easter Bush EH25 9RG, United Kingdom 6Sanford Children’s Health Research Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA * Corresponding author, Email address: [email protected] PHOSPHO1 regulates phosphocholine metabolism, ATP production, and amino acid supply during erythropoiesis 1 Copyright © 2018 American Society of Hematology From www.bloodjournal.org by guest on May 1, 2018. For personal use only. Abstract Red cells contain a unique constellation of membrane lipids. While much is known about regulated protein expression, the regulation of lipid metabolism during erythropoiesis is poorly studied. Here we show that transcription of PHOSPHO1, a phosphoethanolamine and phosphocholine phosphatase that mediates the hydrolysis of phosphocholine to choline, is strongly upregulated during the terminal stages of erythropoiesis of both human and mouse erythropoiesis, concomitant with increased catabolism of phosphatidylcholine and phosphocholine as shown by global lipidomic analyses of mouse and human terminal erythropoiesis. Depletion of PHOSPHO1 impaired differentiation of fetal mouse and human erythroblasts, and in adult mice depletion impaired phenylhydrazine-induced stress erythropoiesis. Loss of PHOSPHO1 also impaired phosphocholine catabolism in mouse fetal liver progenitors and resulted in accumulation of several lipids; ATP production was reduced as a result of decreased oxidative phosphorylation. Glycolysis replaced oxidative phosphorylation in PHOSPHO1 knockout erythroblasts and the increased glycolysis was used for the production of serine or glycine. Our study elucidates the dynamic changes in lipid metabolism during terminal erythropoiesis and reveals the key roles of phosphatidylcholine and phosphocholine metabolism in energy balance and amino acid supply. Introduction The lipid and protein organization of the red cell membrane is critical for the survival and deformability of red cells, as elucidated by the pathological disturbances of red cell membranes in several genetic disorders. 1 While most abnormalities, such as 2 From www.bloodjournal.org by guest on May 1, 2018. For personal use only. spherocytosis and elliptocytosis, are caused by mutant membrane or skeletal proteins, hereditary high red cell membrane phosphatidylcholine hemolytic anemia and spur anemia result from an altered lipid composition. 2,3 Thus, generating and maintaining the specific lipid composition of red cell membranes is essential. 4 Mature red cells can incorporate fatty acids from the circulation and utilize acyl-coenzyme A and an amino- phospholipid translocase to homeostatically maintain the lipid composition. 5 However, the processes by which this specific lipid composition is formed during erythropoiesis remain unclear. Therefore, we performed metabolomic analysis on cells undergoing terminal erythropoiesis to investigate the dynamic changes in lipid metabolism. We delineate a function for catabolism of phosphatidylcholine and its downstream metabolite phosphocholine in directing metabolic activity in terminal erythropoiesis, and show that the PHOSPHO1 gene and its encoded protein is one of the important regulators of these catabolic steps during erythroblast differentiation. These findings expand our understanding of the links between the regulation of lipid composition and energy metabolism during terminal erythropoiesis. Material and Methods Flow cytometry analyses and antibodies All flow cytometry data were acquired on a FACS Fortessa flow cytometer (BD Biosciences) and analyzed using Flowjo software. All stainings were carried out in FACS buffer (100 μM EDTA and 2% FBS in PBS) for 30 minutes at room temperature unless otherwise described. The following are the antibodies used at 1:100 dilution: anti-human CD235A-APC (eBioscience, 17-9987042), anti-mouse Ter119-APC (eBioscience, 17- 3 From www.bloodjournal.org by guest on May 1, 2018. For personal use only. 5921-83), and anti-mouse CD71-PE (Affymetrix, 12-0711-83). Hoechst 33342 (Life Technologies, H1399) was used to visualize nuclei. Antibodies for western blotting Anti-AMPKα (Cell Signaling Technology, 2603), anti-Phospho-AMPKα (Thr172) (Cell signaling Technology, 2535), anti-Phospho1 antibody (Abcam, ab90581), anti-GAPDH (Santa Cruz Biotechnology, sc-32233), anti-beta-actin (BioLegend, 622101) Human CD34+ cell culture Granulocyte-colony stimulating factor (G-CSF)–mobilized CD34+ peripheral blood stem cells were thawed according to the vendor’s protocol. Cells were cultured according to methods published previously.6 Isolation of erythroid progenitors from murine fetal liver cells Enriched erythroid progenitors were purified from E14.5 C57BL/6J mouse embryos, and cultured in vitro for erythroid differentiation following a protocol described in detail previously. 6 Briefly, pregnant C57BL/6J mice at embryonic day 14.5 were sacrificed by CO2 asphyxiation and their embryos were collected. The fetal livers were isolated and suspended in PBS with 2% FBS and 100 μM EDTA. Mature RBCs in the cell suspension were lysed by incubation for 10 min with an ammonium chloride solution (Stemcell). Following the manufacturer’s protocol, lineage negative cells were obtained after magnetic depletion of lineage positive cells using the BD Pharmingen Biotin MouseLineage Panel (559971; BD Biosciences) and BD Streptavidin Particles Plus-DM (557812; BD Biosciences). These lineage negative fetal liver cells were enriched for more than 90% for erythroid progenitors. Viral infection and culture of murine erythroid progenitors 4 From www.bloodjournal.org by guest on May 1, 2018. For personal use only. Following the isolation step, lineage negative fetal liver cells were plated in 24-well plates at 100,000 cells per well, covered by 1 ml virus containing supernatant, and centrifuged at 500xg for 90 min at 30°C. After this spin-infection, the virus supernatant was replaced with erythroid maintenance medium (StemSpan-SFEM; StemCell Technologies) supplemented with 100 ng/mL recombinant mouse stem cell factor (SCF) (R&D Systems), 40 ng/mL recombinant mouse IGF1 (R&D Systems), 100 nM dexamethasone (Sigma), and 2 U/mL erythropoietin (Amgen) and cultured at 37°C. GFP+ cells were sorted by flow cytometry after 16 h and cultured for another 48 h in erythroid differentiation medium (Iscove modified Dulbecco’s medium containing 15% (vol/vol) FBS (Stemcell), 1% detoxified BSA (Stemcell), 500 μg/mL holo-transferrin (Sigma-Aldrich), 0.5 U/mL Epoetin (Epo; Amgen), 10 μg/mL recombinant human insulin (Sigma-Aldrich), and 2 mM L-glutamine (Invitrogen)) at 37 °C. Seahorse assays 800,000 cells/well were plated into 24 well poly-lysine coated Seahorse microplate and cells were plated in XF media (Sigma #D5030 medium powder, 1.85 g NaCl and 600 μl phenol red (0.5%) in 1 L) containing 25 mM glucose, 2 mM L-glutamine and 1mM pyruvate), pH7.4. The microplate was centrifuged at 400xg for 5 mins and incubated at 37°C in a non-CO2 incubator for 45 mins before measuring. Extracellular acidification rates and oxygen consumption rates were measured on a Seahorse Bioscience XF24–3 Extracellular Flux Analyzer. 1 μM oligomycin and FCCP, 0.5 μM Rotenone and antimycin were used during the measurement. For glycolysis assay, XF media containing 2 mM L-glutamine and 1mM pyruvate was used for plating cells, 25 mM glucose, 1 μM oligomycin, and 50 mM 2-Deoxy-Glucose were used during the measurement. 5 From www.bloodjournal.org by guest on May 1, 2018. For personal use only. Mouse study Our mouse studies were conducted under animal protocols approved by the Division of Comparative Medicine at MIT. PHOSPHO1 KO mice were gifted from Professor Colin Farquharson and were re-derived onto C57BL/6J (Jackson Laboratory) strain. PHOSPHO1-R74X mutant mice (referred to as PHOSPHO1 KO) were previously described.7 PHOSPHO1 KO mice were genotyped by performing PCR (primer 1: 5’- TCCTCCTCACCTTCGACTTC-3’; primer 2: 5’-ATGCGGCGGAATAAACTGT-3’) and BsRDI enzyme digestion of the PCR product. The PCR product from wild type mice is uncut and the PCR product from knockout mice is cut into two
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