Thioredoxin-interacting (TXNIP) protein regulates the differentiation of erythroid precursors
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1 Thioredoxin-interacting (TXNIP) protein regulates the differentiation of erythroid precursors Volker Blank Lady Davis Institute for Medical Research McGill University INSTITUT LADY DAVIS DE RECHERCHES MÉDICALES / LADY DAVIS INSTITUTE FOR MEDICAL RESEARCH
2 Hematopoiesis Specification of HSC Expansion/ Maintenance Lineage Selection & Maturation B-lymphocytes Lymphoid progenitor T-lymphocytes Erythrocytes hematopoietic stem cells Megakaryocytes Mast cells Platelets Myeloerythroid progenitor Neutrophil Eosinophil Monocytes/Macrophages Osteoclasts
3 Erythroid maturation - bone marrow Pro- Poly Ortho Basophilimatic matic chro- chro erythroblast Reticulocyte Average cell diameter (µ) Log of cell volume (µ 3 ) Rate of RNA synthesis Rate of DNA synthesis Enzymes from ALA-S to PROTO ALA-synthase Hemoglobin concentration (10 6 µg) 50 hrs 30 hrs 50 hrs 40 hrs bone marrow
4 Regulation of erythroid development External factors IL-3 Stem Cell Factor (SCF) Glucocorticoids (GC) Thyroid hormone (T3) Erythropoietin (Epo) Insulin (Ins) Ingley et al IUBMB Life 56(4): Transcription factors GATA-2 (G2) GATA-1 (G1) STAT5 (S5) FOG (FG) EKLF (EK) Lee et al Blood 108: NF-E2 (NF)
5 NF-E2 (nuclear factor erythroid 2) - heterodimer (two subunits: p45 & small Maf) - basic-leucine zipper - large subunit specifically expressed in erythroid, megakaryocytic and mast cell lineages
6 NF-E2 -/- mice - increase in spleen erythropoiesis - partial block in erythroid progenitor populations - increase in GATA-1 levels - cell cycle: increase of cells in G1
7 Hemoglobin synthesis heme biosynthesis iron uptake globin gene expression hemoglobin α 2 β 2 tetramer α-chain 141aa β-chain 146aa
8 Erythroleukemia cells Hemin or DMSO MEL globin gene transcription hemin 20mM 50mM 100mM dosage response actin β maj -globin hours time course actin β maj -globin
9 Models MEL & G1-ER cells Mice untreated treated ( 96h) cromosoma.cib.csic.es
10 Genechip expression array (Affymetrix 430A) - MEL cells - differentially expressed TXNIP Microarray - potential candidates 20,000 Signal (relative units) 15,000 10,000 5,000 0 Txnip β-globin Alas2 Ctrl (48h) DMSO (48h)
11 TXNIP (Thioredoxin interacting protein) ubiquitously expressed, ~50 kda cytoplasm, nucleus, mitochondria, cell membrane tumor suppressor (expression reduced in many tumors) overexpression induces cell cycle arrest role in oxidative stress by inhibiting activity or bioavailability of thioredoxin (Huang et al, Nature 2013)
12 TXNIP -/- mice Metabolism In fasting conditions: hypoglycemia insulin triglycerides fatty acids in plasma (Huiet al. 2004, Donnelly et al. 2004, Oka et al. 2006) Carcinogenesis hepatocellular carcinoma (40% of mice) (Sheth et al. 2006) hepatocellular and bladder tumors(carcinogen-induced) (Kwon et al. 2010, Nishizawaet al. 2011) Hematopoiesis impaired maturation of NK cells (Lee et al. 2005) impaired function of dendritic cells (Son et al. 2008) altered HSCs (Jeonget al. 2009, Jung et al. 2013)
13 Induction of TXNIP in differentiating MEL cells Relative transcript levels Txnip transcripts in MEL cells * ** 6h 24h 48h * MEL cells 2h 4h 6h 8h DMSO DMSO DMSO DMSO TXNIP 52kDa mthsp70 70kDa 12h 24h 48h 72h DMSO DMSO DMSO DMSO TXNIP 52kDa mthsp70 70kDa
14 Induction of TXNIP in differentiating G1E-ER cells G1E cells G1E-ER cells 6h 12h 24h 48h 6h 12h 24h 48h 1.4 TXNIP in G1E cells TXNIP in G1E-ER cells * Relative TXNIP protein levels h 12h 24h 48h Induced 6h 12h 24h 48h * * Induced Contro l Induce d Contro l Induce d Contro l Induce d Contro l Induce d Contro l Induce d Contro l Induce d Contro l Induce d Contro l Induce d TXNIP 52kDa eif4e 25kDa Relative TXNIP protein levels
15 TXNIP & ROS scavenger NAC - TXNIP upregulation with unchanged MEL cells 6h 6h NAC (nm) TXNIP 52kDa eif4e 25kDa TXNIP induction with in presence of NAC Relative TXNIP protein levels * * *** *** NAC concentration (nm)
16 MAP kinase pathways Liu et al. Nature Reviews Immunology 7, (March 2007)
17 TXNIP Regulation of MAP kinases MEL cells 6h 24h 48h SB TXNIP 52 kda p38 inhibitor eif4e 25 kda MEL cells SB TXNIP 52 kda 6h 24h 48h JNK inhibitor eif4e 25 kda MEL cells UO126 TXNIP 52 kda 6h 24h 48h ERK inhibitor eif4e 25 kda
18 TXNIP overexpression - increases iron uptake - decreases TFR expression Iron uptake (cpm/ug) relative to +DOX Total iron uptake in TXNIP clone S2 * + Dox - Dox Iron incorporation into heme (%) Iron incorporation into heme in TXNIP clone S2 + Dox - Dox TFR TXNIP eif4e TXNIP S2 Dox + - Relative TFR protein levels TFR reduction upon TXNIP overexpression +Dox ** -Dox
19 Analysis of Txnip -/- mice - spleen size - blood parameters 0.4 Spleen size ** Percentage of body weight (%) WT Txnip-/- WT (n=5) Txnip -/- (n=5) p-value RBC count, x10 6 /µl ± ± HGB level, g/dl ± ± HCT, % ± ±
20 Erythroid precursor populations in Txnip -/- mice - spleen ProE 0.54% Ter119 High EryA 1.57% WT Txnip -/- CD71 CD71 ProE 0.57% Ter119 Ter119 High CD71 CD71 EryB 13.42% EryC 84.04% FSC EryA 1.19% EryB 5.85% WT (n=13) Txnip -/- (n=14) p-value Spleen ProE 1.78 ± ± EryA 2.99 ± ± EryB ± ± 0.64 * EryC ± ± Ter119+ cells ± ± Bone marrow ProE 6.40 ± ± EryA ± ± EryB ± ± EryC ± ± Ter119+ cells ± ± EryA 91.49% Ter119 FSC ProE proerythroblasts EryA basophilic erythroblasts EryB late basophilic and polychromatic erythroblasts EryC orthochromatic erythroblasts and erythrocytes
21 Model - Role of Txnip in erythroid differentiation
22 Acknowledgements Marilène Paquet (MUHC, McGill) Gregory Chevillard Isadore Dodard- Friedman Meenakshi Kannan Simon T. Hui (UCLA) Marina Bury Zaynab Nouhi Thibaut Dutruel Prem Ponka (McGill University) Daniel Garcia Santos Marc Mikhael Anna Derjuga Vera Solovieva Jadwiga Gasiorek Mitchell Weiss (Children s Hospital, Philadelphia) The Cancer Research Society
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