Clathrin Assembly Protein CALM Plays a Key Role In Erythroid Differentiation Via Regulating Transferrin Receptor Endocytosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4256-4256
Author(s):  
Yuichi Ishikawa ◽  
Manami Maeda ◽  
Min Li ◽  
Sung-Uk Lee ◽  
Julie Teruya Feldstein ◽  
...  
Keyword(s):  
Transferrin Receptor ◽  
Mammalian Cells ◽  
Research Funding ◽  
Molecular Mechanisms ◽  
Erythroid Differentiation ◽  
Global Changes ◽  
Erythroid Progenitors ◽  
Erythroid Development ◽  
Terminal Erythroid Differentiation

Abstract Abstract 4256 Clathrin assembly lymphoid myeloid leukemia protein (CALM, also known as PICALM) is ubiquitously expressed in mammalian cells and implicated in clathrin dependent endocytosis (CDE). The CALM gene is the target of the t(10;11)(p13;q14-21) translocation, which is rare, but recurrently observed mutation in multiple types of acute leukemia. While the resultant CALM/AF10 fusion gene could act as an oncogene in vitro and in vivo in animal models, molecular mechanisms by which the fusion protein exerts its oncogenic activity remains elusive. Since CDE is implicated in the regulation of growth factor/cytokine signals, we hypothesized that the CALM/AF10 fusion oncoprotein could affect normal Calm function, leading to leukemogenesis. To determine the role of CALM and CDE in normal hematopoiesis, we generated and characterized both conventional (Calm+/−) and conditional (CalmF/F Mx1Cre+) Calm knockout mutants. While we didn't observe a gross defect in the heterozygous mutant (Calm+/−), homozygous deletion of the Calm gene (Calm-/-) resulted in late embryonic lethality. Total numbers of fetal liver (FL) cells were significantly reduced in Calm-/-embryos compared to that of control due to inefficient erythropoiesis. Proportions of mature erythroblasts (CD71-Ter119+) in FL were significantly reduced in the absence of the Calm gene. Furthermore, Calm deficient Megakaryocyte-Erythroid Progenitors (MEPs) gave rise to less CFU-E colonies when seeded in methyl cellulose plates, suggesting that Calm is required for terminal erythroid differentiation in a cell autonomous manner. To determine the role of Calm in adult hematopoiesis, we analyzed peripheral blood (PB), bone marrow (BM) and spleen of CalmF/F Mx1Cre+ mice after pIpC injection. CalmF/F Mx1Cre+ mice demonstrated hypochromic anemia, T-lymphocytopenia and thrombocytosis one month after pIpC injection. Levels of plasma transferrin and ferritin were intact in CalmF/F Mx1Cre+ mice, while plasma iron levels were increased, indicating that iron uptake is impaired in Calm deficient erythroblasts. We observed significant reduction of mature erythroblasts and erythrocytes in both BM and spleen with concomitant increase of immature erythroblasts (CD71+Ter119+) in CalmF/F Mx1Cre+ mice. The increased population mainly consists of CD71+Ter119+CD44+FSCdim polychromatophilic erythroblasts, and Benzidine staining of PB and splenic erythroblasts revealed reduced hemoglobinization in Calm deficient erythroblasts. To examine the global changes in transcriptome of CD71+Ter119+CD44+FSCdim polychromatophilic erythroblasts with or without the Calm gene, we compared mRNA expression profile by gene chip microarray analysis. Over 400 genes, including genes associated with iron metabolism and CDE pathway, were up- or down-regulated more than 1.5-fold in Calm deficient polychromatophilic erythroblasts as compared to control. Genes Set Enrichment Analysis (GSEA) revealed that multiple metabolic pathways were downregulated in Calm deficient polychromatophilic erythroblasts. Calm deficient CD71+Ter119+CD44+FSCdim polychromatophilic erythroblasts demonstrated a defect in cellular proliferation revealed by cell cycle analysis. Transferrin receptor 1 (TFR1, CD71) is highly expressed in rapidly dividing cells and erythroblasts, and uptake of iron-bound transferrin through TFR1 is the main pathway of iron intake to erythroid precursors. Since CDE is implicated in TFR1 endocytosis, we next examined surface expression levels of CD71 in Calm deficient erythroid progenitors and erythroblasts. While CD71 is normally expressed at low level in early stage of megakaryo/erythroid progenitors and highly expressed in CFU-E through polychromatophilic erythroblasts, its expression was dramatically up-regulated throughout the erythroid development in CalmF/F Mx1Cre+ mice. Up-regulation of surface CD71 expression was also evident in K562 erythroid leukemia cell lines upon ShRNA-mediated CALM knockdown. Taken together, our data indicate that CALM plays an essential role in terminal erythroid differentiation via regulating TFR1 endocytosis. Since iron is required for both erythroblast proliferation and hemoglobinization, Calm deficiency significantly impacts erythroid development at multiple levels. Disclosures: Naoe: Chugai Pharm. Co.: Research Funding; Zenyaku-Kogyo Co.: Research Funding; Kyowa-Kirin Co.: Research Funding; Dainippon-Sumitomo Pharm. Co.: Research Funding; Novartis Pharm. Co.: Research Funding; Janssen Pharm. Co.: Research Funding.

Impairment of human terminal erythroid differentiation by histone deacetylase 5 deficiency

Blood ◽  
2021 ◽  
Author(s):  
Yaomei Wang ◽  
Wei Li ◽  
Vince Schulz ◽  
Huizhi Zhao ◽  
Xiaoli Qu ◽  
...  
Keyword(s):  
Histone Deacetylases ◽  
Molecular Mechanisms ◽  
Chromatin Condensation ◽  
Erythroid Differentiation ◽  
Chromatin Accessibility ◽  
Erythroid Cell ◽  
Global Changes ◽  
Human Cd34 ◽  
Wide Range ◽  
Terminal Erythroid Differentiation

Histone deacetylases (HDACs) are a group of enzymes catalyzing the removal of acetyl groups from histone and non-histone proteins. HDACs have been shown to play diverse functions in a wide range of biological processes. However, their roles in mammalian erythropoiesis remain to be fully defined. We show here that of the eleven classic HDAC family members, six of them (HDAC 1,2,3 and HDAC 5,6,7) are expressed in human erythroid cells with HDAC5 most significantly up regulated during terminal erythroid differentiation. Knockdown of HDAC5 by either shRNA or siRNA in human CD34+ cells followed by erythroid cell culture led to increased apoptosis, decreased chromatin condensation, and impaired enucleation of erythroblasts. Biochemical analyses revealed that HDAC5 deficiency resulted in activation of p53 in association with increased acetylation of p53. Furthermore, while acetylation of histone 4 (H4) is decreased during normal terminal erythroid differentiation, HDAC5 deficiency led to increased acetylation of H4 (K12) in late stage erythroblasts. This increased acetylation was accompanied by decreased chromatin condensation, implying a role for H4 (K12) deacetylation in chromatin condensation. ATAC-seq and RNA-seq analyses revealed that HDAC5 knockdown leads to increased chromatin accessibility genome wide and global changes in gene expression. Moreover, pharmacological inhibition of HDAC5 by the inhibitor LMK235 also led to increased H4 acetylation, impaired chromatin condensation and enucleation. Taken together, our findings have uncovered previously unrecognized roles and molecular mechanisms of action for HDAC5 in human erythropoiesis. These results may provide insights into understanding the anemia associated with HDAC inhibitor treatment.

The Role of K-ras Signaling in Erythropoiesis In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3136-3136
Author(s):  
Jing Zhang ◽  
Yangang Liu ◽  
Caroline Beard ◽  
Rudolf Jaenisch ◽  
Tyler Jacks ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Liver Cells ◽  
Ras Signaling ◽  
Erythroid Progenitors ◽  
Akt Activation ◽  
Fetal Liver Cells ◽  
Terminal Erythroid Differentiation

Abstract K-ras plays an important role in hematopoiesis. K-ras-deficient mouse embryos die around E12-E13 with severe anemia. In humans, oncogenic mutations in K-ras gene are identified in ~30% of patients with acute myeloid leukemia. We used mouse primary erythroid progenitors as a model system to study the role of K-ras signaling in vivo. Both Epo- and stem cell factor (SCF) - dependent Akt activation are greatly reduced in K-ras-/- fetal liver cells, whereas other cytokine- induced pathways, including Stat5 and p44/p42 MAP kinase, are activated normally. The reduced Akt activation in erythroid progenitors per se leads to delayed erythroid differentiation. Our data identify K-ras as the major regulator for cytokine-dependent Akt activation, which is important for erythroid differentiation in vivo. Overexpression of oncogenic Ras in primary fetal erythroid progenitors led to their continual proliferation and a block in terminal erythroid differentiation. Similarly, we found that primary fetal liver cells expressing oncogenic K-ras from its endogenous locus undergo abnormal proliferation and terminal erythroid differentiation is partially blocked. We are currently investigating the signal transduction pathways activated by this oncogenic K-ras that underlies these cellular phenotypes.

Epo-Induced Erythroid Maturation Is Dependent On Plcγ1 Signalling

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1182-1182
Author(s):  
Tina M Schnoeder ◽  
Patricia Arreba-Tutusaus ◽  
Inga Griehl ◽  
Lars Bullinger ◽  
Konstanze Doehner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Cell Line ◽  
Erythroid Differentiation ◽  
Global Gene Expression ◽  
Epigenetic Landscape ◽  
Erythroid Progenitors ◽  
I 11 ◽  
Erythroid Development

Abstract Erythropoiesis is a multi-step process in which the development of red blood cells occurs through expansion and differentiation of hematopoietic stem cells (HSCs) into more committed progenitors and finally into erythrocytes. Erythropoietin (Epo) is strictly required for erythropoiesis as it promotes survival and late maturation. In vivo and in vitro studies have pointed out the major role of erythropoietin receptor (EpoR) signalling through JAK2 tyrosine-kinase and STAT5a/b as a central regulator of erythropoiesis. STAT5a/b is essential in regulating early erythroblast survival, however, with regard to differentiation of erythroid progenitors current data are not definitive in establishing a critical, non-redundant role. Phospholipase C gamma 1 (PLCγ1) is known to act as key mediator of calcium-signalling that can substitute for PI3K/AKT in oncogenic models. Interestingly, genetic deletion of murine PLCγ1 in embryonic development using a conventional knockout mouse model resulted in lethality at E9.0 due to generalized growth failure and there was absence of erythrogenesis and vasculogenesis. Here, we revisited the role of Plcγ1 and investigated its function in signalling, differentiation and transcriptomic/epigenetic regulation of erythropoiesis: Upon Epo stimulation, we were able to demonstrate that Plcγ1 is a downstream target of EpoR/Jak2 signalling in lymphoid (Ba/F3) and myeloid (32D) progenitor cell lines (both transfected with EpoR and Jak2-WT) and in a erythroid progenitor (I/11) cell line. In order to specifically assess its role in erythroid development downstream of the EpoR-Jak2 axis, we focused on the murine pro-erythroblast cell line I/11 which is able to differentiate upon dexamethasone-/stem cell factor-withdrawal combined with erythropoietin stimulation. Interestingly, knockdown of Plcγ1 led to a dramatic delay (scr CD44high 21% vs. Plcγ1 shRNA CD44high 64%, p=0.02) in erythroid differentiation and accumulation of immature erythroid progenitors as assessed by flow cytometry technology. Knockdown of Plcγ1 did alter neither proliferation of cells nor the cell cycle distribution and activation of other EpoR downstream molecules as Stat5, Mek and Akt was not impaired. In addition, we analysed the colony-forming potential of Plcγ1-deficient I/11 and fetal liver cells (FLC) compared to controls. Colony formation was dramatically impaired in both - I/11 (scr 138 vs. Plcγ1 shRNA 32, p=0.03) and primary FLC (scr 107 vs. Plcγ1 shRNA 28, p<0.001) - when compared to control cells. Flow cytometry analysis of the colonies revealed a higher amount of immature populations (CD44high, KIT+) in PLCγ1-deficient cells as compared to controls whereas the content of TER119+ cells, reflecting more mature erythroid cells, was higher in controls. To elucidate on the mechanism of Plcγ1-mediated regulation of erythroid development, we performed global gene expression analysis in I/11 cells at various time points of differentiation after knockdown of Plcγ1. Several of the genes that change expression in the absence of Plcγ1 can be classified as transcription/co-transcription factors, epigenetic regulators, metabolic factors or adaptor molecules involved in intracellular signaling. Thus, Plcγ1-deficient cells showed up-regulation of the transcription factor RUNX1 and the adaptor molecule GRAP2 over time compared to controls whereas the epigenetic regulator H2AFY2 was significantly decreased. Stimulated by our observation that profound changes in global gene expression also included the epigenetic machinery (H2afy2), we speculated whether Plcγ1 signalling also modifies the global epigenetic landscape of I/11 pro-erythroblasts. Therefore, we performed genome-wide DNA methylome analysis in I/11 cells upon Plcγ1 knockdown using MCIP-seq (methyl-CpG immunoprecipitation combined with next-generation sequencing). The observed methylation changes were by far dominated by an apparent hypomethylation of differentially methylated regions (DMRs) in Plcγ1 knockdown cells as compared to control cells. In line with this, gene ontology analysis of DMRs revealed a highly significant enrichment of biological terms associated with developmental processes and cell differentiation. Taken together, our findings provide evidence for an essential role of Plcγ1 in regulating erythroid differentiation through alteration of the transcriptomic and epigenetic landscape. Disclosures: No relevant conflicts of interest to declare.

scholarly journals TET2 deficiency leads to stem cell factor–dependent clonal expansion of dysfunctional erythroid progenitors

Blood ◽  
2018 ◽  
Vol 132 (22) ◽  
pp. 2406-2417 ◽  
Author(s):  
Xiaoli Qu ◽  
Shijie Zhang ◽  
Shihui Wang ◽  
Yaomei Wang ◽  
Wei Li ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Factor ◽  
Molecular Mechanisms ◽  
Erythroid Differentiation ◽  
Negative Regulator ◽  
Loss Of Function ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Proliferation And Differentiation

Abstract Myelodysplastic syndromes (MDSs) are clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis. Anemia is the defining cytopenia of MDS patients, yet the molecular mechanisms for dyserythropoiesis in MDSs remain to be fully defined. Recent studies have revealed that heterozygous loss-of-function mutation of DNA dioxygenase TET2 is 1 of the most common mutations in MDSs and that TET2 deficiency disturbs erythroid differentiation. However, mechanistic insights into the role of TET2 on disordered erythropoiesis are not fully defined. Here, we show that TET2 deficiency leads initially to stem cell factor (SCF)–dependent hyperproliferation and impaired differentiation of human colony-forming unit–erythroid (CFU-E) cells, which were reversed by a c-Kit inhibitor. We further show that this was due to increased phosphorylation of c-Kit accompanied by decreased expression of phosphatase SHP-1, a negative regulator of c-Kit. At later stages, TET2 deficiency led to an accumulation of a progenitor population, which expressed surface markers characteristic of normal CFU-E cells but were functionally different. In contrast to normal CFU-E cells that require only erythropoietin (EPO) for proliferation, these abnormal progenitors required SCF and EPO and exhibited impaired differentiation. We termed this population of progenitors “marker CFU-E” cells. We further show that AXL expression was increased in marker CFU-E cells and that the increased AXL expression led to increased activation of AKT and ERK. Moreover, the altered proliferation and differentiation of marker CFU-E cells were partially rescued by an AXL inhibitor. Our findings document an important role for TET2 in erythropoiesis and have uncovered previously unknown mechanisms by which deficiency of TET2 contributes to ineffective erythropoiesis.

scholarly journals Mitochondrial Iron in Human Health and Disease

2019 ◽  
Vol 81 (1) ◽  
pp. 453-482 ◽  
Author(s):  
Diane M. Ward ◽  
Suzanne M. Cloonan
Keyword(s):  
Human Health ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Innate Immune ◽  
Biological Functions ◽  
Mitochondrial Homeostasis ◽  
Innate Immune Signaling ◽  
Mitochondrial Iron ◽  
Health And Disease

Mitochondria are an iconic distinguishing feature of eukaryotic cells. Mitochondria encompass an active organellar network that fuses, divides, and directs a myriad of vital biological functions, including energy metabolism, cell death regulation, and innate immune signaling in different tissues. Another crucial and often underappreciated function of these dynamic organelles is their central role in the metabolism of the most abundant and biologically versatile transition metals in mammalian cells, iron. In recent years, cellular and animal models of mitochondrial iron dysfunction have provided vital information in identifying new proteins that have elucidated the pathways involved in mitochondrial homeostasis and iron metabolism. Specific signatures of mitochondrial iron dysregulation that are associated with disease pathogenesis and/or progression are becoming increasingly important. Understanding the molecular mechanisms regulating mitochondrial iron pathways will help better define the role of this important metal in mitochondrial function and in human health and disease.

Characterization of HOX Gene Expression During Myelopoiesis: Role of HOX A5 in Lineage Commitment and Maturation

Blood ◽  
1999 ◽  
Vol 93 (10) ◽  
pp. 3391-3400 ◽  
Author(s):  
John F. Fuller ◽  
Jeanne McAdara ◽  
Yifah Yaron ◽  
Mark Sakaguchi ◽  
John K. Fraser ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Bone Marrow Cells ◽  
Homeobox Genes ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Effector Cells ◽  
Gm Csf ◽  
Important Regulator ◽  
Macrophage Colony

During the process of normal hematopoiesis, proliferation is tightly linked to maturation. The molecular mechanisms that lead to production of mature effector cells with a variety of phenotypes and functions from a single multipotent progenitor are only beginning to be elucidated. It is important to determine how these maturation events are regulated at the molecular level, because this will provide significant insights into the process of normal hematopoiesis as well as leukemogenesis. Transcription factors containing the highly conserved homeobox motif show considerable promise as potential regulators of hematopoietic maturation events. In this study, we focused on identification and characterization of homeobox genes of the HOX family that are important in regulating normal human myeloid differentiation induced by the hematopoietic growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF). We have identified three homeobox genes, HOX A5, HOX B6, and HOX B7, which are expressed during early myelopoiesis. Treating bone marrow cells with antisense oligodeoxynucleotides to HOX A5 resulted in inhibition of granulocytic/monocytic hematopoiesis and increased the generation of erythroid progenitors. Also, overexpression of HOX A5 inhibited erythroid differentiation of the K562 cell line. Based on these observations, we propose that HOX A5 functions as an important regulator of hematopoietic lineage determination and maturation.

scholarly journals Decreased PGC1β expression results in disrupted human erythroid differentiation, impaired hemoglobinization and cell cycle exit

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Taha Sen ◽  
Jun Chen ◽  
Sofie Singbrant
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Mitochondrial Function ◽  
Iron Homeostasis ◽  
Erythroid Differentiation ◽  
Refractory Anemia ◽  
Cell Cycle Exit ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Terminal Erythroid Differentiation

AbstractProduction of red blood cells relies on proper mitochondrial function, both for their increased energy demands during differentiation and for proper heme and iron homeostasis. Mutations in genes regulating mitochondrial function have been reported in patients with anemia, yet their pathophysiological role often remains unclear. PGC1β is a critical coactivator of mitochondrial biogenesis, with increased expression during terminal erythroid differentiation. The role of PGC1β has however mainly been studied in skeletal muscle, adipose and hepatic tissues, and its function in erythropoiesis remains largely unknown. Here we show that perturbed PGC1β expression in human hematopoietic stem/progenitor cells from both bone marrow and cord blood results in impaired formation of early erythroid progenitors and delayed terminal erythroid differentiation in vitro, with accumulations of polychromatic erythroblasts, similar to MDS-related refractory anemia. Reduced levels of PGC1β resulted in deregulated expression of iron, heme and globin related genes in polychromatic erythroblasts, and reduced hemoglobin content in the more mature bone marrow derived reticulocytes. Furthermore, PGC1β knock-down resulted in disturbed cell cycle exit with accumulation of erythroblasts in S-phase and enhanced expression of G1-S regulating genes, with smaller reticulocytes as a result. Taken together, we demonstrate that PGC1β is directly involved in production of hemoglobin and regulation of G1-S transition and is ultimately required for proper terminal erythroid differentiation.

scholarly journals The centrosome–Golgi apparatus nexus

2014 ◽  
Vol 369 (1650) ◽  
pp. 20130462 ◽  
Author(s):  
Rosa M. Rios
Keyword(s):  
Golgi Apparatus ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Animal Cells ◽  
Critical Feature ◽  
Differentiated Cells ◽  
Ribbon Structure ◽  
Centrosomal Proteins ◽  
Dependent Processes

A shared feature among all microtubule (MT)-dependent processes is the requirement for MTs to be organized in arrays of defined geometry. At a fundamental level, this is achieved by precisely controlling the timing and localization of the nucleation events that give rise to new MTs. To this end, MT nucleation is restricted to specific subcellular sites called MT-organizing centres. The primary MT-organizing centre in proliferating animal cells is the centrosome. However, the discovery of MT nucleation capacity of the Golgi apparatus (GA) has substantially changed our understanding of MT network organization in interphase cells. Interestingly, MT nucleation at the Golgi apparently relies on multiprotein complexes, similar to those present at the centrosome, that assemble at the cis -face of the organelle. In this process, AKAP450 plays a central role, acting as a scaffold to recruit other centrosomal proteins important for MT generation. MT arrays derived from either the centrosome or the GA differ in their geometry, probably reflecting their different, yet complementary, functions. Here, I review our current understanding of the molecular mechanisms involved in MT nucleation at the GA and how Golgi- and centrosome-based MT arrays work in concert to ensure the formation of a pericentrosomal polarized continuous Golgi ribbon structure, a critical feature for cell polarity in mammalian cells. In addition, I comment on the important role of the Golgi-nucleated MTs in organizing specialized MT arrays that serve specific functions in terminally differentiated cells.

Essential Roles for Mef2c in Lymphoid Commitment and B-Cell Function.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 377-377
Author(s):  
Sandra Stehling-Sun ◽  
Rebecca Jimenez ◽  
Andrew Hu ◽  
Fernando D. Camargo
Keyword(s):  
B Cells ◽  
B Cell ◽  
Molecular Mechanisms ◽  
Muscle Development ◽  
Cell Function ◽  
Physiological Role ◽  
Erythroid Differentiation ◽  
Hematopoietic Stem ◽  
Interleukin 7

Abstract MEF2 transcription factors are well-established regulators of muscle development. Recently, work in murine models has identified one of these factors, Mef2c, as an important regulator in the pathogenesis and the development of acute myeloid leukemia (AML). However, little is know about the molecular mechanism and physiological role of Mef2c in hematopoiesis. Using conditional gene ablation, we have discovered an unexpected role for MEF2c in hematopoietic stem cells (HSCs), where it is required for pan-lymphoid commitment. Competitive repopulation experiments using Mef2c-null HSCs deleted by means of the Mx1-Cre/poly(IC) approach, revealed completely normal monocytic, granulocytic and erythroid differentiation capacities by mutant cells. Generation and renewal of myeloid progenitors and HSCs was also normal. However, contribution to lymphoid lineages (T-cells, B-cells and natural killer cells) was dramatically reduced. Mef2c-deleted HSCs were able to generate lymphoid primed multipotent progenitors (LMPPs) and expressed normal levels of Flt-3 and the master lymphoid regulator ikaros. However, expression of the interleukin-7 receptor (IL-7R) and the number of phenotypically defined common lymphoid progenitors (CLPs) were substantially reduced. We have found two conserved Mef2c-binding sites in the promoter of the Il-7R gene, indicating that Mef2c could directly regulate Il-7R transcription. This and other potential molecular mechanisms of Mef2c-mediated lymphoid commitment will be discussed. We have also studied the effects of lineage-specific deletion of Mef2c in both myeloid and lymphoid populations. Whereas deletion in myelomonocytic cells using the LysM-Cre strain resulted in no anomalies, B-cell specific ablation with the CD19-Cre line revealed major phenotypical and functional abnormalities. CD19-Cre:Mef2cf/f mice show impaired germinal center formation and reduced antibody production in response to T-cell dependent antigens. In addition Mef2c-null mature B-cells fail to express the mature marker CD23, the low affinity receptor for IgE, which we show is a direct transcriptional target. As a consequence of CD23 reduction, CD19-Cre:Mef2cf/f mice have increased IgE production, thus indicating a potential role of Mef2c in allergic disease. Our work here sheds new light on the molecular mechanisms of lymphopoiesis and identifies MEF2 factors as critical hematopoietic transcriptional regulators.

HMGB1 Is a Key Modulator Of Stress Erythropoiesis During Sepsis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 8-8 ◽  
Author(s):  
Sergio I Valdés-Ferrer ◽  
Lionel Blanc ◽  
Sebastien Didier ◽  
Johnson M. Liu ◽  
Jeffrey M. Lipton ◽  
...  
Keyword(s):  
Severe Sepsis ◽  
Cord Blood ◽  
Critical Role ◽  
Cecal Ligation ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Stress Erythropoiesis ◽  
Cd34 Cells ◽  
Sepsis Survivors ◽  
Terminal Erythroid Differentiation

Abstract Severe sepsis is a leading cause of death and disability. Anemia in sepsis survivors affects close to 100% of patients after the third day of in-hospital stay, regardless of blood levels on admission. Circulating levels of Erythropoietin (Epo) are low; paradoxically, administration of recombinant Epo is ineffective, and related to increased morbidity. During sepsis, bone Marrow is hypoproliferative. While transfusions can improve outcome in the short term, its use increases the risk of infection and mortality without any sustained beneficial effect. The pathogenesis of anemia during sepsis is unclear. High mobility group box 1 (HMGB1), a cytokine that is a critical mediator of sepsis, is released into circulation a few days after sepsis onset, remaining increased for 8 weeks after severe sepsis. HMGB1 levels are increased for at least 8 weeks in murine models of sepsis survival. To induce severe sepsis, cecal ligation and puncture (CLP) was performed in BALB/c mice. Three days after CLP, mice developed persistent anemia, represented by a significant reduction in hematocrit (Sham=49.8±3.2 vs. CLP=29.7±6.7%; p≤0.001), hemoglobin (16.7±1.2 vs. 9.9±2.4mg/dL; p≤0.001), and red blood cells mass (10.2±0.7 vs. 5.4±1.7 x106/µL; p≤0.001). Anemia persisted for at least 25 days after CLP. In CLP survivors, reticulocyte counts were erratic, and insufficient to the degree and duration of anemia (8.2±0.8 vs. 6.6±2.1%; p=ns). Analysis of terminal erythroid differentiation using CD44 and Ter119 or CD44 and FSC as markers demonstrated a significant decrease in all erythroid progenitors, from proerythroblast to orthochromatic erythroblast. Concomitantly, mice surviving CLP developed splenomegaly. Splenic architecture was disrupted after CLP, with expansion of the red pulp, characteristic of stress erythropoiesis. Analysis of terminal erythroid differentiation demonstrated an increase in the quantity of erythroid progenitors. An anti-HMGB1 mAb (2G7) was administered after CLP. Strikingly, 2G7-treated septic mice were significantly protected from developing anemia, and had levels of hemoglobin and hematocrit similar to sham-operated mice. These results highlight a critical role for HMGB1 as key modulator of stress erythropoiesis in a murine model of sepsis survivors. To get further insight into the function of HMGB1 and translate our findings to the pathophysiology of human erythropoiesis, we used CD34+ cells derived from cord blood. Cord blood-derived CD34+ cells were incubated in MethoCult in the presence or not of HMGB1. HMGB1 induced a dose dependent decrease in CFU-E. In murine sepsis, there is a stepwise elevation of different redox forms of HMGB1, with an early increase in all-thiol (inflammatory), followed by a partially oxidized before a fully oxidized (with no known inflammatory activity) appears. At day 7, all-thiol HMGB1 reduced significantly the number of CFU-E, while the fully oxidized had no significant effect. At day 14, the number of BFU-E was reduced in the presence of HMGB1, and further decreased with all-thiol HMGB1. In conclusion, our findings suggest that CLP is a reproducible model to study anemia of sepsis. In mice surviving sepsis, stress erythropoiesis is consistently found. Administration of anti-HMGB1 monoclonal antibody reverses anemia of murine sepsis, demonstrating that HMGB1 can be a potential target in the anemia of sepsis survivors. Translating the findings to the human system, we found that HMGB1 impairs differentiation of CD34+ cells towards the BFU-E and CFU-E stages in colony formation assays, implying that HMGB1 might play a role early during differentiation. The redox status of HMGB1 is critical for its biological function, since its effects are not retrieved when HMGB1 is fully oxidized. Disclosures: No relevant conflicts of interest to declare.

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