scholarly journals Bcl-xL prevents apoptosis of late-stage erythroblasts but does not mediate the antiapoptotic effect of erythropoietin

Blood ◽  
2005 ◽  
Vol 106 (5) ◽  
pp. 1857-1863 ◽  
Author(s):  
Melissa M. Rhodes ◽  
Prapaporn Kopsombut ◽  
Maurice C. Bondurant ◽  
James O. Price ◽  
Mark J. Koury
Keyword(s):  
Late Stage ◽  
B Cell Lymphoma ◽  
Conditional Knockout ◽  
Ineffective Erythropoiesis ◽  
Erythroid Progenitor ◽  
Heme Synthesis ◽  
Erythroid Progenitor Cells ◽  
Long Form ◽  
Mitochondrial Membrane Protein

Abstract The long form of B-cell lymphoma-x (Bcl-xL), an outer mitochondrial membrane protein, has been proposed to mediate the antiapoptotic action of erythropoietin on erythroid progenitor cells and to be necessary for heme synthesis in erythroblasts. Mice with conditional knockout of Bcl-xL (conditional bcl-x-/- mice) develop severe anemia that has been attributed to hemolysis and is accompanied by splenomegaly. We characterized further the anemia of conditional bcl-x-/- mice and investigated the role of Bcl-xL in the action of erythropoietin and in heme synthesis. We analyzed peripheral blood cells and cultured splenic erythroblasts of conditional bcl-x-/- mice and littermates that were rendered anemic by bleeding. Although they had massive splenic erythroblastosis, conditional bcl-x-/- mice had decreased circulating reticulocytes compared to littermates even prior to bleeding the littermates. Compared to erythroblasts of bled littermates, bcl-x-/- erythroblasts cultured with erythropoietin underwent apoptosis during the later, hemoglobin-synthesizing stages of differentiation. The bcl-x-/- erythroblasts synthesized heme, but at reduced rates compared to bled littermate erythroblasts. When cultured without erythropoietin, bcl-x-/- erythroblasts underwent apoptosis at early stages of differentiation, prior to hemoglobin synthesis. Bcl-xL is not required for heme synthesis and does not mediate the antiapoptotic effects of erythropoietin, but it prevents ineffective erythropoiesis due to apoptosis in late-stage, hemoglobin-synthesizing erythroblasts. (Blood. 2005;106:1857-1863)

Bcl-x Prevents the Apoptosis of Late-Stage, Hemoglobin-Synthesizing Erythroblasts, but It Does Not Mediate the Anti-Apoptotic Effect of Erythropoietin on Early-Stage Erythroblasts.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1260-1260
Author(s):  
Melissa M. Rhodes ◽  
Prapaporn Kopsombut ◽  
Maurice Bondurant ◽  
James O. Price ◽  
Mark J. Koury
Keyword(s):  
Cell Cycle ◽  
Late Stage ◽  
Early Stage ◽  
Erythroid Differentiation ◽  
Conditional Knockout ◽  
Erythroid Progenitor ◽  
Heme Synthesis ◽  
Erythroid Progenitor Cells ◽  
Cell Counts ◽  
Apoptotic Effect

Abstract Bcl-x is a protein in the outer mitochondrial membrane. A member of the Bcl-2 family, Bcl-x protects developing erythroid cells from apoptosis. The exact stage of erythroid development at which Bcl-x exerts its anti-apoptotic effect is not known, but induction of Bcl-x has been proposed to be the mediator of erythropoietin’s (EPO) anti-apoptotic effect in erythroid differentiation. EPO is the principal trophic hormone that controls red blood cell production by regulating apoptosis of erythroid progenitor cells at the CFU-E and early erythroblast stages. Bcl-x has also been reported to be necessary for heme synthesis. In mice, Bcl-x deficiency is embryonically lethal; when Bcl-x deficiency is acquired postnatally by conditional knockout technology, it is associated with splenomegaly, thrombocytopenia, and a profound anemia that is thought to be hemolytic in origin. Objectives: 1)To characterize the defect of erythroid differentiation in conditional Bcl-x −/− mice. 2)To determine whether Bcl-x is the mediator of EPO’s anti-apoptotic action. 3)To determine whether Bcl-x is necessary for heme synthesis. Methods: Phlebotomized or unbled littermate controls and anemic adult Bcl-x −/− mice obtained by cre-lox conditional knockout were bled, sacrificed, and splenectomized. Purified populations of splenic erythroblasts were isolated by sedimentation at unit gravity, cultured with or without EPO, and harvested at 0, 8, 20, 32, and 44 hours for cell counts, cytospin preparations for morphology, flow cytometry analyses for apoptosis (TUNEL) and cell cycle phases, and 59FeCl3 incorporation into heme. Results: Compared to littermate controls, Bcl-x −/− mice were severely anemic (Hgb 2.8 g/dL vs 15.4 g/dL in unbled controls and 7.2 g/dL in bled controls), thrombocytopenic (platelets 23x103/microL vs 905x103/microL in unbled controls and 984x103/microL in bled controls), and reticulocytopenic (82.8x103/microL vs 281x103/microL in unbled controls and 1410x103/microL in bled controls), while WBCs were unaffected. Expanded erythropoiesis led to massive splenomegaly (spleens =4.3gm vs 0.1gm in unbled controls and 0.3gm in bled controls). After 44 hours of culture with EPO, purified erythroblasts from bled controls proliferated 4-fold and differentiated such that the majority enucleated, producing 200–250 reticulocytes per 100 erythroblasts plated, whereas Bcl-x −/− erythroblast numbers doubled during the first 20 hours in culture, but the large majority died by apoptosis between 20 and 44 hours, producing only 9–12 reticulocytes per 100 erythroblasts plated. Bcl-x −/− erythroblast apoptosis occurred after the initiation of heme synthesis and proportionally in all phases of cell cycle. Compared to culture with EPO, Bcl-x −/− erythroblasts cultured without EPO underwent increased apoptosis at earlier times of culture-- at 8 hours (45% vs 29%), 20 hours (71% vs 42%) and 32 hours (83% vs 57%). Conclusions: 1)Bcl-x is required for the survival and differentiation of the late-stage erythroblasts in all phases of cell cycle. Thus, Bcl-x deficiency results in ineffective erythropoiesis rather than hemolytic anemia. 2)Bcl-x is not required for heme synthesis, but has its anti-apoptotic effect during the stage of hemoglobin synthesis. 3)Bcl-x does not mediate EPO’s anti-apoptotic effect in early-stage erythroblasts.

UCP2 Modulates Cell Proliferation through the MAPK/ERK Pathway during Erythropoiesis and Has No Effect on Heme Biosynthesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5372-5372
Author(s):  
Alvaro A Elorza ◽  
Brigham B Hyde ◽  
Hanna Mikkola ◽  
Sheila Collins ◽  
Orian S Shirihai
Keyword(s):  
Cell Proliferation ◽  
Progenitor Cells ◽  
Fetal Liver ◽  
Mitochondrial Protein ◽  
Erythroid Progenitor ◽  
Heme Synthesis ◽  
Erythroid Progenitor Cells ◽  
Deficient Mice

Abstract UCP2, an inner membrane mitochondrial protein, has been implicated in bioenergetics and Reactive Oxygen Species (ROS) modulation. UCP2 has been previously hypothesized to function as a facilitator of heme synthesis and iron metabolism by reducing ROS production. While UCP2 has been found to be induced by GATA1 during erythroid differentiation its role in erythropoiesis in vivo or in vitro has not been reported thus far. Here we report on the study of UCP2 role in erythropoiesis and the hematologic phenotype of UCP2 deficient mouse. In vivo we found that UCP2 protein peaks at early stages of erythroid maturation when cells are not fully committed in heme synthesis and then becomes undetectable at the reticulocyte stage. Iron incorporation into heme was unaltered in erythroid cells from UCP2 deficient mice. While heme synthesis was not influenced by UCP2 deficiency, mice lacking UCP2 had a delayed recovery from chemically induced hemolytic anemia. Analysis of the erythroid lineage from bone marrow and fetal liver revealed that in the UCP2 deficient mice the R3 (CD71high/Ter119high) population was reduced by 24%. The count of BFU-E and CFU-E colonies, scored in an erythroid colony assay, was unaffected, indicating an equivalent number of early erythroid progenitor cells in both UCP2 deficient and control cells. Ex-vivo differentiation assay revealed that UCP2 deficient c-kit+ progenitor cells expansion was overall reduced by 14% with population analysis determining that the main effect is at the R3 stage. No increased rate of apoptosis was found indicating that expansion rather than cell death is being compromised. Reduced expansion of c-kit+ cells was accompanied by 30% reduction in the phosphorylated form of ERK, a ROS dependent cytosolic regulator of cell proliferation. Analysis of ROS in UCP2 null erythroid progenitors revealed altered distribution of ROS resulting in 14% decrease in cytosolic and 32% increase in mitochondrial ROS. Restoration of the cytosolic oxidative state of erythroid progenitor cells by the pro-oxidant Paraquat reversed the effect of UCP2 deficiency on cell proliferation in in vitro differentiation assays. Together, these results indicate that UCP2 is a regulator of erythropoiesis and suggests that inhibition of UCP2 function may contribute to the development of anemia.

scholarly journals TAF10 Interacts with GATA1 Transcription Factor and Controls Mouse Erythropoiesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2912-2912
Author(s):  
Petros Papadopoulos ◽  
Laura Gutierrez ◽  
Jeroen Demmers ◽  
Dimitris Papageorgiou ◽  
Elena Karkoulia ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells ◽  
Fetal Liver Cells ◽  
Gata1 Transcription Factor

Abstract The ordered assembly of a functional preinitiation complex (PIC), composed of general transcription factors (GTFs) is a prerequisite for the transcription of protein coding genes by RNA polymerase II. TFIID, comprised of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs), is the GTF that is thought to recognize the promoter sequences allowing site-specific PIC assembly. Transcriptional cofactors, such as SAGA (Spt-Ada-Gcn5-acetyltransferase), are also necessary to have tightly regulated transcription initiation. However, a new era on the role of the GTFs and specifically on the role of TFIID in tissue specific and promoter specific transcriptional regulation has emerged in the light of novel findings regarding the differentiation programs of different cell types1. TAF10 is a subunit of both the TFIID and the SAGA co-activator HAT complexes2. The role of TAF10 is indispensable for early embryonic transcription and mouse development as knockout (KO) embryos die early in gestation between E3.5 and E5.5, around the stage when the supply of maternal protein becomes insufficient3. However, when analyzing TFIID stability and transcription it was noted that not all cells and tissues were equally affected by the loss of TAF10. The contribution of the two TAF10-containing complexes (TFIID, SAGA) to erythropoiesis remains elusive. Ablation of TAF10 specifically in erythroid cells by crossing the TAF10-Lox with the EpoR-Cre mouse led to a differentiation block at around E13.5 with erythroid progenitor cells accumulating at a higher percentage (26% in the KO embryos vs 16% in the WTs at E12.5) at the double positive stage KIT+CD71+ and giving rise to fewer mature TER119+ cells in the fetal liver. At E13.5 embryos were dead with almost no erythroid cells in the fetal liver. Gene expression analysis of the fetal liver cells of the embryos revealed down-regulation of GATA1 expression and its target genes, bh1&bmaj/min globins and KLF1 transcription factor while expression of other genes known to have a role in mouse hematopoiesis remained unaffected (MYB, GATA2, PU.1). In order to get insight to the role of TAF10 during erythropoiesis we analyzed the composition of both TAF10-containing complexes (TFIID and SAGA) by mass spectrometry. We found that their stoichiometry changes slightly but not fundamentally during erythroid differentiation and development (human fetal liver erythroid progenitors, human blood erythroid progenitors and mouse erythroid progenitor cells) and no major rearrangements were generated in the composition of the TFIID as it was reported in other cell differentiation programs (e.g. skeletal differentiation, hepatogenesis). Additionally, we found GATA1 transcription factor only in the fetal liver and not in the adult erythroid cells in the mass spectrometry data of TAF10 immunoprecipitations (IPs), an interaction that we confirmed by reciprocal IP of TAF10 and GATA1 in MEL and mouse fetal liver cells. Most importantly, we checked whether TAF10 binding is enriched on the GATA1 locus in human erythroid cells during the fetal and the adult stage in erythroid proerythroblasts and we found that there is enriched binding of TAF10 in the palindromic GATA1 site in the fetal stage. Our results support a developmental role for TAF10 in GATA1 regulated genes, including GATA1 itself, during erythroid differentiation emphasizing the crosstalk between the transcriptional machinery and activators in erythropoiesis. References 1. Goodrich JA, Tjian R (2010) Unexpected roles for core promoter recognition factors in cell-type-specific transcription and gene regulation. Nature reviews Genetics 11: 549-558 2 .Timmers HT, Tora L (2005) SAGA unveiled. Trends Biochem Sci 30: 7-10 3. Mohan WS, Jr., Scheer E, Wendling O, Metzger D, Tora L (2003) TAF10 (TAF(II)30) is necessary for TFIID stability and early embryogenesis in mice. Mol Cell Biol 23: 4307-4318 Disclosures No relevant conflicts of interest to declare.

Death Associated Protein Kinase 2 (DAPK2): A Novel Epo-Modulated Apoptotic Regulator of Late Erythropoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 582-582 ◽  
Author(s):  
Madhu Menon ◽  
Bethany Vincent ◽  
Anne Breggia ◽  
Don M. Wojchowski
Keyword(s):  
Protein Kinase ◽  
Progenitor Cells ◽  
Late Stage ◽  
Specific Binding ◽  
Gene Profiling ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells ◽  
Repressive Effect ◽  
Death Associated Protein Kinase ◽  
High Level

Abstract Death-associated kinase-1 (DAPK1) is a broadly expressed pro-apoptotic cytoplasmic S/T protein kinase that regulates cell proliferation and survival, modulates a p53 apoptotic checkpoint, and contributes to ALL, AML, and myelodysplastic syndromes. DAPK2, by comparison, is an under-studied (but likewise pro-apoptotic) DAPK1 orthologue that we have discovered to be expressed (via Affymetrix gene profiling) at high-levels in late-stage erythroid progenitor cells. In particular, and as examined in purified and developmentally-staged proerythroblasts, basophillic erythroblasts and orthrochromatic erythroblasts, DAPK2 gene expression was sharply up-modulated as progenitor cells developed to a Ter119(+), CD71-high stage. Using a DAPK2-specific antibody, this was confirmed at the protein level, and initial analyses of expression in cell lines further indicated lineage-restricted DAPK2 expression (e.g. high level in erythroid GIE2 cells but undetectable in pluripotent EML cells). To advance an initial understanding of DAPK2’s role in erythroid cells, its possible regulation via the Epo receptor (EpoR) also was studied. Here, primary erythroid progenitor cells from mice with knocked-in minimal EpoR alleles were used (PY-null and PY343-only EpoR-HM and EpoR-H forms, respectively). Interestingly, the point-mutation of a PY3434/Stat5 specific binding site (to F343) (EpoR-HM allele) resulted in a clear multi-fold de-repression of DAPK2 that also correlated with a decreased survival potential for EpoR-HM erythroblasts. This repressive effect of EpoR/PY343/Stat5 signaling on DAPK2 expression was highly stage-specific, and was exerted distinctly in advance of an observed EpoR/PY343/Stat5-dependent up-modulation of Bcl-xl expression. In addition, in wild-type EpoR pro-erythroblasts, DAPK2 levels increased with decreasing Epo concentrations. Investigations therefore reveal DAPK2 to be a late-stage erythroid- restricted, (and EpoR/PY343/Stat5- modulated) novel candidate regulator of red cell production.

scholarly journals Survival and proliferative roles of erythropoietin beyond the erythroid lineage

2008 ◽  
Vol 10 ◽  
Author(s):  
Constance Tom Noguchi ◽  
Li Wang ◽  
Heather M. Rogers ◽  
Ruifeng Teng ◽  
Yi Jia
Keyword(s):  
Progenitor Cells ◽  
Proliferative Activity ◽  
Essential Role ◽  
Receptor Expression ◽  
Protective Activity ◽  
Erythroid Progenitor ◽  
Isolation And Purification ◽  
Erythroid Progenitor Cells ◽  
Epor Expression

Since the isolation and purification of erythropoietin (EPO) in 1977, the essential role of EPO for mature red blood cell production has been well established. The cloning of theEPOgene and production of recombinant human EPO led to the widespread use of EPO in treating patients with anaemia. However, the biological activity of EPO is not restricted to regulation of erythropoiesis. EPO receptor (EPOR) expression is also found in endothelial, brain, cardiovascular and other tissues, although at levels considerably lower than that of erythroid progenitor cells. This review discusses the survival and proliferative activity of EPO that extends beyond erythroid progenitor cells. Loss of EpoR expression in mouse models provides evidence for the role of endogenous EPO signalling in nonhaematopoietic tissue during development or for tissue maintenance and/or repair. Determining the extent and distribution of receptor expression provides insights into the potential protective activity of EPO in brain, heart and other nonhaematopoietic tissues.

Role of NF-kappa B in regulation of apoptosis of erythroid progenitor cells

2005 ◽  
Vol 74 (4) ◽  
pp. 315-323 ◽  
Author(s):  
N. Sae-ung ◽  
T. Matsushima ◽  
I. Choi ◽  
Y. Abe ◽  
P. Winichagoon ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Nf Kappa B ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

scholarly journals Late-stage tumors induce anemia and immunosuppressive extramedullary erythroid progenitor cells

2018 ◽  
Vol 24 (10) ◽  
pp. 1536-1544 ◽  
Author(s):  
Lintao Zhao ◽  
Ran He ◽  
Haixia Long ◽  
Bo Guo ◽  
Qingzhu Jia ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Late Stage ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

scholarly journals Role of Erythropoietin Receptor Signaling in Parvovirus B19 Replication in Human Erythroid Progenitor Cells

2010 ◽  
Vol 84 (23) ◽  
pp. 12385-12396 ◽  
Author(s):  
Aaron Yun Chen ◽  
Wuxiang Guan ◽  
Sai Lou ◽  
Zhengwen Liu ◽  
Steve Kleiboeker ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Ex Vivo ◽  
Virus Entry ◽  
Parvovirus B19 ◽  
Erythropoietin Receptor ◽  
Receptor Expression ◽  
Janus Kinase ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

ABSTRACT Parvovirus B19 (B19V) infection is highly restricted to human erythroid progenitor cells. Although previous studies have led to the theory that the basis of this tropism is receptor expression, this has been questioned by more recent observation. In the study reported here, we have investigated the basis of this tropism, and a potential role of erythropoietin (Epo) signaling, in erythroid progenitor cells (EPCs) expanded ex vivo from CD34+ hematopoietic cells in the absence of Epo (CD36+/Epo− EPCs). We show, first, that CD36+/Epo− EPCs do not support B19V replication, in spite of B19V entry, but Epo exposure either prior to infection or after virus entry enabled active B19V replication. Second, when Janus kinase 2 (Jak2) phosphorylation was inhibited using the inhibitor AG490, phosphorylation of the Epo receptor (EpoR) was also inhibited, and B19V replication in ex vivo-expanded erythroid progenitor cells exposed to Epo (CD36+/Epo+ EPCs) was abolished. Third, expression of constitutively active EpoR in CD36+/Epo− EPCs led to efficient B19V replication. Finally, B19V replication in CD36+/Epo+ EPCs required Epo, and the replication response was dose dependent. Our findings demonstrate that EpoR signaling is absolutely required for B19V replication in ex vivo-expanded erythroid progenitor cells after initial virus entry and at least partly accounts for the remarkable tropism of B19V infection for human erythroid progenitors.

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