The function of the bcl-x promoter in erythroid progenitor cells

The protein Bcl-xL is essential for survival of erythroid progenitor cells, and it increases substantially during late erythrocyte differentiation due to an increase of mRNA. We mapped the transcription start sites of bcl-x mRNA in mouse and human erythroblasts, and we analyzed the function of the mousebcl-x promoter by transient and stable transfection assays in a mouse erythroid cell line using plasmids containing thebcl-x promoter fused to a luciferase reporter gene. In mouse erythroblasts, a cluster of start sites at positions −664, −655, and −644 relative to the ATG initiation codon account for almost all transcripts. Human erythroblasts exhibit a start site at −654 that is homologous to the triplet in the mouse. A short sequence element in the mouse bcl-x promoter that includes nucleotides −1804 through −1734 was identified as very important for transcription. This element also showed strong enhancerlike activity in concert with the SV40 promoter in an enhancer test vector. Analyses of mutations indicated that 2 short sequences within the element, about 15 base pair apart, are necessary for full enhancer activity. Gel shift experiments with oligonucleotides representing these sequences revealed specific binding of nuclear proteins from erythroblasts. Some of these proteins are regulated during the late erythroid differentiation.

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Ligand-dependent repression of the erythroid transcription factor GATA-1 by the estrogen receptor.

Molecular and Cellular Biology ◽

10.1128/mcb.15.6.3147 ◽

1995 ◽

Vol 15 (6) ◽

pp. 3147-3153 ◽

Cited By ~ 88

Author(s):

G A Blobel ◽

C A Sieff ◽

S H Orkin

Keyword(s):

Bone Marrow ◽

Estrogen Receptor ◽

Transcription Factor ◽

Progenitor Cells ◽

Erythroid Cell ◽

High Dose ◽

Dependent Manner ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells

High-dose estrogen administration induces anemia in mammals. In chickens, estrogens stimulate outgrowth of bone marrow-derived erythroid progenitor cells and delay their maturation. This delay is associated with down-regulation of many erythroid cell-specific genes, including alpha- and beta-globin, band 3, band 4.1, and the erythroid cell-specific histone H5. We show here that estrogens also reduce the number of erythroid progenitor cells in primary human bone marrow cultures. To address potential mechanisms by which estrogens suppress erythropoiesis, we have examined their effects on GATA-1, an erythroid transcription factor that participates in the regulation of the majority of erythroid cell-specific genes and is necessary for full maturation of erythrocytes. We demonstrate that the transcriptional activity of GATA-1 is strongly repressed by the estrogen receptor (ER) in a ligand-dependent manner and that this repression is reversible in the presence of 4-hydroxytamoxifen. ER-mediated repression of GATA-1 activity occurs on an artificial promoter containing a single GATA-binding site, as well as in the context of an intact promoter which is normally regulated by GATA-1. GATA-1 and ER bind to each other in vitro in the absence of DNA. In coimmunoprecipitation experiments using transfected COS cells, GATA-1 and ER associate in a ligand-dependent manner. Mapping experiments indicate that GATA-1 and the ER form at least two contacts, which involve the finger region and the N-terminal activation domain of GATA-1. We speculate that estrogens exert effects on erythropoiesis by modulating GATA-1 activity through protein-protein interaction with the ER. Interference with GATA-binding proteins may be one mechanism by which steroid hormones modulate cellular differentiation.

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Globin Genes Induction by Nitric Oxide of Stromal Cell Origin.

Blood ◽

10.1182/blood.v110.11.4102.4102 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4102-4102

Author(s):

Vladan P. Cokic ◽

Bojana B. Beleslin-Cokic ◽

Constance Tom Noguchi ◽

Alan N. Schechter

Keyword(s):

Progenitor Cells ◽

Globin Gene ◽

Erythroid Cell ◽

Mrna Levels ◽

Globin Genes ◽

Globin Mrna ◽

Erythroid Progenitor ◽

Gamma Globin ◽

Erythroid Progenitor Cells ◽

Globin Gene Expression

Abstract We have previously shown that nitric oxide (NO) is involved in the hydroxyurea-induced increase of gamma-globin gene expression in cultured human erythroid progenitor cells and that hydroxyurea increases NO production in endothelial cells via endothelial NO synthase (NOS). Here we report that co-culture of human bone marrow endothelial cells with erythroid progenitor cells induced gamma-globin mRNA expression (1.8 fold), and was further elevated (2.4 fold) in the presence of hydroxyurea (40 μM). Based on these results, NOS-dependent stimulation of NO levels by bradykinin and lipopolysaccharide has been observed in endothelial (up to 0.3 μM of NO) and macrophage cells (up to 6 μM of NO), respectively. Bradykinin slightly increased gamma-globin mRNA levels in erythroid progenitor cells, but failed to increase gamma-globin mRNA levels in endothelial/erythroid cell co-cultures indicating that stimulation of endothelial cell production of NO alone is not sufficient to induce gamma-globin expression. In contrast, lipopolysaccharide and interferon-gamma mutually increased gamma-globin gene expression (2 fold) in macrophage/erythroid cell co-cultures. In addition, hydroxyurea (5–100 μM) induced NOS-dependent production of NO in human (up to 0.7 μM) and mouse macrophages (up to 1.2 μM). Co-culture studies of macrophages with erythroid progenitor cells also resulted in induction of gamma-globin mRNA expression (up to 3 fold) in the presence of hydroxyurea (20–100 μM). These results demonstrate a mechanism by which hydroxyurea may induce globin genes and affect changes in the phenotype of hematopoietic cells via the common paracrine effect of bone marrow stromal cells.

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Neuromedin U Peptide Activates STAT5 and S6 in a JAK-2 Dependent Manner and Promotes Erythroid Cell Growth in Primary Erythroid Progenitor Cells

Blood ◽

10.1182/blood.v120.21.1241.1241 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1241-1241

Author(s):

Rebecca Lenzo ◽

Martha Dua-Awereh ◽

Martin Carroll ◽

Susan E. Shetzline

Keyword(s):

Cell Growth ◽

Progenitor Cells ◽

Surrogate Marker ◽

Janus Kinase ◽

Erythroid Cell ◽

Dependent Manner ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Erythroid Progenitor Cells ◽

Pi3k Activation

Abstract Abstract 1241 Erythropoiesis is a multi-step process during which hematopoietic stem cells terminally differentiate into red blood cells (RBCs). Erythropoietin (EPO) is the only known cytokine regulator of terminal erythroid differentiation. Previously, we reported that the neuropeptide, neuromedin U (NmU), which interacts with NmU receptor type 1 (NMUR1), functions as a novel extracellular cofactor with EPO to promote the expansion of early erythroblasts, which are CD34−, CD71+, glycophorin A (GlyA)dim(Gambone et al, Blood. 2011). Here, we describe studies to understand the mechanism whereby NmU augments EPO effects on erythroid cell growth. EPO triggers Janus kinase (Jak)-2 dependent activation of signal transducer and activator of transcription (STAT) 5 and phosphatidylinositol 3-kinase (PI3K) to promote the proliferation and/or survival of erythroid progenitor cells. We hypothesized that NmU peptide would cooperate with EPO to promote the proliferation of early erythroblasts through STAT5 and/or PI3K activation. To address this hypothesis, we cultured primary human CD34+ cells in 2-stage liquid culture with IL-3, IL-6, and stem cell factor (SCF) from day 0 to day 6. On day 6, 2U/mL of EPO was added, and the cells were cultured for an additional 5 days to expand erythroid progenitors. On day 11, cells were briefly serum starved and then stimulated with EPO and/or NmU in the absence or presence of a Jak-1/2 inhibitor. Activation of STAT5 and S6, a surrogate marker for PI3K activation, were assessed by phospho-flow in ERY3 (CD34−, CD71+, GlyA+) and ERY4 (CD34−, CD71dim, GlyA+) cells. As expected, EPO alone activated STAT5 and S6 in ERY3 cells only, and the presence of a Jak-1/2 inhibitor diminished STAT5 activation. Interestingly, STAT5 and S6 were activated by NmU peptide alone in ERY3 and ERY4. Surprisingly, in the presence of a Jak-1/2 inhibitor, NmU peptide, which binds to NMUR1 a G-protein coupled receptor, did not activate STAT5 or S6 in ERY3 or 4 cells, suggesting that NmU functions through a JAK kinase in erythroid cells. No additive or synergistic activation of STAT5 and S6 is observed in the presence of both EPO and NmU peptide when EPO was used at a dose of 2 U/mL. The mechanism whereby NmU activates a JAK dependent signaling pathway is under investigation. Preliminary evidence suggests that EPO induces the physical association of NMUR1 with EPO receptor (EPOR). Taken together, we propose that NmU is a neuropeptide expressed in bone marrow cells that cooperates to regulate erythroid expansion during early erythropoiesis through the activation of cytokine receptor like signaling pathways and perhaps through direct interaction with EPOR. NmU may be useful in the clinical management of anemia in patients unresponsive to EPO or other erythroid-stimulating agents. Disclosures: No relevant conflicts of interest to declare.

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Establishment of an erythroid cell line from primary CD36+ erythroid progenitor cells

Experimental Hematology ◽

10.1016/j.exphem.2010.07.012 ◽

2010 ◽

Vol 38 (11) ◽

pp. 994-1005.e2 ◽

Cited By ~ 10

Author(s):

Susan Wong ◽

Keyvan Keyvanfar ◽

Zhihong Wan ◽

Sachiko Kajigaya ◽

Neal S. Young ◽

...

Keyword(s):

Cell Line ◽

Progenitor Cells ◽

Erythroid Cell ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells

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Abnormal erythroid progenitor cells in human preleukemia

Blood ◽

10.1182/blood.v60.2.362.362 ◽

1982 ◽

Vol 60 (2) ◽

pp. 362-367 ◽

Cited By ~ 43

Author(s):

DH Chui ◽

BJ Clarke

Keyword(s):

Progenitor Cells ◽

Peripheral Blood ◽

Culture Technique ◽

The Other ◽

Erythroid Cell ◽

Erythroid Progenitor ◽

Clonal Culture ◽

Erythroid Progenitor Cells ◽

Marrow Cells

Abstract Ten patients with preleukemia were studied by the erythroid cell clonal culture technique. In nine of these patients, erythroid colonies derived from peripheral blood BFU-E were not observed, while the other patient had markedly decreased peripheral blood BFU-E-derived erythroid colonies in vitro. In three patients, marrow cells were also cultured and no BFU-E-derived erythroid colonies were detected. These studies indicate that immature erythroid progenitor cells, BFU-E, in patients with preleukemia are either markedly decreased in number or grossly defective in their proliferative or differentiative capacities.

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

Blood ◽

10.1182/blood.v104.11.582.582 ◽

2004 ◽

Vol 104 (11) ◽

pp. 582-582 ◽

Cited By ~ 3

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.

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Lineage-Specific Activation of p53 in Response to Ribosomal Haploinsufficiency in Human Bone Marrow Cells.

Blood ◽

10.1182/blood.v114.22.948.948 ◽

2009 ◽

Vol 114 (22) ◽

pp. 948-948

Author(s):

Shilpee Dutt ◽

Anupama Narla ◽

Jeffery Lorne Kutok ◽

Benjamin L. Ebert

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Ribosome Biogenesis ◽

Conflicts Of Interest ◽

Erythroid Cell ◽

Erythroid Progenitor ◽

Intense Staining ◽

Bone Marrow Biopsies ◽

Erythroid Progenitor Cells ◽

Cd34 Cells

Abstract Abstract 948 Haploinsufficiency for the ribosomal protein genes RPS14 and RPS19 have been implicated in the erythroid defect in the 5q- syndrome and Diamond Blackfan Anemia, respectively. However, the mechanism by which defective ribosome biogenesis causes erythroid failure is unknown. In this study, we found that shRNA mediated knockdown of RPS14 or RPS19 in primary human CD34+ cells stabilize TP53 by day 4 after infection with concomitant arrest of these cells at G1 stage of cell cycle. The levels of TP53 attained are comparable to the levels observed following gamma irradiation (5Gy) of the CD34+ cells. Using quantitative PCR, we confirmed that stabilized TP53 activates expression of downstream target genes MDM2, p21, Bax and Wig-1. Furthermore, treatment of the CD34+ cells with Nutlin-3 phenocopies RPS14 or RPS19 knockdown, suggesting that the mechanism of TP53 activation is mediated by MDM2 pathway. Conversely, treatment with pifithrin-alpha, which inhibits the transactivation activity of TP53, rescues the effects of RPS14 or RPS19 knockdown. The in vitro activation of TP53 in CD34+ cells was restricted to erythroid cell lineage, consistent with the clinical phenotype of RPS14 or RPS19 haploinsufficiency. Moreover, immunohistochemical analysis of bone marrow biopsies from patient with the 5q- syndrome demonstrated intense staining of TP53 that was restricted to erythroid progenitor cells. Taken together our study indicates that inhibition of ribosomal biogenesis causes TP53 activation selectively in erythroid progenitor cells. Clinically, TP53 staining of patient samples could be used as a diagnostic marker for some types of MDS. Disclosures: No relevant conflicts of interest to declare.

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Epo-Independent Proliferation and Differentiation of Genetically Modified Erythroid Progenitor Cells Using a Chemical Inducer of Dimerization.

Blood ◽

10.1182/blood.v108.11.1159.1159 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1159-1159

Author(s):

Chris P. Miller ◽

Songmao Zheng ◽

C. Anthony Blau

Keyword(s):

Progenitor Cells ◽

Erythroid Cell ◽

Glycophorin A ◽

Erythroid Cells ◽

Erythroid Progenitor ◽

Cell Production ◽

Erythroid Progenitor Cells ◽

Proliferation And Differentiation ◽

Cells Cultured ◽

Chemical Inducer Of Dimerization

Abstract Recombinant human erythropoietin (Epo) revolutionized the care of anemia in cancer. In light of emerging (albeit controversial) roles for Epo in supporting tumor growth, it may be useful to develop Epo-independent methods for regulating red blood cell production. Previous studies in this laboratory established that marrow transduced with conditional derivatives of fibroblast growth factor receptor-1 (F36VFGFR1) and the thrombopoietin receptor (F36VMpl) can support the chemical inducer of dimerization (CID)-dependent production of erythroid cells in transplanted mice. In the current study, we used human CD34+ cord blood (CB) cells to test whether CID-regulated red cell production might require collaboration between CID-initiated signals and those provided by Epo. CD34+ CB cells cultured in the absence of Epo did not proliferate and retained expression of the myeloid marker CD33. Epo (5U/mL) promoted proliferative expansion (66.2-fold in 12 days) and CD33 expression was lost as the cells differentiated to mature, glycophorin A+ erythroid cells (92.9 +/− 1.8%, n=3). Addition of a soluble human Epo receptor extracellular domain (shEpoR) at a concentration of 3ug/mL completely blocked Epo-dependent proliferation and similar to cells cultured without Epo, they retained expression of CD33. Addition of CID (100nM AP20187) to F36VMpl-transduced CD34+ CB cells in the absence of Epo promoted proliferative expansion (89.8-fold in 12 days) and differentiation as glycophorin A+ erythroid cells (77.2 +/− 4.8%, n=3). These CID responses were not significantly affected by the addition of shEpoR, as CID induced both mitogenic expansion (84.5 fold in 12 days) and differentiation as glycophorin A+ erythroid cells (74.2 +/− 6.7%, n=3) in the presence of concentrations of shEpoR that blocked Epo responses. These data suggest that F36VMpl does not require Epo signaling to support the proliferation and differentiation of human erythroid progenitor cells, and further define an Epo-independent method of erythroid cell production.

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Abnormal erythroid progenitor cells in human preleukemia

Blood ◽

10.1182/blood.v60.2.362.bloodjournal602362 ◽

1982 ◽

Vol 60 (2) ◽

pp. 362-367 ◽

Cited By ~ 2

Author(s):

DH Chui ◽

BJ Clarke

Keyword(s):

Progenitor Cells ◽

Peripheral Blood ◽

Culture Technique ◽

The Other ◽

Erythroid Cell ◽

Erythroid Progenitor ◽

Clonal Culture ◽

Erythroid Progenitor Cells ◽

Marrow Cells

Ten patients with preleukemia were studied by the erythroid cell clonal culture technique. In nine of these patients, erythroid colonies derived from peripheral blood BFU-E were not observed, while the other patient had markedly decreased peripheral blood BFU-E-derived erythroid colonies in vitro. In three patients, marrow cells were also cultured and no BFU-E-derived erythroid colonies were detected. These studies indicate that immature erythroid progenitor cells, BFU-E, in patients with preleukemia are either markedly decreased in number or grossly defective in their proliferative or differentiative capacities.

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