Ligand-Regulated and Developmental Stage-Specific Expression Of The EPO Receptor During Human Erythroid Progenitor Cell Formation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3424-3424
Author(s):  
Nicole Rainville ◽  
David Kuhrt ◽  
Su Su ◽  
Edward Jachimowicz ◽  
Asch Ruth ◽  
...  
Keyword(s):  
Cell Surface ◽  
Progenitor Cell ◽  
Conflicts Of Interest ◽  
Cell Formation ◽  
Cell Surface Receptor ◽  
Specific Expression ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cell ◽  
Insight Into ◽  
Epor Expression

Abstract Upon engagement of its cell surface receptor, EPO sharply regulates erythroid progenitor cell (EPC) development. Little is known concerning the dynamics of EPOR expression, however, due to its rarity in primary cells. Using EPO dosing and response studies together with a highly specific and sensitive hEPOR antibody, we provide new insight into stage and ligand dose-dependent regulation of cell surface EPOR expression among human bone marrow derived primary EPCs. Developmentally, EPOR levels peaked within a KITposCD36posGPAneg proerythroblast cohort. In contrast to prior studies using transfected murine myeloid cell lines, the limiting of EPO substantially increased EPOR levels, demonstrating ligand dependent modulation of endogenous EPOR in hEPCs. EPO dosing and withdrawal studies further showed that in early EPCs EPO efficiently supported survival but not proliferation. In GPApos erythroblasts EPOR levels were diminished, but GPApos cells nonetheless exhibited strong EPO dependency for both survival and growth. Investigations provide novel insight into regulated EPOR surface expression among developing primary EPCs, and may also aid in understanding the action modes and effects of emerging EPOR agonists and erythropoiesis stimulating agents. Disclosures: No relevant conflicts of interest to declare.

scholarly journals "C1ORF150", a Novel Mediator of EPO/EPOR/JAK2 Dependent Human Erythroid Progenitor Cell Formation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 544-544
Author(s):  
Don M. Wojchowski ◽  
Darryl Abbott ◽  
Edward Jachimowicz ◽  
Matthew Held
Keyword(s):  
Progenitor Cells ◽  
Progenitor Cell ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Cell Formation ◽  
Refractory Anemia ◽  
Red Cell ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cell ◽  
Erythroid Progenitor Cells

Abstract An understanding of cellular events that are propagated within erythroid progenitor cells upon HGF-R / JAK complex activation is of basic importance for generating new insight into regulated red cell formation, anemia and myeloproliferative disease. Using the EPO/EPOR/JAK2 system as a paradigm, our group is successfully applying post-translational modification-based proteomics to uncover important new mediators of EPO-dependent human erythropoiesis (certain of which may also relate to EPO's untoward effects on hypertension and cancer progression). Here, we report on the discovery of a novel ORF, "C1ORF150", that is strongly tyrosine phosphorylated in response to EPO, possesses several unique features, and modulates EPO- dependent erythroid progenitor cell formation. In human erythroid progenitor UT7epo cells, EPOR ligation leads to C1ORF150 phosphorylation at tandem tyrosine p-Y69, p-Y89 and p-Y110, p-Y129 sites (up to 10-fold within 15 minutes). For each PTM site, EPOR/JAK2 mediated- phosphorylation was validated in independent LC-MS/MS experiments using Hematide as an EPOR agonist. p-Y69 and p-Y89 are predicted SFK sites, while p-Y110 and p-Y129 are predicted RTK sites (including KIT). Notably, C1ORF150 is conserved in H. sapiens and primates, but is not represented in mouse, rat or lower vertebrates. In addition, C1ORF150 has no obvious orthologues, but within EPO-regulated pY regions exhibits sequence homology with HGAL, an important factor for B cell receptor signaling. To assess C1ORF150's functional effects, we used a lentiviral shRNA loss-of-function approach (80% knockdown efficiency). At physiological EPO levels, the knockdown of C1ORF150 substantially compromised UT7epo erythroid progenitor cell (EPC) survival, including 200% increases in apoptosis observed relative to control sh-NT transduced EPCs (p < 0.01). The ectopic expression of C1ORF150, in contrast, heightened baseline JAK2 activation, and potentiated STAT5 activation following EPO challenge. C1ORF150's subcellular localization proved to be predominantly membrane associated. With regards to expression profiles, C1ORF150 levels were markedly elevated in bone marrow (among 30 human tissues), and during erythroid development were maximal at a CFUe stage. Furthermore, transcriptome profiles of myelodysplastic syndrome (MDS) CD34pos hematopoietic progenitor cells revealed elevated C1ORF150 expression in MDS refractory anemia (p=0.005) and refractory anemia-blast patients (p=0.05) compared to normal controls. In summary, via PTM-proteomics we have discovered "C1ORF150" as a major new pY- regulated EPOR/JAK2 target and membrane associated phosphoprotein that is proposed to have evolved in human erythroid progenitor cells to support EPO's cytoprotective effects, and red cell formation, in part by reinforcing JAK2 and STAT5 activation. In anemia and pre-leukemic contexts, attention also is brought to possible roles for C1ORF150 in the onset and progression of MDS. Disclosures No relevant conflicts of interest to declare.

scholarly journals Direct Generation of Immortalized Erythroid Progenitor Cell Lines from Peripheral Blood Mononuclear Cells

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 523
Author(s):  
Abhirup Bagchi ◽  
Aneesha Nath ◽  
Vasanth Thamodaran ◽  
Smitha Ijee ◽  
Dhavapriya Palani ◽  
...  
Keyword(s):  
Cell Lines ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Progenitor Cell ◽  
Mononuclear Cells ◽  
Red Cell ◽  
Peripheral Blood Mononuclear ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cell ◽  
Blood Mononuclear Cells

Reliable human erythroid progenitor cell (EPC) lines that can differentiate to the later stages of erythropoiesis are important cellular models for studying molecular mechanisms of human erythropoiesis in normal and pathological conditions. Two immortalized erythroid progenitor cells (iEPCs), HUDEP-2 and BEL-A, generated from CD34+ hematopoietic progenitors by the doxycycline (dox) inducible expression of human papillomavirus E6 and E7 (HEE) genes, are currently being used extensively to study transcriptional regulation of human erythropoiesis and identify novel therapeutic targets for red cell diseases. However, the generation of iEPCs from patients with red cell diseases is challenging as obtaining a sufficient number of CD34+ cells require bone marrow aspiration or their mobilization to peripheral blood using drugs. This study established a protocol for culturing early-stage EPCs from peripheral blood (PB) and their immortalization by expressing HEE genes. We generated two iEPCs, PBiEPC-1 and PBiEPC-2, from the peripheral blood mononuclear cells (PBMNCs) of two healthy donors. These cell lines showed stable doubling times with the properties of erythroid progenitors. PBiEPC-1 showed robust terminal differentiation with high enucleation efficiency, and it could be successfully gene manipulated by gene knockdown and knockout strategies with high efficiencies without affecting its differentiation. This protocol is suitable for generating a bank of iEPCs from patients with rare red cell genetic disorders for studying disease mechanisms and drug discovery.

scholarly journals Establishment of an erythroid progenitor cell line capable of enucleation achieved with an inducible c-Myc vector

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Steven Mayers ◽  
Pablo Diego Moço ◽  
Talha Maqbool ◽  
Pamuditha N. Silva ◽  
Dawn M. Kilkenny ◽  
...  
Keyword(s):  
Cell Line ◽  
Progenitor Cell ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cell ◽  
Progenitor Cell Line

Influence of albuterol on erythropoietin production and erythroid progenitor cell activation

1979 ◽  
Vol 236 (3) ◽  
pp. H422-H426 ◽  
Author(s):  
F. Przala ◽  
D. M. Gross ◽  
B. Beckman ◽  
J. W. Fisher
Keyword(s):  
Bone Marrow ◽  
Progenitor Cell ◽  
Cell Activation ◽  
Plasma Clot ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cell ◽  
Adrenergic Blocker ◽  
Rabbit Bone

The effect of albuterol, a potent beta2-adrenergic agonist, on kidney production of erythropoietin (Ep) was studied. Its effects on erythroid colony (CFU-E) formation in vitro in rabbit bone marrow cultures were also assessed. Albuterol produced a significant increase in plasma Ep levels in conscious rabbits following 7 h intravenous infusion (50 (microgram/kg)/min). This effect was blocked by pretreatment of the rabbits with butoxamine (5 mg/kg ip), a potent beta2-adrenergic blocker. Albuterol in doses of 10(-10) to 10(-8) M in combination with Ep was also found to produce a significant increase in the numbers of CFU-E in the plasma clot culture system of rabbit bone marrow. This effect was blocked completely by DL-propranolol (10(-8) M) and by butoxamine (10(-8) M). The data presented suggest that albuterol, a potent activator of beta2-adrenergic receptors, increases kidney production of Ep in vivo and also produces a direct effect in combination with Ep on the proliferation of the erythroid progenitor cell compartment.

scholarly journals RHEX, a novel regulator of human erythroid progenitor cell expansion and erythroblast development

2015 ◽  
Vol 212 (6) ◽  
pp. 971-971
Author(s):  
Rakesh Verma ◽  
Su Su ◽  
Donald J. McCrann ◽  
Jennifer M. Green ◽  
Karen Leu ◽  
...  
Keyword(s):  
Cell Expansion ◽  
Progenitor Cell ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cell

Essential Role for Activated Leukocyte Cell Adhesion Molecule Gene Silencing by GATA-1 in Megakaryocytic Progenitor Cell Biology.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1194-1194
Author(s):  
Fang Tan ◽  
Robert Thomas ◽  
Flaubert Mbeunkui ◽  
Solomon F. Ofori-Acquah
Keyword(s):  
Cell Adhesion ◽  
Cell Lines ◽  
Adhesion Molecule ◽  
Cell Biology ◽  
Progenitor Cell ◽  
Cell Adhesion Molecule ◽  
K562 Cells ◽  
Jurkat Cells ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cell

Abstract Regulation of hematopoietic progenitor cell lineage-commitment, proliferation and differentiation by cell-cell adhesion mechanisms is poorly understood. Activated leukocyte cell adhesion molecule (ALCAM) is a member of the immunoglobulin super family. It is expressed by human hematopoietic stem cells, bone marrow stromal cells, endothelial cells and osteoblasts. Monoclonal anti-ALCAM antibodies inhibit myeloid but not erythroid colony formation, which suggest a lineage-specific role for ALCAM in hematopoiesis. To explore this hypothesis, ALCAM mRNA and protein expression was quantified in human hematopoietic cell lines of myeloid, lymphoid, erythroid, and megakaryocytic lineages by real-time quantitative PCR and western blot analyses. No ALCAM transcripts were detected in K562 and MEG-01 cells, the level of ALCAM mRNA was 2-fold more abundant in HL-60 and THP-1 cells than in U937 and Jurkat cells. This expression pattern was confirmed at the protein level as none of the megakaryocyte-erythroid progenitor cell lines (K562, MEG-01 and HEL) expressed ALCAM. On the contrary, ALCAM was abundantly expressed in THP-1 and HL-60 cells and moderately in U937 and Jurkat cells. GATA-1 was abundantly expressed in megakaryocyte-erythroid progenitor cell lines but not in any of the myeloid cell lines. Thus, there is an inverse relationship between expression of ALCAM and GATA-1 in hematopoietic cells. To test the hypothesis that GATA-1 is involved in silencing ALCAM gene expression, multiple ALCAM-promoter luciferase constructs were studied. A negative regulatory region was identified in the ALCAM promoter containing an inverted GATA-1 cis element at −850 upstream of the translational start site. GATA-1 occupied this canonical element in vivo as determined by chromatin immunoprecipitation experiments. A two-base pair mutation of the −850 GATA-1 cis element increased ALCAM promoter activity 3-fold in K562 and MEG-01 cells, providing direct evidence of GATA-1’s negative regulatory role in ALCAM promoter activity. To test the hypothesis that ALCAM silencing is essential for megakaryocyte-erythroid progenitor cell biology, stable lines of K562 cells were established forcibly expressing ALCAM-GFP or a control GFP. Live cell imaging demonstrated recruitment of ALCAM to sites of cell-cell adhesion in ALCAM-GFP-K562 cells, whereas GFP remained distributed in the cell cytosol in control cells. ALCAM-GFP-K562 cells formed markedly more clusters consisting of significantly more cells than control GFP-K562 cells. Finally, the number of ALCAM-GFP-K562 cells at log-phase growth was significantly higher than GFP-K562 cells over the same time period. Our findings demonstrate for the first time lineage-specific silencing of the cell adhesion molecule ALCAM in megakaryocyte-erythroid progenitor cells, mediated at least in part by GATA-1. That ectopic expression of ALCAM increased proliferation of K562 cells suggests that GATA-1-mediated silencing of ALCAM is essential in slowing down expansion of megakaryocyte-erythroid progenitor cells. Indeed, preliminary studies show an excessive number of erythroid and megakaryocytic cells in the adult spleen of ALCAM-null mice. This model is being used in ongoing studies to confirm our findings in vivo.

The G9A Histone Methyltransferase BIX-01294 Reactivates ε-Globin Expression and Blocks Erythroid Differentiation in Primary Baboon Erythroid Progenitor Cell Cultures

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 129-129 ◽  
Author(s):  
Virryan Banzon ◽  
Vinzon Ibanez ◽  
Kestis Vaitkus ◽  
Tatiana Kousnetzova ◽  
Joseph Desimone ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Cell Cultures ◽  
Progenitor Cell ◽  
Globin Gene ◽  
Histone H3 ◽  
Erythroid Differentiation ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cell

Abstract The development of new therapies to increase fetal hemoglobin (HbF) levels in patients with sickle cell disease and β-thalassemia depends on an increased understanding of the mechanism responsible for the developmental regulation of globin gene expression. A role for epigenetic modifications in the mechanism of of globin gene regulation is suggested by the presence of high levels of DNA methylation near the 5’ regions of developmentally silenced ε- and γ-globin genes and the ability of pharmacological inhibitors of DNA methyltransferase (DNMTase) to reactivate ε- and γ-globin expression in adults. Whether additional epigenetic modifications associated with gene silencing and DNA methylation, such as histone H3 (lys9) dimethylation, are also involved is unknown. To investigate the hypothesis that histone H3 (lys9) dimethylation may function in the mechanism of developmental globin gene silencing, chromatin immunopreciptation assays were performed to determine the distribution of histone H3 (lys9) dimethyl and histone H3 (lys9) acetyl throughout the β-globin gene complex in purified primary baboon bone marrow (BM) erythroid cells from phlebotomized baboons expressing low levels (5–10%) of HbF and purified erythroid cells from erythroid progenitor cell cultures expressing high levels of HbF (30–50%). In BM erythroid cells, the level of histone H3 (lys9) acetyl associated with the β-globin gene was 10–20 fold higher than with the ε- and γ-globin genes, while the level of histone H3 (lys9) dimethyl associated with the ε- and γ-globin genes was 2–4 fold higher than with the β-globin gene. In erythroid cells from day 12 erythroid progenitor cell cultures, the level of histone H3 (lys9) acetyl associated with the highly expressed γ- and β-globin genes was 10–20 fold higher than with the silent ε-globin gene, while the level of histone H3 (lys9) dimethyl associated with the ε-globin gene was 2–4 fold higher than with the γ- and β-globin genes. Therefore a reciprocal relationship was observed between levels of histone H3 (lys9) acetylation and dimethylation associated with active and inactive globin genes. Experiments were performed to further investigate the role of histone H3 (lys9) dimethyl in ε-globin gene silencing by determining the effect of the G9A histone methyltransferase inhibitor BIX-01294 on ε-globin expression. Erythroid progenitor cell cultures derived from CD34+ BM cells of three individual baboons were treated with the varying doses of the DNMTase inhibitor decitabine (0.125–1.0μM), and BIX-01294 (1.25–5μM), alone and in combination. Changes in ε- globin were assessed by real time PCR using the ΔΔCT method with α-globin as the standard. Decitabine (0.5μM) increased ε-globin 25.8±7.7 fold while BIX-01294 (2.5μM) increased ε-globin 3.09±1.16 fold. Decitabine (1μM) and BIX-01294 (2.5μM) in combination increased ε-globin 55.7±24.9 fold. BIX-01294 enhanced ε-globin expression approximately twofold at all decitabine doses ranging from 0.125–1.0μM (mean increase=103± 44.7%). BIX-01294 also blocked terminal erythroid differentiation and allowed expansion of more primitive cells as evidenced by the presence of a large population of basophilic erythroblasts at late stages of culture (day 14). These results demonstrate that BIX-01294 reactivates expression of the silenced ε-globin gene and that synergistic reactivation can be achieved using combinations of BIX-01294 and decitabine. While these results are consistent with the hypothesis that epigenetic modifications are important in the mechanism of developmental globin gene silencing, the observation that BIX-01294 blocks erythroid differentiation suggests the possible involvement of a reprogramming mechanism.

Apolipoprotein(a)-Dependent Inhibition of Pericellular Plasminogen Activation Is Mediated by Specific Cellular Receptors

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2236-2236
Author(s):  
Rocco Romagnuolo ◽  
Michael B Boffa ◽  
Marlys L Koschinsky
Keyword(s):  
Cell Surface ◽  
Plasminogen Activator ◽  
Conflicts Of Interest ◽  
Cell Surface Receptor ◽  
Dependent Manner ◽  
Plasminogen Activation ◽  
Apolipoprotein A ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

Abstract Abstract 2236 Lipoprotein(a) [Lp(a)] has been identified as an independent risk factor for cardiovascular diseases such as coronary heart disease. Lp(a) levels vary over 1000-fold within the human population and Lp(a) possesses both proatherogenic and prothrombotic properties due to the LDL-like moiety and apolipoprotein(a) [apo(a)] components, respectively. Apo(a) is highly homologous to plasminogen and thus can potentially interfere with plasminogen activation. Plasmin generated in the context of fibrin mediates the breakdown of blood clots, which are the causative factors in heart attacks and strokes. Plasmin generated on the surface of vascular cells plays a role in cell migration and proliferation, two of the fibroproliferative inflammatory events that underlie atherosclerosis. Previous studies have suggested that apo(a) may inhibit pericellular plasminogen activation on the basis of observations that apo(a) decreases plasminogen binding to cells. We have undertaken analysis of the mechanism by which apo(a) may interfere with pericellular plasminogen activation to allow for a more definitive description of the role of Lp(a) within the vasculature. Plasminogen activation was found to be markedly inhibited by the recombinant apo(a) variant 17K, in a dose dependent manner, on human umbilical vein endothelial cells (HUVECs), human monocytic leukemia cells (THP-1), THP-1 macrophages, and smooth muscle cells. The strong lysine binding site in kringle IV type 10, as well as kringle V appear to be required for this effect since apo(a) variants lacking these elements (17KΔAsp and 17KΔV, respectively) failed to inhibit activation. However, the role of lysine-dependent binding of apo(a) itself to the cells is not clear. Carboxypeptidase treatment of cells did not decrease apo(a) binding, and apo(a) does not compete directly for plasminogen binding to the cells. Rather, apo(a) and plasminogen may bind to the cells as a complex. We next attempted to identify the cell-surface receptor(s) that mediate plasminogen activation on the cell surface as well as its inhibition by apo(a). Urokinase-type plasminogen activator receptor (uPAR) has been previously shown to bind to urokinase-type plasminogen activator (uPA), vitronectin, and β3 integrins. uPAR is involved in the remodeling of the extracellular matrix (ECM) through regulation of plasminogen activation. We found evidence that uPAR is a potential receptor for both plasminogen and apo(a). Knockdown of uPAR in HUVECs results in decreased binding of plasminogen, 17K and, to a lesser extent, 17KΔAsp and 17KΔV. Similar experiments in SMCs revealed no changes in binding. A decrease in tPA-mediated plasminogen activation following uPAR knockdown occurred in HUVECs, and addition of 17K did not result in any further decrease. Overexpression of uPAR in THP-1 macrophages leads to greater than a two fold increase in 17K and plasminogen binding. Plasminogen activation increases over two-fold as a result of overexpression of uPAR, while 17K blunts the effect of uPAR overexpression. These results indicate that uPAR plays a crucial role in both plasminogen and apo(a) binding to the cell surface of specific cells and inhibition by apo(a) of plasminogen activation. Macrophage-1-antigen (Mac-1) receptor consists of CD11b (αM) and CD18 (β2) integrin and has been previously shown to recognize uPA and control migration and adhesion. Furthermore, αVβ3 has been previously shown to bind to vitronectin and the uPA-uPAR complex which promotes cell adhesion through binding of both vitronectin and αVβ3 integrins. We found that blocking the αM, β2, or αVβ3 receptors with monoclonal antibodies in THP-1 cells leads to a decrease in plasminogen activation, as well as a blunting of the inhibitory effects of apo(a) on plasminogen activation. These results indicate a role for Mac-1 and αVβ3 in apo(a) binding and inhibition of plasminogen activation. In conclusion, we have demonstrated, for the first time, the role of specific receptors in binding of apo(a) to vascular cell surfaces and in mediating the inhibitory effect of apo(a) on pericellular plasminogen activation. Disclosures: No relevant conflicts of interest to declare.

Inhibition of erythroid progenitor cell growth by anti-Ge3

2006 ◽  
Vol 133 (4) ◽  
pp. 443-444 ◽  
Author(s):  
Gregory A. Denomme ◽  
Ali Shahcheraghi ◽  
Douglas P. Blackall ◽  
Krishna K. Oza ◽  
George Garratty
Keyword(s):  
Cell Growth ◽  
Progenitor Cell ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cell
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