Erythropoietin receptor-dependent erythroid colony-forming unit development: capacities of Y343 and phosphotyrosine-null receptor forms

Blood ◽  
2002 ◽  
Vol 99 (3) ◽  
pp. 898-904
Author(s):  
Chris P. Miller ◽  
Destin W. Heilman ◽  
Don M. Wojchowski
Keyword(s):  
Growth Factor Receptor ◽  
Cell Development ◽  
Erythropoietin Receptor ◽  
Janus Kinase ◽  
Erythroid Cell ◽  
Compensatory Mechanisms ◽  
Erythroid Progenitor ◽  
Epo Receptor ◽  
Binding Domains ◽  
Progenitor Cell Proliferation

Red cell development depends on the binding of erythropoietin (EPO) to receptors expressed by erythroid colony-forming units (CFUe) and the subsequent activation of receptor-bound Janus kinase (Jak2). Jak2 then mediates the phosphorylation of receptor tyrosine sites and the recruitment of 25 or more Src homology 2 domain-encoding proteins and associated factors. Previous studies have shown that an EPO receptor form containing Jak2-binding domains plus a single phosphotyrosine343 (PY343)–STAT5-binding site provides all signals needed for erythroid cell development. However, roles for PY343 and STAT5 remain controversial, and findings regarding PY-null receptor activities and erythropoiesis in STAT5-deficient mice are disparate. To study activities of a PY-null EPO receptor in primary cells while avoiding compensatory mechanisms, a form retaining domains for Jak2 binding and activation, but lacking all cytoplasmic tyrosine sites, was expressed in transgenic mice from aGATA1 gene-derived vector as a human epidermal growth factor receptor- murine EPO receptor chimera (EE-T-Y343F). The bio-signaling capacities of this receptor form were investigated in CFUe from thiamphenicol-treated mice. Interestingly, this PY-null EPO receptor form supported CFUe development (in the absence of detectable STAT5 activation) at efficiencies within 3-fold of those levels mediated by either an EE-T-Y343 form or the endogenous EPO receptor. However, EE-T-Y343F–dependent Ter119+ erythroblast maturation was attenuated. In tests of cosignaling with c-Kit, EE-T-Y343F nonetheless retained full capacity to synergize with c-Kit in promoting erythroid progenitor cell proliferation. Thus, EPO receptor PY-dependent events can assist late erythropoiesis but may be nonessential for EPO receptor–c-Kit synergy.

Erythropoietin receptor-dependent erythroid colony-forming unit development: capacities of Y343 and phosphotyrosine-null receptor forms

Blood ◽  
2002 ◽  
Vol 99 (3) ◽  
pp. 898-904 ◽  
Author(s):  
Chris P. Miller ◽  
Destin W. Heilman ◽  
Don M. Wojchowski
Keyword(s):  
Growth Factor Receptor ◽  
Cell Development ◽  
Erythropoietin Receptor ◽  
Janus Kinase ◽  
Erythroid Cell ◽  
Compensatory Mechanisms ◽  
Erythroid Progenitor ◽  
Epo Receptor ◽  
Binding Domains ◽  
Progenitor Cell Proliferation

Abstract Red cell development depends on the binding of erythropoietin (EPO) to receptors expressed by erythroid colony-forming units (CFUe) and the subsequent activation of receptor-bound Janus kinase (Jak2). Jak2 then mediates the phosphorylation of receptor tyrosine sites and the recruitment of 25 or more Src homology 2 domain-encoding proteins and associated factors. Previous studies have shown that an EPO receptor form containing Jak2-binding domains plus a single phosphotyrosine343 (PY343)–STAT5-binding site provides all signals needed for erythroid cell development. However, roles for PY343 and STAT5 remain controversial, and findings regarding PY-null receptor activities and erythropoiesis in STAT5-deficient mice are disparate. To study activities of a PY-null EPO receptor in primary cells while avoiding compensatory mechanisms, a form retaining domains for Jak2 binding and activation, but lacking all cytoplasmic tyrosine sites, was expressed in transgenic mice from aGATA1 gene-derived vector as a human epidermal growth factor receptor- murine EPO receptor chimera (EE-T-Y343F). The bio-signaling capacities of this receptor form were investigated in CFUe from thiamphenicol-treated mice. Interestingly, this PY-null EPO receptor form supported CFUe development (in the absence of detectable STAT5 activation) at efficiencies within 3-fold of those levels mediated by either an EE-T-Y343 form or the endogenous EPO receptor. However, EE-T-Y343F–dependent Ter119+ erythroblast maturation was attenuated. In tests of cosignaling with c-Kit, EE-T-Y343F nonetheless retained full capacity to synergize with c-Kit in promoting erythroid progenitor cell proliferation. Thus, EPO receptor PY-dependent events can assist late erythropoiesis but may be nonessential for EPO receptor–c-Kit synergy.

A Minimal Cytoplasmic Subdomain of the Erythropoietin Receptor Mediates Erythroid and Megakaryocytic Cell Development

Blood ◽  
1999 ◽  
Vol 94 (10) ◽  
pp. 3381-3387 ◽  
Author(s):  
Chris P. Miller ◽  
Zi Y. Liu ◽  
Constance T. Noguchi ◽  
Don M. Wojchowski
Keyword(s):  
Progenitor Cells ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Cell Development ◽  
Erythropoietin Receptor ◽  
Erythroid Progenitor ◽  
Epo Receptor ◽  
Receptor Complexes ◽  
Erythroid Progenitor Cells ◽  
Fetal Liver Cells

Signals provided by the erythropoietin (Epo) receptor are essential for the development of red blood cells, and at least 15 distinct signaling factors are now known to assemble within activated Epo receptor complexes. Despite this intriguing complexity, recent investigations in cell lines and retrovirally transduced murine fetal liver cells suggest that most of these factors and signals may be functionally nonessential. To test this hypothesis in erythroid progenitor cells derived from adult tissues, a truncated Epo receptor chimera (EE372) was expressed in transgenic mice using a GATA-1 gene-derived vector, and its capacity to support colony-forming unit-erythroid proliferation and development was analyzed. Expression at physiological levels was confirmed in erythroid progenitor cells expanded ex vivo, and this EE372 chimera was observed to support mitogenesis and red blood cell development at wild-type efficiencies both independently and in synergy with c-Kit. In addition, the activity of this minimal chimera in supporting megakaryocyte development was tested and, remarkably, was observed to approximate that of the endogenous receptor for thrombopoietin. Thus, the box 1 and 2 cytoplasmic subdomains of the Epo receptor, together with a tyrosine 343 site (each retained within EE372), appear to provide all of the signals necessary for the development of committed progenitor cells within both the erythroid and megakaryocytic lineages.

Lyn kinase plays important roles in erythroid expansion, maturation and erythropoietin receptor signalling by regulating inhibitory signalling pathways that control survival

2014 ◽  
Vol 459 (3) ◽  
pp. 455-466 ◽  
Author(s):  
Neli S. Slavova-Azmanova ◽  
Nicole Kucera ◽  
Alison Louw ◽  
Jiulia Satiaputra ◽  
Adley Handoko ◽  
...  
Keyword(s):  
Cell Survival ◽  
Cell Development ◽  
Erythropoietin Receptor ◽  
Erythroid Cell ◽  
Signalling Pathways ◽  
Erythroid Cells ◽  
Src Family Kinase ◽  
Receptor Signalling ◽  
Lyn Kinase ◽  
Control Survival

In erythroid cells both positive viability signals and feedback inhibitory signalling require the Src family kinase Lyn, influencing cell survival and their ability to differentiate. This illustrates that Lyn is critical for normal erythropoiesis and erythroid cell development.

Both proteasomes and lysosomes degrade the activated erythropoietin receptor

Blood ◽  
2005 ◽  
Vol 105 (2) ◽  
pp. 600-608 ◽  
Author(s):  
Pierre Walrafen ◽  
Frédérique Verdier ◽  
Zahra Kadri ◽  
Stany Chrétien ◽  
Catherine Lacombe ◽  
...  
Keyword(s):  
Cell Surface ◽  
Intracellular Signaling ◽  
Erythropoietin Receptor ◽  
Janus Kinase ◽  
Receptor Internalization ◽  
Down Regulation ◽  
Epo Receptor ◽  
Receptor Complexes ◽  
Regulation Mechanisms ◽  
Rapid Activation

AbstractActivation of the erythropoietin receptor (EpoR) after Epo binding is very transient because of the rapid activation of strong down-regulation mechanisms that quickly decrease Epo sensitivity of the cells. Among these down-regulation mechanisms, receptor internalization and degradation are probably the most efficient. Here, we show that the Epo receptor was rapidly ubiquitinated after ligand stimulation and that the C-terminal part of the Epo receptor was degraded by the proteasomes. Both ubiquitination and receptor degradation by the proteasomes occurred at the cell surface and required Janus kinase 2 (Jak2) activation. Moreover, Epo-EpoR complexes were rapidly internalized and targeted to the lysosomes for degradation. Neither Jak2 nor proteasome activities were required for internalization. In contrast, Jak2 activation was necessary for lysosome targeting of the Epo-EpoR complexes. Blocking Jak2 with the tyrphostin AG490 led to some recycling of internalized Epo-Epo receptor complexes to the cell surface. Thus, activated Epo receptors appear to be quickly degraded after ubiquitination by 2 proteolytic systems that proceed successively: the proteasomes remove part of the intracellular domain at the cell surface, and the lysosomes degrade the remaining part of the receptor-hormone complex. The efficiency of these processes probably explains the short duration of intracellular signaling activated by Epo.

scholarly journals Epobis is a Nonerythropoietic and Neuroprotective Agonist of the Erythropoietin Receptor with Anti-Inflammatory and Memory Enhancing Effects

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Oksana Dmytriyeva ◽  
Stanislava Pankratova ◽  
Irina Korshunova ◽  
Peter S. Walmod
Keyword(s):  
Clinical Signs ◽  
Neuronal Cell ◽  
Erythropoietin Receptor ◽  
Autoimmune Encephalomyelitis ◽  
Erythroid Progenitor ◽  
Epo Receptor ◽  
Antioxidative Effects ◽  
Anti Inflammatory ◽  
Memory Enhancing

The cytokine erythropoietin (EPO) stimulates proliferation and differentiation of erythroid progenitor cells. Moreover, EPO has neuroprotective, anti-inflammatory, and antioxidative effects, but the use of EPO as a neuroprotective agent is hampered by its erythropoietic activity. We have recently designed the synthetic, dendrimeric peptide, Epobis, derived from the sequence of human EPO. This peptide binds the EPO receptor and promotes neuritogenesis and neuronal cell survival. Here we demonstrate that Epobisin vitropromotes neuritogenesis in primary motoneurons and has anti-inflammatory effects as demonstrated by its ability to decrease TNF release from activated AMJ2-C8 macrophages and rat primary microglia. When administered systemically Epobis is detectable in both plasma and cerebrospinal fluid, demonstrating that the peptide crosses the blood-brain barrier. Importantly, Epobis is not erythropoietic, but systemic administration of Epobis in rats delays the clinical signs of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, and the peptide has long-term, but not short-term, effects on working memory, detected as an improved social memory 3 days after administration. These data reveal Epobis to be a nonerythropoietic and neuroprotective EPO receptor agonist with anti-inflammatory and memory enhancing properties.

scholarly journals During EPO or anemia challenge, erythroid progenitor cells transit through a selectively expandable proerythroblast pool

Blood ◽  
2010 ◽  
Vol 116 (24) ◽  
pp. 5334-5346 ◽  
Author(s):  
Arvind Dev ◽  
Jing Fang ◽  
Pradeep Sathyanarayana ◽  
Anamika Pradeep ◽  
Christine Emerson ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Progenitor Cell ◽  
Ex Vivo ◽  
Erythropoietin Receptor ◽  
Erythroid Cell ◽  
Short Term ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells ◽  
Stage Development ◽  
Bcl2 Expression

Abstract Investigations of bone marrow (BM) erythroblast development are important for clinical concerns but are hindered by progenitor cell and tissue availability. We therefore sought to more specifically define dynamics, and key regulators, of the formation of developing BM erythroid cell cohorts. A unique Kit−CD71highTer119− “stage E2” proerythroblast pool first is described, which (unlike its Kit+ “stage E1” progenitors, or maturing Ter119+ “stage E3” progeny) proved to selectively expand ∼ 7-fold on erythropoietin challenge. During short-term BM transplantation, stage E2 proerythroblasts additionally proved to be a predominantly expanded progenitor pool within spleen. This E1→E2→E3 erythroid series reproducibly formed ex vivo, enabling further characterizations. Expansion, in part, involved E1 cell hyperproliferation together with rapid E2 conversion plus E2 stage restricted BCL2 expression. Possible erythropoietin/erythropoietin receptor proerythroblast stage specific events were further investigated in mice expressing minimal erythropoietin receptor alleles. For a hypomorphic erythropoietin receptor-HM allele, major defects in erythroblast development occurred selectively at stage E2. In addition, stage E2 cells proved to interact productively with primary BM stromal cells in ways that enhanced both survival and late-stage development. Overall, findings reveal a novel transitional proerythroblast compartment that deploys unique expansion devices.

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

Blood ◽  
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.

scholarly journals Differentiation and erythropoietin receptor gene expression in human erythroid progenitor cells [see comments]

Blood ◽  
1992 ◽  
Vol 80 (8) ◽  
pp. 1940-1949 ◽  
Author(s):  
A Wickrema ◽  
SB Krantz ◽  
JC Winkelmann ◽  
MC Bondurant
Keyword(s):  
Half Life ◽  
Messenger Rna ◽  
Receptor Gene ◽  
Erythropoietin Receptor ◽  
Northern Analysis ◽  
Rapid Decline ◽  
Mrna Levels ◽  
Surface Binding ◽  
Erythroid Progenitor ◽  
Epo Receptor

Partially purified human burst-forming unit-erythroid (BFU-E) cells from peripheral blood were cultured for 6 to 8 days to obtain colony- forming unit-erythroid (CFU-E) cells. When these BFU-E-derived CFU-E were further purified and recultured in liquid suspension cultures with erythropoietin (EPO), they matured and differentiated into reticulocytes in vitro. A maximum rate of hemoglobin synthesis was observed at day 10 of cumulative culture time by measuring 59Fe incorporation into heme. Withdrawal of EPO from erythroblast cultures at various times during development showed that between day 10 and day 11 (when the majority of the cells are in the polychromatic erythroblast stage), these cells became independent of EPO. The timing of the disappearance of the EPO requirement in these cells coincided with the marked decline in proliferation. Measurement of EPO receptor messenger RNA (mRNA) levels by Northern analysis showed that there is a slight decline during the day 8 to day 10 time period, followed by a rapid decline between days 10 and 14. Binding of 125I-EPO to erythroblasts also showed a steady decline of the cell surface binding during maturation and terminal differentiation. The half-life of the human EPO receptor was 90 minutes in the presence of the transcriptional inhibitor actinomycin D and the half-life measured at two different times during the 8- to 14-day culture period remained constant. These results indicate that human EPO receptor mRNA must be transcribed continuously to maintain the levels seen by Northern analysis. The human cell system described here is well suited for the study of a wide variety of biochemical events during late erythroid differentiation.

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

2014 ◽  
Vol 211 (9) ◽  
pp. 1715-1722 ◽  
Author(s):  
Rakesh Verma ◽  
Su Su ◽  
Donald J. McCrann ◽  
Jennifer M. Green ◽  
Karen Leu ◽  
...  
Keyword(s):  
Cell Expansion ◽  
Homo Sapiens ◽  
Growth Factor Receptor ◽  
Sh2 Domain ◽  
Erythroid Cell ◽  
Erythroid Progenitor ◽  
Lower Vertebrate ◽  
Extracellular Signal ◽  
Src Homology 2 Domain ◽  
Dependent Growth

Ligation of erythropoietin (EPO) receptor (EPOR) JAK2 kinase complexes propagates signals within erythroid progenitor cells (EPCs) that are essential for red blood cell production. To reveal hypothesized novel EPOR/JAK2 targets, a phosphotyrosine (PY) phosphoproteomics approach was applied. Beyond known signal transduction factors, 32 new targets of EPO-modulated tyrosine phosphorylation were defined. Molecular adaptors comprised one major set including growth factor receptor-bound protein 2 (GRB2)–associated binding proteins 1–3 (GAB1-3), insulin receptor substrate 2 (IRS2), docking protein 1 (DOK1), Src homology 2 domain containing transforming protein 1 (SHC1), and sprouty homologue 1 (SPRY1) as validating targets, and SPRY2, SH2 domain containing 2A (SH2D2A), and signal transducing adaptor molecule 2 (STAM2) as novel candidate adaptors together with an ORF factor designated as regulator of human erythroid cell expansion (RHEX). RHEX is well conserved in Homo sapiens and primates but absent from mouse, rat, and lower vertebrate genomes. Among tissues and lineages, RHEX was elevated in EPCs, occurred as a plasma membrane protein, was rapidly PY-phosphorylated >20-fold upon EPO exposure, and coimmunoprecipitated with the EPOR. In UT7epo cells, knockdown of RHEX inhibited EPO-dependent growth. This was associated with extracellular signal-regulated kinase 1,2 (ERK1,2) modulation, and RHEX coupling to GRB2. In primary human EPCs, shRNA knockdown studies confirmed RHEX regulation of erythroid progenitor expansion and further revealed roles in promoting the formation of hemoglobinizing erythroblasts. RHEX therefore comprises a new EPO/EPOR target and regulator of human erythroid cell expansion that additionally acts to support late-stage erythroblast development.

Sign in / Sign up

Export Citation Format

Share Document