Erythropoietin and Friend Virus gp55 Activate Different JAK/STAT Pathways through the Erythropoietin Receptor in Erythroid Cells

ABSTRACT Abnormal erythropoietin (EPO)-independent cell growth is induced after infection of erythroid progenitor cells with a polycythemic strain of Friend virus (FVp). Binding of its Env-related glycoprotein (gp55) to the EPO receptor (EPOR) mimics the activation of the EPOR with EPO. We investigated the gp55-EPOR signaling in erythroblastoid cells from mice infected with FVp and in cells of FVp-induced or gp55-transgenic-mouse-derived erythroleukemia cell lines, comparing it with the EPO-EPOR signaling in EPO-responsive erythroblastoid cells. While the Janus protein tyrosine kinase JAK2 and the transcription factor STAT5 became tyrosine phosphorylated with the EPO stimulation in EPO-responsive erythroblastoid cells from anemic mice, JAK1 and STAT5 were constitutively tyrosine phosphorylated in all of these FVpgp55-induced erythroblastoid or erythroleukemic cells. Moreover, this constitutively tyrosine-phosphorylated STAT5 was unable to bind to its specific DNA sequences and did not translocate to the nucleus. Nuclear translocation and DNA binding of this STAT5 species required EPO stimulation. These findings clearly indicate that the FVpgp55-EPOR signaling is distinct from the EPO-EPOR signaling and suggest that STAT5 may not play an essential role in the transmission of the cell growth signals in FVp gp55-induced erythroleukemia cells.

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Growth suppression of Friend virus-transformed erythroleukemia cells by p53 protein is accompanied by hemoglobin production and is sensitive to erythropoietin

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1456-1463.1993 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1456-1463

Author(s):

P Johnson ◽

S Chung ◽

S Benchimol

Keyword(s):

Cell Line ◽

P53 Protein ◽

Erythroid Differentiation ◽

Erythropoietin Receptor ◽

Temperature Sensitive ◽

Friend Virus ◽

Erythroleukemia Cells ◽

Erythroleukemia Cell ◽

Hemoglobin Production ◽

Striking Effect

The murine allele temperature-sensitive (ts) p53Val-135 encodes a ts p53 protein that behaves as a mutant polypeptide at 37 degrees C and as a wild-type polypeptide at 32 degrees C. This ts allele was introduced into the p53 nonproducer Friend erythroleukemia cell line DP16-1. The DP16-1 cell line was derived from the spleen cells of a mouse infected with the polycythemia strain of Friend virus, and like other erythroleukemia cell lines transformed by this virus, it grows independently of erythropoietin, likely because of expression of the viral gp55 protein which binds to and activates the erythropoietin receptor. When incubated at 32 degrees C, DP16-1 cells expressing ts p53Val-135 protein, arrested in the G0/G1 phase of the cell cycle, rapidly lost viability and expressed hemoglobin, a marker of erythroid differentiation. Erythropoietin had a striking effect on p53Val-135-expressing cells at 32 degrees C by prolonging their survival and diminishing the extent of hemoglobin production. This response to erythropoietin was not accompanied by down-regulation of viral gp55 protein.

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Growth suppression of Friend virus-transformed erythroleukemia cells by p53 protein is accompanied by hemoglobin production and is sensitive to erythropoietin.

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1456 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1456-1463 ◽

Cited By ~ 74

Author(s):

P Johnson ◽

S Chung ◽

S Benchimol

Keyword(s):

Cell Line ◽

P53 Protein ◽

Erythroid Differentiation ◽

Erythropoietin Receptor ◽

Temperature Sensitive ◽

Friend Virus ◽

Erythroleukemia Cells ◽

Erythroleukemia Cell ◽

Hemoglobin Production ◽

Striking Effect

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A constitutively activated erythropoietin receptor stimulates proliferation and contributes to transformation of multipotent, committed nonerythroid and erythroid progenitor cells.

Molecular and Cellular Biology ◽

10.1128/mcb.14.4.2266 ◽

1994 ◽

Vol 14 (4) ◽

pp. 2266-2277 ◽

Cited By ~ 33

Author(s):

G D Longmore ◽

P N Pharr ◽

H F Lodish

Keyword(s):

Cell Line ◽

Progenitor Cells ◽

Cell Lines ◽

Leukemia Virus ◽

Active Form ◽

Erythropoietin Receptor ◽

Mutant Form ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells

If the env gene of spleen focus-forming virus (SFFV) is replaced by a cDNA encoding a constitutively active form of the erythropoietin receptor, EPO-R(R129C), the resultant recombinant virus, SFFVcEPO-R, induces transient thrombocytosis and erythrocytosis in infected mice. Clonogenic progenitor cell assays of cells from the bone marrow and spleens of these infected mice suggest that EPO-R(R129C) can stimulate proliferation of committed megakaryocytic and erythroid progenitors as well as nonerythroid multipotent progenitors. From the spleens of SFFVcEPO-R-infected mice, eight multiphenotypic immortal cell lines were isolated and characterized. These included primitive erythroid, lymphoid, and monocytic cells. Some expressed proteins characteristic of more than one lineage. All cell lines resulting from SFFVcEPO-R infection contained a mutant form of the p53 gene. However, in contrast to infection by SFFV, activation of PU.1 gene expression, by retroviral integration, was not observed. One cell line had integrated a provirus upstream of the fli-1 gene, in a location typically seen in erythroleukemic cells generated by Friend murine leukemia virus infection. This event led to increased expression of fli-1 in this cell line. Thus, infection by SFFVcEPO-R can induce proliferation and lead to transformation of nonerythroid as well as very immature erythroid progenitor cells. The sites of proviral integration in clonal cell lines are distinct from those in SFFV-derived lines.

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

Blood ◽

10.1182/blood.v94.10.3381.422k25_3381_3387 ◽

1999 ◽

Vol 94 (10) ◽

pp. 3381-3387 ◽

Cited By ~ 28

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.

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

Blood ◽

10.1182/blood-2009-12-258947 ◽

2010 ◽

Vol 116 (24) ◽

pp. 5334-5346 ◽

Cited By ~ 17

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.

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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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Diagnosis and Treatment of Erythrocytosis

European Oncology & Haematology ◽

10.17925/eoh.2010.04.0.55 ◽

2010 ◽

Vol 00 (04) ◽

pp. 55 ◽

Cited By ~ 1

Author(s):

Mary Frances McMullin ◽

Keyword(s):

Cell Mass ◽

Erythropoietin Receptor ◽

Congenital Defects ◽

Intrinsic Defect ◽

Haematocrit Level ◽

Polycythaemia Vera ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Number Of Patients ◽

Secondary Causes

An erythrocytosis arises when the red cell mass is increased. This can be due to a primary intrinsic defect in the erythroid progenitor cells or secondary to erythropoietin production from some source. Primary and secondary causes can be congenital or acquired. Rare, primary congenital defects are due to mutations leading to truncation of the erythropoietin receptor. The main acquired, primary erythrocytosis is polycythaemia vera. Among the congenital secondary causes, a number of defects in the genes in the oxygen-sensing pathway have recently been described, which lead to a secondary erythrocytosis. An extensive list of acquired secondary causes needs to be considered. A number of patients do not have an identifiable cause of erythrocytosis and are therefore described as having idiopathic erythrocytosis. Investigation should commence with careful clinical evaluation. Determination of the erythropoietin level is then a first step to guide the further direction of investigation. In those with congenital defects, a number of serious thromboembolic events have been described, but there is little information available about outcomes in these individuals and, therefore, no evidence to guide management. In this group, consideration should be given to the use of venesection to attain an achievable haematocrit level, and also low-dose aspirin therapy.

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Differentiation of erythroid progenitor (CFU-E) cells from mouse fetal liver cells and murine erythroleukemia (TSA8) cells without proliferation.

Molecular and Cellular Biology ◽

10.1128/mcb.8.6.2604 ◽

1988 ◽

Vol 8 (6) ◽

pp. 2604-2609 ◽

Cited By ~ 9

Author(s):

T Noguchi ◽

H Fukumoto ◽

Y Mishina ◽

M Obinata

Keyword(s):

Colony Formation ◽

Fetal Liver ◽

Liver Cells ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Proliferation And Differentiation ◽

Erythroleukemia Cell ◽

Fetal Liver Cells ◽

Murine Erythroleukemia

Erythropoietin (epo) appears to play a significant role in influencing the proliferation and differentiation of erythroid progenitor (CFU-E) cells. To determine the mechanism of action of epo, the effect of drugs on the in vitro colony formation of CFU-E cells induced from a novel murine erythroleukemia cell line, TSA8, was examined. While cytosine arabinoside inhibited colony formation and terminal differentiation of the CFU-E cells responding to epo, herbimycin, which is a drug that inhibits src-related phosphorylation, inhibited colony formation only. The same effect of herbimycin was observed with normal CFU-E cells from mouse fetal liver cells. These results suggest that epo induces two signals, one for proliferation and the other for differentiation, and that the two signals are not linked in erythroid progenitor cells.

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GRB2-mediated recruitment of GAB2, but not GAB1, to SF-STK supports the expansion of Friend virus-infected erythroid progenitor cells

Oncogene ◽

10.1038/sj.onc.1209288 ◽

2005 ◽

Vol 25 (17) ◽

pp. 2433-2443 ◽

Cited By ~ 19

Author(s):

H E Teal ◽

S Ni ◽

J Xu ◽

L D Finkelstein ◽

A M Cheng ◽

...

Keyword(s):

Progenitor Cells ◽

Friend Virus ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells

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Erythropoietin-induced tyrosine phosphorylations in a high erythropoietin receptor-expressing lymphoid cell line

Blood ◽

10.1182/blood.v80.8.1923.1923 ◽

1992 ◽

Vol 80 (8) ◽

pp. 1923-1932 ◽

Cited By ~ 39

Author(s):

J Damen ◽

AL Mui ◽

P Hughes ◽

K Humphries ◽

G Krystal

Keyword(s):

Cell Surface ◽

Cell Line ◽

Tyrosine Phosphorylation ◽

Erythropoietin Receptor ◽

Biologically Active ◽

Erythroid Progenitor ◽

Retroviral Gene Transfer ◽

Molecular Weights ◽

Erythroid Progenitor Cells ◽

Major Species

Abstract Retroviral gene transfer of the murine erythropoietin receptor (EpR) cDNA into the pro-B-cell line, Ba/F3, was used to generate cells expressing high EpR levels. One of the resulting clones, Ba/F3 clone C5, contained 5 integrated copies of the gene and expressed, at the cell surface, a single affinity class of EpRs at 10 to 15 times the level present on spleen cells from phenylhydrazine-treated mice. Cross- linking studies with clone C5, using 125I-Ep, yielded the same two 105- and 88-Kd major species as that seen with typical erythroid cells. This was distinct from that obtained with EpR-transfected COS cells or L cells, which gave species of 88 and 65 Kd. This suggests that the biologically active EpR complex generated in this Ba/F3 cell line may closely resemble that present in native Ep-responsive erythroid progenitor cells. Tyrosine phosphorylation experiments showed that several proteins in clone C5 cells were rapidly phosphorylated on tyrosine residues in response to Ep, one being the EpR itself. The proportion of cell surface EpRs tyrosine phosphorylated in response to Ep was substantial, reaching a maximum of approximately 10% within 30 minutes of incubation at 37 degrees C. A comparison of Ep- and murine interleukin-3 (mIL-3)-induced tyrosine phosphorylation patterns in clone C5 cells showed that both growth factors stimulated the tyrosine phosphorylation of proteins with molecular weights of 135, 93, 70, and 55 Kd. This could suggest that the Ep and mIL-3 receptors are capable of using the same tyrosine kinase in these cells.

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