Gab1 Is Involved in Erythropoietin Receptor-Mediated Survival Signal through Activation of the Erk Pathway.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2207-2207
Author(s):  
Tetsuya Fukumoto ◽  
Yoshitsugu Kubota ◽  
Akira Kitanaka ◽  
Fusako Waki ◽  
Osamu Imataki ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Adaptor Protein ◽  
Annexin V ◽  
Erythropoietin Receptor ◽  
Erk Pathway ◽  
Biological Functions ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells ◽  
Survival Signal ◽  
Survival Signals

Abstract Erythropoietin (EPO) is required for the survival, proliferation and differentiation of erythroid progenitor cells. The scaffolding adaptor protein Grb2-associated binder-1 (Gab1) is tyrosine phosphorylated upon stimulation of EPO in several cell lines and erythroid progenitor cells, and interacts with signaling molecules such as SHP2 phosphatase and the p85 subunit of phosphatidylinositol 3-kinase (PI3K). However, biological functions of Gab1 in EPO receptor (EPOR)-mediated signaling has not yet been established. In this study, to explore the biological functions of Gab1 in vivo, Gab1-deficient F-36P human erythroleukemia cells were generated by means of transfection of the expression vector of siRNA against Gab1. WST-1 assay revealed that growth of Gab1-deficient F-36P cells was reduced to 61% and 77%, respectively, 5 days after incubation with lower concentrations of EPO (0.001 and 0.01 ng/ml), compared with that of mock-transfected F-36P (F-36P-mock) cells. In contrast, growth of Gab1-deficient F-36P cells at sufficient concentration of EPO (10 ng/ml) was similar to that of F-36P-mock cells. Analysis of apoptosis by flow cytometry using FITC-labeled annexin-V showed that the percentage of annexin-V-positive apoptotic cells in Gab1-deficient F-36P and F-36P-mock cells was increased to 19% and 34%, and 8% and 17%, respectively, 72 h after incubation with 0.01 and 0.001 ng/ml of EPO. These results indicate that Gab1 plays a crucial role in transducing EPOR-mediated survival signals. Next, we examined the molecular mechanism of EPOR-mediated signaling involved in survival of erythroid cells through Gab1. Western blot analysis showed that EPO-induced phosphorylation of threonine 202/ tyrosine 204 on Erk-1 and Erk-2 in Gab1-deficient F-36P but not in F-36P-mock cells was significantly suppressed. Interestingly, phosphorylation of serine 473 on Akt in Gab1-deficient F-36P cells in response to EPO was slightly suppressed in comparison with that in F-36P-mock cells. Therefore, Gab1-mediated survival signals appear to be mainly transmitted to downstream through activation of the Erk pathway, although the PI3K/Akt pathway may be involved in EPO-initiated survival signal transduction mediated by Gab1. Furthermore, EPO induced the association of SHP2 with EPOR in Gab1-deficient F-36P cells. Gab1 was associated with SHP2 in EPO-treated F-36P cells. In addition, Gab1 was constitutively associated with Grb2 in F-36P cells. Taken together, EPO induces the recruitment of Gab1 to EPOR through binding of Gab1 to SHP2, which is associated with EPOR. Because the guanine nucleotide exchange factor SOS1 is known to bind to the SH3 domain of Grb2, SOS1-Grb2 complex is recruited to vicinity of Ras at the plasma membrane to activate this GTP-binding protein through the interaction of Grb2 with Gab1, leading to activation of Erk.

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

1994 ◽  
Vol 14 (4) ◽  
pp. 2266-2277 ◽  
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.

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.

scholarly journals Erythropoietin can promote erythroid progenitor survival by repressing apoptosis through Bcl-XL and Bcl-2

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1576-1582 ◽  
Author(s):  
M Silva ◽  
D Grillot ◽  
A Benito ◽  
C Richard ◽  
G Nunez ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Apoptotic Cell ◽  
Ectopic Expression ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Survival Factor ◽  
Erythroid Progenitor Cells ◽  
Survival Signal ◽  
Proliferation And Differentiation

Abstract Erythropoietin (Epo), the hormone that is the principal regulator of red blood cell production, interacts with high-affinity receptors on the surface of erythroid progenitor cells and maintains their survival. Epo has been shown to promote cell viability by repressing apoptosis; however, the molecular mechanism involved is unclear. In the present studies we have examined whether Epo acts as a survival factor through the regulation of the bcl-2 family of apoptosis-regulatory genes. We addressed this issue in HCD-57, a murine erythroid progenitor cell line that requires Epo for proliferation and survival. When HCD-57 cells were cultured in the absence of Epo, Bcl-2 and Bcl-XL but not Bax were downregulated, and the cells underwent apoptotic cell death. HCD-57 cells infected with a retroviral vector encoding human Bcl-XL or Bcl-2 rapidly stopped proliferating but remained viable in the absence of Epo. Furthermore, endogenous levels of bcl-2 and bcl-XL were downregulated after Epo withdrawal in HCD-57 cells that remained viable through ectopic expression of human Bcl-XL, further indicating that Epo specifically maintains the expression of bcl-2 and bcl-XL. We also show that HCD-57 rescued from apoptosis by ectopic expression of Bcl-XL can undergo erythroid differentiation in the absence of Epo, demonstrating that a survival signal but not Epo itself is necessary for erythroid differentiation of HCD-57 progenitor cells. Thus, we propose a model whereby Epo functions as a survival factor by repressing apoptosis through Bcl-XL and Bcl-2 during proliferation and differentiation of erythroid progenitors.

Erythropoietin Increases Erythropoiesis, Metabolism and Weight Loss In Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 946-946
Author(s):  
Constance Tom Noguchi ◽  
Heather Marie Rogers ◽  
Li Wang ◽  
Ruifeng Teng
Keyword(s):  
Body Weight ◽  
Adipose Tissue ◽  
Oxygen Consumption ◽  
Progenitor Cells ◽  
White Adipose Tissue ◽  
Erythropoietin Receptor ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells ◽  
Erythropoietin Treatment ◽  
Expected Increase

Abstract Erythropoietin is required for erythroid progenitor cell survival, proliferation and differentiation. Increasing evidence suggests that erythropoietin treatment in mice can stimulate erythropoiesis and also affect metabolic processes in a dose dependent manner. For example, medium to high dose erythropoietin treatment (600 U/kg or 3000 U/kg) in leptin deficient obese (ob/ob) mice three times a week for three weeks or more results in the expected increase in hematocrit as well as decrease in accumulated body fat and improved glucose tolerance. Phlebotomy to maintain normal hematocrit demonstrated that erythropoietin regulation of body weight was not dependent on increased red cell mass. In non-obese wild type C57BL/6 mice, erythropoietin treatment also demonstrated the expected increase in hematocrit as well as a 15% reduction in body weight and decreased fasting blood glucose. Erythropoietin receptor is expressed at the highest level in erythroid progenitor cells. The link between increased metabolism and erythropoietin stimulated erythroid differentiation was suggested by the increased oxygen consumption rate observed in vitro in primary cultures of erythropoietin stimulated erythroid progenitor cells. Erythropoietin also stimulated glucose uptake in differentiating erythroid progenitor cells in a dose dependent manner. Glucose uptake decreased with the down regulation of erythropoietin receptor during terminal differentiation. Relatively high erythropoietin receptor expression and erythropoietin activity that may also contribute to erythropoietin metabolic activity has been observed in non-hematopoietic mouse tissue including the hypothalamus and white adipose tissue (Teng R, Gavrilova O et al., Nat Commun 2011). The hypothalamus contributes importantly to appetite regulation and mice treated with erythropoietin exhibited a decrease in food intake compared with saline control. We found that pair-feeding decreased body weight and fat mass, and improved glucose tolerance, but no more than half that observed with erythropoietin treatment, providing evidence that erythropoietin regulation of food intake accounts for only part of the metabolic response observed with erythropoietin treatment. Adipocytes isolated from white adipose tissue in erythropoietin treated mice showed an increase in oxygen consumption compared with vehicle treated or pair-fed mice. To assess the role of direct erythropoietin response of white adipose tissue in regulation of fat mass accumulation, we engineered mice with targeted deletion of erythropoietin receptor in adipose tissue. Erythropoietin treatment gave rise to the expected increase in hematocrit but resulted in a reduced decrease in body weight compared with saline treatment. These data show that erythropoietin treatment can stimulate cell oxygen consumption and can contribute to regulation of metabolism and body weight in mice. Erythropoietin receptor expression on erythroid progenitor cells provides for erythropoietin response to promote erythropoiesis and increase cell metabolic activity including glucose uptake and oxygen consumption. In non-hematopoietic tissue, erythropoietin receptor expression further contributes to erythropoietin regulated metabolic activity such as control of food intake attributed in part to hypothalamus response and modulation of fat mass affected by direct erythropoietin response in white adipose tissue. Therefore, in addition to its critical role in promoting erythropoiesis, erythropoietin can contribute to metabolic homeostasis via its activity in erythroid tissue and beyond. Disclosures: No relevant conflicts of interest to declare.

Erythropoietin Receptor Mutations Associated With Familial Erythrocytosis Cause Hypersensitivity to Erythropoietin in the Heterozygous State

Blood ◽  
1999 ◽  
Vol 94 (7) ◽  
pp. 2530-2532 ◽  
Author(s):  
Stephanie S. Watowich ◽  
Xiaoling Xie ◽  
Ursula Klingmuller ◽  
Juha Kere ◽  
Mikael Lindlof ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Negative Control ◽  
Erythropoietin Receptor ◽  
Heterozygous State ◽  
Wild Type ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells ◽  
Swedish Family ◽  
Inherited Mutations ◽  
Low Serum

Inherited mutations in the erythropoietin receptor (EPOR) causing premature termination of the receptor cytoplasmic region are associated with dominant familial erythrocytosis (FE), a benign clinical condition characterized by hypersensitivity of erythroid progenitor cells to EPO and low serum EPO (S-EPO) levels. We describe a Swedish family with dominant FE in which erythrocytosis segregates with a new truncation in the negative control domain of the EPOR. We show that cells engineered to concomitantly express the wild-type (WT) EPOR and mutant EPORs associated with FE (FE EPORs) are hypersensitive to EPO-stimulated proliferation and activation of Jak2 and Stat5. These results demonstrate that FE is caused by hyperresponsiveness of receptor-mediated signaling pathways and that this is dominant with respect to WT EPOR signaling.

Interferon gamma delays apoptosis of mature erythroid progenitor cells in the absence of erythropoietin

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3742-3749 ◽  
Author(s):  
Ilseung Choi ◽  
Koichiro Muta ◽  
Amittha Wickrema ◽  
Sanford B. Krantz ◽  
Junji Nishimura ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Interferon Gamma ◽  
Human Peripheral Blood ◽  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells ◽  
Flow Cytometric ◽  
Ifn Γ ◽  
Protease Assay

Based on the hypothesis that interferon gamma (IFN-γ) may have stimulating effects on survival of hematopoietic progenitor cells, we examined the effect of IFN-γ on apoptosis of mature erythroid colony-forming cells (ECFCs) derived from human peripheral blood obtained from normal, healthy volunteers. When the cells were cultured in the presence of IFN-γ, even without erythropoietin (EPO), the viability of the cells was maintained for at least 36 hours. When apoptosis of ECFCs was assessed by flow cytometric analysis', using annexin V, IFN-γ reduced the extent of apoptosis of the cells, as well as EPO. DNA fragmentation of ECFCs was also reduced by IFN-γ. In cells cultured with IFN-γ alone, expression of Bcl-x was detected but the level of expression decreased gradually during incubation for 36 hours, and the expression level was lower than incubation with EPO. Fas expression and activation of downstream caspases were assessed by flow cytometric analysis or fluorometric protease assay. IFN-γ induced Fas expression of the cells without the activation of caspase8 or caspase3 during 16 hours of incubation, while deprivation of EPO induced expression of Fas and the activation of both caspase8 and caspase3. We propose that IFN-γ produces a stimulating signal for the survival of mature erythroid progenitor cells by reducing apoptosis through a mechanism other than modulating Fas and one related to the expression of Bcl-x.

A mouse model for visualization and conditional mutations in the erythroid lineage

Blood ◽  
2004 ◽  
Vol 104 (3) ◽  
pp. 659-666 ◽  
Author(s):  
Achim C. Heinrich ◽  
Roberta Pelanda ◽  
Ursula Klingmüller
Keyword(s):  
Stem Cell ◽  
Mouse Model ◽  
Progenitor Cells ◽  
Molecular Mechanisms ◽  
Regulatory Elements ◽  
Erythropoietin Receptor ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Erythroid Progenitor Cells ◽  
Recombination System

AbstractHematologic disorders can be caused by sporadic or inherited mutations. However, the molecular mechanisms that lead to pathogenicity are only partially understood. An accurate method to generate mouse models is conditional gene manipulation facilitated by the Cre-loxP recombination system. To enable identification and genomic manipulation of erythroid progenitor cells, we established a knock-in mouse model (ErGFPcre) that expresses an improved GFPcre fusion protein controlled by the endogenous erythropoietin receptor (EpoR) promoter. We show that ErGFPcre mice enable the identification of GFP-positive erythroid progenitor cells and the highly specific genomic manipulation of the erythroid lineage. Analysis of GFP-positive erythroid progenitor cells suggests a developmental switch in lineage progression from the hematopoietic stem cell compartment to early erythroid progenitor cells that are stem cell antigen-1–negative (Sca-1–) and c-kithigh. Within the hematopoietic system, Cre-mediated recombination is limited to erythroid progenitor cells and occurs in the adult bone marrow at a frequency of up to 80% and in the fetal liver with an efficiency close to 100%. Differential transcriptional activity of the wild-type and the knock-in locus was observed in nonhematopoietic tissues. Thus, our ErGFPcre mouse model could promote the identification of regulatory elements controlling nonhematopoietic EpoR expression and facilitates the characterization and genomic manipulation of 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.

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

1994 ◽  
Vol 14 (4) ◽  
pp. 2266-2277
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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