Alteration of nuclear Proto‐Oncogene expression by Erythropoietin (Epo) in Epo‐responsive murine cell lines

Tsuda, Hiroyuki; N, Aso; Sawada, Tomohiro; Hata, Hiroyuki; Kawakita, Makoto; Mori, Koichi; Takatsuki, Kiyoshi

doi:10.1002/stem.5530090203

Cited by 10 publications

(10 citation statements)

References 34 publications

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“…This finding regarding c-fos and c-jun contrasts with several reports that EPO induces mRNA for c-fos in erythroleukemia cell lines or primary isolates of human erythroid progenitors (Tsuda et al, 1991;Umemura et al, 1988) or that the c-fos gene is constitutively expressed in early and late erythroblasts (Caubet et al, 1989). While it is possible that there are different responses in the experimental cell systems used by various investigators, our results show that mRNAs for c-fos and c-jun are transiently induced in FVA cells by equilibration in basal medium without growth factors, and such effects could occur during other cell handling procedures.…”

Section: Discussioncontrasting

confidence: 66%

c-myc expression affects proliferation but not terminal differentiation or survival of explanted erythroid progenitor cells

et al. 1996

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The expression of c-myc was analyzed in murine and human erythroblasts throughout their differentiation in vitro into reticulocytes. The murine cells were splenic erythroblasts from animals infected with the anemia strain of Friend virus (FVA cells). In FVA cells cultured without EPO, the c-myc mRNA and protein levels decrease sharply within 3 to 4 h, showing that continual EPO stimulation is required to maintain c-myc expression. When cultured with EPO, the c-myc mRNA level of FVA cells is raised within 30 min of exposure. The c-myc mRNA and protein reach maxima at 1 to 3 h, then decline slowly to very low levels by 18 h. In contrast, c-fos and c-jun mRNA levels are not regulated by EPO in FVA cells. The human cells analyzed were colony-forming units-erythroid, CFU-E, derived in vitro by the culture of peripheral blood burst-forming units-erythroid (BFU-E). When grown in EPO and insulin-like growth factor 1 (IGF-1) these cells differentiate into reticulocytes over 6 days rather than the 2 days required for murine cells, but the c-myc mRNA kinetics and response to EPO parallel those of mouse cells at similar stages of differentiation. Both IGF-1 and c-kit ligand (SCF) cause an additive increase in c-myc mRNA in human CFU-E in conjunction with EPO. These additive effects suggest that EPO, IGF-1, and SCF affect c-myc mRNA accumulation by distinct mechanisms. Addition of an antisense oligonucleotide to c-myc in cultures of human CFU-E specifically inhibited cell proliferation but did not affect erythroid cell differentiation or apoptosis. When human cells were grown in high SCF concentrations, an environment which enhances proliferation and retards differentiation, antisense oligonucleotide to c-myc strongly inhibited proliferation, but such inhibition did not induce differentiation. This latter result indicates that differentiation requires signals other than depression of c-Myc and resultant depression of proliferation.

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Section: Discussioncontrasting

confidence: 66%

c-myc expression affects proliferation but not terminal differentiation or survival of explanted erythroid progenitor cells

et al. 1996

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“…Epo up-regulation of c-fos was demonstrated several years ago in ELM-1-1 cells (30). This action of Epo on Epo-dependent cells was confirmed by Miura et al (51) who demonstrated an up-regulation of c-fos, c-myc, and egr1.…”

Section: Discussionmentioning

confidence: 67%

“…However, other investigators used an Epo-sensitive cell line and demonstrated that, in contrast to the effect of Me 2 SO on c-myc, Epo treatment up-regulated expression of the proto-oncogene concomitant with induction of differentiation (29). Although up-regulation of c-myc by Epo was not detected in studies of Epo-sensitive ELM-1-1 cells (30), it was detected in a novel J2E cell line (31) and was later shown also in erythroid cells derived from the spleens of Friend virus-infected mice (32).…”

Section: Discussionmentioning

confidence: 97%

Erythropoietin Activates Two Distinct Signaling Pathways Required for the Initiation and the Elongation of c-myc

Chen¹,

Sytkowski²

2001

Journal of Biological Chemistry

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Erythropoietin (Epo) stimulation of erythroid cells results in the activation of several kinases and a rapid induction of c-myc expression. Protein kinase C is necessary for Epo up-regulation of c-myc by promoting elongation at the 3-end of exon 1. PKC⑀ mediates this signal. We now show that Epo triggers two signaling pathways to c-myc. Epo rapidly up-regulated Myc protein in BaF3-EpoR cells. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 blocked Myc up-regulation in a concentrationdependent manner but had no effect on the Epo-induced phosphorylation of ERK1 and ERK2. LY294002 also had no effect on Epo up-regulation of c-fos. MEK1 inhibitor PD98059 blocked both the c-myc and the c-fos responses to Epo. PD98059 and the PKC inhibitor H7 also blocked the phosphorylation of ERK1 and ERK2. PD98059 but not LY294002 inhibited Epo induction of ERK1 and ERK2 phosphorylation in normal erythroid cells. LY294002 blocked transcription of c-myc at exon 1. PD98059 had no effect on transcription from exon 1 but, rather, blocked Epo-induced c-myc elongation at the 3-end of exon 1. These results identify two Epo signaling pathways to c-myc, one of which is PI3K-dependent operating on transcriptional initiation, whereas the other is mitogenactivated protein kinase-dependent operating on elongation.Erythroid progenitor cell growth and differentiation are regulated by the hematopoietic growth factor erythropoietin (Epo).1 The interaction of Epo with its cognate receptor (EpoR) initiates a cascade of signaling pathways that mediate Epo's growth-promoting, anti-apoptotic, and differentiation-inducing actions. Previously, we reported that Epo up-regulates c-myc as a primary (immediate/early) response gene via a protein kinase C (PKC)-dependent pathway (1, 2) and that this PKC-dependent signal increased c-myc expression by reducing transcriptional attenuation (arrest) at the 3Ј-end of exon 1, thereby inducing transcriptional elongation (3). Later, we showed that the PKC⑀ isoform mediates this signal and that down-regulation of PKC⑀ inhibits Epo's growth-promoting action but not its induction of ␤-globin expression (differentiation) (4).We have now investigated Epo up-regulation of c-myc further. We report that Epo's PKC-dependent signal to the transcriptional attenuation site in exon 1 of c-myc utilizes the MEK/ERK pathway and, simultaneously, that this pathway leads to up-regulation of c-fos. In addition, we have discovered that Epo independently up-regulates c-myc expression by increasing transcriptional initiation through a phosphoinositide 3-kinase (PI3K)-dependent pathway. EXPERIMENTAL PROCEDURESCell Culture and Epo Treatment-Two types of Epo-responsive cells were used in these studies. First, BaF3 cells stably transfected with the human EpoR cDNA (BaF3-EpoR cells, generous gift of M. Showers) (5-9) were maintained in a humidified atmosphere of 95% air/5% CO 2 at 36.5°C in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (HyClone Laboratories) and 1 unit of Epo/ml (Elanex Pharmaceutic...

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“…Spangler et al (1991Spangler et al ( , 1992 presented evidence suggesting that the increase in c-myc mRNA is dependent on the activation of protein kinase C. Also, in these erythroleukemic cell lines a decrease in mRNA for the c-myb gene occurs following EPO administration (Todokoro et al, 1988;Spangler et al, 1991). A report of increased c-fos gene expression within 30 min of EPO exposure in murine erythroleukemia cell lines is of uncertain significance since this study involved the uncontrolled replenishment of fresh medium and serum to starved cells at the same time as exposure to EPO (Tsuda et al, 1991).…”

Section: Cellular Responses To Epo Stimulation and The Nature Of Epo mentioning

confidence: 99%

The molecular mechanism of erythropoietin action

Koury

Bondurant

1992

European Journal of Biochemistry

265

108

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Erythropoietin (EPO) is the glycoprotein hormone which controls the production of erythrocytes in mammals. Erythrocytes contain hemoglobin which delivers oxygen from the lungs to other tissues throughout the body. The amount of oxygen delivered to the tissues is controlled by the number of erythrocytes in the bloodstream. Erythrocytes have a finite life span, and, therefore, maintenance of normal oxygen delivery requires continuous replacement of those aged erythrocytes normally lost from the circulation. Adjustments in this basal rate of erythrocyte production are made when tissue oxygenation becomes either decreased or increased compared to the normal level. Development of anemia due to blood loss or inspiration of decreased atmospheric oxygen at high altitude results in decreased oxygen delivery to the tissues. In response to this tissue hypoxia, plasma EPO levels increase due to increased EPO production by the kidney, erythrocyte production increases and the resulting increase in circulating erythrocytes permits more oxygen delivery. Conversely, when tissue oxygenation is increased above normal, which occurs during a sudden change from high altitude to sea level or in a transfusion of erythrocytes to greater than normal numbers, plasma EPO levels decrease, erythrocyte production is decreased and the resultant decline in circulating erythrocytes decreases oxygen delivery to the tissues. Thus, a negativefeedback mechanism exists in which the amount of oxygen delivered by erythrocytes to the body tissues determines the plasma EPO concentration, and, in turn, the EPO concentration determines the number of circulating erythrocytes by controlling the rate of erythrocyte production.Significant progress in understanding the central role of EPO in the oxygenation/EPO/erythrocyte cycle has been made in the last few years. This progress is the result of multiple research efforts, which have resulted in the purification of EPO and the cloning of the EPO gene, an increased understanding of how EPO production is controlled by oxygen in the kidney and liver, the development of in vitro model systems Abbreviations. BFU-E, burst-forming-unit erythroid; CFU-E, colony-forming-unit erythroid; CHO, Chinese hamster ovary; EPO, erythropoietin; FVA cells, splenic erythroblasts from mice infected with Friend leukemia virus (anemia-inducing strain); GATA-I, transcription factor that binds DNA sequence GATA in erythroid cells; GM-CSF, granulocyte-macrophage colony-stimulating factor; HCD cells, an erythropoietin-dependent murine erythroleukemia cell line; rh-EPO, recombinant human erythropoietin; SFFVp, Friend spleen focus-forming virus (polycythemia-inducing); uh-EPO, urinary human erythropoietin; IL, interleukin. of erythropoiesis which provide EPO-responsive cell populations, and the cloning of the EPO receptor gene. These scientific advances have also had a dramatic impact on clinical medicine. Commercially produced, recombinant human EPO is used routinely in the care of patients with renal failure. Patients with renal failure are...

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Alteration of nuclear Proto‐Oncogene expression by Erythropoietin (Epo) in Epo‐responsive murine cell lines

Cited by 10 publications

References 34 publications

c-myc expression affects proliferation but not terminal differentiation or survival of explanted erythroid progenitor cells

c-myc expression affects proliferation but not terminal differentiation or survival of explanted erythroid progenitor cells

Erythropoietin Activates Two Distinct Signaling Pathways Required for the Initiation and the Elongation of c-myc

The molecular mechanism of erythropoietin action

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