EPO-EPOR Signaling Research Articles

Important protective role of endogenous erythropoietin mediated signaling in myocardial ischemia and pressure-overload induced cardiac hypertrophy

Recent studies have suggested that erythropoietin (Epo) receptors (EpoR) are expressed not only in the hematopoietic lineage cells but also in the non-hematopoietic cells including the heart, and that the administration of Epo elicits cardioprotective effects in myocardial ischemia. We investigated whether endogenous Epo–EpoR system plays an important protective role in myocardial ischemia/reperfusion and pressure-overload induced cardiac hypertrophy using transgene-rescued EpoR null mutant mice (EpoR−/− rescued), which express EpoR exclusively in the hematopoietic lineage cells. After 30 min of myocardial ischemia followed by 24 h of reperfusion, the infarct size in EpoR−/− rescued was larger than that in wild-type mice (WT). The caspase-3 activity and number of TUNEL-positive cardiomyocytes in the ischemic area were increased in EpoR−/− rescued compared with WT. The extents of p38 and JNK phosphorylations in the ischemic area were significantly increased in WT, but not in EpoR−/− rescued as compared with corresponding sham-operated mice. We then investigated whether the deficiency of EpoR gene in the non-hematopoietic cells deteriorates cardiac function and survival in LV pressure overload. At one week after transverse aortic banding, the LV end-diastolic diameter was increased, LV fractional shortening was decreased, LV weight was increased, and the survival rate was reduced in EpoR−/− rescued compared with WT. These data suggest that the endogenous Epo–EpoR signaling system plays an important protective role in both myocardial ischemia/reperfusion and pressure-overload induced cardiac hypertrophy.

Erythropoietin Biology in Cancer

Erythropoietin (Epo) has long been known to be the principal hematopoietic growth factor that regulates cellular proliferation and differentiation along the erythroid lineage. Recent studies have shown that Epo is a pleiotropic cytokine that is proangiogenic and exerts broad tissue-protective effects in diverse nonhematopoietic organs. Recombinant Epo (rEpo) has been widely used in the clinic to prevent or treat malignancy-associated anemia. A series of clinical trials have documented the efficacy of rEpo in reducing RBC transfusion requirements and improving quality of life in cancer patients, and a recent meta-analysis suggested a positive effect on survival. However, two randomized trials reported negative outcomes with rEpo, as patients in the rEpo arm fared worse than their placebo-treated counterparts with respect to progression-free survival. The expression of Epo receptor (EpoR) in cancer cells has raised the possibility that exogenous rEpo may exert direct effects on tumor cells associated with the potential for stimulation of proliferation, inhibition of apoptosis, or modulation of sensitivity to chemoradiation therapy. The presence of an autocrine-paracrine Epo-EpoR system in tumors and potential effects of Epo on tumor microenvironment and angiogenesis are consistent with a complex biology for Epo-EpoR signaling in cancer that requires further research. This review describes Epo and EpoR biology, focusing on the pleiotropic effects of Epo on nonhematopoietic tissues as well as the expression and function of EpoR in cancer cells.

The Expression of Erythropoietin and Its Receptor in Breast Cancer Is Associated with In Vivo Tumor Hypoxia.

Erythropoietin (EPO) is primarily produced in the adult kidney where hypoxia is the major stimulus for EPO expression under the control of an oxygen-sensing mechanism. A series of previous studies in our laboratories and others have demonstrated the expression of EPO and its receptor (EPOR) in breast cancer cells. In previous work, we found that the administration of antagonists of EPO-EPOR signaling was associated with delayed tumor growth in a rodent syngeneic breast cancer model, suggesting the presence of a functional EPO-EPOR system in cancer. The mechanisms of EPO and EPOR expression in tumor cells requires further study. In vitro experiments using monolayer cultures of breast cancer cells have suggested that EPO and EPOR expression in tumor cells may be hypoxia-regulated. The objective of our current studies was to determine the relationship between the expression of EPO, EPOR and tumor hypoxia in breast cancer to test the hypothesis that EPO or EPOR expression in malignant cells may be associated with the presence of in vivo tumor hypoxia, an important factor involved in resistance to radiation treatment, tumor aggressiveness and poor prognosis. Thirty-eight patients with primary breast cancer were enrolled in a tumor hypoxia study under a research protocol approved by the Institutional Review Board at the University of North Carolina Hospitals. All patients provided signed informed consent. The patients received an intravenous infusion of the hypoxia marker pimonidazole hydrochloride (Hypoxyprobe-1™) prior to tumor biopsy. Two or more biopsies were obtained from 33 tumors and 5 primary tumors had a single biopsy available. Contiguous sections from a total of 93 biopsies were analyzed by immunohistochemistry using monoclonal antibodies for the expression of EPO and EPOR and pimonidazole binding. We found EPO expression in tumor cells in 94% of the biopsies. Focal EPO expression pattern was observed in many tumors. EPOR expression was present in 93% of biopsies. The pattern of EPOR immunoreactivity was predominantly cytoplasmic but was found to be localized to the membrane in some sections. In many tumors, co-localization of EPO and EPOR expression in tumor cells was present when contiguous sections were examined. Tumor hypoxia was detected in 83% of the biopsies at variable levels and did not always co-localize with EPO or EPOR expression in tumor cells. Semi-quantitative analyses for EPO immunostaining and tumor hypoxia on a section-by-section basis revealed a significant positive correlation between levels of micro-regional EPO expression and pimonidazole binding (r = 0.6, P < 0.0001, n=93 by two-tailed Spearman's rank correlation analysis). A similar significant positive correlation was found between levels of EPOR expression and pimonidazole binding (r = 0.63, P < 0.0001, n=93). In addition, there was a significant association between the semi-quantitative EPO score in tumor cells and the EPOR score (r = 0.6, P < 0.0001, n=93). We also determined the micro-vessel density (MVD), a marker of tumor angiogenesis, in 35 biopsies using factor VIII immunostaining. There was no correlation between EPO or EPOR expression and MVD in these samples. In conclusion, these data demonstrate for the first time that EPO and EPOR expression in breast cancer cells correlates with in vivo tumor hypoxia in clinical specimens of primary breast cancer.

Erythropoietin and Friend virus gp55 activate different JAK/STAT pathways through the erythropoietin receptor in erythroid cells.

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 FVp gp55-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 FVp gp55-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.

Activated Ki-Ras complements erythropoietin signaling in CTLL-2 cells, inducing tyrosine phosphorylation of a 160-kDa protein.

We have previously shown that expression of erythropoietin (EPO) receptor (EPOR) alone failed to confer EPO responsiveness on the interleukin 2-dependent T-cell line CTLL-2, whereas the introduction of the EPOR into interleukin 3-dependent pro-B-cell lines, such as BAF-B03, allowed the cells to proliferate in response to EPO. Here, we report that additional expression of v-Ki-Ras conferred EPO-dependent growth on CTLL-2 cells expressing the EPOR, with additional formation of a high-affinity EPOR. To investigate possible mechanisms of EPOR downstream signaling induced by v-Ki-Ras expression in these CTLL-2-derived cells, we carried out anti-phosphotyrosine immunoblot analysis of the EPOR complex immunoprecipitated with anti-EPOR antibody from lysates of cells with and without cytokine stimulation, revealing two 160-kDa and 130-kDa phosphotyrosyl proteins. An anti-JAK2 antibody did not react with these proteins, suggesting that they may represent cellular components involved in an EPO-EPOR signaling pathway induced by v-Ki-Ras. Similar phosphotyrosyl proteins were present among Friend erythroleukemia cell lines, in which the Friend virus gp55/EPOR complex on the cell surface constitutively sends signals for cell growth.

Both Megakaryocytopoiesis and Erythropoiesis Are Induced in Mice Infected With a Retrovirus Expressing an Oncogenic Erythropoietin Receptor

Increasing direct and indirect evidence suggests that erythropoietin (Epo) promotes both erythropoiesis and megakaryocytopoiesis. Here we report that, in mice infected with a recombinant spleen focus-forming retrovirus (SFFV) expressing an oncogenic erythropoietin receptor (EpoR), there was an increase in platelet count preceding the ensuing erythrocytosis. Concurrently, there was a substantial increase in splenic megakaryocytes. Culture of the bone marrow and spleen cells from infected mice showed enhanced numbers of multipotent megakaryocytic progenitors. DNA polymerase chain reaction analysis of individual megakaryocyte-containing colonies showed recombinant SFFV (SFFVcEpoR) proviral integration. Immunofluorescence of spleen sections showed overexpression of EpoR protein in the megakaryocytes. Mice infected with a strain of SFFV also developed splenic megakaryocytosis without activating overexpression of the EpoR in megakaryocytes. This in vivo system shows that a relationship between erythropoiesis and thrombopoiesis can exist at the level of the Epo-EpoR signaling pathway. Also, SFFV-based vectors may be excellent vehicles for the introduction of genes into multipotent, hematopoietic progenitors, in vitro.

Both megakaryocytopoiesis and erythropoiesis are induced in mice infected with a retrovirus expressing an oncogenic erythropoietin receptor [see comments

Increasing direct and indirect evidence suggests that erythropoietin (Epo) promotes both erythropoiesis and megakaryocytopoiesis. Here we report that, in mice infected with a recombinant spleen focus-forming retrovirus (SFFV) expressing an oncogenic erythropoietin receptor (EpoR), there was an increase in platelet count preceding the ensuing erythrocytosis. Concurrently, there was a substantial increase in splenic megakaryocytes. Culture of the bone marrow and spleen cells from infected mice showed enhanced numbers of multipotent megakaryocytic progenitors. DNA polymerase chain reaction analysis of individual megakaryocyte-containing colonies showed recombinant SFFV (SFFVcEpoR) proviral integration. Immunofluorescence of spleen sections showed overexpression of EpoR protein in the megakaryocytes. Mice infected with a strain of SFFV also developed splenic megakaryocytosis without activating overexpression of the EpoR in megakaryocytes. This in vivo system shows that a relationship between erythropoiesis and thrombopoiesis can exist at the level of the Epo-EpoR signaling pathway. Also, SFFV-based vectors may be excellent vehicles for the introduction of genes into multipotent, hematopoietic progenitors, in vitro.