Erythropoietin is known for its role in erythroid differentiation, and animals lacking the erythropoietin receptor die due to severe anemia at embryonic day 13.5. In addition to being required for definitive erythropoiesis, EpoR expression plays a role in normal heart and brain development. Mice lacking the erythropoietin receptor exhibit severe anemia and defective cardiac development, and die at embryonic day 13.5. We observed that during normal heart development, EpoR is expressed in paricardium, myocardium and endocardium. The erythropoietin receptor null mouse exhibits apoptosis in corresponding areas of the heart with defects in the formation of the compact zone and trabeculae, but not in the cardiac cushion that lack erythropoietin expression in normal animals. These data suggest that cardiac myocytes and endothelium may require erythropoietin signaling for survival/proliferation. In other studies we demonstrated that erythropoietin can also stimulate the proliferation and survival of primary skeletal muscle progenitor cells and expand the cell population during myocyte differentiation into myotubes in culture, providing another example of erythropoietin stimulation of myoblasts. We have previously observed high level of erythopoietin receptor expression in embryonic mouse brain at mid-gestation. During development, in addition to apoptosis in fetal liver, endocardium and myocardium by embryonic day 12.5, the erythropoietin receptor null mouse shows extensive apoptosis in fetal brain. Lack of erythropoietin receptor affects brain development as early as embryonic day 10.5, resulting in a reduction in the number of neural progenitor cells and increased apoptosis. Corresponding in vitro cultures of cortical cells showed increased sensitivity to low oxygen tension with no surviving neurons in cortical cultures of the erythropoietin receptor null mouse after 24 hr exposure to hypoxia. In contrast exposure of normal cultures to hypoxia induced a 10 fold increase in erythropoietin receptor expression and erythropoietin stimulatio, increased cell survival and decreased apoptosis. These data demonstrate that the neuroprotective activity of erythropoietin is observed as early as E10.5 in the developing brain, and that induction of erythropoietin and its receptor by hypoxia may contribute to selective cell survival in the brain. Expression of the erythropoietin receptor is also present during human brain development. Screening of a human brain cDNA library and quantitative analysis of erythropoietin receptor transcripts indicate that the erythropoietin receptor gene locus is transcriptionally active in human brain. In transgenic mice expressing a human erythropoietin receptor transgene, anemic stress induces expression of the transgene and endogenous erythropoietin receptor gene in hematopoietic tissue and brain. In contrast to erythropoiesis where erythroid progenitor cells express high levels of erythropoietin receptor and are directly responsive to erythropoietin stimulation, the neuroprotective effect of erythropoietin and its receptor may require two molecular events: the induction of erythropoietin production by hypoxia and an increase in its receptor expression in neuronal cells resulting in increased sensitivity to erythropoietin. Our studies also provide evidence of the similarities and differences between erythropoietin receptor transcription regulation in erythroid and non-erthroid tissues. We found that no species barrier existed between mouse and human erythropoietin receptor and that the human erythropoietin receptor transgene was able to provide specific expression in hematopoietic and other selected tissues to rescue erythropoiesis and other organ defects observed in the erythropoietin receptor null mouse. Animals expressing only the human erythropoietin receptor survived through adulthood with normal hematologic parameters and appeared to respond appropriately to induced anemic stress. In addition to restoration of erythropoiesis during development, the cardiac defect associated with embryos lacking the erythropoietin receptor was corrected and the increased apoptosis in fetal liver, heart, and brain in the erythropoietin receptor null phenotype was markedly reduced. In clinical application, long-term administration of recombinant erythropoietin in anemic patients with renal insufficiency has been associated with hypertension, attributed in part to the increased hemoglobin concentration that may trap nitric oxide (NO). We found that erythropoietin also stimulated cultured endothelial cells to proliferate in an oxygen dependent manner. Erythropoietin receptor expression and erythropoietin responsiveness increased at low oxygen tension. The hypoxia induction of endothelial nitric oxide synthase was further enhanced by erythropoeitin stimulation, suggesting that the increase eNOS production by endothelium stimulated with erythropoietin may counter the hypertensive effects of NO binding by increased hemoglobin mass. The common effect of erythropoietin to promote cell viability and provide for progenitor cell proliferation may relate to the plasticity of tissue derived stem cells, including that from hematopoietic, muscle, neural and endothelial tissues which can also contribute to multiple tissue types when transplanted in vivo. These data suggest that rather than acting in an instructive manner, erythropoietin increases select cell survival allowing for proliferation of specific progenitor cell populations during differentiation and may have a potential role in development, stress response or tissue repair.
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