Erythropoietin (EPO), the principal hematopoietic cytokine that regulates mammalian erythropoiesis, exhibits diverse cellular effects in non-hematopoietic tissues. The physiologic functions of EPO are mediated by its specific cell-surface receptor EPOR. In this study, we demonstrate EPOR expression in adult rat cardiac myocytes and examine the direct effects of EPO on the heart to investigate whether recombinant EPO may exert an acute cardioprotective effect during ischemia-reperfusion injury. To determine whether EPO is cardioprotective, isolated rat hearts were perfused for 10 min in the Langendorff-mode with Krebs-Henseleit buffer in the absence or presence of brief recombinant EPO treatment while left-ventricular-developed pressure (LVDP) was measured continuously to assess contractile function. The hearts were then subjected to 20 min of normothermic global ischemia followed by 25 min of reperfusion. The post-ischemic recovery of LVDP in the untreated control hearts was 26 +/- 5% of their baseline LVDP, whereas hearts pretreated with EPO exhibited significantly improved post-ischemic recovery to 57 +/- 7%. We used 31P nuclear magnetic resonance (NMR) spectroscopy to determine whether modulation of intracellular pH and/or high-energy phosphate levels during ischemia contributed to EPO-mediated cardioprotection. These experiments revealed that the rapid cardioprotective effect of EPO during ischemia-reperfusion injury was associated with preservation of ATP levels in the ischemic myocardium.

In view of current uncertainty regarding the optimum route for iron supplementation in patients receiving recombinant human erythropoietin (EPO), a prospective randomized controlled study was designed to investigate this issue. All iron-replete renal failure patients commencing EPO who had a hemoglobin concentration < 8.5 g/dl and an initial serum ferritin level of 100 to 800 micrograms/liter were randomized into three groups with different iron supplementation: Group 1, i.v. iron dextran 5 ml every 2 weeks; Group 2, oral ferrous sulphate 200 mg tds; Group 3, no iron. All patients were treated with 25 U/kg of EPO thrice weekly subcutaneously. The hemoglobin concentration, reticulocyte count, serum ferritin, transferrin saturation, and EPO dose were monitored every two weeks for the first four months. Thirty-seven patients entered the study (12 i.v., 13 oral, 12 no iron). The three groups were equivalent with regard to age, sex, and other demographic details. Even allowing for dosage adjustments, the hemoglobin response in the group receiving i.v. iron (7.3 +/- 0.8 to 11.9 +/- 1.2 g/dl) was significantly greater than that for the other two groups (7.2 +/- 1.1 to 10.2 +/- 1.4 g/dl and 7.3 +/- 0.8 to 9.9 +/- 1.6 g/dl for Groups 2 and 3, respectively; P < 0.005 for both groups vs. Group 1 at 16 weeks). There was no difference between the groups supplemented with oral iron and no iron. Serum ferritin levels remained constant in those receiving i.v. iron (345 +/- 273 to 359 +/- 140 micrograms/liter), in contrast to the other two groups in which ferritin levels fell significantly (309 +/- 218 to 116 +/- 87 micrograms/liter and 458 +/- 206 to 131 +/- 121 micrograms/liter for Groups 2 and 3, respectively; P < 0.0005 for Group 1 vs. Group 2, and P < 0.005 for Group 1 vs. Group 3 at 16 weeks). Dosage requirements of EPO were less in Group 1 (1202 +/- 229 U/kg/16 weeks) than in Group 2 (1294 +/- 314 U/kg/16 weeks) or Group 3 (1475 +/- 311 U/kg/16 weeks; P < 0.05 vs. Group 1). The results of this study suggest that, even in iron-replete patients, those supplemented with i.v. iron have an enhanced hemoglobin response to EPO with better maintenance of iron stores and lower dosage requirements of EPO, compared with those patients receiving oral iron and no iron supplementation.

The kidney is sensitive to changes in oxygen delivery. This sensitivity has the merit of facilitating the kidneys in their adjustment of erythropoietin (EPO) production to changes in oxygen supply. The main determinant of EPO synthesis is the transcriptional activity of its gene in kidneys, which is related to local oxygen tensions. Regulation of EPO production is mediated by hypoxia-inducible factor (HIF). When local oxygen tension decreases, accumulated HIF binds to the key sequence of the EPO gene, the hypoxia-responsive element (HRE), and activates transcription of EPO. HIF consists of a constitutive beta-subunit and one of alternative oxygen-regulated HIF alpha-subunits (HIF-1alpha, HIF-2alpha, and HIF-3alpha), and HIF-2alpha is responsible for erythropoietin production. However, the high sensitivity to changes in oxygen tension also makes the kidney prone to hypoxic injury. Severe energy depletion and subsequent activation of a number of critical alterations in metabolism occurs under hypoxic conditions. Hypoxia is also a profibrogenic stimulus. In addition to ischemic acute renal failure, hypoxia can also play a crucial role in the development of nephrotoxic acute kidney injury, radiocontrast nephropathy, and acute glomerulonephritis. Furthermore, accumulating evidence suggests that chronic hypoxia is a final common pathway to end-stage kidney failure in chronic kidney disease. Given that renal hypoxia has pivotal roles on the development and progression of both acute and chronic kidney disease, hypoxia can be a valid therapeutic target for chronic kidney disease. Activation of HIF leads to expression of a variety of adaptive genes in a coordinated manner. Studies utilizing HIF-stimulating agents proved efficacy in various kidney disease models, suggesting that HIF activation is an ideal target of future therapeutic approaches.

BACKGROUND

Treatment with recombinant human erythropoietin (rHuEPO) has been a major advance for the management of anemia in patients on hemodialysis. Therapy, however, is often observed to be associated with recurrent cyclic fluctuations in hemoglobin levels. The purpose of this analysis was to describe the phenomenology of hemoglobin cycling during rHuEPO treatment.

METHODS

Data were analyzed for 281 hemodialysis patients treated at Winthrop-University Hospital Dialysis Centers between 1998 and 2003. Eligible patients' first full 1-year period with less than 10 hospital days was studied. Hemoglobin cycling (cycles with amplitude >1.5 g/dL and duration >8 weeks) and excursions (half of one full cycle) were analyzed.

RESULTS

Greater than 90% of patients experienced hemoglobin cycling. The mean number of hemoglobin excursions was 3.1 +/- 1.1 per patient/year. The mean amplitude per hemoglobin excursion was 2.51 +/- 0.89 g/dL. The mean duration of hemoglobin excursions was 10.3 +/- 5.1 weeks. Factors associated with initiation of up excursions included increases in rHuEPO dose (84%), intravenous iron treatment initiation or increase in dose (27%), posthospital discharge (36%), factors associated with down excursions included rHuEPO dose hold (15%) or dose reduction (62%), infection (6%), discontinuation of intravenous iron therapy (5%), and hospitalization (14%). Patients with frequent hemoglobin cycling >>two full cycles per year) were characterized as being more responsive to rHuEPO [index of EPO responsiveness (ERI) 1036 +/- 659 compared to 1992 +/- 701 for other patients] (P = 0.02).

CONCLUSIONS

Hemoglobin cycling is a common occurrence in rHuEPO-treated hemodialysis patients. It is most closely associated with frequent rHuEPO dose changes, hospitalization, and iron treatment practices.

Thrombopoietin (Tpo), the ligand for the c-mpl receptor, is a major regulator of platelet production in vivo. Treatment of mice with purified recombinant Tpo increases platelet count fourfold and expands colony-forming unit-megakaryocyte (CFU-Meg) numbers. Other cytokines including interleukin-3 (IL-3), IL-6, IL-11, erythropoietin (Epo), and stem cell factor (SCF) can stimulate megakaryopoiesis. Therefore, we examined the effects of recombinant murine Tpo in combination with these cytokines on megakaryopoiesis in vitro. Murine marrow cells were cultured in agar in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% horse serum and beta-mercaptoethanol in the presence of recombinant growth factors, and CFU-Meg colonies were counted on day 5. Megakaryocyte ploidy was analyzed using murine marrow cells cultured for 5 days in IMDM supplemented with 1% nutridoma-SP and recombinant growth factors. Megakaryocytes were identified by labeling with the 4A5 antibody and ploidy was analyzed by flow cytometry. Tpo supported the growth of CFU-Meg in a dose-dependent manner. Although the addition of SCF (50 ng/mL), Epo (2 U/mL), or IL-11 (50 ng/mL) alone exerted only a modest effect on CFU-Meg growth, the combination of SCF plus Tpo, Epo plus Tpo, or IL-11 plus Tpo resulted in a synergistic enhancement of the number of CFU-Meg colonies. IL-3 alone supported CFU-Meg colony growth, and the effects of IL-3 plus Tpo or IL-6 plus Tpo on colony growth appeared to be approximately additive. Fifty percent of megakaryocytes generated in cultures containing IL-3 or Epo displayed < or = 16 N ploidy. In contrast, cultures containing Tpo uniquely generated large numbers (30% to 35% of the total) of megakaryocytes with>> or = 64N ploidy. These results show that Tpo stimulates both proliferation of committed megakaryocytic progenitor cells and maturation of megakaryocytes, and that two multipotent cytokines, SCF and IL-11, as well as a late-acting erythroid cytokine, Epo, can synergize with Tpo to stimulate proliferation of CFU-Meg.

OBJECTIVE

This study sought to understand the prevalence and clinical relevance of iron deficiency in patients with idiopathic pulmonary arterial hypertension (IPAH).

BACKGROUND

Iron availability influences the pulmonary vascular response to hypoxia in humans and may be significant in the pathogenesis of IPAH.

METHODS

Iron deficiency, defined by raised levels of soluble transferrin receptor (sTfR), was investigated in 98 patients with IPAH. Hepcidin and erythropoietin (EPO) levels were also measured. The effect of bone morphogenetic protein (BMP) receptor knockdown on BMP-6-stimulated hepcidin production was assessed in human hepatoma HepG2 cells. Relationships between sTfR and exercise capacity, functional class, and all-cause mortality were analyzed.

RESULTS

Circulating sTfR levels were raised in 63% of IPAH patients, indicating significant iron deficiency. Consistent with this, iron, ferritin, and transferrin saturation levels were reduced and red cell distribution width increased, without overt anemia. Hepcidin correlated inversely with sTfR and positively with increasing ferritin. Hepcidin was inappropriately raised in IPAH independent of the inflammatory marker interleukin-6. EPO levels were also raised and correlated inversely with hepcidin. BMP receptor-type 2 (BMPR2) knockdown in HepG2 cells increased BMP-6-stimulated hepcidin expression. sTfR increased with World Health Organization functional class (p < 0.05), correlated negatively with exercise capacity (p = 0.027), and values >28.1 nmol/l independently predicted survival (p = 0.011).

CONCLUSIONS

Iron deficiency is common in IPAH patients and associated with disease severity and poor clinical outcome. Inappropriately raised hepcidin levels, which impair iron absorption from the gut, may be a factor.

Erythropoietin (Epo)-producing cells were identified in the murine hypoxic kidney by in situ hybridization. Profound anemia was induced in order to greatly increase Epo production. This resulted in high levels of Epo mRNA in the kidney. 35S-labeled DNA fragments of the murine Epo gene were used as probes for in situ hybridization. Control experiments conducted in parallel included kidneys of nonanemic mice, RNase-treated hypoxic kidney sections, and 35S-labeled non-Epo-related DNA. The Epo probe gave a specific hybridization signal in the hypoxic kidney in the cortex and to a lesser extent in the outer medulla. Glomerular and tubular cells were not labeled. All positive cells were identified as peritubular cells. Using immunofluorescence, we showed that cells with the same topography contained Factor VIII-related antigen. These data demonstrated that peritubular cells, most likely endothelial cells, constitute the major site of Epo production in the murine hypoxic kidney.

Although erythropoietin (Epo) has been shown to possess in vitro angiogenic activity, its physiological significance has not been demonstrated. Normally angiogenesis does not occur actively in adults but an exception is the female reproductive organ. In the uterine endometrium, angiogenesis takes place actively for supporting the endometrial growth that occurs during transition from the diestrus to estrous stage. This transition is under control of 17beta-estradiol (E2), an ovarian hormone, and can be mimicked by injection of E2 to ovariectomized (OVX) mouse. Thus, the uterus is a pertinent site to examine the Epo function in angiogenesis. We found that Epo protein and its mRNA were produced in an E2-dependent manner, when the uterus from OVX mouse was cultured in vitro. The de novo protein synthesis was not needed for E2 induction of Epo mRNA. Administration of E2 to OVX mouse induced a rapid and transient increase in Epo mRNA in the uterus. Injection of Epo into the OVX mouse uterine cavity promoted blood vessel formation in the endometrium. Furthermore, injection of the soluble Epo receptor capable of binding with Epo into the uterine cavity of non-OVX mouse in diestrus stage inhibited the endometrial transition to proestrus stage, whereas heat-inactivated soluble Epo receptor allowed the transition to occur. These results, combined with our finding that the endothelial cells in uterine endometrium express Epo receptor, strongly suggest that Epo is an important factor for the E2-dependent cyclical angiogenesis in uterus.

BACKGROUND

It is well established that bleeding activates the hematopoietic system to regenerate the loss of mature blood elements. We have shown that hematopoietic stem cells (HSCs) isolated from animals challenged with an acute bleed regulate osteoblast differentiation from marrow stromal cells. This suggests that HSCs participate in bone formation where the molecular basis for this activity is the production of BMP2 and BMP6 by HSCs. Yet, what stimulates HSCs to produce BMPs is unclear.

RESULTS

In this study, we demonstrate that erythropoietin (Epo) activates Jak-Stat signaling pathways in HSCs which leads to the production of BMPs. Critically, Epo also directly activates mesenchymal cells to form osteoblasts in vitro, which in vivo leads to bone formation. Importantly, Epo first activates osteoclastogenesis which is later followed by osteoblastogenesis that is induced by either Epo directly or the expression of BMPs by HSCs to form bone.

CONCLUSIONS

These data for the first time demonstrate that Epo regulates the formation of bone by both direct and indirect pathways, and further demonstrates the exquisite coupling between hematopoiesis and osteopoiesis in the marrow.

Schwann cells provide trophic support and in some cases, insulation to axons. After injury, Schwann cells undergo phenotypic modulation, acquiring the capacity to proliferate, migrate, and secrete soluble mediators that control Wallerian degeneration and regeneration. Amongst the soluble mediators are pro-inflammatory cytokines that function as chemoattractants but also may sensitize nociceptors. At the same time, Schwann cells produce factors that counterbalance the pro-inflammatory cytokines, including, for example, interleukin-10 and erythropoietin (Epo). Epo and its receptor, EpoR, are up-regulated in Schwann cells after peripheral nerve injury. EpoR-dependent cell signaling may limit production of TNF-alpha by Schwann cells within the first five days after injury. In addition, EpoR-dependent cell signaling may reduce axonal degeneration and facilitate recovery from chronic pain states. Other novel factors that regulate Schwann cell phenotype in nerve injury have been recently identified, including the low-density lipoprotein receptor related protein (LRP-1). Our recent studies indicate that LRP-1 may be essential for Schwann cell survival after peripheral nerve injury. To analyze the function of specific Schwann cell gene products in nerve injury and sensory function, conditional gene deletion and expression experiments in mice have been executed using promoters that are selectively activated in myelinating or non-myelinating Schwann cells. Blocking ErbB receptor-initiated cell-signaling in either myelinating or non-myelinating Schwann cells results in unique sensory dysfunctions. Data obtained in gene-targeted animals suggest that sensory alterations can result from changes in Schwann cell physiology without profound myelin degeneration or axonopathy. Aberrations in Schwann cell biology may lie at the foundation of neuropathic pain and represent an exciting target for therapeutic intervention.

The injured brain can be stimulated to amplify its intrinsic restorative processes to improve neurological function. Thus, after stroke, both cell and pharmacological neurorestorative treatments, amplify the induction of brain neurogenesis and angiogenesis, and thereby reduce neurological deficits. In this manuscript, we describe the use of bone marrow mesenchymal cells (MSCs) and erythropoietin (EPO) as examples of cell-based and pharmacological neurorestorative treatments, respectively, for both stroke and a mouse model of experimental autoimmune encephalomyelitis (EAE). We demonstrate that these therapies significantly improve neurological function with treatment initiated after the onset of injury and concomitantly promote brain plasticity. The application of MRI to monitor changes in the injured brain associated with reduction of neurological deficit is also described.

Erythropoietin (Epo) initiates its cellular response by binding to the Epo receptor, which triggers the activation of signal transducer and activator of transcription (Stat) 5 protein. Cell culture studies of erythroid progenitors have suggested that Epo functions as a survival factor by repressing apoptosis at least in part through Bcl-x(L), an anti-apoptotic protein of the Bcl-2 family. In this report, we examine whether Stat5 can induce transactivation of the bcl-x gene in response to Epo. Two Epo-responsive progenitor cell lines, HCD-57 and Bcl-2-transfected Ba/F3-Epo receptor (Ba/F3-EpoR-Bcl-2), were used in this study. After Epo stimulation, we observed a correlation between expression of bcl-x(L) and activation of Stat5 as assessed by the expression of oncostatin M, a direct target of Stat5, and the phosphorylation and nuclear translocation of Stat5. Moreover, a Stat binding element in the bcl-x promoter was found to be active in response to Epo, a finding that was further confirmed because mutagenesis of this sequence motif abrogated its promoter activity and overexpression of a dominant negative Stat5 protein blocked transactivation. When DNA-protein binding analyses were performed, we found that Stat5, not Stat1 or Stat3, was the protein bound to the bcl-x promoter in response to Epo. These data suggest that Epo-dependent activation of Stat5 is a transcriptional pathway that can be used by Epo-responsive progenitor cells to induce the expression of bcl-x(L) and consequently to inhibit apoptosis.

The megakaryocytic (MK) and erythroid lineages are tightly associated during differentiation and are generated from a bipotent megakaryocyte-erythroid progenitor (MEP). In the mouse, a primitive MEP has been demonstrated in the yolk sac. In human, it is not known whether the primitive MK and erythroid lineages are generated from a common progenitor or independently. Using hematopoietic differentiation of human embryonic stem cells on the OP9 cell line, we identified a primitive MEP in a subset of cells coexpressing glycophorin A (GPA) and CD41 from day 9 to day 12 of coculturing. This MEP differentiates into primitive erythroid (GPA(+)CD41(-)) and MK (GPA(-)CD41(+)) lineages. In contrast to erythropoietin (EPO)-dependent definitive hematopoiesis, KIT was not detected during erythroid differentiation. A molecular signature for the commitment and differentiation toward both the erythroid and MK lineages was detected by assessing expression of transcription factors, thrombopoietin receptor (MPL) and erythropoietin receptor (EPOR). We showed an inverse correlation between FLI1 and both KLF1 and EPOR during primitive erythroid and MK differentiation, similar to definitive hematopoiesis. This novel MEP differentiation system may allow an in-depth exploration of the molecular bases of erythroid and MK commitment and differentiation.

We defined erythropoietin (EPO) resistance by the ratio of the weekly EPO dose to hematocrit (Hct), yielding a continuously distributed variable (EPO/Hct). EPO resistance is usually attributed to iron or vitamin deficiency, hyperparathyroidism, aluminum toxicity, or inflammation. Activation of the acute-phase response, assessed by the level of the acute-phase C-reactive protein (CRP), correlates strongly with hypoalbuminemia and mortality in both hemodialysis (HD) and peritoneal dialysis (PD) patients. In this cross-sectional study of 92 HD and 36 PD patients, we examined the contribution of parathyroid hormone (PTH) levels, iron indices, aluminum levels, nutritional parameters (normalized protein catabolic rate [PCRn]), dialysis adequacy (Kt/V), and CRP to EPO/Hct. Albumin level serves as a measure of both nutrition and inflammation and was used as another independent variable. Serum albumin level (deltaR2 = 0.129; P < 0.001) and age (deltaR2 = 0.040; P = 0.040) were the best predictors of EPO/Hct in HD patients, and serum albumin (deltaR2 = 0.205; P = 0.002) and ferritin levels (deltaR2 = 0.132; P = 0.015) in PD patients. When albumin was excluded from the analysis, the best predictors of EPO/Hct were CRP (deltaR2 = 0.105; P = 0.003) and ferritin levels (deltaR2 = 0.051; P = 0.023) in HD patients and CRP level (deltaR2 = 0.141; P = 0.024) in PD patients. When both albumin and CRP were excluded from analysis in HD patients, low transferrin levels predicted high EPO/Hct (deltaR2 = 0.070; P = 0.011). EPO/Hct was independent of PTH and aluminum levels, PCRn, and Kt/V. High EPO/Hct occurred in the context of high ferritin and low transferrin levels, the pattern expected in the acute-phase response, not in iron deficiency. In well-dialyzed patients who were iron replete, the acute-phase response was the most important predictor of EPO resistance.

We describe a new two-step culture method for mass production in vitro of erythroid cells from either CD34+ (10(5) cells/mL) or light-density (10(6) cells/mL) cells purified from the blood of normal donors and thalassemic patients. The method includes (i) culture of the cells in the presence of dexamethasone and estradiol (10(-6) M each) and (ii) the growth factors SCF (50 ng/mL), IL-3 (1 ng/mL), and EPO (1 U/mL). In their proliferative phase, these cultures generated approximately 1.2 x 10(7) erythroblasts for each milliliter of blood collected from normal donors or thalassemic patients. They were composed mostly (90%) of CD45(low)/glycophorin (GPA)(neg)/CD71(1ow) cells at day 7, 50-60\% of which became CD45(neg)/GPA+/CD71high by days 15-20. However, when cells from days 7 to 12 of the proliferative phase were transferred in differentiation medium containing EPO and insulin, they progressed to mature erythroblasts (g90% benzidine(pos) and CD45(neg)/GPA+/CD71medium) in 4 days. Because of the high number of erythroid cells that are generated from modest volumes of blood, this method will prove useful in donor-specific studies of erythroid differentiation.

OBJECTIVE

We assessed the effects of erythropoietin (EPO) treatment in a rat model of post-myocardial infarction (MI) heart failure.

BACKGROUND

Erythropoietin, traditionally known as a hematopoietic hormone, has been linked to neovascularization. Whereas administration of EPO acutely after MI reduces infarct size and improves cardiac function, its role in the failing heart is unknown.

METHODS

Rats underwent coronary ligation or sham surgery. Rats with MI were randomly assigned to: untreated (MI), a single bolus of EPO immediately after MI induction (MI-EPO-early), EPO treatment immediately after MI and once every three weeks (MI-EPO-early+late), and EPO treatment starting three weeks after induction of MI, once every three weeks (MI-EPO-late). After nine weeks, hemodynamics, infarct size, myosin heavy chain (MHC) isoforms, myocyte hypertrophy, and capillary density were measured.

RESULTS

Erythropoietin treatment started immediately after MI (MI-EPO-early and MI-EPO-early+late) resulted in a 23% to 30% reduction in infarct size (p < 0.01) and, accordingly, hemodynamic improvement. Erythropoietin treatment, started three weeks after MI (MI-EPO-late), did not affect infarct size, but resulted in an improved cardiac performance, reflected by a 34% reduction in left ventricular end-diastolic pressure (p < 0.01), and 46% decrease in atrial natriuretic peptide levels (p < 0.05). The improved cardiac function was accompanied by an increased capillary density (p < 0.01), an increased capillary-to-myocyte ratio (p < 0.05), and a partial reversal of beta-MHC (p < 0.05) in all treated groups.

CONCLUSIONS

In addition to its effect on infarct size reduction, EPO treatment improves cardiac function in a rat model of post-MI heart failure. This observation may be explained by neovascularization, associated with an increased alpha-MHC expression.

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.

Although the formation of terminally differentiated erythroid cells has been shown to require the presence of a functional GATA-1 gene in vivo, the role of this transcription factor and other members of the GATA family at earlier stages of erythroid differentiation is unclear. In this report, the expression of GATA-1, GATA-2, and GATA-3 has been examined in enriched peripheral blood progenitors before and after culture in a well-characterized liquid culture system. In addition primary leukemic cells as well as several erythroleukemic and nonerythroid cell lines were analyzed for GATA factor expression. The results show that the profile of GATA factor expression in erythroid cells is distinct from that of myeloid or lymphoid lineages. Erythroleukemic cell lines express little or no GATA-3, but high levels of GATA-1 and GATA-2. When they are induced to display the terminal erythroid phenotype, little change in the level of GATA-1 is detected but a significant decline in the levels of GATA-2 is observed commensurate with the degree of maturation achieved by the cells. Enrichment of erythroid progenitors from peripheral blood leads to selection of cells that express both GATA-1 and GATA-2. As the enriched populations are cultured in suspension in the presence of multiple cytokines, the levels of both GATA-1 and GATA-2 initially increase. However, in cultures containing only erythropoietin, which show exclusive erythroid differentiation, the levels of GATA-1 continue to increase, whereas GATA-2 expression declines as erythroid maturation progresses. In contrast, cultures lacking Epo (ie, with interleukin-3 and kit ligand) display limited progression towards both the myeloid and erythroid pathways, and high levels of expression of both GATA-1 and GATA-2 are maintained. Despite the initial upregulation of GATA-1 expression in the latter cultures, terminal erythroid differentiation does not occur in the absence of erythropoietin. These results indicate that GATA-1 upregulation is associated with both the initiation and the maintenance of the erythroid program, but that these two processes appear to be under separate regulatory control. Thus, the dynamic changes in the levels of different GATA factors that occur during primary erythroid differentiation suggest that the levels of these factors may influence the progression to specific hematopoietic pathways.

Erythropoietin (EPO) reduced Ca(2+)-induced glutamate (Glu) release from cultured cerebellar granule neurons. Inhibition was also produced by EPO mimetic peptide 1 (EMP1), a small synthetic peptide agonist of EPO receptor (EPO-R), but not by iEMP1, an inactive analogue of EMP1. EPO and EMP1 induced autophosphorylation of Janus kinase 2 (JAK2), a tyrosine kinase that associates with EPO-R. Furthermore, genistein, but not genistin, antagonized both the phosphorylation of JAK2 and the suppression of Glu release induced by EPO and EMP1. During chemical ischemia, substantial amounts of Glu were released from cultured cerebellar and hippocampal neurons by at least two distinct mechanisms. In the early phase, Glu release occurred by exocytosis of synaptic vesicle contents, because it was abolished by botulinum type B neurotoxin (BoNT/B). In contrast, the later phase of Glu release mainly involved a BoNT/B-insensitive non-exocytotic pathway. EMP1 inhibited Glu release only during the early exocytotic phase. A 20-min exposure of hippocampal slices to chemical ischemia induced neuronal cell death, especially in the CA1 region and the dentate gyrus, which was suppressed by EMP1 but not iEMP1. However, EMP1 did not attenuate neuronal cell death induced by exogenously applied Glu. These results suggest that activation of EPO-R suppresses ischemic cell death by inhibiting the exocytosis of Glu.

Given the cytoprotective ability of erythropoietin (EPO) in cerebral microvascular endothelial cells (ECs) and the invaluable role of ECs in the central nervous system, it is imperative to elucidate the cellular pathways for EPO to protect ECs against brain injury. Here we illustrate that EPO relies upon the modulation of SIRT1 (silent mating type information regulator 2 homolog 1) in cerebral microvascular ECs to foster cytoprotection during oxygen-glucose deprivation (OGD). SIRT1 activation which results in the inhibition of apoptotic early membrane phosphatidylserine (PS) externalization and subsequent DNA degradation during OGD becomes a necessary component for EPO protection in ECs, since inhibition of SIRT1 activity or diminishing its expression by gene silencing abrogates cell survival supported by EPO during OGD. Furthermore, EPO promotes the subcellular trafficking of SIRT1 to the nucleus which is necessary for EPO to foster vascular protection. EPO through SIRT1 averts apoptosis through activation of protein kinase B (Akt1) and the phosphorylation and cytoplasmic retention of the forkhead transcription factor FoxO3a. SIRT1 through EPO activation also utilizes mitochondrial pathways to prevent mitochondrial depolarization, cytochrome c release, and Bad, caspase 1, and caspase 3 activation. Our work identifies novel pathways for EPO in the vascular system that can govern the activity of SIRT1 to prevent apoptotic injury through Akt1, FoxO3a phosphorylation and trafficking, mitochondrial membrane permeability, Bad activation, and caspase 1 and 3 activities in ECs during oxidant stress.

G-CSF (Granulocyte-colony stimulating factor) is a hematopoietic growth factor that has been known for 20 years, and has been named for its role in the proliferation and differentiation of cells of the myeloic lineage. We have uncovered a novel spectrum of activities of G-CSF in the central nervous system. G-CSF and its receptor are expressed by neurons in many brain regions, and are upregulated upon experimental stroke. In neurons, G-CSF acts anti-apoptotically by activating several protective pathways. In vivo, G-CSF decreases infarct volumes in acute stroke models in rodents. Moreover, G-CSF stimulates neuronal differentiation of adult neural stem cells in the brain, and improves long-term recovery in more chronic stroke models. Thus, G-CSF is a novel neurotrophic factor, and a highly attractive candidate for the treatment of neurodegenerative conditions. Here we discuss this new property of G-CSF in contrast to its known functions in the hematopoietic system, summarize data from other groups on G-CSF's actions in cerebral ischemia, compare G-CSF to Erythropoietin (EPO) in the CNS, and highlight clinical implications.

The aim of the present study is to better understand oxygen-sensitive adaptative pathways underlying the hypoxic preconditioning-induced protection of the brain against ischemia. Using oligonucleotide microarrays, we examined the brain genomic response of adult mice following hypoxia preconditioning (8% O2 for 1 or 6 h of hypoxia with reoxygenation 12, 18, 24 h or 72 h) and ischemia (6 h), preceeded (tolerant state) or not, by preconditioning. Real-time PCR was used to validate the results. Most gene expression increases occurred during hypoxia, including those of HIF-1-dependent genes (RTP801, AM, VEGF, p21, GLUT-1), early response genes (IER3) and transcriptional factors (ATF3, C/EBPdelta). A second wave of changes occurred 24 h after reoxygenation (S100A5, TH, Calretinin, PBX3). A third one occurred during ischemia itself, revealing that hypoxic preconditioning modifies the brain genomic response to ischemia. In addition, we show that some identical genes are overexpressed by hypoxia in both neonatal and adult brains (VEGF, EPO, GLUT-1, AM, MTs, C/EBPdelta).

The hematopoietic cytokine erythropoietin (Epo) exerts cytoprotective effects on several types of neuronal cells both in vivo and in culture. Detailed molecular mechanisms underlying this phenomenon have not been elucidated and even the identity of the cytoprotective Epo receptors in neuronal cells is controversial. Here we show that Epo prevents staurosporine-induced apoptosis of differentiated human neuroblastoma SH-SY5Y cells, and activates the STAT5, AKT and MAPK signaling pathways. Differentiated SH-SY5Y cells have fewer than 50 high affinity Epo surface binding sites per cell, which could not be detected by standard assays measuring binding of 125I-labeled Epo. However, by measuring endocytosis of 125I-Epo, we could reliably quantify very small numbers of high-affinity Epo surface binding sites. Using SH-SY5Y cells stably expressing an Epo receptor (EpoR) shRNA and thus lacking detectable EpoR expression, we show that high affinity binding of Epo to these neuronal cells is mediated by the hematopoietic EpoR, and that this EpoR is also essential for the antiapoptotic activity of Epo. In contrast, a mutant Epo that has an intact binding site 1 but a non-functional binding site 2 and hence binds only to one cell surface EpoR molecule ("site 2" Epo mutant) displays significantly lower antiapoptotic activity than wild-type Epo. Furthermore, expression of the GM-CSF/IL-3/IL-5 receptor common beta chain, which was proposed to be responsible for the cytoprotective activity of Epo on certain types of neuronal cells, was undetectable in differentiated SH-SY5Y cells. Epo also alleviated staurosporine-induced apoptosis of rat PC-12 pheochromocytoma cells while the R103A "site 2" Epo mutant did not, and we could not detect expression of the common beta chain in PC-12 cells. Together our results indicate that Epo exerts its antiapoptotic effects on differentiated SH-SY5Y and PC-12 cells through the standard stoichiometry of one molecule of Epo binding to two EpoR subunits, comprising the "classical" Epo receptor signaling complex.

The V617F activating point mutation in Jak2 is associated with a proportion of myeloproliferative disorders. In normal hematopoietic cells, Jak2 signals only when associated with a growth factor receptor, such as the erythropoietin receptor (EpoR). We sought to identify the molecular requirements for activation of Jak2V617F by introducing a point mutation in the FERM domain (Y114A), required for receptor binding. Whereas BaF3.EpoR cells are readily transformed by Jak2V617F to Epo independence, we found that the addition of the FERM domain mutation blocked transformation and the induction of reactive oxygen species. Further, while cells expressing Jak2V617F had constitutive activation of STAT5, cells expressing Jak2V617F/Y114A did not, suggesting that signaling is defective at a very proximal level. In addition, expression of the Myc and Pim proto-oncogenes by Jak2V617F was found to be FERM domain dependent. An inducible constitutively active STAT5 mutant expressed in BaF3 cells was sufficient to induce Myc and Pim. Finally, the FERM domain in Jak2V617F was also required for abnormal hematopoiesis in transduced primary murine fetal liver cells. Overall, our results suggest that constitutive activation of Jak2 requires an intact FERM domain for a transforming phenotype, and is necessary for activation of the major target of Jak2, STAT5.