Apoptosis Of Erythroid Progenitor Cells Research Articles

N6-methyl-2′-deoxyadenosine promotes self-renewal of BFU-E progenitor in erythropoiesis

Red blood cells supply the oxygen required for all human cells and are in demand for emerging blood-loss therapy. Here we identified N6-methyl-2'-deoxyadenosine (6mdA) as an agonist that promotes the hyperproliferation of burst-forming unit erythroid (BFU-E) progenitor cells. In addition, 6mdA represses the apoptosis of erythroid progenitor cells (EPCs). Combined use of with SCF and EPO enabled cultures of isolated BFU-E to be expanded up to 5,000-fold. Transcriptome analysis showed that 6mdA upregulates the expression of the EPC-associated factors c-Kit, Myb, and Gata2 and downregulates that of the erythroid maturation-related transcription factors Gata1, Spi1, and Klf1. Mechanistic studies suggested that 6mdA enhances and prolongs the activation of erythropoiesis-associated master gene c-Kit and its downstream signaling, leading to expansion and accumulation of EPCs. Collectively, we demonstrate that 6mdA can efficiently stimulate the EPC hyperproliferation and provide a new regenerative medicine recipe to improve exvivo generation of red blood cells.

Investigation of the molecular causes underlying physical abnormalities in Diamond‐Blackfan anemia patients with RPL5 haploinsufficiency

Diamond-Blackfan anemia (DBA) is a genetic disorder caused by mutations in genes encoding ribosomal proteins and characterized by erythroid aplasia and various physical abnormalities. Although accumulating evidence suggests that defective ribosome biogenesis leads to p53-mediated apoptosis in erythroid progenitor cells, little is known regarding the underlying causes of the physical abnormalities. In this study, we established induced pluripotent stem cells from a DBA patient with RPL5 haploinsufficiency. These cells retained the ability to differentiate into osteoblasts and chondrocytes. However, RPL5 haploinsufficiency impaired the production of mucins and increased apoptosis in differentiated chondrocytes. Increased expression of the pro-apoptotic genes BAX and CASP9 further indicated that RPL5 haploinsufficiency triggered p53-mediated apoptosis in chondrocytes. Murine double minute 2 (MDM2), the primary negative regulator of p53, plays a crucial role in erythroid aplasia in DBA patient. We found the phosphorylation level of MDM2 was significantly decreased in RPL5 haploinsufficient chondrocytes. In stark contrast, we found no evidence that RPL5 haploinsufficiency impaired osteogenesis. Collectively, our data support a model in which RPL5 haploinsufficiency specifically induces p53-mediated apoptosis in chondrocytes through MDM2 inhibition, which leads to physical abnormalities in DBA patients.

Role of Erythropoietin in Cerebral Glioma: An Innovative Target in Neuro-Oncology

Erythropoietin (EPO) is a cytokine primarily involved in the regulation of erythropoiesis. In response to hypoxia-ischemia, hypoxia-inducible factor 1 induces EPO production, which, in turn, inhibits apoptosis of erythroid progenitor cells. By the same mechanism and acting through other signaling pathways, EPO exerts neuroprotective effects. Increased resistance to hypoxia and decreased apoptosis are thought to be important mechanisms for tumor progression, including malignant glioma. Because recent studies have demonstrated that EPO and its receptor (EPOR) are expressed in several tumors and can promote tumor growth, in the present study, we investigated EPO and EPOR expression in human glioma and the effect of EPO administration in a rat model of glioma implantation. Using Western blotting and immunohistochemical analysis, we examined the expression of EPO, EPOR, platelet endothelial cell adhesion molecule, and Ki-67 in human glioma specimens and experimentally induced glioma in rats. In the experimental setting, a daily dose of recombinant human EPO (rHuEPO) or saline solution were administered for 21 days in Fischer rats subjected to 9L cell line implantation. In both human and animal specimens, we found an increase in EPOR expression as long as the lesion presented with an increasing malignant pattern. A significant direct correlation was found between the expression of EPOR and Ki-67 and EPOR and platelet endothelial cell adhesion molecule in low- and high-grade gliomas. The rats treated with rHuEPO presented with significantly larger tumor spread compared with the saline-treated rats. The results of our study have shown that the EPO/EPOR complex might play a significant role in the aggressive behavior of high-grade gliomas. The larger tumor spread in rHuEPO-treated rats suggests a feasible role for EPO in the aggressiveness and progression of malignant glioma.

Loss of ASXL1 triggers an apoptotic response in human hematopoietic stem and progenitor cells

ASXL1 is frequently mutated in myelodysplastic syndrome and other hematological malignancies. It has been reported that a loss of ASXL1 leads to a reduction of H3K27me3 via the polycomb repressive complex 2 (PRC2). To determine the role of ASXL1 loss in normal hematopoietic stem and progenitor cells, cord blood CD34+ cells were transduced with independent small hairpin interfering RNA lentiviral vectors against ASXL1 and cultured under myeloid and erythroid permissive conditions. Knockdown of ASXL1 led to a significant reduction in stem-cell frequency and a reduced cell expansion along the myeloid lineage. Cell expansion along the erythroid lineage was also reduced significantly and was accompanied by an increase in apoptosis of erythroid progenitor cells throughout differentiation and by an accumulation of cells in the G0/G1 phase. Bone marrow stromal cells supported the growth of immature erythroid cells, but did not alter the adverse phenotype of ASXL1 knockdown. Chromatin immunoprecipitation revealed no loss of H3K27me3 in myeloid progenitor cells, but demonstrated a loss of H3K27me3 on the HOXA and the p21 locus in erythroid progenitors. We conclude that ASXL1 is essential for erythroid development and differentiation and that the aberrant differentiation is, at least in part, facilitated via PRC2.

Interleukin-1 involved in Apoptosis of Beta-Thalassemia/Hemoglobin E Erythroid Progenitor Cells

Objective: The major pathophysiological features of β-thalassemia are anemia and ineffective erythropoiesis. Ineffective erythropoiesis has been shown by an intense marrow erythroid hyperplasia and increased apoptosis during basophilic to orthochromatic normoblast stages. Some cytokines like interferon-γ (IFN-γ), tumor necrosis factor α (TNF-α) and interleukin-1 (IL-1) have been found to involved in apoptosis. Although the pro-apoptotic activity of IFN-γ and TNF-α is well documented, there are only few studies on IL-1, especially on erythroid lineage. In this in vitro study, the role of cytokine IL-1α and IL-1β in apoptosis of erythroid progenitor cells from β- thalassemia/HbE patients was assessed. Methods: Erythroid progenitor cells were isolated from peripheral blood of healthy subjects and β-thalassemia/HbE patients. Cells were then cultured, with and without 20 ng/ml IL-1α and IL-1β. Total cells and percent cell viability were performed by using trypan blue staining. Percent cell apoptosis was analyzed by using flow cytometer. Results: Both IL-1α and IL-1β were significantly decreased erythroid progenitor cells. IL-1 at 20 ng/ml reduced the glycophorin A positive cells and percent cell viability of erythroid progenitor cells from β-thalassemia/HbE patients, while there was increased apoptosis in this group. The highest percent apoptosis was observed in 20 ng/ml IL-1β treated β-thalassemia/HbE erythroid progenitor cells. Conclusion: IL-1β could be involved in apoptosis of erythroid progenitor cells from β-thalassemia/HbE patients which might be related with ineffective erythropoiesis of the disease. DOI: http://dx.doi.org/10.3126/ajms.v3i4.6805 Asian Journal of Medical Science Vol.3(4) 2012 pp.8-14

How to approach chronic anemia.

We present herein an approach to diagnosing the cause of chronic anemia based on a patient's history and complete blood cell count (CBC). Four patterns that are encountered frequently in CBCs associated with chronic anemias are considered: (1) anemia with abnormal platelet and/or leukocyte counts, (2) anemia with increased reticulocyte counts, (3) life-long history of chronic anemia, and (4) anemia with inappropriately low reticulocytes. The pathophysiologic bases for some chronic anemias with low reticulocyte production are reviewed in terms of the bone marrow (BM) events that reduce normal rates of erythropoiesis. These events include: apoptosis of erythroid progenitor and precursor cells by intrinsic and extrinsic factors, development of macrocytosis when erythroblast DNA replication is impaired, and development of microcytosis due to heme-regulated eIF2α kinase inhibition of protein synthesis in iron-deficient or thalassemic erythroblasts.

Interferon-gamma induced nitric oxide-mediated apoptosis of anemia of chronic disease in rheumatoid arthritis

Proinflammatory cytokines play a role in the pathogenesis of anemia of chronic disease (ACD), which is a common cause of anemia in rheumatoid arthritis (RA). Anemia in RA is associated with increased apoptosis of erythroid cells. However, there is unclear information on the mechanism of ACD in the disease. The purpose of this study is to investigate the role of cytokines on nitric oxide-mediated apoptosis in erythroid progenitor cells of ACD in RA patients. Erythroid progenitor cells from healthy subjects and RA patients with ACD were treated with cytokines like interleukin-1β, tumor necrosis factor-α, and interferon-γ at concentrations of 2, 20, and 40 ng/ml for 14 days. Cell viability and cell apoptosis were analyzed by trypan blue staining and flow cytometry, respectively. The results showed that the highest effect of cytokines on reduction cell viability and induction cell apoptosis was found in 20 ng/ml IFN-γ-treated cells of RA patients. In addition, IFN-γ showed significantly increased nitric oxide production and iNOS mRNA expression, which was measured by Griess assay and real-time PCR, respectively. The percentage of cell apoptosis and NO production was reduced after an inducible nitric oxide synthase inhibitor, SMT, treatment. In conclusion, IFN-γ could induce nitric oxide production-mediated apoptosis process, which might be involved in the pathogenesis of ACD in RA patients.

Bench-to-bedside review: Erythropoietin and its derivatives as therapies in critical care

Erythropoietin (EPO) is known to have numerous biological functions. Its primary function in the body is to increase red blood cell numbers by way of preventing the apoptosis of erythroid progenitor cells via the homodimeric EPO receptor. The discovery that the local production of EPO within the brain in response to hypoxia or ischemia protects neurons against injury via an anti-apoptotic effect formed the basis of the hypothesis that the local generation of EPO limits the extent of injury. Although the hypothesis proved to be true in pre-clinical models of ischemia/reperfusion injury and inflammation, the randomized, controlled clinical trials that followed demonstrated serious adverse events of EPO due to activation of the hematopoietic system. Consequently, derivatives of EPO that lacked erythropoietic activity were discovered to reduce injury in many pre-clinical models associated with ischemia and inflammation. Unfortunately, there are no published clinical trials to determine the efficacy of non-erythropoietic derivatives of EPO in humans.

Erythropoietin in the intensive care unit: beyond treatment of anemia

Erythropoietin (EPO) is the major hormone stimulating the production and differentiation of red blood cells. EPO is used widely for treating anemia of critical illness or anemia induced by chemotherapy. EPO at pharmacological doses is used in this setting to raise hemoglobin levels (by preventing the apoptosis of erythroid progenitor cells) and is designed to reduce patient exposure to allogenic blood through transfusions. Stroke, heart failure, and acute kidney injury are a frequently encountered clinical problem. Unfortunately, in the intensive care unit advances in supportive interventions have done little to reduce the high mortality associated with these conditions. Tissue protection with EPO at high, nonpharmacological doses after injury has been found in the brain, heart, and kidney of several animal models. It is now well known that EPO has anti-apoptotic effects in cells other than erythroid progenitor cells, which is considered to be independent of EPOs erythropoietic activities. This review article summarizes what is known in preclinical models of critical illness and discusses why this does not correlate with randomized, controlled clinical trials.

Cytokine-Induced Apoptosis of Beta-Thalassemia/Hemoglobin E Erythroid Progenitor Cells via Nitric Oxide-Mediated Process in vitro

Background/Aim: β-Thalassemia/hemoglobin E (β-thal/HbE) is a common hereditary anemia in Thailand. Ineffective erythropoiesis due to apoptosis and decreased lifespan of circulating thalassemic red blood cells are the major causes of anemia. Changes to bone marrow microenvironment could contribute to apoptotic events. This study examined the effects of cytokines interleukin-1β, tumor necrosis factor-α and interferon-γ on apoptosis of β-thal/HbE erythroid progenitor cells in vitro, including nitric oxide-mediated apoptotic processes. Methods: Percent apoptosis of erythroid progenitor cells from 5 β-thal/HbE patients and 5 normal control subjects was examined using flow cytometry. In addition, the inducible nitric oxide synthase (iNOS) mRNA level and nitrite production were measured using quantitative PCR and the Griess method, respectively. Results: Upon cytokine treatment, a higher percent apoptosis was obtained with β-thal/HbE erythroid progenitor cells compared with control, and the maximum effect was observed using 20 ng/ml interferon-γ on day 14 of culture. There was an increase in iNOS mRNA level and a concomitant elevation of nitrite concentration in culture medium. Apoptosis and nitrite level were abrogated when β-thal/HbE and control cells were treated with S-methylisothiourea sulfate, an iNOS inhibitor. Conclusion: The marked sensitivity of erythroid progenitor cells from β-thal/HbE patients to cytokine-induced apoptosis via an NO-mediated process reflects a proapoptotic status of such thalassemic red blood cells.

Elongin B/C Recruitment Regulates Substrate Binding by CIS

SOCS proteins play a major role in the regulation of cytokine signaling. They are recruited to activated receptors and can suppress signaling by different mechanisms including targeting of the receptor complex for proteasomal degradation. The activity of SOCS proteins is regulated at different levels including transcriptional control and posttranslational modification. We describe here a novel regulatory mechanism for CIS, one of the members of this protein family. A CIS mutant deficient in recruitment of the Elongin B/C complex completely failed to suppress STAT5 activation. This deficiency was not caused by altered turnover of CIS but by loss of cytokine receptor interaction. Intriguingly, no such effect was seen for binding to MyD88. The interaction between CIS and the Elongin B/C complex, which depends on the levels of uncomplexed Elongin B/C, was easily disrupted. This regulatory mechanism may be unique for CIS, as similar mutations in SOCS1, -2, -3, -6, and -7 had no functional impact. Our findings indicate that the SOCS box not only plays a role in the formation of E3 ligase complexes but, at least for CIS, can also regulate the binding modus of SOCS box-containing proteins.

P59 Involvement of CD9 in Erythroid Progenitor Cell Apoptosis

Introduction The study of erythroid progenitor cell proliferation and differentiation in vitro is essential if we are to achieve our goal of producing therapeutic quantities of red cells by cell culture. The mechanisms of cell regulation need to be appreciated and a cell culture environment developed for optimal cell renewal and growth. Changes in cell surface molecules and understanding of their function form an important part of the study together with knowledge about the genes involved in the erythropoietic process, including those genes involved in apoptosis. CD9, a member of the tetraspanin super family, is reportedly involved in tumour progression and metastasis and has been implicated in apoptosis of cancer cells.Methods Apoptosis was induced in erythroid progenitor cells by depriving the cultures of erythropoietin (EPO). CD34+ cells were isolated from the peripheral blood of adults and cultured for 5 days in serum free medium with IL‐3 (1 ng mL−1), SCF (10 ng mL−1) and EPO (3 IU mL−1). On day 6 the culture was divided and half the cells deprived of EPO; both cultures were analysed on day 8. Total RNA was extracted and run on Affymetrix microarray chips to compare the mRNA profile of the apoptotic and control cells.Results One gene that was consistently upregulated in the apoptotic cells was CD9. This result was confirmed by real‐time PCR. Immunofluorescent staining and confocal microscopy revealed that CD9 protein expression changed during apoptosis from low level expression within the cell membrane to a much higher level, which was cytoplasmic as well as membranous. To determine whether this CD9 upregulation was an initiator of apoptosis or a consequence, CD34+ cells were cultured with anti‐CD9 (ALB6) antibody added to the culture medium. Ligation of CD9 by anti‐CD9 did not change the apototic profile of the cultures.Conclusion It can be concluded that although CD9, by itself, is not an initiator of apoptosis in erythroid progenitor cells it is obviously an important target in the subsequent apoptotic pathway. Continued research into CD9 and other mechanisms of apoptosis may allow manipulation of cells in culture with a possible extension of their life span and increase in the number of cells produced.

Possible Neuroprotective Effect of Erythropoietin in Acute Inflammation Induced by Exhaustive Exercise.

Erythropoietin is a glycoprotein hormone produced mainly by interstitial fibroblasts in the kidneys of the adult and by hepatocytes of the foetus. Released into the circulation, erythropoietin makes its way to the bone marrow, where it regulates red cell production by preventing apoptosis of erythroid progenitor cells. Recently, erythropoietin has emerged as a multifunctional growth factor that plays a significant role in the central nervous system (CNS). Both erythropoietin and its receptor are expressed throughout the brain in glial cells, neurons and endothelial cells. Hypoxia and ischaemia have been recognised as important stimuli of erythropoietin expression in the brain. Erythropoietin has potent neuroprotective properties in vivo and in vitro and appears to act in a dual way by directly protecting neurons from ischaemic damage and by stimulating endothelial cells. S-100b, a glial-derived protein is a well-established biomarker for severity of neurological injury and prognosis for recovery. Cell-based and clinical studies have implicated S-100b protein in the initiation and maintenance of a pathological, glial-mediated pro-inflammatory state in the CNS. We studied the possible association between erythropoietin and S-100b in 18 athletes participated in the ultra-distance foot race of the 246Km “Sparthathlon”. This race consists of continuous, prolonged, brisk exercise. We have reported that IL-6, CRP SAA and free plasma DNA levels markedly increased (by 8000-, 152- 108- and 10-fold, respectively) over the baseline at the end of the race1. However, IL-6 levels returned to normal by 48h, while CRP, SAA and free plasma DNA remained elevated. Erythropoietin and S-100b protein levels were determined by means of chemiluminescence (Nichols Institute Diagnostics, CA, USA and Sangtec Medical AB, Bromma, Sweden respectively). The measurements were performed before (phase I), at the end (phase II) and 48h post-race (phase III). Erythropoietin levels at phase I (13.5±6.3 IU/L), were increased significantly at phase II (52.0±52.0 IU/L) and subsequently decreased at phase III (21.0±14.6 IU/L). At the same time period S-100b protein followed the same pattern (phase I: 0.13±0.01 mg/L, phase II: 0.29±0.01 and phase III: 0.13±0.01mg/L). A significantly positive correlation between erythropoietin and S-100b protein was found at phase II (r=0.521, p<0.03), while this correlation was absent in the other two phases (p>0.40 and p>0.08 respectively). Simultaneous measurements of haemoglobin levels showed only a significant drop of haemoglobin at phase III (143.4±12.4, 140.4±13.0 and 124.3±9.5 g/L respectively). Exhaustive exercise induces inflammation and CNS injury, promoting an erythropoietin anti-inflammatory response. We suggest that in this particular inflammatory process, erythropoietin secretion is associated with brain lesions, as they are expressed by the S-100b protein release in the circulation. This is strongly supported by the lack of any association between erythropoietin values and haemoglobin levels.

Deficient Ribosomal Protein S19 in Diamond-Blackfan Anemia Causes a Reduction in bcl-2 and Bad and Leads to Apoptosis in Erythroid Progenitor Cells.

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein (RP) S19 gene. It is not known how the RPS19 deficiency impairs erythopoiesis and proliferation of hematopoietic progenitors. We have established an in vitro models for RPS19 deficient DBA using lentiviral vector mediated doxycycline (Dox) inducible small interfering RNA (siRNA) against RPS19 (Mol Ther. 11:627–637. 2005). Suppression of cell growth and erythroid colony formation correlated with the suppression level of RPS 19, indicating that these cell lines are useful to determine the mechanisms of RPS19 deficient DBA. To elucidate molecular mechanisms in RPS19 deficient DBA, we analyzed cell cycle of Dox induced RPS19 deficient TF-1 cells. RPS19 deficient TF-1 cells showed G0/G1 arrest (82% vs 58%, p<0.05) together with accumlation of p21 and p27, and apoptotic cells detected by Annexin-V analysis also increased compared to control Dox induced TF-1 cells (13% vs 3.1%, p<0.05). Increase of apoptotic cells in RPS19 deficient cells was confirmed by TUNEL assay. Western blot analysis of apoptotic related protein showed that the level of bcl-2 and Bad was decreased in RPS19 deficient TF1 cells compared to control cells. This down-regulation of apoptotic related protein was improved by transduction with lentiviral vector expressing modified RPS19, which is not affected by siRNA but produce normal functional RPS19 protein and rescues the DBA phenotype. Moreover, primary CD34 positive cells from DBA patients detected by Annexin-V analysis also generate a high number of apoptotic cells compared to normal CD34 positive cells during in vitro culture (38% vs 8.9%, n=5, p<0.001). These findings indicate that erythroid progenitor cells are more sensitive to apoptosis than other hematopoietic progenitors and that RPS19 deficiency causes apotosis and accelerated loss of erythroid progenitors in RPS19 deficient DBA.

Role of NF‐κB in regulation of apoptosis of erythroid progenitor cells

Erythropoietin (EPO) and interferon-gamma (IFN-gamma) added to human erythroid progenitor cells purified from peripheral blood (erythroid colony-forming cells; ECFC) significantly reduces apoptosis as assessed by flow cytometry (FCM) using annexin V. To clarify the role of NF-kappaB in the regulation of the apoptosis of erythroid progenitor cells, cyclosporin A (CsA), which blocks dissociation of the NF-kappaB complex, was added to serum-free cultures of ECFC. CsA induced the apoptosis of ECFCs in the presence of EPO or IFN-gamma, but at different magnitudes. In the presence of a relatively low concentration of CsA (10 microm), apoptosis was induced only in cultures with EPO. The direct involvement of NF-kappaB was then assessed by Western blotting and confocal microscopy. In the presence of EPO, NF-kappaB was abundant both in the cytoplasm and in the nucleus, and nuclear expression was diminished after adding CsA. In contrast, NF-kappaB was undetectable in the nucleus in the presence of IFN-gamma. The effect of CsA on mitochondrial function was investigated by determining the DeltaPsim and reactive oxygen species production. CsA disturbed the transmembrane potential in the presence of either EPO or IFN-gamma, although the viability of the cells was maintained in the presence of IFN-gamma plus CsA. These results indicate that IFN-gamma reduced the apoptosis of erythroid progenitor cells through a unique signaling pathway that is independent of NF-kappaB translocation, and which is not mediated by modulating mitochondrial function, whereas EPO reduced apoptosis through NF-kappaB translocation to the nucleus.

Ex Vivo Treatment of Erythroid Cells with Glycolytic Intermediates for Metabolic Correction of Pyruvate Kinase Deficiency.

Glycolysis is an essential metabolic pathway for survival of red blood cells (RBC), and mutations in the RBC-type of pyruvate kinase (R-PK) cause hereditary non-spherocytic hemolytic anemia. We have shown that R-PK deficiency induced apoptosis in erythroid progenitor cells (Blood 96: 596a, 2000; Am J Hematol 74: 68, 2003). In this study, we examined whether ex vivo treatment of erythroid cells with high concentration of glycolytic intermediates ameliorated apoptosis of R-PK deficiency. For this purpose, we used the R-PK deficient Friend erythroleukemic cell, SLC3, which has been established from a mice model of R-PK deficiency (Jpn J Cancer Res 90: 171, 1999). SLC3 and a control Friend cell were cultured with 0, 1, 3, and 5mM phosphoenolpyruvate (PEP) or pyruvate (PA). Supplementations of either PEP or PA resulted in about 2-fold accumulation of intracellular PA; however, ATP concentration was not significantly changed. Flow cytometric analysis showed that 24-hours treatment with 5mM PEP significantly reduced annexinV (+)/PI (−) cells. These data suggested that exogenously supplemented glycolytic intermediates increased intracellular PA concentration, resulting in amelioration of erythroid apoptosis. We have shown that a glutathione precursor, N-acetyl cysteine, reduced apoptosis of SLC3. Several studies revealed that PA, which effectively maintained the redox state and also prevented the loss of intracellular antioxidants, antagonized the apoptotic cell death. Taken together, oxidative stress caused by decreased supply of PA might account for erythroid apoptosis due to R-PK deficiency.

Bcl-x Prevents the Apoptosis of Late-Stage, Hemoglobin-Synthesizing Erythroblasts, but It Does Not Mediate the Anti-Apoptotic Effect of Erythropoietin on Early-Stage Erythroblasts.

Bcl-x is a protein in the outer mitochondrial membrane. A member of the Bcl-2 family, Bcl-x protects developing erythroid cells from apoptosis. The exact stage of erythroid development at which Bcl-x exerts its anti-apoptotic effect is not known, but induction of Bcl-x has been proposed to be the mediator of erythropoietin's (EPO) anti-apoptotic effect in erythroid differentiation. EPO is the principal trophic hormone that controls red blood cell production by regulating apoptosis of erythroid progenitor cells at the CFU-E and early erythroblast stages. Bcl-x has also been reported to be necessary for heme synthesis. In mice, Bcl-x deficiency is embryonically lethal; when Bcl-x deficiency is acquired postnatally by conditional knockout technology, it is associated with splenomegaly, thrombocytopenia, and a profound anemia that is thought to be hemolytic in origin.Objectives: 1)To characterize the defect of erythroid differentiation in conditional Bcl-x −/− mice. 2)To determine whether Bcl-x is the mediator of EPO's anti-apoptotic action. 3)To determine whether Bcl-x is necessary for heme synthesis.Methods: Phlebotomized or unbled littermate controls and anemic adult Bcl-x −/− mice obtained by cre-lox conditional knockout were bled, sacrificed, and splenectomized. Purified populations of splenic erythroblasts were isolated by sedimentation at unit gravity, cultured with or without EPO, and harvested at 0, 8, 20, 32, and 44 hours for cell counts, cytospin preparations for morphology, flow cytometry analyses for apoptosis (TUNEL) and cell cycle phases, and 59FeCl3 incorporation into heme.Results: Compared to littermate controls, Bcl-x −/− mice were severely anemic (Hgb 2.8 g/dL vs 15.4 g/dL in unbled controls and 7.2 g/dL in bled controls), thrombocytopenic (platelets 23x103/microL vs 905x103/microL in unbled controls and 984x103/microL in bled controls), and reticulocytopenic (82.8x103/microL vs 281x103/microL in unbled controls and 1410x103/microL in bled controls), while WBCs were unaffected. Expanded erythropoiesis led to massive splenomegaly (spleens =4.3gm vs 0.1gm in unbled controls and 0.3gm in bled controls). After 44 hours of culture with EPO, purified erythroblasts from bled controls proliferated 4-fold and differentiated such that the majority enucleated, producing 200–250 reticulocytes per 100 erythroblasts plated, whereas Bcl-x −/− erythroblast numbers doubled during the first 20 hours in culture, but the large majority died by apoptosis between 20 and 44 hours, producing only 9–12 reticulocytes per 100 erythroblasts plated. Bcl-x −/− erythroblast apoptosis occurred after the initiation of heme synthesis and proportionally in all phases of cell cycle. Compared to culture with EPO, Bcl-x −/− erythroblasts cultured without EPO underwent increased apoptosis at earlier times of culture-- at 8 hours (45% vs 29%), 20 hours (71% vs 42%) and 32 hours (83% vs 57%).Conclusions: 1)Bcl-x is required for the survival and differentiation of the late-stage erythroblasts in all phases of cell cycle. Thus, Bcl-x deficiency results in ineffective erythropoiesis rather than hemolytic anemia. 2)Bcl-x is not required for heme synthesis, but has its anti-apoptotic effect during the stage of hemoglobin synthesis. 3)Bcl-x does not mediate EPO's anti-apoptotic effect in early-stage erythroblasts.

Erythropoietin and the hypoxic brain.

Normal tissue function in mammals depends on adequate supply of oxygen through blood vessels. A discrepancy between oxygen supply and consumption (hypoxia) induces a variety of specific adaptation mechanisms at the cellular, local and systemic level. These mechanisms are in part governed by the activation of hypoxia-inducible transcription factors (HIF-1, HIF-2), which in turn modulate expression of hypoxically regulated genes such as those encoding vascular endothelial growth factor (VEGF) and erythropoietin (EPO). EPO is a glycoprotein that is produced mainly by interstitial fibroblasts in the kidneys of the adult and in hepatocytes in the foetus. Released into the circulation, EPO makes its way to the bone marrow, where it regulates red cell production by preventing apoptosis of erythroid progenitor cells. Recently, EPO has emerged as a multifunctional growth factor that plays a significant role in the nervous system. Both EPO and its receptor are expressed throughout the brain in glial cells, neurones and endothelial cells. Hypoxia and ischaemia have been recognised as important driving forces of EPO expression in the brain. EPO has potent neuroprotective properties in vivo and in vitro and appears to act in a dual way by directly protecting neurones from ischaemic damage and by stimulating endothelial cells and thus supporting the angiogenic effect of VEGF in the nervous system. Thus, hypoxia-induced gene products such as VEGF and EPO might be part of a self-regulated physiological protection mechanism to prevent neuronal injury, especially under conditions of chronically reduced blood flow (chronic ischaemia). In this review, I will briefly summarize the recent findings on the molecular mechanisms of hypoxia-regulated EPO expression in general and give an overview of its expression in the central nervous system, its action as a growth factor with non-haematopoietic functions and its potential clinical relevance in various brain pathologies.

Non-erythroid functions of erythropoietin.

The oxygen-dependent, renal cytokine eythropoietin (Epo) is well known to increase red cell production. Binding of Epo to the Epo receptor (EpoR) represses apoptosis of erythroid progenitor cells, thereby allowing their final maturation. We and others showed that Epo and its receptor are expressed in many other tissues, including brain, spinal cord, retina and testis. The presence of a blood barrier suggests that Epo plays a local role in these organs. Indeed, therapeutically applied or hypoxically induced Epo has been shown to reduce the infarct volume in various stroke animal models, to prevent retinal degeneration, and to ameliorate spinal cord injury. In a study conducted by Ehrenreich and colleagues, stroke patients treated with Epo showed reduced infarct volume, fast neurological recovery and improved clinical outcome. In analogy to its function on erythroid progenitor cells, this neuroprotective effect of Epo might be explained by repression of programmed cell death. Apart from neuroprotection, there is an assumption that Epo present in breast milk has the potential to protect against mother-to-infant transmission of HIV. When using Epo at high doses for longer time periods; however, care has to be taken to control the resulting chronic polycythemia that most probably caused enlarged cerebral infarct volumes in a transgenic mouse model that due to Epo-overexpression reached hematocrit levels of about 0.8. Overall, these data strongly support the notion that Epo will soon find new applications in the clinic.

The Cytokine-inducible Scr Homology Domain-containing Protein Negatively Regulates Signaling by Promoting Apoptosis in Erythroid Progenitor Cells

The small cytokine-inducible SH2 domain-containing protein (CIS) has been implicated in the negative regulation of signaling through cytokine receptors. CIS reduces growth of erythropoietin receptor (EpoR)-dependent cell lines, but its role in proliferation, differentiation, and survival of erythroid progenitor cells has not been resolved. To dissect the function of CIS in cell lines and erythroid progenitor cells, we generated green fluorescent protein (GFP)-tagged versions of wild type CIS, a mutant harboring an inactivated SH2 domain (CIS R107K), and a mutant with a deletion of the SOCS Box (CISDeltaBox). Retroviral expression of the GFP fusion proteins in BaF3-EpoR cells revealed that both Tyr-401 in the EpoR and an intact SH2 domain within CIS are prerequisites for receptor recruitment. As a consequence, both are essential for the growth inhibitory effect of CIS, whereas the CIS SOCS box is dispensable. Accordingly, the retroviral expression of GFP-CIS but not GFP-CIS R107K impaired proliferation of erythroid progenitor cells in colony assays. Erythroid differentiation was unaffected by either protein. Interestingly, apoptosis of erythroid progenitor cells was increased upon GFP-CIS expression and this required the presence both of an intact SH2 domain and the SOCS box. Thus, CIS negatively regulates signaling at two levels, apoptosis and proliferation, and thereby sets a threshold for signal transduction.