Control Of Erythropoiesis Research Articles

Increased angiotensin II formation in the brain modulates cardiovascular homeostasis and erythropoiesis.

In spite of the fact that the modulatory effects of angiotensin II (Ang II) on the sympathetic nerve activity to targeted organs involved in blood pressure (BP) regulation is well acknowledged, the local production of this peptide in the brain and the consequences of enhanced central Ang II beyond the cardiovascular system are not yet well comprehended. In the present study, we generated and validated a new transgenic mouse line overexpressing the rat full-length angiotensinogen (Agt) protein specifically in the brain (Agt-Tg). Adult Agt-Tg mice presented overall increased gene expression of total Agt in the brain including brainstem and hypothalamus. In addition, the excess of Agt led to abundantly detectable brain Ang II levels as well as increased circulating copeptin levels. Agt-Tg displayed raised BP in acute recordings, while long-term telemetrically measured basal BP was indistinguishable from wild-types. Agt-Tg has altered peripheral renin-angiotensin system and vasomotor sympathetic tone homeostasis because renal gene expression analysis, plasma Ang II measurements and ganglionic blockade experiments revealed suppressed renin expression and reduced Ang II and higher neurogenic pressure response, respectively. Plasma and urine screens revealed apparently normal fluid and electrolyte handling in Agt-Tg. Interestingly, hematological analyses showed increased hematocrit in Agt-Tg caused by enhanced erythropoiesis, which was reverted by submitting the transgenic mice to a long-term peripheral sympathectomy protocol. Collectively, our findings suggest that Agt-Tg is a valuable tool to study not only brain Ang II formation and its modulatory effects on cardiovascular homeostasis but also its role in erythropoiesis control via autonomic modulation.

Hypoxia inducible factor stabilizers: a promising treatment for chronic kidney disease

Anemia in chronic kidney disease (CKD) is a very common complication. The two main factors contributing to the development of anemia in CKD is decreased erythropoietin production and iron deficiency. Other factors that might play a role in the pathogenesis of renal anemia are: chronic inflammation leading to increased hepcidin, uremic toxins, and shorter red blood cell life span. The mainstay of treatment is iron supplementation, blood transfusion and erythropoietin stimulating agents (ESA). The discovery of hypoxia inducible factor (HIF) pathway has opened a new chapter in the treatment of anemia in CKD. The oxygen-sensitive HIF pathway plays a prominent role in the control of erythropoiesis and iron metabolism. HIF stabilizers are a new set of drugs that inhibits prolyl hydroxylase domain (PHD) proteins which are key regulators of HIF activity. Several such compounds are being developed to revolutionize the treatment of renal anemia.

An Erythrocytosis-Associated Mutation in the Zinc Finger of PHD2 Provides Insights into Its Binding of p23.

BackgroundLoss of function mutations in the EGLN1 gene are a cause of erythrocytosis. EGLN1 encodes for prolyl hydroxylase domain protein 2 (PHD2). PHD2 hydroxylates and downregulates hypoxia-inducible factor-2α (HIF-2α), a transcription factor that regulates erythropoiesis. While the large majority of erythrocytosis-associated EGLN1 mutations occur within its catalytic domain, rare mutations reside in its zinc finger. This zinc finger binds a Pro-Xaa-Leu-Glu motif in p23, an HSP90 cochaperone that facilitates hydroxylation of HIF-α, an HSP90 client. Essentially nothing is known about the specific interactions between the PHD2 zinc finger and p23.ResultsHere, we characterize an erythrocytosis-associated mutation in the zinc finger, K55N, that abolishes interaction with p23. We provide evidence that the affected residue, Lys-55, interacts with Asp-152 of p23. We also present results that indicate that PHD2 Arg-32 interacts with p23 Glu-160.ConclusionThese studies not only reinforce the importance of the PHD2 zinc finger in the control of erythropoiesis, but also lead to a model in which a peptide motif in p23 binds in a specific orientation to a predicted groove in the zinc finger of PHD2.

Therapeutic targeting of the HIF oxygen-sensing pathway: Lessons learned from clinical studies

The oxygen-sensitive hypoxia-inducible factor (HIF) pathway plays a central role in the control of erythropoiesis and iron metabolism. The discovery of prolyl hydroxylase domain (PHD) proteins as key regulators of HIF activity has led to the development of inhibitory compounds that are now in phase 3 clinical development for the treatment of renal anemia, a condition that is commonly found in patients with advanced chronic kidney disease. This review provides a concise overview of clinical effects associated with pharmacologic PHD inhibition and was written in memory of Professor Lorenz Poellinger.

Treatment with erythropoietin in neonatology

The article presents the basics and con-trol of erythropoiesis in the fetus and the newborn, the development of anaemia of prematurity and its treatment, with an em-phasis on the use of human recombinant erythropoietin. The Intensive Care Unit of the Paediatric Clinic Maribor began treat-ing anaemia of prematurity with eryth-ropoietin in 2000. After introducing the treatment, the clinic found that the num-ber of blood product transfusions and the needed blood volume decreased. In addi-tion to erythropoietin, this was the result of stricter criteria for applying transfusion of concentrated erythrocytes.

New genetic and physiological factors for excessive erythrocytosis and Chronic Mountain Sickness.

In the last few years, genetic and functional studies have provided important insight on the pathophysiology of excessive erythrocytosis (EE), the main sign of Chronic Mountain Sickness (CMS). The recent finding of the association of the CMS phenotype with a single-nucleotide polymorphism (SNP) in the Sentrin-specific Protease 1 (SENP1) gene, and its differential expression pattern in Andean highlanders with and without CMS, has triggered large interest in high-altitude studies because of the potential role of its gene product in the control of erythropoiesis. The SENP1 gene encodes for a protease that regulates the function of hypoxia-relevant transcription factors such as Hypoxia-Inducible Factor (HIF) and GATA, and thus might have an erythropoietic regulatory role in CMS through the modulation of the expression of erythropoietin (Epo) or Epo receptors. The different physiological patterns in the Epo-EpoR system found among Andeans, even among highlanders with CMS, together with their different degrees of erythropoietic response, might indicate specific underlying genetic backgrounds, which in turn might reflect different levels of adaptation to lifelong high-altitude hypoxia. This minireview discusses recent genetic findings potentially underlying EE and CMS, and their possible physiological mechanisms in Andean highlanders.

Lipopolysaccharide-induced cross-tolerance against renal ischemia–reperfusion injury is mediated by hypoxia-inducible factor-2α-regulated nitric oxide production

Although the protective effect of lipopolysaccharide (LPS) pretreatment on renal ischemia/reperfusion injury is known, a link to hypoxia-inducible factors (HIFs) has not been established. Here we show that LPS treatment led to HIF-2α accumulation in mouse kidneys and endothelial cells, a result of nuclear factor-κB activation. Inactivation of HIF-2α, rather than HIF-1α, completely negated LPS-mediated protection against renal ischemia/reperfusion injury. LPS-stimulated renoprotection was related to inducible/endothelial nitric oxide synthase (iNOS/eNOS) expression, increased production of nitric oxide, and enhanced postischemic microcirculatory recovery. All these effects were lost in HIF-2α knockout mice. Preischemic administration of a nitric oxide donor, rather than erythropoietin, restored the lost preconditioning effect of LPS in HIF-2α knockout mice. In vitro and in vivo studies demonstrated that HIF-2α in endothelial cells, rather than myeloid cells or hepatocytes, was responsible for the LPS-mediated effects. Thus, our results demonstrated that LPS preconditioning protected against renal ischemia/reperfusion injury by HIF-2α activation in endothelial cells that subsequently improved renal microvascular perfusion and reduced ischemic tubular damage.

BH3-only protein Noxa contributes to apoptotic control of stress-erythropoiesis

Apoptosis plays an essential role in the control of erythropoiesis under normal and pathological conditions. However, the contribution of individual proteins within cell death signalling pathways remains poorly defined. Here, we investigated the role of the pro-apoptotic Bcl-2 family member Noxa in the regulation of erythropoiesis. We found that expression of Noxa is induced during erythroid differentiation of human and murine precursor cells. Using in vitro model systems for erythroid progenitors, we observed rapid induction of Noxa upon cytokine deprivation. Knockdown or deletion of Noxa conferred significant protection against apoptosis upon cytokine withdrawal. In vivo, Noxa deficiency did not affect hematological blood parameters or erythroid progenitor composition of bone marrow and spleen under steady-state conditions. In contrast, in a model of acute haemolytic anemia, Noxa-deficiency enhanced hematocrit recovery. Moreover, in a model of chronic inflammation-induced anemia, Noxa-ablation resulted in a dramatic increase of erythroblast expansion. Our data indicate that induction of Noxa in erythroid progenitors sets a survival threshold that limits expansion beyond the number of cells that can be sustained by the available cytokines, which becomes apparent under conditions of induced anemia.

Oscillations of Numerical Solutions for Nonlinear Delay Differential Equations in the Control of Erythropoiesis

We consider the oscillations of numerical solutions for the nonlinear delay differential equations in the control of erythropoiesis. The exponentialθ-method is constructed and some conditions under which the numerical solutions oscillate are presented. Moreover, it is proven that every nonoscillatory numerical solution tends to the equilibrium point of the continuous system. Numerical examples are given to illustrate the main results.

Association between polymorphisms in erythropoietin gene and upper limit haematocrit levels among regular blood donors

Purpose of the studyErythropoietin (EPO) is a glycoprotein hormone that functions primarily on the stimulation and control of erythropoiesis in bone marrow. In this study, polymorphisms in EPO gene; C3434T, G3544T (rs551238) and rs1617640 were evaluated to determine their frequencies and genotype distribution patterns among blood donors with upper-limit haematocrit level. Subjects and methodsA total of 298 subjects, 181 blood donors with haematocrit level greater or equal to 48% and 117 donors with haematocrit between 42–47.5% as control were recruited. All subjects were genotyped for C3434T, rs551238 polymorphisms and for rs1617640 using restriction fragment length polymorphism method (PCR-RFLP) and sequencing techniques. ResultsA significant difference was found in rs1617640 and rs551238 genotype frequencies in blood donors with upper-haematocrit compared to the control group (P<0.05). In accordance with genotype frequencies, G allele in these two polymorphisms were found at higher frequency among upper-haematocrit group compared to the control (P<0.05). On the other hand, C3434T polymorphism was not significantly different between the two groups, neither for genotype frequencies nor for allele frequencies. ConclusionResults suggest a strong association between rs551238 and rs1617640 polymorphisms in the EPO gene and upper-limit haematocrit level among blood donors.

Érythropoïétine et neuroprotection

Erythropoietin (Epo) has long been recognised for its role in the control of erythropoiesis and therefore in the treatment of anemia including anemia of prematurity. The erythropoietin receptor (Epo-R) though is expressed in many other organs including the CNS. This review focuses on the role of erythropoietin during the development of the CNS and its potential role as a neuroprotective agent. Epo-R is expressed in many different cellules of the CNS during development including neural progenitor cells, neurons, astrocytes and oligodendrocytes. In the event of hypoxia CNS cells respond with increase of erythropoietin release with subsequent stimulation of neurogenesis through Epo-R on neural progenitor cells. In an Epo-R knock-out model therefore cerebral development is severely impaired. In models of hypoxia-ischemia exogenous Epo has been shown to reduce lesion size and improve structural and functional recovery. Human studies are emerging using Epo as a neuroprotective agent both for the term infant with hypoxia-ischemia as well as for the extremely preterm infant.

Chest pain and small red cells: size does matter

A 63-year-old woman was admitted to our hospital in June, 2008, with a 24-h history of central chest pain radiating to her neck. In the 7 days before admission, she had experienced intermittent chest tightness without any obvious precipitating cause. Her medical history was unremarkable. She did not smoke and was on no regular medication. There were no abnormalities on clinical examination. ECG showed inversion of T waves in leads III, V2, and V3. Serum troponin T concentration after 12 h was 1·22 μg/L (normal range [NR] 0–0·01 μg/L) indicating an inferior non-STelevation myocardial infarction. We started our patient on aspirin, clopidogrel, and dalteparin. Serum cholesterol was slightly high (5·3 mmol/L; normal up to 5 mmol/L) but no additional risk factors for coronary artery disease were evident. Blood tests showed haemoglobin 137 g/L (NR 120–160 g/L), leuco cytes 14·5×109/L (NR 4–11×109/L), neutrophils 12×109/L (NR 2·0–7·5×109/L), and platelets 614×109/L (NR 150–400×109/L), with normal eosinophil (0·08×109/L; NR 0–0·4×109/L) and basophil counts (0×109/L; NR 0–1×109/L). C-reactive protein concentration was 4 mg/L (normal up to 5 mg/L). After 24 h, leucocyte count had normalised, and the platelets decreased to 528×109/L. Coronary angiography showed normal left coronary arteries. The right coronary artery was dominant with a long layer of fresh thrombus in the proximal/mid vessel (fi gure A), clearly the cause of her symptoms. Because of the risk of distal embolisation, angioplasty was deferred and the patient was started on an infusion of the glycoprotein IIb/IIIa inhibitor, tirofi ban. Repeat angiography after 72 h of tirofi ban showed almost complete resolution of the thrombus (fi gure B) and we managed our patient conservatively thereafter, with the initiation of dual antiplatelet therapy. On closer scrutiny of the presenting blood count, despite normal haemoglobin and packed-cell volume, there was micro cytosis with reduced mean corpuscular volume (74·5 fL; NR 80–96 fL) and hypochromia (mean corpuscular haemoglobin 23 pg; NR 27–32 pg). The serum ferritin concentration (12 μg/L; NR 13–150 μg/L) confi rmed iron defi ciency. This conclusion was supported by the microscopic appearance of the blood, which showed anisopoikilocytosis with microcytic hypochromic cells, elliptocytes, and pencil cells (fi gure C). In addition, platelet anisocytosis and giant platelets were seen (fi gure D). Paradoxically, despite iron defi ciency, there was erythrocytosis (red cell count 5·96×1012/L; NR 3·8–4·8×1012/L) indicating loss of homoeostatic control of erythropoiesis. The suspicion of an iron-defi cient myelo proliferative disorder underlying the myocardial infarction was confi rmed by the presence of a heterozygous mutation (V617F) in the JAK2 gene, detected in 90–95% of patients with myeloproliferative polycythaemia. We started our patient on hydroxy carbamide, resulting in satisfactory control of the platelet count. When last seen in May, 2009, our patient was asymptomatic with no further angina. Myeloproliferative disorders are associated with thrombotic complications with predominance of arterial over venous events. The recognition of the somatic mutation JAK2V617F in most patients with myeloproliferative polycythaemia has improved the diagnostic accuracy and bone-marrow examination is not mandatory. Diagnosis of myeloproliferative polycyth ae mia is therefore straight forward in the setting of an elevated haemoglobin and packed-cell volume. Some patients may present with a normal haemoglobin and only subtle changes in red cell indices, as a result of iron-defi ciency; identifi cation of these patients is important because of the potential for life-threatening thrombosis through mechanisms independent of haemoglobin and packedcell volume. Despite iron defi ciency, these patients should not be given iron-replacement therapy unless closely supervised, because it could stimulate a rapid and potentially catastrophic increase in haemoglobin.

Gfi-1B Promoter Remains Associated with Active Chromatin Marks Throughout Erythroid Differentiation of Human Primary Progenitor Cells

Growth Factor Independent-1B (Gfi-1B) is a transcriptional repressor that plays critical roles in the control of erythropoiesis and megakaryopoiesis. Gfi-1B expression was described to be repressed by an autoregulatory feedback control loop. Here, we show that Gfi-1 transcription is positively regulated early after induction of erythroid differentiation and remains highly active to late erythroblasts. Using chromatin immunoprecipitation assays in CD34+ cells from human cord blood, we found that Gfi-1 and GATA-2 in immature progenitors and then Gfi-1B and GATA-1 in erythroblasts are bound to the Gfi-1B promoter as well as to the promoter of c-myc, a known Gfi-1B target gene. Surprisingly, this Gfi-1/GATA-2–Gfi-1B/GATA-1 switch observed at erythroblast stages is associated to an increase in the Gfi-1B transcription whereas it triggers repression of c-myc transcription. Accordingly, analysis of chromatin modification patterns shows that HDAC, CoREST, and LSD1 are recruited to the c-myc promoter leading to appearance of repressive chromatin marks. In contrast, the Gfi-1B promoter remains associated with a transcriptionally active chromatin configuration as highlighted by an increase in histone H3 acetylation and concomitant release of the LSD1 and CoREST corepressors. The repressive function of Gfi-1B therefore depends on the nature of the proteins recruited to the target gene promoters and on chromatin modifications. We conclude that Gfi-1B behaves as a lineage-affiliated gene with an open chromatin configuration in multipotent progenitors and sustained activation as cells progress throughout erythroid differentiation.

Reflections on the European Union Eurythron Network Meeting “Molecular Control of Erythropoiesis,” September 22–23, 2005, Istituto Superiore Di Sanità, Rome, Italy

Red blood cells (RBCs) mediate oxygen transport throughout the body, a function that is essential for life. RBCs are continuously produced via a process called erythropoiesis. Anemias (insufficient numbers of functional RBCs), caused by failure of erythropoiesis, are a major cause of disease worldwide. Hereditary anemias constitute the most common human genetic disorders; they have no effective cure yet. The European research training network Eurythron follows a multidisciplinary approach to clarify the important molecular mechanisms in normal and pathological erythropoiesis, with a view to develop novel therapies to cure the anemias. The aim is to generate a comprehensive molecular description of mechanisms governing specification of hematopoietic stem cells in embryogenesis, lineage commitment, differentiation, and postmitotic maturation of RBCs. We report on the Eurythron meeting in Rome, in which novel approaches in stem cell and erythroid cell biology, including in vitro expansion of primary cells, biochemistry of receptor/signal transduction complexes and transcription factors, and (epi)genetics, were discussed.

Apoptotic mechanisms in the control of erythropoiesis.

Erythropoiesis is a complex multistep process encompassing the differentiation of hemopoietic stem cells to mature erythrocytes. The steps involved in this complex differentiation process are numerous and involve first the differentiation to early erythoid progenitors (burst-forming units-erythroid, BFU-E), then to late erythroid progenitors (colony-forming units-erythroid) and finally to morphologically recognizable erythroid precursors. A key event of late stages of erythropoiesis is nuclear condensation, followed by extrusion of the nucleus to produce enucleated reticulocytes and finally mature erythrocytes. During the differentiation process, the cells became progressively sensitive to erythropoietin that controls both the survival and proliferation of erythroid cells. A normal homeostasis of the erythropoietic system requires an appropriate balance between the rate of erythroid cell production and red blood cell destruction. Growing evidences outlined in the present review indicate that apoptotic mechanism play a relevant role in the control of erythropoiesis under physiologic and pathologic conditions. Withdrawal of erythropoietin or stimulation of death receptors such as Fas or TRAIL-Rs leads to activation of a subset of caspase-3, -7 and -8, which then cleave the transcription factors GATA-1 and TAL-1 and trigger apoptosis. In addition, there is evidence that a number of caspases are physiologically activated during erythroid differentiation and are functionally required for erythroid maturation. Several caspase substrates are cleaved in differentiating cells, including the protein acinus whose activation by cleavage is required for chromatin condensation. The studies on normal erythropoiesis have clearly indicated that immature erythroid precursors are sensitive to apoptotic triggering mediated by activation of the intrinsic and extrinsic apoptotic pathways. These apoptotic mechanisms are frequently exacerbated in some pathologic conditions, associated with the development of anemia (ie, thalassemias, multiple myeloma, myelodysplasia, aplastic anemia). The considerable progress in our understanding of the apoptotic mechanisms underlying normal and pathologic erythropoiesis may offer the way to improve the treatment of several pathologic conditions associated with the development of anemia.

Progression through key stages of haemopoiesis is dependent on distinct threshold levels of c-Myb.

The c-Myb transcription factor is expressed in immature haemopoietic cells and at key stages during differentiation. Loss of the c-myb gene results in embryonic lethality because mature blood cells fail to develop, although commitment to definitive haemopoiesis occurs. We have generated a knockdown allele of c-myb, expressing low levels of the protein, which has enabled us to investigate further the involvement of c-Myb in haemopoiesis. Low levels of c-Myb are sufficient to allow progenitor expansion but, importantly, we show that progression of progenitors towards terminal differentiation is significantly altered. Suboptimal levels of c-Myb favour differentiation of macrophage and megakaryocytes, while higher levels seem to be particularly important in the control of erythropoiesis and lymphopoiesis. We provide evidence that the transition from the CFU-E to erythroblasts is critically dependent on c-Myb levels. During thymopoiesis, c-Myb appears to regulate immature cell numbers and differentiation prior to expression of CD4 and CD8. Overall, our results point to a complex involvement of c-Myb in the regulation of proliferation and commitment within the haemopoietic hierarchy.

The v-erbA oncogene blocks expression of alpha2/beta1 integrin a normal inhibitor of erythroid progenitor proliferation.

T2EC are chicken erythrocytic progenitors that balance between self-renewal and differentiation as a function of response to specific growth factors. Their transformation by the v-erbA oncogene locks them into the self-renewal program. We show here that the expression of the VLA-2 integrin alpha2 subunit mRNA is downregulated by v-erbA and that VLA-2 engagement and clustering, brought about by treatment with an alpha2-specific antibody or by culture on the VLA-2 ligand collagen I, inhibits T2EC proliferation. From competition studies using antibodies, VLA-2 was shown to be involved in the collagen-induced response. While engagement of VLA-2 inhibited proliferation, it was not sufficient to induce differentiation. The transformation of T2EC by v-erbA decreased their interaction with collagen I and the VLA-2 brake on cell proliferation, which may account for the increased proliferation potential of transformed erythrocytic progenitors and for their shedding into the blood of infected chickens. Our data suggest that the interaction between erythroid progenitors and collagen, mediated by VLA-2, play a major role in the control of erythropoiesis in vitro and that this pathway is a target of the v-erbA oncogene.

Erythropoietin receptor expression in human melanoma cells.

Erythropoietin is well known for its role in the control of erythropoiesis, where it acts by binding to its cognate receptor (EpoR) on the surface of erythroid progenitor cells. Here we present the novel finding that the EpoR is also expressed in cells of the melanocytic lineage. It is expressed in transformed cell lines established from normal melanocytes and also in established human melanoma cell lines derived from melanoma metastases, but not in normal primary human melanocytes. The analysis of individual subclones isolated from spontaneously transformed melanocytes revealed that approximately 50% of all the clones examined expressed the EpoR. Further analysis of the individual growth characteristics of EpoR-positive and EpoR-negative clones indicated that, under standard cell culture conditions, expression of the receptor did not affect cell growth. Expression of this receptor is consequently most likely driven by an event that is associated with, but not absolutely required for, the transformed phenotype. While the definite function of this receptor in melanoma cells is still unknown and additional studies are required, our findings support the hypothesis that the EpoR may serve as a progression marker for human melanoma. This observation might be useful in the early diagnosis of melanoma.

The Roles of Bcl-XL and Apopain in the Control of Erythropoiesis by Erythropoietin

Erythropoietin (EP) is required by late-stage erythroid progenitor cells to prevent apoptosis. Several lines of evidence suggest that it is this action of EP that regulates erythrocyte production in vivo. To study the control of apoptosis in mouse and human erythroblasts, the expression of members of the Bcl-2 family of proteins and the expression and activation of the apoptosis-linked cysteine protease Yama/CPP32/apopain were examined. These proteins have been implicated as regulators of apoptosis in several cell models. The Bcl-2 family members analyzed were Bcl-2, Bcl-X, Bax, Bad, Bak, A1, and Mcl-1. Bcl-X expression in proerythroblasts was highly EP-dependent. Bcl-X was strongly increased during the terminal differentiation stages of human and mouse erythroblasts, reaching maximum transcript and protein levels at the time of maximum hemoglobin synthesis. This increase in Bcl-X expression led to an apparent level of approximately 50 times the level in proerythroblasts. In contrast, neither mouse nor human erythroblasts expressed Bcl-2 transcript or protein. Bax and Bad proteins remained relatively constant throughout differentiation, but diminished near the time of enucleation. Bak protein was present in early erythroblasts, but diminished progressively during differentiation. EP deprivation in both mouse and human erythroblasts led to activation of the cysteine protease, apopain, as was indicated by cleavage of the proenzyme into its proteolytically active fragments. Apopain activation was detectable within 2 hours of EP deprivation in mouse erythroblasts. These findings suggest an important role for Bcl-X in late erythroid differentiation and for apopain in apoptosis of erythroblasts caused by deprivation of EP.

The Roles of Bcl-XL and Apopain in the Control of Erythropoiesis by Erythropoietin

AbstractErythropoietin (EP) is required by late-stage erythroid progenitor cells to prevent apoptosis. Several lines of evidence suggest that it is this action of EP that regulates erythrocyte production in vivo. To study the control of apoptosis in mouse and human erythroblasts, the expression of members of the Bcl-2 family of proteins and the expression and activation of the apoptosis-linked cysteine protease Yama/CPP32/apopain were examined. These proteins have been implicated as regulators of apoptosis in several cell models. The Bcl-2 family members analyzed were Bcl-2, Bcl-X, Bax, Bad, Bak, A1, and Mcl-1. Bcl-X expression in proerythroblasts was highly EP-dependent. Bcl-X was strongly increased during the terminal differentiation stages of human and mouse erythroblasts, reaching maximum transcript and protein levels at the time of maximum hemoglobin synthesis. This increase in Bcl-X expression led to an apparent level of approximately 50 times the level in proerythroblasts. In contrast, neither mouse nor human erythroblasts expressed Bcl-2 transcript or protein. Bax and Bad proteins remained relatively constant throughout differentiation, but diminished near the time of enucleation. Bak protein was present in early erythroblasts, but diminished progressively during differentiation. EP deprivation in both mouse and human erythroblasts led to activation of the cysteine protease, apopain, as was indicated by cleavage of the proenzyme into its proteolytically active fragments. Apopain activation was detectable within 2 hours of EP deprivation in mouse erythroblasts. These findings suggest an important role for Bcl-X in late erythroid differentiation and for apopain in apoptosis of erythroblasts caused by deprivation of EP.