The physiologically obligatory growth factor for erythroid development is EPO, a 35-kDa glycoprotein produced mainly by the peri tubular cells of the kidney in response to the oxygen need sensed by a heme-containing protein. Through the interaction of EPO with receptor-bearing cells within the bone marrow, physiologic oxygen demands are translated into increased red cell production. Thus, EPO is a true hormone, manufactured at one anatomic site and transported through the bloodstream to the site of activity.

According to the prevailing model of hematopoiesis, progenitor cells committed to erythroid differentiation are generated in a stochastic fashion from pluripotent stem cells.59 According to this model, neither EPO nor other lineage-restricted regulators play any role in determining lineage commitment. This view is supported by the observation that BFU-E and CFU-E can be generated in vitro and in vivo, in the absence of EPO or EPOR (in EPO or EPOR^null mice). At the progenitor level, EPO influences erythroid differentiation by rescuing (from apoptosis) cells that express its receptor, EPOR, and amplifying them further. In addition to the permissive role of EPO ascribed by the stochastic theory, experiments in vivo, in anemic states, or after pharmacologic doses of EPO suggest that high levels of EPO hasten the transition from BFU-E to hemoglobin-synthesizing cells by decreasing either the number of divisions required for this transition or the resting periods between cell divisions.

Autoradiographic studies of purified BFU-E populations indicate that EPORs are detectable only in a subset of BFU-E and that their number increases as BFU-E mature to CFU-E, with the highest level observed at the CFU-E/proerythroblast boundary. That the transition from BFU-E to CFU-E occurs under the influence of EPO suggests ligand (EPO)-induced receptor upregulation. Whether the magnitude of such upregulation is dependent on EPO dose and whether it can modulate the rate of entry of these cells into the maturing compartment is unclear.

For CFU-E, EPO seems to affect their survival and not their cycling status since CFU-E are irrevocably lost after one cycle of DNA synthesis if EPO is not present. It also stimulates all the biochemical processes required to initiate the terminal maturation process (i.e., heme synthesis, globin synthesis, and synthesis of cytoskeletal proteins).

EPORs decrease progressively (from approximately 1000 to <300 receptors per cell) as proerythroblasts mature, and they are undetectable at the reticulocyte level. Through these receptors, EPO exerts its proliferative influence on proerythroblasts and basophilic erythroblasts, but maturation beyond these stages can proceed in the absence of EPO and is instead regulated by thyroid hormone. The reduced number of EPO receptors observed in the most mature erythroid cells is probably instrumental to facilitate enucleation, a process that would be inhibited by the oxidative stress induced by EPO in these cells.

The exquisite role of EPO in determining red cell numbers in the circulation has been clearly established by direct correlations between hematocrit and EPO plasma concentrations in individuals exposed to hypoxia and in patients with compromised kidney functions. However, the variabilities around the mean of hematocrit and EPO plasma levels found in normal individuals under steady-state conditions are not correlated, indicating that other factors (sex and age) cooperate with EPO in determining the fluctuations in red cell mass under steady-state hematopoiesis.

The EPOR polypeptide is a 66-kDa membrane protein that is a member of the cytokine receptor superfamily which includes the receptors for IL-3, GM-CSF, and IL-5. Like other members of this superfamily, EPOR contains four conserved cysteine residues and a WSXWS motif in the extracellular region. The extracytoplasmic region of EPOR contains the EPO binding activity of the receptor. The cytoplasmic region of EPOR does not contain a tyro sine kinase catalytic domain; instead, it interacts with cytoplasmic tyrosine kinases. Cross-linking of radiolabeled EPO to cell surface EPOR results in formation of at least two major cross-linked protein complexes of 140 and 120 kDa. The molecular composition of these complexes remains unsolved but suggests that EPOR contains additional subunits or accessory proteins, one of which is represented by second gene for TfR (TfR2), a potential therapeutic target for β-thalassemia.

The dimeric structure of the activated receptor has been confirmed by crystallization of the extracytoplasmic region of EPOR. These crystallization studies have also allowed the identification of small synthetic peptides capable of inducing EPOR dimerization, suggesting a profitable avenue to design EPO-mimetic and EPO-antagonist drugs possibly more effective than the native protein, especially with regard to the activity of the growth factor in nonhematopoietic tis sues. In addition to EPO mimetics, erythroid stimulating agents under development are represented by modulators of HIF-1α expression, IgA2, an immunoglobin that selectively increases under conditions of anemic stress and TGF-β superfamily ligand traps.

EPOR mRNA, originally isolated from murine and human erythroid cell lines, has been found in nonerythroid cells as well. EPO promotes the differentiation of megakaryocytes and chemotaxis of endothelial cells, at physiologic concentrations of hormone, suggesting the presence of functional cell surface EPORs in these cells. In addition, cell surface EPOR, detected by radiolabeled EPO cross linking, has been found in rat and mouse placenta and in neural and smooth muscle cells. The presence of EPOR in the placenta may be related to the fact that this organ is an erythropoietic site in first trimester fetuses. Adverse effects in cancer patients treated with EPO have been attributed to the effects of EPO on tumor cells.

The existence of naturally occurring splice variants of the EPOR gene encoding EPOR polypeptides of variable length and activity has been shown. The soluble secreted form of EPOR binds EPO and thereby competes with the cell surface receptor isoform. The biologic function of these alternative forms, including a truncated form of EPOR found in early progenitors, remains unknown but may be related either to differential EPO signaling and responses (survival, proliferation, differentiation, and epigenomic modeling) at different stages in erythroid development or to the establishment of erythroid-specific versus myeloid-specific niches in the marrow microenvironment.

Considerable progress has been made in our understanding of EPOR-mediated signal transduction. As mentioned above, EPO induces homodimerization of EPOR and phosphorylation of its first signaling element, Janus-activated kinase 2 (JAK2), a tyrosine kinase constitutively docked at the membrane proximal domain of the receptor required for its appropriate Golgi processing and cell sur face expression. The essential role played by JAK2 in delivering EPO signaling has been demonstrated by the observation that JAK2^null mice die at an early embryonic stage. Phosphorylation activates the catalytic domain of JAK2 that next phosphorylates several tyrosine residues of the cytoplasmic tail of EPOR downstream to its docking site. Once phosphorylated, these tyrosine residues serve as docking sites for other cytoplasmic effector proteins containing Src homology 2 (SH2) domains, such as the p85 subunit of phosphatidylinositol 3-kinase,72 the adaptor protein Shc, and the signal transducer and activator of transcription 5 (STAT5). Once docked on EPOR, these proteins become tyrosine phosphorylated and engage other downstream signaling events. In addition, JAK2 directly activates the Ras/Raf/MAPK (mitogen-activated protein kinase) pathway, further contributing to the EPO-induced mitogenic signal. The most carboxyterminal tyrosine is a negative regulatory domain that recruits the phosphatase SHP1. Once activated, SHP1 rapidly dephosphorylate JAK2, downregulating its activity, and EPOR, setting the system to rest. Failure to recruit the SHP1 phosphatase results in increased EPOR signaling and polycythemia.

Activation of the JAK2/STAT5 signaling has been studied in considerable detail. Upon EPOR tyrosine phosphorylation, STAT5 binds to a specific phosphorylated tyrosine residue of EPOR. Following EPOR binding, STAT5 itself becomes phosphorylated at amino acid Y694. Activated STAT5 then disengages from EPOR, under goes homodimerization, and translocates to the cell nucleus, where it activates transcription of EPO-inducible genes. Some EPO-inducible genes, such as MYC and FOS, are common to other hematopoietic growth factor signaling pathways. Other EPO-inducible genes are specifically expressed in erythroid cells and are not shared by other growth factor responses.

Other signal transduction pathways downstream from cytokine receptors have been identified. For instance, EPO (and IL-3) activates the signaling protein CBL and the subsequent binding and activation of CrkL. Inositide-specific phospholipases C (PLCs) and the protein kinase C (PKC) pathways also are involved in EPO signaling. PLCs catalyze hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol 3,4,5-bisphosphate, a well-known intracellular messenger for PKC activation and intracellular Ca2+ mobilization. In fact, the involvement of PLCs in erythroid differentiation was suggested for the first time by studies demonstrating that stimulation of EPOR in primary erythroid cells results in increased calcium ion flux. PLCs are classified into four families (α, β, γ, and δ), each one with multiple isoforms. More recent studies demonstrated that primary erythroblasts express only some (i.e., PLC β1 , β2 , β 3 , δ1 , γ1 , and γ2 ) PLC isoforms. Among these, PLCβ1 most likely is involved in EPO signaling since its expression is induced within 6 hours of stimulation with the growth factor.

PKC represents a family of nine different serine-threonine kinases genes, encoding a total of 12 different isoforms, involved in the regulation of many cellular functions. These enzymes exert their biologic functions as a cytoplasmic-nuclear shuttle of the transduction machinery and become phosphorylated, and hence activated, in response to a variety of stimuli. Human CD34+ cells express all of the PKC isoforms. Commitment of these cells along the erythroid lineage is associated with suppression of some isoforms. Of those, PKCε exerts positive control on erythropoiesis, because its inhibitors specifically impair the ability of erythroid cells to respond to EPO. It is also possible that different PKC isoforms are active at different ontogenic stages, because differentiation of neonatal or adult erythroblasts is associated with activation of PKCα or PKCδ, respectively.

EPO signaling also activates Lyn, a tyrosine kinase of the Src family physically associated with EPOR that acts upstream to both STAT5 and PLCγ2/PI3K.75 Failure to activate Lyn prevents erythroid differentiation of the J2E cell line and Lyn^null mice have a phenotype remarkably similar to that of mice hypomorphic for GATA1 (normal hematocrit thanks to extramedullary spleen hematopoiesis). Lyn also activates Liar, a Lyn-binding nuclear/cytoplasmic shuttling protein specifically responsible for downregulating KIT expression in response to EPO. In humans, Lyn is responsible for the phosphorylation of several membrane proteins, and failure to activate Lyn results in the formation of acanthocytic red cells, a diagnostic marker of chorea-acanthocytosis, a rare autosomal recessive neurodegenerative disorder.

The development of robust methods for unbiased phosphopro teomic investigations has recently permitted the identification of as many as 22 novel transducers of EPOR signaling in human erythroid cells, including thioredoxin-interacting protein (TXNIP) and protein tyrosine phosphatase non-receptor type 18 (PTPN18).

A critical question in the field of EPOR signaling is the mechanism of erythroid specificity since most, if not all, of the signaling pathways activated by EPOR are also activated by other hematopoietic cytokine receptors. Several models are possible. First, EPOR may activate unique but unknown signaling pathways specific to EPOR and dis tinct from other cytokine receptors. Alternatively, EPOR may activate pathways in common with other cytokine receptors but the specificity of the signal is provided not by EPOR itself but from interactions with other developmentally programmed events, such as expression of erythroid-specific transcription factors. Experiments exist in support of the latter hypothesis. Activation of EPOR in the murine IL-3–dependent cell line Ba/F3 induce both mitogenesis and globin accumulation while activation of other cytokine receptors, such as IL-3R and IL-2R, drive proliferation but not β-globin synthesis. By contrast, the murine IL-2–dependent cell line CTLL-2 engineered to express EPOR grows in EPO but does not differentiate into globin bearing cells. Therefore, EPOR may be necessary for erythroid differentiation but not sufficient alone. Other erythroid-specific markers, such as the transcription factors GATA1 or EKLF, are likely required for cells to differentiate down the erythroid pathway. Taken together, these results suggest that EPOR generates a differentiation-specific signaling within the context of a proper transcriptional environment.

The tyrosine residues involved in EPOR signaling were identified by structure-function studies in cell lines expressing mutant forms of the receptor. Expression of the full-length, wild-type EPOR poly peptide in these cells resulted in EPO-dependent growth and partial EPO-induced erythroid differentiation. By contrast, expression of its truncated forms resulted in variable growth responses. For instance, truncation of the membrane proximal region of EPOR demonstrated a critical positive regulatory domain of EPOR required for mitogenesis. Furthermore, truncation of the carboxy-terminal (C-terminal) 40 amino acids of EPOR resulted in increased EPO-dependent growth, providing indication that the C-terminal region contain the negative regulatory domain required for down-modulating EPOR signals.

The detailed analysis of the EPOR receptor domain and chromosomal mapping of EPOR (to chromosome 9 in mice and chromo some 19p in men) led to the identification of EPOR mutation in the rare congenital disease familial erythrocytosis. Familial erythrocytosis is a heterogeneous group of hereditary conditions characterized by erythrocytosis in the setting of low serum EPO levels. A few families that demonstrate autosomal dominant inheritance have been identified. The linkage between the EPOR gene and familial erythrocytosis was first established by the observation that a mutant EPOR allele segregates with the disease in one familial erythrocytosis kindred. This allele contains a nonsense mutation in the coding region of the gene that results in synthesis of a truncated EPOR that lacks the negative regulatory domain of its C-terminal region. Since that report, several other frameshift and deletion EPOR gene mutations, all encoding C-terminal–truncated forms of the protein pro viding EPO hypersensitivity and resulting in familial erythrocytosis, have been reported.

Congenital polycythemia may arise not only from gene mutations leading to abnormal EPOR signaling but also from gene mutations altering EPO production. The T598T mutation in one of the genes controlling oxygen sensing (von Hippel-Lindau [VHL] gene), which leads to increased EPO production by the kidney, is associated with congenital erythrocytosis, the Chuvash polycythemia. The C598T VHL mutation is endemic in Chuvashia, Russian Federation, and in the small island of Ischia, in southern Italy.

Polycythemia may also arise from somatic mutations leading to constitutive EPOR signaling. Several investigators have reported a mutation in the JAK2 gene resulting in a valine→phenylalanine substitution at position 617 of the protein (JAK2^V617F mutation) in patients with Ph-negative myeloproliferative disorders.

Studies have suggested that EPO functions synergistically with other multilineage growth factors, such as KL and IL-3, produced by the microenvironment. EPO and KL function together, resulting in increased erythroid colony cell growth in methylcellulose culture. Studies with the EPOR polypeptide suggest a molecular mechanism for such synergy. In fact, in erythroid cells EPOR and KIT are physically associated and activation of KIT by KL results in transphosphorylation of EPOR at the cell surface. Physical interaction between EPOR and the β common chain of the IL-3 receptor has been demonstrated and may be involved in the neuroprotective action exerted by EPO. In fact, a carbamylated derivative of EPO prevents motoneuron degeneration in vitro and in vivo and promotes recovery in several in vivo models of brain and heart injuries, such as chronic autoimmune encephalomyelitis in mice, radiosurgery- or ischemia-induced brain injury, and myocardium ischemia-reperfusion injury in rats. (For a review of the nonhematopoietic activity of EPO, see reference Papayannopoulou and Migliaccio). Taken together, these results suggest that receptor cross-talk at the cell sur face may account, at least in part, for the physiologic interaction of some cytokines in controlling hematopoietic versus non-hematopoietic effects of EPO.

Proliferation and/or survival of early erythroid progenitors in vitro are dependent on the presence of numerous cytokines, elaborated by stromal and accessory cells within the microenvironment. Among the cytokines, KL, which is produced by stromal cells, and IL-3, which is produced by a subset of T cells, alone and in synergy, promote proliferation of BFU-E and its progeny. Other cytokines, such as GM-CSF, IL-11, and TPO, stimulate a subset of BFU-E. In addition to proliferation, cytokines also control BFU-E survival since most of them die when cultured for more than a few days in the absence of cytokines. Cytokines exert their effects through interaction with specific receptors present on the BFU-E surface. These receptors are also expressed by the leukemic counterparts of normal BFU-E and by leukemic cell lines. Additional important positive effects on erythropoiesis are exerted by vitamins (such as vitamin A and D) and hormones, such as insulin growth factor 1 (IGF1), which facilitate the cell entry into the S phase of the cycle. A polymorphism at the IGF1 locus is associated with anemia in men. In addition, targeted ablation and naturally existing mutations have disclosed the importance, even if indirect, played by the vascular endothelial growth factor (VEGF)/ Flk-1 receptor in the control of erythropoiesis, since Flk-1 deletion affects both endothelial and hematopoietic development through its presumed presence in the hemangioblast, the common endothelial/ hematopoietic stem cell.

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