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Review

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Vol.15 No.3 March 2005

Turning cells red: signal transduction mediated by erythropoietin


Terri D. Richmond1,3, Manprit Chohan1,3 and Dwayne L. Barber1,2,3
Department of Medical Biophysics, University of Toronto, Toronto, Ontario, M5G 2M9, Canada Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, M5G 2M9, Canada 3 Division of Cellular and Molecular Biology, Ontario Cancer Institute, University Health Network, Toronto, Ontario, M5G 2M9, Canada
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Erythropoietin (EPO) is the crucial cytokine regulator of red blood-cell production. Since the discovery of EPO in 1985 and the isolation of its cognate receptor four years later, there has been signicant interest in understanding the unique ability of this ligandreceptor pair to promote erythroid mitogenesis, survival and differentiation. The development of knockout mice has elucidated the precise role of the ligand, receptor and downstream players in murine erythroid development. In this review, we summarize EPO-mediated signaling pathways and examine their signicance in vivo.

the plethora of signaling proteins that are recruited to and/or activated downstream of the EPO-R. The signicance of these proteins in mediating erythropoiesis is known because of the availability of knockout mice. In this review, we discuss EPO-mediated signaling pathways with an emphasis on recent developments that exploit knockout mice to delineate crucial steps in erythroid maturation. It updates our knowledge

Hematopoietic stem cell

Introduction The identication of erythropoietin (EPO) dramatically improved the quality of life of patients suffering from chronic kidney disease and EPO is the worlds leading biopharmaceutical [1]. EPO is the crucial regulator of red blood-cell production and delivers essential growth, differentiation and survival signals to erythroid progenitors in the foetal liver, bone marrow and spleen (Figure 1; Box 1). The biosynthesis of EPO is regulated by oxygen tension and many details of this cascade have been elucidated recently (reviewed in [2]). The EPO receptor (EPO-R), which was described initially in 1989 [3], is a member of the cytokine receptor superfamily. The EPO-R is expressed in bone marrowderived erythroid progenitors and several non-hematopoietic tissues including myocytes, cortical neurons, and prostatic, breast and ovarian epithelia. The EPO-R is believed to exist in an unliganded, dimeric state. On binding EPO, it undergoes a conformational change that activates the pre-bound, cytoplasmic, tyrosine kinase, Janus kinase 2 (JAK2) [4]. In turn, JAK2 (and, potentially, other tyrosine kinases) phosphorylate several cytoplasmic tyrosine residues in the cytoplasmic tail of the EPO-R that act as docking sites for proteins that contain Src-homology 2 (SH2) domains. EPO activates tyrosine phosphorylation of the SH2 domain-containing transcription factors, signal transducer and activator of transcription 1 (STAT1), STAT3 and STAT5a/b. The number of citations that involve the EPO-R is O1000 and we know much about
Corresponding author: Barber, D.L. (dbarber@uhnres.utoronto.ca).

Myeloid stem cell

CFU-GEMM

BFU-E

CFU-E

Proerythroblast

Erythroblast

Reticulocyte

Erythrocyte
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Figure 1. Erythropoiesis is the process by which multipotential hematopoietic stem cells differentiate into mature, non-nucleated erythrocytes. The requirement for erythropoietin starts between the BFU-E and CFU-E stages as the cell begins to express the erythropoietin receptor, and diminishes with erythroblast differentiation. Erythropoietin dependence is represented by red italics. Abbreviation: CFU-GEMM, colony forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte.

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Box 1. Erythropoiesis
Erythropoiesis is the process by which multipotential hematopoietic stem cells differentiate into mature, non-nucleated erythrocytes (Figure 1). EPO responsiveness correlates with the expression of the EPO-R, which begins at the earliest progenitor stage, the burst forming unit-erythroid (BFU-E). However, dependence on EPO is not observed until the colony forming unit-erythroid (CFU-E) stage because mice that are decient in either EPO or the EPO-R can generate committed erythroid BFU-E and CFU-E progenitors [13]. BFU-E and CFU-E are observed in vitro using clonogenic assays. These assays require progenitor cells to be isolated and plated in a semi-solid media containing factors that promote erythroid growth and differentiation. BFU-E and CFU-E are enumerated 79 days and 2 days after plating, respectively. The normal distribution of murine erythroid progenitors is 90% in the bone marrow and 10% in the spleen. However, the spleen can expand up to 10-fold during times of erythropoietic stress. Murine models allow us to observe gene-specic phenotypes under different conditions: erythroid precursors in foetal liver describe embryonic erythropoiesis, bone marrow and splenic precursors illustrate adult erythropoiesis, and the spleen can expand to show signaling under conditions of erythroid stress (induced by anaemia). Hidden phenotypes might only be seen during erythroid stress. Human erythropoiesis occurs solely in the bone marrow. The difference between mouse and human erythropoiesis indicates that data from mouse models should be extrapolated with caution and it is crucial that investigators analyze all facets of murine erythropoiesis. The comprehensive examination of existing studies and scrutiny of future developments promise the establishment of a denitive picture of the pathways required for EPO-dependent erythroid development.

about the relevant players in mouse erythroid development in vivo and is relevant for those interested in hematopoiesis, cytokine biology and signal transduction. Earlier work is discussed in several excellent reviews [57]. Mechanism of activation of the EPO-R In this section, we discuss the domain architecture and regulation of the murine EPO-R, which is a membrane protein with 507 amino-acids and a single transmembrane domain (Figure 2). The extracellular domain of the EPO-R has two bronectin type II domains. Crystallographic studies demonstrate that the EPO-R extracellular domain exists as a dimer in the absence of EPO. A high-afnity receptor is generated when one EPO molecule binds to a preformed EPO-R dimer. Binding of EPO (or other
WSAWS LILTLSLILVLISLLLTVLALLS

D1

D2

TM

IWPGIPSP BOX1

PAHLEVLS EPR BOX2


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Figure 2. Domain structure of the erythropoietin receptor (EPO-R). The extracellular domain consists of a bronectin type III repeat. It can be divided into two subdomains, termed D1 (amino acids 11-113) and D2 (amino acids 118218) as illustrated. Each subdomain expresses two cysteine residues that form an intradomain disulde bond. Residues 208212 form the WS!WS box. The sequence of the transmembrane domain (TM) includes L240 and L241 (pink), which are essential for activation. The juxtamembrane sequence includes the Box 1 (amino acids 257264) and Box 2 (amino acids 302312) regions. Several residues (L253, I257, W258 and D287) are important for EPO-dependent activation of JAK2. I257 and W258 in the Box 1 region are indicated in red.
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small-molecule mimetics) generates conformational changes in the D1 and D2 domains, which brings together receptor dimer and JAK2 that are associated with the intracellular domains, thereby activating the enzymes (reviewed in [8]). Attempts to crystallize the full-length EPO-R have been unsuccessful. However, several elegant mutagenesis studies have identied residues and motifs in the transmembrane domain and juxtamembrane regions that are essential for EPO-R function. Two leucine residues at positions 240 and 241 in the transmembrane domain are crucial for receptor dimerization and downstream signaling [9]. Hematopoietic cells transfected with EPO-R that is mutated at these two sites have impaired EPO-dependent growth and differentiation into erythroid cells. It is signicant that substitution of only one of the leucine residues with alanine impairs neither EPO-R dimerization nor downstream signaling, illustrating the importance of the dual leucine motif in EPO-R activation. The EPO-R transmembrane domain also contains regions that are important in its orientation, activation and regulation of downstream signal transducers. To identify these regions, Constantinescu and colleagues [10] generated a series of mutants in which they fused the EPO-R transmembrane domain to a dimerization domain. Two active fusions, which have similar transmembrane interfaces and orientation, confer growth and differentiation through tyrosine phosphorylation of JAK2 and STAT5. This illustrates the specicity of the transmembrane domain sequence and orientation in activating downstream signaling pathways. The juxtamembrane region between the transmembrane domain and Box 1 region is conformationally rigid and contains a highly conserved hydrophobic motif that is dependent on its a-helical orientation rather than distance from the membrane bilayer. This helical model has been tested by introducing sequential alanine residues into the EPO-R starting at Y252 [11]. Introduction of either one or two alanines destroys the register of the a-helical domain and reduces EPO-R activation. However, the addition of three alanines (i.e. one helical turn) restores EPO-dependent growth and tyrosine phosphorylation. These results indicate that signals might be transmitted through the transmembrane a-helix to the cytosolic juxtamembrane domain and, furthermore, that the transmembrane domain is crucial in maintaining the orientation between the extracellular and cytosolic domains. The cytoplasmic tail of the EPO-R contains two regions that are required for JAK2 activation [12]. Mutation of the Box 1 region and amino acids extending to Q284 allow association between EPO-R and JAK2, but the receptor fails to trafc to the membrane. Mutations of the hydrophobic motif between the transmembrane domain and Box 1 (L253A, I257A, W258A and D287A) permit membrane localization, but fail to activate JAK2 on EPO binding. These mutants might cause a crucial conformational change in EPO-R. Gene targeting reveals non-redundant roles for EPO, EPO-R and JAK2 in erythropoiesis The physiological role of EPO [13] and EPO-R [1315] in erythropoiesis has been determined, in part, using

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knockout mice. Mice with null mutations in genes encoding either EPO or EPO-R die during embryogenesis at embryonic day E12.5E13.5, a stage of massive erythroid expansion. However, intact colony forming unit-erythroid (CFU-E) cells are obtained, indicating that EPO signaling is dispensable at earlier stages of hematopoiesis including the burst forming unit-erythroid (BFU-E) (Figure 1). JAK2-knockout mice have a phenotype that is almost identical, with embryonic lethality observed one day earlier, probably because of activation of JAK2 by other cytokines including thrombopoietin, interleukin 3, granulocyte colony stimulating factor and interferon g [16,17]. These studies illustrate that there is a linear pathway from a single ligand (EPO) to a single receptor (EPO-R) and downstream tyrosine kinase (JAK2). Several laboratories have focussed on understanding the SH2 effectors recruited to the EPO-R (Table 1). Early studies indicated that Y343 of the EPO-R is crucial for downstream signaling. This hypothesis has been addressed by creating knock-in alleles of two truncated EPO-R constructs. One strain, EPO-R H, contains a truncation of the distal half of the cytoplasmic domain (which lacks the distal 108 amino acids) whereas the second strain, EPO-R HM, expresses the identical truncation plus the Y343F mutation [18]. Both strains are viable, fertile and have normal growth and hematopoietic parameters, which indicates that the distal region and the cytoplasmic tyrosines are not essential for viability in mice. Erythroid progenitor cells from EPO-R HM mice have delayed erythroid differentiation and enhanced apoptosis at low concentrations of EPO, which indicates that compensatory mechanisms associated with the wild-type EPO-R are compromised in these animals [19]. No studies have compared signaling in primary erythroblasts from wild-type, EPO-R H and EPO-R HM mice. Recent reports have suggested new roles for EPO in non-hematopoietic tissues including brain, heart and muscle. For example, EPO-R-knockout mice have reduced numbers of neural progenitor cells and enhanced sensitivity to hypoxia [20]. Cardiomyocytes from EPO-Rknockout mice do not respond to EPO stimulation and analysis reveals signicant myocardial hypoplasia in a non-autonomous manner [21]. Embryonic lethality in EPO-R-knockout mice can be corrected by introducing an EPO-R transgene that rescues expression exclusively in the hematopoietic lineage using a GATA-binding protein 1 promoter [22]. Despite the lack of EPO-R expression in non-hematopoietic tissues, these mice are
Table 1. Association between EPO-R and SH2-domain-containing proteins

normal, which indicates that lethality in EPO-R-knockout mice can be attributed solely to the hematopoietic function of EPO. Efforts to understand murine erythropoiesis in vivo have been hampered by the embryonic lethality of EPO, EPO-R and JAK2 knockouts. Two recent developments provide signicant advances in this area. Klingmuller and colleagues have developed a Cre transgenic-mouse model to promote gene inactivation exclusively in the erythroid lineage. The GFP-Cre knock-in mouse line is engineered to express GFP-Cre under the control of the endogenous EPO-R promoter [23]. Mice expressing this construct express GFP efciently in the foetal liver, bone marrow and spleen. These animals, used in conjunction with conditional-knockout mice, will facilitate accurate analysis of gene-specic, erythroid phenotypes. In addition to EPO, EPO-R and JAK2, a similar approach could be taken with STAT3, SH2-domain protein tyrosine phosphatase 2 (Shp2) and Suppressor of cytokine signaling-3 (SOCS-3) because knockout of the genes encoding these proteins is also embryonic lethal. Another challenge in analysing erythroid phenotypes in vivo is generating sufcient cells for signal transduction experiments. Beug and colleagues maintain long-term culture of erythroid progenitors in a model system that mimics stress erythropoiesis, dened as erythropoiesis induced by anaemia. Erythroid progenitors are maintained in an undifferentiated state in media containing EPO, stem cell factor (SCF) and the synthetic glucocorticoid dexamethasone (Dex) [24]. Erythroid differentiation is induced by removing SCF and Dex. This methodology can be adapted to embryonic stem cells to yield a population of adult erythroid progenitors [25]. The role of specic proteins in erythrogenesis might be studied more easily using this in vitro assay and embryonic stem cells with targeted, null mutations in genes that encode these proteins. EPO-dependent tyrosine kinase activation: JAK2 and friends Higher mammals express four JAKs. EPO stimulation leads to robust tyrosine phosphorylation of JAK2, which is time and dose dependent [4]. All JAK kinases have seven unique domains, termed JAK-homology (JH) domains (Figure 3). A catalytically active tyrosine kinase domain (JH1) and adjacent pseudokinase domain (JH2) are located at the C terminus. Although the pseudokinase domain has the architecture of a tyrosine kinase domain, key catalytic residues are not conserved and it might have a negative regulatory role [26]. Domains JH3JH7 are
Y1007 Y1008 JH2 JH1
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Y221

EPO-R tyrosine Y343 Y401 Y429 Y431 Y443 Y460 Y464 Y479

SH2 associated proteins STAT5 STAT5, SHIP-1, Shp2, Cis, SOCS-3 Shp1 Shp1, STAT3 None identied Couples to EPO-dependent TRPC2 activation Grb2 p85 a subunit of PI-3 kinase

Refs [5] [5,41,42,64,65,66] [74], [74,34] [58,59] [40] [5]

JH7

JH6 FERM domain

JH4

Figure 3. Domain architecture of JAK2. The seven JH domains are indicated. The JH1 domain is the functional kinase domain and the JH2 domain is the pseudokinase or kinase-like domain. The positions of tyrosine residues (Y221, Y570, Y1007 and Y1008) that are involved in regulation of catalysis are shown. The position of the FERM domain is indicated by a red line.

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required for binding to the EPO-R and appropriate Golgi processing [12]. It is hypothesized that the band 4.1, ezrin, radixin, moesin-homology (FERM) domain (a 300-aminoacid domain in JH4JH7) is responsible for Golgi trafcking the EPO-RJAK2 complex [12]. Determining the crystal structure of JAK2 including the pseudokinase and kinase domains would be a signicant advance. Several phosphotyrosine residues in JAK2 have important roles in its enzymatic regulation and substrate phosphorylation. All JAK kinases contain a di-tyrosine motif in the activation loop. An early report demonstrated that Y1007 is required for tyrosine kinase activity [27]. Two recent studies illustrate that phosphorylation of Y221 regulates JAK2 positively, whereas phosphorylation of Y570 decreases JAK2 kinase activity [28,29]. These data indicate that JAK2 preferentially phosphorylates substrates with the Yxx[L/I/V] motif. It is signicant that four of the eight tyrosine residues in the cytoplasmic region of the EPO-R express this motif and it is also conserved in several other downstream substrates that are recruited to the EPO-R following activation (Table 2). However, not all substrates of JAK2, including STAT5a/b, contain this consensus motif. In addition, it is unclear whether JAK2 and other tyrosine kinases, including Lck/Yes-related novel tyrosine kinase (Lyn) and Brutons tyrosine kinase (Btk), are responsible for phosphorylation of Y460, Y464 and Y479 of the EPO-R. Other substrates, known to be tyrosine phosphorylated in an EPO-dependent manner, including SH2-domain inositol phosphatase 1 (SHIP-1), Grb2-associated binder 1 (Gab2), Shp2 and Shc, contain the consensus JAK2 phosphorylation motif (Table 2). JAK2 plays a vital role in mediating signals downstream of EPO. However, we know little of the proteins
Table 2. Putative JAK2 phosphorylation substrates
Tyrosine Surrounding sequence SH2 and PTB-associated proteins

that interact with JAK2. Elucidation of bona de substrates of JAK2 and their biochemical regulation is a priority, as is determining the precise role of the JH2 domain. Lyn Lyn and other Src family tyrosine kinases are substrates of EPO signaling. Evidence of their involvement has emerged following the isolation of a human erythroid cell line, J2E-NR, which is Lyn-decient and has impaired erythroid differentiation [30]. The J2E-NR cell line has decreased concentrations of the erythroid transcription factors GATA-1 and erythroid kruppel-like factor (EKLF), and STAT5a [31]. Similarly, erythroblasts from Lyndecient mice do not express GATA-1 and EKLF, and expression of STAT5a and STAT5b are decreased [31]. Extramedullary hematopoiesis, which is dened as hematopoietic development outside the bone marrow, occurs in Lyn-decient mice because there is a dramatic increase in BFU-E and CFU-E in the spleen. In addition, these mice respond more quickly to anaemic stress, develop anaemia with aging and have defective clearance of apoptotic erythroid cells. Foetal erythropoiesis was not examined in this study [31]. These data demonstrate that other tyrosine kinases play an important role in erythropoiesis downstream of JAK2. Btk Several reports have examined the activation of Tec family tyrosine kinases, including Btk, following EPO-receptor activation. Although Btk-knockout mice have no overt defect in erythropoiesis, evidence indicates that Btk is crucial for EPO-mediated signaling. Erythroblasts isolated from Btk-decient mice have enhanced erythroid differentiation, even when cultured in media that supports self-renewal rather than differentiation [32]. Tyrosine phosphorylation of STAT5 is delayed following EPO stimulation of primary erythroblasts isolated from Btk-knockout mice. In addition, tyrosine phosphorylation of the EPO-R and JAK2 is diminished in Btk-null erythroid lines, and tyrosine phosphorylation of the Btk target, phospholipase Cg1, is not observed. Reconstitution experiments in COS cells indicate that activation of either Jak or Lyn is upstream of Btk, which has led to the suggestion that Btk is downstream of JAK2 and that it might regulate the recruitment of signaling proteins with pleckstrin homology domains. Activation of EPO and STAT Seven members of the STAT family are expressed in mammals. These proteins are SH2-domain-containing transcription factors that play an integral role in cytokine signaling. Studies utilizing cells in culture show that EPO activates STAT1, STAT3 and STAT5a/b (Figure 4) [57]. Analysis of erythropoiesis is complete in STAT1-decient and STAT5a/b-knockout mice. Studies of cell lines illustrate that EPO-dependent activation of STAT1 is dependent on JAK2 but does not require cytoplasmic tyrosines of the EPO-R. STAT1decient mice show a shift in erythropoiesis from the bone marrow to the spleen, which often indicates erythroid stress [33]. STAT1-decient erythroblasts

Tyrosine phosphorylation sites on JAK2a Y221 AKIQDY(P)HILTRKRI Y570 REVGDY(P)GQLHKTEV Y1007 PQDKEY(P)YKVKEPGE Y1008 QDKEYY(P)KVKEPGES Tyrosine phosphorylation sites on the EPO-Rb Y343 HAQDTY(P)LVLDKWLL STAT5 Y401 AASFEY(P)TILDPSSQ STAT5, SHIP-1, Shp2, CIS, SOCS-3 Y429 PPHLKY(P)LYLVVSDS Shp1 Y431 HLKYLY(P)LVVSDSGI Shp1 Tyrosine phosphorylation sites on substrates that are phosphorylated after EPO stimulationc SHC Y313 FDDPSY(P)VNIQNLDK Grb2 Shp2 Y546 RKGHEY(P)TNIKYSLV Grb2 Y580 DSARVY(P)ENVGLMQQ Grb2 Gab2 Y193 SAPQEY(P)LYLHQCIS Shp2 SHIP-1 Y1021 FENPLY(P)GSVSSFPK DOK1
Adapted from [28]. The EPO-R cytoplasmic tyrosines that match the consensus Y(P)xx[L/I/V] phosphorylation motif preferred by JAK2 are indicated and the SH2 interacting proteins at each site. c Phosphoproteins known to be tyrosine phosphorylated by EPO are shown that express the consensus Y(P)xx[L/I/V] phosphorylation motif. These sites are phosphorylated in vivo. The SH2 and PTB domain-associated proteins are illustrated.
b a

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EPO

EPO-R Plasma membrane P JAK2 STAT1 Y343 Y401 STAT5 STAT1 P STAT1 STAT5 P STAT5 P P Y443 Y460 Y464 Y479 P STAT3 JAK2 Y343 Y401 Y429 Y431 STAT3 P

STAT3 P

STAT5a/b-decient mice with a RAG genetic background (devoid of B and T cells) do not show this phenotype [38]. These studies used targeting vectors that delete exons 1 and 2 of STAT5a and exon 1 of STAT5b, which raises the possibility that these are hypomorphic alleles rather than complete knockouts. A recent report demonstrates that conditional inactivation of the entire STAT5a/b locus generates mice with perinatal lethality. The hematocrit of E18.5 embryos and neonatal STAT5a/b-null mice is lowered signicantly [39]. Detailed analysis of foetal erythropoiesis has not been performed in these animals, but it is unlikely that STAT5a/b plays an essential role in embryogenesis because embryos survive to birth. Although we know much about the roles of STAT5a and STAT5b in vivo, thorough analysis of STAT5a/b-null mice will help resolve the ongoing controversy about the role of these transcription factors in the regulation of erythropoiesis. Probing other pathways EPO activates Erk1/2, SAP kinase/Jun kinase and p38 EPO activates the catalytic activity of several mitogenactivated protein kinases, including extracellular-regulated kinases 1/2 (Erk1/2), SAP kinase (SAPK)/Jun kinase (Jnk) and p38. Erk1/2 is thought to participate in mitogenesis, whereas the roles of SAPK/Jnk and p38 are more complicated. Many growth factors and cytokines activate Ras by recruiting Grb2-Sos to their receptors. There is signicant redundancy in how the Grb2-Sos exchange complex is recruited to the EPO-R. For example, Grb2 can bind directly to Y464 of the EPO-R [40], and can associate indirectly through binding to either SHIP-1 [41] or Shp2 [40,42]. When Ras is activated EPO stimulates the canonical kinase cascade, culminating in activation of Erk1/2 [43]. SAPK/Jnk and p38 were described originally as integrators of stress responses. Several studies have investigated the activation of SAPK/Jnk and p38 by the EPO-R. Conicting data concerning this pathway have been published, caused largely by the use of different experimental models. Many cytokines, including EPO, cause a dosedependent activation of SAPK/Jnk and p38 in several murine cell lines [5,43,44]. SAPK/Jnk requires a distal region of the EPO-R for activation, whereas p38 and Erk1/2 are downstream of a proximal region of the EPO-R that lacks cytoplasmic tyrosine residues [43]. Expression of the isoforms of p38 is regulated differentially in primary human erythroid cells. p38a is expressed at high levels throughout erythroid differentiation, whereas p38d is upregulated during late erythroid differentiation in human primary erythroid cells [45]. Interestingly, p38a-decient mice have a fatal, foetal anaemia because this isoform increases the stability of mRNA that encodes EPO [46]. EPO stimulates cell survival by activating phosphatidylinositol 3 kinase EPO stimulation activates the phosphatidylinositol 3 kinase (PI 3-kinase) signaling cascade directly by recruiting the p85 regulatory subunit directly to EPO-R Y479, and indirectly through binding to Casitas B lymphoma (Cbl), Gab1, Gab2, insulin-receptor substrate 2 (IRS-2)

Nuclear membrane

GAS

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Figure 4. A schematic representation on the JAKSTAT signal transduction pathways that are initiated on EPO binding and activation of the erythropoietin receptor (EPO-R). The positions of the eight cytoplasmic tyrosine (Y) residues in the EPO-R are indicated. STAT5 associates with Y343 and Y401 (red) and STAT3 binds to Y431 (green). STAT1 is activated downstream of JAK2, but does not require tyrosine phosphorylation of the EPO-R. STAT1STAT3 heterodimers can form but are not shown. STAT dimers shuttle to the nucleus and activate promoters that contain GAS sequence elements. Circled P represents a phosphorylation event.

also have elevated phosphorylation of several substrates of the EPO-R. Activation of STAT3 is uncommon in cell lines and primary cells following stimulation with EPO. However, EPO causes tyrosine phosphorylation of STAT3 in a human cell line, which requires Y432 of the human EPO-R (equivalent to Y431 in murine EPO-R) [34]. Although detailed analysis of STAT3-null mice is not possible, the availability of conditionally targeted STAT3 mice will allow the role of STAT3 to be evaluated in erythroid development [35]. STAT5a/b is activated by several growth factors and cytokines. Consequently, there has been signicant interest in characterizing STAT5a/b double-knockout mice. STAT5a/b-null mice have defects in lactation, growth and peripheral T-cell function that are attributed to aberrant prolactin, growth hormone and interleukin 2 signaling, respectively [36]. Although that initial analysis of these mice indicated no hematopoietic abnormalities, subsequent analyses revealed an underlying, non-fatal, foetal anaemia [18,37]. The spleens of STAT5a/b-null mice enlarge with age, but this partially penetrant phenotype might be caused by secondary cytokine secretion because
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EPO

p27Kip1 [49]. Elucidating the relevant downstream substrates of PKB/Akt will increase insight into the signicance of this pathway in erythroid homeostasis. Cell survival mediated through the Bcl family Proteins of the B cell lymphoma 2 (Bcl2) family are key effectors of survival signals mediated by growth factor receptors. Several studies have shown that Bcl2 and B cell lymphoma-XL (Bcl-XL) protect erythroid cell lines and primary erythroid cells from apoptosis. Bcl2-knockout mice have no overt erythroid abnormalities, probably because Bcl2 is not expressed in erythroid progenitors. However, Bcl-XL-knockout mice display embryonic lethality at E12.5 because of brain defects and severe anaemia [53], and disruption of Bcl-XL in the haematopoietic lineage results in severe haemolytic anaemia [54]. Although both models generate normal numbers of BFU-E, CFU-E and erythroblasts, defective maturation leads to anaemia. Exogenous expression of human Bcl-XL results in EPO-independent differentiation of wild-type murine erythroblasts [55], which indicates that expression of Bcl-XL alone can drive erythroid differentiation. EPO stimulates the synthesis of Bcl-XL in a JAK2dependent manner [56], but whether it is a direct transcriptional target of STAT5a/b is controversial [37,56,57]. Further experiments are required to determine whether Bcl-XL is the crucial downstream target of JAK2 in erythroid signal-transduction pathways. EPO and Ca2C Unlike antigen receptors, cytokine receptors do not have a rapid, phospholipase Cg-mediated Ca2C ux on receptor engagement. However, Ca2C does play a role in erythroid development: Ca2C inux is required for murine erythroleukemia cell differentiation and intracellular Ca2C increases in human BFU-E culture (reviewed in [7]). Utilizing a series of deletion and tyrosine mutants of the EPO-R, Miller and colleagues have demonstrated that Y460 of the EPO-R is necessary and sufcient for EPOdependent Ca2C ux [58]. Screening murine cell lines and splenic erythroblasts reveals that two members of the transient receptor potential channel (TRPC) family, TRPC2 and TRPC6, are expressed highly in erythroid cells [59]. Coexpression of TRPC2 and EPO-R in CHO cells results in a dramatic rise in intracellular Ca2C in response to EPO, whereas TRPC6 inhibits this response [60]. The function of the vomeronasal organ is defective in TRPC2decient mice [61,62], but erythropoiesis has not been examined. It will be interesting to determine whether loss of TRPC2 has adverse effects on erythroid homeostasis. Turning signals off Although the signals that regulate mitogenesis are well understood, less is known about the mechanisms of negative regulation. This discussion is limited to SOCS, tyrosine phosphatases and inositol phosphatases because these are the best-studied negative regulators (Figure 6). SOCS The SOCS family of proteins, which was discovered in 1997, plays a role in negative regulation of cytokine signaling. The

EPO-R Plasma membrane P JAK2 P JAK2 Y343 Y401 Y429 Y431 Y443 Y460 Y464 Y479 p85 p110 ? Foxo3a ? P P PKB/ AKT P P 3 PI PKB/ AKT PDK1 P

p27kip1 BTG1 Proliferation

Differentiation
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Figure 5. A schematic representation of the phosphoinositide 3-kinase signal transduction pathways initiated on EPO binding and activation of the erythropoietin receptor (EPO-R). The p85 a subunit of PI 3-kinase either binds directly to Y479 (blue) of the EPO-R or is recruited indirectly to the EPO-R via CbI, Gab1, Gab2, Irs-2 and Vav (not shown). Activation of PI 3-kinase results in phosphorylation of PKB/Akt. One of the substrates of PKB/Akt is the Foxo3a transcription factor that regulates the expression of p27kip1 and BTG1, and phosphorylation of Foxo3a reduces its transcriptional activity. Circled P represents a phosphorylation event 125.

and Vav (Figure 5) (reviewed in [5]). Studies in an apoptosis-resistant erythroid cell line indicate that activation of PI 3-kinase is necessary but not sufcient to protect against apoptosis [47]. Mice that do not express the p85 a subunit of PI 3-kinase have markedly reduced erythropoiesis with reductions in the number of BFU-E and CFU-E precursors [48]. Protein kinase B (PKB)/Akt is activated downstream of PI 3-kinase, and many downstream substrates of PKB/Akt have been proposed, including the forkhead box O3A (Foxo3a) transcription factor, which is important in erythropoiesis [49,50]. The transcriptional activity of Foxo3a is inhibited by phosphorylation. Foxo3a-decient mice are anaemic because of enhanced numbers of reticulocytes, which indicates that this transcription factor has a role in regulating erythroid differentiation [51]. A recent report demonstrates that the concentration of Foxo3a increases during erythroid differentiation and used microarray strategies to identify B cell translocation gene 1 (BTG1) as one of several, novel, Foxo3a targets [52]. BTG1 is one of six genes whose transcription is regulated by Foxo3a and have putative anti-proliferative functions. Another important Foxo3a target is the p27Kip1 cyclindependent kinase inhibitor, and activating PI 3-kinase in primary human erythroblasts decreases the expression of
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eight family members have a unique N-terminal region, a central SH2 domain and a C-terminal SOCS box. SOCS proteins are transcriptionally regulated immediate early genes that feedback on EPO-signaling pathways. Induction of cytokine-inducible SH2 domain (Cis), SOCS-1 and SOCS-3 mRNA transcripts is observed in cell lines and primary cells that have been stimulated with EPO [63]. Cis [64] and SOCS-3 [65,66] bind to Y401 of the EPO-R, thereby competing for binding of STAT5a/b, Shp2 and SHIP-1. SOCS proteins also play a role in ubiquitination of their bound substrates. SOCS-1 binds to the activation loop of JAK2 [67], which recruits ubiquitination machinery and targets of JAK2 for degradation [67]. The concentration of SOCS mRNA varies during erythroid differentiation. Specically, Cis mRNA decreases, and SOCS-1 and SOCS-3 mRNA increases within 48 h of exposure to EPO [68]. Analysis of transgenic and knockout mice indicates that SOCS proteins are not vital to the regulation of erythropoiesis. Cis-null mice have no detectable phenotype [69] whereas SOCS-1-knockout mice have a low hematocrit and delayed erythroid differentiation [70]. CFU-E in SOCS-1-decient foetal liver are hyper-responsive to EPO [68]. SOCS-3-null mice display increased erythroid progenitors during embryogenesis [71], but this

is a secondary defect caused by defective signaling by leukemia inhibitory factor [72]. The availability of a conditionally targeted SOCS-3 knockout will allow analysis in the erythroid lineage. Tyrosine phosphatases Four tyrosine phosphatases are involved in regulating erythropoiesis. The SH2-domain containing phosphatases, Shp1 and Shp2, cluster designation 45 (CD45) and protein tyrosine phosphatase 1B (PTP-1B) all modulate activity of JAK2 kinase (Figure 6). The motheaten (me) mouse has germline mutations in Shp1 (reviewed in [73]). Erythroid progenitors from mev/mev mice are either hypersensitive to EPO or show EPO independent-growth. Importantly, Shp1 associates with Y429 and Y431 of the EPO-R [74]. The Shp2 tyrosine phosphatase is a crucial regulator of signaling downstream of several growth factor and cytokine receptors. Shp2 is recruited to Y401of the EPO-R [42] and several studies have shown that Shp2 is a robust substrate for tyrosine phosphorylation by EPO-R, which leads to the recruitment of Grb2-Sos [40,42]. Shp2 is essential for gastrulation during murine embryogenesis [75]. Although analysis of Shp2-decient embryonic stem cells reveals decreased formation of erythroid

EPO

EPO-R Plasma membrane P SOCS 1/3 STAT P Y401 CIS/ SOCS3 Y429 Y431 Y443 Y460 Y464 Y479 STAT P STAT CIS STAT P STAT Nuclear membrane P SOCS1 P Y401 Y429 Y431 Y443 Y460 Y464 Y479 JAK2 Y343 JAK2 Y343 ? ? SHP-1 PTB-1B PtdIns(3,4)P2 PtdIns(3,4,5)P2 PtdIns(4,5)P2 P PI3K SHIP1 CD45

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Figure 6. Negative regulation of erythropoietin receptor (EPO-R) signaling. JAK2 is regulated negatively by PTP-1B and CD45 tyrosine phosphatases. Shp1 tyrosine phosphatase binds to Y429 and Y431 of the EPO-R and, potentially, negatively regulates JAK2. EPO-dependent activation of STATs leads to the expression of the genes encoding CIS and SOCS1. CIS and SOCS3 compete for STAT5 binding at Y401, whereas SOCS1 and SOCS3 bind to the activation loop of JAK2. For simplicity, STAT represents STAT1, STAT3 and STAT5 proteins. Regulation of phosphoinositide metabolism by PI 3-kinase and SHIP1 is also indicated. Circled P represents a phosphorylation event.
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progenitors [76], a denitive analysis of Shp2 function in erythropoiesis awaits conditional-targeting strategies. Impaired maturation of B and T cells was described originally in CD45-knockout mice and further analysis revealed that CD45 is a JAK phosphatase [77]. Importantly, the numbers of bone marrow-derived BFU-E increase in CD45-knockout mice, but the numbers of CFU-E progenitors in bone marrow, and splenic BFU-E and CFU-E progenitors have not been examined. These results are surprising given the lymphoid phenotype of CD45-null mice, but underscore the importance of CD45 in regulating antigen and cytokine-receptor signaling. Elucidation of the relevant substrates of tyrosine phosphatases has been enhanced by the generation of mutant phosphatases with precise mutations in catalytic residues. These substrate-trapping mutants are resistant to hydrolysis and allow identication of relevant tyrosine phosphatase substrates. Study of the related tyrosine phosphatases, PTP-1B and T cell tyrosine phosphatase (TC-PTP) reveals that these enzymes trap substrates with a di-tyrosine motif. PTP-1B targets Tyk2 and Jak2 [78], whereas the related TC-PTP binds JAK1 and JAK3 [79]. These data indicate that this clade of tyrosine phosphatases has unique reactivity towards JAK kinase dephosphorylation. Inositol phosphatases Another manner of desensitization involves the regulation of metabolic intermediates of inositol. Phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] activates many crucial signaling events. Several enzymes dephosphorylate PtdIns(3,4,5)P3, including phosphatase and tensin homolog (which dephosphorylate the 3 0 phosphate) and SH2-inositol phosphatase, SHIP-1 and SHIP-2 (which dephosphorylate the 5 0 phosphate). SHIP-1 is recruited to Y401 of the EPO-R [41] and SHIP-1 null mice show elevated formation of BFU-E and CFU-E in bone marrow [80]. SHIP-1 negatively regulates B cell, macrophage and mast cell signal transduction pathways (reviewed in [81]). Further analysis of the erythroid lineage of SHIP-1decient mice is needed to determine whether loss of SHIP-1 affects Erk1/2 and PKB/Akt activation, as postulated in other lineages. Concluding remarks Many studies provide details of the intricacies of signaling events that are activated by EPO. Analysis of erythropoiesis in gene-targeted mice has allowed determination of the signaling events that are required for normal erythropoiesis. Although the importance of EPO, EPO-R and JAK2 is undeniable in foetal erythropoiesis, other signaling proteins might have important roles in signal integration in adults. These underlying effects might only be observed under conditions of erythroid stress. Therefore, investigators must analyze foetal and adult erythropoiesis under normal conditions as well as when exposed to stressinducing agents including EPO and/or phenylhydrazine. In this manner we will obtain a clear picture of pathways required for EPO-dependent erythroid development. It is evident that JAK2 is crucial in fetal erythropoiesis but many aspects of the activation and regulation of JAK2
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remain mysterious, and the crucial downstream substrates remain elusive. With the current ambivalence regarding the importance of STAT5 in erythropoiesis, future studies will address whether other substrates are vital in erythropoiesis. Results from a recent paper that characterizes STAT5a/b-null mice indicate that these transcription factors facilitate neonatal development, but it remains to be investigated whether this involves erythropoiesis. Similarly, analysis of erythropoiesis in a conditional knockout of STAT3 will determine whether STAT3 is crucial to this process. Last, the generation of compound knockout mice, targeted for multiple combinations of STAT proteins, might be the only denitive method to determine which STATs are necessary for facilitating normal erythroid development. Although several tyrosine phosphatases negatively regulate EPO signaling, the regulation of these phosphatases has not been studied in detail. For example, how do these enzymes access their substrates? How is PTP-1B recruited to its substrate? What are the kinetics of dephosphorylation? How are Shp1, Shp2, CD45 and PTP-1B regulated in primary erythroid cells? Moreover, the signaling studies described in this review focus on events that are downstream of the EPO-R in isolation. However, the EPO-R associates with other membrane receptors including the interleukin 3 bc chain, c-kit and stem-cell tyrosine kinase. The nature and stoichiometry of these complexes is unknown and the downstream signaling effects should be examined. The EPO-R couples to the TRPC2 Ca2C channel. The mechanism by which this occurs remains to be elucidated. Several studies have examined EPO-regulated genes using subtraction hybridization and microarray experiments. In addition, some reports have also examined gene regulation during erythroid differentiation. The results from these investigations are not included here because of space restraints, but no clear trends have emerged, probably because of the unique experimental systems used by each investigator. It is important to determine whether gene regulation is different during foetal and adult erythropoiesis. In addition, whether normal and stress erythropoiesis result in distinct patterns of gene regulation should be evaluated. Ultimately, identication of ligand-induced targets is challenging because of the distal nature of the stimulus. Elucidation of direct transcriptional targets via chromatin immunoprecipitation in conjunction with CpG island microarrays might better identify genes that are required for erythroid development. Nearly 20 years have passed since the discovery of EPO. Extensive research has identied the essential roles of EPO, EPO-R and JAK2 in mediating foetal erythroid development, but much work remains to understand the crucial signals downstream of JAK2 that regulate erythropoiesis. Identifying genetic regulatory events are vital to determine the roles of EPO in haematopoiesis and other tissues, most notably in neuronal survival. Onwards ho!
Acknowledgements
Research in the authors laboratory is supported by Canadian Institutes of Health Research, National Cancer Institute of Canada and Leukemia and Lymphoma Society. DLB is a National Cancer Institute of Canada Research Scientist. We thank Sam Benchimol, Martin Carroll and

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Madeleine Bonnard for helpful comments on the manuscript and apologize to many colleagues whose contributions we were unable to list because of reference citation limitations.

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