Trends in Cell Biology
Turning cells red: signal transduction mediated by erythropoietin
Introduction
The identification of erythropoietin (EPO) dramatically improved the quality of life of patients suffering from chronic kidney disease and EPO is the world's 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 marrow-derived 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 >1000 and we know much about the plethora of signaling proteins that are recruited to and/or activated downstream of the EPO-R.
The significance 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 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 5, 6, 7.
Section snippets
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 fibronectin type II domains. Crystallographic studies demonstrate that the EPO-R extracellular domain exists as a dimer in the absence of EPO. A high-affinity receptor is generated when one EPO molecule binds to a preformed EPO-R dimer. Binding of EPO (or other
Gene targeting reveals non-redundant roles for EPO, EPO-R and JAK2 in erythropoiesis
The physiological role of EPO [13] and EPO-R 13, 14, 15 in erythropoiesis has been determined, in part, using knockout mice. Mice with null mutations in genes encoding either EPO or EPO-R die during embryogenesis at embryonic day E12.5–E13.5, 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).
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
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-deficient 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 Lyn-deficient mice do not express GATA-1 and EKLF, and
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-deficient 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
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) 5, 6, 7. Analysis of erythropoiesis is complete in STAT1-deficient 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
EPO activates Erk1/2, SAP kinase/Jun kinase and p38
EPO activates the catalytic activity of several mitogen-activated 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 significant redundancy in how the Grb2-Sos exchange complex is recruited to the EPO-R. For example, Grb2 can
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
EPO and Ca2+
Unlike antigen receptors, cytokine receptors do not have a rapid, phospholipase Cγ-mediated Ca2+ flux on receptor engagement. However, Ca2+ does play a role in erythroid development: Ca2+ influx is required for murine erythroleukemia cell differentiation and intracellular Ca2+ 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 sufficient for
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 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]
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
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′ phosphate) and SH2-inositol phosphatase, SHIP-1 and SHIP-2 (which dephosphorylate the 5′ phosphate). SHIP-1 is recruited to Y401 of the EPO-R [41] and SHIP-1 null mice show
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.
Acknowledgements
Research in the author's 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 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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