Page 371 - Hematology_ Basic Principles and Practice ( PDFDrive )

Chapter 26 Biology of Erythropoiesis, Erythroid Differentiation, and Maturation 307

involvement of PLCs in erythroid differentiation was suggested by the erythroid cell, such as expression of erythroid-specific transcrip-
early studies demonstrating that stimulation of EPOR in primary tion factors.
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erythroid cells results in increased calcium ion flux. More recent Activation of EPOR in the murine IL-3–dependent cell line Ba/
studies demonstrated that primary erythroblasts express only some F3 results in induction of both mitogenesis and globin accumula-
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(i.e., PLC β 1, β 2, β 3, δ 1, γ 1, and γ 2) PLC isoforms. Among these, tion. In contrast, the murine IL-2–dependent cell line CTLL-2,
PLCβ 1 most likely is involved in EPO signaling, based on findings when engineered to express the heterologous EPOR, grows in EPO
that its expression is induced within 6 hours of stimulation with but does not differentiate into globin-bearing cells. These data suggest
the growth factor. 26,199,269 On the other hand, PKC represents a that expression of EPOR is necessary for erythroid differentiation
family of nine different serine-threonine kinases genes, encoding a but not sufficient alone. Other erythroid-specific markers, such as
total of 12 different isoforms, involved in the regulation of many GATA1 and NF-E2, or EKLF, are likely required for cells to dif-
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cellular functions. These enzymes exert their biologic functions ferentiate down the erythroid pathway. Other cytokine receptors,
as a cytoplasmic-nuclear shuttle of the transduction machinery and such as IL-3R and IL-2R, do not drive β-globin synthesis in these
become phosphorylated, and hence activated, in response to a variety cell lines. Taken together, these results suggest that EPOR generates
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of stimuli. Human multipotent CD34 progenitor cells express all a differentiation-specific signaling within the context of a proper
of the PKC isoforms. 271,272 Commitment of these cells along the transcriptional environment.
erythroid lineage requires suppression of PKCε. 271,273 PKCε exerts a Regardless of the mechanism of cytokine specificity, each cytokine
positive control on erythropoiesis, because its inhibitors specifically receptor activates a similar but not identical pattern of signaling
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impair the ability of erythroid cells to respond to EPO and to events. For instance, EPOR shows a preferential activation of the
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phosphorylate EPOR, STAT5, GAB1, ERK1/2, and AKT. It also is JAK2/STAT5 pathway in cultured erythroid cells in vitro. In con-
possible that different PKC isoforms are active at different ontogenic trast, IL-2R shows preferential activation of the JAK1/JAK3/STAT6
stages, because PKCα and PKCδ are differentially phosphorylated, pathway. 264,290,291 Interestingly, although EPO activates STAT5a and
and hence activated, during differentiation of neonatal and adult STAT5b in cultured cells, knockout of the STAT5a or STAT5b gene
erythroblasts. 276 by homologous recombination results in a mouse phenotype with
EPO signaling activates also Lyn, a tyrosine kinase member of the slightly impaired stem cell activity but apparently normal baseline
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Src family physically associated with EPOR. Lyn acts upstream erythroid development. 292,293 More extensive analysis of this pheno-
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to both the STAT5 and the PLCγ2/PI3K pathways. Failure to type has revealed that the mice experience increased apoptotic rates
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activate Lyn prevents erythroid differentiation of the J2E cell line at erythroblast levels that are compensated by a cellular compensatory
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and Lyn mice have a phenotype remarkably similar to that of mechanism very similar to that described for GATA1 LOW mutants,
GATA1 LOW mice (normal hematocrit in spite of reduced levels of involving expansion of hemopoietic progenitors in the marrow and
GATA1, erythroid Krüppel-like factor [EKLF], and STAT5 expres- recruitment of the spleen as an additional hemopoietic site. These
sion because of development of extramedullary hematopoiesis in results suggest that, in vivo, other STAT proteins are at least partially
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spleen). In addition to STAT5 and PLCγ2/PI3K signaling, Lyn capable of substituting for STAT5 and functioning downstream of
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activates Liar, a Lyn-binding nuclear/cytoplasmic shuttling protein the EPOR. These findings emphasize the importance of in vivo
specifically responsible for downregulating KIT expression in response studies in confirming the phenotypic relevance of in vitro studies.
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to EPO. In humans, Lyn is responsible for the phosphorylation Studies have suggested that EPO functions synergistically with
of several membrane proteins, and failure to activate Lyn results other multilineage growth factors, such as KL and IL-3. EPO and KL
in the formation of acanthocytic red cells, a diagnostic marker of function together, resulting in increased erythroid colony cell growth
chorea-acanthocytosis, a rare autosomal recessive neurodegenerative in methylcellulose culture. Studies with the EPOR polypeptide
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disorder. 283 suggest a molecular mechanism for such synergy. Activation of the
Erythroblasts generated under conditions of stress retain C-KIT KIT receptor by KL results in transphosphorylation of EPOR at the
expression. Several studies have investigated the relationship between cell surface. A direct interaction between EPOR and the KIT receptor
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KIT signaling and erythroid cell fate. In human and murine erythroid has been demonstrated. Physical interaction between EPOR and
progenitors, KL induces rapid (within 15 minutes) ERK activation, the β common chain of the IL-3 receptor in erythroid cells has been
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which lasts only 1 hour. 284,285 In human erythroleukemic K562 and demonstrated. This interaction might be involved in the neuropro-
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myeloid MO7e cells, the rapid KL-dependent ERK activation is tective action exerted by EPO. In fact, a carbamylated derivative
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associated with proliferation, whereas the late sustained ERK activa- of EPO prevents motoneuron degeneration in vitro and in vivo
tion is responsible for differentiation. 286,287 Whether KL activates and ameliorates recovery in several in vivo models of brain and heart
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the STAT5 pathway in erythroid cells is controversial. Although injuries, such as chronic autoimmunoencephalomyelitis in mice,
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KL was found to be unable to activate STAT5 in prospectively radiosurgery- or ischemia-induced brain injury, and myocardium
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isolated human erythroid progenitor cells, more recent single-cells ischemia-reperfusion injury in rats. (For a review of the nonhema-
fluorescence-activated cell sorter (FACS) analyses indicate that KL topoietic activity of EPO, see reference 301.) Taken together, these
activates STAT5 in bipotent erythroid/megakaryocytic but not in results suggest that receptor cross-talk at the cell surface may account,
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myelomonocytic progenitor cells. KL has also been described to at least in part, for the physiologic interaction of some cytokines in
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activate the PI3K/AKT pathway in murine erythroid progenitors controlling hematopoietic versus nonhematopoietic effects of EPO.
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and in human MO7e cells. Finally, coexpression of KIT and EPOR
deletion mutants in 32D cells have identified that KIT intracellular
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tyrosines play an essential role in EPOR cosignaling, providing a ALTERATIONS IN EPOR AND ITS SIGNALING IN
mechanism for the signaling synergy observed between KL and EPO. DISORDERS OF ERYTHROPOIESIS
A critical question in the field of EPOR signal transduction is the
mechanism of EPO specificity. Most, if not all, of the signal transduc- As discussed earlier, the normal role of EPO is to stimulate cell surface
tion pathways activated by EPOR (i.e., Ras/Raf/MAPK and JAK/ EPOR in developing erythroid cells. The latter cells respond to EPO
STAT) are shared by other hematopoietic cytokine receptors, such via a proliferative and differentiation response. EPO-activated signal
as the receptors for IL-3, GM-CSF, and IL-5. How EPOR triggers a transduction of EPOR is quickly downregulated in the cell, and
specific growth factor response resulting in erythroid differentiation is continuing presence of EPO is required for optimal differentiation.
unclear. Several models are possible. First, EPOR may activate unique In some cells, the EPOR may become constitutively activated.
but unknown signaling pathways specific to EPOR and distinct from In these cases, erythroid progenitor cells are placed into a sustained
other cytokine receptors. Alternatively, EPOR may activate identical proliferative state. Interestingly, these mechanisms underlie several
pathways, activated by other cytokine receptors. In the latter model, murine and human examples of erythrocytosis (erythroid overpro-
the specificity of the EPO signal is derived not from EPOR itself but duction). Multiple mechanisms exist by which EPOR may become
from interactions with other developmentally programmed events in constitutively activated. First, the Friend spleen focus-forming virus