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484 Part VI: The Erythrocyte Chapter 32: Erythropoiesis 485

screen for GATA-1 interacting proteins. It binds to the amino zinc fin- signaling (via its receptor tyrosine kinases). Gas6 receptors are expressed
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ger of GATA-1. FOG-1−/− mice die during embryonic days 10.5 to 11.5 in hematopoietic tissue, megakaryocytes, myelomonocytic precursors,
from severe anemia with arrest in erythroid maturation at a stage simi- and marrow stromal cells. Gas6 amplifies the erythropoietic response
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lar to that observed in the GATA-1− mice. FOG-1 physically interacts to EPO using a mouse model of Gas6 knockout. Gas6 is known to
with GATA-1 to augment or inhibit its transcriptional activity depend- downregulate the expression of inflammatory cytokines such as tumor
ing on the promoter context. necrosis factor-α by macrophages. 74
GATA-2 was initially cloned as a GATA motif-binding factor is Figure 32-5 outlines the interrogation of the molecular mecha-
present in all erythroid cells; and, targeted deletion of GATA-2 resulted nisms that regulate lineage-specific differentiation and commitment
in embryonic lethality at day 10.5 from ablation of blood cell develop- reveals the existence of separate megakaryocytic/erythroid progenitors
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ment. GATA-1 and GATA-2 directly regulate GATA-2 transcription versus both myeloid and lymphoid lineages. 75
in a reciprocal fashion during erythroid differentiation. 61,62 GATA-2
autoregulates its transcription by binding to its own regulatory elements ERYTHROPOIETIN, OXYGEN SENSING, AND
in the promoter region. This autoregulation is abolished by the displace-
ment of GATA-2 by GATA-1 (GATA-2/GATA-1 switch), an interaction HYPOXIA-INDUCIBLE FACTOR
facilitated by FOG-1. Chromatin immunoprecipitation studies indicate Erythropoietin
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that FOG-1 facilitates occupancy by GATA-1 at selected cis-regulatory The principal hormone regulating erythropoiesis is EPO, which is pro-
chromatin elements. Double knockout of GATA-1 and GATA-2 results duced principally in the kidney. Erythroid progenitors express their
7
in embryonic lethality with complete absence of primitive erythropoie- own EPO. Different levels of kidney-produced EPO are optimal for
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sis. The severity of this phenotype compared to either single GATA-1 various stages of erythroid maturation. Purification of EPO provided
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or GATA-2 knockout suggests overlapping functions of these two tran- a partial protein sequence that led to cloning of the gene and permitted
scription factors in primitive erythropoiesis. mass production of the recombinant protein. EPO and its recombi-
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nant form are heavily glycosylated α-globulins with a molecular mass
Kruppel-like Factor of 34,000 daltons and a specific activity of approximately 200,000 IU/
EKLF is a zinc finger protein identified by subtractive hybridization mg. 79,80 Sixty percent of the molecular weight of the recombinant protein
of the mRNA of erythroid cells with common messages in a myeloid is contributed by amino acids; the remaining 40 percent is composed
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cell line. It interacts with CACCC sequence in the β-globin pro- of carbohydrate. Using molecular probes for EPO, mRNA enabled the
moter, where it modifies chromatin structure permitting β-globin gene localization of the synthesis of EPO to renal cortical interstitial cells 81,82
transcription. EKLF-deficient mice die at embryonic day 14.5 to 15 of endothelial or fibroblastic lineage. The cells appear to function in an
from severe anemia from defective definitive erythropoiesis. There all-or-none fashion, with the overall production of mRNA dependent
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is a marked decrease in β-globin mRNA and protein levels in EKLF- on the number of cells activated. 83
deficient erythroid cells. Large amounts of iron accumulate in the Certain 5′ sequences located 6000 to 12,000 bp upstream also affect
reticuloendothelial system of EKLF-deficient mice, consistent with an EPO gene transcription. These sequences are not hypoxia sensitive but
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ineffective erythropoiesis. appear necessary for tissue and cellular specificity. Hepatic production
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is contributed primarily by hepatocytes but is a much less important
STEM CELL LEUKEMIA/T-CELL ACUTE source than is the kidney. During fetal life, however, hepatic EPO pro-
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LYMPHOBLASTIC LEUKEMIA 1 duction is of major importance for red cell production (Chap. 7). 86,87
EPO production is regulated exclusively at the level of its transcription
SCL/TAL1 is a member of basic helix-loop-helix transcription fac- by hypoxia at the transcription level. The transcriptional activation
tors essential for maturation of the erythroid and megakaryocytic of the EPO gene is controlled by a specific sequence located in the 3′
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lineages. Knockout of SCL/TAL1 leads to failure of hematopoiesis. flanking region termed hypoxia-responsive element. 88–90 The core of the
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Selective rescue of SCL/TAL1 null embryonic stem cells under the con- enhancer is constituted by the sequence CACGTGCT and mutations in
trol of stem cell enhancer revealed differentiation blocks in erythroid this core sequence abolish hypoxia responsiveness.
and megakaryocytic maturation. Conditional knockout studies have EPO is not stored; it is secreted immediately. 81–83 Circulating
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revealed that erythroid and megakaryocytic precursors do not develop recombinant EPO and presumably native EPO have a half-life (T ) of
1/2
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in the marrow of mice upon deletion of SCL/TAL1. Heterodimeriza- 4 to 12 hours, with a volume of distribution slightly larger than that of
tion of SCL with other transcription factors, such as E2A, is a prerequi- the plasma volume. EPO is degraded after it binds to EPOR (see “Ery-
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site for its functions. 71 thropoietin Receptor” below). 92
BCL11A, a transcription factor initially identified in lymphoid cells,
regulates erythroid differentiation, especially in switching from fetal to Erythropoietin Receptor
adult hemoglobin. Fetal hemoglobin (HbF) levels decline after birth and Interaction of EPO with its receptor EPOR results in (1) stimulation
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are then replaced by adult hemoglobin A. The molecular mechanisms of erythroid cell division, (2) erythroid differentiation by induction
responsible for this switch are not completely known. Genome-wide of erythroid-specific protein expression, and (3) prevention of ery-
association findings have provided a major breakthrough in understand- throid progenitor apoptosis. Earlier models of this interaction were
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ing this phenomenon. There is an inverse correlation between BCL11A based on the ligand (EPO)-induced homodimerization of EPOR. In
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and HbF expression in erythroid cells. BCL11A occupies several discrete reality, EPOR is a preformed homodimer that undergoes a major con-
sites in the β-globin gene cluster and likely plays an important role in formational change upon binding, which initiates the EPO-specific
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hemoglobin switching during erythroid differentiation. erythroid signal transduction cascade (Fig. 32–6). The cytoplasmic
portion of EPOR contains a positive regulatory domain that inter-
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GROWTH ARREST-SPECIFIC 6 PROTEIN acts with Janus kinase 2 (JAK2). Immediately after EPO binding,
JAK2 cross-phosphorylates the EPOR itself, and other proteins such
Growth arrest-specific 6 (Gas6) protein is a secreted vitamin K–dependent as STAT5 (signal transducer and activator of transcription 5), thus
protein that interacts with cell membranes and leads to intracellular initiating a cascade of erythroid-specific signaling. JAK2/STAT5
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