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Chapter 26 Biology of Erythropoiesis, Erythroid Differentiation, and Maturation 303

cell, HbF or γ-globin constitutes 14% to 25% of total globin. (2) approximately 1000 to <300 receptors per cell) as proerythroblasts
The number of F cells is genetically determined, and the gene(s) mature, and they are undetectable at the reticulocyte level. 67,173
linked or nonlinked to the β-locus is responsible for F-cell formation. Through these receptors, EPO exerts its proliferative influence on
(3) F cells do not display other features of “fetalness” because proerythroblasts and basophilic erythroblasts, but maturation beyond
their membrane components and enzymes are characteristically these stages can proceed in the absence of EPO.
adult. (4) Synthesis of HbF peaks earlier than that of HbA, so TfRs are found in characteristic abundance in erythroid cells
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the proportion of HbF is higher in immature cells compared with (300,000 to 800,000 TfRs per cell). This composition reflects
mature, fully hemoglobinized cells. (5) F cells and cells that contain not only the proliferative needs of erythroid cells but also their
only HbA are not derived from distinct stem cell populations but extreme requirements for iron uptake for hemoglobin synthesis. For
from a common adult stem cell. Whether the latter will form F or this reason, TfRs persist in maturing nondividing erythroblasts and in
non-F (i.e., A) cells is determined at the BFU-E level and throughout reticulocytes. TfRs belong to a large group of receptors that internal-
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the CFU-E level. In vitro the great majority of BFU-E have the ize their ligand through receptor-mediated endocytosis. This cycle
potential to express HbF, whereas in vivo only a very small proportion allows for reuse both of the ligand (transferrin) for resaturation with
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of red cells contain HbF. This potential appears to be lost during iron and of the receptor for entering another route of endocytosis.
normal cell differentiation and maturation in vivo. This concept The density of TfRs decreases with maturation. After the reticulocyte
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links the potential for HbF expression to the pathway of erythroid stage, receptors appear to be shed as small lipid vesicles. An inverse
differentiation and thus may have implications for interpreting the relationship exists between receptor density and iron availability.
reactivation of HbF that occurs in adults under diverse circumstances Deprivation of iron results in receptor induction, and excess iron
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(e.g., after chemotherapy or with acute bleeding). Many of these results in receptor suppression. However, the mechanisms that
circumstances seem to influence HbF levels by directly or indirectly regulate the number of TfRs throughout the maturation of precursors
modifying the kinetics of the normal differentiation/maturation (even within progenitors) are largely unknown. Erythroid precursor
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process, 157,158 or the result of changes in stress signaling molecules. cells differ from nonerythroid cells not only by requiring a higher
HbF levels in red cells can be increased by exposing the cells during number and higher occupancy of TfRs, but also by displaying immu-
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maturation to chemical inhibitors of HDAC. This class of enzymes nologically distinct receptor isoforms. A second gene for transferrin
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suppresses gene transcription by catalyzing deacetylation of histones, receptor (TfR2) has been identified, and monoclonal antibodies
and consequently inducing chromatin condensation, and of tran- recognizing distinct receptor isoforms are useful in isolating erythroid
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scription factors altering their DNA binding ability. Therefore cells from bone marrow. 12,72 TfR1 and TfR2 are members of a family
they can directly activate transcription of γ-globin genes in vitro and of genes encoding at least seven different homologous proteins in
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in vivo 161–163 and in a number of patients with β-thalassemia. 164,165 primates. TfR1 is a type II membrane glycoprotein that, as a
Recently new insights have been revealed about transcription factors cell surface homodimer, binds iron-loaded transferrin as part of
that are responsible for the physiologic silencing of fetal Hb in adult the process of iron transfer and uptake. In addition to providing
life. Most prominent among these are BCL11A, KLF1, and c-Myb erythroid cells with the much needed iron directly, data in mouse
along with chromatin modifiers. These novel discoveries may be models have recently indicated that expression of TfR1 on erythroid
considered as therapeutic targets in the future. 166 marrow cells may favor iron uptake also indirectly by establishing
Synthesis of globin appears to be coordinated with synthesis of an ERFE-independent feedback mechanism that suppresses hep-
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heme throughout erythroid maturation so that functional hemo- cidin expression in the liver. TfR2 is expressed in two forms,
globin tetramers are formed rapidly and spontaneously after release membrane-bound (TfR2-α) and nonmembrane (TfR2-β), both of
of newly synthesized globins from polysomes. Information about which bind transferrin with low affinity. The specific role of TfR2
the accumulation of heme and its synthetic intermediaries has been in hematopoietic cells is unclear. TfR2 may also play a prominent
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provided by crude biochemical approaches (see Fig. 26.2). However, role in the liver as the key regulator of iron metabolism. In cells
now that the genes for several enzymes in the heme synthetic pathway from 67 patients with de novo acute myeloid leukemia (AML), high
(e.g., δ-5-aminolevulinic acid synthase, porphobilinogen deaminase, levels of TfR2-α expression were correlated with better prognosis,
ferrochelatase) have been cloned, information about their regulation and higher levels of both TfR2-α and TfR2-β were associated with
is rapidly emerging. 73,75 longer survival, suggesting that TfR-independent iron uptake plays
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An important role in coordinating heme and globin chain assem- a role in in vivo proliferation of AML cells. Recent data in TfR2-
bly during hemoglobin production is exerted by α-hemoglobin- deficient mouse models suggest that this receptor may modulate the
stabilizing protein (AHSP). AHSP is a protein abundantly expressed erythroblast sensitivity to EPO. 182
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in erythroid cells whose function is to bind free α-chains, Maintenance of stable extracellular iron concentrations requires
stabilizing their structure and limiting their ability to participate the coordinated regulation of iron transport into plasma from dietary
in chemical reactions that generate ROS. 168,169 In addition, AHSP sources in the duodenum, recycled senescent red cells in macro-
binding increases the affinity of α-chains for β-chains, accelerating phages, and storage in hepatocytes. Diferric transferrin is present
the formation of Hb tetramers. The essential role exerted by this gene in the liver because of complex machinery involving the product
in erythroid development has been demonstrated by the fact that its of the hereditary hemochromatosis (HFE) gene (a protein of the
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deletion in normal mice impairs red cell production. AHSP red major histocompatibility complex class I), TfR2, and the product of
cells have a decreased half-life, contain Hb precipitates, and exhibit the hemojuvelin (HJV) gene (also known as HFE2). Given that the
null
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signs of oxidative damage. The observation that double AHSP levels of TfR2 expression are exclusively regulated by holotransfer-
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β-thalassemic mutant mice have an exacerbated phenotype suggests rin, TfR2 expressed by hepatocytes is likely the first element of
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that AHSP is a gene modifier that, like the hereditary persistence of the iron sensory pathway in the liver. Hepatocytes respond to
HbF mutations, ameliorates the phenotype of thalassemic patients. iron sensoring by modulating hepcidin expression and secretion.
However, the search for AHSP polymorphisms that might correlate Hepcidin, a 25-amino acid disulfide-rich peptide, acts as a systemic
with milder clinical phenotypes in thalassemia has not provided iron regulatory hormone that regulates both dietary iron absorption
consistent results. Gene mapping, direct genomic sequencing, and by the enterocytes and iron recycling by the macrophages. Because
extended haplotype analysis did not reveal any mutation or specific ferroportin shuttles iron from the enterocytes to the macrophages
association between haplotypes of AHSP in 120 β-thalassemic and hepcidin is required for ferroportin internalization and degrada-
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patients. On the other hand, a polymorphism in the putative tion, decreased hepcidin expression blocks iron export in the two
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AHSP promoter leading to a threefold higher expression of the gene cell types. Each gene involved in iron metabolism has a role in
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in reticulocytes has been observed in the normal population, but regulating the expression of the other genes. In particular, reduced
the clinical consequences of this observation are unknown. expression of HEF, TfR2, and HJV reduces expression of hepcidin.
Crucial to the functional response of erythroid precursors is the It is not surprising then that mutations altering the function of all
expression of EPORs and TfRs. EPORs decrease progressively (from of these genes have been found to be associated with hereditary