Erythropoeisis

The production of erythrocytes is a tightly regulated process. During steady state hematopoiesis,
approximately 1010 red blood cells are produced per hour in the bone marrow to maintain the
hemoglobin level within fairly narrow limits. Production can be rapidly increased in the setting of
ongoing blood loss or hemolysis.

Erythropoiesis begins with the differentiation of multipotent hematopoietic stem cells (HSC) into the
most primitive erythroid progenitors In the classical hematopoietic hierarchy, HSC progeny can
differentiate into a common myeloid progenitor (CMP), which in turn differentiates into the common
megakaryocyte-erythroid progenitor cell (MEP).

Studies in humans and mouse models using transplantation and single-cell lineage tracing have
shown that the MEPs may derive directly from HSC. MEPs give rise to the erythroid progenitor
cells, which subsequently follow a differentiation program that culminates in the emergence of
mature erythrocytes.

The existence of an MEP explains why iron deficiency is associated not only with anemia but
frequently with thrombocytosis as well. Iron metabolism is an evolutionarily highly conserved
process. Data from mouse models show that an iron-deficient diet or knockout of the gene that is
altered in iron-refractory iron deficiency anemia (IRIDA), TMPRSS6, have thrombocytosis [3]. The
mechanism was found to involve increased flux of progenitor cells through MEPs, with evidence
for attenuation of extracellular signal-regulated kinase (ERK) phosphorylation. This mechanism
illustrates an example of extracellular influences on progenitor cell fate.

●This process is driven by successive combinations of transcription factors that dictate the
expression of adhesion and hematopoietic growth factor receptors (HGFRs).
●Adhesion receptors play an important role in the localization and release of maturing cells
from specific niches in bone marrow.
●Hematopoietic growth factors (HGFs), such as interleukin 3 (IL-3), granulocyte-macrophage
colony-stimulating factor (GM-CSF), and Stem Cell Factor (SCF, also called Kit ligand or
Steel factor), are important for the amplification of progenitor cells.

Erythropoietin (EPO) is a growth factor essential for the amplification and terminal differentiation of
erythroid progenitors and precursors. Information concerning the control of EPO expression by
hypoxia has provided new insight into the regulation of erythropoiesis.

ERYTHROID PROGENITOR CELLS

The erythroid progenitor compartment of the bone marrow is invisible upon inspection by light
microscopy. These committed single lineage progenitors are derived from the stochastic
differentiation of bipotential or multipotential progenitors, which emerged from a tiny population of
stem cells . The two sets of erythroid progenitors (BFU-E and CFU-E) described below cannot be
identified by specific morphological features, although they have been purified and analyzed using
flow cytometric methods. Evidence suggests that all hematopoietic progenitors or stem cells
resemble lymphoblasts .

Erythroid burst-forming unit — In humans, the most primitive single lineage committed
erythroid progenitor is the erythroid burst-forming unit (BFU-E). These cellular clusters are so
named because they have the following characteristics in semisolid in vitro cultures:

●The colonies have a burst-like morphology.

●In response to the combination of EPO and one of SCF, IL-3, or GM-CSF, the progeny of
the first few cellular divisions are motile and form subpopulations of erythroid colony-forming
units (CFU-E) . Each of these units subsequently forms a large colony of proerythroblasts,
which mature into erythroblasts and a few enucleated erythrocytes.

The entire process requires approximately two weeks in vitro.

Erythroid colony-forming unit — Bone marrow also contains the more mature erythroid
colony-forming unit (CFU-E) that, under the influence of EPO, form small colonies of erythroblasts
in seven days of culture.
PRECURSORS AND MATURE CELLSThe erythroid precursor or erythroblast pool
represents about one-third of the marrow cell population in the normal child (above the age of
three) and the adult. Proerythroblasts are the earliest recognizable forms . These cells divide and
mature through basophilic, polychromatic, and orthochromatic normoblast cells to form the
reticulocyte and finally to the circulating mature erythrocyte; this process involves a reduction in
cell size, nuclear condensation and extrusion, and hemoglobin accumulation

●On average, each proerythroblast can form approximately eight reticulocytes. The mean
transit time from proerythroblast to the emergence of the reticulocyte into the circulation is
approximately five days .
●In the circulation, the reticulocyte takes approximately one day to become the mature
erythrocyte .
●The lifespan in the circulation of the mature erythrocyte is approximately four months.

Acute anemia — In acute anemia, the length of this transit time may decrease to as little as
one or two days because of skipped divisions . In this setting, the red cells that emerge are
macrocytic; they may also bear surface i antigen and other fetal characteristics, such as
increased fetal hemoglobin (HbF), because they have had insufficient time to have
converted i antigen to I antigen or to have acquired other adult characteristics . The cells
also contain excessive burdens of the debris that normally accumulates during cell
assembly . As a result, stress erythropoiesis is associated with circulating Pappenheimer
bodies (iron granules), basophilic stippling (ribosomes), Heinz bodies (hemoglobin
inclusions), and Howell-Jolly bodies (nuclear remnants)

STROMAThe bone marrow stroma consists of fibroblastoid cells, endothelial cells, and
macrophages that comprise the hematopoietic microenvironment [15,16]. Interaction between
receptors on erythrocyte precursors and elements of the stromal matrix are important determinants
of erythroid maturation. As an example, murine erythroleukemia cells bind specifically to the
extracellular matrix protein fibronectin [17]; induction of differentiation of these cells subsequently
leads to loss of adhesion. Binding to fibronectin involves very late antigen (VLA)-5, an erythroid
integrin that is expressed on erythroid cells and is lost at the reticulocyte stage of development.
VLA-5 binds to a specific peptide in the fibronectin molecule, Arg-Gly-Asp-Ser (RGDS) [18].

Normal BFU-E bind preferentially to a stromal fibroblast cell strain [19]; after binding, these cells
proliferate and differentiate in the presence of EPO alone. This interaction is blocked by antibodies
specific for the cell binding domain of fibronectin, which contains the RGDS sequence [19].

Macrophages — Interactions between developing erythroid cells and macrophages are also
important . This is classically exemplified by the "erythroblastic island", composed of developing
erythroblasts surrounding a central macrophage . These macrophages make physical contacts
with developing erythroblasts, enabling signaling and the transfer of growth factors and nutrients
(eg, iron) to these cells.

Deoxyribonuclease II from these macrophages may be required for the process through which
mature red cells lose their nuclei . In addition, Rac GTPases play an essential role in enucleation
through activation of the formin mDia2, which nucleates unbranched actin filaments leading to
formation of the contractile actin ring.

TRANSCRIPTION FACTORS
Much has been learned about the essential roles of several transcription factors via the study of
their activation in leukemic translocations and the effect of gene disruption in embryonic stem cells
[29]. Important factors that most likely act at the level of hematopoietic stem cells include
TAL1/SCL, LMO2/RBTN2, and GATA2, while GATA1, FOG1 and EKLF are more important for
erythropoiesis.

TAL1/SCL — T-cell acute lymphocytic leukemia 1 (TAL1; also called stem cell leukemia [SCL])
transcription factor is expressed in leukemias (biphenotypic [lymphoid/myeloid] and T
cell) ,primitive hematopoietic progenitors, and more mature erythroid, mast, megakaryocyte, and
endothelial cells. Targeted disruption of this gene in mice leads to death in utero from the absence
of blood formation. This finding, together with lack of in vitro myeloid colony formation, and data
indicating that TAL1 contribute to angiogenesis [36], suggest a very early role for TAL1, most likely
at the pluripotent or myeloid-erythroid stem cell level. Interestingly, conditional gene targeting in
adult mice shows that TAL1 is not required for engraftment and self-renewal of adult stem cells,
but is required for differentiation of erythroid and megakaryocyte lineages. Overexpression of this
factor in erythroid or bipotent cell lines produces an increase in erythroid differentiation, but there
are no data on overexpression in adult hematopoietic stem cells.

LMO2/RBTN2 — Another transcription factor implicated in T cell acute lymphoblastic leukemia is

the LIM-only domain nuclear protein lim domain only 2 (LMO2; also called rhombotin 2 [RBTN2])
[40,41]. Mice that lack this factor die in utero and have the same bloodless phenotype as animals
that lack TAL1 [42]. Although LMO2 does not show sequence specific DNA binding,
immunoprecipitates in erythroid cells reveal that LMO2 exists in a complex with TAL1 [43,44],
thereby suggesting a physiologic interaction in vivo.

GATA2 — GATA2 is expressed in the regions of Xenopus and Zebrafish embryos that are fated
to become hematopoietic, and is highly expressed in progenitor cells. Overexpression of GATA2 in
chicken erythroid progenitors leads to proliferation at the expense of differentiation [48].

On the other hand, targeted disruption of the GATA2 gene leads to reduced primitive
hematopoiesis in the yolk sac and embryonic death by day 10 to 11 . Definitive hematopoiesis in
liver and bone marrow is profoundly reduced with loss of virtually all lineages, and in vitro
differentiation data reveal a marked deficiency of SCF-responsive definitive erythroid and mast cell
colonies and reduced macrophage colonies. This finding suggests that GATA2 serves as a
regulator of genes that control hematopoietic growth factor responsiveness or proliferation of stem
and/or early progenitor cells.

The importance of GATA2 to hematopoiesis and lymphopoiesis has been brought into sharp focus
by the discovery that heterozygous mutations in GATA2 are the cause of a broad spectrum of
previously described syndromes including monocytopenia and non-tuberculous mycobacterial
infections, usually Mycobacterium avium complex, (monoMAC syndrome); dendritic cell, monocyte,
B and NK lymphoid deficiency; familial myelodysplastic syndrome (MDS) and acute myeloid
leukemia; and Emberger syndrome (lymphedema and MDS). In addition, susceptibility to human
papilloma virus, chronic neutropenia, aplastic anemia, and pulmonary alveolar proteinosis have
also been described.

The finding of markedly decreased expression of GATA2 mRNA in CD34+ cells in patients with
aplastic anemia suggests that an aberrant expression of this transcription factor may have been an
early description of this disease.

GATA1 — GATA1 expression is limited to multipotent progenitors and to erythroid,

megakaryocyte, mast, and eosinophil lineages. Analysis of chimeric mice injected at the blastocyst
stage with GATA1-/- embryonic stem cells reveals a failure of such cells to contribute to erythrocyte
development; however, they can contribute to other hematopoietic lineages and tissues.
Differentiation assays in vitro show proerythroblast arrest and apoptosis of these cells.

Mouse embryos lacking GATA1 die because of severe anemia secondary to arrested maturation
of primitive erythroid cells, while GATA1 loss in adult mice results in a condition resembling pure
red cell aplasia in humans.

Clinically, a constitutive mutation in GATA1 that interferes with the interaction with its essential
cofactor FOG1 (friend of GATA1) is associated with familial dyserythropoietic anemia, defective
megakaryocyte maturation, and macrothrombocytopenia [62]. Furthermore, in the context of
trisomy 21, infants with Down syndrome (DS) and transient myeloproliferative disorder (TMD) or
acute megakaryoblastic leukemia (AMKL) have acquired mutations in the 5' region of GATA1 that
lead to a N-terminal truncated GATA1 protein [63,64]. These mutations are almost always present
at birth, and therefore acquired in utero, and have also been noted in approximately 10 percent of
neonates with DS and no evidence of TMD or AMKL [65]. It remains to be seen whether such
infants will go on to develop AMKL.

Interestingly, germ line mutations in GATA1 that also lead to loss of exon 2 and truncation of the
N-terminus of GATA1 were discovered in a small proportion of males with Diamond-Blackfan
anemia. These mutations lead to synthesis of a short form of GATA1 that is impaired in its ability to
drive erythroid differentiation [66]. Whether the more common ribosomal protein gene mutations
that account for approximately 60 percent of cases are related in some way to GATA1 deficiency
was an open question, but compelling evidence from a 2014 in vitro study demonstrates that
GATA1 translation is impaired in cells from Diamond-Blackfan anemia patients with different
ribosomal protein gene mutations because ribosomes are limiting [67]. Ribosome deficiency
adversely affects the translation of complex structured mRNAs that have low initiation rates
[68]. GATA1 has a complex 5' untranslated region (UTR) that predicts poor translation initiation
rates, and such mRNAs are more sensitive to ribosome deficiency than mRNAs with high initiation
rates.

Whether precursor stem cells commit to erythroid or myeloid lineages may depend in part upon
interactions between the GATA proteins (both GATA1 and GATA2) and PU.1 (Spi-1), a
transcription factor essential for the development of myeloid cells. PU.1 appears to interact with a
zinc finger domain of the GATA proteins via a helix-turn-helix wing [70]; this interaction appears to
inhibit PU.1 activation of critical myeloid genes, as well as GATA transactivating functions, thereby
suggesting that these effects have a role in the commitment of cells to either the erythroid or
myeloid lineage.

Krϋppel-like factor 1 — The Krϋppel-like factor 1 (KLF1) transcription factor is a master

regulator of terminal erythroid differentiation, controlling expression of many key erythroid
pathways and structures, including cell division, the cell membrane and cytoskeleton, iron
metabolism, and heme and globin synthesis. KLF1 was found to bind to the beta-globin promoter
[71]; it controls globin expression and some aspects of erythroid development. In addition to
activating beta-globin expression, KLF1 plays a role in silencing gamma-globin expression,
probably by regulating BCL11A and its interaction with Sox6 at the level of the gamma-globin gene.

Heterozygous mutations in KLF1 are characterized by hereditary persistence of fetal hemoglobin

synthesis [72] and/or congenital dyserythropoietic anemia type IV [75]. In contrast, compound
heterozygous mutations of the KLF1 gene have been associated with an unusual hereditary
persistence of embryonic globin synthesis, as well as a wide variety of hematologic abnormalities
including a thalassemic phenotype with hypochromia, microcytosis, target cells, fragments and
anisopoikilocytosis; a picture suggestive of chronic non-spherocytic hemolytic anemia with
schistocytes, fragmented cells and acanthocytes, and pyruvate kinase deficiency; and hydrops
fetalis in a neonate with compound heterozygosity for null mutations in KLF1 [76-78]. However,
most monoallelic KLF1 mutations give rise to benign phenotypes, including significant increases in
levels of HbF and HbA2, which may ameliorate the clinical severity of beta thalassemia.

GROWTH FACTORSThe regulation of the proliferation and maturation of erythroid

progenitors depends upon interaction with a number of growth factors. The availability of pure
recombinant growth factors, enrichment of target progenitor cells, use of defined "serum-free"
culture conditions, and the targeted disruption of factors and their receptors have provided insights
into the role of these factors during hematopoiesis.

Erythropoietin — Erythropoietin (EPO) is essential for the terminal maturation of erythroid cells
[80]. Its major effect appears to be at the level of the CFU-E during adult erythropoiesis;
recombinant preparations are as effective as the natural hormone .

CFU-E do not survive in vitro in the absence of EPO. Since the majority of CFU-E are cycling, their
survival in the presence of EPO may be tightly linked to their proliferation and differentiation to
mature erythrocytes.

EPO also acts upon a subset of presumptive mature BFU-E, which requires EPO for survival and
terminal maturation. A second subset of BFU-E, presumably less mature, survive EPO deprivation
if other hematopoietic growth factors such as IL-3 or GM-CSF are present.

Since EPO is crucial for the terminal differentiation of erythroid progenitors, mice with homozygous
null mutations of the EPO or EPO receptor (EPOR) genes form BFU-E and CFU-E normally but
these BFU-E and CFU-E fail to differentiate into mature erythrocytes [86]. Both the EPO-/-
and EPOR-/- mutations are embryonically lethal due to failure of definitive (fetal liver)
erythropoiesis. However, yolk sac erythropoiesis is only partially impaired, indicating the existence
of a population of EPO-independent primary erythropoietic precursors.

Insight into the molecular mechanism by which EPO prevents apoptosis in erythroid cells comes
from studies of the signal transducer and activator of transcription 5 (STAT 5) proteins. STAT5A-/-
STAT5B-/- mouse embryos are severely anemic during fetal development; the erythroid
progenitors in fetal liver are reduced in number and show increased apoptosis. This result was
explained by the finding that STAT5 mediates the early induction of the anti-apoptotic
gene BCLXL through direct binding to its promoter. Although adult animals were thought to be
hematologically normal, about half have chronic anemia with splenomegaly, due to an increase in
early erythroid precursors; erythropoiesis is ineffective because of an increase in apoptosis in early
normoblasts showing reduced levels of BCLXL. In contrast, dominant gain-of-function mutations in
the EPOR due to truncation of the cytoplasmic portion of the receptor lead to familial erythrocytosis
(type 1) because of elimination of the negative regulatory domain of the receptor that binds an
inhibitory phosphatase SH-PTP-1.

Insight into biased signaling downstream of the EPOR comes from study of a recessively inherited
R150Q mutation of EPO [91]. This mutation was found in a transfusion-dependent child thought
initially to have Diamond-Blackfan anemia who unexpectedly did not experience improvement after
a matched related hematopoietic stem cell transplant. The mutation results in alterations in the
kinetics of binding of EPO to the EPOR, with an increased on-rate and a markedly increased off-
rate. This results in suboptimal JAK2 phosphorylation, which is essential for STAT5 activation.
Perhaps surprisingly, STAT5 activation by JAK2 showed equivalent phosphorylation in response to
wild type and mutant EPO, whereas STAT1 and STAT3 activation were both reduced, indicating a
bias in the signal output from the mutant receptor.

Stem Cell Factor — Although Stem Cell Factor (SCF) alone has no colony-forming ability, it has
marked synergistic effects on BFU-E cultured in the presence of EPO. SCF is crucial for the
normal development of CFU-E, since mice that lack SCF (Steel mutants) or its receptor KIT (White
spotting or W mutants) are severely anemic and have a reduction in fetal liver CFU-E. Studies of
cell lines that express both KIT and EPOR (HCD57 cells) or are transduced with cDNAs for these
two receptors (32D cells) demonstrate that SCF supports the proliferation of these cells only if the
EPOR is also present.

In vitro, SCF induces tyrosine phosphorylation of the EPOR and KIT associates with the
cytoplasmic domain of the tyrosine phosphorylated EPOR. Whether such an interaction between
KIT and the EPOR occurs during normal erythropoiesis is not known.

IL-3 and GM-CSF — Interleukin 3 (IL-3) and GM-CSF receptors share a common beta chain.
Both cytokines enhance erythropoietin-dependent in vitro erythropoiesis by primary hematopoietic
progenitors and factor-dependent cells. The beta chain interacts functionally and physically with
the EPOR, thereby providing an explanation for the synergistic effects of IL-3 and GM-CSF on
EPO-driven erythropoiesis.

Insulin and insulin-like growth factor 1 — Factors distinct from the classical colony-
stimulating factors may positively regulate erythropoiesis, either directly or indirectly. Limiting
dilution studies of highly purified CFU-E in serum-free culture show that insulin and insulin-like
growth factor 1 (IGF-1) act directly on these cells in the presence of EPO. By comparison, earlier
murine studies of unfractionated cells found that CFU-E respond to IGF-1 or insulin in the absence
of EPO.

TGF-beta gene family — This gene family comprises several TGF-beta and bone morphogenic
proteins (BMPs), growth and differentiation factors (GDFs), and activin and inhibin. Their receptors
comprise two members of the serine/threonine kinase gene family. Activin enhances both BFU-E
and CFU-E colony formation. This protein dimer, also known as follicle stimulating hormone-
releasing protein, appears to have a lineage-specific effect on erythropoiesis that is indirect, since
removal of monocytes and/or T lymphocytes abrogates its activity [101]. Activin is the factor
produced by vegetal cells during blastogenesis that induces animal ectodermal cells to form
primary mesoderm.

Murine data suggest that TGF family members may play an intriguing regulatory role during
erythropoiesis [103]. Under normal circumstances, GDF11, produced by developing erythroid
progenitor cells, inhibits the differentiation of more mature erythroid precursors. In thalassemia and
myelodysplastic syndromes (MDS), ineffective erythropoiesis is markedly increased, with
increased production of GDF11 and consequent decreased differentiation of erythroid cells. Activin
and GDF11 ligand traps consist of the extracellular domain of the activin receptor 2A or B linked to
an immunoglobulin Fc domain. One possible explanation of the mechanism of action is
that luspatercept and other ligand traps alleviate the anemia by removing the GDF11-mediated
inhibition of erythroid differentiation. However, SMAD2/3 signaling also decreases the availability
of GATA1. Ligand traps may also act by alleviating the sequestration of GATA1 by HSP40, thereby
increasing its availability. Luspatercept is approved for the treatment of transfusion-dependent
thalassemia patients and individuals with very low- to intermediate-risk MDS with ring sideroblasts
who are transfusion dependent or myelodysplastic/myeloproliferative neoplasm with ring
sideroblasts and thrombocytosis .

Use of these agents in disorders with ineffective erythropoiesis is discussed separately:

●Thalassemia
●MDS

MicroRNAsMicroRNAs (miRNAs) are 18 to 25 nucleotide non-protein encoding RNAs that play

important roles in the regulation of post transcriptional gene expression by incorporation into an
RNA-induced silencing complex (RISC) that contains the Argonaute proteins. The miRNA strand
guides RISC typically to complementary sequences in the 3’ untranslated region of target mRNA
by Watson Crick base-pairing, and leads to either mRNA degradation or translational repression.

There is strong evidence that miRNAs are important for erythropoiesis. Many miRNAs are
expressed in developing erythroid cells ,most appear to decline in expression level as precursors
mature, suggesting that they might regulate the timing of genes important for the synthesis of late
erythroid proteins. For example, miR-191 is downregulated, and overexpression in mouse fetal
liver cells results in a block in enucleation, a late event in erythropoiesis. Other miRNAs that
decrease in expression during differentiation act at an earlier stage of erythropoiesis. Both miR-
221 and miR-222 are encoded on a common pre-miRNA; they target and repress KIT expression,
so overexpression impairs the proliferation of erythroid precursors.

Other miRNAs, such as miR-144 and miR-451, increase in expression level during erythropoiesis;
both are co-expressed in a bi-cistronic pri-miRNA that is activated by GATA1. Enforced expression
of these two miRs enhances erythropoiesis while depletion of miR-451 has the opposite effect
GATA2 is a target of miR-451 in zebrafish, and since down-regulation of GATA2 is required for
normal erythropoiesis, this might explain the effect of miR-451 depletion. Other experiments
suggest that the miR-144/451 effect is more complex, and different mouse models show different
phenotypes ranging from an erythroblast block to a hemolytic anemia with an increase in
reticulocytes.

HYPOXIA AND EPO EXPRESSIONEPO has long been recognized as the physiologic
regulator of red cell production. It is produced in the kidney and the fetal liver in response to
hypoxia or exposure to cobalt chloride.

Hypoxia-inducible factor and the response to hypoxia — Specialized interstitial cells in

the inner cortex and outer medulla of the kidney respond to hypoxia by producing and secreting
EPO. Downstream events from the oxygen sensor involved in activation of EPO gene expression
require stabilization and activation of specific transcription factors. One of these is the
heterodimeric transcription factor complex hypoxia-inducible factor (HIF-1), comprising both HIF-1-
alpha and HIF-1-beta subunits (the dimer is called HIF-1).

While HIF-1-beta is ubiquitously expressed independent of oxygen tension, HIF-1-alpha is only

detectable under hypoxic conditions. Above a critical oxygen concentration HIF-1-alpha is
ubiquitinated and rapidly degraded in proteosomes. This oxygen sensor in mammalian cells
appears to be a proline hydroxylase (prolyl hydroxylase domain proteins PHD1, PHD2, and PHD3)
that adds a hydroxyl moiety in an oxygen- and iron-dependent manner to P-564 of HIF-1-alpha; P-
564 lies within a highly conserved region of HIF-1-alpha that binds the von Hippel-Lindau (VHL)
protein. When P-564 hydroxylated HIF-1-alpha binds VHL, an ubiquitin E3 ligase complex is
activated, leading to ubiquitination and subsequent destruction of HIF-1-alpha. A second related
protein, HIF-2-alpha, is also subject to oxygen dependent prolyl hydroxylation, and also dimerizes
with HIF-1-beta to form HIF-2. HIF-1-alpha is expressed in all nucleated cells, while HIF-2-alpha
expression is restricted to vascular endothelial cells, renal interstitial cells, hepatocytes,
cardiomyocytes and astrocytes.

A homozygous R200W mutation of the VHL gene leads to reduction in the interaction of VHL with
hydroxylated HIF, accumulation of HIF, and excessive erythrocyte production in Chuvash
polycythemia (familial erythrocytosis, type 2). A different set of dominantly inherited mutations is
found in the von Hippel-Lindau syndrome, with loss of the second normal allele in the tumors that
characterize this disease.
An autosomal dominant form of erythrocytosis occurs with mutations the EGLN1 gene that
encodes PHD2 (familial erythrocytosis, type 3). These mutations may have dominant negative
effects because PHD2+/- heterozygous mice do not have erythrocytosis.

Additionally, mutations in the EPAS1 gene that encodes HIF-2-alpha can also cause familial
erythrocytosis (type 4). These mutations interfere with the interaction of HIF-2-alpha with PHD2,
thereby reducing hydroxylation. Mice with targeted disruption of the EPAS1 gene are anemic,
emphasizing the importance of HIF-2-alpha in EPO regulation .

HIF-1 activity is induced in a variety of cell types in response to hypoxia or cobalt; this finding
suggests that, while HIF-1 is important in the activation of EPO expression, it also plays a role in
the activation of other hypoxia-inducible genes, including angiogenesis and glycolysis, and may
play a role in the regulation of more than 2 percent of all human genes . HIF-1-alpha may also bind
to and negatively regulate the hepcidin promoter, leading to decreased levels of hepcidin .
Through coordinate downregulation of hepcidin and upregulation of ferroportin and erythropoietin,
the VHL pathway effectively mobilizes iron in support of increased red cell production.

The activity of HIF-1 is essential for survival. Mice that lack HIF-1 die at midgestation, while
heterozygotes, although developmentally normal and similar to normal mice in normoxic conditions,
fail to respond adequately to chronic hypoxia.

HIF-1 binds to an enhancer sequence located approximately 130 base pairs downstream from the
poly-A addition signal of the EPO gene. This enhancer segment renders other promoter-reporter
gene constructs hypoxia-responsive with typical inductions in the range of 4 to 15 fold; this degree
of stimulation is significantly less than the 50 to 100 fold induction observed with the chromosomal
EPO gene in Hep 3B cells.

Other studies have identified a 53 base pair sequence in the EPO promoter with important
regulatory characteristics. This sequence confers oxygen-sensitivity (6 to 10-fold inducibility) to a
luciferase reporter gene. The combination of both the enhancer and promoter sequences results in
cooperative (50-fold) inducibility of transcription of EPO in response to hypoxia, enhancements
approaching that observed in vivo.

HYPOXIA AND EPO EXPRESSIONEPO has long been recognized as the physiologic
regulator of red cell production [116-119]. It is produced in the kidney and the fetal liver in
response to hypoxia or exposure to cobalt chloride.

Hypoxia-inducible factor and the response to hypoxia — Specialized interstitial cells in

the inner cortex and outer medulla of the kidney respond to hypoxia by producing and secreting
EPO. Downstream events from the oxygen sensor involved in activation of EPO gene expression
require stabilization and activation of specific transcription factors . One of these is the
heterodimeric transcription factor complex hypoxia-inducible factor (HIF-1), comprising both HIF-1-
alpha and HIF-1-beta subunits (the dimer is called HIF-1).

While HIF-1-beta is ubiquitously expressed independent of oxygen tension, HIF-1-alpha is only

detectable under hypoxic conditions . Above a critical oxygen concentration HIF-1-alpha is
ubiquitinated and rapidly degraded in proteosomes. This oxygen sensor in mammalian cells
appears to be a proline hydroxylase (prolyl hydroxylase domain proteins PHD1, PHD2, and PHD3)
that adds a hydroxyl moiety in an oxygen- and iron-dependent manner to P-564 of HIF-1-alpha; P-
564 lies within a highly conserved region of HIF-1-alpha that binds the von Hippel-Lindau (VHL)
protein. When P-564 hydroxylated HIF-1-alpha binds VHL, an ubiquitin E3 ligase complex is
activated, leading to ubiquitination and subsequent destruction of HIF-1-alpha . A second related
protein, HIF-2-alpha, is also subject to oxygen dependent prolyl hydroxylation, and also dimerizes
with HIF-1-beta to form HIF-2. HIF-1-alpha is expressed in all nucleated cells, while HIF-2-alpha
expression is restricted to vascular endothelial cells, renal interstitial cells, hepatocytes,
cardiomyocytes and astrocytes.

A homozygous R200W mutation of the VHL gene leads to reduction in the interaction of VHL with
hydroxylated HIF, accumulation of HIF, and excessive erythrocyte production in Chuvash
polycythemia (familial erythrocytosis, type 2) . A different set of dominantly inherited mutations is
found in the von Hippel-Lindau syndrome, with loss of the second normal allele in the tumors that
characterize this disease.
An autosomal dominant form of erythrocytosis occurs with mutations the EGLN1 gene that
encodes PHD2 (familial erythrocytosis, type 3). These mutations may have dominant negative
effects because PHD2+/- heterozygous mice do not have erythrocytosis.

Additionally, mutations in the EPAS1 gene that encodes HIF-2-alpha can also cause familial
erythrocytosis (type 4). These mutations interfere with the interaction of HIF-2-alpha with PHD2,
thereby reducing hydroxylation. Mice with targeted disruption of the EPAS1 gene are anemic,
emphasizing the importance of HIF-2-alpha in EPO regulation .

HIF-1 activity is induced in a variety of cell types in response to hypoxia or cobalt; this finding
suggests that, while HIF-1 is important in the activation of EPO expression, it also plays a role in
the activation of other hypoxia-inducible genes, including angiogenesis and glycolysis, and may
play a role in the regulation of more than 2 percent of all human genes. HIF-1-alpha may also bind
to and negatively regulate the hepcidin promoter, leading to decreased levels of hepcidin .
Through coordinate downregulation of hepcidin and upregulation of ferroportin and erythropoietin,
the VHL pathway effectively mobilizes iron in support of increased red cell production.

The activity of HIF-1 is essential for survival. Mice that lack HIF-1 die at midgestation, while
heterozygotes, although developmentally normal and similar to normal mice in normoxic conditions,
fail to respond adequately to chronic hypoxia.

HIF-1 binds to an enhancer sequence located approximately 130 base pairs downstream from the
poly-A addition signal of the EPO gene. This enhancer segment renders other promoter-reporter
gene constructs hypoxia-responsive with typical inductions in the range of 4 to 15 fold; this degree
of stimulation is significantly less than the 50 to 100 fold induction observed with the chromosomal
EPO gene in Hep 3B cells.

Other studies have identified a 53 base pair sequence in the EPO promoter with important
regulatory characteristics. This sequence confers oxygen-sensitivity (6 to 10-fold inducibility) to a
luciferase reporter gene [143]. The combination of both the enhancer and promoter sequences
results in cooperative (50-fold) inducibility of transcription of EPO in response to hypoxia,
enhancements approaching that observed in vivo.

Hepatocyte nuclear factor 4 and p300 — Because both promoter and enhancer sequence
elements include the consensus hexanucleotide nuclear hormone receptor response elements,
various orphan members of this gene family have been examined for binding to the EPO promoter
and enhancer and for their presence in transcription complexes isolated from Hep 3B nuclear
extracts. One of these, hepatocyte nuclear factor 4 (HNF-4) is present in these extracts and plays
a critical role in hypoxia-induced activation of EPO gene expression in Hep 3B cells [144]. HIF-1a
and -1b also bind to a large protein p300 (homologous to the cyclic AMP response element (CREB)
binding protein [CBP]) that acts as a general transcriptional activator for a number of genes [145].

The net effect is that HIF-1a, which accumulates in response to hypoxia, combines with its partner
HIF-1b and with HNF-4 and p300 to activate EPO transcription. The role of HIF-2 in this process is
under active investigation.

STRESS AND ERYTHROPOIESISThe level of oxyhemoglobin and the rate of delivery of

oxygen to the tissues are the fundamental regulators of erythropoiesis. In species that package
hemoglobin in erythrocytes, erythropoietin mediates the response to oxygen demand; it performs
this function by interacting with specific receptors on the surface of mature committed erythroid
BFU-E and CFU-E (erythroid burst-forming units and colony-forming units, respectively). As noted
above, hypoxia induces a transcriptional increase in expression of the EPO gene via HIF-1a and
HIF-1b.

BFU-E mature to single CFU-E that divide, and, under the influence of lower concentrations of
erythropoietin, form single, relatively small colonies of proerythroblasts in about one week in vitro.
The bone marrow of an adult mouse contains about 500 CFU-E per 105 nucleated cells. In
response to anemic stress, as in hemorrhage or hemolysis, almost the entire burden of
accelerated reticulocyte production is borne by the rapid erythropoietin-dependent influx from the
progenitor compartment into the proerythroblast pool, resulting in an expanded proerythroblast
pool.

Anemic stress produces little or no increase in the mitotic rate of recognizable erythroid
precursors . Instead, the following changes are seen:
 ●The late BFU-E and CFU-E proliferate (because of EPO binding to its receptors) and
differentiate to proerythroblasts and beyond. In normal murine marrow, the CFU-E frequency
increases from about 500 per 105 nucleated cells to 1000 to 2000 CFU-E per 105 nucleated
cells following experimental hemorrhage or hemolysis. By comparison, hypertransfused
mice, in which erythropoiesis is completely suppressed, show an 80 to 90 percent reduction
in number of CFU-E.
●The orderly progression from immature BFU-E through CFU-E to proerythroblast is
interrupted. High erythropoietin levels permit or induce differentiation of progenitors to
proerythroblasts. In the rhesus monkey, this premature terminal differentiation may account
for the marked increase in fetal hemoglobin content and F cells observed during stress
erythropoiesis [154]. In contrast, progenitors in humans have a lesser ability to generate
erythroblasts capable of synthesizing large quantities of fetal hemoglobin. As a result, the
accumulation of F cells in peripheral blood in response to anemia is relatively small.

Red cells produced under such situations are usually macrocytic and carry the i antigen. However,
these two characteristics may relate to a short transit time through the marrow in response to
erythropoietin rather than an intrinsic characteristic of fetal hemoglobin-containing cells themselves.
Fetal hemoglobin-containing cells (F cells) are also present in very small numbers in the blood of
normal individuals, but these cells are not macrocytic and do not bear i antigen. F cell progenitors
can be easily detected in the bone marrow of patients with various dyspoieses and even in normal
marrows.

In mice, stress erythropoiesis appears to occur largely in the spleen, where "stress" BFU-E
respond to EPO, SCF, and bone morphogenic protein 4 (BMP4), which signals via the Smad5
pathway; hypoxia increases both BMP expression and the BMP4/Smad5 signaling response. This
contrasts with "steady state" bone marrow BFU-E that require only SCF, IL-3/GM-CSF and
become responsive to EPO as they mature to CFU-E and proerythroblasts. There is also evidence
that Hedgehog signaling replenishes splenic stress BFU-E from the bone marrow BFU-E pool.

Glucocorticoids play an important role in the erythropoietic response to stress and are also the
principal treatment for Diamond-Blackfan anemia (DBA).

Mouse and human studies have shown that glucocorticoids have a greater effect on BFU-E than
CFU-E proliferation. A major insight into the mechanism of this effect comes from single-cell
transcriptome profiling of purified mouse fetal liver erythroid progenitors. These studies show that
glucocorticoids enhance erythropoiesis by slowing the transit of early through late BFU-E, without
affecting the rate of the cell cycle or asymmetric self-renewal divisions. Rather, the slowed rate of
progression allows an increase in the number of cell divisions, thereby increasing output into the
CFU-E pool. The mechanism by which this occurs is through prolonged expression of genes such
as GATA2 that antagonize expression of genes such as GATA1 that drive terminal differentiation.