Hypoxic regulation of erythropoiesis and iron metabolism
Volker H. Haase
Departments of Medicine, Cancer Biology, and Molecular Physiology and Biophysics, Vanderbilt School of Medicine,
Submitted 29 March 2010; accepted in final form 29 April 2010
Haase VH. Hypoxic regulation of erythropoiesis and iron metabolism. Am J
Physiol Renal Physiol 299: F1–F13, 2010. First published May 5, 2010;
doi:10.1152/ajprenal.00174.2010.—The kidney is a highly sensitive oxygen sensor
and plays a central role in mediating the hypoxic induction of red blood cell
production. Efforts to understand the molecular basis of oxygen-regulated eryth-
ropoiesis have led to the identification of erythropoietin (EPO), which is essential
for normal erythropoiesis and to the purification of hypoxia-inducible factor (HIF),
the transcription factor that regulates EPO synthesis and mediates cellular adapta-
tion to hypoxia. Recent insights into the molecular mechanisms that control and
integrate cellular and systemic erythropoiesis-promoting hypoxia responses and
their potential as a therapeutic target for the treatment of renal anemia are discussed
in this review.
erythropoietin; hypoxia; hypoxia-inducible factors; iron; red blood cells
ONE OF THE MOST EXTENSIVELY studied systemic adaptations to
hypoxia is the stimulation of red blood cell (RBC) production.
Over 100 years ago, Paul Bert and Denis Jourdanet observed
the association between reduced atmospheric oxygen pressure
and elevated RBC numbers in the blood of animals and humans
(12, 13, 64). Francois-Gilbert Viault then demonstrated that
ascent to a high altitude provided an acute and direct physio-
logical stimulus for RBC production during his 1890 expedi-
tion to the Peruvian Andes by measuring the RBC increases in
his own and the blood of his companions (156). It was the
interest in understanding the physiological and molecular basis
of this erythropoietic response that led to the discovery of
erythropoietin (EPO) and that paved the way for the identifi-
cation of the molecular machinery that senses oxygen and
controls a wide spectrum of tissue-specific and systemic re-
sponses to hypoxia.
The hypoxic induction of EPO serves as a paradigm of
oxygen-dependent gene regulation, and the search for the
transcription factor that mediates this induction led to the
discovery of the hypoxia-inducible factor (HIF) as a key
mediator of cellular adaptation to low oxygen. Recent experi-
mental evidence suggests that HIF promotes erythropoiesis
through coordinated cell type-specific hypoxia responses,
which include increased EPO production in the kidney and
liver, enhanced iron uptake and utilization, as well as changes
in the bone marrow microenvironment that facilitate erythroid
progenitor maturation and proliferation. Because of its central
role in the hypoxic regulation of erythropoiesis, pharmacolog-
ical targeting of the HIF oxygen-sensing pathway has the
potential to become an effective, novel therapy in the treatment
of anemia that is associated with inadequate EPO production.
This review provides an overview of recent insights into the
molecular mechanisms that underlie oxygen-dependent regu-
lation of EPO synthesis, iron metabolism, and erythroid pro-
genitor maturation and discusses their relevance to clinical
Oxygen-Dependent Regulation of EPO Synthesis: A
Paradigm of Hypoxic Gene Regulation
The human EPO gene encodes a glycoprotein hormone,
which consists of 165 amino acids in its circulating form.
Serum EPO is heavily glycosylated and has a molecular mass
of ?30 kDa, 40% of which is derived from its carbohydrate
portion. Its major action is the prevention of apoptosis in
EPO-dependent colony-forming unit-erythroid cells and eryth-
roblasts that have not begun hemoglobin synthesis. Its receptor
(EPO-R), which is also hypoxia inducible (26, 91, 167), lacks
intrinsic enzymatic function and associates with the tyrosine
kinase Janus kinase 2 (JAK2), which phosphorylates EPO-R at
multiple sites upon ligand binding, thus providing docking
sites for signal-transducing molecules that contain src homol-
ogy 2 domains. EPO-R signals through multiple pathways.
These include the signal transduction and activator of tran-
scription (STAT) 5 pathway, the phosphatidylinositol 3-kinase/
protein kinase B (PI-3K/AKT) and MAPK/ERK pathways, and
Hypoxia is the primary physiological stimulus for EPO
production, which, depending on the hypoxic condition, in-
creases serum EPO levels up to several hundred-fold (33).
Studies in hepatoma cells aimed at isolating the transcriptional
activator responsible for the hypoxic induction of EPO iden-
tified the heterodimeric basic helix-loop-helix transcription
factor HIF-1 as the transcriptional regulator that binds to the
hypoxia-sensitive enhancer located in the 3=-prime region of
the EPO gene (159, 160). HIF-1 belongs to the PAS [PER/aryl
hydrocarbon receptor nuclear translocator (ARNT)/single
minded (SIM)] family of transcription factors and consists of
Address for reprint requests and other correspondence: V. H. Haase, Dept.
of Medicine, Division of Nephrology and Hypertension, Vanderbilt Univ.
Medical Center, C-3119A, MCN, 1161 21st Ave, Nashville TN 37232 (e-mail:
Am J Physiol Renal Physiol 299: F1–F13, 2010.
First published May 5, 2010; doi:10.1152/ajprenal.00174.2010.
0363-6127/10 Copyright © 2010 the American Physiological Societyhttp://www.ajprenal.org F1
an oxygen-sensitive ?-subunit and a constitutively expressed
?-subunit, also known as ARNT (69, 138, 163). Together with
HIF-2? (also known as EPAS-1 or HLF), HIF-1? facilitates
oxygen delivery and cellular adaptation to hypoxia by stimu-
lating multiple biological processes, such as erythropoiesis,
angiogenesis, and anaerobic glucose metabolism (137). HIFs
regulate gene expression by binding to specific DNA recogni-
tion sequences, referred to as hypoxia-response elements
(HREs) (Fig. 1). All three known HIF ?-subunits, HIF-1?,
HIF-2?, and HIF-3?, are targeted for rapid proteasomal deg-
radation under normoxia by the von Hippel-Lindau tumor
suppressor pVHL, which acts as the substrate recognition
component of an E3 ubiquitin ligase complex (99, 101).
Whereas HIF-1? and HIF-2? heterodimers function as tran-
scriptional activators, splice variants of HIF-3? have been
shown to be inhibitory (90, 100). Although HIF-1 and HIF-2
share many common transcriptional targets, they also regulate
unique targets and have specific biological functions. Anaero-
bic glycolysis, for example, appears to be predominantly con-
trolled by HIF-1 (55), whereas HIF-2 has emerged as the main
regulator of EPO production in the adult (48, 107, 126, 135). In
addition to HRE-mediated transcriptional regulation, which
requires heterodimerization with ARNT, HIF-? modulates cel-
lular signaling pathways through functional interaction with
proteins that do not contain PAS domains. These include,
among others, tumor suppressor protein p53, the c-Myc proto-
oncogene, and the Notch intracellular domain (2, 49, 72, 127).
Under normal oxygen conditions, HIF-?-subunits are rap-
idly degraded following ubiquitylation by the pVHL-E3 ubiq-
uitin ligase, which precludes the formation of transcriptionally
active heterodimers. pVHL-mediated polyubiquitylation of
HIF-? requires hydroxylation of specific proline residues
(Pro402 and Pro564 in human HIF-1?; Pro405 and Pro531 in
human HIF-2?) within its oxygen-dependent degradation do-
main (20, 35, 53, 56, 58, 94, 169). Hydroxylation of HIF-? is
carried out by three major 2-oxoglutarate-dependent dioxyge-
Fig. 1. Hypoxia-inducible factor (HIF)-2 regulates erythropoietin (EPO). Shown is an overview of EPO gene regulation by the von Hippel-Lindau
(VHL)/HIF/prolyl-4-hydroxylase domain (PHD) oxygen-sensing pathway. Proteasomal degradation of HIF-2? by the VHL tumor suppressor (pVHL)-E3-
ubiquitin ligase complex (shown are key components of this complex) requires hydroxylation by oxygen- and iron-dependent PHDs. Binding to hydroxylated
HIF-? occurs at the ?-domain of pVHL, which spans amino acid residues 64–154. The C-terminal ?-domain links the substrate recognition component pVHL
to the E3 ubiquitin ligase via elongin C. In the absence of molecular oxygen, HIF-2? is not degraded and translocates to the nucleus where it forms a heterodimer
with HIF-?, also known as the aryl hydrocarbon receptor nuclear translocator (ARNT). HIF-2?/? heterodimers bind to the HIF consensus binding site
5=-RCGTG-3= and increase EPO transcription in the presence of transcriptional coactivators, such as CREB-binding protein (CBP) and p300. Hypoxic induction
of EPO in the liver is mediated by the liver-inducibility element located in the 3=-end of the EPO gene and in renal interstitial fibroblast-like cells by the
5=-kidney-inducibility element, which is located 6–14 kb upstream of its transcription start site. Nitric oxide, reactive oxygen species, Krebs cycle metabolites
succinate and fumarate, cobalt chloride (CoCl2), and iron chelators such as desferrioxamine inhibit HIF PHDs in the presence of oxygen, resulting in increased
EPO transcription. EPO mRNA is encoded by 5 exons depicted by boxes. Coding sequences are shown in red. Nontranslated regions are shown in blue, and
numbers indicate distance from the transcription start site in kb (not drawn to scale). Also shown are binding sites for hepatocyte nuclear factor (HNF)-4 in the
3=-liver-inducibility region. Fe2?, ferrous iron; NO, nitric oxide; ROS, reactive oxygen species; ub, ubiquitin.
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