Erythropoietin is a glycoprotein hormone/cytokine that controls red blood cell production in the bone marrow.
The circulating peptide core of mature EPO is composed of 165 amino acids which form two bisulphide bridges (Cys7-Cys161, Cys29-Cys33). The carbohydrate (glycosid bridges) portion (40% of molecule) comprises three tetraantennary N-linked (Asn24, Asn38 and Asn83) and one small O-linked (Ser126) glycans.
Endogenous circulating Epo have several glycosylation isoforms. The N-glycans are essential for its biological activity in vivo. Of major importance are the terminal sialic acid residues of these glycans. Like other asialo-glycoproteins, asialo-Epo is rapidly removed via galactose receptors of hepatocytes (The in vivo metabolism of recombinant human erythropoietin in the rat, 1989). On the contrary, the introduction of additional N-glycans into recombinant Epo by site-directed mutagenesis results in a prolonged in vivo survival of the molecules (Enhancement of therapeutic protein in vivo activities through glycoengineering, 2003).
Human EPO has a molecular weight of 30,4 kDa.
The human Epo gene is located on the long arm of chromosome 7 (q11–q22) (73–75). It contains five exons, which encode a 193-amino acid prohormone, and four introns. The amino acid leader sequence of 27 residues is cleaved prior to secretion
Protein amino acids percentage
Cellular localization of EPO
Experiments in animals in which various organs were surgically removed identified the kidney as the major site of EPO production, where interstitial peritubular fibroblast-like cells in the inner cortex and outer medulla synthesize EPO (approximately 90% of systemic erythropoietin in adults) (Erythropoietin production by interstitial cells of hypoxic monkey kidneys, 1998).
The isolation and establishment of EPO-producing cells from the kidney have been largely unsuccessful, although hypoxic induction of EPO synthesis was reported mesenchymal cell clones with stromal cell characteristics isolated from the adult kidney. EPO-producing renal cells, however, have been successfully tagged with GFP, using BAC transgenes, in which a segment of the murine EPO gene was replaced with GFP cDNA, bringing GFP under the control of EPO-regulatory elements (Repression via the GATA box is essential for tissue-specific erythropoietin gene expression, 2008). GFP expression in transgenic mice was induced by anemia in renal peritubular interstitial cells specifically, and in hepatocytes surrounding the central vein, supporting the notion that these two cell types are the major sources of EPO production under hypoxic conditions.
In the kidney, GFP-positive interstitial cells were unique in their morphological appearance, possessed dendritelike processes, and expressed neuronal-specific markers, as microtubule-associated protein 2 and neurofilament protein, light polypeptide. This finding suggested that renal EPO producing cells might be derived from progenitor cells related to the neural lineage.
While the kidney is the primary physiological site of adult EPO synthesis, the liver is the main source during embryonic development. In adults, however, the liver is the major source of extrarenal EPO production following stimulation with moderate to severe hypoxia. While hepatocytes have been identified as the primary cell type responsible for EPO synthesis in the liver, EPO has also been detected in hepatic stellate cells, also known as ITO cells (Expression of a homologously recombined erythopoietin-SV40 T antigen fusion gene in mouse liver: evidence for erythropoietin production by Ito cells,1994).
In the adult, liver EPO mRNA levels, which are very difficult to detect at baseline, rise substantially following stimulation with moderate to severe hypoxia and account for most, if not all, physiologically relevant systemic EPO of extrarenal origin. However, hypoxia-stimulated liver EPO production is usually not sufficient to compensate for the loss of renal EPO in the setting of nephrectomy or advanced chronic renal failure.
Aside from the kidney and liver as the two major sources of synthesis, EPO mRNA expression has also been detected in the brain (neurons and glial cells), lung, heart, bone marrow, spleen, hair follicles, and the reproductive tract. Of those cell types, glial cells in the mouse brain have recently been shown to contribute to the serum EPO pool in response to acute anemia (Erythropoietin after a century of research: younger than ever, 2007).
EPO synthesized in other organs appears to act locally, modulating, for example, regional angiogenesis and cellular viability and does not seem to contribute to erythropoiesis.
Concentration in blood
Erythropoietin levels in blood are quite low in the absence of anemia, at around 10 mU/ml. However, in hypoxic stress, EPO production may increase 1000-fold, reaching 10,000 mU/ml of blood.
Primary role in red blood cell production (Erythropoiesis)
Erythropoietin is an essential hormone for red cell production. Without it, definitive erythropoiesis does not take place. Under hypoxic conditions, the kidney will produce and secrete erythropoietin to increase the production of red blood cells by targeting CFU-E, proerythroblast basophilic erythroblast subsets in the differentiation. Erythropoietin has its primary effect on red blood cell progenitors and precursors (which are found in the bone marrow in humans) by promoting their survival through protecting these cells from apoptosis. The primary mechanism by which Epo maintains erythropoiesis is prevention of apoptosis. CFU-Es express abundant GATA-1, which is an important transcription factor in erythrocytic development. The balance between GATA-1 and caspases determines the balance between apoptosis, proliferation and differentiation of erythrocytic progenitors.
Erythropoietin is the primary erythropoietic factor that cooperates with various other growth factors (e.g. IL-3, IL-6, and glucocorticoids) involved in the development of erythroid lineage from multipotent progenitors. The burst-forming unit-erythroid (BFU-E) cells start erythropoietin receptors expression and are sensitive to erythropoietin. Subsequent stage, the colony-forming unit-erythroid (CFU-E), expresses maximal erythropoietin receptor density and is completely dependent on erythropoietin for further differentiation. Precursors of red cells, the proerythroblasts and basophilic erythroblasts also express erythropoietin receptor and are therefore affected by it.
Additional nonhematopoietic roles
Epo-R mRNA is expressed in various non-haematopoietic tissues.
Functional Epo-R in non-erythrocytic cells were first demonstrated in endothelial cell cultures(Erythropoietin has a mitogenic and positive chemotactic effect on endothelial cells, 1990 and Human erythropoietin induces a pro-angiogenic phenotype in cultured endothelial cells and stimulates neovascularization in vivo, 1999).
In vitro, Epo promotes the proliferation and migration of endothelial cells, stimulates the production of modulators of vascular tone, such as endothelin (Effects of erythropoietin on endothelin-1 synthesis and the cellular calcium messenger system in vascular endothelial cells, 1997) and NO (Nitric oxide mediates renal vasodilation during erythropoietin-induced polycythemia, 1993), favours a pro-angiogenic phenotype and induces neovascularization (Recombinant human erythropoietin stimulates angiogenesis in vitro, 1995).
In vivo, Epo increases the number of circulating endothelial progenitor cells(Erythropoietin regulates endothelial progenitor cells, 2004). Vascular smooth muscle cells also express Epo-R. Here, Epo causes Ca2+ mobilisation (Erythropoietin increases cytosolic free calcium concentration in vascular smooth muscle cells, 1993), phospholipase C activation (Erythropoietin induces Ca2+ mobilization and contraction in rat mesangial and aortic smooth muscle cultures, 1996) and smooth muscle contraction (Erythropoietin induces Ca2+ mobilization and contraction in rat mesangial and aortic smooth muscle cultures1996). Other effects include the activation of the MAPK and PI-3K/Akt pathways , which mediate the inhibition of apoptosis (Signal transduction in the erythropoietin receptor system, 1999).
Furthermore, Epo-R is expressed by human cardiomyocyte cell lines and in human adult cardiac tissue(Recombinant human erythropoietin protects the myocardium from ischemia-reperfusion injury and promotes beneficial remodeling, 2003). Probably due to anaemia and tissue hypoxia the complete knock-out of the Epo-R gene results in a phenotype of severe cardiac malformations and foetal death at day 13.5 in mice. Non-anaemic mice with a tissue-specific Epo-R knock-out outside the haemopoietic tissue exhibit no abnormalities.
With respect to the central nervous system both Epo-R and Epo become detectable at week 5 after conception in the brain of human embryos (Immunohistochemical localization of erythropoietin and its receptor in the developing human brain, 1999). Epo-R is expressed by neurones and astrocytes (Effects of erythropoietin on glial cell development; oligodendrocyte maturation and astrocyte proliferation, 2002) and by brain capillary endothelial cells (Brain capillary endothelial cells express two forms of erythropoietin receptor mRNA, 1996.) and exerts neuroprotective and neurotrophic effects (Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury, 2000.).
Epo-R mRNA and/or Epo-R protein have been detected in various types of cancer. Breast carcinoma, lung carcinoma, renal carcinoma, tumours of the cervix and of other organs of the female reproductive tract, and various paediatric tumours. However, many investigators failed to demonstrate any relationship between Epo-R expression and Epo signaling or tumour growth (Recombinant epoetins do not stimulate tumor growth in erythropoietin receptor-positive breast carcinoma models, 2006).
Epo also can increase iron absorption by suppressing the hormone hepcidin (Erythropoietin administration in humans causes a marked and prolonged reduction in circulating hepcidin, 2010)
Epo-R belongs to the cytokine class I receptor superfamily whose members are characterised by an extracellular N-terminal domain, a single hydrophobic transmembrane segment and a cytosolic domain that lacks enzymatic activity.
The mature human Epo-R is a 484-amino acid glycoprotein with 1 N-glycan. The calculated mass of human Epo-R of 52.6 kDa increases to about 60 kDa because of glycosylation and phosphorylation.
Two of the membrane-spanning Epo-R molecules form a homodimer that binds one Epo molecule. Most of the ligand/Epo-R interaction occurs in a hydrophobic flat region of the Epo-R (Phe93, Met150, Phe205).
With respect to novel pharmacological compounds it is of interest that the affinity of Epo analogues for Epo-R decreases with glycosylation: the carbohydrate portion of the glycoprotein prevents receptor binding through electrostatic forces(Development and characterization of novel erythropoiesis stimulating protein (NESP), 2001).
Binding of Epo induces a conformational change and a tight connection of the two monomeric Epo-R molecules. Thereby, two Janus kinases 2 which are in contact with the cytoplasmic region of Epo-R are activated. As a result, several tyrosine residues of the Epo-R are phosphorylated to provide docking sites for signalling proteins containing SRC homology 2 (SH2) domains(JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin, 1993). The pathways through which Epo signals include phosphatidyl-inositol 3-kinase (PI-3K)/Akt, STAT5, MAP kinase and protein kinase C.
The effect of Epo is terminated by the action of the haematopoietic cell phosphatase (HCP) which catalyses JAK2 de-phosphorylation (Hematopoietic cell phosphatase associates with erythropoietin (Epo) receptor after Epo-induced receptor tyrosine phosphorylation: identification of potential binding sites, 1995). Mutations of the cytoplasmic C-terminal regions of Epo-R and functional deficiencies of HCP may lead to erythrocytosis (Genetic heterogeneity of primary familial and congenital polycythemia, 2001).
The Epo/ Epo-R complex is internalised following de-phosphorylation of Epo-R. Gross and Lodish have recently shown that 60% of internalised Epo is re-secreted, while 40% is intracellularly degraded.
Feedback regulation of EPO
EPO regulation follows a feedback mechanism measuring blood oxygenation. The controlled variable is not the concentration of erythrocytes or of haemoglobin in blood, but the tissue O2 pressure (pO2), which depends on the haemoglobin concentration, the arterial pO2, the O2 affinity of the haemoglobin and the rate of blood flow. Because the tissue pO2 is the controlled variable, residence at high altitudes leads to a stimulation of erythropoiesis, and in the long term to erythrocytosis (increase in red blood cells mass).
The kidney is very appropriate for controlling Epo production because the pO2 in the renal cortex is little affected by the rate of blood flow as the renal O2 consumption changes in proportion with the glomerular filtration rate (Why the kidney?, 1985).
Oxygen-Dependent Regulation of EPO Synthesis
Hypoxia is the primary physiological stimulus for EPO production, which, depending on the hypoxic condition, increases serum EPO levels up to several hundred-fold. The transcriptional activator responsible for the hypoxic induction of EPO is the heterodimeric basic helix-loop-helix transcription factor HIF that binds to the hypoxia-sensitive enhancer located in the 3’-prime region of the EPO gene.
Hypoxia Inducible Factors HIFs in EPO Regulation
HIFs belongs to the PAS [Per-ARNT-Sim proteins (bHLH-PAS)] family of transcription factors and consists of an oxygen-sensitive α-subunit and a constitutively expressed β-subunit, also known as ARNT. HIFαs regulate, upon heterodimerization with HIF-β, gene expression by binding to specific DNA recognition sequences, referred to as hypoxia-response elements (HREs) (Fig. 1).
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-2α 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- α occurs at the β-domain of pVHL, which spans amino acid residues 64–154. The C-terminal α-domain links the substrate recognition component pVHL to the E3 ubiquitin ligase via elongin C. In the absence of molecular oxygen, HIF-2α is not degraded and translocates to the nucleus where it forms a heterodimer with HIF-β(or ARNT). HIF-2 α/β 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. 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. 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.
There are three known HIF α-subunits: HIF-1 α, HIF-2 α and HIF-3 α. HIF-1 α and HIF-2 α heterodimers function as transcriptional activators, splice variants of HIF-3 α have been shown to be inhibitory(Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression, 2001). HIF-1α (120 kDa) and HIF-2α (91–94 kDa) facilitates oxygen delivery and cellular adaptation to hypoxia by stimulating multiple biological processes, such as erythropoiesis, angiogenesis, and anaerobic glucose metabolism(HIF-1 and mechanisms of hypoxia sensing, 2001). However, although HIF-1 and HIF-2 share many common transcriptional targets, they also regulate unique targets and have specific biological functions. Anaerobic glycolysis, for example, appears to be predominantly controlled by HIF-1, whereas HIF-2 has emerged as the main regulator of EPO production in the adult (Acute postnatal ablation of Hif-2alpha results in anemia, 2007).
Mechanism of action
All three known HIF α-subunits, HIF-1 α, HIF-2 α, and HIF-3 α, are targeted for rapid proteasomal degradation under normoxia by the von Hippel-Lindau tumor suppressor pVHL, which acts as the substrate recognition component of an E3 ubiquitin ligase complex.
Under normal oxygen conditions, HIF-α subunit is rapidly degraded, prevent it from entering the nucleus, following ubiquitylation by the pVHL-E3 ubiquitin ligase, which precludes the formation of transcriptionally active heterodimers. pVHL-mediated polyubiquitylation of HIF- α requires hydroxylation of specific proline residues (Pro402 and Pro564 in human HIF-1 α; Pro405 and Pro531 in human HIF-2 α) within its oxygen-dependent degradation domain.
Hydroxylation of HIF- α is carried out by three major 2-oxoglutarate-dependent dioxygenases [prolyl-4-hydroxylase domain (PHD) proteins: PHD1, PHD2, and PHD3] and requires molecular oxygen, ferrous iron, and ascorbate (Oxygen sensing by HIF hydroxylases, 2004). To add complexity to the regulation of this pathway, HIF increases transcription of PHD2 and PHD3. Furthermore, protein turnover of PHD1 and PHD3 is hypoxically regulated by Siah proteins, which themselves are hypoxia inducible (Siah2 regulates stability of prolyl-hydroxylases, controls HIF1alpha abundance, and modulates physiological responses to hypoxia, 2004).
An additional hypoxic switch operates in the carboxy-terminal transactivation domain of HIF-α with oxygen-dependent asparagine (Asn803 in HIF-1α and Asn851 in HIF-2α) hydroxylation via factor inhibiting HIF (FIH), which blocks binding of transcriptional coactivators CREB-binding protein (CBP) and p300. Conversely, FIH inactivation facilitates CBP/p300 recruitment to the HIF transcriptional complex and results in increased HIF target gene expression under hypoxia or in VHL-deficient cell lines (FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor, 2002).
Prolyl-4-hydroxylase domain (PHD) proteins
On binding to Fe2+ (a non-haem iron), O2 is split with one O-atom being transferred to the amino acid residue of HIF-α and the other to α-oxoglutarate, forming CO2 and succinate.
α-Ketoglutarate antagonists, which inhibit the hydroxylation of HIF-a (HIF stabilisers), stimulate Epo production in the kidney and the liver (Mouse model for noninvasive imaging of HIF prolyl hydroxylase activity: assessment of an oral agent that stimulates erythropoietin production, 2006).
Erythropoietin is available as a therapeutic agent produced by recombinant DNA technology in mammalian cell culture and it is kown as Epoetin alfa . Authorised by the European Medicines Agency on 28 August 2007, it stimulates erythropoiesis (increases red blood cell levels) and is used to treat anemia.
In chronic kidney disease (CKD) (Management of renal anemia in 2013, 2013), anemia is believed to be a surrogate index for tissue hypoxia that continues preexisting renal tissue injury. Constant inflammatory cell infiltration and pericyte-myofibroblast transition lead to renal fibrosis and insufficiency which result in decreased production of erythropoietin. Thus far, therapeutic efforts to treat patients with chronic renal failure by administering erythropoietin have been made only to correct anemia and putative hypoxic tissue damage. The introduction of recombinant human erythropoietin (rhEPO) has marked a significant advance in the management of anemia associated with chronic renal failure. With an increasing number of patients with chronic renal failure receiving erythropoietin treatment, emerging evidence suggests that erythropoietin not only has an erythropoietic function, but also has renoprotective potential. In fact, in recent years, the additional non-erythropoietic tissue/ organ protective efficacy of erythropoietin has become evident, especially in the kidneys (Renal Cell Protection of Erythropoietin beyond Correcting The Anemia in Chronic Kidney Disease Patients, 2013).
At the same time, experimental studies have unveiled its potential neuroprotective and cardioprotective properties, occurring independently of its hematopoietic action. As with other cytoprotective agents, administration of exogenous rhEPO can confer cerebral and myocardial protection against ischemia-reperfusion injury in terms of reduction in cellular apoptosis and necrosis, as well as improvement in functional recovery(Cellular protection by erythropoietin: new therapeutic implications?, 2007).
In addition to this protective actions, the treatment with recombinant Epo or its analogues can prevent the anaemias associated with cancer, acquired immunodeficiency syndrome (AIDS), hepatitis C infection, bone marrow transplantation, myelodysplastic syndromes, autoimmune diseases. The concentration of circulating Epo is relatively low in many adult cancer patients, when related to the blood haemoglobin concentration. The primary goals of Epo therapy in tumour patients are to maintain the haemoglobin values above the transfusion trigger, increase the exercise tolerance, prevent fatigue and improve quality-of-life parameters. Antianaemic therapy is considered beneficial for the outcome of radiotherapy and chemotherapy.
In the surgical setting, Epo may be administered pre-operatively in order to stimulate erythropoiesis in phlebotomy programmes for autologous re-donation or for correction of a pre-existing anaemia, and post-operatively for recovery of red blood cell mass.
Erythropoietin has beneficial effects also in certain neurological diseases, like schizophrenia (Erythropoietin: a candidate compound for neuroprotection in schizophrenia, 2004)
Erythropoietin after a century of research: younger than ever. Wolfgang Jelkmann - Institute of Physiology, University of Luebeck, Luebeck, Germany
Hypoxic regulation of erythropoiesis and iron metabolism. Volker H. Haase - Departments of Medicine, Cancer Biology, and Molecular Physiology and Biophysics, Vanderbilt School of Medicine,Nashville, Tennessee