(Post updated on June 21st 2017)
Erythropoietin (EPO), a 34kD 166 amino acid polypeptide, is the main regulator of erythrocyte production. It acts via the erythropoietin receptor (EPOR).
Importance of Erythropoietin Signalling
No inactivating mutations of EPO signalling pathway are known. Mice with deletion of EPO or EPOR gene die of anaemia at a gestational age of 12-13 days. They have some haematopoiesis in the yolk sac but none in in foetal liver. CFU-E do not survive in the absence of erythropoietin. Erythropoietin is also essential for survival of the more mature population of BFU-E. EPO reduces apoptosis. The effects are mediated by STAT5 (signal transducer and activator of transcription 5). STAT5 induces the production of the anti-apoptotic protein bcl- x by binding to it’s promoter. As will be discussed below the non-receptor tyrosine kinase Janus kinase 2 (JAK2) activates STAT. Erythropoietin has effect on a range on non-errythroid tissue. These are beyond the scope of this article and is discussed elsewhere (JEM 2013;210:205).
Control of EPO Production
Erythropoietin is produced in response to hypoxia by the interstitial fibroblasts of the kidney. The induction of erythropoietin secretion by hypoxia involves
- Hypoxia inducible factor (HIF) that promotes the transcription of genes induced by hypoxia. There are three HIF, HIF1, HIF2 and HIF3
- HIF prolyl hydroxylase (PHD) an enzyme that uses oxygen as a substrate and is inactive in hypoxia. It promoted the degradation of HIF.
- pVHL (von Hipple-Lindau tumour suppressor gene product) is involved in degradation of HIF
Protein synthesis needs energy. It may appeal to common sense to synthesise proteins only when needed and conserve energy. Increasing protein synthesis is a slow process making it unsuitable for situations where a rapid response is needed. Hypoxia needs a rapid response. The alternate strategy adopted by the body is to synthesise a protein continuously and control the levels of the protein by controlling degradation. A peptide may be made inactive by breaking just one critical bond. Synthesis needs building tens to hundred bonds. Degradation can be stopped almost instantaneously resulting in a rapid rise of the desired molecule. HIFs are synthesised and degraded continuously. The degradation mechanism is hypoxia sensitive allowing for a rapid rise of HIFs and consequently in the transcription of genes under control of HIFs as the partial pressure of oxygen falls.
There are three HIFs, HIF-1, HIF-2 and HIF-3. HIF-2 controls erythropoietin production. HIF-2 has two units, α and β. The levels of the β subunit are constant. The levels of the α vary inversely with oxygen availability and determine the HIF 2 concentration activity. The α subunit is continuously being synthesized. Synthesis is matched by degradation by a proteolytic system known as proteasome. Proteasome only degrades peptides marked for destruction. Peptides are marked for destruction by tagging them by a multiple molecules of a protein ubiquitin. Ubiquitin is transferred by an enzyme complex known as ubiquitin E3 ligase. This complex consists of pVHL (product of the von Hipple-Lindau tumour suppressor gene), elongins B and C, cullin 2 and ring box 1 (Rbx1). pVHL identifies targets giving the ubiquitin E3 ligase specificity for HIF-1 and HIF-2. HIF needs to be hydroxylated at proline residues before polyubiquitination. The hydroxylation is brought about at proline residues by HIF prolyl hydroxylase (PHD). Oxygen is one of the substrates for PHD. In hypoxic conditions hydroxylation and consequently polyubiquitination does not take place. Proteasomal destruction stops and the levels of HIF-2α levels rapidly rise. HIF-2 translocates to the nucleus where it combines with HIF-2β. The heterodimer (HIF 2) acts on segments of DNA, known as hypoxia response elements, flanking the erythropoietin gene finally leading to erythropoietin synthesis. HIF 2 along with HIF 1, which is regulated by mechanisms identical to those regulating HIF 2, promotes the expression of multiple genes of proteins involved in response to hypoxia.
Mechanism of Action
EPO acts via the EPOR. Binding of erythropoietin has proliferative and anti-apoptotic effects. Receptor activation results in activation of a cascade of enzymes by phosphorylation. These include STATs (a family of seven proteins), Pi3K and RAS/MAPK/Erk. Phosphorylation can either be brought about by the tyrosine kinase activity of the receptor or, as is the case with the erythropoietin receptor, by a non-receptor tyrosine kinase associated with the receptor. The Janus kinase 2 (JAK2) is the non-receptor tyrosine kinase that associates with the erythropoietin receptor. The EPO receptor is a homodimer (a dimmer made from similar monomers). Each monomer associates with a JAK2 molecule. Binding of EPO to EPOR brings about a conformational change in the receptor bringing the two JAK2 moleclues in proximity that results in transautophosphorylation and activation of the JAK2 kinases. Activated JAK2 phosphorylates tyrosine residues on the receptor forming docking sites for molecules that activate the following pathways:
- Dimerization and translocation of STAT5 to the nucleus where it induces transcription of genes involved in proliferation and cell survival. STAT pathway appears to be the most important pathway for EPO action.
- Phophoinositide-3-kinase (PI3-K) mediated induction of several anti-apoptotic proteins e.g. Bcl-2 abd BclX. As mentioned above STAT5 is also involved in induction of bcl-x.
- Activation of Ras/extracellular-signal-regulated kinase mitogen-activated protein (RAS/Erk/MAP) kinase pathway that sustains proliferation.
The EPO signalling is short lasting and the activated EPOR signal pathway returns to normal levels in 30-60 minutes. The cytoplasmic portion of the receptor is polyubiquitinated and degraded by proteasome. The extracellular portion bound to EPO is internalized and degraded. Regulators of cytokine activity can inhibit EPOR.
Mutations and Therapeutic Manipulation of EPO Signalling
Acquired (somatic) mutations of the JAK2 kinase are associated with myeloproliferative disease. The JAK2V617F mutation is seen in about 95% of the patients of polycythaemia vera and about 50% the patients with essential thrombocytosis and idiopathic myelofibrosis. JAK2V617F causes the JAK2 molecule to be constitutively (continuously, irrespective of EPO binding to EPOR) active eliminating the need for binding of EPO to EPOR for activation pathways stimulated by EPOR. JAK2 exon 12 mutations are found in patients with JAK2V617F negative polycythemia vera. These patients, unlike those with JAK2V617F positive patients, do not show thrombocytosis or leucocytosis. Inherited (germline) mutations in the EPOR, HIF 2α, VHL gene and the PHD gene have been associated with congenital erythrocytosis. The erythrocytosis seen in patients with EPOR mutations is primary, i.e. with low EPO levels. Other mutations result in secondary erythrocytosis, i.e. with inappropriately high EPO levels. Inhibitor of HIF prolyl hydroxylase FG-2216 and FG-4592 are under evaluation for treatment of anaemia associated with chronic kidney disease.