The Erythropoietin Receptor Signalling

Erythropoietin (EPO), a 34kD 166 amino acid polypeptide, is the main regulator of erythrocyte production. It acts via the erythropoietin receptor (EPOR). EPO signalling pathway appears to be critical for survival as no inactivating mutations of EPO or the EPOR are known. Mice with deletion of EPO or EPOR gene die of anaemia at a gestational age of 12-13 days. EPO is anti-apoptotic. The target cell is a stage between CFU-E and pronormoblast. These cells can not survive in the absence of EPO. It has been suggested that EPO has proliferative effects but the evidence is less compelling. The non-erythroid targets of EPO for which therapeutic efficacy of EPO is under evaluation include the heart and the brain.

 


 

Control of EPO Production
Erythropoietin is produced in response to hypoxia by the interstitial fibroblasts of the kidney. Hypoxia increases the levels of factors known as hypoxia inducible factors (HIF). 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. The α subunit is continuously being synthesized and destroyed. This apparently futile exercise is aimed at mounting a rapid response to hypoxia. Hypoxia prevents destruction of the α subunit allowing an almost instantaneous rise in concentration of HIF 2.

HIF 2α is destroyed by a proteolytic system known as proteasome. Proteasome destroys any peptide tagged for destruction. The tagging is done by transferring multiple molecules of a protein ubiquitin (polyubiquitination) 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 it specificity for HIF-1 and HIF-2. HIF-2α is needs to be hydroxylated at proline residues before polyubiquitination. The hydroxylation is brought about at proline residues by HIF prolyl hydroxylase (PHD) with oxygen as one of the substrates. In hypoxic conditions hydroxylation and the subsequent polyubiquitination does not take place. Proteasomal destruction stops and the levels of HIF-2α levels rapidly rise. The molecule 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 and promotes 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 EPO receptor. The EPO receptor is a homodimer (a dimer made of two similar units). A cascade of molecules that links the receptor to its action. Phosphorylation of tyrosine or serine/threonine residues on the peptides plays an important role receptor signalling. EPO receptor signals by phosphorylation of tyrosine residues. EPO receptor, unlike other receptors like insulin receptor, lacks tyrosine kinase activity. EPO receptor overcomes this limitation by associating with a cytoplasmic kinase, Janus kinase 2 (JAK2). Each peptide of EPO receptor is associated with a JAK2 molecule. Binding of EPO brings about a conformational change in the receptor bringing the two JAK2 moleclues in proximity. The proximity allows transautophosphorylation and activation of the JAK2 kinases. Activated JAK2 phosphorylates tyrosine residues on the receptor forming docking sites for molecules resulting in activation of the following pathways.

  1. 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.
  2. Phophoinositide-3-kinase (PI3-K) mediated induction of several anti-apoptotic proteins e.g. Bcl-2 abd BclX,
  3. 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 activation of EPOR 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 half the patients with essential thrombocytosis and idiopathic myelofibrosis. JAK2v617F causes the JAK2 molecule to be constitutively active eliminating the erythropoietin dependence of erythropoiesis. 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.

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