To watch the companion video to this series, click here.
To read the introduction to this series, click here.
WHAT EPO IS
In 1906, French researchers first postulated that a humoral factor regulated the production of red blood cells[1]. Erythropoietin (EPO) was finally isolated from the urine of anemic patients in the 1970’s[2]. EPO is the main and crucial[3] hormonal regulator of red blood cell (erythrocyte) production in the bone marrow and spleen, which is called erythropoiesis. It is a 165-amino acid glycoprotein that belongs to the family of type I cytokines, produced in adulthood by fibroblasts in the renal cortex, which stimulate the survival, proliferation, and differentiation of erythrocytic progenitors[4].
EPO is synthesized most prominently when hypoxic conditions are sensed. When that occurs, hypoxia-inducible factors (HIFs) induce the expression of genes that are normally suppressed by GATA-2 and NF-kB, particularly the EPO gene located on chromosome 7[5]. In the nervous system, it is also synthesized during infection[6], mechanical damage[7], intense neural activity[8], elevated temperature[9], and metabolic stress. It is tempting to speculate that EPO is synthesized during sauna practice, though this has yet to be studied.
THE EPO RECEPTOR
EPO is structurally defined by 4 a-helices, named A, B, C, and D. EPO binds to the EPO receptor (EPOR) at two binding sites on its molecule[10], the high-affinity first (site 1; binding affinity of 1 nM) located in helices A, B, D, and a part of the loop connecting A and B, and the low-affinity second (site 2; binding affinity of 1 mM) located in helices A and C[11]. Although the molecules affinity for site 2 is 1000x weaker than its affinity for site 1, binding at both sites appears necessary for full signal transduction, as mutation at one site is sufficient to impair signaling[12][13].
The EPOR has three isoforms that interact with each other and with EPO to dictate the effect of EPO’s eventual agonism of the EPORs. There is a full-length protein, a soluble protein lacking a transmembrane and lacking intracellular domains, and a shortened protein lacking large amounts of intracellular domains[14]. Soluble EPORs have been found in the brain, though in contrast to the full-length EPOR, its expression is downregulated in hypoxic conditions[15]. It is speculated that soluble EPORs function to modulate circulating EPO, dictating the availability of EPO to bind to full-length EPORs. Dopaminergic neurons of the substantia nigra co-express the truncated and full-length isoforms, where it appears that the truncated form may interfere with EPO signaling to the full-length form[16]. Consequently, the levels of co-expression of the truncated form may modulate sensitivity of full-length EPORs to EPO.
Overall, EPORs are expressed on progenitor but not mature cells of skeletal muscle and blood, and they are also expressed in the heart[17], kidney[18], pancreas[19], uterus[20], and brain[21]. In the brain, EPORs isoforms are expressed on neurons, astrocytes, oligodendrocytes, and cerebral endothelial cells[22].
EPO RECEPTOR SIGNALLING
When agonized by EPO at sites 1 and 2, EPOR then activates signaling pathways including the Janus kinase (JAK-2[23]) and the signal transducer and activator of transcription 5A and 5B (STAT-5[24]) pathways[25]. The EPOR contains intracellular domains associated with members of the JAK-2 pathway, without which it would be unable to phosphorylate[26]. Binding at EPOR activates JAK-2 by transphosphorylation, and once activated JAK-2 phosphorylates receptors on tyrosines, which then become docking sites for the STAT-5 transcription factors. STAT-5 transcription factors located at the receptor are then phosphorylated by JAK-5, causing their disassociation from the receptor, translocation to the nucleus of the cell, and transcription of cytokine-responsive genes that regulate cell proliferation, differentiation, and apoptosis[27]. Note that the STAT-5 pathway is also phosphorylated by other cytokines, including growth hormone, interleukin-2, and interleukin-3[28].
Additionally, there is evidence that the EPOR also activate the phosphoinositide-3-kinase (PI3K-AKT) and mitogen-activated protein kinase (MAPK) pathways[29].
ALTERNATIVE RECEPTORS FOR EPO
There is evidence that EPO exerts some of its biological effects independent of its namesake receptor. The splice variant of EPO that lacks the third exon of the full-length EPO, EV-3, does not activate the EPOR, does not stimulate erythropoiesis, and yet induces neuroprotection in rodents[30]. Moreover, human engineered variants including CEPO, Epobis, and HBSP (discussed in detail in the third article of this series) do not induce erythropoiesis but also produce neuroprotection[31][32][33][34]. While EPO has a greater affinity for the hematopoietic EPOR than it does for non-hematopoietic, tissue-protective receptors, it also appears to require longer exposure to EPORs than it requires for the non-hematopoietic receptors in order to exert an effect[35]. Thus far, candidate receptors for EPO’s non-erythropoietic, neuroprotective effect are the common b chain receptor, the ephrin B4 receptor, and the cytokine receptor-like factor 3.
COMMON BETA CHAIN RECEPTORS (BCR)
Also known as CD131, the BCR is a receptor from the cytokine type I receptor family that forms heteromeric receptors with other cytokine receptors involved in hematopoiesis, including those of interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF)[36]. It is co-expressed with the EPOR in a tissue protective heteroreceptor[37] that is developed even in the absence of EPO[38], though it is EPOR is expressed 3x more than BCR in hippocampal regions of rodents[39].
BCR’s activity is upregulated by hypoxic conditions, inflammation, and metabolic stress[40][41]. EPO induces phosphorylation of the BCR[42] and the EPOR/BCR complexes activate JAK similarly to the homomeric EPORs, producing similar downstream responses. The man-made EPO variants CEPO[43] and pHBSP[44] have been shown to produce selective agonism of heteromeric BCR/EPOR units.
Because BCR/EPOR heteromeric units are not expressed in all brain regions that have been observed to receive a neuroprotective effect, and because the EPO splice variant EV-3 is neuroprotective but neither agonizes EPOR homomeric units nor BCR/EPO heteromeric units[45], the neuroprotection offered by EPO is mediated by more than EPOR and BCR.
EPHRIN B4 RECEPTOR (EPHB4)
Ephrins are proteins that characteristically agonize the ephrin receptors (EPH), with mammalian A-ephrins binding to 9 EPH-A receptors and B-ephrins binding to 5 EPH-B receptors. The ephrin signaling to EPH is involved in not only hematopoiesis, angiogenesis, and cancer cell regulation[46], but also neurogenesis[47], axon growth[48], synapse formation and plasticity[49], dendritic morphology[50], and memory development[51].
In studies on cancer cells, it was shown that EPH-B4 receptors are agonized by both ephrin B2 and EPO, and that they are co-expressed with EPORs on heteromeric units, as EPO is co-expressed with BCRs in heteromeric units. Interestingly, EPH-B4 receptors are over 30x less sensitive to EPO than EPORs, indicating that their role likely only predominates under a paucity of EPOR receptors. It was also shown that expression of EPH-B4s in cancer cells was correlated to higher mortality rates, though expression of EPORs was not, and that EPO treatment worsened patient outcomes, likely through agonism of the EPH-B4 receptors. The neurons of rodents also co-express EPH-B4 receptors and EPORs, indicating that some of the neurotrophic and memory-enhancing effects of EPO may be mediated by the EPH-B4 receptors[52].
CYTOKINE RECEPTOR-LIKE FACTOR 3 (CRLF3)
The CRLF3 is a little-studied cytokine receptor that has a docking site for JAK[53]. An in vitro study on beetle neurons has shown that cell death resulting from hypoxia and serum deprivation can be averted by administration of both EPO and EV-3, which does not agonize the EPO receptor. However, knock-down of the CRLF3 receptor abolishes EV-3’s protective effect on neuronal survival, implying that CRLF3 mediates EV-3’s neuroprotective effect in beetles, although the exact mechanisms are yet unknown[54].
Having discussed endogenous EPO and provided a summary of its action on known receptors, the next article will summarize its effects on health, with a particular focus on cognitive health.
To read the second article in the series, click here.
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