EPO: From Cytokine to Speed

Background

Last fall, I was enrolled in a metabolism course at Middlebury.  We were assigned a 40 minute presentation on a metabolic disease.  To the eleven other students in the class who were planning on going to medical school, this was an interesting topic and was designed with their interests in mind.  Metabolism is a fascinating field of study with many aspects, so I asked my professor if I could do my presentation on a different metabolic topic: PEDs.  After some preliminary research, I settled on presenting about EPO.  The following is what I came up with (there is some overlap with a previous post).

What is this "EPO" thing, anyway?

EPO--short for erythropoietin (derived from erythropoiesis, combining the Greek erythro meaning red and poiesis meaning generation; an erythrocyte is a red blood cell, or RBC--the cells responsible for transporting oxygen in the blood)--is a cytokine (protein signaling molecule) that induces red blood cell production by preventing red blood cell progenitors from committing apoptosis (cellular suicide or death) and allows them to proliferate and differentiate.  EPO occurs naturally in your body!  It is a 34 kilodalton glycoprotein that is about 165 amino acids long.  Roughly 40% of its weight comes from glycosylation, which is the addition of sugar groups to amino acid residues on the peptide chain (in the case of EPO, this is mostly N-linked glycosylations, mainly occuring on asparagine residues).  In addition to its primary role as an inducer of RBC production, EPO has a variety of secondary functions, including neural production during stroke (EPO can cross the blood-brain barrier) and implication in apoptotic pathways through the PI3-kinase pathway.  In the fetus, the EPO gene is expressed in the liver and after birth it is expressed in the kidney.  In addition to its well documented use as a PED, synthetic EPO is an incredibly successful drug in the clinic, used mainly to treat patients with anemia (RBC deficiency).  It has also been used in patients who have diabetes, Alzheimer's and cardiovascular disease.  There are some drawbacks, though, particularly in cancerous patients (many of whom are prescribed EPO): synthetic EPO has been shown to block tumor cell apoptosis, enhance tumor progression rates, increase the metastatic rate of cancer, and negating radiation treatment by assisting in tumor angiogenesis (for those of you keeping count, that's 4 out of the 6 Hall Marks of Cancer--original paper and follow-up; some of the more generally interesting and accessible journal articles you'll find).

How does EPO get produced in the body?

http://www.sciencedirect.com/science/article/pii/S0006295211004291

I'm going to start large and then get smaller.  EPO production is dependent on physiologic conditions: hypoxia in arterial blood running through the kidney or anemia.  Under these conditions, the EPO gene is expressed in the kidney and produces the EPO protein (this is known as the Central Dogma of Molecular Biology: DNA is transcribed into RNA, which, after processing, is translated into a protein).  EPO is then directed to cells expressing its receptor, EPOR.  Remember that EPO is a signaling molecule: that means it "swims" around the extracellular matrix and blood until it finds a receptor protein on the surface of a cell specifically tailored for it to bind to.  Compounds of EPO's size cannot move directly through the cell membrane to catalyze chemistry in the cell; instead, they bind to a receptor that can then catalyze many things inside the cell.  In this case, EPOR is a Type I transmembrane receptor protein (example).  Type I means that it is composed of alpha-helicesand transmembrane means that the protein sits directly in the plasma membrane and has parts of it exposed to the outside of the cell and into the inside of the cell.  EPOR is a member of the cytokine receptor superfamily, which is a group of cytokine receptors that have four conserved cysteine residues and a conserved Trp - Ser - X - Trp - Ser motif in the extracellular domain (conservation, in this sense, means those amino acid residues appear in roughly the same place in the amino acid sequence in many different proteins that have similar functions.  Conserved sequences are usually pretty important for function.  Wikipedia page).  EPOR is mainly expressed in red blood cell progenitors as they are nearing death either to rescue them from apoptosis or to induce their proliferation.  There has also been research showing EPOR being involved in RBC differentiation (specialization of a cell's function).

All right, we have EPO, and it's found a receptor.  What does it do now?

Previously, I briefly mentioned that EPO works via a JAK-STAT signaling mechanism.  I'm now going to go through that in more detail.  I truly believe that one does not need a background in biology or chemistry to understand what I'm about to explain.  All you need is a healthy dose of common sense, because when you think about what's going on, it does make sense.

http://www.jbc.org/content/282/28/20059.short

Signaling that occurs via cytokine receptors is promoted by enzymes in the cell known as protein tyrosine kinases (PTKs) and is thought to be terminated by enzymes known as protein tyrosine phosphatases (PTPs; I will revisit this idea in a bit).  Like I said before, EPO is directed to cells expressing EPOR.  EPOR sits in the membrane as a homodimer (a dimer is two proteins associated with one another; thus, a homodimer is two of the same protein associated with one another) in an inactive conformation.  EPO comes in and binds to the receptor dimer and causes it to change conformation, activating it.  This idea of active and inactive conformations dependent on ligand binding or ambient conditions is a huge theme in biology; it provides a simple mechanism to turn proteins/enzymes/receptors/etc. on and off.  Upon the conformation change, JAK2 (a random kinease--seriously, JAK stands for Just Another Kinase.  Kinases are enzymes which phosphorylate things), which is associate with EPOR, is activated via an autophosphorylation mechanism (phosphorylating itself).  Phosphorylation is the addition of a phosphate group (PO4(3-)) to a moiety.  Phosphorylation is the way by which just about everything in the cell is activated; when you hear about ATP (adenosine triphosphate) being the energy currency of the cell, it activates things by putting one of its phosphate groups on that thing and becomes ADP (adenosine diphosphate).  This autophosphorylation, in addition to activating that particular JAK, promotes further autophosphorylation of other JAKs and promotes further kinase activity.  This is a positive feedback loop, which means when this thing happens, it makes more of either itself or another thing happen.  Upon the autophosphorylation of JAK2, eight tyrosine residues on the cytoplasmic side of the cell (the inside) are phosphorylated (tyrosine phosphorylation happens a lot in protein signaling pathways).  The tyrosine phosphorylation leads to SH2-dependent recruitment of STAT5.  SH2 is a conserved protein domain (in larger proteins, different parts of the protein will fold up and form their own ball-like structures that act basically independently from the other domains/rest of the protein) that is roughly 100 amino acids long that selectively binds or docks to phosphorylated tyrosine residues on other proteins.  STAT5 (Signal Transducer and Activator of Transcription 5) is activated by this selective docking.  Once STAT5 has been activated, presumably via phosphorylation, it forms an antiparallel dimer with itself (proteins aren't symmetrical and have some form of directionality, so in this case, the two STATs are running in opposite directions of one another, kind of like a two-way street) and translocates to the nucleus.  The nucleus of the cell has its own membrane (well, it really has two, but that's besides the point), so the STAT dimer manages its way through those membranes into the body of the nucleus.  Once it's in the nucleus, it acts as a transcription factor for important erythro-regulation genes as well as possibly activating GATA-1, another transcription factor for erythro-regulating genes.  Transcription factors are proteins that promote the expression of a gene into a protein (running through the Central Dogma: DNA --> RNA --> protein).

References

1. Bodary, P. F.; Pate, R. R.; Wu, Q. F.; McMillan, G. S. Effects of acute exercise on plasma erythropoietin levels in trained runners. Med. Sci. Sports Exerc. 1999, 31, 543-546.

2. Chateauvieux, S.; Grigorakaki, C.; Morceau, F.; Dicato, M.; Diederich, M. Erythropoietin, erythropoiesis and beyond. Biochem. Pharmacol. 2011, 82, 1291-1303.

3. Klingmüller, U.; Lorenz, U.; Cantley, L. C.; Neel, B. G.; Lodish, H. F. Specific recruitment of SH-PTP1 to the erythropoietin receptor causes inactivation of JAK2 and termination of proliferative signals. Cell 1995, 80, 729-738.

4. Kretz, A.; Happold, C. J.; Marticke, J. K.; Isenmann, S. Erythropoietin promotes regeneration of adult CNS neurons via Jak2/Stat3 and PI3K/AKT pathway activation. Molecular and Cellular Neuroscience 2005, 29, 569-579.

5. Maiese, K.; Chong, Z. Z.; Shang, Y. C. Raves and risks for erythropoietin. Cytokine Growth Factor Rev. 2008, 19, 145-155.

6. Roels, B.; Bentley, D. J.; Coste, O.; Mercier, J.; Millet, G. P. Effects of intermittent hypoxic training on cycling performance in well-trained athletes. Eur. J. Appl. Physiol. 2007, 101, 359-368.

7. Sasaki, A.; Yasukawa, H.; Shouda, T.; Kitamura, T.; Dikic, I.; Yoshimura, A. CIS3/SOCS-3 suppresses erythropoietin (EPO) signaling by binding the EPO receptor and JAK2. J. Biol. Chem. 2000, 275, 29338-29347.

8. Schindler, C.; Levy, D. E.; Decker, T. JAK-STAT signaling: from interferons to cytokines. J. Biol. Chem. 2007, 282, 20059-20063.

9. Smith, J. A. Exercise, training and red blood cell turnover. Sports medicine 1995, 19, 9-31.

10. Tong, W.; Zhang, J.; Lodish, H. F. Lnk inhibits erythropoiesis and Epo-dependent JAK2 activation and downstream signaling pathways. Blood 2005, 105, 4604-4612.

11. Yao, Z.; Cui, Y.; Watford, W. T.; Bream, J. H.; Yamaoka, K.; Hissong, B. D.; Li, D.; Durum, S. K.; Jiang, Q.; Bhandoola, A.; Hennighausen, L.; O'Shea, J. J. Stat5a/b are essential for normal lymphoid development and differentiation. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 1000-1005.