Erythropoietin

EPO is an additional haemopoietic growth factor. It is primarily responsible for stimulating and regulating erythropoiesis (i.e. erythrogenesis, the production of red blood cells) in mammals.

The erythron is a collective term given to mature erythrocytes, along with all stem-cell-derived progeny that have committed to developing into erythrocytes. It can thus be viewed as a disperse organ whose primary function relates to transport of oxygen and carbon dioxide (haemoglobin constitutes up to one-third of the erythrocyte cytoplasm), as well as maintaining blood pH. An average adult contains in the region of 2.3 X 1013 erythrocytes (weighing up to 3 kg). They are

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Figure 10.3 Three-dimensional structure of EPO. Structural details courtesy of the Protein Data Bank, http://www.rcsb.org/pdb/

synthesized at a rate of about 2.3 million cells per second and have a circulatory life of approximately 120 days, during which they travel almost 200 miles.

EPO is an atypical cytokine, in that it acts as a true (endocrine) hormone and is not synthesized by any type of white blood cell. It is encoded by a single copy gene, located on (human) chromosome 7. The gene consists of four introns and five exons. The mature EPO gene product contains 165 amino acids and exhibits a molecular mass in the region of 36 kDa (Figure 10.3). EPO is a glycoprotein, almost 40 per cent of which is carbohydrate. Three N-linked and one O-linked gly-cosylation sites are evident. The O-linked carbohydrate moiety appears to play no essential role in the (in vitro or in vivo) biological activity of EPO. Interestingly, removal of the N-linked sugars, although having little effect on EPO's in vitro activity, all but destroys its in vivo activity. The sugar components of EPO are likely to contribute to the molecule's solubility, cellular processing and secretion, as well as its in vivo metabolism.

Incomplete (N-linked) glycosylation prompts decreased in vivo activity due to more rapid hepatic clearance of the EPO molecule. Enzymatic removal of terminal sialic acid sugar residues from oligosaccharides exposes otherwise hidden galactose residues. These residues are then free to bind specific hepatic lectins, which promote EPO removal from the plasma. The reported plasma t1/2 value for native EPO is 4-6 h. The t1/2 for desialated EPO is 2 min. Comparison of native human EPO with its recombinant form produced in CHO cells reveals very similar glycosylation patterns.

EPO in the human adult is synthesized almost exclusively by specialized kidney cells (peritubu-lar interstitial cells of the kidney cortex and upper medulla). Minor quantities are also synthesized in the liver, which represents the primary EPO-producing organ of the foetus.

EPO is present in serum and (at very low concentrations) in urine, particularly of anaemic individuals. This cytokine/hormone was first purified in 1971 from the plasma of anaemic sheep, and small quantities of human EPO were later purified (in 1977) from over 2500 l of urine collected from anaemic patients. Large-scale purification from native sources was thus impractical. The isolation (in 1985) of the human EPO gene from a genomic DNA library facilitated its transfection into CHO cells. This now facilitates large-scale commercial production of the recombinant human product (rhEPO), which has found widespread medical application.

EPO stimulates erythropoiesis by:

• increasing the number of committed cells capable of differentiating into erythrocytes;

• accelerating the rate of differentiation of such precursors;

• increasing the rate of haemoglobin synthesis in developing cells.

An overview of the best-characterized stages in the process of erythropoiesis is given in Figure 10.4. The erythroid precursor cells, BFU-E (burst forming unit-erythroid), display EPO receptors on their surface. The growth and differentiation of these cells into CFU-Es (where E stands for erythroid) require the presence of not only EPO, but also IL-3 and/or GM-CSF. CFU-E cells display the greatest density of EPO cell surface receptors. These cells, not surprisingly, also display the greatest biological response to EPO. Progressively more mature erythrocyte precursors display progressively less EPO receptors on their cell surfaces. Erythrocytes themselves are devoid of EPO receptors. EPO binding to its receptor on CFU-E cells promote their differentiation into proerythroblasts and the rate at which this differentiation occurs appears to determine the rate of erythropoiesis. CFU-E cells also are responsive to IGF-1.

Although the major physiological role of EPO is certainly to promote red blood cell production, EPO mRNA has also been detected in bone marrow macrophages, as well as some multipotential haemopoietic stem cells. Although the physiological relevance is unclear, it is possible that EPO produced by such sources may play a localized paracrine (or autocrine) role in promoting erythroid differentiation. The level of EPO production in the kidneys (or liver) is primarily regulated by the oxygen demand of the producer cells, relative to their oxygen supply.

The EPO receptor is a member of the haemopoietic cytokine receptor superfamily. Its in-tracellular domain displays no known catalytic activity, but it appears to couple directly to the JAK2 kinase (Chapter 8) that likely promotes the early events of EPO signal transduction. Other studies have implicated additional possible signalling mechanisms, including the involvement of G proteins, protein kinase C and Ca2+. The exact molecular events underlining EPO signal trans-duction remain to be elucidated in detail.

10.2.6.1 Therapeutic applications of erythropoietin

A number of clinical circumstances have been identified which are characterized by an often profoundly depressed rate of erythropoiesis (Table 10.7). Many, if not all, such conditions could

Haemopoietic stem cell

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Immature BFU-E

!

Mature BFU-E

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!

CFU-E

!

Proerythroblast

V

!

Basophilic erythroblast

\

!

Polychromatophilic erythroblast

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Figure 10.4 Stages in the differentiation of haemopoietic stem cells, yielding mature erythrocytes. The EPO-sensitive cells are indicated. Each cell undergoes proliferation as well as differentiation; thus, greater numbers of the more highly differentiated daughter cells are produced. The proliferation phase ends at the reticulocyte stage; each reticulocyte matures over a 2-day period, yielding a single mature erythrocyte

Figure 10.4 Stages in the differentiation of haemopoietic stem cells, yielding mature erythrocytes. The EPO-sensitive cells are indicated. Each cell undergoes proliferation as well as differentiation; thus, greater numbers of the more highly differentiated daughter cells are produced. The proliferation phase ends at the reticulocyte stage; each reticulocyte matures over a 2-day period, yielding a single mature erythrocyte be/are responsive to administration of exogenous EPO. The prevalence of anaemia, and the medical complications that ensue, prompts tremendous therapeutic interest in this haemopoietic growth factor. EPO has been approved for use to treat various forms of anaemia (Table 10.2). It was the first therapeutic protein produced by genetic engineering whose annual sales value topped US$1 billion. Current combined annual sales value of commercialized recombinant EPO products is now close to US$10 billion.

Table 10.7 Diseases (and other medical conditions) for which anaemia is one frequently observed symptom

Renal failure

Rheumatoid arthritis

Cancer

AIDS

Infections

Bone marrow transplantation

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