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Development of bioassays and radioimmunoassays, along with the later development of specific mRNA probes, allowed determination of the sites of production of EPO in the body. It has now been established that EPO in the human adult is synthesized almost exclusively by specialized kidney cells (peritubular 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 fetus.
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, while small quantities of human EPO was later purified (in 1977) from over 2500 litres 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 Chinese hamster ovary (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 6.5.
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 (colony forming unit-erythroid), require the presence not only of EPO but also of 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 fewer EPO receptors on their cell surfaces. Erythrocytes themselves are devoid of EPO receptors. EPO binding to its receptor on CFU-E cells promotes their differentiation into pro-erythroblasts and the rate at which this differentiation occurs
Haemopoietic stem cell
Figure 6.5. 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
appears to determine the rate of erythropoiesis. CFU-E cells are also responsive to insulin-like growth factor 1 (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.
HAEMOPOIETIC GROWTH FACTORS 267
The EPO receptor and signal transduction
The availability of biologically active 125I-labelled EPO facilitated the detection and study of cell surface receptors. In addition to erythroid precursors, various other cell lines were shown to express EPO receptors, at least when cultured in vitro. Many harboured two classes of receptors: a high-affinity and a low-affinity form. Most appeared to express between 1000 and 3000 receptors/cell. Radiotracer experiments illustrated that the EPO receptor is rapidly internalized after ligand binding and the EPO-receptor complex is subsequently degraded within lysosomes.
The human EPO receptor is encoded by a single gene on chromosome 19. The gene houses eight exons, the first five of which appear to code for the 233 amino acid extracellular receptor portion. The sixth encodes a single 23 amino acid transmembrane domain, while the remaining two exons encode the 236 amino acid cytoplasmic domain. The mature receptor displays a molecular mass of 85-100 kDa. It is heavily glycosylated through multiple O-linked (and a single N-linked) glycosylation site. High- versus low-affinity receptor variants may be generated by self-association.