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The EPO receptor is a member of the haemopoietic cytokine receptor superfamily. Its intracellular domain displays no known catalytic activity but it appears to couple directly to the JAK 2 kinase (Chapter 4), which probably 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 transduction remain to be elucidated in detail.
Binding of EPO to its receptor stimulates the proliferation of BFU-E cells and triggers CFU-Es to undergo terminal differentiation. As well as such stimulatory roles, EPO may play a permissive role, in that it also appears to inhibit apoptosis (programmed cell death) of these cells. With this scenario, normal serum EPO levels permit survival of a specific fraction of BFU-E and CFU-Es, which dictates the observed rate of haemopoiesis. Increased serum EPO concentrations permit survival of a greater fraction of these progenitor cells, thus increasing the number of red blood cells ultimately produced. The relative physiological importance of EPO stimulatory versus permissive activities has yet to be determined.
Regulation of EPO production
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. Under normal conditions, when the producer cells are supplied with adequate oxygen via the blood, EPO (or EPO mRNA) levels are barely detectable. However, the onset of hypoxia (a deficiency of oxygen in the tissues) results in a very rapid increase of EPO mRNA in producer cells. This is followed within 2 h by an increase in serum EPO levels. This process is prevented by inhibitors of RNA and protein synthesis, indicating that EPO is not stored in producer cells, but synthesized de novo when required.
Interestingly, hypoxia prompts increased renal and hepatic EPO synthesis in different ways. In the kidney, the quantity of EPO produced by an individual cell remains constant, while an increase in the number of EPO-producing cells is evident. In the liver, the quantity of EPO produced by individual cells appears to simply increase in response to the hypoxic stimulus.
A range of conditions capable of inducing hypoxia stimulate enhanced production of EPO, thus stimulating erythropoiesis. These conditions include:
moving to a higher altitude;
increased renal sodium transport;
decreased renal blood flow;
increased haemoglobin oxygen affinity;
chronic pulmonary disease;
some forms of heart disease.
On the other hand, hyperoxic conditions (excess tissue oxygen levels) promote a decrease in EPO production.
The exact mechanism by which hypoxia stimulates EPO production remains to be elucidated. This process has been studied in vitro using an EPO-producing cancerous liver (hepatoma) cell line as a model system. These studies suggest the existence of a haem protein (probably membrane-bound), which effectively acts as an oxygen sensor (Figure 6.6). Adequate ambient oxygen concentration retains the haem group in an oxygenated state. Hypoxia, however, promotes a deoxy-configuration, which alters the haem conformation. The deoxy- form of haem is postulated to be capable of generating an active transcription factor which, upon migration to the nucleus, enhances transcription of the EPO gene. Evidence cited to support such a theory includes the fact that cobalt promotes erythropoiesis (cobalt can substitute for the iron atom in the haem porphyrin ring; Cobalt-haem, however, remains in the deoxy-conformation, even in the presence of a high oxygen tension). In addition to oxygen levels, a number of other regulatory factors can stimulate EPO synthesis, either on their own or in synergy with hypoxia (Table 6.6).
Therapeutic applications of EPO
A number of clinical circumstances have been identified which are characterized by an often profoundly depressed rate of erythropoiesis (Table 6.7). Many, if not all, such conditions could be/are responsive to administration of exogenous EPO. The prevalence of anaemia, and the medical complications which ensue, prompts tremendous therapeutic interest in this haemopoietic growth factor. EPO has been approved for use to treat various forms of anaemia (Table 6.8). It was the first therapeutic protein produced by genetic engineering, whose annual sales value topped $1 billion. Its current annual sales value is now close to $2 billion. EPO used therapeutically is produced by recombinant means in CHO cells.
Neorecormon is one such product. Produced in an engineered CHO cell line constitutively expressing the EPO gene, the product displays an amino acid sequence identical to the native human molecule. An overview of its manufacturing process is presented in Figure 6.7. The final freeze-dried product contains urea, sodium chloride, polysorbate, phosphate buffer and several amino acids as excipients. It displays a shelf-life of 3 years when stored at 2-8 °C. A pre-filled syrine form of the product (in solution) is also available, which is assigned a 2 year shelf-life at 2-8 °C.