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IFGP family protein 3
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[erythropoietin] also abbr. Ep.
ECSA (erythroid colony stimulating activity);
ESF (erythropoiesis stimulating factor).
Epo is predominantly synthesized and secreted by tubular and juxtatubular capillary endothelial and interstitial cells of the kidney. Approximately 10-15 % of the total amount of Epo comes from extrarenal sources and is predominantly produced by hepatocytes and Kupffer cells of the liver.
In the fetus the liver is the main source of Epo. It appears that the switch in the synthesis from liver to kidney which takes place after birth is genetically determined. The synthesis of erythropoietin, in liver and kidney is inducible by anemia of various origins, a fall of the arterial oxygen tension caused by either cardiopulmonary disorders or by a decrease of the oxygen tension in the inspiratory gas. Hepatic synthesis of Epo is enhanced after hepatic viral infections and exposure to hepatotoxic substances.
Macrophages have been described also to produce Epo. The factor is produced also by some kidney and liver tumors, fibroleiomyomas of the uterus, cerebral hemangioblastomas, and dermoid cysts of the ovary.
The synthesis of Epo in vitro can be inhibited in hepatoma cells and also in serum-free perfused rat kidneys by IL1 and TNF-alpha. This synthesis is not affected by IL6, TGF-beta and IFN-gamma.
Epo is a relatively heat- and pH-stable acidic (pI = 4.5) protein of 34-37 kDa. It is N-glycosylated at Asn24, Asn36, Asn83 and O-glycosylated at Ser126. Several differently glycosylated variants are synthesized. In addition Epo is also sialylated. Epo contains two disulfide bonds (positions 7/161; 29/33). The protein is formed as a precursor of 193 amino acids that yields a mature protein of 166 amino acids. The sequences of simian, murine, and human Epo show a sequence identity of 95 % and 85 %, respectively.
Approximately 40 % of the molecular mass of Epo is due to its glycosylation. Glycosylation is an important factor determining the pharmacokinetic behavior of Epo in vivo. Non-glycosylated Epo has an extremely short biological half life. It still binds to its receptor and may even have a higher specific activity in vitro (see also: Response elements).
Wrighton et al (1996) have described a 20-residue cyclic peptide unrelated in sequence to Epo, which binds to the Epo receptor with high affinity and displays the bioactivities of Epo (see also: Peptide mimetics). For a recombinant modified human erythropoietin see also: darbepoetin alfa.
The human Epo gene maps to chromosome 7q21-q22. It contains at least five exons. The DNA sequences of the human and the murine factor show 82 % homology. Transcriptional response of the Epo gene to hypoxia is mediated in part by promoter sequences and to a greater extent by a 24 bp sequence 3' to the human Epo gene functioning as a hypoxia-responsive transcriptional enhancer.
The biological activity of Epo is mediated by specific receptors expressed at 300-3000 copies/cell (approved gene symbol: EPOR). The receptor is expressed also by cell types not responding to Epo. Pluripotent embryonic stem cells and early multipotent hematopoietic cells (see also: hematopoiesis, hematopoietic stem cells) express receptor transcripts. The commitment to non-erythroid lineages (for example, macrophages and lymphocytes) is accompanied by the cessation of receptor expression.
The murine receptor is a protein of 507 amino acids with a single membrane-spanning domain. The cytoplasmic domain has a length of 236 amino acids. A point mutation at codon 129 of the murine Epo receptor gene results in constitutive activation. Mice expressing the aberrant receptor develop erythrocytosis and splenomegaly. Clonal growth factor-independent (see also: Factor-dependent cell lines), proerythroblast cell lines that express Epo receptor have been isolated from the spleen of these animals.
Heterologous expression of the human cDNA in COS cells yields a protein of about 66 kD (508 amino acids). Both the cDNA and the protein sequence of the human receptor are 82 % homologous to the murine receptor. The human Epo receptor gene consists of 8 exons and has a length of approximately 6 kb. It maps to chromosome 19p13.3. It is a member of a cytokine receptor family including receptors for Growth hormone, IL6, and IL2.
The Epo receptor is internalized after Epo has bound. The details of post receptor signal transduction processes are largely unknown. The activation of adenylate or guanylate cyclase does not seem to be involved although cAMP and cGMP may modulate receptor signals. Binding of Epo to its receptor activates phospholipases A2 and C. This leads to a release of membrane phospholipids (enhancement of lipoxygenase mediated arachidonic acid metabolism), the synthesis of diacyl glycerol, an increase in intracellular calcium levels (see also: Calcium ionophore) and intracellular pH. Phospholipase C induction also leads to an increased expression of the fos oncogene and the myc oncogene.
A point mutation in codon 129 of the murine Epo receptor has been shown to cause the constitutive activation of the receptor. Mice infected with recombinant viruses carrying the mutated receptor develop a pronounced erythrocytosis. Cells expressing the mutated receptor cause an erythroleukemia following their injection into experimental mice.
Epo receptor signaling has been shown to involve the tyrosine kinase JAK2 (see: Janus kinases). For a protein involved in signal transduction see also: CIS (cytokine inducible SH2-containing protein).
Human Epo is biologically active in rodents. Its synthesis is subject to a complex control circuit which links kidney and bone marrow in a feedback loop. Synthesis depends on venous oxygen partial pressure and is increased under hypoxic conditions. The oxygen sensor in the kidney is believed to be a heme protein. Epo production is influenced also by a variety of other humoral factors, including, among others, testosterone, thyroid hormone, Growth hormone, and catecholamines.
Several immunomodulatory cytokines such as IL1, TNF-alpha and IL6 have been shown to reduce the synthesis of Epo in vitro.
Epo is mainly a differentiation factor for late determined and differentiated progenitor cells of erythropoiesis. It determines their differentiation and maturation into erythrocytes. In addition Epo also regulates the proliferation of erythropoietic progenitor cells. The Epo sensitivity progressively increases with differentiation of immature progenitor cells (see: BFU-E, CFU-E. Epo has not been shown to act on pluripotent hematopoietic stem cells (see also: hematopoiesis).
The pathophysiological excess of Epo leads to erythrocytosis. This is accompanied by an increase in blood viscosity and cardiac output and may lead in some cases also to heart failure and pulmonary hypertension.
To a certain extent Epo is also a costimulating factor of megakaryocytopoiesis. The activity of Epo is synergised by IL4. The suppression of erythropoiesis induced by TNF-alpha can be abolished by exogenous Epo.
Epo has been shown to act on certain human erythroleukemic cells in an autocrine manner. In addition, Epo is a mitogen and a chemoattractant for endothelial cells (see also: Chemotaxis). Epo also directly stimulates activated and differentiated B-cells and enhances B-cell immunoglobulin production and proliferation.
TRANSGENIC ANIMALS, KNOCK-OUT, AND ANTISENSE STUDIES
Studies of Epo transgenic mice have revealed that different DNA sequences flanking the Epo gene control liver versus kidney expression of the gene and that some of these sequences are located 3' to the gene. Some 3' flanking sequences of approximately 50 nucleotides also function as an enhancer which can mediate transcriptional induction in response to hypoxia.
The consequences of a deregulated expression of Epo have been demonstrated in transgenic mice expressing increased levels of human Epo in all transgenic tissues analyzed. Overexpression of Epo leads to a polycythemia and a general increase in numbers of erythrocytes, hematocrit values, and hemoglobin values. A significant reduction of platelets is observed also in these animals.
DETECTION AND ASSAY METHODS
Epo can be assayed by employing cell lines such as HCD57, NFS-60, TF-1 and UT-7, which respond to the factor (see also: bioassays). Epo activity can be assessed also in a colony formation assay by determining the number of CFU-E from bone marrow cells. An alternative and entirely different detection method is RT-PCR quantitation of cytokines. For further information see also subentry "Assays" in the reference section. For further information on assays for cytokines see also: bioassays, cytokine assays.
CLINICAL USE AND SIGNIFICANCE
Chronical kidney disease causes the destruction of Epo-producing cells in the kidney. The resulting lack of Epo frequently induces hyporegenerative normochrome normocytic anemias. The main clinical use of Epo is therefore the treatment of patients with severe kidney insufficiency (hematocrit below 0.3) who usually also receive transfusions. Renal anemia is seen frequently as a complication of terminal kidney insufficiency occurring in approximately 50 % of dialysis patients. While dialysis is a means to overcome disturbances of water, electrolyte, and acid-base balance in uremic patients, it cannot balance the loss of endocrine kidney functions. Prolonged bleeding times have been shown to be improved by Epo treatment of uremic patients. In uremic patients on chronic maintenance hemodialysis treatment with recombinant Epo (see also: Recombinant cytokines) also improves platelet adhesion and aggregation in addition to and independent of its effect on the hematocrit.
The most important complication in the treatment of renal anemia with Epo is hypertony. Increases in urea, potassium, and phosphate levels are also possible. An increase in blood viscosity must be considered. Iron deficiency is the main reason for insufficient response to recombinant Epo therapy (see also: Recombinant cytokines). It can be overcome by concomitant intravenous iron supply.
Epo treatment has been described also to lead to an expansion of thrombopoietic progenitor cells and circulating platelets (see also: hematopoiesis).
One important application of Epo may be the pre-surgical activation of erythropoiesis, allowing the collection of autologous donor blood.
The use of Epo has been suggested also for non-renal forms of anemia induced, for example, by chronic infections, inflammatory processes, radiation therapy, and cytostatic drug treatment, and encouraging results in patients with non-renal anemia have been reported.
See REFERENCES for entry Epo.
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