Eur. J. Biochem. 210,649 - 663 (1 992)

0FEBS 1992

Review The molecular mechanism of erythropoietin action Mark J. KOURY and Maurice C. BONDURANT Division of Hematology, Vanderbilt University School of Medicine and Veteran’s Administration Medical Center, Nashville, USA (Received July 2, 1992)

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EJB 92 0929

Erythropoietin (EPO) is the glycoprotein hormone which controls the production of erythrocytes in mammals. Erythrocytes contain hemoglobin which delivers oxygen from the lungs to other tissues throughout the body. The amount of oxygen delivered to the tissues is controlled by the number of erythrocytes in the bloodstream. Erythrocytes have a finite life span, and, therefore, maintenance of normal oxygen delivery requires continuous replacement of those aged erythrocytes normally lost from the circulation. Adjustments in this basal rate of erythrocyte production are made when tissue oxygenation becomes either decreased or increased compared to the normal level. Development of anemia due to blood loss or inspiration of decreased atmospheric oxygen at high altitude results in decreased oxygen delivery to the tissues. In response to this tissue hypoxia, plasma EPO levels increase due to increased EPO production by the kidney, erythrocyte production increases and the resulting increase in circulating erythrocytes permits more oxygen delivery. Conversely, when tissue oxygenation is increased above normal, which occurs during a sudden change from high altitude to sea level or in a transfusion of erythrocytes to greater than normal numbers, plasma EPO levels decrease, erythrocyte production is decreased and the resultant decline in circulating erythrocytes decreases oxygen delivery to the tissues. Thus, a negativefeedback mechanism exists in which the amount of oxygen delivered by erythrocytes to the body tissues determines the plasma EPO concentration, and, in turn, the EPO concentration determines the number of circulating erythrocytes by controlling the rate of erythrocyte production. Significant progress in understanding the central role of EPO in the oxygenation/EPO/erythrocyte cycle has been made in the last few years. This progress is the result of multiple research efforts, which have resulted in the purification of EPO and the cloning of the EPO gene, an increased understanding of how EPO production is controlled by oxygen in the kidney and liver, the development of in vitro model systems Correspondence to M. J. Koury, Room C-3101, Medical Center North, Vanderbilt University School of Medicine, Nashville, TN, 37232-2281, USA Fax: + 1 615 343 4589. Abbreviations. BFU-E, burst-forming-unit erythroid; CFU-E, colony-forming-unit erythroid; CHO, Chinese hamster ovary; EPO, erythropoietin; FVA cells, splenic erythroblasts from mice infected with Friend leukemia virus (anemia-inducing strain); GATA-I, transcription factor that binds DNA sequence GATA in erythroid cells; GM-CSF, granulocyte-macrophage colony-stimulating factor; HCD cells, an erythropoietin-dependent murine erythroleukemia cell line; rh-EPO, recombinant human erythropoietin; SFFVp, Friend spleen focus-forming virus (polycythemia-inducing); uh-EPO, urinary human erythropoietin; IL, interleukin.

of erythropoiesis which provide EPO-responsive cell populations, and the cloning of the EPO receptor gene. These scientific advances have also had a dramatic impact on clinical medicine. Commercially produced, recombinant human EPO is used routinely in the care of patients with renal failure. Patients with renal failure are usually very anemic, and a major factor contributing to their anemia is insufficient production of endogenous EPO. Almost all patients respond to the administration of recombinant EPO, and frequent transfusions of blood which had been required previously in their care, are not now needed. EPO therapy is being tested in patients with anemias due to a wide variety of causes other than renal failure, and its role in clinical medicine will almost certainly increase in the next few years. This review will concern the mechanism of the action of EPO: the structure and function of EPO and its receptor; the biochemical effects of EPO on its primary targets, the erythroid-progenitor cells: the cellular responses which permit the regulation of erythroidprogenitor-cell differentiation into erythrocytes. Structure of the EPO molecule EPO was purified from the urine of anemic patients (Miyake et al., 1977),and partial amino acid sequences derived from this purified EPO led to the cloning of the human EPO gene (Jacobs et al., 1985; Lin et al., 1985). Subsequently, monkey (Lin et al., 1986) and mouse (Shoemaker and Mitsock, 1986; McDonald et al., 1986) EPO genes were cloned. In each species, EPO is encoded by a single-copy gene which has five exons. The human and mouse EPO genes have 90% similar sequences immediately upstream of the transcription start site, 80% in the coding regions and 65% in the first intron (McDonald et al., 1986; Shoemaker and Mitsock, 1986). The locations of introns and splice donor and acceptor sites are conserved between human and mouse EPO genes. The mRNA for EPO contains both 5’ and 3’ untranslated regions and codes for a leader peptide sequence and a predicted mature EPO protein of 166 amino acids for human and mouse, and 168 amino acids for monkey. The secreted form of human EPO, both the naturally occurring EPO recovered from urine (uh-EPO) or the recombinant EPO (rh-EPO) expressed in Chinese hamster ovary (CHO) cells, lacks the Cterminal arginine, which is presumably removed by posttranslational cleavage (Recny et al., 1987). The amino acid sequence of human EPO is shown in Fig. IA. Human and murine EPO have four cysteines and monkey EPO has five. Internal disulfide bridges exist in human EPO, between Cys7 and Cyslbl, and between Cys29 and Cys33 (Lai et al., 1986; Davis et al., 1987). At least one of these disulfide bridges

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Fig. 1. Amino acid sequence (A) and predicted tertiary structure (B) of human EPO. (A) The site of U-glycosylation is represented by an open hexagon; sites of N-glycosylation are represented by filled hexagons. Rectangles show the a-helical sequences predicted by the model of Bazan (1990b). Dashed lines indicate disulfide bonds. (B) The predicted tertiary structure of human EPO based on folded, antiparallel arrangements of a-helices (Bazan 1990b). Glycosylation sites are designated as in (A). @-helicesare labeled A through D in order from the N-terminus to the C-terminus with intervening loops shown with an arrow directed toward the C-terminus. Disulfide bonds are also indicated. (B) was reproduced with slight modification from Krantz (1990).

is important in the secondary structure, since the biological activity of human EPO can be inactivated reversibly by reduction or irreversibly by alkylation (Sytkowski, 1980; Wang et al., 1985). Sedimentation-equilibrium studies with recombinant human EPO show a molecular mass of 30.4 kDa (Davis et al., 1987). The difference between the predicted molecular mass of 18 240 Da for the 165-amino-acid protein lacking C-terminal arginine and this experimentally determined molecular mass indicates that human EPO is 40% carbohydrate. Human EPO has four glycosylation sites: a single 0-linked site at Ser126 and three N-linked sites at Asn24, Am38 and Am83 (Lai et al., 1987; Recny et al., 1987). The N-linked glycosylation sites are conserved in murine and monkey EPO (McDonald et al., 1986; Shoemaker and Mitsock, 1986). Analysis of the isolated oligosaccharide chains from human EPO show that the majority are fucose-containing, sialylated tetraantennary oligosaccharides, some of which contain repeated N-acetyllactosamines (Sasaki et al., 1987a; Takeuchi et al., 1988). The remaining N-linked oligosaccharides are triantennary and biantennary oligosaccharides. Comparison of uh-EPO and rhEPO produced in CHO cells shows that they have the same glycosylation structure except for some slight differences in the number of sialic acids. rh-EPO molecules produced in baby hamster kidney cells or human B-lymphoblastic cells also have a very similar carbohydrate structure to uh-EPO, but some differences are found in some of the tetraantennary and triantennary oligosaccharides (Tsuda et al., 1988; Yanagi et al., 1989). The processing and secretion of EPO by the human hepatoblastoma cell line, Hep G2, is greatly retarded by tunicamycin, an inhibitor of N-linked glycosylation (Neilsen et al., 1987; Ueno et al., 1989). Similarly, processing and secretion of the rh-EPO by CHO cells is inhibited by sitedirected mutagenesis at Ser126, Am38 and Am83 (Dube et al., 1988). Subsequent studies, however, have demonstrated that Ser126 glycosylation is not necessary for correct processing or secretion (Wasley et al., 1991). The role of glycosylation in the EPO molecule appears to involve solubility, cellular processing and secretion, and in vivo metabolism. Partially glycosylated uh-EPO has a decreased solubility (Dordal et al., 1985). Structural stability of rh-EPO has been associated with glycosylation (Narhi et al., 1991). Totally deglycosylated rh-EPO has been reported to have vari-

able activities in vitro. rh-EPO produced in insect cells lacks glycosylation but is fully active in vitro (Wojchowski et al., 1987). Likewise, when rh-EPO produced in CHO cells is Ndeglycosylated it retains full activity in vitro (Tsuda et al., 1990; Sytkowski et al., 1991). However, rh-EPO produced in baby hamster kidney cells lacks activity in vitro following Ndeglycosylation (Takeuchi et al., 1990). Whereas the role of glycosylation in the in vitro activity of rh-EPO may vary depending upon the cells used to produce it, the glycosylation state of rh-EPO clearly plays an important role in EPO activity in vivo. EPO molecules produced in CHO cells which are enriched in biantennary oligosaccharides are only about 15% as active in vivo as normally glycosylated EPO or tetraantennary-enriched EPO (Takeuchi et al., 1989). Incomplete glycosylation of the N-linked oligosaccharides leads to decreased activity in vivo through more rapid hepatic clearance (Dordal et al., 1985; Takeuchi et al., 1989; Wasley et al., 1991; Yamaguchi et al., 1991). Enzymic removal of terminal sialic acids from oligosaccharides of uh-EPO or rh-EPO exposes galactose residues which bind to specific hepatocyte lectins, which in turn leads to prompt removal of the EPO molecules from the plasma (Goldwasser et al., 1974; Fukuda et al., 1989; Spivak and Hogans, 1989; Tsuda et al., 1990; Imai et al., 1990). Similarly, EPO with oligosaccharides containing more than three lactosamine repeats are rapidly bound and removed by the liver (Fukuda et al., 1989). The protein portion of EPO appears to be responsible for the specific interaction with receptors on target cells. Attempts to determine the receptor-binding sequence(s) of EPO have been made using EPO mutants and antibodies to specific sequences of the molecule. Since antibodies may block a binding site by steric hindrance, and mutant molecules can block binding sites by causing abnormal secondary or tertiary structure from altered protein folding, these results have not been conclusive. Antibodies to the N-terminal region of rh-EPO do not affect its activity (Sytkowski and Fisher, 1985; D'Andrea et al., 1990). The Cys33+Pro mutant has extremely low activity in vitro (Lin, 1987),but since the disulfide bridge disrupted by this mutation does not exist in murine EPO (McDonald et al., 1986; Shoemaker and Mitsock, 1986), the low activity may be due to an effect other than disruption of the Cys29Cys33 bridge. Among antibodies prepared to multiple peptide sequences encompassing the entire 165 amino acids of mature

EPO, only those directed against amino acids 99- 118 and 111 - 129 were able to inhibit the biological activity of EPO (Sytkowski and Donahue, 1987). Further studies with deletion mutants indicate that a critical region for biological function is at amino acids 99-110 (Chern et al., 1991). In another study, from among seven sequential deletion mutants of rhEPO involving segments for almost all of the mature protein, only the deletion of amino acids 111 -119 left EPO activity intact (Boissel and Bunn, 1990). Since EPO has not been crystallized, only predicted models of secondary and tertiary structure are available. About 50% of its secondary structure is predicted to be a-helical by circular dichroism (Lai et al., 1986; Davis et al., 1987). Bazan (1990a) has proposed that EPO belongs to a group of hormones, hematopoietic growth factors and cytokines, which have similar spacing of predicted a-helical segments. Initially, the recognition that the factors had related areas of secondary structure arose from the observation that their receptors were related in primary structure and in conserved locations of cysteine residues. In addition to EPO, these hormones and hematopoietic factors include growth hormone, prolactin, interleukins (IL) 2 - 7, granulocyte- macrophage colony-stimulating factor (GM-CSF), granulocyte colony-simulating factor (G-CSF). A model for tertiary folding for each of these hormone growth factors, including EPO, has been based on the crystallographically determined structure of growth hormone, IL-2 and GM-CSF (Bazan, 1990b; Manavalan et al., 1992). The predicted tertiary structure of EPO according to this model is shown in Fig. 1B. In this model, four antiparallel helices are joined by variable-size loops with the proposed receptor-binding portion occurring on the surface of the helix closest to the C-terminus. In EPO, this putative binding helix is comprised of amino acids 136-160. Antibodies raised against a synthetic peptide corresponding to amino acids 131 - 150 reacted with EPO but did not neutralize EPO activity (Sytkowski and Donahue, 1987), while antibodies to a peptide containing amino acids 152- 166 neutralized EPO activity in vitro (Fibi et al., 1991). These results would suggest that if the proposed four-a-helix model is correct, and the receptor-binding site is in the helix closest to the C-terminus, then a likely binding site is the helical structure formed by amino acids 152- 160. Production of EPO Although at any one time the oxygen delivered to each tissue is similar, the kidney and the liver are the specific organs which respond to reduced oxygenation by producing EPO. Many years before the purification of EPO and the cloning of its gene, whole-animal experiments had identified the kidney as the major source of EPO in adults (Jacobson et al., 1957). Whole-animal studies also identified the liver as a primary source in the fetus (Zanjani et al., 1977) and the secondary source in adults (Fried, 1972). The availability of the cloned EPO gene permitted the recent confirmation of the relative roles of kidney and liver in EPO production through quantitative analysis of EPO mRNA (Bondurant and Koury, 1986; Beru et al., 1986; Schuster et al., 1987; Eckardt et al., 1992). By in situ hybridization of EPO mRNA, the EPO- producing cells in the kidney have been shown to be a subset of peritubular, interstitial cells in the cortex in mice (Koury et al., 1988b; Lacombe et al., 1988) and rats (Schuster et al., 1992). It is not yet known whether EPO-producing cells are capillary endothelial cells or one of the several types of ’true’ interstitial cells which lie outside the capillaries. In situ hy-

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Fig. 2. Map of the human EPO gene. Exons are blocks with translated regions filled solid and untranslated regions hatched. Flanking sequences and introns are shown as lines. The indicated areas contain the following: (a) kidney regulatory element; (b) liver regulatory element; (c) binding sites in 5’-flanking region for transacting factors; (d) sequence that encodes a 3’ untranslated region of EPO mRNA which binds a putative cytoplasmic stabilization factor; (e) 3’-flanking sequence with transcription-enhancer activity.

bridization has also shown that hepatocytes as well as some interstitial, sinusoid-associated cells, are the hepatic sources of EPO (Koury et al., 1991; Schuster et al., 1992). Following blood loss or reduced atmospheric oxygen, the kidneys can increase their basal level of EPO production in an exponential manner depending upon the severity of oxygen reduction (Schuster et al., 1987; Koury et al., 1989a). This massive increase in EPO production appears to be the result of expanding focal areas of hypoxia in the renal cortex with ensuing recruitment of more interstitial cells to transcribe EPO mRNA (Koury et al., 1989a). Conversely, an exponential decline in EPO production occurs as the oxygen-carrying capacity of the blood is replenished and tissue hypoxia is relieved (Koury et al., 1989a; Schuster et al., 1987). The cells that sense these changes in oxygen delivery reside in the kidney (Ratcliffe et al., 1990; Page1 et al., 1991; Scholz et al., 1991). Furthermore, although a pure population of EPO-producing cells from a renal source has not been available, studies in vitro with the human hepatoma cell line, Hep 3B, show that the same cells which sense hypoxia can produce EPO (Goldberg et al., 1987). Both Hep 3B cells and renal interstitial cells appear to produce EPO in an all-or-none fashion after a critical threshold of hypoxia is met (Goldberg et al., 1987; Koury et al., 1989a). EPO mRNA accumulates in renal EPO-producing cells within 1- 2 h following the hypoxia stimulus (Bondurant and Koury, 1986; Schuster et al., 1987). EPO is translated and secreted without any evidence of intracellular storage (Schuster et al., 1987; Goldberg et al., 1987). Nuclear run-off studies using renal cell nuclei (Schuster et al., 1989) and Hep 3B cells (Goldberg et al., 1991) indicate that hypoxia regulates EPO production mainly through effects on the rate of EPO gene transcription. A map of the human EPO genomic sequence with indicated areas involved in regulation of the EPO gene expression is shown in Fig. 2. In Hep 3B cells placed under hypoxic conditions, a cis- acting DNA sequence with transcription-enhancer activity has been identified in a sequence located 120- 270 bp 3’ to the human EPO gene polyadenylation signal (Beck et al., 1991). An enhancer element of approximately 70 bp has also been found in the same 3’ location in the murine gene (Pugh et al., 1991). Presumably, this DNA region functions as a hypoxia-related enhancer sequence in vivo in liver and kidney, because there is evidence of induction by hypoxia of two proteins which bind to this region in transgenic mice carrying the human EPO gene (Semenza et al., 1991a). The cis-acting DNA sequences determining tissue and cell-type specificity of EPO production have been studied in transgenic mice carrying a series of human EPO gene constructs which have variable lengths of 5’flanking sequences. These studies have demonstrated a sequence in the 5’-flanking region 0.4-6 kb upstream of the

652 transcription-start site (-0.4 to -6 kb) which restricts expression to the liver and kidney, and a sequence at -14 kb to -6 kb which is necessary for expression in the kidney (Semenza et al., 1991b). Studies of possible trans-acting transcriptional factors within the EPO-producing cells have been performed with Hep 3B cells and with murine kidney and liver cells. Although its mechanism is unknown, cobalt as well as hypoxia induces EPO production in vivo (Goldwasser et al., 1958).Experiments with metallic ions show that cobalt, nickel and manganese can also induce EPO production in Hep 3B cells (Goldberg et al., 1988). Carbon monoxide inhibits the hypoxia-induced EPO production by Hep 3B cells. These results have been interpreted as suggesting that a heme protein plays a role in the response of EPO-producing cells to hypoxia (Goldberg et al., 1988). By analogy to hemoglobin, this putative heme protein would have a deoxy conformation which is mimicked when a metallic ion such as cobalt replaces iron in the porphyrin ring of the heme component, and an oxy conformation which is mimicked when carbon monoxide binds to the heme moiety. The activity of this heme protein would vary with its oxy versus deoxy conformation which, in turn, would be determined by the oxygen concentration in the EPO-producing cell. Whether such a heme protein would act directly in the induction of EPO gene transcription or would be part of a more complex mechanism of induction is uncertain. Nuclear extracts from EPO-producing cells have been used to identify factors which can interact with cis- acting DNA sequences of the EPO gene. Nuclear extracts from hypoxic Hep 3B cells have greatly enhanced activity in the in vitro transcription of an EPO gene construct, compared to extracts of normal Hep 3B cells (Costa-Giomi et al., 1990). Two factors in nuclear extracts of liver and kidney of transgenic mice are induced by anemia and bind to specific nucleotide sequences in the above-described 3’ enhancer region of the human EPO gene (Semenza et al. 1991a). With regard to 5’-flanking sequences of the EPO gene, a trans-acting factor found in nuclear extracts of murine kidney cells is induced by anemia (Tsuchiya et al., 1992). The factor binds within 200 bp of the transcription-start site of the human EPO gene. In other studies, Beru et al. (1990) have reported that nucleotides - 61 to -45 in the highly conserved 5’-flanking region of the murine EPO gene bind a protein and a ribonucleoprotein. Comparisons of kidney extracts from normal and cobalt-treated mice suggest that the ribonucleoprotein is a negative transcription factor, since cobalt decreased the levels of RNA binding components. In Hep 3B cells, the increased transcription rate observed in nuclear run-off assays does not appear to account for the increases in EPO mRNA induced by hypoxia (Goldberg et al., 1991). Increased stabilization of EPO mRNA in hypoxic Hep 3B cells has been proposed, and a cytoplasmic protein which binds to a 120-base sequence in the 3’ untranslated region of EPO mRNA has been identified in Hep 3B cells by gel-shift assays (Rondon et al., 1991). However, the proposed role of this protein in stabilization of EPO mRNA is tenuous since the protein/nucleotide band remains unchanged by hypoxia in cytoplasmic extracts from Hep 3B, murine kidney and murine liver cells. EPO-responsive cells in normal differentiation The target cells for EPO are the progenitors of erythrocytes found in the hematopoietic organs. These organs include the bone marrow, spleen and fetal liver. In addition to erythroidprogenitor cells, megakaryocytes (Fraser et al., 1989),lympho-

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1 Reticulocyte Fig. 3. Stages of normal erythroid differentiation.The stages are parts of a continuous process of proliferation and differentiation with varying numbers of cell divisions between different stages. Thus, each stage has less proliferative potential than the preceding stage, but the number of cells is much greater than in the preceding stage. The period of differentiation in which erythroid cells are dependent upon EPO is indicated.

cytes (Kimata et al., 1991) and endothelial cells (Anagnostou et al., 1990) have been reported to have surface receptors for EPO. However, these other cell types have not been studied further with regard to the receptors or effects of EPO. EPO receptors have also been identified in placenta of mice and rats (Sawyer et al., 1989) but not in placenta of sheep and man (Widness et al., 1991). These placental receptors are similar in EPO affinity and estimated structure (as determined by chemical cross-linking of ‘251-EPO) to the EPO receptors found in rodent erythroid progenitors. In mice, the maternal to fetal transfer of EPO indicates a possible function for these placental EPO receptors (Koury et al., 1988a). EPO responsiveness in erythroid-progenitor cells has been found at specific stages of differentiation. Erythroid differentiation occurs over a continuum of stages from the pluripotent hematopoietic stem cell to the mature erythrocyte. In the process of differentiation, a few rare hematopoietic stem cells give rise to the billions of new erythrocytes which enter the human bloodstream each day. Hematopoietic stem cells give rise to each of the different hematopoietic cell lineages through a poorly understood process termed commitment. The committed progenitor cells of each lineage then proliferate and differentiate through a series of stages until they become mature cellular components of the blood. The stages of erythroid lineage differentiation are shown in Fig. 3. The later stages of erythroid differentiation, which encompasses the proerythroblasts through reticulocytes, can be recognized microscopically. The earlier stages of erythroid differentiation have been operationally defined by short-term tissue culture of bonemarrow or spleen cells in semisolid medium in the presence of EPO. The colony-forming-unit erythroid (CFU-E; Stephenson et al., 1971) is defined as a cell which multiplies and differentiates in vitro into a colony of 8 - 64 hemoglobinized erythroblasts after 2 days for mice or 7 days for humans.

653 CFU-E are the immediate predecessors of proerythroblasts. A more primitive erythroid progenitor termed the burstforming-unit erythroid (BFU-E; Axelrad et al., 1973) gives rise to a cluster of colonies or a very large single colony of hundreds to thousands of hemoglobin-containing erythroblasts over a culture period of 7 - 10 days for murine cells and about 14-21 days for human cells (Eaves et al., 1979; Eaves and Eaves, 1984). An intermediate stage between CFU-E and BFU-E has been defined and is referred to as the mature BFUE (Gregory, 1976; Gregory and Eaves, 1977). Extensive work has been done to define the erythroid progenitor stages which are responsive to EPO and which are dependent on EPO. Studies in adult mice show that reducing or elevating EPO levels in vivo does not affect the numbers of BFU-E (Iscove, 1977; Hara and Ogawa, 1977); even the numbers of mature BFU-E, which form mature colonies at 3 days of in vitro culture, are unaffected by EPO levels (Gregory and Eaves, 1978). However, CFU-E numbers in mice are strongly affected by in vivo EPO levels (Iscove, N. N., 1977; Hara and Ogawa, 1977; Gregory and Eaves, 1978). These studies in mice suggest that the mature murine BFU-E, defined by in vitro colony formation at 3 days, is the earliest progenitor which responds to EPO in vivo by progressing to the next stage of development, the CFU-E. In vitro studies of erythroidcolony formation using cells from mice and humans have generally supported the view that mature BFU-E represent the differentiation stage of the erythroid lineage at which most cells acquire responsiveness to EPO (Eaves and Eaves, 1984; Umemura et al., 1989; Sawada et al., 1990). However, two reports (Peschle et al., 1980; Dessypris and Krantz, 1984) suggest that EPO can perturb the cell cycle kinetics of a population of more immature BFU-E derived from bone marrow, indicating that EPO can have effects on at least a subset of these BFU-E. Emerson et al., (1989) provided evidence that BFU-E from human fetal liver respond to EPO, and thus differ from blood-derived BFU-E which show no evidence of EPO response. Furthermore, Valtieri et al. (1989) showed EPO responsiveness in human embryonic BFU-E. However, BFUE derived from adult human bone marrow appear to be heterogeneous in that EPO can support early phases of growth of a small fraction of them, but it does not support early survival or growth of the majority (Sieff et al., 1986; Emerson et al., 1989). Finally, there is a report that the EPO receptor gene is expressed in embryonal stem cells (ES cells), pluripotent cell lines derived from mouse embryos which have a capacity for multifaceted differentiation in vitro (Heberlein et al., 1992). The physiological role that EPO receptors might play in cells before the mature BFU-E stage remains unknown. Evidence suggests that other factors such as IL-3, granulocytemacrophage colony-stimulating factor, c-kit ligand (Metcalf et al., 1980; Emerson, et al., 1988; Sonada et al., 1988; Dai et al., 1991), and perhaps other multilineage growth factors, may be able to supply regulatory functions for immature BFU-E in postnatal animals, and that factors other than EPO are normally of prime importance for immature BFU-E in vivo. However, EPO may play some role in the early stages of hematopoiesis under particular circumstances, such as in prenatal erythropoiesis. Experimental EPO-responsive cell systems

While in vitro assays of colony-forming cells are useful in defining the stages of erythroid differentiation, the use of these cells in biochemical studies of EPO is limited by their rarity in hematopoietic tissue. In normal bone marrow, CFU-E rep-

resent less than 0.5% of all cells and BFU-E are rarer than 0.1% of cells. Furthermore, the bone-marrow erythroid-progenitor cells are at all different stages of differentiation. HOWever, experiments concerning the molecular mechanism of EPO action require large numbers of pure erythroid cells which are EPO-responsive and relatively homogeneous in their stage of differentiation. Thus, several in vitro cell systems are being used for biochemical studies of EPO and its receptor: erythroid-progenitor cells freshly explanted from hematopoietic organs; established cell lines, which normally express EPO receptors and are generally derived from malignant cells of tumor-bearing animals; established cell lines which do not express the EPO receptor normally but are engineered to do so by artificial introduction and expression of the EPO receptor gene. The biological responses to EPO of these in vitro cell systems differ according to their origins. Purified, erythroidprogenitor cell systems explanted from animals, or derived by short-term tissue culture of explanted cells, typically proliferate and differentiate in vitro into reticulocytes in response to EPO (Koury et al., 1987, 1989b; Wickrema et al. 1992). In the absence of EPO, these events do not occur. Unpurified cell populations used to study EPO have included those explanted from fetal livers of mice or rats, or from the spleens of mice which have been made anemic by injection of phenylhydrazine. As mentioned above, these cell populations are contaminated with non-erythroid cells, and the erythroid population contains all stages of erythroid differentiation. Three systems of highly pure, EPO-responsive, erythroid-progenitor cells have been derived from explanted cells: the murine erythroblasts derived from spleens of mice infected with the anemiainducing strain of Friend virus (FVA cells; Koury et al., 1984); regenerating mouse CFU-E derived by purification from spleens of mice treated first with thiamphenicol, then with phlebotomy (Nijhof and Wierenga, 1983); human CFU-E purified from an initial culture of partially purified human peripheral blood BFU-E (Sawada et al., 1987). These systems yield relatively pure populations of cells at the CFU-E stage or the slightly, more mature proerythroblast stage of erythroid differentiation. Cell lines which bear EPO receptors have been established from mice and humans. Many murine erythroid cell lines have been established from eythroleukemic mice which had been infected with either the Friend leukemia virus complex, the Friend murine leukemia virus or the Rauscher leukemia virus complex. Other murine cell lines of hematopoietic, but not necessarily erythroid, origin have been derived from mice with leukemia or lymphoma due to causes other than an erythroleukemic virus. A few human lines have been derived from patients with leukemia. For an extensive listing of cell lines or cell types which bear EPO receptors, see the reviews by Sawyer (1990), D’Andrea and Zon (1990) and Spivak (1992). While many cell lines have EPO receptors, only a few exhibit biological responses to EPO, and none exhibit uniform and complete differentiation into reticulocytes such as those seen in the explanted cell systems described above. Many of the murine erythroleukemia cell lines show no apparent response to EPO. Other lines show limited evidence of differentiation such that a portion of the cell population may synthesize small amounts of hemoglobin or other erythroid proteins in response to EPO (Sytkowski et al., 1980; Todokoro et al., 1987; Broudy et al., 1990). Some cell lines are dependent upon EPO for survival. These include lines which exhibit other limited indications of erythroid character, such the as HCD 57 cell line (Spivak et al., 1991) which was established from leukemic mice by continuous culture in the presence of EPO.

654 Also, several cell lines are dependent on EPO for survival only in the absence of other growth factors. These lines were derived by selection in EPO-containing medium of a nonerythroid line which had previously been dependent upon a hematopoietic growth factor other than EPO. Examples include 32D-EPO cells (Migliaccio et al., 1989), B6Sut. EP cells (Quelle and Wojchowski, 1991b) and DA-1 ER cells (Branch et al., 1987; Sakaguchi et al., 1987). Many of these cell lines express proteins which have been thought to be associated with hematopoietic cell lineages other than the erythroid lineage. Whether such patterns of expression of these presumed non-erythroid proteins actually occurs in vivo in certain hematopoietic progenitors is not known. One problem with both explanted cell systems and continuous cell lines expressing natural EPO receptors is that they have 3000 or less receptors in each cell. Until recently, few reports had described early biochemical events following EPO binding to such cells. This problem has been overcome in cell lines which can be engineered to produce much larger numbers of EPO receptors and to require EPO for survival. Four murine cell lines that are dependent upon IL-3 for survival, BA/ F3 (D’Andrea at al., 1991), DA-3 (Miura et al., 1991), FDCP1 (Quelle and Wojchowski, 1991a; Carroll et al., 1991) and LyD9 (Chiba et al., 1992) have been used as recipients for transfected expression vectors containing the EPO receptor gene. By introducing and expressing the EPO receptor gene in the above cell lines, they are converted into lines which can survive with either EPO or IL-3. These cell lines have been used to analyze the effects of specific mutations of the EPO receptor and also to analyze the early intracellular effects of EPO receptor activation.

normal concentrations of EPO in vivo or in vitro. After having been placed in tissue culture briefly without EPO, cells coming from a high-EPO environment are found to display a second class of high-affinity receptors (Sawada et al., 1988; Landschulz et al., 1989). The modulation of surface receptor number is physiologically controlled by an immediate effect due to internalization of the EPO/EPO-receptor complex and a longer-term effect due to the loss of EPO receptors as terminal differentiation proceeds. Within 1 min of binding at 37”C, 1251-EP0is lost from the cell surface and lZ5I accumulates within the cell indicating internalization of EPO (Sawyer et al., 1987a, 1989; D’Andrea et al., 1989). Degraded lZ5I-EPO,mostly in the form of 1251-tyrosine,progressively accumulates in the cell culture medium indicating intracellular degradation of EPO (Sawyer et al., 1987a). Inhibition of this degradation by ammonium chloride or chloroquine indicates lysomal involvement in this immediate effect. Culturing primary explanted erythroblasts and CFU-E in medium containing EPO results in their differentiation and, upon reexamining these cell populations after various culture periods, the number of EPO receptors in each cell declines. Indeed, erythrocytes have no EPO receptors. This longer-term decrease in receptor numbers during terminal differentiation is discussed below in the section on EPO receptor expression. In continuous erythroid cell lines, EPO receptors are present in similar numbers and have EPO affinities similar to the primary explanted erythroid-progenitor cells. A few cell lines have more EPO receptors in each cell, but, in general, the range is 1000- 3000 receptors/cell (Sawyer 1990, D’Andrea and Zon, 1990; Spivak, 1992). The large majority of cell lines displaying EPO receptors also have a single class of receptors with binding constants similar to the low-affinity class found The EPO receptor in primary explanted cells. Two classes of receptors with typical higher and low-affinity EPO dissociation constants are The EPO-binding component found in some pluripotent cell lines. The Friend murine The cloning and production of recombinant EPO along erythroleukemia cell line, clone 745, found to have a single with the development of homogeneous populations of class of the low-affinity receptors (Sawyer et al., 1987a; erythroid-progenitor cells and erythroid cell lines has permit- Mayeux et al., 1987; Sasaki et al., 1987b) is unresponsive to ted direct study of specific EPO receptors. 1251labeling of EPO. However, an expression library from this cell line has EPO without loss of biological activity (Sawyer, 1990), as well been used to clone a murine EPO receptor cDNA (D’Andrea as the commercial marketing of lZ5I-EPO,have led to studies et al., 1989). This cloned EPO receptor cDNA and its protein of EPO receptors in a wide variety of cells. Tabular listings of product has led to a dramatic expansion of studies concerned experimentally determined EPO receptor numbers and bind- with the EPO receptor in particular, and the mechanism of ing constants in a wide variety of erythroid cells, both freshly EPO action in general. Also, the cloned cDNA for the murine explanted cells and continuous cell lines, are included in three EPO receptor has been used to clone a homologous human recent reviews by Sawyer (1990), D’Andrea and Zon (1990) cDNA (Winkleman et al. 1990, Jones et al., 1990) as well as and Spivak (1992). The first demonstration of specific EPO the genomic sequences (Youssoufian et al., 1990; Kuramochi binding to erythroid-progenitor cells was in FVA cells (Krantz et al., 1990a). The EPO receptor is encoded by a single-copy gene conand Goldwasser, 1984; Sawyer et al., 1987a). In FVA cells, binding of lZ5I-EPOshows a biphasic curve on a Scatchard taining eight exons. The mouse and human genes are 82% plot (Sawyer et al., 1987a). Studies of the release of 1251-EP0 similar in the coding regions and have conserved intron-exon bound to FVA cells demonstrate that the biphasic curve is not boundaries. The murine and human genes encode 507-aminoa result of negative cooperativity but rather that FVA cells acid and 508-amino-acid proteins, respectively; both have a have two classes of receptors (Sawyer 1990). One class has a single 23-amino-acid transmembrane domain, a 24-aminohigher affinity for EPO (Kd = 90 pM) while the other has acid signal peptide and a predicted molecular mass of 5 5 kDa a lower affinity (Kd = 570 pM). Approximately 1000 total (D’Andrea et al., 1989). A model of the cloned murine EPO receptors (300 high affinity and 700 low affinity) are found in receptor is shown in Fig. 4. The first five exons encode the each cell. Similar total numbers of EPO receptors and range 233-amino-acid extracellular part of the receptor, the sixth of affinities for EPO receptors are found in most erythroid exon encodes the single transmembrane domain and the sevcell populations which have been freshly explanted or grown enth and eighth exons encode the 236-amino-acid cytoplasmic in short-term primary tissue cultures after explantation. domain (Youssoufian et al., 1990). Multiple sites for potential Although some populations appear to have only a single class 0-linked glycosylations exist in the EPO receptor protein for of receptors similar to the low-affinity class of FVA cells, in both species, while the murine and human receptors each have several cases these cells have been exposed to higher than one N-linked glycosylation site. Antibodies reacting with the

655

NHZ

COOH EXTRACELLULAR DOMAIN

ITMI

CYTOPLASMIC DOMAIN

Fig. 4. Model of murine EPO receptor cloned by D’Andrea et al. (1989). Transmembrane region (TM) is hatched. The filled hexagon indicates the single N-glycosylation site. Sites of conserved cysteines (C) and Trp-Ser repeated sequence (WSAWS), which indicate membership of the hemopoietin/cytokine family, are identified. The point mutation Argl29+Cys, that leads to constitutive receptor action, is shown as (R+C). Amino acid sequences in the cytoplasmic domain: (a) sequence essential for receptor action which has extensive similarity to a sequence in the IL-2 receptor; (b) other sequence essential for EPO receptor action; (c) negative regulatory sequence.

cloned EPO receptor immunoprecipitate a 66-kDa band on SDSjPAGE from COS cells transfected with either receptor. Also, EPO receptors of approximately 66 kDa can be demonstrated by affinity purification of radiolabeled, solubilized EPO receptors from transfected COS cells using bound, biotinylated EPO (Wognum et al., 1990) The immunoprecipitated 66-kDa bands can be reduced in size to 62 kDa with endoglycosidase F (N-glycanase; Yoshimura et al., 1990) demonstrating N-linked glycosylation of the EPO receptor. In addition to detection with antibodies, chemical cross-linking of 1251-EP0bound to COS cells expressing the transfected cloned EPO receptor (D’Andrea et al., 1989), or to murine erythroleukemic cell lines (Todokoro et al., 1987; Mayeux et al., 1991), have demonstrated proteins in the 62- 66-kDa range. However, proteins in the 62 - 66-kDa range appear to be products of proteolytic cleavage of the 105-kDa and 90-kDa proteins (Sawyer, 1989) found in most cross-linking experiments (see below). The EPO receptor is a member of the hemopoietin/ cytokine receptor superfamily of proteins which have several structural features in common (Bazan, 1990a). Each member of the family binds a specific hormone or growth factor at the surface of a cell. The receptor proteins in this superfamily have a single transmembrane domain and are oriented with their N-terminus outside cell. They have four conserved cysteine residues in the N-terminal half of the extracellular domain and a conserved 20-amino-acid sequence in the extracellular domain near the plasma membrane which contains the specific amino acid sequence Trp-Ser-Xaa-Trp-Ser. Their cytoplasmic domains are much more variable in size, and no known structure associated with signal transduction is found in these domains. Within this family of receptors, however, the cytoplasmic domain of the EPO receptor is most closely related to the p chain of the IL-2, IL-3 and IL-4 receptors, in that all have a conserved region which is rich in proline, serine and acidic amino acids (D’Andrea and Zon, 1990). Mutational analysis of the cloned EPO receptor sequence has permitted some evaluation of the function of various portions of the receptor. Substitutions, insertions and deletions within the Trp-Ser-Xaa-Trp-Ser sequence result in inactivation of EPO binding and a lack of biological responsiveness to EPO due to improper folding and processing of the mutant receptor molecules retained in the endoplasmic reticulum (Chiba et al., 1992; Yoshimura et al., 1992). Deletion mutations in the cytoplasmic portion of the EPO receptor indicate that a negative regulatory sequence exists in the Cterminal 42 amino acids, since Ba/F3 cells expressing such truncated mutants respond to much lower concentrations of

EPO than those expressing the full-length, wild-type receptor (D’Andrea et al., 1991). Additional studies show that a sequence of 21 amino acids (amino acids 280 - 301 of the mature EPO receptor) from a region near the plasma membrane, which is similar to a sequence found in the IL-2-receptor p chain, is essential to the function of the EPO receptor in DA3 cells (Miura et al., 1991). Another region of the cytoplasmic domain which has been found to be essential for biological function of the mature EPO receptor by deletion-mutation analyses has been mapped to amino acids 337 - 375 by Miura et al. (1991) and amino acids 328-372 by Quelle and Wojchowski (1991a). Other studies suggest that the C-terminal domain of the cloned EPO receptor can interact with, and may be a substrate for, a kinase (Yoshimura and Lodish, 1992). Immunoprecipitation studies indicate that a very minor percentage of the total number of EPO receptors has a molecular mass of 72 kDa and is located in the plasma membrane where it is phosphorylated at tyrosine residues following EPO binding (Miura et al., 1991; Yoshimura and Lodish, 1992). However, the biological significance of this receptor phosphorylation remains unknown. In a human cell line, UT-7, tyrosine phosphorylation of the EPO receptor is more prominent (Dusanter-Fourt et al., 1992) than in the murine cell lines examined. A point mutation resulting in a substitution of Argl29+Cys results in constitutive action of the EPO receptor, in that it abrogates the need for EPO in dependent cells and, when expressed in a retroviral vector, leads to erythroleukemia in infected mice (Longmore and Lodish, 1991). These mutant receptors appear to form homodimers through disulfide linkages, and this dimerized form of the receptor appears to be the constitutively active form (Watowich et al., 1992). These results suggest that non- covalent homodimerization of wild-type receptors may be involved in signal transduction following EPO binding. Additional receptor components In addition to the evidence for self-association of the cloned EPO receptor protein, results of chemical cross-linking studies suggest that proteins other than the cloned EPO-binding protein may be involved in a receptor complex. Other members of the hemopoietin/cytokine receptor superfamily such as GM-CSF, IL-2, IL-3, IL-5 and IL-6, have each been shown to have an additional subunit which appears to play a role in stabilizing the binding of ligand to the receptor complex and transducing a signal from the membrane to the interior of the cell (Leonard et al., 1984; Niaido et al., 1984; Cosman, 1984; Hayashida, 1990; Hibi, 1990). When 1251-EP0is bound to cells, then chemically cross-linked to nearby proteins, most cells have two cross-linked proteins that are approximately 105 kDa and 90 kDa by SDSjPAGE analysis (Sawyer, 1987b; Sawyer, 1989). These two proteins are found not only in explanted cells and erythroid cell lines which bind EPO, but the 105-kDa protein is found in non-erythroid cells in which the cloned EPO receptor gene has been transfected and expressed (D’Andrea et al., 1989). The two proteins found with crosslinking are similar by proteolytic peptide mapping, and the use of multiple proteolytic inhibitors during experiments suggests that the 90-kDa protein is a proteolytic product of the 105-kDa protein (Sawyer, 1989). The relationship of these two proteins found in cross-linking studies to the cloned EPObinding protein is uncertain. The two cross-linked proteins have very little or no glycosylation (Mayeux et al., 1990). To determine whether these two proteins are an artifact of a bifunctional cross-linker, Hosoi et al. (1991) used a radio-

656 labeled, heterobifunctional cross-linker which was first linked to unlabeled EPO. After these EPO/cross-linker Complexes were bound to FVA cells, they were subsequently linked to other proteins and cleaved so that the cross-linked proteins could be identified in ligand-free form. This approach also found the same 105-kDa and 90-kDa proteins, confirming the previous cross-linking results. Mayeux et al. (1991) used antibodies to the cloned receptor to immunoprecipitate preparations of solubilized cells to which '251-EP0 was bound and cross-linked. When the immunoprecipitation was performed after denaturation and reduction of the cell preparations, the 105-kDa and 90-kDa proteins were not precipitated, whereas they were precipitated using non-denatured, non-reduced cell preparations. These results have been interpreted as showing that the 251-EP0cross-linked proteins are immunologically unrelated to the cloned EPO-binding protein but that they form a complex with it. Whereas the functions of the proteins identified by chemical cross-linking are unknown and their relationship to the cloned EPO-binding protein is unclear, the interaction of the cloned EPO receptor with the 55-kDa envelope glycoprotein of the polycythemia-inducing strain of the Friend spleen focus-forming virus (SFFVp; Axelrad and Steeves, 1964) has been demonstrated. This retroviral protein is found in membranes of infected cells and, in susceptible mice, it causes an acute proliferation of non-malignant erythroblasts with an accompanying polycythemia (Tambourin et al., 1973). Freshly explanted erythroid-progenitor cells, infected in vitro with SFFVp, undergo terminal differentiation without the addition of EPO to their culture medium (Hankins et al., 1978). In cells expressing both the cloned EPO receptor and gp55 of SFFV, gp55 immunoprecipitates with antibodies against the cloned EPO receptor and vice versa (Li et al., 1990; Yoshimura et al., 1990; Casadevall et al., 1991). The expression of SSFVp gp55 in EPO-dependent cell lines results in their constitutive survival and growth (Ruscetti et al., 1990; Yoshimura et al., 1990). The specific region of SSFVp gp55 which leads to this EPO-like effect appears to be contained in a portion of its transmembrane domain (Chung et al., 1989). Also, Zon et al. (1992) showed that activation of the EPO receptor by gp55 specifically requires the EPO receptor transmembrane region. The gp55/EPO-receptor interaction occurs in the rough endoplasmic reticulum, and very little of the EPO receptor reaches the cell surface in Ba/F3 cells which express both gp55 and EPO receptor (Yoshimura et al., 1990). Thus, interaction in the endoplasmic reticulum of SFFVp gp55 and EPO receptor appears to result in an effect on erythroid-progenitor cells which mimics that of chronic, maximal EPO stimulation. In fact, when retroviruses encoding EPO or gp55 are used to superinfect cells infected previously with a retrovirus coding for the EPO receptor, both have the same effect as exposing the EPO-receptor-expressing cells to EPO itself (Hoatlin et al., 1990). Indeed, when mice are infected with the retrovirus encoding EPO, they develop the same erythroblastosis/ polycythemia type of disease that occurs with SFFVp infection. Furthermore, mice which are genetically resistant to Friend virus disease (Fv-2') have the same resistance to the EPO retrovirus (Hoatlin et al., 1990). This observation implies that the retroviruses do not infect the target erythroid-progenitor cells in Fv-2' mice, or that the gp55 or EPO retroviral products are not produced in infected Fv-2' erythroid progenitors or that the Fv-2' gene product prevents activation of the infected progenitor cell by the EPO viral product, just as it does with the gp55. In fact, other evidence indicates that the erythroid progenitors of Fv-2' mice are not resistant to

infection or to production of gp.55 (Bondurant et al., 1985a). Thus, the third possibility that the Fv-2' product prevents the action of both gp55 and retrovirally produced EPO suggests that the Fv-2 product is directly involved in the signal-transduction mechanism of the EPO receptor. Since the EPO receptor and Fv-2 genes are not identical or linked, the Fv-2 gene product has been proposed to play a role in EPO receptor signalling (Hoatlin et al., 1990). While this hypothesis is interesting, it does not account for the apparently normal erythropoiesis and EPO responsiveness of Fv-2' strains such as C57BL/6 mice.

EPO receptor expression Expression of the EPO receptor varies according to the stage of erythroid differentiation. Explanted erythroid-progenitor cells around the CFU-E stage of differentiation appear to have more EPO receptors than at other stages. As CFU-E undergo terminal differentiation, the number of EPO receptors progressively declines (Landschulz, 1989; Sawada, 1990; Sawyer, 1990; Broudy et al., 1991). During terminal differentiation, the EPO receptor mRNA declines in parallel with the loss of EPO binding, while the half-life of EPO mRNA is unchanged (Wickrema et al., 1991, 1992). These results indicate that the decline in EPO receptors seen with terminal differentiation is due to decreased EPO receptor gene transcription. As cells differentiate from stem cells to CFU-E, the changes in EPO receptors are much more difficult to study due to a lack of pure progenitor cell populations at the BFUE stages and earlier. Studies with autoradiography of '"1EPO binding to partially purified human BFU-E and their descendants formed during tissue culture indicate that the number of EPO receptors increases to a maximum on CFUE and proerythroblasts (Sawada et al., 1990). Using a population of immature hematopoietic progenitor cells isolated from human blood, Nakamura et al. (1992) found mRNA for several forms of EPO receptors. The mRNA in progenitor cells arose from alternative splicing of transcripts of the cloned human gene. One type of mRNA, which was prevalent in immature progenitors, encoded an EPO receptor with a truncated cytoplasmic domain and greatly reduced function compared to full-length receptors prevalent in later erythroidprogenitor cells. Studies with chemically induced differentiation of murine erythroleukemic cell lines indicate that EPO receptor numbers increase with differentiation (Sasaki et al., 1987b; Broudy et al., 1988; Tojo et al., 1988) but these cells do not complete terminal differentiation. Cell lines of diverse differentiation states, including embryonal stem cells and embryonal carcinoma cells, so-called 'multipotential' hematopoietic lines, and finally erythroleukemic lines, have all been shown to express EPO receptor mRNA (Heberlein et al., 1992). The levels of EPO receptor mRNA, however, are 10- 100-fold more in murine erythroleukemic cell lines as opposed to the embryonal or multipotential lines. These levels of EPO receptor mRNA correlate with the presence of a specific DNase-hypersensitive site in the first intron of the cloned EPO receptor gene and involvement of the erythroid transcription factor, GATA-1, at this site (Heberlein et al., 1992). In addition, GATA-1 binds to the 5' promoter region (Chiba et al., 1991; Zon et al., 1991). Thus, an increase in EPO receptor mRNA which may occur as early erythroid progenitors differentiate could be controlled in part by the expression of the lineage restricted transcription factor, GATA-1. While EPO receptor mRNA levels and EPO binding correlate in terminally differentiating explanted cells (Wick-

657 rema et al., 1991), some multipotential hematopoietic cell sublines contain EPO receptor mRNA and yet they do not bind EPO. In such a cell line, 32D, the determining factor for the binding of and response to EPO appears to be the translocation of the EPO receptor to the cell surface (Migliaccio et al., 1991). Thus, the question about whether EPO receptors are expressed at very early stages of erythroid differentiation is complex and has yet to be resolved.

EPO-mediated signal transduction The intracellular events which follow the binding of EPO to its cell-surface receptor are unknown. Signal-transduction studies with EPO have often yielded contradictory results. Some of these contradictions are undoubtedly due to differences in the cell lines used or the purity and developmental homogeneity of primary explanted EPO-responsive cells. Another particular problem involves the purity of the EPO used. Some frequently used commercial preparations of recombinant EPO contain less than 10% EPO, with the remaining 90% of protein being from tissue culture supernatants of the cells used to produce the rh-EPO. Likewise, some preparations of human urinary EPO have been 1% or less EPO and 99% other urinary proteins. These impure preparations can lead to erroneous results and incorrect conclusions, especially when the cell population contains non-erythroid cells. For the purposes of this review, an effect is considered relevant to the cellular signal-transduction mechanism only when it can be detected within 10 rnin after exposure to EPO. Among the possible signal-transducing events which have been studied in EPO-responsive cells is the change in phosphorylation state of specific proteins. The tyrosine phosphorylation of the cloned EPO receptor (Miura et al., 1991; Dusanter-Fourt et al., 1992; Yoshimura et al., 1992) was discussed in the previous section. Depending upon the cells used, several groups have reported a variety of unknown cellular proteins to be phosphorylated within 10 min of EPO exposure (Im et al., 1990; Quelle and Wojchowski, 1991b; Miura et al., 1991; Bailey et al., 1991; Kuramochi et al., 1991; DusanterFourt et al., 1992; Sawada et al., 1992; Torti et al., 1992). However, some cell lines show no evidence of protein phosphorylation in response to EPO despite having a subsequent biological response (Quelle and Wojchowski, 1991b). Two proteins which do become rapidly phosphorylated have been identified with specific antibodies: the protooncogene product, Raf-1 (Carroll et al., 1991) in murine erythroleukemia cells, HCD-57, following exposure to physiological concentrations of EPO; the 120-kDa GTPase-activating protein (GAP) in a human erythroleukemia cell line, HEL, following exposure to extremely high concentrations of EPO (Torti et al., 1992). Both Raf-1 and GAP have been associated with signal transduction in cells treated with growth factors in other contexts. Following EPO exposure, Raf-1 is phosphorylated on serine and tyrosine residues and the serinelthreonine kinase activity of Raf-1 itself is stimulated 2-3-fold (Carroll et al., 1991). The transient phosphorylation of GAP following EPO exposure inactivates the GAP and is temporally associated with a fivefold increase in GTP bound to the protooncogene product, ~ 2 1 ' "(Torti ~ et al., 1992). p2lraS,which is associated with growth-factor-induced signal transduction, is activated by the binding of GTP as opposed to GDP. A third protein found to be rapidly phosphorylated following EPO exposure of murine erythroleukemia cells is an 80-kDa protein with the

same p l a s an 80-kDa substrate of the known signal transducer of hormones and growth factors, protein kinase C (Spangler et al., 1991). Thus, there have been numerous reports of rapid phosphorylation of many different cellular proteins, and identification of those which may be critical for the signal transduction of EPO binding will require many more experiments. The two proteins reported to show dephosphorylation following EPO exposure, a 43-kDa membrane protein (Choi et al.) and an 80-kDa cytoplasmic protein (Bailey et al., 1991), both require EPO treatment for more than 10 rnin for the dephosphorylation to be detected, suggesting that they are further along in any signal-transduction pathway of EPO than the rapid phosphorylation described above. Two studies using pure EPO provide a possible mechanism for protein kinase C activation via diacylglycerol. In an EPOresponsive murine erythroleukemia cell line (Kuramochi et al., 1990b) and in murine fetal liver cells (Mason-Garcia et al., 1992), EPO increases diacylglycerol about 1.5-fold in 10 rnin while inositol triphosphates remain unchanged. In fetal liver cells, the arachidonate metabolites, leukotriene B4 and 12hydroicosatetraenoic acid, which can function in signal transduction, are reported to be rapidly induced by EPO exposure (Mason-Garcia et al., 1992). Evidence for rapid increases in cAMP has been reported with very impure EPO preparations (Bonanou-Tzedaki et al., 1986; Setchenska et al., 1988), but other studies found that similar impure EPO preparations had no effect on cyclic nucleotides (Graber et al., 1974; 1979). Using a highly purified EPO preparation, Kuramochi et al. (1990b) reported that cAMP increased about 1.5-fold in the EPO-sensitive, murine erythroleukemia cell line, SKT6. However, this has not been confirmed in several other studies using pure EPO, in the EPO-dependent DA-1 cell line (Tsuda et al., 1989), the EPOsensitive murine erythroleukemia cell line, TSA8 (Fukumoto et al., 1989) and in FVA cells (Koury and Bondurant, unpublished results). Another potential signal-transduction mechanism which has been actively investigated is a change in free intracellular calcium concentration following EPO exposure. Using Friend virus infected erythroblasts, Sawyer and Krantz (1984) found a rapid exchange of intracellular and extracellular Ca2 following EPO exposure. Also, reticulocyte membranes exposed to EPO were reported to show a progressive decline in Ca2+ATPase activity (Lawrence et al., 1987). Since these results suggest that EPO may affect intracellular Ca2+, several spectrofluorimetric studies have been performed with fluorescent, Ca2+-bindingdyes. Mladenovic and Kay (1988) reported a twofold increase in intracellular Ca2+ in mononuclear human bone-marrow cells within 8 min of exposure to pure EPO. In spleen cells from Friend-virus-infected mice, Worthington (1989) reported a transient increase in intracellular Ca2+ following exposure to an EPO preparation of unspecified purity. With impure EPO preparations, erythroidprogenitor cells derived by in vitro culture of human blood BFU-E, have been reported to show rapid increases in intracellular Ca2+with higher Ca2+concentrations in the nucleus than the cytoplasm (Miller et al., 1988, 1989, 1991; Yelamarty et al., 1990). However, using the same impure EPO preparation, Jones et al. (1992) found no change in intracellular Ca2+ in human fetal liver cells. In fact, when pure EPO is used, no evidence of any change in intracellular Ca2+ can be found for partially purified murine CFU-E (Imagawa et al., 1989), for SKT6 erythroleukemia cells (Kuramochi et al., 1990b), or for FVA cells (Kelley et al., 1992). +

658 Cellular responses to EPO stimulation and the nature of EPO regulation

GATA-1 action would necessarily be very protracted since some erythrocyte proteins such as carbonic anhydrase I and spectrins are present in erythroid-progenitor cells before the Some cellular responses occur within the first 1 - 3 h fol- CFU-E stage (Villeval et al., 1985; Koury et al., 1987), while lowing EPO exposure. Certain protooncogenes, which in others such as the globins, the anion transporter and the other biological systems are associated with rapidly dividing membrane skeleton protein, band 4.1, are not produced until cells or with early responses to growth factors, have been after the CFU-E stage (Bondurant et al., 1985b; Koury et al., examined in cells exposed to EPO. A 3-4-fold increase in 1987; Nijhof et al., 1987). EPO may yet be found to control mRNA for the c-myc gene occurs within 1 h of exposure to directly some critical differentiation event, but currently a pure EPO in two murine erythroleukemia cell lines (Todokoro series of events of unknown relationship to the action of EPO et al., 1988; Spangler et al., 1991) and in spleen erythroid cells appear to control the erythroid-differentiation program. from anemicmice (Spangler et al., 1992). Spangler et al. (1991, If EPO does not induce a program of erythroid-specific 1992) presented evidence suggesting that the increase in c-myc differentiation, then what other possible effects does it have mRNA is dependent on the activation of protein kinase C. on the erythroid-progenitor cells? The two most studied possiAlso, in these erythroleukemic cell lines a decrease in mRNA bilities are that EPO either induces progenitor-cell mitosis or for the c-myb gene occurs following EPO administration that it permits survival of progenitor cells that would other(Todokoro et al., 1988; Spangler et al., 1991). A report of wise die. The hypothesis that EPO acts as a mitogen appears increased c-fos gene expression within 30 min of EPO exposure to have developed from the in vitro growth patterns of in murine erythroleukemia cell lines is of uncertain signifi- hematopoietic cells in colony assays and from concepts decance since this study involved the uncontrolled replenishment veloped from the culture of fibroblast-like cell lines. Since of fresh medium and serum to starved cells at the same time progenitors such as CFU-E and BFU-E are defined by their as exposure to EPO (Tsuda et al., 1991). growth into colonies of mature erythroblasts in the presence While changes in mRNA levels for c-myc and c-myb occur of EPO, their responses to EPO have been considered as a within the first hour following EPO exposure, other cellular stimulation of proliferation and differentiation. Cell lines with responses occur within 2 - 3 h following EPO exposure. After which many of the concepts of cell cycle and mitogens were exposure to pure EPO, FVA cells show increased RNA syn- developed usudlly grow on a surface until they become confluthesis (Bondurant et al., 1985b), increased glucose transport ent and stop proliferating due to contact inhibition from their (Koury et al., 1987) and prevention of DNA degradation, neighboring cells. They may also stop proliferating due to thought to be a forerunner of cell death (Koury et a]., 1990a). deprivation of growth factors or nutrients. This non-proThese changes within the first 2-3 h after EPO exposure liferating, latent state is termed G o and represents a phase that precede by several hours any observed changes in specific is outside the normal cell cycle. Exit from the G o state and erythroid effects, such as an increased rate of globin gene reentry into cell cycle can be induced by either disruption of transcription (Bondurant et al., 1985b). Beyond these first few neighboring cell contact, by repleting serum growth factors or hours of in vitro culture, EPO-dependent, explanted erythroid by supplying deficient nutrients. progenitors differentiate into reticulocytes in a process which The early erythroid progenitors such as immature BFU-E involves an elaborate series of erythroid-specific cellular may have either a protracted cell cycle or a latent G o phase, events (Koury et al., 1987). EPO appears to be required con- since a minority of them are in DNA synthesis at a given time tinuously for a specific portion of this differentiation process (Iscove, 1977; Hara and Ogawa, 1977; Eaves et al., 1979). It (see below). appears possible that EPO may induce these progenitors to To understand how EPO regulates erythroid-progenitor proliferate. The concept of a Go or latent phase may not cells, the fundamental intracellular functions which are modu- apply, however, to later stages of erythroid-progenitor differlated directly by EPO must be identified. One possibility is entiation. The majority of CFU-E in mice are in DNA synthat EPO specifically initiates or controls an erythroid differ- thesis (i.e. in S-phase of the cell cycle) regardless of the EPO entiation program. In such a scenario, erythroid-progenitor levels in the animals (Iscove, 1977; Hara and Ogawa, 1977); cells do not produce the proteins characteristic of mature indeed, they have not been observed to survive in vitro without erythrocytes until they are induced by EPO to do so. A puta- proliferating. Thus, EPO does not appear to stimulate protive regulatory factor which controls the expression of these liferation of CFU-E from a latent, Go state. erythroid-specific genes would be induced in cells exposed to A mitogenic effect of EPO is suggested from studies of EPO. Attempts to identify a single erythroid-specific factor human BFU-E (Dessypris and Krantz, 1984), the murine cell which might control the components of the erythroid-specific line DA-1 ER (Tsuda et al., 1989) and the murine erythroleudifferentiation program have not been successful. A candidate kemia cell line, HCD 57 (Spivak et al., 1991). Human bonefor such an EPO-induced regulatory factor would be the tran- marrow BFU-E increase their percentage in DNA synthesis scription factor, GATA-1, which binds to the DNA sequence after 1 day in culture with EPO, compared to those cultured T,GATA& This sequence is found in many genes expressed in without EPO (Dessypris and Krantz, 1984). However, a subset erythroid cells such as the genes coding for globins, carbonic of BFU-E are lost during the 1 day in culture without EPO. anhydrase I and heme-synthetic enzymes. One problem with Thus, in human BFU-E, EPO appears to have a mitogenic implicating GATA-1 as the regulatory factor for effect, but it also acts by increasing cell survival. HCD 57 cells erythropoiesis is that GATA-1 is expressed in mast cells and deprived of EPO for 24 h accumulate in a G1/Gophase of the megakaryocytes as well as erythroid cells (Martin et al., 1990; cell cycle as determined by DNA content with flow cytometry Romeo et al., 1990). Also, the timing of GATA-1 expression (Spivak et al., 1991). In the same cells, EPO restimulation, in erythroid cells is complex, and it has not yet been proven following 24 h deprivation, leads to a marked increase in to be induced by EPO in a variety of cell systems which [3H]thymidine incorporation into DNA. Using the DA-1 ER differentiate in response to EPO. Nevertheless, there is one cell line, Tsuda et al. (1989) has reported results very similar report of GATA-1 induction by EPO in the murine cell line, to those described above for HCD 57 cells. However, after SKT6 (Chiba et al., 1991). However, the time period for 24 h without EPO, HCD 57 cells begin to die, indicating the

659 EPO also acts to permit their survival. Since HCD 57 and DA1 ER cells do not undergo terminal erythroid differentiation, a corresponding stage of normal erythroid differentiation cannot be assigned to them. In the late stages of erythroid differentiation, the effect of EPO in preventing cell death is very evident, and this effect obscures any putative mitotic effect during the terminal differentiation phases. FVA cells deprived of EPO undergo a process of programmed death which has the morphological and biochemical characteristics of apoptosis (Koury and Bondurant, 1990a; Kelley et al., 1992). These characteristics include decreased cell size, homogeneous nuclear condensation and internucleosomal cleavage of DNA, as described in many other cell systems (Arends and Wyllie, 1991). DNA cleavage begins in EPO deprived FVA cells by 2-4 h and reaches levels of 40% by 6 h and 80% by 20 h. DNA breakdown also occurs in erythroblasts from spleens of anemic mice when placed in culture without EPO (Koury and Bondurant, 1990a). The prevention of apoptosis by EPO in FVA cells is unrelated to any mitotic effect because DNA synthesis remains undiminished in the cells deprived of EPO up until they undergo apoptosis (Koury and Bondurant, 1988). Furthermore, FVA cells undergo DNA cleavage at various times throughout the cell cycle (Kelley, L., Bondurant, M. and Koury, M., unpublished results). The continued DNA synthesis found in FVA cells over a prolonged period without EPO does not appear to occur in murine CFU-E and EPOdependent erythroblasts which are not virus infected (Krystal, 1983). Thus, intracellular signals resulting from the Friend leukemia virus infection may be responsible for continued DNA synthesis in FVA cells following EPO withdrawal. However, this proliferative signal is not in itself sufficient to ensure survival and differentiation. Rather, the survival effect of EPO during terminal differentiation appears to be separate from any mitotic signal. The question remains, does EPO cause any mitotic signal in uninfected CFU-E and early erythroblasts? In the [3H]thymidine-incorporation assay for EPO developed by Krystal (1983), the incorporation in cells without EPO decreases dramatically during the early hours of deprivation, whereas incorporation also decreases with EPO but not nearly as much. Thus, the difference in thymidine incorporation with EPO as opposed to without EPO may be due to the rapid death of the EPO-deprived controls. Regulation of erythrocyte production

After an erythroid-progenitor cell with EPO receptors is exposed to the hormone, the effects of EPO may well have different consequences depending upon the stage of differentiation of the cell. In the case of immature erythroid progenitors, BFU-E siages and earlier, the cells may have EPO receptors, but as discussed above they also have receptors for, and are responsive to, other hematopoietic growth factors such as IL-3, granulocyte-macrophage colony-stimulating factor and c-kit ligand. In other words, some of these early stage erythroid-progenitor cells may be considered EPO responsive, rather than strictly EPO dependent. However, at later stages of erythroid development, such as CFU-E and proerythroblasts, the progenitor cells appear to lose their ability to respond to these other factors while retaining or greatly increasing their EPO receptors. Thus, cells at these late stages of erythroid differentiation have become dependent upon EPO for their survival. The period of continuous EPO dependence in late-stage progenitor cells has been estimated from experiments using

FVA cells and regenerating murine CFU-E. The erythroblasts appear to lose their EPO dependence between their penultimate and final cell divisions (Nijhof et al., 1987; Koury and Bondurant, 1988). This stage would correspond to basophilic erythroblasts. In other words, a murine erythroid cell that has begun hemoglobin synthesis appears to have lost its EPO dependence. The acquisition of EPO dependence is more difficult to assess, but EPO-removal experiments with elutriated, regenerating CFU-E (Landschulz et al., 1992) indicate that they acquire EPO dependence when they have four remaining divisions. These results would predict that EPO dependence in murine erythroid progenitors extends over approximately three generations of cells beginning at the early CFU-E stage and ending prior to the last cell division. While all of the erythroid progenitors in the dependent stages require EPO for their survival, individual progenitor cells require different EPO concentrations. Indeed, the concentrations of EPO required by individual FVA cells to prevent apoptosis vary by several-hundredfold (Kelley, L., Bondurant, M. and Koury, M. unpublished results). This varying EPO requirement of individual progenitors is consistent with the EPO dose responsiveness of CFU-E for survival and growth in vitro. CFU-E demonstrate dose responsiveness to EPO in concentrations ranging over 0.01 -2.0 U/ml(2.5 500 pM; Eaves et al., 1979; Eaves and Eaves, 1984). This concentration range is equal to that encountered in the plasma in vivo and extends from concentrations found in normal individuals to those found in severely anemic states (Koury et al., 1989a; Erslev, 1991). Thus, significant evidence suggests that individual erythroid-progenitor cells in the CFU-E and proerythroblast stages can have similar differentiation potentials while requiring markedly different amounts of EPO to survive and develop. A model has been proposed to explain the control of erythrocyte production by EPO based on apoptosis in the EPO-dependent stage of erythroid differentiation, and on individual cell heterogeneity in the concentration of EPO required to prevent the apoptosis (Koury and Bondurant 1990a; 1990b). This model is depicted in Fig. 5. In this model, most erythroid progenitors succumb to apoptosis during the EPOdependent period such that the normal production of billions of erythrocytes each day is accomplished with a minority of erythroid-progenitor cells surviving the EPO-dependent period. In anemic or hypoxic states, the increased EPO produced by the kidneys permits the survival of many EPOdependent progenitors that would normally have died in the presence of normal EPO concentrations. Conversely, in hypertransfused or hyperoxic states, the decreased EPO that results leads to the apoptosis of the large majority of EPO-dependent progenitor cells, including some which would have survived in the presence of normal EPO concentrations. Outlook

Most of the information contained in this review has come from the results of research performed in the seven years since the EPO gene was cloned. This dramatic expansion of research on EPO and its function has been accompanied by the successful introduction of recombinant human EPO into standard medical practice. Although these research accomplishments and their medical applications have been remarkable, many questions remain unanswered and provide the basis for future research. Questions arise in each area discussed in the review. Concerning the relationship between hypoxia and EPO gene transcription: how does cellular hypoxia result in EPO

660 A. NORMAL ERYTHROPOIETIN PRE EPO DEPENDENT CELLS

DEPENDENT CELLS

--__--POST DEPENDENT CELLS

I I I I I l l 1

sg:g

B. INCREASED ERYTHROPOIETIN PRE EPO DEPENDENT CE

______

The authors thank Stephen T. Sawyer and Stephen T. Koury for reviewing the manuscript and for helpful discussions. They thank Mary J. Rich for preparing the manuscript. The authors are supported by research grants from the National Institutes of Health (DK-31513) and the Department of Veterans Affairs.

REFERENCES

DEPENDENT

C. DECREASED ERYT H RO PO lETl N PR E

DEPENDENT CELLS POST DEPENDENT CELLS

In terms of the EPO-responsive cells: at which stage of early erythroid cell differentiation does EPO first have a role? Is EPO involved in the process of commitment of pluripotent hematopoietic cells to erythroid differentiation? What cellular events lead to EPO dependence? What is the basis of the heterogeneity among EPO-dependent cells for the amount of EPO required to permit survival? Answers to these questions will have implications for other hematopoietic growth factors in particular, and development and differentiation in general.

f I 1

EQ

Fig.5. Model of erythropoiesis based on EPO suppression of programmed cell death (apoptosis). Erythroid-progenitor cells enter a period of development in which they are dependent upon EPO for survival (EPO DEPENDENT CELLS). See Fig. 3 for the relationship to the EPO-dependent period and the stages of erythroid differentiation. ( 0 )Surviving viable cells; ( 0 )cells undergoing programmed cell death owing to insufficient EPO. Before entering the EPO-dependent period, the progenitors can survive without EPO (PRE EPO DEPENDENT CELLS). Cells surviving transit through the EPOdependent period can complete maturation into reticulocytes without EPO and ultimately become red cells (POST EPO DEPENDENT CELLS). Only one division appears to occur in murine erythropoiesis in the post-EPO-dependent stages. Reproduced with slight modification from Koury and Bondurant (1990b).

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