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EPO’s Alter Ego: Erythropoietin Has Multiple Actions

^a Haematology,
^b Nephrology, and
^c Oncology, Cancer Research Centre, Belfast City Hospital, Queen’s University, Belfast, Northern Ireland

Key Words. Anemia • Drug target • Erythropoietin receptor • Pleiotropic effects • Quality of life • Recombinant human erythropoietin

Terence R. Lappin, Ph.D., Department of Haematology, Cancer Research Centre, Queen’s University, Belfast, University Floor, Tower Block, Belfast City Hospital, Lisburn Road, Belfast BT7 9AB, Northern Ireland. Telephone: 44-2890-329241, ext 2013; Fax: 44-2890-263927; e-mail: t.lappin{at}qub.ac.uk

	ABSTRACT

Top Abstract Introduction EPO as a Hormone EPO as a Paracrine... Role of EPO in... Angiogenesis Neural Tissue Gonadal Function Mucosal Tissue Other Tissues The Role of EPO... References

 
Many cancer patients suffer from anemia, which has a major detrimental effect on their quality of life. Recombinant human erythropoietin (rHuEPO) is now widely used in cancer patients, as it improves hematocrit, lowers blood transfusion requirements, and improves quality of life. Recent research indicates that EPO has pleiotropic effects on the body well beyond the maintenance of red cell mass, but the mechanisms involved in relieving fatigue and improving quality of life in cancer patients are poorly understood. EPO receptors (EPO-Rs) have been detected in many different cells and tissues, providing evidence for autocrine, paracrine, and endocrine functions of EPO. Apart from its endocrine function, EPO may have a generalized role as an antiapoptotic agent that is associated with enhancement of muscle tone, mucosal status, and gonadal and cognitive function. The recent discovery of EPO-Rs in breast tumor vasculature, while raising important questions about the possible effects of pharmacological doses of rHuEPO on tumor cells, also suggests that the receptors could provide a useful target for drugs attached to EPO.

	INTRODUCTION

Top Abstract Introduction EPO as a Hormone EPO as a Paracrine... Role of EPO in... Angiogenesis Neural Tissue Gonadal Function Mucosal Tissue Other Tissues The Role of EPO... References

 
Many cancer patients suffer from anemia, which is a major contributor to cancer-associated disease. Chemotherapy-induced anemia causes reductions in a patient’s energy level and quality of life [1]. Recombinant human erythropoietin (rHuEPO) is now widely used in cancer patients, as it improves hematocrit, lowers blood transfusion requirements, and improves quality of life. There is now clear evidence that EPO has pleotropic effects on the body well beyond the maintenance of red cell mass, but the mechanisms involved in relieving fatigue and improving quality of life in cancer patients are poorly understood. A major question is whether EPO exerts its effects exclusively through increased oxygen delivery via increased red cell mass or through its actions on diverse cells and tissues. The question has important physiological and clinical implications because EPO receptors (EPO-Rs) have been discovered on many different cells and recently have been found in breast tumor vasculature [2]. Clearly, it is relevant to establish the effect of pharmacological doses of rHuEPO on tissues outside the erythroid compartment and to assess the possible effects of EPO on tumor growth.

Erythropoietin production is regulated in order to maintain an optimal red cell mass under physiological conditions. In the functional feedback model proposed by Erslev and Gabuzda [3], the rate of red cell production is closely linked to the demand for oxygen by the tissues (Fig. 1). Normal red cell production is dependent on basal levels of secreted EPO, which are equivalent to 0.8 to 4.0 pM/l of EPO (5-25 U/l) in plasma. Decreases in oxygen delivery to the kidney result in increased EPO production and stimulation of erythropoiesis. Conversely, EPO levels are downregulated by increased oxygen supply to the kidneys. The inappropriately low levels of EPO found in persons with anemia secondary to chronic renal failure provided a major incentive to produce an exogenous source. The widespread use of rHuEPO for renal patients arguably represents the most significant advance in clinical nephrology over the past 20 years [4]. Parenteral rHuEPO therapy for patients with the anemia of chronic renal failure currently aims to maintain the hemoglobin above 11g/dl [5] and within the hematocrit range of 33%-36% [6].

View larger version (25K): [in this window] [in a new window]   Figure 1. The EPO endocrine feedback loop.

	EPO AS A HORMONE

Top Abstract Introduction EPO as a Hormone EPO as a Paracrine... Role of EPO in... Angiogenesis Neural Tissue Gonadal Function Mucosal Tissue Other Tissues The Role of EPO... References

 
The foundations of our present understanding of the hormonal role of EPO were laid by a succession of French scientists who visited South America during the second half of the nineteenth century. Paul Bert and his collaborator Denis Jourdanet showed that the physiological effects of gases depend upon their partial pressure, and they established the link between tissue hypoxia and the production of erythrocytes [9, 10]. They realized that the symptoms of mountain sickness experienced by climbers in the Andes, and the anemia suffered by Jourdanet’s patients in Paris, could both be attributed to hypoxemia. On a visit to Peru in 1890, Viault took blood samples from himself at sea level in Lima, and, a few weeks later, after climbing 15,000 feet to Morococha, found a substantial increase in his red blood cell count [11]. More than a century earlier in 1774, Joseph Priestley had demonstrated that oxygen is required to support both life and combustion in his famous "mouse and candle in a bell-jar" experiment. Then, toward the end of the nineteenth century, Louis Pasteur showed that there was a substantial increase in carbohydrate consumption at low oxygen tension. Against this background and a gathering interest in the effects of hormones, Carnot and Deflandre hypothesized in 1906 that a circulating factor, hemopoietine, was responsible for red cell production and that its concentration in the blood increased in response to anemia or high altitude [12]. The evocative name hemopoietine was replaced in 1948 by the term erythropoietin [13].

EPO is produced in the adult kidney and fetal liver in response to hypoxia. Although only present in plasma at picomolar concentrations, EPO regulates the production of approximately 2.3 million cells per second. The task of isolating EPO was accomplished by Goldwasser’s group at the University of Chicago in 1977, the result of a continuous effort spanning 20 years [14]. This remarkable achievement paved the way for cloning the EPO gene and the subsequent production of rHuEPO for treatment of patients with EPO-deficiency anemias [15, 16]. This research also provided the necessary tools to explore the structure of the EPO molecule and its range of functions.

Erythropoietin is a glycoprotein with a molecular weight of 30.4 kilodaltons; 39% of its mass consists of carbohydrate structures. The EPO gene encodes a protein precursor of 193 amino acids. Cleavage of a 27-amino acid leader sequence yields a mature protein that undergoes N-linked glycosylation at three asparagine residues at amino acid positions 24, 38, and 83 [17], and O-linked glycosylation at a serine residue, amino acid position 126 [18]. The C-terminal arginine is removed, possibly by an intracellular carboxypeptidase [19], to produce the final circulating form of 165 amino acids. The tertiary structure of EPO is defined by four antiparallel -helices with adjoining loops. Intact single molecules bind to two adjacent EPO-Rs on the membrane of target cells and trigger an intracellular signaling cascade that regulates cell survival, proliferation, and differentiation.

	EPO AS A PARACRINE AND AUTOCRINE FACTOR

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For many years, EPO was thought to act exclusively on erythroid progenitor cells. This assumption was challenged when EPO gene expression was detected in the testes and spleen [20]. The subsequent detection of the EPO-Rs by immunocytochemistry and in situ hybridization in a wide range of cells, including neurons, astrocytes, microglia, and developing heart and cancer cell lines, provided further evidence for a potential autocrine or paracrine role for EPO in some tissues. Of particular interest was the observation that some cells (e.g., astrocytes) are capable of producing both EPO and EPO-Rs. Utilizing radiolabeled EPO, specific EPO binding sites have been identified in the hippocampus and the cerebral cortex of the adult mouse [21]. These regions are vulnerable to ischemia, and evidence is accumulating that EPO protects neurons from ischemic insult. Sakanaka and colleagues [22] infused EPO into the lateral ventricles of gerbils and found that it prevented ishemic-induced learning disability and rescued hippocampal CA1 neurons from lethal insult. Furthermore, when the animals were subjected to a mild ischemic insult, insufficient to produce neuronal damage, infusion of soluble EPO-R (the extracellular domain of EPO-R) caused neuronal degeneration and impaired learning ability. In contrast, infusion of heat-denatured soluble EPO-R was not detrimental, confirming that EPO can prevent neuronal death in the hippocampus following an ischemic event.

Infusion of EPO into the cerebral ventricles of stroke-prone spontaneously hypertensive rats in which the left middle cerebral artery was permanently occluded alleviated the ischemia-induced place navigation disability as judged by the results of a specific water maze test [23]. This preliminary evidence from a rat model suggests that EPO may have a potential therapeutic role in limiting the sequelae of stroke.

Circulating EPO produced in the kidney does not cross the blood-brain barrier because of its large molecular weight. Nevertheless, there is evidence for a separate EPO paracrine system in brain that is supported by three lines of investigation. First, astrocytes from rat fetuses produce EPO in an oxygen-dependent manner in vitro [24], and exposure to low oxygen levels led to elevated EPO mRNA levels in monkey brain in vivo [25]. Of interest, the EPO produced by the rat astrocytes had a lower molecular weight compared to the circulating form, by virtue of its lower sialic acid content [24]. Second, immunoreactive EPO is present in human ventricular cerebrospinal fluid [26]. Third, EPO-Rs are expressed in fetal brain, as early as day 7 in the neural plate of mouse embryos [27] and in the central nervous system of human fetuses [28].

One experimental approach to characterize endocrine and paracrine functions of EPO is to assess the effect of hypoxia on EPO production in various tissues over time. In mice subjected to sustained hypoxic stimulation, the levels of EPO gene expression in the kidney and circulating EPO increase and peak at about 4 hours, returning to near normal values after 24 hours [29, 30]. In contrast, the same hypoxic stimulus causes an increase in EPO production in the brain, which remains at a plateau for as long as the hypoxia remains. Of further interest, the combined stimuli of estrogen and hypoxia act synergistically to increase EPO expression in the oviducts of immature mice [30]. Placentas from mice and rats were found to have low affinity receptors for EPO [31]. These different responses suggest that EPO is acting as a true endocrine hormone in the kidney/bone marrow system but has a paracrine mode of action in the oviduct and in the brain, possibly providing protection against the deleterious effects of hypoxia.

	ROLE OF EPO IN NONERYTHROID SYSTEMS

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There are now several lines of evidence indicating that the action of EPO is not restricted to the erythroid compartment. The detection of EPO-Rs on numerous nonerythroid cell types over the past decade has led to a major revision of the biological role of EPO (Fig. 2). EPO/EPO-R interactions have been reported to induce a range of cellular responses, including mitogenesis, chemotaxis, angiogenesis, mobilization of intracellular calcium, and inhibition of apoptosis.

View larger version (30K): [in this window] [in a new window]   Figure 2. The expanding portfolio of EPO target cells and tissues.

	ANGIOGENESIS

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EPO also enhances endothelin-1 production [32, 33] by transcriptional upregulation [39], and the induction is augmented by two potent stimulators of ET-1 release, thrombin and angiotensin [32]. By utilizing rat aortic rings, EPO was found to stimulate angiogenesis by a mechanism involving endothelin [40]. Since hematopoietic and endothelial cell lines share common progenitors, it is reasonable to expect that cytokines and growth factors usually associated with hematopoiesis may also have a role in angiogenesis. Ribatti and colleagues [41] have shown that the human EA.hy926 endothelial cell line expresses EPO-R and responds to EPO by differentiating into vascular structures when seeded on Matrigel^TM. This process was associated with stimulation of JAK2 phosphorylation, cell proliferation, and matrix metalloproteinase-2 production. Furthermore, rHuEPO induced a potent angiogenic response in the chick embryo chorioallantoic membrane, strongly suggesting that EPO acts directly as a bona fide angiogenic factor [41].

	NEURAL TISSUE

Top Abstract Introduction EPO as a Hormone EPO as a Paracrine... Role of EPO in... Angiogenesis Neural Tissue Gonadal Function Mucosal Tissue Other Tissues The Role of EPO... References

 
Anemic patients treated with rHuEPO often experience an improvement in cognitive function. This has been attributed to better oxygen supply to the brain as a result of improved hematocrit. Cognitive function may also be enhanced by the increased level of circulating EPO, because the EPO-R has been detected in a wide range of neural tissues, including neuronal cell lines PC12 and SN6 [42]; NT2 and hNT cells [28]; rat brain capillary endothelial cells [43]; rat hippocampal and cortical neurons [44, 45], and human neurons, astrocytes, and microglia [46]. PC12 cells have a single class of EPO-R binding sites with a low affinity (K_d 16 nM) and are considerably weaker than the two classes of binding sites found in erythroid cells (K_d 95 pM and 1.9 nM) [42]. EPO also stimulated mitogen-activated protein kinase (MAPK) activity in PC12 cells [47] and caused an increase in intracellular Ca²⁺ [40]. EGTA had no effect on binding of EPO to its receptor but completely inhibited the increase in intracellular Ca²⁺, indicating that there is calcium influx from outside the cells [42].

Rat brain capillary endothelial cells have a single class of EPO-Rs with low affinity and approximately 10,000 sites per cell. As well as authentic EPO-R mRNA, they also contain an elongated isoform [43]. These observations suggest that EPO acts in brain capillary cells as a competence factor. Protein and mRNA for EPO and EPO-R are expressed by human neurons and glial cells in spinal cord and brain during fetal development [28]. Human neurons were found to undergo an increase of EPO-R expression when stimulated with TNF-, whereas human astrocytes showed a decrease in EPO-R expression when stimulated by IL-1ß, IL-6, or TNF- [46].

Neural cells also produce EPO. Astrocytes produce EPO in an oxygen-dependent manner [24], and EPO mRNA expression in human fetal neuronal cells doubled under conditions of hypoxia [28]. EPO increases both cellular Ca²⁺ uptake and the intracellular Ca²⁺ concentration in differentiated PC12 cells, effects that are abrogated by the calcium channel antagonist nicardipine or anti-EPO antibody. EPO improved cell viability in serum and nerve growth factor-deficient medium by activation of Ca²⁺ channels [47]. The EPO produced in the brain has a lower molecular mass, possibly as the result of reduced sialylation [24]. This supports the concept of a paracrine function for brain EPO because plasma EPO requires extensive sialylation in order to prolong its survival in the circulation.

Erythropoietin protects neurons from glutamate toxicity [44], but the protective effect is blocked by the simultaneous addition of EGTA, indicating the transient rise in intracellular Ca²⁺ elicited by EPO is a key event in the neuroprotective mechanism. The neuroprotective effect of EPO in reducing hypoxia-induced cell death has been investigated in rat postnatal hippocampal neurons [45]. Hypoxia-induced neuronal cell death was inhibited by addition of EPO or cycloheximide. This suggests that neuronal cell death triggered by hypoxia is the result of an active protein-dependent process that can be prevented by EPO.

	GONADAL FUNCTION

Top Abstract Introduction EPO as a Hormone EPO as a Paracrine... Role of EPO in... Angiogenesis Neural Tissue Gonadal Function Mucosal Tissue Other Tissues The Role of EPO... References

 
rHuEPO improves sexual function in patients with end-stage renal disease. In isolated, intact adult rat Leydig cells, rHuEPO bound to two classes of receptors with different affinities. EPO stimulated testosterone production, and this effect was completely blocked by the addition of anti-EPO antibody [48]. The calcium channel blocker verapamil did not affect rHuEPO-induced testosterone secretion, indicating that the mechanism does not entail Ca²⁺ transmembrane flux. Staurosporine blocked rHuEPO-stimulated testicular steroidogenesis in a dose-dependent manner, suggesting that the mechanism involves protein kinase C [49].

Foresta and colleagues [50] catheterized the peripheral and left spermatic vein in a group of young adults. Injection of rHuEPO (60 IU/kg) into the cubital vein had no effect on luteinizing hormone or follicle-stimulating hormone levels in either the peripheral or spermatic veins, but increased spermatic testosterone levels by approximately 400%. Thus, rHuEPO influences testicular steroidogenesis in man by directly stimulating testosterone production by Leydig cells in a process that appears to be independent of gonadotrophin secretion.

	MUCOSAL TISSUE

Top Abstract Introduction EPO as a Hormone EPO as a Paracrine... Role of EPO in... Angiogenesis Neural Tissue Gonadal Function Mucosal Tissue Other Tissues The Role of EPO... References

 
Gastric mucosal lesions are common in patients with end-stage renal disease and are often improved after treatment with rHuEPO. Utilizing the rat gastric mucosal cell line, RGM-1, rHuEPO was found to bind specifically and to increase cell proliferation in a dose-dependent manner [51]. To determine whether rHuEPO affects response to injury, Juul and colleagues [52] treated IEC-6 cells with TNF- plus cycloheximide and found that cells pretreated with rHuEPO migrated faster than controls left untreated with EPO. EPO-treated cells also showed decreased cytokine-induced apoptosis. Since EPO-Rs are expressed on enterocytes in the small bowel of human fetuses, the EPO present in human milk may modulate intestinal function in the neonate.

	OTHER TISSUES

Top Abstract Introduction EPO as a Hormone EPO as a Paracrine... Role of EPO in... Angiogenesis Neural Tissue Gonadal Function Mucosal Tissue Other Tissues The Role of EPO... References

 
Clearly, EPO can no longer be regarded as a hormone whose actions are restricted to erythroid cells in bone marrow. In addition to the EPO functions already discussed, several other tissues have been reported to express the EPO-R or EPO itself. For instance, EPO induces myocyte proliferation, probably through signaling pathways involving tyrosine kinase, phospholipase C, and phosphokinase C [53]. This smooth muscle cell turnover may also be a factor in modifying peripheral vascular resistance.

EPO appears to have a role in alleviating oxidative stress and inflammation. Kristal and colleagues [54] have reported that incubation of polymorphonuclear leukocytes (PMNLs) from hemodialysis patients and healthy controls showed a significant reduction of superoxide release. More recently, EPO-R gene expression was detected in PMNLs and the cells specifically bound ¹²⁵I-EPO [55]. Furthermore, the percentage of cells expressing EPO-R correlated with the serum EPO level, suggesting upregulation of the receptor by the EPO.

	THE ROLE OF EPO IN CANCER

Top Abstract Introduction EPO as a Hormone EPO as a Paracrine... Role of EPO in... Angiogenesis Neural Tissue Gonadal Function Mucosal Tissue Other Tissues The Role of EPO... References

 
Recently, Acs and colleagues [2] reported basal and hypoxia-stimulated expression of EPO and EPO-R in human breast cancer cells. EPO stimulated proliferation of breast cancer cells. In clinical specimens of breast carcinoma, high levels of EPO and EPO-R were associated with the tumor vasculature but not with normal breast, benign papilloma, or fibrocystic tissue [2]. Hypoxic tumor regions displayed the highest level of EPO and EPO-R expression. In addition, exposure to hypoxia stimulated both EPO mRNA and EPO-R mRNA expression in two breast cancer cell lines, MCF-7 and BT549.

These observations provoke two major questions: First, does EPO therapy affect the rate of proliferation of the cancer? Second, does the expression of EPO-R on tumor cell membranes provide a target for cancer therapy? Although EPO-R has been detected in tumor cells, there is no compelling evidence at present that exogenous rHuEPO confers a proliferative advantage on these cells. Specific cell surface receptors provide an ideal therapeutic target and it may be possible to utilize EPO-R in novel chemotherapeutic approaches for eradicating tumor cells (Fig. 3). Alternatively, it may be possible to identify therapeutic targets within the signaling pathway of tumor cells bearing EPO-R. EPO activates two major signaling cascades, the JAK2/STAT5 and the MAPK pathways. In erythroid progenitor cells, the JAK2/STAT5 pathway is predominant. In contrast, vascular smooth muscle cells (implicated in hypertension, a complication of rHuEPO treatment) treated with EPO showed strong MAPK activation, but EPO-mediated phosphorylation of proteins involved in the EPO-R/JAK2/STAT5 cascade was undetectable [56]. Although Acs et al. [2] demonstrated EPO-stimulated tyrosine phosphorylation in breast cancer cells, they did not identify the signaling cascade involved. Careful investigation of the EPO-R signaling cascades present in specific tumor cells may reveal further potential targets.

View larger version (12K): [in this window] [in a new window]   Figure 3. Proposed model for potential chemotherapy directed to EPO-R on tumor cells. A) EPO-R expressed on tumor cells; B) EPO-R interactions with MEK/MAPK and JAK2/STAT5 signaling; C) Blockade of EPO-R signaling in tumor cells leads to apoptosis. Abbreviation: MAPK = mitogen-activated kinase; ERK = extracellular signal-regulated kinase; MEK = MAPK/ERK kinase.

The effectiveness of rHuEPO as an adjunct to chemotherapy was assessed in a prospective nonrandomized multicenter community trial conducted by Demetri and colleagues [61]. More than 2,000 patients receiving cytotoxic therapy were given 10,000 U of epoetin alfa three times per week for up to 16 weeks. Patients who showed an increase of hemoglobin of less than 1g/dl had their dose escalated to 20,000 U three times per week. The patients reported significant enhancement of quality of life related to energy level, activity level, and functional status. The improvements were significantly correlated with hemoglobin levels but were unrelated to antitumor response. The results of this study and others confirm the benefits of rHuEPO for anemic cancer patients but do not elucidate the mechanisms responsible for the changes. It is tempting to speculate that the pharmacological doses used have direct effects on cells and tissues over and above the correction of hypoxemia. Since EPO-Rs have been detected in many different organ systems, EPO may have a generalized role as an antiapoptotic agent that is associated with enhancement of muscle tone, mucosal status, and gonadal and cognitive function. In addition, rHuEPO may exert an antitumor effect indirectly by improving the general health and well-being of the patient.

Apart from its classical role as a hormone, recent revelations of novel physiological functions and tissue-specific regulation clearly show that EPO has an alter ego. Obviously, time is required to develop an understanding of this side of EPO’s character, and there are likely to be more surprises along the way.

	ACKNOWLEDGMENT

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These studies constitute a scientific product of the All Ireland Fatigue Coalition and the NCI-All Ireland Cancer Consortium.

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Top Abstract Introduction EPO as a Hormone EPO as a Paracrine... Role of EPO in... Angiogenesis Neural Tissue Gonadal Function Mucosal Tissue Other Tissues The Role of EPO... References

 

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