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News Physiol Sci 16: 110-113, 2001;
1548-9213/01 $5.00
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News in Physiological Sciences, Vol. 16, No. 3, 110-113, June 2001
© 2001 Int. Union Physiol. Sci./Am. Physiol. Soc.

Pleiotropic Functions and Tissue-Specific Expression of Erythropoietin

Ryuzo Sasaki, Seiji Masuda and Masaya Nagao

R. Sasaki, S. Masuda, and M. Nagao are in the Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.

   Abstract
 
Erythropoietin (EPO) is produced in the brain, uterus, and oviduct. Brain EPO plays a neuroprotective role, and uterine EPO is likely involved in estrogen-dependent angiogenesis. Hypoxic induction of brain EPO markedly differs from that in the kidney. EPO in the uterus and oviduct is estrogen inducible.


   Introduction
Top
 Introduction
Expression of EPO and...
 Neuroprotective role of EPO
 Angiogenic role of EPO
 Regulation of EPO production
 References
 
Erythropoietin (EPO), which is produced by the liver in the fetus and by the kidney in the adult, is the primary stimulator of red blood cell formation. A major signal regulating EPO production in these tissues is the oxygen concentration. EPO production is markedly enhanced under hypoxia, mainly through transcriptional activation of the EPO gene (see citations in Ref. 18). A hypoxia-induced increase of EPO in the blood stimulates the formation of red blood cells, resulting in improvement of the oxygen supply and eventually repression of the activated gene transcription.

Erythropoietic stimulation has been believed to be the sole physiological function of EPO. More recently, however, the central nervous system (CNS) and female reproductive organs have been found to produce EPO. In this review, we deal with possible roles of EPO in these new sites and characteristics of regulation of EPO production, which markedly differ from those of the kidney.


   Expression of EPO and its receptor in the brain
Top
 Introduction
 Expression of EPO and...
Neuroprotective role of EPO
 Angiogenic role of EPO
 Regulation of EPO production
 References
 
A survey of rat organs has revealed hypoxia-inducible expression of EPO mRNA in the testis, brain, liver, and kidney (19). Subsequently, astrocytes have been shown to be responsible for production of brain EPO (11, 14). More recently, it has been shown that neurons also produce EPO (2).

Expression of the functional EPO receptor (EPOR) has been found in neuronal cell lines such as PC12. Binding of EPO causes a rapid and transient Ca2+ influx via plasma membrane Ca2+ channels (1, 8, 13). EPO also increases the intracellular monoamines, dopamine release, and tyrosine hydroxylase activity (8, 13) and supports cell survival when cultured without serum and nerve growth factor (8). EPO augments choline acetyltransferase in mouse embryonic primary septal neurons (7). EPOR is expressed in primary cultured neurons prepared from hippocampus and cerebral cortex of rat embryos (15). Specific binding sites of radioiodinated EPO are present in some defined areas of the adult mouse brain, including hippocampus and cerebral cortex, in which neurons highly vulnerable to ischemia exist (5). EPOR mRNA is abundantly expressed in the brain of mouse early embryos, and its level is dramatically reduced during development (9), suggesting that EPO might play an unidentified role in brain development. The CNS of primates, including humans, also expresses EPO and EPOR (6, 10, 11). It is unlikely that the renal EPO crosses the blood-brain barrier under physiological conditions. Thus CNS has a paracrine EPO/EPOR system that is independent of the endocrine system (kidney/bone marrow and spleen) for erythropoiesis.


   Neuroprotective role of EPO
Top
 Introduction
 Expression of EPO and...
 Neuroprotective role of EPO
Angiogenic role of EPO
 Regulation of EPO production
 References
 
In vivo neuroprotective action.
In a gerbil forebrain ischemia model, intracerebroventricular infusion of EPO ameliorates the ischemia-induced loss of synapses in the hippocampal CA1 field, which contains neurons highly vulnerable to ischemic insult, and eventually alleviates neuron death and learning disability (Fig. 1AGo; see Ref. 17). The use of soluble EPOR (sEPOR), an extracellular domain capable of binding with EPO, has provided evidence that the endogenous brain EPO is critical for neuronal survival. Infusion of sEPOR yielded neurons containing fragmented DNA (TUNEL positive) in brain subjected to a mild ischemia, under which brain damage is undetectable. Infusion of sEPOR also caused neuron death and impaired learning ability, whereas infusion of the heat-denatured sEPOR was not detrimental (Fig. 1BGo; see Ref. 17). Focal cerebral ischemia by permanent middle cerebral artery occlusion is thought to be a better model for stroke. Infusion of EPO into the cerebroventricles attenuates place navigation disability and cortical infarction induced by permanent occlusion of middle cerebral artery (2, 16).



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  		FIGURE 1. Neuroprotective effect of erythropoietin (EPO) and neurotoxic effect of soluble EPO receptor (sEPOR), which is capable of binding with EPO and thereby inhibits EPO function (see Ref. 17). A: intracerebroventricular infusion of EPO prevents ischemia-induced death of neurons in the hippocampal CA1 region in gerbils. Impairment of learning ability is also alleviated. B: intracerebroventricular infusion of sEPOR causes neuron death in the hippocampal CA1 region in animals subjected to a mild ischemia and decreases learning ability, whereas infusion of the heat-denatured sEPOR is not detrimental.

 
In addition to ischemia, the exogenous EPO has been shown to be neuroprotective in the brain exposed to a variety of brain insults. EPO injected into the lateral ventricle supports the survival of septal cholinergic neurons in adult rats that have been subjected to fimbria-fornix transection (7). Interestingly, intraperitoneal administration of EPO diminishes the extent of ischemic neuron death, concussive brain injury, immune damage in experimental autoimmune encephalomyelitis, and kainate-induced seizures (3). Brines et al. (3) proposed that translocation of peripheral EPO across the blood-brain barrier might take place through transcytosis by using EPOR expressed abundantly in brain capillaries. Since a massive dose of EPO (5,000 U/kg) was used, however, their findings do not indicate that the renal EPO crosses the blood-brain barrier under normal physiological conditions and thereby exhibit a neuroprotective effect in the CNS. Nevertheless, the neuroprotective effect of systemically administered EPO encourages a novel therapeutic approach.

In vitro neuroprotective action.
Glutamate is a principal excitatory amino acid neurotransmitter in the mammalian CNS and also mediates pathological neuronal injury (see citations in Ref. 18). Activation of the N-methyl-D-aspartate (NMDA) receptor, a glutamate receptor expressed in neurons, opens a channel permeable to both Na+ and Ca2+. Prolonged NMDA receptor activation because of insufficient recovery of glutamate released as a neurotransmitter has been thought to be mainly responsible for ischemia-induced neuronal death. A massive increase of intracellular Ca2+ by NMDA receptor activation leads to neuron death. EPO protected primary cultured neurons of the hippocampus and cerebral cortex from glutamate toxicity (2, 15).

A glutamate-mediated increase in intracellular Ca2+ activates neuronal nitric oxide synthase that requires the Ca2+-calmodulin complex, resulting in increased nitric oxide that is rapidly converted to highly toxic compounds such as peroxynitrite. Addition of EPO in neuron cultures rescued neurons from death induced by nitroprusside, a nitric oxide generator (17). Thus EPO may prevent neuronal death by mitigating the toxicity of nitric oxide-derived compounds. EPO supports the survival of erythroid precursor cells by inducing the expression of Bcl-X, a member of the Bcl-2 family that acts as an antiapoptotic protein. The mechanism by which EPO supports neuron survival is unknown.


   Angiogenic role of EPO
Top
 Introduction
 Expression of EPO and...
 Neuroprotective role of EPO
 Angiogenic role of EPO
Regulation of EPO production
 References
 
A number of investigators have reported that EPOR is expressed in cultured endothelial cells and that EPO exerts angiogenic activity on these cells (see citations in Ref. 20). However, whether endothelial EPOR is physiologically functional or is only a vestige reflecting a common developmental lineage between endothelial cells and hematopoietic cells remained unknown. Angiogenesis occurs actively in embryos, but it is repressed in healthy adults. An exception in adults is the female reproductive organs. In the uterine endometrium, angiogenesis takes place to support the endometrial growth that occurs during transition from the diestrus to estrous stage. Injection of sEPOR into the mouse uterine cavity in the diestrus stage inhibited endometrial growth (20). Activation of the uterine angiogenesis is triggered by 17ß-estradiol (E2), an ovarian hormone, and therefore this activation can be mimicked by injection of E2 to ovariectomized (OVX) mouse. EPO injected into the OVX mouse uterine cavity stimulated endometrial growth and angiogenesis, although the alterations induced by EPO differed somewhat from those induced by E2 (20). These results, combined with the finding that endothelial cells in the uterine endometrium express EPOR, suggest that EPO is one of the E2-regulated angiogenic factors, including vascular endothelial growth factor, that are required for completion of the cyclic uterine angiogenesis in the estrus cycle.


   Regulation of EPO production
Top
 Introduction
 Expression of EPO and...
 Neuroprotective role of EPO
 Angiogenic role of EPO
 Regulation of EPO production
References
 
Brain and kidney.
Exposure of mice to severe hypoxia (7% O2) markedly elevates serum EPO and EPO mRNA in the kidney and cerebrum. However, the levels of serum EPO and renal EPO mRNA are quickly lowered despite continuous hypoxia. Surprisingly, brain EPO mRNA is sustained at a high level for at least 24 h (Fig. 2AGo). Since hematocrit values are unchanged during experiments, the rapid decline of renal EPO mRNA is not due to the operation of the classic negative-feedback inhibition (EPO gene expression is repressed through the improved O2 delivery by the increased erythrocytes). Although the mechanism for the rapid decrease in the kidney remains unknown, this notable difference in the temporal patterns of hypoxia inducibility of EPO mRNA between kidney and brain seems to well reflect the tissue-specific functions of EPO. In the brain, EPO supports neuron survival under ischemia, and therefore a high level of EPO expression is required as long as hypoxia continues, whereas the continuous activation of EPO gene expression in the kidney would result in overproduction of erythrocytes, causing various disorders. Therefore, the downregulation of EPO gene expression must operate even under hypoxia in the kidney but not in the brain. EPO mRNA in the cerebellum is also hypoxia inducible, with a temporal pattern similar to that in the cerebrum, but its role in the cerebellum is not known (18). E2 induces EPO production in the female reproductive organs as described below, but E2 shows little effect on the kidney and brain (4).



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  		FIGURE 2. Temporal patterns of stimuli [hypoxia and 17ß-estradiol (E2)]-induced EPO and EPO mRNA. A: EPO in the serum and EPO mRNA in the kidney and cerebrum of mice under hypoxia. Eight-week-old mice were exposed to hypoxia (7% O2). {circ}, Cerebrum EPO mRNA; •, kidney EPO mRNA; {blacktriangleup}, serum EPO. The left ordinate shows the fold induction of EPO mRNA over the basal level, defined as 1. Values are means ± SE (n = 3). B: EPO mRNA in the uterus of ovariectomized (OVX) mouse. •, Mice exposed to hypoxia (7% O2) immediately after E2 injection; {circ}, mice exposed to hypoxia after olive oil (solvent for E2); {blacksquare}, mice left under normoxia after E2. Values are means ± SE (n = 5). C: EPO mRNA in the oviduct. •, 3-wk-old mice exposed to hypoxia (7% O2) immediately after E2; {circ}, mice exposed to hypoxia after olive oil; {blacksquare}, mice left under normoxia after E2. Values are means ± SE (n = 4). Modified from Refs. 4 and 12.

 
Female reproductive organs.
The role of EPO in uterine angiogenesis raises a possibility that the uterus produces EPO in an E2-dependent manner, because it is hard to speculate that the renal EPO is responsible for the periodic angiogenesis in the uterus. In vitro cultured uterus from OVX mouse expressed EPO protein and its mRNA in an E2-dependent manner (20). Administration of E2 to the OVX mouse induced a rapid and transient increase in EPO mRNA in the uterus (Fig. 2BGo). Interestingly, the uterine EPO mRNA is hypoxia inducible only in the presence of E2. Furthermore, the hypoxia-induced increase of EPO mRNA is far smaller in the uterus (2.5-fold) than in the kidney or brain (30-fold) (Fig. 2AGo). The rapid reduction in the uterine EPO mRNA level after E2 administration is not due to the metabolic depletion of E2 but due to the loss of responsiveness of the uterine EPO-producing cells to E2 (4). Although the molecular mechanism is not known, this downregulation may be very important for preventing uterine angiogenesis in an estrous cycle stage in which it should not occur.

The oviduct also produces EPO in an E2-inducible manner (12). Administration of E2 to a 3-wk-old non-OVX mouse before commencement of cyclic E2 synthesis in the ovary causes a transient increase in the oviductal EPO mRNA (Fig 2CGo). Notably, hypoxia induces oviductal EPO mRNA not only in mouse given E2 but also in mouse without E2 administration. As in the uterus of the OVX mouse (see Fig. 2BGo), exposure of a 3-wk-old non-OVX mouse to hypoxia did not induce uterine EPO mRNA when E2 was not given (12). Thus the EPO-producing cells in the uterus and oviduct differ in requirement of E2 for hypoxia inducibility of EPO mRNA. The possible physiological functions of EPO and tissue-specific regulatory features of EPO production are summarized in Table 1Go.


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  		TABLE 1. Possible roles of EPO and tissue-specific regulation of expression
 


   Acknowledgments
 
This work was supported by Grants-in-Aid from the Ministry of Education, Science, Culture and Sports of Japan and from the "Research for the Future" program of The Japan Society for the Promotion of Science.


   References
Top
 Introduction
 Expression of EPO and...
 Neuroprotective role of EPO
 Angiogenic role of EPO
 Regulation of EPO production
 References
 

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  3. Brines ML, Ghezzi P, Keenan S, Agnello D, de Lanerolle NC, Cerami C, Itri LM, and Cerami A. Erythropoietin crosses the blood-brain barrier to protect against experimental injury. Proc Natl Acad Sci USA 97: 10526–10531, 2000.[Abstract/Free Full Text]
  4. Chikuma M, Masuda S, Kobayashi T, Nagao M, and Sasaki R. Tissue-specific regulation of erythropoietin production in the murine kidney, brain, and uterus. Am J Physiol Endocrinol Metab 279: E1242–E1248, 2000.[Abstract/Free Full Text]
  5. Digicaylioglu M, Bichet S, Marti HH, Wenger RH, Rivas LA, Bauer C, and Gassmann M. Localization of specific erythropoietin binding sites in defined areas of the mouse brain. Proc Natl Acad Sci USA 92: 3717–3720, 1995.[Abstract/Free Full Text]
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  10. Marti HH, Gassmann M, Wenger RH, Kvietikova I, Morganti-Kossmann MC, Kossmann T, Trentz O, and Bauer C. Detection of erythropoietin in human liquor: intrinsic erythropoietin production in the brain. Kidney Int 51: 416–418, 1997.[Web of Science][Medline]
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  12. Masuda S, Kobayashi T, Chikuma M, Nagao M, and Sasaki R. The oviduct produces erythropoietin in an estrogen- and oxygen-dependent manner. Am J Physiol Endocrinol Metab 278: E1038–E1044, 2000.[Abstract/Free Full Text]
  13. Masuda S, Nagao M, Takahata K, Konishi Y, Gallyas F Jr, Tabira T, and Sasaki R. Functional erythropoietin receptor of the cells with neural characteristics: comparison with receptor properties of erythroid cells. J Biol Chem 268: 11208–11216, 1993.[Abstract/Free Full Text]
  14. Masuda S, Okano M, Yamagishi K, Nagao M, Ueda M, and Sasaki R. A novel site of erythropoietin production: oxygen-dependent production in cultured rat astrocytes. J Biol Chem 269: 19488–19493, 1994.[Abstract/Free Full Text]
  15. Morishita E, Masuda S, Nagao M, Yasuda Y, and Sasaki R. Erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons, and erythropoietin prevents in vitro glutamate-induced neuronal death. Neuroscience 76: 105–116, 1997.[Web of Science][Medline]
  16. Sadamoto Y, Igase K, Sakanaka M, Sato K, Otsuka H, Sakaki S, Masuda S, and Sasaki R. Erythropoietin prevents place navigation disability and cortical infarction in rats with permanent occlusion of the middle cerebral artery. Biochem Biophys Res Commun 253: 26–32, 1998.[Web of Science][Medline]
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  19. Tan CC, Eckardt KU, Firth JD, and Ratcliffe PJ. Feedback modulation of renal and hepatic erythropoietin mRNA in response to graded anemia and hypoxia. Am J Physiol Renal Fluid Electrolyte Physiol 263: F474–F481, 1992.[Abstract/Free Full Text]
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