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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 4, 2005 as doi:10.1096/fj.05-3975fje. |
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Laboratory of Animal Molecular Evolution, Institute of Evolution, University of Haifa, Mt. Carmel, Haifa, Israel
3 Correspondence: Laboratory of Animal Molecular Evolution, Institute of Evolution, University of Haifa, Mt. Carmel, Haifa 31905, Israel. E-mail: aaron{at}esti.haifa.ac.il
SPECIFIC AIMS
The blind subterranean mole rat of the Spalax ehrenbergi superspecies that inhabits underground burrow systems provides a unique experimental system. In field measurements of Spalax’s natural underground habitat in flooded heavy clay soils during the rainy season, levels of 7.2% O2 and 6.1% CO2 were recorded. Great differences in the structure and expression of the erythropoietin gene in subterranean Spalax when compared with aboveground Rattus have been shown. In the present study we report the DNA sequence and the expression patterns of two forms of the Spalax’s erythropoietin receptor (sEpoR) in different developmental stages and hypoxic conditions compared with Rattus.
PRINCIPAL FINDINGS
1. Cloning
Full-length sEpoR mRNA was amplified and sequenced from the four Spalax species in Israel. The only potential glycosylation site in the tentative EpoR protein (amino acids NYS at position 77-79 in Spalax) that is common to all four Israeli Spalax species and to the rat, human, and mouse is preceded by a deletion of four amino acids. Two of the eight potential Tyr phosphorylation sites in the intracellular domain of the EpoR molecule, common to the human, rat, and mouse are substituted in all four Spalax species (Tyr
Phe at position 481, and TyrGly at position 499). A novel truncated form of sEpoR was found in the bone marrow (BM) of the four Israeli Spalax species. It contains the 3' proximal 91 bp from the total 1238 bp that comprises intron 6. The insert causes a frameshift and introduces a termination codon 13 bp after the insertion point. The truncated form produces a tentative 276-amino acid premature protein that includes the extracellular domain, the transmembrane domain and only 7 amino acids of the intracellular part, the last three of which are different from the authentic amino acids.
2. Expression of EpoR in the spleen of Spalax and Rattus
Spalax total EpoR (the complete and truncated forms) is constitutively expressed in the spleen of newborn and adult animals, but with no clear response to hypoxic stress (Fig. 1
). The total EpoR expression in Rattus spleen, especially in newborns, was higher than in Spalax. In Rattus spleen total EpoR showed a 25% increase in response to hypoxia in 7-day-old newborns and a slight increase in 14-day-old newborns. The level of EpoR transcripts of Rattus normoxic adult was lower than in newborns, but responded dramatically to severe short-term hypoxia with a 3-fold increase, while no response to long-term mild hypoxia was noticed (Fig. 1)
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The Rattus intron 5-inserted truncated form was expressed in the spleen in all conditions studied. Levels of this truncated form appeared to decrease gradually toward maturation. Similar to the pattern noticed for total Rattus EpoR expression, the truncated form also responded to short-term severe hypoxia with an 
5-fold increase in adults, but it did not respond to long-term mild hypoxia.
Expression of the Rattus intron 5-inserted truncated form in the spleens varies from 6% in normoxic adults to 17% in 7-day-old normoxic newborns from the levels of total EpoR expression. The Spalax intron 6-inserted form was not detected in any spleen samples studied.
3. Expression of sEpoR in bone marrow
We could not detect any expression of the truncated sEpoR in the spleen, so we tested the expression of both sEpoR forms in the BM. Total sEpoR in the BM was expressed at similar levels and with no dramatic changes at different developmental stages or under different physiological conditions imposed by hypoxic stress. The maximum expression was noticed in the normoxic adult BM with a decrease to half-fold in both severe short-term and mild long- hypoxia. In contrast with the spleen, EpoR is up-regulated by hypoxia in the BM of 7-day-old Spalax newborns; no such response was observed in 14-day-old newborns.
Compared with the total sEpoR expression, the quantities of the Spalax’s truncated form ranged between 1% in normoxia and short-term severe hypoxia to 6% in long-term mild hypoxia. This truncated form was undetectable in Spalax newborns both under normoxia and after hypoxic stress. In adults the truncated form was detectable, but comprised only 1% of the total sEpoR mRNA levels in normoxia. mRNA levels of the truncated sEpoR form decreased to half-fold in short-severe hypoxia whereas a 3-fold increase was noticed in long-term mild hypoxia (Fig. 2
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CONCLUSIONS AND SIGNIFICANCE
The EpoR mRNA was cloned from the blind subterranean mole rat, Spalax ehrenbergi, a model organism for hypoxia tolerance that spends its entire life in underground sealed burrows. The expression in hematopoietic tissues in development under normal and hypoxic conditions was also studied. In addition to the cloning of the full-length Epo-R, we identified a novel truncated form of the Spalax EpoR that is detectable in the Spalax BM but not in the spleen.
There are eight highly conserved tyrosine phosphorylation sites in the intracellular part of the EpoR protein of the human, mouse, rat, and pig. The Phe481 residue in Spalax EpoR corresponds to Tyr460 residue in the mouse. Substitution of Tyr460 to Phe abolishes the Epo-stimulated calcium influx into the cells and thus is required for calcium channel activation. Epo has been reported to affect calcium homeostasis by regulating Ca2+ influx. High Ca2+ influx contributes to cell damage induced by hypoxia and ischemia. Ca2+ entry blockers protect cells against hypoxic damage in different tissues, and the protective effect has been related to prevention of hypoxia-induced overload of the Ca2+ ion. Therefore, the Tyr to Phe substitution in the sEpoR could be an important adaptation that reduces this damage. Gly residue at position 499 in sEpoR corresponds to Tyr479 in the mouse and has been reported to play a role in phosphoinositide 3-kinase (PI 3-kinase) Epo-dependent activation through direct association with it. PI 3-kinase has been shown to play a major role in Epo-induced proliferation of human erythroid progenitors. The biological significance of this substitution in Spalax awaits elucidation.
Previous studies showed that Rattus Epo mRNA is highly expressed in neonatal liver and kidney and adult kidney after severe-short hypoxia whereas in long-mild hypoxia, after 24 h of stress, it declines to levels comparable with normoxia. Total Rattus spleen EpoR shows a similar pattern: high levels in normoxic, and short-severe hypoxic newborns and short-severe hypoxic adults, and a decrease in adult normoxia and long-mild hypoxia.
Spalax generally expresses lower levels of EpoR than Rattus in all samples studied; the highest levels were detected in stages where Epo levels were minimal. Thus, relatively high levels of EpoR are noticed in adult normoxic BM, when Epo expression in the kidney was relatively very low. High Epo levels might be correlated with the low expression of Epo-R in BM. EpoR mRNA was quantified on total BM cells and not only on erythroid progenitors. Therefore, the measured levels of EpoR mRNA do not necessarily indicate lower Epo-R expression in the expressing cells. The Epo-induced differentiation of erythrocyte progenitor cells in BM, due to the enhanced responsiveness of Spalax to Epo, might decrease the amount of sEpoR-expressing cells in the BM rather than reduce sEpoR expression within the cells. The differences in EpoR expression between Spalax and Rattus reported here may reflect the dramatic differences in Epo expression patterns that showed the adaptation of Spalax to short- and long-term hypoxia. Unlike Rattus, Spalax showed a unique ability to respond to hypoxia in a stress-dependent manner by strong and long-term expression of Epo.
Several splice variants of EpoR mRNA have been reported. In this study, a Spalax-specific alternatively spliced variant with 91 bp fragment inserted from intron 6 was found. The insert introduces four novel amino acids and a termination codon and results in a 276-amino acid premature EpoR putative protein with 7 amino acids of the cytoplasmic domain (NRRsvgv*). As this alternatively spliced sEpoR form lacks boxes-1 and -2, it is probably unable to confer Epo-mediated signal transduction. Expression of the Spalax truncated form in the BM was increased after 24 h mild hypoxia, at the same stage when Epo mRNA levels were maximal, which may support the hypothesis that the truncated form modulates the function of Epo by competing with its binding to the authentic receptor. The Rattus intron 5-inserted truncated form studied here comprises 6% to 17% of the total EpoR; the truncated form in Spalax comprises 1% to 6% of the total EpoR. If we assume that the truncated EpoR forms of both Spalax and Rattus are unable to elicit mitogenesis and signal transduction, which still awaits proof, but play a modulator role, then our results may suggest that the negative effect of the truncated form on the intact EpoR appears to be more emphasized in Rattus, but reduced in Spalax BM and absent in the Spalax spleen. This may increase the responsiveness of the intact receptor to Epo and explain the higher and time- and stress-dependent response of Spalax Epo to hypoxia as yet another mechanism to tolerate the stress in its niche.
Epo and EpoR are the focus of increasing interest as they have recently been shown to modulate cellular signal transduction pathways that extend beyond the erythropoietic function of Epo. Many of these activities are influenced by hypoxia. For example, in the CNS, up-regulation of both Epo and EpoR has been demonstrated after ischemia in the rat brain, which appears to protect the neurons from ischemic damage by inhibiting their apoptosis. Hypoxia-associated up-regulation of EpoR expression has also been demonstrated in several tumor types, mainly in breast and renal cancer.
The findings presented here open new avenues to unravel the functional implications of the unique structural features of sEpoR forms, which may well extend beyond the hemaopoietic system. Further studies, comparing the sEpoR with that of other mammals, will contribute to our understanding of the function of Epo and its receptor under physiological and pathophysiological conditions where oxygen delivery drops below what is necessary for the tissue. The mechanisms underlying these responses will be instrumental in the design of novel treatment modalities for hypoxia.
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FOOTNOTES
1 In partial fulfillment of Ph.D. studies. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3975fje;
This article has been cited by other articles:
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