The Role Of Erythropoietin And Its Receptor In Hypoxia In Gansu Zokor And Its Pathway

Eospalax cansus (E. cansus) is an excellent model of a mammal toward hypoxic tolerance which lives on the Loess Plateau of China. E. cansus belong to the genus Myospalax family of Rodentia, Spalacidae, Myospalacinae, Eospalax. They living long term in the hypoxia underground environment (low O2 and high CO2), is an ideal animal to study hypoxia adaptation.It has been recently reported that erythropoietin (EPO) and its receptor (EpoR) are involved in against hypoxic. Erythropoietin (Epo) is a 34.4 kDa circulating glycoprotein hormone, apart from its hematopoietic activity, also known as a tissue-protective cytokine. EpoR is a transmembrane protein that belongs to a superfamily of cytokine receptors. Epo binds to its receptor (Epo:EpoR2), inducing conformational changes in the receptor, followed by the activation of downstream signaling cascades. EpoR signaling in vitro was recently shown to increase Bcl-xL and decrease Bim mRNA. The downregulation of Bcl-xL induced apoptotic cell death. Bim down-regulation is a key component of survival signaling. Highly dynamic activation of the Bcl-xL and Bim survival pathways, having a clear segregation of function during the acute and chronic phases of the stress response.Studies have shown that low oxygen is huge damage to brain tissue. Results shown Epo/EpoR expression is associated with hypoxia stress, and they has recently been evaluated in tissue protection. We used real-time polymerase chain reaction (PCR) quantification to determine the expression of the Epo and EpoR gene in brain, liver and kidney tissues in E. cansus and SD rats. Our aimed at providing the hypoxic protection of Epo and EpoR on the molecule mechanism in E. cansus. And, we quantitatively Bcl-xL and Bim to they role in EpoR signal. And, full-length cDNA of EpoR gene is coloned from total RNA of E. cansus, and and to analyze the bioinformatics of EpoR protein/gene sequences. Eighteen E. cansus were randomly divided into three groups, each consisting of six rats:(i) oxygen content of 21%, (ii) oxygen content of 10.5%, and (iii) oxygen content of 6.5% conditions. We used quantitative real-time PCR to determine the tissue-specific expression of the Epo, EpoR, Bcl-xL and Bim mRNA in tissues under normoxic and hypoxic conditions. The major experiment results are as follow:1 Epo?EpoR?Bcl-xL and Bim mRNA expression of results in E. cansus and SD rat:Brain:Epo mRNA expression in the 21% O2 brain is substantially lower in E. cansus than in SD rat. EpoR mRNA response to 21% O2 in brain is similar in both species. However, mRNA levels of Epo and EpoR in E. cansus brain are higher than in SD rat under 10.5% and 6.5% O2. Hypoxia causes an up-regulation of Epo, EpoR and Bcl-xL mRNA in E. cansus brain, but downregulation of Bim.6.5% O2 additionally increased the Epo mRNA than 10.5% O2 in E. cansus.10.5% O2 additionally increased the EpoR and Bcl-xL mRNA than 6.5% O2 in E. cansus. No significant differences in Bim mRNA between 10.5% and 6.5% O2 in E. cansus. SD rats Epo and Bim of E. cansus are similar, but no significant differences in EpoR and Bcl-xL mRNA between 10.5% and 6.5% O2 in E. cansus.Liver:E. cansus liver produce higher Epo mRNA than SD rat under 21%,10.5% and 6.5% O2. EpoR levels in 21% and 10.5%O2 livers of E. cansus are similar, however, SD rat showed higher EpoR mRNA levels than E. cansus in 6.5% O2. Hypoxia (10.5% and 6.5% O2) causes an up-regulation of Epo, EpoR and Bcl-xL mRNA in E. cansus liver, but downregulation of Bim.10.5% O2 response is more apparent.6.5% O2 additionally increased the Epo mRNA than 10.5% O2 in E. cansus.10.5% O2 additionally increased the Bcl-xL mRNA than 6.5% O2 in E. cansus. No significant differences in EpoR and Bim mRNA between 10.5% and 6.5% O2 in E. cansus. Under the condition of low oxygen concentration, causes an up-regulation of Epo and EpoR mRNA in SD rat liver, but downregulation of Bcl-xL. Only under the 6.5% O2 up-regulation of Bim.Kidney:Epo mRNA expression in the 21% O2 kidney is substantially higher in E. cansus than in SD rat, but lower than SD rat in hypoxia. EpoR in E. cansus kidney are higher than in SD rat under 21%,10.5% and 6.5% O2 and hypoxia.6.5% O2 causes an up-regulation of Epo, EpoR, Bcl-xL and Bim mRNA in E. cansus kidney.10.5% O2 enhanced Bcl-xL expression in E. cansus kidney, but no significant differences in Epo, EpoR mRNA between 21% and 10.5% O2. under the condition of low oxygen concentration, causes an up-regulation of Epo and Bim mRNA in SD rat kidney, but downregulation of EpoR. but no significant differences in Bim mRNA between 21%, 10.5% and 6.5% O2 in SD rat.2 Here, we cloned E. cansus EpoR:a full-length EpoR cDNA sequence, containing a 1575bp open reading frame (ORF) that encoded 524 amino acids and termination codon. It's cDNA and protein sequence homology was high up to 90% with spalax using BLAST.3 Bioinformatics analysis the EpoR protein sequences. The study was found for EpoR in E. cansus, suggesting stabilize of transmembrane protein. Its chemical formula was C2546H3929N677O769S22, the molecular weigh was 57kDa, and theoretical isoelectric point was 4.89. The EpoR protein have a signal peptidemand and two transmembrane domain, positioning in the cell membrane. Fibronectin type 3 domain (FN3) is E. cansus EpoR protein conservative domain structure. The secondary structure of EpoR protein was a kind of mixed protein, composed of alpha helix and random coil. The E. cansus EpoR encompasses five tyrosine motifs on its cytoplasmic tail. Phylogenetic tree of EpoR protein sequence showed that E. cansus and spalax had the minimum genetic distance and far away from the Mus, Rattus and Homo etc, and the "furthest genetic distance with Ochotona princeps. E. cansus EpoR reveals unique amino acid changes, which may be functionally important for this transcription factor and indicate positive selection for hypoxia. The deduced EpoR proteins of the E. cansus contain characteristic structures and important domains similar to EpoR from other taxa.Epo and EpoR under the condition of the low oxygen concentration organized protection. We found that Epo and EpoR mRNA levels responses are species and tissue specific to hypoxia. The differences between tissues (brian, liver and kidney) in the kidneys. The observed differences help to elucidate E. cansus's outstanding capacity to cope with extreme environmental stress. The results described above suggested that hypoxia to increase EpoR mRNA expression in E. cansus brain to adapt to the hypoxic environment. Liver and kidney has a similar situation, but was more effectively increased in brain than liver and kidney. EpoR signal are important targets for adaptive change during evolution of hypoxia tolerance in E. cansus. We contrasted Bcl-xL with Bim, these results show that Bcl-xL and Bim are involved in the EpoR signal-mediated of the hypoxia stress in E. cansus brain and brain.10.5% O2 response is more apparent. Bcl-xL and Bim contrasting dynamic responses suggest a clear segregation of function during the acute and chronic phases of the stress response. We found a similar, highly dynamic activation of the Bcl-xL pathways in E. cansus kidney that was dependent on 6.5% O2, Bim are not involved in the EpoR signal-mediated. We deduced that the E.cansus more effectively mobilize the feedback mechanism to adapt to hypoxic environment than SD rat. And EpoR signal has to protect brain tissue from the effects of hypoxia. But more information is still necessary to fully understand the mechanism of EpoR biology. We can determine that the gained sequence was the cDNA sequence of E. cansus EpoR gene, and it can encode a complete EpoR protein. Not conservative intracellular area may be a characteristic of hypoxia adaptation, Five tyrosine phosphorylation site may downstream pathway is an important site. Conservative such release area and across the membrane area may be EpoR protein in common, such as localization in the cell membrane and extracellular combined with Epo, etc.