Biological Significance Of Signal Transducer And Activator Of Transcription3, Hypoxia Inducible Factor1Alpha And Hypoxia Inducible Factor2Alpha In High Altitude Hypoxic Adaptation Species-Tibetan Antelope

The maintaining of oxygen homeostasis is the fundamental precondition ofaerobic organisms' normal life activities. The hypoxic uniqueness of theQinghai-Tibet Plateau environment has a great impact on the distribution, structure,function, adaptation, and evolutionary patterns of various organisms. Tibetan antelope(Pantholops hodgsonii), is a rare wild species of the Qinghai-Tibet Plateau, whichhas the strongest sports ability and hypoxia resistant ability among aboriginal animalsof the Qinghai-Tibet Plateau. It perennially lives in alpine meadow, prata, alpine-colddesert areas at the altitude of4300~5500m. During long term evolutions, the specieshas already achieved genetic features of hypoxia adaptation in high altitude atphysiology, morphology and gene level, and has already formed unique adaptationmechanisms. The establishment of the adaptation mechanism due to expression levelsof hypoxia-adaptive genes can be adjusted to change. In a large number ofhypoxia-adaptive genes, Signal transduction and activator of transcription factor3(STAT3), Hypoxia inducible factor-1(HIF-1), Hypoxia inducible factor-2(HIF-2) isthe core transcription factors of the oxygen signal transduction system. They play akey role in regulating cell hypoxia adaptive response to maintain body oxygenequilibrium and energy balance status.STAT3is the key signaling cascade components which integrate signals ofmultiple signaling pathways, having functions of signal transducing and transcriptioncontrolling. It regulate gene expressions of hypoxia response in various ways, such ascell proliferation, survival and apoptosis, angiogenesis, energy metabolism, whichhif-1is an important downstream target genes of STAT3. As a criticaloxygen-dependent transcriptional activator, HIF-1through the induction of glucosemetabolism, angiogenesis, erythropoiesis, many of hypoxic response gene expressionin oxygen uptake and transport pathway involved in the regulation of the intracellularoxygen equilibrium. HIF-2is also a critical oxygen-dependent transcriptionalactivator. With family members as hypoxia inducible factors (HIFs),HIF-1and HIF-2are compose of the oxygen regulation of subunit alpha and the structure of subunitbeta. Alpha subunit is the regulation of HIF-1and HIF-2activity. HIF-1α, HIF-2α,48%of the amino acid sequence homology, have very similar structural domains.HIF-1and HIF-2regulation of downstream target genes both overlap and each withspecificity. HIF-2target genes that mainly involves the bone marrow, blood vessel growth, vascular contraction, energy metabolism, catecholamine synthesis and ironmetabolism. Under hypoxic conditions, HIF-1and HIF-2are in a functioncomplementary, synergistic manner that promote cell adaptation to hypoxia regulateand maintain the body′s oxygen equilibrium. In summary, as important transcriptionfactors of the oxygen signal transduction system, STAT3, HIF-1and HIF-2play animportant role in the regulation of cell hypoxia adaptive responses. We speculate thatSTAT3, HIF-1and HIF-2plays a very important role in Tibetan antelopes′adaptionmechanisms to hypoxia. But there are few reports on the cloning and tissue expressioncharacters of stat3,hif-1α and hif-2α cDNA, which are the important content of theresearch on the hypoxia adaption molecular mechanism of Tibetan antelope. So thisresearch analyzed the differences of the tissue expression and hypoxia adaption ofstat3,hif-1α and hif-2α, with Tibetan antelope, Tibetan sheep and sheep asmaterials, further uncovered the meaning of these genes expression character in natureand the physiological significance of stat3,hif-1α and hif-2α for Tibetan antelope tosurvive.In this study, the cloning of stat3,hif-1α and hif-2α gene cDNA of Tibetanantelope, using RT-PCR and RACE, was applied, as well as the comparative analysisof the tissue-specific expressions of stat3,hif-1α and hif-2α among Tibetan antelope,Tibetan sheep and sheep(Ovis aries), using Real-time PCR and Western blot.Themain achievement of this study is listed as follows:1)Sequence analysis revealed that (a)The full-length Tibetan antelope stat3cDNA,which cloned by gene sequence analysis, is4288bp, comprising a2312bp openreading frame (ORF), a29bp5′UTR and a1947bp3′UTR. The similarity between itscoding sequence, predicted amino acid sequence and stat3of other mammalsexceeded86%, in which the similarity with cow was up to more than98%.(b)ThecDNA sequences acquired by cloning from the hif-1α gene of Tibetan antelopecomprised a2471bp ORF and a1911bp3′UTR. The similarity between its codingsequence, predicted amino acid sequence and hif-1α of other mammals exceeded87%,in which the similarity with cow was up to more than98%.(c)Partial coding regionsequence of Tibetan antelope′s hif-2α gene is515bp. Similarity between its codingsequence and hif-2α of other mammals is about88%~97%, in which the similaritywith cow is the highest. The results indicate that cDNA sequences of Tibetanantelope′s stat3and hif-1α and partial coding region sequence of Tibetan antelope′shif-2α gene were obtained. 2)Real-time PCR and Western blot analysis revealed that (a)Both the mRNA level orprotein level, stat3,hif-1α and hif-2α of Tibetan antelope′s were significantlyexpressed in the lung, liver, kidney, cardiac muscle, skeletal muscle tissue and thereare organizational differences in expression. The compare of the same tissues amongthe three kind of animals shows that the expression of STAT3, HIF-1α and HIF-2αprotein in the five tissues of Tibetan antelope and Tibetan sheep are all more than thatof sheep.(b)Sheep as control, there were not consistent between the expression ofstat3, hif-1α and hif-2α mRNA in the five tissues of Tibetan antelope and them ofTibetan sheep.The expression of stat3,hif-1α and hif-2α mRNA in the five tissues ofTibetan antelope are all more than that of sheep. However, there were variouscharacteristics about the expression of stat3,hif-1α and hif-2α mRNA in the fivetissues of Tibetan sheep, in which expression of these factors′mRNA was nosignificant difference in skeletal muscle. We speculated that the tissue specificexpression and higher expression of stat3,hif-1α and hif-2α is molecular basis ofTibetan antelope and Tibetan sheep′s adaptability and genetic features. The expressionof stat3,hif-1α and hif-2α gene differences between Tibetan antelope and Tibetansheep, although they live in the same altitude. It indicates Tibetan antelope andTibetan sheep establish their own unique hypoxia adaptation mechanisms based ontheir living environment. It further announced that Tibetan antelope and Tibetan sheepcomply with the laws of optimizing, long-term evolution and survival of the fittest inthe highland environment.