The Cloning And Comparison Of CRF/UCN/CRFR1 Varivation From Myospalax Cansus

Since its initial identification and characterization,corticotropin-releasing factor(CRF)has been shown to have a major role in coordinating the endocrine,autonomic and behavioural response to stress.CRF is released into the periphery from the paraventricular nucleus of the hypothalamus(PVN)and steers the activation of the hypothalamic-pituitary—adrenal(HPA)axis by triggering the release of adrenocorticotropic hormone from the anterior pituitary,which in turn stimulates the synthesis and secretion of glucocorticoids from the adrenal gland.The family of corticotropin-releasing factor related peptides includes CRF,urocortin(UCN1,UCN2,UCN3),They act by binding to two different G protein-coupled receptors:CRFR1 and CRFR2.Small mammals in the Qinghai-Tibet live in cold and hypoxic environments for a long time.Myospalax baileyi lived in underground caves of 3000-4500 m above the plateau,whereas Myospalax cansus also live in the ground and the altitude is about 800-1500 m,both animals are faced with different hypoxia and high CO2 environments.Our previous studies found that the HPA axis of these animals was relatively low reactivity for hypoxia stress.In order to give an insight into the plateau environment adaptation mechanism of these small mammals,especially the effect of CRFR1 function on regulating hypoxic stress responses,the study adopts many laboratory techniques,such as molecular biology,cell biology,enzyme linked immunosorbent assay and other means.We cloned CRF,UCN and CRFR1 coding sequence of Myospalax Cansus,comparied with the plateau mammals Myospalax Baileyi.Further,rats were exposed to hypoxia environment,and the changes of CRF,Ucn and CRFR1 mRNA in brain tissue were observed.Hypoxia(7000 m,8h)stress could significantly up-regulate the expression of CRF,UCN and CRFR1 mRNA in the prefrontal cortex of rats.The expression levels of CRF,UCN and CRFR1 mRNA in the prefrontal cortex of Myospalax Baileyi were also higher than those in Myospalax Cansus.CHO cells were used to study the second messenger expression levels change after ligand-receptor binding via stimulating different receptors.When stimulated by different concentrations of CRF,the cAMP production levels of CHO cells transfected with CRFR1 receptors are dose-dependent and the EC50 are different.The highest is Myospalax baileyi,and then followed by Myospalax cansus and rat.We investigated the transcriptional mechanism of hypoxia in the promoter region of CRFR1 receptor by bioinformatics analysis and ChIP experiments.We identified that there are five AP-1 and eight NF-?B binding sites on the mouse CRFR1 receptor promoter,respectively.The levels of CRF of serum and saliva after exposure to plateau environment(3860 m,1-3 days)were tested by ELISA kit.The CRF level of saliva and serum in AMS are significant higher than that of control group,and the CRF level of saliva and serum in subject with pulmonary edema,and pulmonary edema+cerebral edema is significantly higher than that of AMS group.Variation of CRFR1 in plateau mammals might one of the molecular adaptation mechanisms to plateau hypoxic environment,and contributes to low HPA axis function.Native animals have demonstrated the mutil-model to adapt the plateau hypoxic environment by regulating the mRNA of CRF and CRFR1,and different transcription factors,HIF-lalpha,NF-kB,and AP-1 binding to the promoter to regulate the CRFR1 transcription.Elevated CRF levels in human maybe alert AMS occurred,and there is markedly higher saliva and serum CRF level in severe AMS with brain edema and pulmonary edema.