Involvements Of NF-kB And AP-1in Hypoxia-induced CRHR1Transcription Of Rats

Hypoxia is one of stresses. To adapt to hypoxia and survive, a large number of physiologically relevant genes are therefore regulated in response to the wide variety of oxygen tension. Corticotropin-releasing hormone (CRH) plays a crucial role in maintaining homeostasis via coordinating the hypothalamus-pituitary-adrenal (HPA) axis, neuroendocrine, autonomic, immune responses to hypoxia stress. The physiological actions of CRH are mediated mainly through corticotropin releasing hormone receptor-1(CRHR1), the gene expression of which is a pivotal step in triggering HPA axis response. Our lab had previously demonstrated that exposure to intermittent hypoxia (5km) for2days triggered HPA axis and up-regulated CRHR1mRNA expression in rat anterior pituitary. Studies on the binding sites of transcription factors, which participate in regulation of CRHR1gene, may provide insights into the hypoxia-induced transcriptional regulation mechanism.MethodsThe isolated rat pituitary CRHR15'-upstream flanking region from-2161～+347was subcloned into a promoterless luciferase vector pGL3to generate p2161Luc. To investigate the hypoxia-induced CRHR15'-upstream flanking region transcriptional activity in vitro, p2161Luc was transfected into AtT20and Dual-luciferase activity assay was performed. Animal model treated with intermittent hypoxia,4hours per day, was established to investigate the expression of rat pituitary CRHR1gene. EMS A experiments were performed to show the binding ability of the transcription factors to the binding sites on the CRHR1gene in vitro. The detection of CRHR1mRNA expression in vivo was performed by Real-time PCR.Results1. The5'-upstream flanking region from-2161to+347of CRHR1gene from rat pituitary was cloned. We searched the CRHR1gene promoter region and identified several DNA elements by bioinformatic analysis, including five c-jun/AP-1binding sites, three GR binding sites, four NF-κB binding sites and eight HIF-1binding sites.2. p2161-18T and p2161Luc were constructed, respectively. According to distribution of the binding sites of transcription factors in the rat pituitary CRHR1gene5'-upstream flanking region from-2161to+347, a series of deletion constructs including p1833Luc, p1795Luc, p1692Luc, p1289Luc, p1248Luc, p1218Luc, p1140Luc, p838Luc, p687Luc and p360Luc were generated in a similar manner. Two sets of mutants with the NF-κB site positioned at-809--800and the AP-1site positioned at-1277-1271mutation were constructed by site-directed mutagenesis to create p838mLuc and p1289mLuc respectively. The c-jun expression plasmid pcDNA3.1-c-jun was constructed.3. Transfection of plasmids in AtT20cells resulted in time-dependent increases in the activity of p2161Luc, p1289Luc and p838Luc, and the maximal response was observed after24h of hypoxia treatment. The increment of the activity of p2161Luc and p1289Luc descended from8to12hours, while p838Luc did not.4. After exposure to hypoxia for24hours, the activity of p838Luc in AtT20cells markedly increased in contrast to normoxia, which could be eliminated by the NF-κB inhibitor PDTC. The mutant p838mLuc, however, completely abolished the hypoxia-induced increase. Using an oligonucleotide containing the NF-κB binding sequence positioned at-809～-800as a labeled probe, we confirmed that the in vitro NF-κB extracted from nuclear of AtT20cells specifically binds to the labeled probe. Hypoxia treatment can cause a significant enhancement of the binding, while NF-κB antibody, PDTC and addition of an excess amount (100-fold) of unlabeled probe abolished it. So did the mutated label positioned at-809～-800. Exposure to intermittent hypoxia (5km) caused a biphasic change in CRHR1mRNA expression in rat anterior pituitary, which being an initial significant decline on day2and then an enhancement by day5and could be eliminated by PDTC. Exposure to continual hypoxia (7km) for8hours resulted in greater decrease in CRHR1mRNA expression in rat anterior pituitary, which could also be eliminated by PDTC.5. Using an oligonucleotide containing the c-jun/AP-1binding sequence positioned at-1277～1271as a labeled probe, we confirmed that AP-1extracted from the nuclear of AtT20cells specifically binds to the labeled probe. Hypoxia treatment can cause a significant enhancement of the binding, while c-jun/AP-1antibody or addition of an excess amount (100-fold) of unlabeled probe abolished it. So did the mutated label positioned at-1277-1271. AtT20cells were transfected together with pcDNA3.1-c-jun and pl289Luc. Hypoxia treatment for24hours markedly increased the activity of the rat pituitary CRHR15'-upstream flanking region positioned at-1289-+347to the peak. However, pcDNA3.1-c-jun expressions can completely abolished the hypoxia-induced increase.Conclusion1. After exposure to hypoxia for24hours, the activity of the5'-upstream flanking region from-2161to+347of rat CRHR1gene in AtT20cells markedly increased in contrast to normoxia.2. NF-κB mediates hypoxia-induced CRHRl gene expression by selectively interacting with the κB site positioned at-809～800.3. AP-1specifically binds to the labeled probe containing the c-jun/AP-1binding sequence positioned at-1277～1271in vitro. Hypoxia treatment may cause an inhibitory effect on the transcriptional activity from8to12hours.