NF-κB和AP-1参与低氧诱导大鼠CRHR1基因转录调节

发布时间：2018-05-19 05:14

本文选题：CRH + CRHR1　；参考：《浙江大学》2012年博士论文

【摘要】：低氧应激是应激的重要形式之一。为适应低氧环境、进行自我保护,机体形成了在不同氧环境下的基因表达调控机制。促肾上腺皮质激素释放激素(Corticotropin-releasing hormone, CRH)家族在机体低氧应激过程中通过调节下丘脑-垂体-肾上腺皮质(hypothalamus pituitary adrenal, HPA)轴、神经内分泌系统、自主神经系统和免疫系统功能,参与调节机体的低氧应激过程,进而稳定机体内环境。促肾上腺皮质激素释放激素1型受体(Corticotropin releasing hormone receptor1, CRHR1)介导CRH生理作用的发挥,其基因表达调控是应激诱导HPA轴反应的关键环节。我们实验室前期研究发现低氧应激激活HPA轴,诱导大鼠等动物脑不同部位以及腺垂体CRHR1mRNA表达上调,提示低氧下转录因子可能通过与其作用元件结合,在转录水平调控CRHR1mRNA的表达。本研究旨在探讨低氧应激下NF-κB和AP-1参与CRHR1转录调节的机制。研究方法将大鼠垂体CRHR1基因5’调控区-2161～+347段不同长度DNA序列亚克隆入不含启动子的萤光素酶载体pGL3中,构建各种质粒,转染AtT20细胞,检测其活性与内参pRL-TK萤光素酶活性比值；利用整体间歇低氧应激动物模型,研究低氧诱导大鼠垂体CRHR1基因表达变化；应用EMSA方法检测转录因子在体外与靶序列结合活性；应用定量RealtimePCR方法检测整体水平CRHR1mRNA的表达。研究结果如下： 1.获得大鼠垂体CRHR1基因5’调控区-2161～+347段DNA序列,全长为2508bp,经过生物信息学分析,发现该序列分别含有5个c-jun/AP-1,3个GR,4个NF-κB以及8个HIF-1结合位点。 2.成功构建质粒p2161-18T和p2161Luc,分别用于测序和检测CRHR1基因5’端调控区转录活性。根据转录因子结合元件在CRHR1基因5’调控区-2161～+347段的分布,又分别构建了p1833Luc、p1795Luc、p1692Luc、p1289Luc、p1248Luc、 p1218Luc、p1140Luc、p838Luc、p687Luc、p360Luc等系列缺失体质粒。进一步构建了含-809～-800位点的NF-κB结合元件的突变体质粒p838mLuc和含有-1277～1271位点的c-jun/AP-1结合元件的突变体质粒p1289mLuc和以及c-jun的真核表达质粒pcDNA3.1-c-jun. 3.发现p2161Luc、p1289Luc、p838Luc在AtT20细胞中的活性随低氧时间延长而呈现增强的趋势,在24小时达到最大值。P2161Luc和p1289Luc的活性在低氧暴露8-12小时期间曲线平缓。 4.发现低氧暴露24小时,p838Luc在AtT20细胞中的活性与常氧对照组相比显著增强,这一现象可以被NF-κB抑制剂(PDTC)抑制；而突变体p838mLuc活性无明显增强。大鼠垂体CRHR1基因5’调控区-809～-800区域的结合元件在体外能与AtT20细胞以及大鼠垂体的核蛋白中的NF-κB结合,且低氧进一步增强二者结合活性,此结合可以被PDTC、p65抗体、100倍浓度的冷探针消除；突变探针(-809～-800)不再与NF-κB结合。在整体低压舱模拟5km间歇低氧(10.8%02,4h/d)条件下,CRHR1mRNA表达表现为一个先下降后增强的过程,在第2天表达显著下降,第5天表达上升,二者均可被PDTC反转；7km(7.8%02)连续低氧8h,CRHR1mRNA表达下降幅度更大,此结果同样可被PDTC反转。 5.发现大鼠垂体CRHR1基因5’调控区-1277～1271区域的结合元件在体外能与AtT20细胞核蛋白中的c-jun/AP-1结合,低氧暴露进一步增强二者结合活性,此结合可以被c-jun磷酸化抗体、100倍浓度的冷探针消除。突变探针(-1277～1271)不再与c-jun/AP-1结合。p1289Luc转染AtT20细胞,低氧暴露24h后,CRHR1基因5’调控区-1289～+347段的转录活性较常氧对照组增幅达到最大,此增幅可以被2μg pcDNA3.1-c-jun的共表达完全抑制。结论 1CRFR1基因5’调控区-2161～+347参与低氧上调CRFR1基因转录。 2.证明了大鼠垂体CRHR1基因5’调控区-809～-800位点的NF-κB结合元件促进低氧诱导的大鼠垂体CRHR1基因的转录。 3.大鼠垂体CRHR1基因5’调控区-1277～1271位点的结合元件与AP-1在低氧暴露下结合活性增强,在AtT20细胞低氧暴露8～12小时AP-1可能参与抑制CRHR1基因的转录活性。
[Abstract]:Hypoxia stress is one of the important forms of stress. In order to adapt to the hypoxic environment, self protection, the body forms the regulation mechanism of gene expression in different oxygen environment. The Corticotropin-releasing hormone, CRH family regulates the hypothalamus pituitary adrenal cortex in the process of hypoxic stress. Hypothalamus pituitary adrenal (HPA) axis, neuroendocrine system, autonomic nervous system and immune system function, participate in regulating the body's hypoxia stress process, and then stabilize the body environment. Adrenocorticotropin releasing hormone 1 receptor (Corticotropin releasing hormone receptor1, CRHR1) mediates the physiological role of CRH. The regulation of gene expression is the key link in stress induced HPA axis response. In our previous laboratory, we found that hypoxia stress activates the HPA axis and induces the up regulation of CRHR1mRNA expression in different parts of the brain and adenohypophysis in rats, suggesting that the hypoxic transcription factors may be combined with its components and regulate the table of CRHR1mRNA at the transcriptional level. The aim of this study is to explore the mechanism of NF- kappa B and AP-1 involved in CRHR1 transcriptional regulation under hypoxia stress.
The -2161 ~ +347 segment of the rat pituitary CRHR1 gene 5 'gene was subcloned into the fluoro enzyme carrier pGL3 without promoter, and various plasmids were constructed and transfected to AtT20 cells to detect the activity ratio of the activity to the internal parameter pRL-TK luciferase activity, and the hypoxic induced rat pituitary C was studied by using the whole intermittent hypoxia stress animal model. RHR1 gene expression changes; EMSA method was used to detect the binding activity of transcription factors to target sequences in vitro; quantitative RealtimePCR method was used to detect the overall level of CRHR1mRNA expression. The results were as follows:
1. the DNA sequence of -2161 to +347 segment of the rat pituitary CRHR1 gene was obtained. The total length of the DNA sequence was 2508bp. Through bioinformatics analysis, it was found that the sequence contained 5 c-jun/AP-1,3 GR, 4 NF- kappa B and 8 HIF-1 binding sites.
2. the plasmid p2161-18T and p2161Luc were successfully constructed and used to sequence and detect the transcriptional activity of the 5 'terminal regulatory region of the CRHR1 gene, respectively. According to the distribution of the transcription factor binding element in the -2161 to +347 segment of the CRHR1 gene 5' regulatory region, the p1833Luc, p1795Luc, p1692Luc, p1289Luc, and p1248Luc were constructed, respectively. A series of missing body plasmids. The mutant body p838mLuc of the NF- kappa B binding element containing -809 to -800 and the c-jun/AP-1 binding element containing -1277 to 1271, and the eukaryotic expression plasmids pcDNA3.1-c-jun. of c-jun, are constructed.
3. it was found that the activity of p2161Luc, p1289Luc and p838Luc in AtT20 cells increased with the prolongation of hypoxia time, and the maximum value of.P2161Luc and p1289Luc at 24 hours was slow during 8-12 hours of hypoxia exposure.
4. after 24 hours of hypoxia exposure, the activity of p838Luc in AtT20 cells was significantly enhanced compared with that of the normal oxygen control group. This phenomenon could be inhibited by NF- kappa B inhibitor (PDTC), but the activity of the mutant p838mLuc was not significantly enhanced. The binding element in the -809 to -800 region of the pituitary CRHR1 gene 5 'regulation region could be in vitro with AtT20 cells and rats. The combination of NF- kappa B in the nuclear protein of the pituitary, and the hypoxia further strengthens the two binding activity, which can be eliminated by the cold probe of PDTC, p65, and 100 times; the mutant probe (-809 to -800) is no longer combined with NF- kappa B. Under the condition of the analog 5km intermittent hypoxia (10.8% 02,4h/d) in the whole hypobaric chamber, the CRHR1mRNA expression is a first descending. The expression of the enhanced process decreased significantly at second days, the fifth day expression rose and the two could be reversed by PDTC; 7km (7.8%02) was a continuous hypoxic 8h, and the CRHR1mRNA expression decreased significantly, and this result could also be reversed by PDTC.
5. it was found that the binding element in the -1277 ~ 1271 region of the CRHR1 gene 5 'region of the rat pituitary could be combined with the c-jun/AP-1 in the AtT20 nuclear protein in vitro, and the hypoxia exposure further enhanced the two binding activity, which could be eliminated by the c-Jun phosphorylated antibody and the cold probe at 100 times the concentration. The mutant probe (-1277 ~ 1271) was no longer with the c-jun/AP-1 junction. After transfection of.P1289Luc to AtT20 cells, after hypoxia exposure to 24h, the transcription activity of -1289 to +347 segment of the CRHR1 gene 5 'regulatory region was the largest, and the increase could be completely suppressed by co expression of 2 mu g pcDNA3.1-c-jun.
conclusion
1CRFR1 gene 5 'regulatory region -2161 ~ +347 participates in hypoxia and upregulates CRFR1 gene transcription.
2. it is proved that the NF- kappa B binding element of the -809 to -800 locus in the 5 'regulatory region of the pituitary CRHR1 gene promotes the transcription of CRHR1 gene in rat pituitary induced by hypoxia.
The binding element of the -1277 ~ 1271 loci of the pituitary CRHR1 gene 5 'regulatory region of the 3. rats was enhanced by the binding activity of AP-1 under hypoxia exposure, and AP-1 may be involved in inhibiting the transcriptional activity of the CRHR1 gene in the AtT20 cell hypoxia exposure for 8~12 hours.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R346

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