GCLC基因上游AHR/ARNT元件负性调控GCLC基因在大鼠支气管上皮细胞的表达

Expression Inhibition of GCLC Gene by Proximal AHR/ARNT Element in Rat Bronchial Epithelial Cells

研究谷氨酰半胱氨酸合成酶催化亚单位(GCLC)基因上游调控序列中2个AHR/ARNT元件的功能,从而了解γ-谷氨酰半胱氨酸合成酶(γ-GCS)基因转录调节特征.分别构建缺失2个位点AHR/ARNT元件的GCLC基因上游近端序列的萤光素酶报道基因载体以及含有2个AHR/ARNT元件核心序列的萤光素酶报道基因载体.转染大鼠支气管上皮细胞(RTE),比较检测野生与缺失报道载体的基因转录调控效率;利用电泳迁移率变动实验(EMSA)和超级迁移率变动实验检测AHR/ARNT元件与AHR以及ARNT因子的特异性结合;通过转染AHR因子真核表达质粒进一步确定AHR/ARNT元件与AHR结合在GCLC基因表达中的最终作用.结果显示,相比其野生序列,缺失AHR/ARNT元件(-1 090^-1 085)和双缺失AHR/ARNT元件(-1 090^-1 085,-215^-210)的GCLC上游调控序列报道载体在RTE显著提高萤光素酶表达(均P<0.05),而缺失AHR/ARNT元件(-215^-210)则未见显著影响(P>0.05);独立AHR/ARNT元件(-1 090^-1 085)具有转录促进作用(P<0.05)而独立AHR/ARNT元件(-215^-210)无明显影响(P>0.05).转染CMV2-AHR能够抑制野生型和缺失型报道载体的萤光素酶表达(P<0.05).EMSA证实GCLC基因上游调控区域的2个AHR/ARNT元件均有核蛋白结合,并且超级迁移率变动实验显示结合的蛋白主要含有转录因子AHR以及ARNT.因此,2个AHR/ARNT元件均可以与异源二聚体AHR/ARNT结合,AHR/ARNT元件(-1 090^-1 085)是GCLC基因中重要的抑制元件.

To characterize the promoter of rat γ-glutamyl cysteine synthetase（γ-GCS）, either or both of the two AHR/ARNT elements upstream of glutamate-cysteine ligase catalytic subunit （GCLC） were deleted to construct luciferase reporter vectors based on the rat GCLC 1.8 kb proximal regulatory sequence previously reported by point-directed PCR. The ability of transcriptional regulation between wild-type and AHR/ARNT deletion mutant were compared by luciferase activities in transfected rat bronchial epithelial （RTE） cells. Electrophoretic mobility shift assays （EMSA） with antibody supershift were used to confirm the specific binding of the transcription factor AHR or ARNT. Co-transfection of rat AHR expression vector（CMV2-AHR）with the reporter vectors indicated the involvement of AHR in GCLC transcription regulation. The result showed that deletion of one AHR/ARNT element （ - 1 090 - - 1 085） or both elements （ - 1 090 - - 1 085, - 215 - 210）significantly increased the luciferase activity in RTE cells compared with the wild-type（ P 〈 0.05） ; but not in AHR/ARNT element （ - 215 - - 210） single deletion （ P 〉 0.05）. The AHR/ARNT（ - 1 090 - - 1 085 ） core and neighboring sequence was able to increased luciferase activity in the reporter compared with control（P 〈0.05）, while AHR/ARNT（ -215- -210） had no such effect（ P 〉 0.05）. Transfection of a CMV2-AHR vector could repress luciferase expression in wild type and deletion mutants（ P 〈 0.05）. EMSA showed that the probes with complete AHR/ARNT element could bind nuclear proteins from RTE, which contained the transcription factor AHR and ARNT as confirmed by supershift assays. We concluded that the transcription factor AHR and ARNT could bind the two AHR/ARNT element ofT-GCS gene, the one at - 1 090 - - 1 085 was important for transcriptional suppression.