Zou Zhao-xia, Ran Pi-xin
Guangzhou Institute of Respiratory Disease, First Affiliated Hospital, Guangzhou Medical College, Guangzhou 510120, China.
Zhonghua Yi Xue Za Zhi. 2005 Dec 7;85(46):3289-92.
To investigate the effects of cigarette smoking coacervate (CSC) on the expression and activation of gamma-glutamylcysteine synthetase (GCS), a rate-limitating enzyme in the synthesis of glutathione (reduced form).
Rat alveolar epithelial cells of the line CCL149 were cultured and exposed to CSC of the concentrations of 10, 1, and 0.1 microg/ml for 1, 4, 8, 12, 24, and 48 hours respectively. RT-PCR was used to detect the mRNA expression of gamma-GCS, and Western blotting was used to detect the protein expression of gamma-GCS. CCL149 cells were transfected with pGL3/gamma-GCS or blank pGL3 plasmid. The luciferase activity was examined Gel retardation assay was used to detect the binding level of activator protein (AP)-1 with the region of the GCLC promoter in CCL-149 cell.
The gamma-GCS mRNA expression levels of the CCL149 cells exposed to CSC > 1 microg/ml for 12, 24, and 48 hours were significantly higher than that of the control group (all P < 0.05). The gamma-GCS protein expression levels of the CCL149 cells exposed to CSC > 1 microg/ml for 12, 24, and 48 hours were significantly higher than that of the control group (all P < 0.05). The gamma-GCS protein activity of the CCL149 cells treated with CSC of the concentrations of 10 microg/ml and 1 microg/ml decreased 1, 4, and 8 hours after and then increased in comparison with the control group (all P < 0.05). The gamma-GCS protein activity levels of the CCL149 cells treated with CSC of the concentration of 0.1 microg/ml for less than 48 hours was not significantly different from those of the control group (all P > 0.05), and the gamma-GCS protein activity level of the CCL149 cells treated with CSC of the concentration of 0.1 microg/ml for 48 hours was significantly higher than that of the control group (P < 0.05). The activity of the luciferase with the plasmids containing 5-flanking regulatory region of rat GCLC gene and the activity of gamma-GCS in the CCL149 cells significantly increased after stimulation of CSC for 12, 24 and 48 hours (all P < 0.05). The binding levels of AP-1 with the region of the GCLC promoter in the CCL149 cells treated with CSC for 12, 24, and 48 hours were significantly increased.
CSC up-regulates the expression of gamma-GCS by activation of the redox-sensitive transcription factor AP-1.
研究香烟凝聚物（CSC）对γ-谷氨酰半胱氨酸合成酶（GCS，谷胱甘肽（还原型）合成中的限速酶）表达及激活的影响。
培养大鼠肺泡上皮细胞系CCL149，分别用浓度为10、1和0.1μg/ml的CSC处理1、4、8、12、24和48小时。采用逆转录聚合酶链反应（RT-PCR）检测γ-GCS的mRNA表达，蛋白质免疫印迹法检测γ-GCS的蛋白表达。用pGL3/γ-GCS或空白pGL3质粒转染CCL149细胞。检测荧光素酶活性，凝胶阻滞试验检测激活蛋白（AP）-1与CCL-149细胞中GCLC启动子区域的结合水平。
暴露于浓度>1μg/ml CSC 12、24和48小时的CCL149细胞的γ-GCS mRNA表达水平显著高于对照组（均P<0.05）。暴露于浓度>1μg/ml CSC 12、24和48小时的CCL149细胞的γ-GCS蛋白表达水平显著高于对照组（均P<0.05）。用浓度为10μg/ml和1μg/ml的CSC处理的CCL149细胞的γ-GCS蛋白活性在处理1、4和8小时后降低，随后与对照组相比升高（均P<0.05）。用浓度为0.1μg/ml的CSC处理48小时以内的CCL149细胞的γ-GCS蛋白活性水平与对照组无显著差异（均P>0.05），用浓度为0.1μg/ml的CSC处理48小时的CCL149细胞的γ-GCS蛋白活性水平显著高于对照组（P<0.05）。用含大鼠GCLC基因5'-侧翼调控区的质粒转染的荧光素酶活性及CCL149细胞中γ-GCS的活性在CSC刺激12、24和48小时后显著升高（均P<0.05）。用CSC处理12、24和48小时的CCL149细胞中AP-1与GCLC启动子区域的结合水平显著升高。
CSC通过激活氧化还原敏感转录因子AP-1上调γ-GCS的表达。
