[Cigarette smoking coacervate up-regulates the expression of gamma-glutamylcysteine synthetase in alveolar epithelial cells mediated by activator protein-1].

Objective To investigate the effects of cigarette smoking coacervate(CSC) on the expression and activation of γ glutamylcysteine synthetase(GCS),a rate-limitating enzyme in the synthesis of glutathione(reduced form).Methods Rat alveolar epithelial cells of the line CCL149 were cultured and exposed to CSC of the concentrations of 10,1,and 0.1 μg/ml for 1,4,8,12,24,and 48 hours respectively.RT-PCR was used to detect the mRNA expression of γ-GCS,and Western blotting was used to detect the protein expression of γ-GCS.CCL149 cells were transfected with pGL3/γ-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,Results The γ-GCS mRNA expression levels of the CCL149 cells exposed to CSC 1 μg/ml for 12,24,and 48 hours were significantly higher than that of the control group(all P0.05).The γ-GCS protein expression levels of the CCL149 cells exposed to CSC 1 μg/ml for 12,24,and 48 hours were significantly higher than that of the control group(all P0.05).The γ-GCS protein activity of the CCL149 cells treated with CSC of the concentrations of 10 μg/ml and 1 μg/ml decreased 1,4,and 8 hours after and then increased in comparison with the control group(all P0.05).The γ-GCS protein activity levels of the CCL149 cells treated with CSC of the concentration of 0.1 μg/ml for less than 48 hours was not significantly different from those of the control group(all P0.05),and the γ-GCS protein activity level of the CCL149 cells treated with CSC of the concentration of 0.1 μg/ml for 48 hours was significantly higher than that of the control group(P0.05).The activity of the luciferase with the plasmids containing 5-flanking regulatory region of rat GCLC gene and the activity of γ-GCS in the CCL149 cells significantly increased after stimulation of CSC for 12,24 and 48 hours(all P0.05).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.Conclusion CSC up-regulates the expression of γ-GCS by activation of the redox-sensitive transcription factor AP-1.