Objective: 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).
Methods: 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.
Results: 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.
Conclusion: CSC up-regulates the expression of gamma-GCS by activation of the redox-sensitive transcription factor AP-1.
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