[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 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.