Figure 3
Effect of cimetidine on TNF-α-mediated NF-κB activation in HSG cells. A, RelA protein was localized mainly in the cytoplasm of HSG cells and transported to the nucleus upon stimulation by TNF-α; it reached a peak at 30 min and then decreased at 60 min. B, the intensity of RelA expression in the HSG cell membrane was decreased by treatment with cimetidine in a time-dependent manner, whereas the intensity of RelA in the cytoplasm after treatment with 10-4 M cimetidine gradually increased up to 60 min. C, RelA transport was detected in the nucleus of HSG cells stimulated with 10 ng/ml of TNF-α and 10-4 M cimetidine for 30 min. In comparison with treatment with TNF-α alone, however, the intensity of RelA expression was markedly inhibited. D, left, maximum κB-dependent transcription was observed with 10 ng/ml of TNF-α at 4 h, which induced a 5-fold increase in luciferase activity compared with cells not exposed to TNF-α, and did not further increase with time. However, luciferase activities in HSG cells were time-dependently decreased in response to 10-4 M cimetidine from 4 h after cimetidine treatment. Half-maximal inhibition of luciferase activity was detected at 24 h after stimulation with 10-4 M cimetidine. Induction of luciferase activity by TNF-α was inhibited by cimetidine in a time-dependent fashion. D, right, the increase or decrease in luciferase activity was completely dependent on the presence of κB sites, since the control plasmid lacking the κB elements did not respond to TNF-α or cimetidine. Relative luciferase activities are shown as -fold induction compared with the activity of cimetidine-treated samples at 24 h (D, left) or untreated (D, right) samples. Each column and bar represents the mean ± S.E.M. of three independent experiments.