ACTIVATION OF NF-KAPPA-B AND ELEVATION OF MNSOD GENE-EXPRESSION BY THIOL REDUCING AGENTS IN LUNG ADENOCARCINOMA (A549) CELLS

The effect of reducing agents, including N-acetylcysteine (NAG), dithi othreitol (DTT), and 2-mercaptoethanol (2-ME) on nuclear transcription factor-kappa B (NF-kappa B) activation and manganese superoxide dismu tase (MnSOD) expression was investigated in a pulmonary adenocarcinoma (A549) cell line. NAG, DTT, and 2-ME each activated the transcription factor NF-kappa B and increased steady-state levels of MnSOD mRNA and enzyme activity in these cells. In addition, NAG, DTT, and 2-ME incre ased chloramphenicol acetyftransferase (CAT) activity in cells transfe cted with a construct containing the CAT gene under the control of the rat MnSOD promoter. SOD and catalase (500 U/ml) plus ethanol (1 mM) d id not inhibit activation of NF-kappa B or elevation of steady-state M nSOD mRNA levels by NAG, DTT, or 2-ME. Controls in which comparable am ounts of O-2(-). to those produced by thiols were generated by hypoxan thine and xanthine oxidase, or in which H2O2 was added directly, had n either activated NF-kappa B nor elevated MnSOD mRNA. This shows that r eactive oxygen intermediates, which may be formed during autooxidation , may not contribute to activation of NF-kappa B. Because the MnSOD pr omoter also contains potential binding sites for other transcription f actors, such as promoter-selective transcription factor-1 (SP-1), acti vator protein-1 (AP-1), AP-2, adenosine 3',5'-cyclic monophosphate-reg ulator element binding factor (CREB), and transcription factor IID com plex (TFIID), the effect of thiols on their activation also were evalu ated. In contrast to findings with NF-kappa B, there was only minor ac tivation of AP-1 by thiols, and none of the other transcription factor s were activated by thiols. AP-1 activation was inhibited by catalase (500 U/ml) plus SOD plus ethanol (1 mM). Addition of 700 mu M H2O2 als o activated AP-1, and catalase at 500 U/ml prevented this activation. This indicates that H2O2 produced as a result of autooxidation of thio ls can activate AP-1 but not NF-kappa B. Thus a close association betw een exposure to reducing agents, activation of NF-kappa B, and elevati on of MnSOD gene expression is demonstrated.