ACTIVATION OF NF-KAPPA-B AND ELEVATION OF MNSOD GENE-EXPRESSION BY THIOL REDUCING AGENTS IN LUNG ADENOCARCINOMA (A549) CELLS
ACTIVATION OF NF-KAPPA-B AND ELEVATION OF MNSOD GENE-EXPRESSION BY THIOL REDUCING AGENTS IN LUNG ADENOCARCINOMA (A549) CELLS
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DOI：
10.1152/ajplung.1995.269.5.l588
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发表时间：
1995-11-01
影响因子：
4.9
通讯作者：
WHITE, CW
中科院分区：
文献类型：
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作者：
DAS, KC;LEWISMOLOCK, Y;WHITE, CW
研究了还原剂N-乙酰半胱氨酸（NAG）、二硫苏糖醇（DTT）和2-巯基乙醇（2-ME）对肺腺癌（A549）细胞核转录因子-κ B（NF-κ B）活化和锰超氧化物歧化酶（MnSOD）表达的影响。NAG、DTT和2-ME分别激活转录因子NF-κ B B，并增加这些细胞中MnSOD mRNA和酶活性的稳态水平。此外，NAG，DTT，和2-ME增加氯霉素乙酰转移酶（CAT）的活性在细胞转染含有CAT基因的大鼠MnSOD启动子的控制下的构建体。SOD和过氧化氢酶（500 U/ml）加乙醇（1 mM）不能抑制NAG、DTT或2-ME对NF-κ B的激活或稳态MnSOD mRNA水平的升高。与次黄嘌呤和黄嘌呤氧化酶产生巯基的细胞相比，或直接加入H_2O_2的细胞，既没有激活NF-κ B B，也没有升高MnSOD mRNA。这表明在自氧化过程中可能形成的活性氧中间体可能对NF-κ B的活化没有贡献。由于MnSOD启动子还含有其他转录因子的潜在结合位点，如启动子选择性转录因子-1（SP-1）、激活蛋白-1（AP-1）、AP-2、腺苷3 '，5'-环单磷酸调节元件结合因子（CREB）和转录因子IID复合物（TFIID），因此还评估了硫醇对其激活的影响。与NF-κ B的研究结果相反，巯基仅轻微激活AP-1，其他转录因子均未被巯基激活。过氧化氢酶（500 U/ml）加SOD加乙醇（1 mM）抑制AP-1激活。加入700 μ M H2 O2也激活AP-1，500 U/ml的过氧化氢酶阻止了这种激活。这表明，由于硫醇的自氧化而产生的H2 O2可以活化AP-1，但不能活化NF-κ B。因此，暴露于还原剂，NF-κ B的激活，和MnSOD基因表达的升高之间的密切关系被证明。
The effect of reducing agents, including N-acetylcysteine (NAG), dithiothreitol (DTT), and 2-mercaptoethanol (2-ME) on nuclear transcription factor-kappa B (NF-kappa B) activation and manganese superoxide dismutase (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 increased chloramphenicol acetyftransferase (CAT) activity in cells transfected 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) did not inhibit activation of NF-kappa B or elevation of steady-state MnSOD mRNA levels by NAG, DTT, or 2-ME. Controls in which comparable amounts of O-2(-). to those produced by thiols were generated by hypoxanthine and xanthine oxidase, or in which H2O2 was added directly, had neither activated NF-kappa B nor elevated MnSOD mRNA. This shows that reactive oxygen intermediates, which may be formed during autooxidation, may not contribute to activation of NF-kappa B. Because the MnSOD promoter also contains potential binding sites for other transcription factors, such as promoter-selective transcription factor-1 (SP-1), activator protein-1 (AP-1), AP-2, adenosine 3',5'-cyclic monophosphate-regulator element binding factor (CREB), and transcription factor IID complex (TFIID), the effect of thiols on their activation also were evaluated. In contrast to findings with NF-kappa B, there was only minor activation of AP-1 by thiols, and none of the other transcription factors 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 also activated AP-1, and catalase at 500 U/ml prevented this activation. This indicates that H2O2 produced as a result of autooxidation of thiols can activate AP-1 but not NF-kappa B. Thus a close association between exposure to reducing agents, activation of NF-kappa B, and elevation of MnSOD gene expression is demonstrated.