Activation of transcription factors activator protein-1 and nuclear factor-κB by 2,3,7,8-Tetrachlorodibenzo- p-dioxin

Abstract

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD; dioxin), the prototype agonist of the aromatic hydrocarbon (Ah) receptor, is a potent tumor promoter as well as a complete liver carcinogen that produces an oxidative stress response in rodents and in cultured cell lines. It has been proposed that TCDD promotes neoplastic transformation through oxidative signal transduction pathways, which results in activation of immediate-early response transcription factors. To set the stage for a test of this hypothesis, we evaluated the effect of TCDD treatment on the activation of several transcription factors, including those in the nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) families, which are activated by changes in the redox state of cells. In an extension of prior results, we found that TCDD treatment produced a sustained overexpression of AP-1 for at least 72 hr in wild-type mouse hepatoma Hepa-1 cells, but not in the Ah receptor-deficient derivative c35 or in cytochrome P450-1A1 (CYP1A1)-negative c37 cells. In addition, TCDD treatment caused a significant increase in the DNA binding activity of NF-κB, but not in the activities of the other transcription factors tested. AP-1 and NF-κB activation were blocked by the thiol antioxidant N-acetylcysteine and by nordihydroguaiaretic acid, an antioxidant and lipooxygenase inhibitor and an inhibitor of the epoxygenase activity of CYP1A1, and did not take place in c35, c37, or in Ah nuclear translator-deficient c4 cells. Hence, sustained activation of these two transcription factors by TCDD is likely to result from a CYP1A1-dependent and Ah receptor complex-dependent oxidative signal. Electrophoretic mobility supershift analyses with specific antibodies showed that most of the increase in NF-κB binding activity could be accounted for by increases in p50/p50 complexes. Since these complexes are known to repress NF-κB-dependent gene transcription, our results delineate a second molecular mechanism, in addition to the recently found block of tumor necrosis factor-α-mediated p50/p65 activation, that may be responsible for the immunosuppresive effects of TCDD.