Nuclear factor p65 interacts with Keap1 to repress the Nrf2-ARE pathway

Keap1 is an inhibitor of Nrf2 involved in Nrf2-dependent antioxidant response. However, the mechanisms on how Keap1 regulates Nrf2-ARE signaling pathway remains to be determined. Here, by using a yeast two-hybrid technology, p65 subunit of NF-kappa B transcription factor was identified as a partner of Keap1. We show that Keap1 physically associated with p65 in vivo and in vitro. Overexpression of p65 inhibited Nrf2-dependent transcription induced by diethylmaleate (DEM) or tert-butyl hydroxyquinone (tBHQ). Knock down of Keap1 by RNA interference partially blocked the repression of Nrf2-mediated activation by p65. It was demonstrated that p65 decreased Nrf2 binding to its cognate DNA sequences and enhanced Nrf2 ubiquitination. The N-terminal region of p65 is necessary for both the interaction with Keap1 and its transcriptional suppression activity. Moreover, nuclear translocation of Keap1 was augmented by p65. Taken together, our findings suggest that NF-kappa B signaling inhibits Nrf2-ARE pathway through the interaction of p65 and Keap1. (C) 2011 Elsevier Inc. All rights reserved.

Keap1是一种参与Nrf2依赖性抗氧化反应的Nrf2抑制剂。然而，Keap1如何调节Nrf2 - ARE信号通路的机制仍有待确定。在此，通过使用酵母双杂交技术，NF - κB转录因子的p65亚基被鉴定为Keap1的一个伴侣蛋白。我们表明Keap1在体内和体外都与p65有物理性结合。p65的过表达抑制了由马来酸二乙酯（DEM）或叔丁基对苯二酚（tBHQ）诱导的Nrf2依赖性转录。通过RNA干扰敲低Keap1可部分阻断p65对Nrf2介导的激活的抑制作用。研究表明，p65降低了Nrf2与其同源DNA序列的结合，并增强了Nrf2的泛素化。p65的N末端区域对于其与Keap1的相互作用及其转录抑制活性都是必需的。此外，p65增强了Keap1的核转位。综上所述，我们的研究结果表明，NF - κB信号通过p65和Keap1的相互作用抑制Nrf2 - ARE通路。（C）2011爱思唯尔公司。保留所有权利。