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-κB 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-κB signaling inhibits Nrf2-ARE pathway through the interaction of p65 and Keap1.
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