Keap1 silencing boosts lipopolysaccharide-induced transcription of interleukin 6 via activation of nuclear factor κB in macrophages

Interleukin-6 (IL6) is a multifunctional cytokine that regulates immune and inflammatory responses. Multiple transcription factors, including nuclear factor kappa B (NF-kappa B) and nuclear factor E2-related factor 2 (Nrf2), regulate IL6 transcription. Kelch-like ECH-associated protein 1 (Keap1) is a substrate adaptor protein for the Cullin 3-dependent E3 ubiquitin ligase complex, which regulates the degradation of many proteins, including Nrf2 and I kappa B kinase beta (IKK beta). Here, we found that stable knockdown of Keap1 (Keap1-KD) in RAW 264.7 (RAW) mouse macrophages and human monocyte THP-1 cells significantly increased expression of 116, and Nrf2-target genes, under basal and lipopolysaccharide (LPS, 0.001-0.1 mu g/ml)-challenged conditions. However, Nrf2 activation alone, by tert-butylhydroquinone treatment of RAW cells, did not increase expression of Il6. Compared to cells transduced with scrambled non-target negative control shRNA, Keap1-KD RAW cells showed enhanced protein levels of IKK beta and increased expression and phosphorylation of NF-kappa B p65 under non-stressed and LPS-treated conditions. Because the expression of 116 in Keap1-KD RAW cells was significantly attenuated by silencing of Ikk beta, but not Nrf2, it appears that stabilized IKK beta is responsible for the enhanced transactivation of 116 in Keap1-KD cells. This study demonstrated that silencing of Keap1 in macrophages boosts LPS-induced transcription of 116 via NF-kappa B activation. Given the importance of IL6 in the inflammatory response, the Keap1-IKK beta-NF-kappa B pathway may be a novel target for treatment and prevention of inflammation and associated disorders. (C) 2013 Elsevier Inc. All rights reserved.