High Impact Short Article Keap1 silencing boosts lipopolysaccharide-induced transcription of interleukin 6 via activation of nuclear factor κB in macrophages Peng Lv a,b , Peng Xue b , Jian Dong b , Hui Peng b,c , Rebecca Clewell b , Aiping Wang a , Yue Wang d , Shuangqing Peng c , Weidong Qu e , Qiang Zhang b , Melvin E. Andersen b , Jingbo Pi b, ⁎ a Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China b Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709, USA c Evaluation and Research Center for Toxicology, Institute of Disease Control and Prevention, Academy of Military Medical Sciences, China d Institute for Medical Device Standardization Administration, National Institutes for Food and Drug Control, Beijing, China e Key Laboratory of the Public Health Safety, Ministry of Education, School of Public Health, Fudan University, Shanghai, China abstract article info Article history: Received 28 April 2013 Revised 3 July 2013 Accepted 19 July 2013 Available online 29 July 2013 Keywords: IL6 Nrf2 Keap1 IKKβ NF-κB Macrophage Interleukin-6 (IL6) is a multifunctional cytokine that regulates immune and inflammatory responses. Multiple transcription factors, including nuclear factor κB (NF-κB) 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 IκB kinase β (IKKβ). 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 Il6, and Nrf2-target genes, under basal and lipopolysaccharide (LPS, 0.001–0.1 μg/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 IKKβ and increased expression and phosphorylation of NF-κB p65 under non-stressed and LPS-treated conditions. Because the expression of Il6 in Keap1-KD RAW cells was significantly attenuated by silencing of Ikkβ, but not Nrf2, it appears that stabilized IKKβ is responsible for the enhanced transactivation of Il6 in Keap1-KD cells. This study demonstrated that silencing of Keap1 in macrophages boosts LPS-induced transcription of Il6 via NF-κB activation. Given the importance of IL6 in the inflammatory response, the Keap1–IKKβ–NF-κB pathway may be a novel target for treatment and prevention of inflammation and associated disorders. © 2013 Elsevier Inc. All rights reserved. Introduction Interleukin 6 (IL6) is a multifunctional cytokine that acts as both a pro- and anti-inflammatory factor regulating immune and inflammato- ry responses (Gabay, 2006; Kishimoto, 2005; Schuett et al., 2009). IL6 is produced and secreted in response to various acute insults, such as in- fection and trauma, and also involved in the pathogenesis of many chronic disorders, including cancer, vascular diseases and metabolic syndrome (Brasier, 2010; Gabay, 2006; Kundu and Surh, 2012; Odegaard and Chawla, 2013; Schuett et al., 2009). Transcription of IL6 is coordinately regulated by a variety of transcription factors, including nuclear factor κB (NF-κB), CCAAT/enhancer binding protein β (C/EBPβ), cAMP response element-binding protein (CREB), activator protein 1 (AP1) and Notch1 (Dendorfer et al., 1994; Plaisance et al., 1997; Wongchana and Palaga, 2012; Zhang et al., 2013). Among these, the NF-κB family of proteins appears to be the most important, whereas others may cooperate with NF-κB to regulate IL6 transcription (Wongchana and Palaga, 2012; Xiao et al., 2004). Nuclear factor E2-related factor 2 (Nrf2) is a cap “n” collar basic leu- cine zipper (bZIP) transcription factor that coordinately regulates the constitutive and inducible expression of many antioxidant and phase II detoxification enzymes via the antioxidant response element (ARE) (Giudice et al., 2010; Maher and Yamamoto, 2010; Niture et al., 2013). Under normal homeostatic conditions, a low constitutive amount of Nrf2 protein is maintained by the Kelch-like ECH-associated protein 1 (Keap1)-mediated ubiquitination and proteasomal degradation system (Giudice et al., 2010; Hayes and McMahon, 2009). Upon oxidative and/ or electrophilic stress, the enzymatic activity of the Keap1-Cullin3 E3 ubiquitin ligase is compromised, resulting in stabilization of Nrf2 followed by its nuclear accumulation. In the nucleus, Nrf2 partners with small Maf proteins and/or other bZIP proteins to activate the tran- scription of ARE-dependent genes (Hayes and McMahon, 2009; Wang et al., 2008). Because a large number of antioxidant and phase II detox- ification genes contain functional AREs, Nrf2 is considered the master regulator of cellular defense against oxidative/electrophilic stress (Giudice et al., 2010; Niture et al., 2013). Toxicology and Applied Pharmacology 272 (2013) 697–702 ⁎ Corresponding author. Fax: +1 919 558 1305. E-mail addresses: jpi@thehamner.org, jingbopi@gmail.com (J. Pi). 0041-008X/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.taap.2013.07.012 Contents lists available at ScienceDirect Toxicology and Applied Pharmacology journal homepage: www.elsevier.com/locate/ytaap