title: Functional dissection of Nrf2-dependent phase II genes in vascular inflammation and endotoxic injury using Keap1 siRNA.
abstract: Keap1 is a cytoplasmic repressor of the transcription factor Nrf2, and its degradation induces Nrf2 activation, leading to upregulation of antioxidant phase II genes. We investigated the roles of phase II genes in vascular inflammation and septic injury using Keap1 siRNA and elucidated its underlying mechanism. Selective knockdown of Keap1 with siRNA promoted Nrf2-dependent expression of phase II genes in endothelial cells, such as heme oxygenase-1 (HO-1), glutamate-cysteine ligase (GCL), and peroxiredoxin-1 (Prx1), resulting in the elevation of cellular glutathione levels and suppression of tumor necrosis factor (TNF)-α-induced intracellular H(2)O(2) accumulation. Keap1 knockdown inhibited TNF-α-induced expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) by suppressing NF-κB activation via inhibition of its upstream modulators, Akt, NIK, and IKK, resulting in the elevation of monocyte adhesion to endothelial cells. Importantly, these events were reversed by HO-1 and GCL inhibitors and Prx1-specific siRNA. Keap1 knockdown also inhibited endotoxin-induced expression of inducible nitric oxide synthase (iNOS) and TNF-α by upregulating HO-1, GCL, and Prx1 expression in macrophages. Moreover, in vivo Keap1 knockdown increased the expression of phase II genes and suppressed the expression of ICAM-1, VCAM-1, iNOS, and TNF-α in an endotoxemic mouse model, resulting in significant protection against liver and lung injuries and lethality. Our results indicate that Keap1 knockdown prevents NF-κB-mediated vascular inflammation and endotoxic shock by suppressing NF-κB-mediated inflammatory gene expression via upregulation of Nrf2-mediated antioxidant genes. Thus, siRNA targeting Keap1 may provide a new therapeutic approach for inflammation-associated vascular diseases and sepsis.
authors.affiliation: Vascular Homeostasis Laboratory and Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chunchon, Kangwon-do, Korea.
authors.forename: Ji-Hee
authors.initials: JH
authors.lastname: Kim
authors.forename: Yoon Kyung
authors.initials: YK
authors.lastname: Choi
authors.forename: Kwang-Soon
authors.initials: KS
authors.lastname: Lee
authors.forename: Dong-Hui
authors.initials: DH
authors.lastname: Cho
authors.forename: Yi-Yong
authors.initials: YY
authors.lastname: Baek
authors.forename: Dong-Keon
authors.initials: DK
authors.lastname: Lee
authors.forename: Kwon-Soo
authors.initials: KS
authors.lastname: Ha
authors.forename: Jongseon
authors.initials: J
authors.lastname: Choe
authors.forename: Moo-Ho
authors.initials: MH
authors.lastname: Won
authors.forename: Dooil
authors.initials: D
authors.lastname: Jeoung
authors.forename: Hansoo
authors.initials: H
authors.lastname: Lee
authors.forename: Young-Guen
authors.initials: YG
authors.lastname: Kwon
authors.forename: Young-Myeong
authors.initials: YM
authors.lastname: Kim
mesh_terms: D000818:Animals; D061745:Antioxidant Response Elements; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D019610:Cytoprotection; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D005721:Glutamate-Cysteine Ligase / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D051547:Heme Oxygenase-1 / Q000235:genetics / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells / Q000276:immunology / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D000072019:Kelch-Like ECH-Associated Protein 1; D008070:Lipopolysaccharides / Q000494:pharmacology; D008099:Liver / Q000276:immunology / Q000378:metabolism; D008168:Lung / Q000276:immunology / Q000378:metabolism; D008264:Macrophages / Q000276:immunology / Q000378:metabolism; D008297:Male; D050217:Metabolic Detoxication, Phase II / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D051267:NF-E2-Related Factor 2 / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D054464:Peroxiredoxins / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology; D014657:Vasculitis / Q000276:immunology / Q000378:metabolism* / Q000473:pathology
chemical_list: D015815:Cell Adhesion Molecules; D005721:Glutamate-Cysteine Ligase; D005982:Glutathione Transferase; D051547:Heme Oxygenase-1; D047908:Intracellular Signaling Peptides and Proteins; D000072019:Kelch-Like ECH-Associated Protein 1; D008070:Lipopolysaccharides; D051267:NF-E2-Related Factor 2; D016328:NF-kappa B; D054464:Peroxiredoxins; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; C431364:KEAP1 protein, human; C495635:NFE2L2 protein, human; C515940:PRDX1 protein, human; C496541:HMOX1 protein, human