Abstract
Nuclear factor erythroid 2-related factor 2 (Nrf2) is an oxidant-responsive transcription factor known to induce detoxifying and antioxidant genes. Cigarette smoke, with its large oxidant content, is a major stress on the cells of small airway epithelium, which are vulnerable to oxidant damage. We assessed the role of cigarette smoke in activation of Nrf2 in the human small airway epithelium in vivo. Fiberoptic bronchoscopy was used to sample the small airway epithelium in healthy-nonsmoker and healthy-smoker, and gene expression was assessed using microarrays. Relative to nonsmokers, Nrf2 protein in the small airway epithelium of smokers was activated and localized in the nucleus. The human homologs of 201 known murine Nrf2-modulated genes were identified, and 13 highly smoking-responsive Nrf2-modulated genes were identified. Construction of an Nrf2 index to assess the expression levels of these 13 genes in the airway epithelium of smokers showed coordinate control, an observation confirmed by quantitative PCR. This coordinate level of expression of the 13 Nrf2-modulated genes was independent of smoking history or demographic parameters. The Nrf2 index was used to identify two novel Nrf2-modulated, smoking-responsive genes, pirin (PIR) and UDP glucuronosyltransferase 1-family polypeptide A4 (UGT1A4). Both genes were demonstrated to contain functional antioxidant response elements in the promoter region. These observations suggest that Nrf2 plays an important role in regulating cellular defenses against smoking in the highly vulnerable small airway epithelium cells, and that there is variability within the human population in the Nrf2 responsiveness to oxidant burden.
Highlights
In cigarette smokers, phase II detoxifying and antioxidant enzymes are an important mechanism in the protection of the small airway epithelium from the more than 1014 oxidants present in each puff of cigarette smoke [1,2,3,4]
Based on the knowledge that cigarette smoke with its large oxidant content is a major stressor of the small airway epithelium, with cells that are vulnerable to oxidant damage and its associated malignant transformation [2,5,6,7], we asked the questions: does cigarette smoking induce activation of Nuclear factor erythroid 2–related factor 2 (Nrf2) in the human small airway epithelium, is there an associated coordinate control of Nrf2-modulated genes, and are the highly-responsive Nrf2-modulated genes the same or different from those observed in Nrf2 murine models? Fiberoptic bronchoscopy was used to sample pure populations of small airway epithelium in 38 healthy nonsmokers and 45 healthy smokers, and gene expression of human homologs of 201 known murine Nrf2-modulated genes were assessed using Affymetrix Human Genome U133 Plus 2.0 microarrays
In the human small airway epithelium of healthy normal smokers compared with healthy nonsmokers, Nrf2 protein was significantly activated in the cells, and 13 Nrf2modulated genes were identified as highly smoking responsive
Summary
Phase II detoxifying and antioxidant enzymes are an important mechanism in the protection of the small airway epithelium from the more than 1014 oxidants present in each puff of cigarette smoke [1,2,3,4]. Defense against oxidants is a critical cellular function, and oxidant-mediated somatic mutations play an important role in the mechanisms of malignant transformation of the airway epithelium in association with cigarette smoking [2,5,6,7]. Central to the cellular oxidant defense mechanism is the nuclear factor erythroid 2–related factor 2 (NFE2L2, Nrf2), a member of the “Cap ‘n’ Collar” family of the basic leucine transcription factors, known to coordinate the induction of phase II detoxifying and antioxidant enzymes [8,9,10,11]. Microarray analyses of lungs from Nrf knockout mice and wild-type controls have helped to identify Nrf2modulated genes, for example, genes that help protect mice from the development of cigarette smoke–induced lung injuries [23]
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