Coordinate control of expression of Nrf2-modulated genes in the human small airway epithelium is highly responsive to cigarette smoking

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.

Figure 1

Localization of Nrf2 in small airway epithelium of healthy nonsmokers and healthy smokers. Cytospin preparations of small airway epithelium were assessed by immunohistochemical analysis and Western analysis. (A–F) Immunohistochemistry. (A, B) Healthy nonsmokers, + Nrf2 antibody; (C) healthy nonsmoker, + Nrf2 antibody + blocking peptide; (D–E) healthy smokers, + Nrf2 antibody; (F) healthy smoker, + Nrf2 antibody + blocking peptide. For (D–E), arrows indicate nucleoli positive for Nrf2 staining. For (A–F), bar = 10 μm. (G–J) Western analyses. For the Western analyses, small airway epithelial cells of four healthy nonsmokers and four healthy smokers provided nuclear and cytoplasmic extracts, which were normalized to equal protein concentration. (G) Western analysis of Nrf2 in cytoplasmic extracts from small airway epithelial cells of healthy nonsmoker and smoker (upper panel) and β-tubulin as an equal loading control (lower panel). Lane 1–4, examples of nonsmokers; lanes 5–8, examples of smokers. (H) Western analysis of Nrf2 in nuclear extract from small airway epithelium from healthy nonsmoker and healthy smoker (upper panel) and lamin B as loading control (lower panel). Lanes 9–12, examples of non-smokers; lanes 13–16, examples of smokers. (I) Quantitative analyses of band densities for Nrf2 in cytoplasm (normalized to β-tubulin). (J) Quantitative analysis of band densities for Nrf2 in nucleus (normalized to lamin B). Data shown are mean ± standard error.

Figure 2

Expression levels of Nrf2-modulated genes in small airway epithelium. A list of murine Nrf2-modulated genes was compiled and used to identify the human homologs. The gene expression in small airway epithelium was examined to generate a list of putative human Nrf2-modulated genes that are highly responsive to smoking (Supplemental Table 1). Shown is the normalized expression level for each individual (38 healthy non-smokers [○], 45 healthy smokers [●]). The gene names and the P values are located on the abscissa, the relative gene expression levels are on the ordinate as a log scale.

Figure 3

Index for Nrf2-modulated genes in the small airway epithelium of healthy smokers. We created an Nrf2 index that ranks the expression levels of the 13 Nrf2-modulated genes significantly modulated by smoking for all 45 healthy smokers. For each gene, individuals were divided into quartiles on the basis of the level of gene expression. Each individual was assigned a quartile designation of one, two, three, or four for each of the 13 genes. To calculate the Nrf2 index, for each individual the quartile designations for the individual were averaged across all genes. (A) Cluster analysis of Nrf2-modulated genes (from left to right) for 45 healthy smokers (from top to the bottom). The number in each object represents the quartile based on the gene expression, first quartile bright green, second dark green, third dark red, fourth bright red. The mean ± SD of the Nrf2 index is presented at the right. Note that all of the healthy smokers tend to have the same quartile for all genes, with some smokers showing low expression (that is, all green bars) of the 13 genes, some smokers intermediate expression (mixed colors), and some smokers with all 13 genes showing high Nrf2 expression (that is, all red bars). (B,C) The Nrf2 index distribution for the 45 healthy smokers was compared with an index generated in the same way by using 13 “random genes” or 13 “random smoking-responsive genes” (see Supplemental Table 2). (B) Comparison of frequency distribution of smokers among index values. Shown are the indices for the 13 Nrf2-modulated genes, the 13 random genes and the 13 “random smoking-response genes.” For each, the data is grouped by index values of 1–2, 2–3, and 3–4. Note that smokers ranked by random genes or random smoking-responsive genes have indices mostly distributed between 2 and 3, but smokers ranked by the Nrf2 genes are almost evenly distributed. (C) Comparison of SDs of individual smokers of the 13 Nrf2-modulated genes, 13 random genes, and 13 random smoking-responsive genes. Data shown are mean ± standard error. Note that the Nrf2-modulated genes have significantly less variation.

Figure 4

Correlation of expression of the Nrf2-modulated genes in small airway epithelium with the Nrf2 index. Shown are four examples of highly correlated genes. Each point in each panel represents a healthy smoker. (A) Transaldolase 1 (TALDO1); (B) aldehyde dehydrogenase 3 family, memberA1 (ALDH3A1); (C) NAD(P)H dehydrogenase, quinone 1 (NQO1); (D) thioredoxin reductase 1 (TXNRD1).

Figure 5

Quantitative TaqMan RT-PCR confirmation of the microarray data. Taq-Man RT-PCR was used to assess three Nrf2-modulated genes by use of RNA samples from six healthy smokers classified as low expressers (Nrf2 index 1–2) and six healthy smokers who were categorized as high expressers (Nrf2 index 3–4). The normalized average gene expression level is represented on the ordinate, and the individual genes are represented on the abscissa. (A) Microarray data; (B) Taq-Man RT-PCR data. Data shown are mean ± standard error. TALDO1, transaldolase 1; ME1, malic enzyme 1; TXNRD1, thioredoxin reductase 1.

Figure 6

Identification of human Nrf2-modulated genes not previously linked to Nrf2 control. Shown are genes not previously reported as transcriptionally induced by Nrf2, but whose expression in the small airway epithelium is strongly associated with the Nrf2 index. Each point in each panel represents a healthy smoker with Nrf2 index plotted against expression level for: (A) Pirin (PIR ); (B) ATP-binding cassette, sub-family B, member 6 (ABCB6); (C) UDP gluconosyltransferase 1 family, polypeptide A4 (UGT1A4 ); (D) Aldo-keto reductase family 1, member C3 (AKR1C3).

Figure 7

Validation of newly identified Nrf2-responsive genes by electrophoretic mobility shift assay. Nuclear extract from small airway epithelium was incubated with ³²P-labeled antioxidant response element (ARE) oligonucleotides and specifically competed with a fifty-fold excess of ARE oligonucleotides from the same gene or with ARE oligonucleotides from putative Nrf2-modulated genes. (A) NAD(P )H dehydrogenase, quinone 1 (NQO1) ARE, located at −386 bp upstream of the transcription site. Lane 1, no protein; lane 2, nuclear protein alone; lane 3, nuclear protein + specific competitor (NQO1 unlabeled ARE); lane 4, nuclear protein + nonspecific competitor (von Wille-brand [vWF] oligonucleotides); lane 5, no protein; lane 6, nuclear protein alone; lane 7, nuclear protein + anti-Nrf2 antibody; lane 8, nuclear protein + IgG control; lane 9, no protein; lane 10, nuclear protein alone; lane 11, nuclear protein + specific competitor (NQO1 unlabeled ARE); lane 12, nuclear protein + pirin (PIR) unlabeled ARE at position + 33 bp (downstream of the transcription start site); lane 13, nuclear protein + PIR unlabeled ARE at position −3209 bp (upstream of the transcription start site); lane 14, nuclear protein + PIR unlabeled ARE at position −3480 bp; lane 15, nuclear protein + PIR unlabeled ARE at position −5566 bp; lane 16, nuclear protein + nonspecific competitor (vWF oligonucleotides); lane 17, no protein; lane 18, nuclear protein alone; lane 19, nuclear protein + specific competitor (NQO1 unlabeled ARE); lane 20, nuclear protein + ABCB6 unlabeled ARE at position −7575 bp; lane 21, nuclear protein + nonspecific competitor (vWF oligonucleotides); lane 22, no protein; lane 23, nuclear protein alone; lane 24, nuclear protein + specific competitor (NQO1 unlabeled ARE); lane 25, nuclear protein + UGT1A4 unlabeled ARE at −3043 bp upstream of the transcription start site; lane 26, nuclear protein + UGT1A4 unlabeled ARE at position −3159; lane 27, nuclear protein + UGT1A4 unlabeled ARE at position −3885 bp; lane 28, nuclear protein + UGT1A4 unlabeled ARE at position −5523 bp; lane 29, nuclear protein + nonspecific competitor (von Willebrand factor (vWF) oligonucleotides). For lane 13, lane 15, lane 27, and lane 28, disappearance of gel-shift band indicates functional ARE. (B) Pirin (PIR) ARE. Radioactively labeled PIR-ARE at position −3209 bp (lane 30–37). Lane 30, no protein; lane 31, nuclear protein alone; lane 32, nuclear protein + specific competitor (PIR unlabeled ARE); lane 33, nuclear protein + nonspecific competitor (vWF oligonucleotides); lane 34, no protein; lane 35, nuclear protein alone, lane 36, nuclear protein + anti-Nrf2 antibody; lane 37, nuclear protein + IgG control; (C) radioactively labeled UGT1A4-ARE at position −3885 bp (lane 38–45). Lane 38, no protein; lane 39, nuclear protein alone; lane 40, nuclear protein + specific competitor (UGT1A4 unlabeled ARE); lane 41, nuclear protein + nonspecific competitor (vWF oligonucleotides); lane 42, no protein; lane 43, nuclear protein alone, lane 44, nuclear protein + anti-Nrf2 antibody; lane 45, nuclear protein + IgG control.