Smoking-induced upregulation of AKR1B10 expression in the airway epithelium of healthy individuals

Abstract

Background: The aldo-keto reductase (AKR) gene superfamily codes for monomeric, soluble reduced nicotinamide adenine dinucleotide phosphate-dependent oxidoreductases that mediate elimination reactions. AKR1B10, an AKR that eliminates retinals, has been observed as upregulated in squamous metaplasia and non-small cell lung cancer and has been suggested as a diagnostic marker specific to tobacco-related carcinogenesis. We hypothesized that upregulation of AKR1B10 expression may be initiated in healthy smokers prior to the development of evidence of lung cancer.

Methods: Expression of AKR1B10 was assessed at the mRNA level using microarrays with TaqMan confirmation in the large airway epithelium (21 healthy nonsmokers, 31 healthy smokers) and small airway epithelium (51 healthy nonsmokers, 58 healthy smokers) obtained by fiberoptic bronchoscopy and brushing.

Results: Compared with healthy nonsmokers, AKR1B10 mRNA levels were significantly upregulated in both large and small airway epithelia of healthy smokers. Consistent with the mRNA data, AKR1B10 protein was significantly upregulated in the airway epithelium of healthy smokers as assessed by Western blot analysis and immunohistochemistry, with AKR1B10 expressed in both differentiated and basal cells. Finally, cigarette smoke extract mediated upregulation of AKR1B10 in airway epithelial cells in vitro, and transfection of AKR1B10 into airway epithelial cells enhanced the conversion of retinal to retinol.

Conclusions: Smoking per se mediates upregulation of AKR1B10 expression in the airway epithelia of healthy smokers with no evidence of lung cancer. In the context of these observations and the link of AKR1B10 to the metabolism of retinals and to lung cancer, the smoking-induced upregulation of AKR1B10 may be an early process in the multiple events leading to lung cancer.

Figure 1.

Aldo-keto reductase (AKR) 1B10 gene expression levels in large and small airway epithelia from healthy nonsmokers and healthy smokers. A, Average normalized gene expression levels of AKR1B10 assessed with microarray HG-U133 Plus 2.0 in the large airway epithelium of 21 healthy nonsmokers and 31 healthy smokers. B, Average normalized gene expression levels of AKR1B10 assessed using microarray HG-U133 Plus 2.0 in the small airway epithelium of 51 healthy nonsmokers and 58 healthy smokers. C, TaqMan real-time polymerase chain reaction (PCR) confirmation of AKR1B10 gene expression in the large airway epithelium in a subset of 14 healthy nonsmokers and 14 healthy smokers. D, TaqMan real-time PCR confirmation of AKR1B10 gene expression in the small airway epithelium in a subset of 18 healthy nonsmokers and 18 healthy smokers.

Figure 2.

Western blot analysis of AKR1B10 protein expression in large and small airway epithelia. A, Proteins were extracted from large airway epithelial cells of four healthy nonsmokers and four healthy smokers, and the same gel was probed with anti-β-actin antibody, a control for protein loading (bottom). B, Proteins were extracted from small airway epithelial cells of five healthy nonsmokers and five healthy smokers, with the same gel probed with anti-β-actin antibody for comparison. C, Quantification by densitometry of the ratio of AKR1B10 to β-actin comparing nonsmokers to smokers in large airway epithelium. D, Quantification by densitometry of the ratio of AKR1B10 to β-actin comparing nonsmokers to smokers in small airway epithelium. Error bars represent the SE. See Figure 1 legend for expansion of abbreviation.

Figure 3.

Immunohistochemical assessment of large airway epithelium for expression of AKR1B10 in healthy nonsmokers and healthy smokers. A, Isotype mouse IgG control subjects for a healthy nonsmoker and healthy smoker. B, AKR1B10 for healthy nonsmokers. C, AKR1B10 for healthy smokers. Bar = 10 μm. See Figure 1 legend for expansion of abbreviation.

Figure 4.

In vitro upregulation of AKR1B10 expression in 16 human bronchial epithelial (16HBE) airway cells induced by exposure to CSE. The 16HBE cells were treated with different concentrations of CSE for 3 days. MTT and AKR1B10 gene expression then were quantified using TaqMan real-time PCR. A, MTT assessment showed the effect of CSE on 16HBE viability. B, Effect of CSE on AKR1B10 expression in 16HBE cells using 0.1% and 1% CSE concentrations that do not affect viability. AKR1B10 gene expression was upregulated when treated with both 0.1% and 1% CSE. Cells treated with 0.1% and 1% CSE for 3 days were changed with fresh medium without CSE and cultured for another 7 days. AKR1B10 gene expression was quantified again using TaqMan real-time PCR. *P < .01 compared with no-CSE control subjects. Error bars represent the SD. CSE = cigarette smoke extract; MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. See Figure 1 legend for expansion of other abbreviations.