Genetic ablation of Nrf2 enhances susceptibility to cigarette smoke–induced emphysema in mice
Tirumalai Rangasamy, Chung Y. Cho, Rajesh K. Thimmulappa, Lijie Zhen, Sorachai S. Srisuma, Thomas W. Kensler, Masayuki Yamamoto, Irina Petrache, Rubin M. Tuder, Shyam Biswal
Tirumalai Rangasamy, Chung Y. Cho, Rajesh K. Thimmulappa, Lijie Zhen, Sorachai S. Srisuma, Thomas W. Kensler, Masayuki Yamamoto, Irina Petrache, Rubin M. Tuder, Shyam Biswal
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Article Genetics

Genetic ablation of Nrf2 enhances susceptibility to cigarette smoke–induced emphysema in mice

Abstract

Although inflammation and protease/antiprotease imbalance have been postulated to be critical in cigarette smoke–induced (CS-induced) emphysema, oxidative stress has been suspected to play an important role in chronic obstructive pulmonary diseases. Susceptibility of the lung to oxidative injury, such as that originating from inhalation of CS, depends largely on its upregulation of antioxidant systems. Nuclear factor, erythroid-derived 2, like 2 (Nrf2) is a redox-sensitive basic leucine zipper protein transcription factor that is involved in the regulation of many detoxification and antioxidant genes. Disruption of the Nrf2 gene in mice led to earlier-onset and more extensive CS-induced emphysema than was found in wild-type littermates. Emphysema in Nrf2-deficient mice exposed to CS for 6 months was associated with more pronounced bronchoalveolar inflammation; with enhanced alveolar expression of 8-oxo-7,8-dihydro-2′-deoxyguanosine, a marker of oxidative stress; and with an increased number of apoptotic alveolar septal cells — predominantly endothelial and type II epithelial cells — as compared with wild-type mice. Microarray analysis identified the expression of nearly 50 Nrf2-dependent antioxidant and cytoprotective genes in the lung that may work in concert to counteract CS-induced oxidative stress and inflammation. The responsiveness of the Nrf2 pathway may act as a major determinant of susceptibility to tobacco smoke–induced emphysema by upregulating antioxidant defenses and decreasing lung inflammation and alveolar cell apoptosis.

Authors

Tirumalai Rangasamy, Chung Y. Cho, Rajesh K. Thimmulappa, Lijie Zhen, Sorachai S. Srisuma, Thomas W. Kensler, Masayuki Yamamoto, Irina Petrache, Rubin M. Tuder, Shyam Biswal

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Activation of Nrf2 in CS-exposed Nrf2+/+ lungs. (A) EMSA to determine th...
Activation of Nrf2 in CS-exposed Nrf2+/+ lungs. (A) EMSA to determine the DNA binding activity of Nrf2. For gel shift analysis, 10 μg of nuclear protein from the lungs of air-and CS-exposed mice was incubated with the labeled human NQO1 ARE sequence and analyzed on a 5% non-denaturing polyacrylamide gel. For supershift assays, the labeled NQO1 ARE was first incubated with 10 μg of nuclear extract and then with 4 μg of anti-Nrf2 antibody for 2 hours. Nuclear protein of Nrf2+/+ lungs showed increased binding to the ARE-containing sequence (lower arrow) after CS exposure, with a supershifted band caused by preincubation with anti-Nrf2 antibody, thus confirming the binding of Nrf2 to the ARE sequence (upper arrow). Ra–IgG1, rabbit IgG1. (B) Nuclear accumulation of Nrf2. Western blot analysis with anti-Nrf2 antibody showed the nuclear accumulation of the transcription factor Nrf2 in the lungs of Nrf2+/+ mice in response to CS exposure (lanes 1 and 3: air-exposed Nrf2–/– and Nrf2+/+ mice, respectively; lanes 2 and 4: CS-exposed Nrf2–/– and Nrf2+/+ mice, respectively; lamin B1 was used as the loading control). Western blot analysis was carried out 3 times with the nuclear proteins isolated from the lungs of 3 different air- or CS-exposed Nrf2+/+ and Nrf2–/– mice.