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American Journal of Respiratory and Critical Care Medicine Vol 178. pp. 552-554, (2008)
© 2008 American Thoracic Society
doi: 10.1164/rccm.200806-956ED

Editorials

Defective Antioxidant Gene Regulation in COPD: A Case for Broccoli

Peter J. Barnes, D.M., F.R.S.

National Heart and Lung Institute
Imperial College
London, United Kingdom

Oxidative stress in the lungs is a critical feature of lung inflammation and may account for the amplification of inflammation in patients with chronic obstructive pulmonary disease (COPD) and for their characteristic resistance to the antiinflammatory effects of corticosteroids (1). There is abundant evidence that oxidative stress is increased in the lungs of patients with COPD, with increased concentrations of hydrogen peroxide, 8-isoprostane, and ethane in exhaled breath (2–4) and increased lipid peroxidation in lung tissue (5). The increase in oxidative stress may be due to the burden of oxidants in cigarette smoke, air pollutants, or biomass fuels, but may also be generated by activated inflammatory cells, such as neutrophils and macrophages, providing a mechanism which accounts for why oxidative stress remains high in patients with COPD even after quitting smoking (3).

There are several adverse consequences of oxidative stress. Oxidative stress activates the proinflammatory transcription factor nuclear factor-{kappa}B (NF-{kappa}B), which switches on multiple inflammatory genes, such as CXCL1, CXCL8, matrix metalloproteinase-9, and transforming growth factor-β, which together contribute to neutrophilic inflammation, emphysema, and small airway fibrosis. Oxidative stress also inhibits the activation and expression of histone deacetylase-2 (HDAC2), a key nuclear enzyme that suppresses activated inflammatory genes. HDAC2 also mediates the anti-inflammatory effects of corticosteroids through switching off NF-{kappa}B–activated inflammatory genes. The inhibitory effects of oxidative stress on HDAC2 thereby results in amplified inflammation and corticosteroid resistance—the key molecular defects of COPD (6). Oxidative stress may also be critical to accelerated nonprogrammed aging of the lung through inhibitory effects on anti-aging molecules, such as the sirtuin SIRT1, thus resulting in accelerated decline in lung function (7).

Oxidative stress in the lungs is counteracted by exogenous and endogenous antioxidants. These include dietary antioxidants, such as vitamins C and E, which are only weakly effective, and endogenous antioxidants, such as superoxide dismutases, glutathione peroxidase, glutathione reductase, catalase, and heme oxygenase-1 (HO-1). Many of the genes encoding these endogenous antioxidants are regulated by the basic leucine zipper transcription factor nuclear factor erythroid 2–related factor 2 (Nrf2). Nrf2 in the cytoplasm is associated with the cysteine-rich protein Keap1, an adapter for the enzyme Cul3 ubiquitin ligase, which ubiquitinates Nrf2 and thus targets it for destruction by the proteasome. Thus, under normal conditions Nrf2 is undetectable (Figure 1A). Oxidants oxidize cysteine residues on Keap1, so that it dissociates from Nrf2, thereby preventing its degradation and allowing it to translocate to the nucleus. Within the nucleus Nrf2 combines with other transcription factors (small Maf proteins) and binds to antioxidant response elements (ARE), which are found on over 200 antioxidant enzyme and detoxifying enzyme genes, including HO-1, glutathione peroxidase, glutathione-S-transferase, glutathione reductase, {gamma}-glutamylcysteine synthetase, thioredoxin, catalase, and NAD(P)H:quinone oxidoreductase 1 (8). The protein DJ-1 (or PARK7), which has previously been implicated in early onset Parkinson's disease, acts as a stabilizer of Nrf2 to facilitate its effects (9). In this way oxidant exposure up-regulates endogenous antioxidants to counteract the increased oxidative stress and restore normal oxidant–antioxidant balance (Figure 1B).


Figure 1
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  		Figure 1. Regulation of nuclear factor erythroid 2–related factor 2 (Nrf2) in normal subjects, normal smokers, and patients with COPD. (A) Under normal conditions Nrf2 is associated with the inhibitory protein Keap1, which is an adapter protein for the ubiquitin ligase Cul3, resulting in ubiquitination of Nrf2 and its degradation by the proteasome, so that there is no effect on antioxidant genes. (B) In normal smokers, oxidative stress oxidizes cysteine (Cys) residues on Keap1, which dissociates from Nrf2 and is stabilized by the protein DJ-1. Nrf2 translocates to the nucleus, where it switches on several antioxidant genes that counteract the increased oxidative stress. (C) In patients with COPD, oxidative stress dissociates Nrf2 from Keap1, but DJ-1 is reduced in COPD cells so that it is degraded by the proteasome before it reaches the nucleus and compensatory antioxidant genes are not activated, thus resulting in persistent oxidative stress.

 
Nrf2 plays an important role in defense of the lung against oxidative stress, and defective Nrf2 function has been implicated in several pulmonary diseases, including COPD, asthma, pulmonary fibrosis, and acute respiratory distress syndrome (10). Disruption of the Nrf2 gene in mice results in increased susceptibility to emphysema after cigarette exposure (11, 12), indicating that Nrf2 plays an important role in defending against oxidative stress in the lungs. These findings in animals have now been translated to patients with COPD in an article published in this issue of the Journal (see Malhotra and colleagues, pp. 592–604). The authors show that Nrf2 activity and expression are reduced in peripheral lungs of patients with COPD and related to disease severity and increased expression of Nrf2-regulated antioxidant genes (13). This defect in patients with COPD appears to be associated with reduction in the stabilizing protein DJ-1, resulting in degradation of Nrf2, reduced antioxidant responses, and persistent oxidative stress (Figure 1C). The reason why DJ-1 is defective in patients with COPD is not yet certain, but previous studies have shown that DJ-1 itself is reduced by oxidative stress. The reason why DJ-1 is reduced in COPD lungs but not those of normal smokers is not yet understood, but might be due to a genetic variability in the regulation of this protein, thereby providing the basis for the susceptibility in the approximately 25% of smokers who develop COPD.

Defective function of Nrf2 may also contribute to the increased susceptibility of patients with COPD to lung cancer, since Nrf2 plays an important role in defense against certain carcinogens in tobacco smoke by regulating the expression of several detoxifying enzymes (10). However, increased Nrf2 activation due to genetic mutations of Keap1 has been reported in lung cancer cells, so the role of Nrf2 in carcinogenesis is currently uncertain (14).

Reduction in oxidative stress in patients with COPD should provide clinical benefit through reducing inflammation and reversing corticosteroid resistance, but currently available antioxidants, such as N-acetyl cysteine, have proved to be disappointing in reducing progression and exacerbations of COPD (15). However, these glutathione-based antioxidants are consumed by oxidative stress and so may not be efficient in the face of continued high oxidant exposure. It has been difficult to find new more effective antioxidants that are not toxic. A more attractive approach may be to restore Nrf2 levels to normal through inhibiting the action of Keap1. This has been achieved in vitro and in vivo by isothiocyanate compounds, such as sulforaphane, which occur naturally in broccoli, and a related compound that is present in the Japanese horseradish (wasabi) (16).

In the present study, sulforaphane was able to restore antioxidant gene expression in a human bronchial epithelial cell line in which DJ-1 had been reduced by siRNA (13). This interaction of isothiocyanates and Keap-1 prevents the degradation of Nrf2 and might form the basis for the development of novel Nrf2 activators in the future. Furthermore, increasing Nrf2 may also restore important detoxifying enzymes to counteract other effects of tobacco smoke. These new insights into the regulation of oxidative stress in COPD may thus lead to novel therapeutic approaches to COPD.

FOOTNOTES

Conflict of Interest Statement: The author has no financial relationship with a commercial entity that has an interest in the subject of this manuscript.

REFERENCES

  1. Bowler RP, Barnes PJ, Crapo JD. The role of oxidative stress in chronic obstructive pulmonary disease. COPD 2004;2:255–277.
  2. Dekhuijzen PNR, Aben KHH, Dekker I, Aarts LPHJ, Wielders PLM, van Herwarden CLA, Bast A. Increased exhalation of hydrogen peroxide in patients with stable and unstable chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1996;154:813–816.[Abstract]
  3. Montuschi P, Collins JV, Ciabattoni G, Lazzeri N, Corradi M, Kharitonov SA, Barnes PJ. Exhaled 8-isoprostane as an in vivo biomarker of lung oxidative stress in patients with COPD and healthy smokers. Am J Respir Crit Care Med 2000;162:1175–1177.[Abstract/Free Full Text]
  4. Paredi P, Kharitonov SA, Leak D, Ward S, Cramer D, Barnes PJ. Exhaled ethane, a marker of lipid peroxidation, is elevated in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2000;162:369–373.[Abstract/Free Full Text]
  5. Rahman I, van Schadewijk AA, Crowther AJ, Hiemstra PS, Stolk J, Macnee W, de Boer WI. 4-Hydroxy-2-nonenal, a specific lipid peroxidation product, is elevated in lungs of patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2002;166:490–495.[Abstract/Free Full Text]
  6. Barnes PJ. Reduced histone deacetylase in COPD: clinical implications. Chest 2006;129:151–155.[CrossRef][Medline]
  7. Ito K, Barnes PJ. COPD as a disease of accelerated lung aging. Chest 2008; (in press).
  8. Kobayashi M, Yamamoto M. Nrf2-Keap1 regulation of cellular defense mechanisms against electrophiles and reactive oxygen species. Adv Enzyme Regul 2006;46:113–140.[CrossRef][Medline]
  9. Clements CM, McNally RS, Conti BJ, Mak TW, Ting JP. DJ-1, a cancer- and Parkinson's disease-associated protein, stabilizes the antioxidant transcriptional master regulator Nrf2. Proc Natl Acad Sci USA 2006;103:15091–15096.[Abstract/Free Full Text]
  10. Cho HY, Reddy SP, Kleeberger SR. Nrf2 defends the lung from oxidative stress. Antioxid Redox Signal 2006;8:76–87.[CrossRef][Medline]
  11. Rangasamy T, Cho CY, Thimmulappa RK, Zhen L, Srisuma SS, Kensler TW, Yamamoto M, Petrache I, Tuder RM, Biswal S. Genetic ablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in mice. J Clin Invest 2004;114:1248–1259.[CrossRef][Medline]
  12. Iizuka T, Ishii Y, Itoh K, Kiwamoto T, Kimura T, Matsuno Y, Morishima Y, Hegab AE, Homma S, Nomura A, et al. Nrf2-deficient mice are highly susceptible to cigarette smoke-induced emphysema. Genes Cells 2005;10:1113–1125.[Abstract/Free Full Text]
  13. Malhotra D, Thimmulappa R, Navas-Acien A, Sandford A, Elliott M, Singh A, Chen L, Zhuang X, Hogg J, Pare P, et al. Decline in NRF2- regulated antioxidants in chronic obstructive pulmonary disease lungs due to loss of its positive regulator, DJ-1. Am J Respir Crit Care Med 2008;178:592–604.[Abstract/Free Full Text]
  14. Ohta T, Iijima K, Miyamoto M, Nakahara I, Tanaka H, Ohtsuji M, Suzuki T, Kobayashi A, Yokota J, Sakiyama T, et al. Loss of Keap1 function activates Nrf2 and provides advantages for lung cancer cell growth. Cancer Res 2008;68:1303–1309.[Abstract/Free Full Text]
  15. Decramer M, Rutten-van Molken M, Dekhuijzen PN, Troosters T, van Herwaarden C, Pellegrino R, Van Schayck CP, Olivieri D, Del Donno M, De Backer W, et al. Effects of N-acetylcysteine on outcomes in chronic obstructive pulmonary disease (Bronchitis Randomized on NAC Cost-Utility Study, BRONCUS): a randomised placebo-controlled trial. Lancet 2005;365:1552–1560.[CrossRef][Medline]
  16. Morimitsu Y, Nakagawa Y, Hayashi K, Fujii H, Kumagai T, Nakamura Y, Osawa T, Horio F, Itoh K, Iida K, et al. A sulforaphane analogue that potently activates the Nrf2-dependent detoxification pathway. J Biol Chem 2002;277:3456–3463.[Abstract/Free Full Text]

Related articles in AJRCCM:

Decline in NRF2-regulated Antioxidants in Chronic Obstructive Pulmonary Disease Lungs Due to Loss of Its Positive Regulator, DJ-1
Deepti Malhotra, Rajesh Thimmulappa, Ana Navas-Acien, Andrew Sandford, Mark Elliott, Anju Singh, Linan Chen, Xiaoxi Zhuang, James Hogg, Peter Pare, Rubin M. Tuder, and Shyam Biswal
AJRCCM 2008 178: 592-604. [Abstract] [Full Text]  




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