HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 18/15/1897 04-1506fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by MOODIE, F. M. Articles by RAHMAN, I. Search for Related Content PubMed PubMed Citation Articles by MOODIE, F. M. Articles by RAHMAN, I.

Oxidative stress and cigarette smoke alter chromatin remodeling but differentially regulate NF-B activation and proinflammatory cytokine release in alveolar epithelial cells

FIONA M. MOODIE^*, JOHN A. MARWICK^*, CHARLOTTE S. ANDERSON^*, PATRYK SZULAKOWSKI^*, SAIBAL K. BISWAS^*, MARK R. BAUTER, IAIN KILTY and IRFAN RAHMAN^,*

^* ELEGI and Colt Research Laboratories, MRC Centre for Inflammation Research, University of Edinburgh Medical School, Edinburgh, UK;
Pfizer Global Research and Development, Sandwich, UK; and
Department of Environmental Medicine, Division of Lung Biology and Disease, University of Rochester Medical Center, Rochester, New York, USA

¹Correspondence: Department of Environmental Medicine, Division of Lung Biology and Disease Program, University of Rochester Medical Center, Box 850, 601 Elmwood Ave., Rochester, NY 14642, USA. E-mail: irfan_rahman{at}urmc.rochester.edu

SPECIFIC AIMS

Cigarette smoking causes oxidative stress and an abnormal inflammatory response in the lungs, which are the hallmarks of chronic obstructive pulmonary disease (COPD). The molecular mechanism of cigarette smoke-mediated inflammatory gene transcription is not well understood. In this study, we investigated the role of cigarette smoke condensate (CSC) and hydrogen peroxide (H₂O₂) on histone acetylation:deacetylation, activation of NF-B, and cytokine release, and modulation of oxidant-mediated proinflammatory response by a thiol compound, N-acetyl-L-cysteine (NAC) in human alveolar epithelial cells (A549).

PRINCIPAL FINDINGS

1. H₂O₂ and CSC alter the balance of histone acetylation:deacetylation by increasing histone acetyltransferase and decreasing histone deacetylase activity in alveolar epithelial A549 cells
Changes in gene transcription depend on chromatin remodeling and the relative activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). To study the effect of oxidative stress and cigarette smoke condensate (CSC) on chromatin remodeling, A549 alveolar epithelial cells were treated with various concentrations of CSC (1, 2.5 or 5, and 10%), H₂O₂ (100 µM), and the histone deacetylase inhibitor trichostatin A (100 ng/mL). H₂O₂ and CSC significantly induced intrinsic HAT activity, and acetylation of H4 protein assessed by immunocytochemistry, leading to increased acetylation of histone proteins (H4) after 1, 4, and 24 h incubations. Trichostatin A (TSA) treatment also caused an increase in histone acetylation in A549 cells. Cell viability remained between 80–90% after all of the above treatments as assessed by the trypan blue exclusion test.

To determine if decreased deacetylation is involved in net increased acetylation in A549 epithelial cells, we measured HDAC activity using the substrate Boc-Lys(Ac)-pNA, and the levels of HDAC2 by Western blot analysis, in response to H₂O₂, CSC, and TSA. We found that H₂O₂ and CSC (1, 2.5, and 5%) significantly decreased HDAC activity, which was associated with the decreased levels of HDAC2 protein at 4 and 24 h in A549 cells. However, the reduction in HDAC2 protein levels is unlikely to fully account for the degree of inhibition of HDAC activity observed.

2. Decreased HDAC activity is associated with covalent modification of HDAC2 by cigarette smoke
In view of the pro-oxidative components present in CSC, we investigated whether protein modification is involved in the observed reduction in HDAC2 activity. Covalent modification of the HDAC2 protein was assessed by immunoprecipitation, followed by Western blot analysis using monoclonal antibodies for 4-HNE and nitrated tyrosine. There was a significant increase in the tyrosine nitration and 4-HNE modification of HDAC2 after 4 h of cigarette smoke exposure compared with untreated cells (Fig. 1 ). To directly assess the impact of these modifications on HDAC activity, the activity of immunoprecipitated HDAC2 was measured. There was a substantial decrease in HDAC2 activity with all the treatments compared with the control nontreated cells. This supports the concept that decreased HDAC2 activity by cigarette smoke exposure was in part a result of covalent modification of HDAC2 protein by components of cigarette smoke.

View larger version (21K): [in this window] [in a new window]   Figure 1. Cigarette smoke increases HDAC2 protein modification. 4-HNE and tyrosine nitration modification of HDAC2 protein is increased after CSC exposure in A549 cells, as assessed by immunoprecipitation of HDAC2 with an anti-HDAC2 antibody followed by immunoblot analysis using anti-4-HNE anti-nitrotyrosine, and anti-HDAC2 antibodies (n=3). Histograms represent means and bars the SEM of the ratio of HDAC2 modified protein to total HDAC2 protein. *P < 0.05 and **P < 0.01, compared with control values.

3. H₂O₂, but not CSC, activates NF-B and induces cytokine release in A549 epithelial cells
We examined the trans-activation of NF-B in response to H₂O₂ and CSC in A549 cells stably transfected with the NF-B-dependent promoter since it is known that NF-B possesses an intrinsic HAT activity. H₂O₂ and TNF- treatments significantly increased NF-B trans-activation, compared with the control values at 24 h in A549 cells (Fig. 2 ). Inhibition of HDACs by TSA also caused increased NF-B trans-activation in epithelial cells. CSC (1, 2.5, and 5%) treatments did not cause any appreciable increase in NF-B trans-activation at 24 h in A549 cells. However, TNF- and IL-1ß-induced NF-B activation was inhibited by CSC cotreatment compared with TNF- or IL-1ß alone in A549 cell transfected with the NF-B-dependent promoter.

View larger version (12K): [in this window] [in a new window]   Figure 2. Effect of CSC (1, 2.5, and 5%), H2O2 (100 µM), TSA (100 ng/mL) and TNF- on NF-B trans-activation at 24 h. *P < 0.05 and ***P < 0.001, compared with control values.

Increased translocation of the p65 subunit of NF-B from the cytoplasm to nucleus was induced by H₂O₂, TNF-, and IL-1ß, whereas CSC did not result in similar translocation of p65. Furthermore, CSC inhibited IL-1ß-induced p65 nuclear translocation in a dose-dependent manner, suggesting that CSC constituents (mainly electrophilic compounds) have a direct inhibitory effect on translocation of p65 from the cytoplasm to the nucleus. CSC also inhibited TNF--induced phosphorylation of IB-, suggesting that CSC-mediated inhibition of NF-B involves reduction of IKK activity.

To determine the role of histone acetylation and NF-B on IL-8 protein release and IL-8 mRNA expression, A549 cells were treated with CSC and H₂O₂. H₂O₂, but not CSC, resulted in increased IL-8 mRNA expression and release after 24 h compared with control. Similar data was obtained for IL-6. Furthermore, treatment of cells with the HDAC inhibitor TSA also increased the release of IL-8. Concomitant exposure of cells to CSC along with TNF- treatment significantly decreased the IL-8 expression and release compared with TNF- or H₂O₂ alone. Similar results for the inhibition of IL-8 secretion were obtained with rat type II epithelial (L2) cells. This suggests that lung alveolar epithelial cells do not produce IL-8 protein in response to CSC and that CSC can indeed inhibit production of this cytokine induced by other mediators via inhibition of NF-B.

4. Thiol compound NAC attenuates oxidant-mediated decrease in HDAC activity, inhibits NF-B trans-activation, and IL-8 release
To determine the role of intracellular thiol compound on oxidant-mediated regulation of HDAC, NF-B trans-activation, and IL-8 release, we pre-treated A549 cells with NAC (to increase intracellular levels of thiols) prior to addition of CSC or H₂O₂. NAC significantly attenuated H₂O₂–mediated decrease in HDAC activity, significantly inhibited NF-B activation (H₂O₂ 12.5±3.1, H₂O₂+NAC 6.1±2 vs. control 7.1±1.2 U luciferase/ß-gal, n=3) and IL-8 release (H₂O₂ 2.0±0.3, H₂O₂+NAC 0.75±0.45 vs. control 0.6±0.04 ng/mL, n=3). Similarly, CSC-mediated inhibition of HDAC activity was restored by NAC pretreatment suggesting that electrophilic agents present in CSC can be conjugated directly by elevated intracellular thiol. However, TSA-mediated decrease in HDAC activity was not reversed by NAC pretreatment (TSA 58±12, TSA±NAC 62±16 vs. control 122±8 µM deacetylated substrate/mg protein, P<0.001).

CONCLUSIONS AND SIGNIFICANCE

Acetylation of histones that form the nucleosome core around which DNA is coiled, may cause unwinding of condensed chromatin. Decondensation of the chromatin allows access of transcription factors to consensus sites on DNA, leading to the transcription of inflammatory mediators and thereby enhancing the inflammatory response. In this study we show for the first time that exposure of alveolar A549 epithelial cells to H₂O₂ and CSC results in altered chromatin, whereas they have differential effects on NF-B activation and transcription of proinflammatory genes. Both H₂O₂ and CSC cause an increase in histone acetylation as assessed by HAT activity and increased H4 acetylation, in addition to decreased deacetylation by a decrease in HDAC activity in A549 alveolar epithelial cells. The mechanism of increased histone acetylation in response to these agents is not clear. It is possible that activation of MAPK-signaling pathways may regulate activator transcription factor-2 (ATF-2), and CREB binding protein (CBP) coactivators, which possess intrinsic HAT activity. Our data show that the decreased HDAC activity was associated with decreased HDAC-2 levels. This suggests that oxidative stress is involved in increased histone H4 acetylation and corresponding decrease in HDAC activity/HDAC2 levels. H₂O₂ and CSC may inhibit HDAC levels and/or HDAC activity by oxidation and or nitrosylation of the protein or further post-translational modifications, and subsequent degradation by the ubiquitin-proteasome pathway. We have shown a covalent post-translational modification of HDAC2 by 4-HNE and increased tyrosine nitration of HDAC2 after CSC exposure. This protein modification after smoke condensate exposure may be responsible for the decrease in HDAC2 activity. Furthermore, our data show that increasing the intracellular thiol levels attenuated the oxidant- and CSC-mediated decrease in HDAC activity suggesting that HDAC enzyme is at least in part redox regulated. It is known that HDAC2 is recruited by glucocorticoids in the transcriptional initiation complex to inhibit the transcription of proinflammatory mediators. Therefore, post-translational modification of HDAC2 (aldehyde-adduct formation and tyrosine nitration) may represent at least part of the mechanism for poor glucocorticoid anti-inflammatory efficacy in response to smoking.

The induction of inflammatory mediators can be regulated by the activation of redox-sensitive transcription factors, such as NF-B stimulated in response to oxidants and inflammatory cytokines, such as TNF-. In this study, we show that H₂O₂ and TNF- induced an increase in IL-8 and IL-6 release with a corresponding increase in the activation (trans-activation and nuclear translocation) of NF-B in A549 cells. CSC exposure did not cause NF-B trans-activation, translocation, or cytokine expression/release from A549 cells, suggesting that although cigarette smoke increases histone acetylation it does not cause NF-B-dependent signaling in alveolar epithelial cells. Our data shows that CSC inhibited TNF- and IL-1ß-induced NF-B trans-activation or translocation. We also show the CSC-mediated inhibition of TNF--induced NF-B activation was due to inhibition of phosphorylation of IB-, suggesting that CSC inhibits IB kinase (IKK) activity. This is possibly due to protein modification of IKK by CSC. IKK contains cysteine and tyrosine residues in its active site, which may be covalently conjugated/modified with the components (electrophilic compounds/aldehydes and/or NO radicals) of CSC, potentially leading to direct inhibition of this kinase.

Our data showing no effect of CSC on NF-B activation and the release of proinflammatory mediators in A549 epithelial cells suggests that CSC-mediated effects may be cell specific, since we recently found that CSC triggers release of proinflammatory mediators (TNF and IL-8) associated with NF-B activation in a macrophage cell line (Mono-Mac6), and blood derived monocytes obtained from healthy nonsmokers (unpublished). This highlights the importance of stimuli- and cell-specific effects on the ability of cigarette smoke and oxidative stress to induce cytokines and chemokines. This study provides novel data on an important molecular mechanism by which cigarette smoke and H₂O₂ affect gene transcription and augment an inflammatory response in alveolar epithelial cells. CSC may prime the lung epithelial cells by unwinding the chromatin allowing subsequent signaling and gene transcription by inflammatory mediators (Fig. 3 ). This may be important in amplification of chronic inflammatory response by cigarette smoke in the lungs.

View larger version (23K): [in this window] [in a new window]   Figure 3. The hypothesized mechanism of hydrogen peroxide and cigarette smoke-induced chromatin remodeling through a decrease in HDAC2 and increase in NF-B activation resulting in an increase in histone 4 acetylation in lung epithelial cells. The increased DNA binding of the redox sensitive transcription factor results in an increased transcription of specific proinflammatory genes. CSC may prime transcriptional machinery for trans-activation by altering histone acetylation:deacetylation balance in favor of acetylation. (X) shows the pathway is inhibited.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1506fje;

This article has been cited by other articles:

				H. Yao, S.-R. Yang, I. Edirisinghe, S. Rajendrasozhan, S. Caito, D. Adenuga, M. A. O'Reilly, and I. Rahman Disruption of p21 Attenuates Lung Inflammation Induced by Cigarette Smoke, LPS, and fMLP in Mice Am. J. Respir. Cell Mol. Biol., July 1, 2008; 39(1): 7 - 18. [Abstract] [Full Text] [PDF]

				I. Edirisinghe, S.-R. Yang, H. Yao, S. Rajendrasozhan, S. Caito, D. Adenuga, C. Wong, A. Rahman, R. P. Phipps, Z.-G. Jin, et al. VEGFR-2 inhibition augments cigarette smoke-induced oxidative stress and inflammatory responses leading to endothelial dysfunction FASEB J, July 1, 2008; 22(7): 2297 - 2310. [Abstract] [Full Text] [PDF]

				K. F. Chung and I. M. Adcock Multifaceted mechanisms in COPD: inflammation, immunity, and tissue repair and destruction Eur. Respir. J., June 1, 2008; 31(6): 1334 - 1356. [Abstract] [Full Text] [PDF]

				S.-R. Yang, S. Valvo, H. Yao, A. Kode, S. Rajendrasozhan, I. Edirisinghe, S. Caito, D. Adenuga, R. Henry, G. Fromm, et al. IKK{alpha} Causes Chromatin Modification on Pro-Inflammatory Genes by Cigarette Smoke in Mouse Lung Am. J. Respir. Cell Mol. Biol., June 1, 2008; 38(6): 689 - 698. [Abstract] [Full Text] [PDF]

				H. Yao, I. Edirisinghe, S. Rajendrasozhan, S.-R. Yang, S. Caito, D. Adenuga, and I. Rahman Cigarette smoke-mediated inflammatory and oxidative responses are strain-dependent in mice Am J Physiol Lung Cell Mol Physiol, June 1, 2008; 294(6): L1174 - L1186. [Abstract] [Full Text] [PDF]

				H. Yao, I. Edirisinghe, S.-R. Yang, S. Rajendrasozhan, A. Kode, S. Caito, D. Adenuga, and I. Rahman Genetic Ablation of NADPH Oxidase Enhances Susceptibility to Cigarette Smoke-Induced Lung Inflammation and Emphysema in Mice Am. J. Pathol., May 1, 2008; 172(5): 1222 - 1237. [Abstract] [Full Text] [PDF]

				S. Rajendrasozhan, S.-R. Yang, V. L. Kinnula, and I. Rahman SIRT1, an Antiinflammatory and Antiaging Protein, Is Decreased in Lungs of Patients with Chronic Obstructive Pulmonary Disease Am. J. Respir. Crit. Care Med., April 15, 2008; 177(8): 861 - 870. [Abstract] [Full Text] [PDF]

				A. Churg, M. Cosio, and J. L. Wright Mechanisms of cigarette smoke-induced COPD: insights from animal models Am J Physiol Lung Cell Mol Physiol, April 1, 2008; 294(4): L612 - L631. [Abstract] [Full Text] [PDF]

				M. F. Mian, N. M. Lauzon, M. R. Stampfli, K. L. Mossman, and A. A. Ashkar Impairment of human NK cell cytotoxic activity and cytokine release by cigarette smoke J. Leukoc. Biol., March 1, 2008; 83(3): 774 - 784. [Abstract] [Full Text] [PDF]

				A. Kode, S. Rajendrasozhan, S. Caito, S.-R. Yang, I. L. Megson, and I. Rahman Resveratrol induces glutathione synthesis by activation of Nrf2 and protects against cigarette smoke-mediated oxidative stress in human lung epithelial cells Am J Physiol Lung Cell Mol Physiol, March 1, 2008; 294(3): L478 - L488. [Abstract] [Full Text] [PDF]

				H. Dombrowsky and S. Uhlig Steroids and histone deacetylase in ventilation-induced gene transcription Eur. Respir. J., November 1, 2007; 30(5): 865 - 877. [Abstract] [Full Text] [PDF]

				M. Y. Yeh, E. L. Burnham, M. Moss, and L. A. S. Brown Chronic Alcoholism Alters Systemic and Pulmonary Glutathione Redox Status Am. J. Respir. Crit. Care Med., August 1, 2007; 176(3): 270 - 276. [Abstract] [Full Text] [PDF]

				T. Yoshida and R. M. Tuder Pathobiology of Cigarette Smoke-Induced Chronic Obstructive Pulmonary Disease Physiol Rev, July 1, 2007; 87(3): 1047 - 1082. [Abstract] [Full Text] [PDF]

				S. Qanungo, D. W. Starke, H. V. Pai, J. J. Mieyal, and A.-L. Nieminen Glutathione Supplementation Potentiates Hypoxic Apoptosis by S-Glutathionylation of p65-NF{kappa}B J. Biol. Chem., June 22, 2007; 282(25): 18427 - 18436. [Abstract] [Full Text] [PDF]

				T. H. Thatcher, S. B. Maggirwar, C. J. Baglole, H. F. Lakatos, T. A. Gasiewicz, R. P. Phipps, and P. J. Sime Aryl Hydrocarbon Receptor-Deficient Mice Develop Heightened Inflammatory Responses to Cigarette Smoke and Endotoxin Associated with Rapid Loss of the Nuclear Factor-{kappa}B Component RelB Am. J. Pathol., March 1, 2007; 170(3): 855 - 864. [Abstract] [Full Text] [PDF]

				S.-R. Yang, J. Wright, M. Bauter, K. Seweryniak, A. Kode, and I. Rahman Sirtuin regulates cigarette smoke-induced proinflammatory mediator release via RelA/p65 NF-{kappa}B in macrophages in vitro and in rat lungs in vivo: implications for chronic inflammation and aging Am J Physiol Lung Cell Mol Physiol, February 1, 2007; 292(2): L567 - L576. [Abstract] [Full Text] [PDF]

				L. A. Espinoza, F. Tenzin, A. O. Cecchi, Z. Chen, M. L. Witten, and M. E. Smulson Expression of JP-8-Induced Inflammatory Genes in AEII Cells Is Mediated by NF-{kappa}B and PARP-1 Am. J. Respir. Cell Mol. Biol., October 1, 2006; 35(4): 479 - 487. [Abstract] [Full Text] [PDF]

				C.-Y. Zhong, Y. M. Zhou, J. P. Joad, and K. E. Pinkerton Environmental Tobacco Smoke Suppresses Nuclear Factor-{kappa}B Signaling to Increase Apoptosis in Infant Monkey Lungs Am. J. Respir. Crit. Care Med., August 15, 2006; 174(4): 428 - 436. [Abstract] [Full Text] [PDF]

				Z. Yin, L. Gonzales, V. Kolla, N. Rath, Y. Zhang, M. M. Lu, S. Kimura, P. L. Ballard, M. F. Beers, J. A. Epstein, et al. Hop functions downstream of Nkx2.1 and GATA6 to mediate HDAC-dependent negative regulation of pulmonary gene expression Am J Physiol Lung Cell Mol Physiol, August 1, 2006; 291(2): L191 - L199. [Abstract] [Full Text] [PDF]

				I. Rahman and I. M. Adcock Oxidative stress and redox regulation of lung inflammation in COPD. Eur. Respir. J., July 1, 2006; 28(1): 219 - 242. [Abstract] [Full Text] [PDF]

				P. Szulakowski, A. J. L. Crowther, L. A. Jimenez, K. Donaldson, R. Mayer, T. B. Leonard, W. MacNee, and E. M. Drost The Effect of Smoking on the Transcriptional Regulation of Lung Inflammation in Patients with Chronic Obstructive Pulmonary Disease Am. J. Respir. Crit. Care Med., July 1, 2006; 174(1): 41 - 50. [Abstract] [Full Text] [PDF]

				S.-R. Yang, A. S. Chida, M. R. Bauter, N. Shafiq, K. Seweryniak, S. B. Maggirwar, I. Kilty, and I. Rahman Cigarette smoke induces proinflammatory cytokine release by activation of NF-{kappa}B and posttranslational modifications of histone deacetylase in macrophages Am J Physiol Lung Cell Mol Physiol, July 1, 2006; 291(1): L46 - L57. [Abstract] [Full Text] [PDF]

				H. Slevogt, B. Schmeck, C. Jonatat, J. Zahlten, W. Beermann, V. van Laak, B. Opitz, S. Dietel, P. D. N'Guessan, S. Hippenstiel, et al. Moraxella catarrhalis induces inflammatory response of bronchial epithelial cells via MAPK and NF-{kappa}B activation and histone deacetylase activity reduction Am J Physiol Lung Cell Mol Physiol, May 1, 2006; 290(5): L818 - L826. [Abstract] [Full Text] [PDF]

				V. Regueiro, M. A. Campos, J. Pons, S. Alberti, and J. A. Bengoechea The uptake of a Klebsiella pneumoniae capsule polysaccharide mutant triggers an inflammatory response by human airway epithelial cells Microbiology, February 1, 2006; 152(2): 555 - 566. [Abstract] [Full Text] [PDF]

				M. J. Walters, M. J. Paul-Clark, S. K. McMaster, K. Ito, I. M. Adcock, and J. A. Mitchell Cigarette Smoke Activates Human Monocytes by an Oxidant-AP-1 Signaling Pathway: Implications for Steroid Resistance Mol. Pharmacol., November 1, 2005; 68(5): 1343 - 1353. [Abstract] [Full Text] [PDF]

				P. J. Barnes, I. M. Adcock, and K. Ito Histone acetylation and deacetylation: importance in inflammatory lung diseases Eur. Respir. J., March 1, 2005; 25(3): 552 - 563. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 18/15/1897 04-1506fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by MOODIE, F. M. Articles by RAHMAN, I. Search for Related Content PubMed PubMed Citation Articles by MOODIE, F. M. Articles by RAHMAN, I.