Se-Ran Yang

SIRT1, an Antiinflammatory and Antiaging Protein, Is Decreased in Lungs of Patients with Chronic Obstructive Pulmonary Disease

RATIONALE Abnormal inflammation and accelerated decline in lung function occur in patients with chronic obstructive pulmonary disease (COPD). Human sirtuin (SIRT1), an antiaging and antiinflammatory protein, is a metabolic NAD(+)-dependent protein/histone deacetylase that regulates proinflammatory mediators by deacetylating histone and nonhistone proteins. OBJECTIVES To determine the expression of SIRT1 in lungs of smokers and patients with COPD, and to elucidate the regulation of SIRT1 in response to cigarette smoke in macrophages, and its impact on nuclear factor (NF)-kappaB regulation. METHODS SIRT1 and NF-kappaB levels were assessed in lung samples of nonsmokers, smokers, and patients with COPD. Human monocyte-macrophage cells (MonoMac6) were treated with cigarette smoke extract (CSE) to determine the mechanism of CSE-mediated regulation of SIRT1 and its involvement in RelA/p65 regulation and IL-8 release. MEASUREMENTS AND MAIN RESULTS Peripheral lungs of smokers and patients with COPD showed decreased levels of nuclear SIRT1, as compared with nonsmokers, associated with its post-translational modifications (formation of nitrotyrosine and aldehyde carbonyl adducts). Treatment of MonoMac6 cells with CSE showed decreased levels of SIRT1 associated with increased acetylation of RelA/p65 NF-kappaB. Mutation or knockdown of SIRT1 resulted in increased acetylation of nuclear RelA/p65 and IL-8 release, whereas overexpression of SIRT1 decreased IL-8 release in response to CSE treatment in MonoMac6 cells. CONCLUSIONS SIRT1 levels were reduced in macrophages and lungs of smokers and patients with COPD due to its post-translational modifications by cigarette smoke-derived reactive components, leading to increased acetylation of RelA/p65. Thus, SIRT1 plays a pivotal role in regulation of NF-kappaB-dependent proinflammatory mediators in lungs of smokers and patients with COPD.

Differential effects of cigarette smoke on oxidative stress and proinflammatory cytokine release in primary human airway epithelial cells and in a variety of transformed alveolar epithelial cells.

BackgroundCigarette smoke mediated oxidative stress and inflammatory events in the airway and alveolar epithelium are important processes in the pathogenesis of smoking related pulmonary diseases. Previously, individual cell lines were used to assess the oxidative and proinflammatory effects of cigarette smoke with confounding results. In this study, a panel of human and rodent transformed epithelial cell lines were used to determine the effects of cigarette smoke extract (CSE) on oxidative stress markers, cell toxicity and proinflammatory cytokine release and compared the effects with that of primary human small airway epithelial cells (SAEC).MethodsPrimary human SAEC, transformed human (A549, H1299, H441), and rodent (murine MLE-15, rat L2) alveolar epithelial cells were treated with different concentrations of CSE (0.2–10%) ranging from 20 min to 24 hr. Cytotoxicity was assessed by lactate dehydrogenase release assay, trypan blue exclusion method and double staining with acridine orange and ethidium bromide. Glutathione concentration was measured by enzymatic recycling assay and 4-hydroxy-2-nonenal levels by using lipid peroxidation assay kit. The levels of proinflammatory cytokines (e.g. IL-8 and IL-6) were measured by ELISA. Nuclear translocation of the transcription factor, NF-κB was assessed by immunocytochemistry and immunoblotting.ResultsCigarette smoke extract dose-dependently depleted glutathione concentration, increased 4-hydroxy-2-nonenal (4-HNE) levels, and caused necrosis in the transformed cell lines as well as in SAEC. None of the transformed cell lines showed any significant release of cytokines in response to CSE. CSE, however, induced IL-8 and IL-6 release in primary cell lines in a dose-dependent manner, which was associated with the nuclear translocation of NF-κB in SAEC.ConclusionThis study suggests that primary, but not transformed, lung epithelial cells are an appropriate model to study the inflammatory mechanisms in response to cigarette smoke.

Cigarette smoke-mediated inflammatory and oxidative responses are strain-dependent in mice

A variety of mouse models have been used to study the pathogenesis of pulmonary emphysema/chronic obstructive pulmonary disease. The effect of cigarette smoke (CS) is believed to be strain dependent, because certain mouse strains are more susceptible or resistant to development of emphysema. However, the molecular basis of susceptibility of mouse strains to effects of CS is not known. We investigated the effect of CS on lungs of most of the commonly used mouse strains to study the molecular mechanism of susceptibility to effects of CS. C57BL/6J, A/J, AKR/J, CD-1, and 129SvJ mice were exposed to CS for 3 consecutive days, and various parameters of inflammatory and oxidative responses were assessed in lungs of these mice. We found that the C57BL/6J strain was highly susceptible, the A/J, AKR/J, and CD-1 strains were moderately susceptible, and the 129SvJ strain was resistant to lung inflammatory and oxidant responses to CS exposure. The mouse strain that was more susceptible to effects of CS showed augmented lung inflammatory cell influx, activation of NF-kappaB and p38 MAPK, and increased levels of matrix metalloproteinase-9 and NF-kappaB-dependent proinflammatory cytokines compared with resistant mouse strains. Similarly, decreased levels of glutathione were associated with increased levels of lipid peroxidation products in susceptible mouse strains compared with resistant strains. Hence, we identified the susceptible and resistant mouse strains on the basis of the pattern of inflammatory and oxidant responses. Identification of sensitive and resistant mouse strains could be useful for studying the molecular mechanisms of effects of CS on inflammation and pharmacological interventional studies in CS-exposure mouse models.

Redox regulation of lung inflammation: role of NADPH oxidase and NF-κB signalling

Regulation of reduction/oxidation (redox) state is critical for cell viability, activation, proliferation and organ function, and imbalance of oxidant/antioxidant balance is implicated in various chronic respiratory inflammatory diseases, such as asthma, pulmonary fibrosis and chronic obstructive pulmonary disease. CS (cigarette smoke) is a complex mixture of various noxious gases and condensed tar particles. These components elicit oxidative stress in lungs by continuous generation of ROS (reactive oxygen species) and various inflammatory mediators. In the present review, we have discussed the role of oxidative stress in triggering the inflammatory response in the lungs in response to CS by demonstrating the role of NADPH oxidase, redox-sensitive transcription factors, such as pro-inflammatory NF-kappaB (nuclear factor kappaB) and antioxidant Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2), as well as HDAC (histone deacetylase) in pro-inflammatory cytokine release by disruption of HDAC-RelA/p65 NF-kappaB complex.

Deacetylases and NF-kappaB in redox regulation of cigarette smoke-induced lung inflammation: epigenetics in pathogenesis of COPD.

Oxidative stress has been implicated in the pathogenesis of several inflammatory lung disorders including chronic obstructive pulmonary disease (COPD), due to its effect on pro-inflammatory gene transcription. Cigarette smoke-mediated oxidative stress activates NF-kappaB-dependent transcription of pro-inflammatory mediators either through activation of inhibitor kappaB-alpha kinase (IKK) and/or the enhanced recruitment and activation of transcriptional co-activators. Enhanced NF-kappaB-co-activator complex formation results in targeted increase in chromatin modifications, such as histone acetylation leading to inflammatory gene transcription. NF-kappaB-dependent gene expression, at least in part, is regulated by changes in deacetylases such as histone deacetylases (HDACs) and sirtuins. Cigarette smoke and oxidants also alter the activity of HDACs and sirtuins by post-translational modifications by protein carbonylation and nitration, and in doing so further induce gene expression of pro-inflammatory mediators by chromatin modifications. In addition, cigarette smoke/oxidants can reduce glucocorticoid sensitivity by attenuating HDAC2 activity and expression, which may account for the glucocorticoid insensitivity in patients with COPD. Understanding the mechanisms of NF-kappaB regulation, and the balance between histone acetylation and deacetylation may lead to the development of novel therapies based on the pharmacological manipulation of IKK and deacetylases in lung inflammation and injury.

Genetic Ablation of NADPH Oxidase Enhances Susceptibility to Cigarette Smoke-Induced Lung Inflammation and Emphysema in Mice

Cigarette smoke (CS) induces recruitment of inflammatory cells in the lungs leading to the generation of reactive oxygen species (ROS), which are involved in lung inflammation and injury. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a multimeric system that is responsible for ROS production in mammalian cells. We hypothesized that NADPH oxidase-derived ROS play an important role in lung inflammation and injury and that targeted ablation of components of NADPH oxidase (p47(phox) and gp91(phox)) would protect lungs against the detrimental effects of CS. To test this hypothesis, we exposed p47(phox-/-) and gp91(phox-/-) mice to CS and examined inflammatory response and injury in the lung. Surprisingly, although CS-induced ROS production was decreased in the lungs of p47(phox-/-) and gp91(phox-/-) mice compared with wild-type mice, the inflammatory response was significantly increased and was accompanied by development of distal airspace enlargement and alveolar destruction. This pathological abnormality was associated with enhanced activation of the TLR4-nuclear factor-kappaB pathway in response to CS exposure in p47(phox-/-) and gp91(phox-/-) mice. This phenomenon was confirmed by in vitro studies in which treatment of peritoneal macrophages with a nuclear factor-kappaB inhibitor reversed the CS-induced release of proinflammatory mediators. Thus, these data suggest that genetic ablation of components of NADPH oxidase enhances susceptibility to the proinflammatory effects of CS leading to airspace enlargement and alveolar damage.

IKKα Causes Chromatin Modification on Pro-Inflammatory Genes by Cigarette Smoke in Mouse Lung

Cigarette smoke (CS) induces abnormal and sustained lung inflammation; however, the molecular mechanism underlying sustained inflammation is not known. It is well known that activation of I kappaB kinase beta (IKK beta) leads to transient translocation of active NF-kappaB (RelA/p65-p50) in the nucleus and transcription of pro-inflammatory genes, whereas the role of IKK alpha in perpetuation of sustained inflammatory response is not known. We hypothesized that CS activates IKK alpha and causes histone acetylation on the promoters of pro-inflammatory genes, leading to sustained transcription of pro-inflammatory mediators in mouse lung in vivo and in human monocyte/macrophage cell line (MonoMac6) in vitro. CS exposure to C57BL/6J mice resulted in activation of IKK alpha, leading to phosphorylation of ser10 and acetylation of lys9 on histone H3 on the promoters of IL-6 and MIP-2 genes in mouse lung. The increased level of IKK alpha was associated with increased acetylation of lys310 RelA/p65 on pro-inflammatory gene promoters. The role of IKK alpha in CS-induced chromatin modification was confirmed by gain and loss of IKK alpha in MonoMac6 cells. Overexpression of IKK alpha was associated with augmentation of CS-induced pro-inflammatory effects, and phosphorylation of ser10 and acetylation of lys9 on histone H3, whereas transfection of IKK alpha dominant-negative mutants reduced CS-induced chromatin modification and pro-inflammatory cytokine release. Moreover, phosphorylation of ser276 and acetylation of lys310 of RelA/p65 was augmented in response to CS extract in MonoMac6 cells transfected with IKK alpha. Taken together, these data suggest that IKK alpha plays a key role in CS-induced pro-inflammatory gene transcription through phospho-acetylation of both RelA/p65 and histone H3.

VEGFR-2 inhibition augments cigarette smoke-induced oxidative stress and inflammatory responses leading to endothelial dysfunction

Vascular endothelial growth factor (VEGF) induces phosphorylation of VEGF receptor‐2 (VEGFR‐2) and activates the downstream signaling pathway resulting in endothelial cell migration, proliferation, and survival. Cigarette smoking is associated with abnormal vascular and endothelial function, leading to airspace enlargement. Herein, we investigated the mechanism of cigarette smoke (CS) ‐induced endothelial dysfunction by studying the VEGF‐VEGFR‐2 signaling in mouse lung and human endothelial cells. CS exposure caused oxidative stress, as shown by increased levels of 4‐hydroxy‐2‐nonenal‐adducts in mouse lung and reactive oxygen species generation in human lung microvascular endothelial cells (HMVEC‐Ls). Inhibition of VEGFR‐2 by a specific kinase inhibitor (NVP‐AAD777) enhanced the CS‐induced oxidative stress, causing augmented inflammatory cell influx and proinflammatory mediators release in mouse lung. The levels of endothelial nitric oxide synthase (eNOS) and phosphorylated (p) ‐eNOS in the lungs of mice exposed to CS and/or treated with VEGFR‐2 inhibitor were decreased. CS down‐regulated VEGFR‐2 expression, eNOS levels, and VEGF‐induced VEGFR‐2 phosphorylation in HMVEC‐Ls, resulting in impaired VEGF‐induced endothelial cell migration and angiogenesis. Overall, these data show that inhibition of VEGFR‐2 augmented CS‐induced oxidative stress and inflammatory responses leading to endothelial dysfunction. This explains the mechanism of endothelial dysfunction in smokers and has implications in understanding the pathogenesis of pulmonary and cardiovascular diseases.—Edirisinghe, I., Yang, S.‐E., Yao, H., Rajendrasozhan, S., Caito, S., Adenuga, D., Wong, C., Rahman, A., Phipps, R. P., Jin, Z.‐G., Rahman, I. VEGFR‐2 inhibition augments cigarette smoke‐induced oxidative stress and inflammatory responses leading to endothelial dysfunction. FASEB J. 22, 2297–2310 (2008)

Disruption of p21 Attenuates Lung Inflammation Induced by Cigarette Smoke, LPS, and fMLP in Mice

The cyclin-dependent kinase inhibitor p21(CIP1/WAF1/SDI1) (p21) is an important inhibitory checkpoint regulator of cell cycle progression in response to oxidative and genotoxic stresses. It is known that p21 potentiates inflammatory response and inhibits apoptosis and proliferation, leading to cellular senescence. However, the role of endogenous p21 in regulation of lung inflammatory and injurious responses by cigarette smoke (CS) or other pro-inflammatory stimuli is not known. We hypothesized that p21 is an important modifier of lung inflammation and injury, and genetic ablation of p21 will confer protection against CS and other pro-inflammatory stimuli (lipopolysacchride [LPS] and N-formyl-methionyl-leucyl-phenylalanine [fMLP])-mediated lung inflammation and injury. To test this hypothesis, p21-deficient (p21-/-) and wild-type mice were exposed to CS, LPS, or fMLP, and the lung oxidative stress and inflammatory responses as well as airspace enlargement were assessed. We found that targeted disruption of p21 attenuated CS-, LPS-, or fMLP-mediated lung inflammatory responses in mice. CS-mediated oxidative stress and fMLP-induced airspace enlargement were also decreased in lungs of p21-/- mice compared with wild-type mice. The mechanism underlying this finding was associated with decreased NF-kappaB activation, and reactive oxygen species generation by decreased phosphorylation of p47(phox) and down-modulating the activation of p21-activated kinase. Our data provide insight into the mechanism of pro-inflammatory effect of p21, and the loss of p21 protects against lung oxidative and inflammatory responses, and airspace enlargement in response to multiple pro-inflammatory stimuli. These data may have ramifications in CS-induced senescence in the pathogenesis of chronic obstructive pulmonary disease/emphysema.

Cigarette smoke-mediated oxidative stress induces apoptosis via the MAPKs/STAT1 pathway in mouse lung fibroblasts.

Cigarette smoking is the major aetiologic factor in chronic obstructive pulmonary disease (COPD). Lung fibroblasts are key participants in the maintenance of the extracellular matrix within the lung parenchyma. However, it still remains unknown how pulmonary fibroblasts are affected by cigarette smoking. Therefore, in this study, we isolated lung fibroblasts from mice and determined the apoptotic mechanism in response to cigarette smoke extract (CSE). When the lung fibroblasts were exposed to CSE, the generation of ROS was increased as shown by H2-DCFDA staining and Flow Cytometry. By immunocytochemistry, Ki67 expressing cells gradually decreased in a dose-dependent manner. The nitrite concentration in the supernatants increased, while the SOD activity and GSH recycling decreased in response to CSE. CSE increased the mRNA levels of TNF-α and COX-2, and the secretory proteins TNF-α and IL-6 increased as measured by ELISA. We next determined whether this inflammatory process is associated with the Bax/Bcl-2 apoptosis pathway. The Bax/Bcl-2 mRNA ratio increased, and cleaved caspase-3 protein was activated in the lung fibroblasts treated with CSE. Moreover, CSE induced the phosphorylation of STAT1 at Tyr701/Ser727 and increased the activation of ERK1/2, p38, and JNK in the MAPK pathway. Taken together, these data suggest that CSE-mediated inflammation alters the redox regulation via the MAPK-STAT1 pathway, leading to intrinsic apoptosis of lung fibroblasts.