Samuel Caito

Regulation of SIRT1 in cellular functions: role of polyphenols.

Sirtuin 1 (SIRT1) is known to deacetylate histones and non-histone proteins including transcription factors thereby regulating metabolism, stress resistance, cellular survival, cellular senescence/aging, inflammation-immune function, endothelial functions, and circadian rhythms. Naturally occurring dietary polyphenols, such as resveratrol, curcumin, quercetin, and catechins, have antioxidant and anti-inflammatory properties via modulating different pathways, such as NF-kappaB- and mitogen activated protein kinase-dependent signaling pathways. In addition, these polyphenols have also been shown to activate SIRT1 directly or indirectly in a variety of models. Therefore, activation of SIRT1 by polyphenols is beneficial for regulation of calorie restriction, oxidative stress, inflammation, cellular senescence, autophagy/apoptosis, autoimmunity, metabolism, adipogenesis, circadian rhythm, skeletal muscle function, mitochondria biogenesis and endothelial dysfunction. In this review, we describe the regulation of SIRT1 by dietary polyphenols in various cellular functions in response to environmental and pro-inflammatory stimuli.

SIRT1 is a redox-sensitive deacetylase that is post-translationally modified by oxidants and carbonyl stress

Sirtuinl (SIRT1) deacetylase levels are decreased in chronic inflammatory conditions and aging where oxidative stress occurs. We determined the mechanism of SIRT1 redox post‐translational modifications leading to its degradation. Human lung epithelial cells exposed to hydrogen peroxide (150–250 µM), aldehyde‐acrolein (10–30 µM), and cigarette smoke extract (CSE;0.1–1.5%) in the presence of intracellular glutathione‐modulating agents at 1–24 h, and oxidative post‐translational modifications were assayed in cells, as well as in lungs of mice lacking and overexpressing glutaredoxin‐1 (Glrx1), and wild‐type (WT) mice in response to cigarette smoke (CS). CSE and aldehydes dose and time dependently decreased SIRT1 protein levels, with EC50 of 1% for CSE and 30 µM for acrolein at 6 h, and >80% inhibition at 24 h with CSE, which was regulated by modulation of intracellular thiol status of the cells. CS decreased the lung levels of SIRT1 in WT mice, which was enhanced by deficiency of Glrx1 and prevented by overexpression of Glrx1. Oxidants, aldehydes, and CS induced carbonyl modifications on SIRT1 on cysteine residues concomitant with decreased SIRT1 activity. Proteomics studies revealed alkylation of cysteine residue on SIRT1. Our data suggest that oxidants/aldehydes covalently modify SIRT1, decreasing enzymatic activity and marking the protein for proteasomal degradation, which has implications in inflammatory conditions.—Caito, S., Rajendrasozhan, S., Cook, S., Chung, S., Yao, H., Friedman, A. E., Brookes, P. S., Rahman, I. SIRT1 is a redox‐sensitive deacetylase that is post‐translationally modified by oxidants and carbonyl stress. FASEB J. 24, 3145–3159 (2010). www.fasebj.org

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 SIRT1 in inflammation and cellular senescence.

Sirtuin 1 (SIRT1) regulates inflammation, aging (life span and health span), calorie restriction/energetics, mitochondrial biogenesis, stress resistance, cellular senescence, endothelial functions, apoptosis/autophagy, and circadian rhythms through deacetylation of transcription factors and histones. SIRT1 level and activity are decreased in chronic inflammatory conditions and aging, in which oxidative stress occurs. SIRT1 is regulated by a NAD(+)-dependent DNA repair enzyme, poly(ADP-ribose) polymerase-1 (PARP1), and subsequent NAD(+) depletion by oxidative stress may have consequent effects on inflammatory and stress responses as well as cellular senescence. SIRT1 has been shown to undergo covalent oxidative modifications by cigarette smoke-derived oxidants/aldehydes, leading to posttranslational modifications, inactivation, and protein degradation. Furthermore, oxidant/carbonyl stress-mediated reduction of SIRT1 leads to the loss of its control on acetylation of target proteins including p53, RelA/p65, and FOXO3, thereby enhancing the inflammatory, prosenescent, and apoptotic responses, as well as endothelial dysfunction. In this review, the mechanisms of cigarette smoke/oxidant-mediated redox posttranslational modifications of SIRT1 and its roles in PARP1 and NF-κB activation, and FOXO3 and eNOS regulation, as well as chromatin remodeling/histone modifications during inflammaging, are discussed. Furthermore, we have also discussed various novel ways to activate SIRT1 either directly or indirectly, which may have therapeutic potential in attenuating inflammation and premature senescence involved in chronic lung diseases.

SIRT1 regulates oxidant- and cigarette smoke-induced eNOS acetylation in endothelial cells: role of resveratrol.

Endothelial nitric oxide synthase (eNOS) plays a crucial role in endothelial cell functions. SIRT1, a NAD(+)-dependent deacetylase, is shown to regulate endothelial function and hence any alteration in endothelial SIRT1 will affect normal vascular physiology. Cigarette smoke (CS)-mediated oxidative stress is implicated in endothelial dysfunction. However, the role of SIRT1 in regulation of eNOS by CS and oxidants are not known. We hypothesized that CS-mediated oxidative stress downregulates SIRT1 leading to acetylation of eNOS which results in reduced nitric oxide (NO)-mediated signaling and endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) exposed to cigarette smoke extract (CSE) and H(2)O(2) showed decreased SIRT1 levels, activity, but increased phosphorylation concomitant with increased eNOS acetylation. Pre-treatment of endothelial cells with resveratrol significantly attenuated the CSE- and oxidant-mediated SIRT1 levels and eNOS acetylation. These findings suggest that CS- and oxidant-mediated reduction of SIRT1 is associated with acetylation of eNOS which have implications in endothelial dysfunction.

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.

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.