Cigarette smoke-induced autophagy is regulated by SIRT1-PARP-1-dependent mechanism: Implication in pathogenesis of COPD

Autophagy is a fundamental cellular process that eliminates long-lived proteins and damaged organelles through lysosomal degradation pathway. Cigarette smoke (CS)-mediated oxidative stress induces cytotoxic responses in lung cells. However, the role of autophagy and its mechanism in CS-mediated cytotoxic responses is not known. We hypothesized that NAD⁺-dependent deacetylase, sirtuin 1 (SIRT1) plays an important role in regulating autophagy in response to CS. CS exposure resulted in induction of autophagy in lung epithelial cells, fibroblasts and macrophages. Pretreatment of cells with SIRT1 activator resveratrol attenuated CS-induced autophagy whereas SIRT1 inhibitor, sirtinol, augmented CS-induced autophagy. Elevated levels of autophagy were induced by CS in the lungs of SIRT1 deficient mice. Inhibition of poly(ADP-ribose)-polymerase-1 (PARP-1) attenuated CS-induced autophagy via SIRT1 activation. These data suggest that the SIRT1-PARP-1 axis plays a critical role in the regulation of CS-induced autophagy and have important implications in understanding the mechanisms of CS-induced cell death and senescence.     

Nicotinamide phosphoribosyltransferase/sirtuin 1 pathway is involved in human immunodeficiency virus type 1 Tat‐mediated long terminal repeat transactivation

Tat is a multifunctional transactivator encoded by human immunodeficiency virus type 1 (HIV‐1). Tat transactivating activity is controlled by nicotinamide adenine nucleotide⁺ (NAD⁺)‐dependent deacetylase sirtuin 1 (SIRT1). Nicotinamide phosphoribosyltransferase (Nampt) is a rate‐limiting enzyme in the conversion of nicotinamide into NAD⁺, which is crucial for SIRT1 activation. Thus, the effect of Nampt on Tat‐regulated SIRT activity was studied in Hela‐CD4‐β‐gal (MAGI) cells. We demonstrated that Tat caused NAD⁺ depletion and inhibited Nampt mRNA and protein expression in MAGI cells. Resveratrol reversed Tat‐induced NAD⁺ depletion and inhibition of Nampt mRNA and protein expression. Further investigation revealed that Tat‐induced inhibition of SIRT1 activity was potentiated in Nampt‐knockdown by Nampt siRNA compared to treatment with Tat alone. Nampt siRNA potentiated Tat‐induced HIV‐1 transactivation in MAGI cells. Altogether, these results indicate that Nampt is critical in the regulation of Tat‐induced inhibition of SIRT1 activity and long terminal repeat (LTR) transactivation. Nampt/SIRT1 pathway could be a novel therapeutic tool for the treatment of HIV‐1 infection. J. Cell. Biochem. 110: 1464–1470, 2010. © 2010 Wiley‐Liss, Inc.     

SIRT1 deficiency attenuates MPP+-induced apoptosis in dopaminergic cells

One of the functions mediated by sirtuin 1 (SIRT1), the NAD⁺-dependent protein deacetylase, has been suggested to be neuroprotective since resveratrol, a SIRT1 activator, inhibits 1-methyl-4-phenylpyridinium ion (MPP⁺)-induced cytotoxicity. In this study, we show that SIRT1 siRNA transfection blocks MPP⁺-induced apoptosis in SH-SY5Y cells. The ratio of potential pro-apoptotic BNIP2 to antiapoptotic BCL-xL was attenuated in SIRT1-deficient cells following MPP⁺ treatment. In addition, BNIP2 shRNA-transfected cells showed reduced cleavage of PARP-1, while BNIP2 overexpression intensified the cleavage in MPP⁺-treated SH-SY5Y cells, suggesting that BNIP2 participates in the MPP⁺-induced apoptosis. Overall, these data imply that SIRT1 may mediate MPP⁺-induced cytotoxicity, possibly through the regulation of BNIP2.     

Structural and Functional Analysis of Human SIRT1

SIRT1 is a NAD⁺-dependent deacetylase that plays important roles in many cellular processes. SIRT1 activity is uniquely controlled by a C-terminal regulatory segment (CTR). Here we present crystal structures of the catalytic domain of human SIRT1 in complex with the CTR in an open apo form and a closed conformation in complex with a cofactor and a pseudo-substrate peptide. The catalytic domain adopts the canonical sirtuin fold. The CTR forms a β hairpin structure that complements the β sheet of the NAD⁺-binding domain, covering an essentially invariant hydrophobic surface. The apo form adopts a distinct open conformation, in which the smaller subdomain of SIRT1 undergoes a rotation with respect to the larger NAD⁺-binding subdomain. A biochemical analysis identifies key residues in the active site, an inhibitory role for the CTR, and distinct structural features of the CTR that mediate binding and inhibition of the SIRT1 catalytic domain.     

A fluorometric assay of SIRT1 deacetylation activity through quantification of nicotinamide adenine dinucleotide

Sirtuins are nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylases that catalyze the deacetylation of proteins such as histones and p53. A sensitive and convenient fluorometric assay for evaluating the SIRT1 enzymatic activity was developed here. Specifically, the remaining NAD⁺ after the deacetylation was determined by converting NAD⁺ to a highly fluorescent cyclized α-adduct compound. By this assay, we found that nicotinamide, Cu²⁺, and Zn²⁺ antagonize the activity of SIRT1. Resveratrol stimulates the enzymatic activity specifically with 7-amino-4-methylcoumarin (AMC)-labeled acetylated peptide. Epigallocatechin galate (EGCG) inhibits SIRT1 activity with both AMC-labeled and unlabeled peptide. However, a combination of vitamin C with EGCG can reverse the inhibition of EGCG with the unlabeled peptide or stimulate the deacetylation of AMC-labeled peptide by SIRT1. The assay does not require any isotopic material and thus is biologically safe. It can be adapted to a 96-well microplate for high-throughput screening. Notably, the acetylated peptides with or without fluorescent labels may be used in the assay, which facilitates the substrate specificity study of SIRT1 activators or inhibitors in vitro.     

GABA protects pancreatic beta cells against apoptosis by increasing SIRT1 expression and activity

We have previously shown that GABA protects pancreatic islet cells against apoptosis and exerts anti-inflammatory effects. Notably, GABA inhibited the activation of NF-κB in both islet cells and lymphocytes. NF-κB activation is detrimental to beta cells by promoting apoptosis. However, the mechanisms by which GABA mediates these effects are unknown. Because the above-mentioned effects mimic the activity of sirtuin 1 (SIRT1) in beta cells, we investigated whether it is involved. SIRT1 is an NAD⁺-dependent deacetylase that enhances insulin secretion, and counteracts inflammatory signals in beta cells. We found that the incubation of a clonal beta-cell line (rat INS-1) with GABA increased the expression of SIRT1, as did GABA receptor agonists acting on either type A or B receptors. NAD⁺ (an essential cofactor of SIRT1) was also increased. GABA augmented SIRT1 enzymatic activity, which resulted in deacetylation of the p65 component of NF-κB, and this is known to interfere with the activation this pathway. GABA increased insulin production and reduced drug-induced apoptosis, and these actions were reversed by SIRT1 inhibitors. We examined whether SIRT1 is similarly induced in newly isolated human islet cells. Indeed, GABA increased both NAD⁺ and SIRT1 (but not sirtuins 2, 3 and 6). It protected human islet cells against spontaneous apoptosis in culture, and this was negated by a SIRT1 inhibitor. Thus, our findings suggest that major beneficial effects of GABA on beta cells are due to increased SIRT1 and NAD⁺, and point to a new pathway for diabetes therapy.     

Mechanism of Inhibition of the Human Sirtuin Enzyme SIRT3 by Nicotinamide: Computational and Experimental Studies

Sirtuins are key regulators of many cellular functions including cell growth, apoptosis, metabolism, and genetic control of age-related diseases. Sirtuins are themselves regulated by their cofactor nicotinamide adenine dinucleotide (NAD⁺) as well as their reaction product nicotinamide (NAM), the physiological concentrations of which vary during the process of aging. Nicotinamide inhibits sirtuins through the so-called base exchange pathway, wherein rebinding of the reaction product to the enzyme accelerates the reverse reaction. We investigated the mechanism of nicotinamide inhibition of human SIRT3, the major mitochondrial sirtuin deacetylase, in vitro and in silico using experimental kinetic analysis and Molecular Mechanics-Poisson Boltzmann/Generalized Born Surface Area (MM-PB(GB)SA) binding affinity calculations with molecular dynamics sampling. Through experimental kinetic studies, we demonstrate that NAM inhibition of SIRT3 involves apparent competition between the inhibitor and the enzyme cofactor NAD⁺, contrary to the traditional characterization of base exchange as noncompetitive inhibition. We report a model for base exchange inhibition that relates such kinetic properties to physicochemical properties, including the free energies of enzyme-ligand binding, and estimate the latter through the first reported computational binding affinity calculations for SIRT3:NAD⁺, SIRT3:NAM, and analogous complexes for Sir2. The computational results support our kinetic model, establishing foundations for quantitative modeling of NAD⁺/NAM regulation of mammalian sirtuins during aging and the computational design of sirtuin activators that operate through alleviation of base exchange inhibition.     

Tankyrase-1 overexpression reduces genotoxin-induced cell death by inhibiting PARP1

Poly(ADP-ribose) polymerases or PARPs are a family of NAD⁺-dependent enzymes that modify themselves and other substrate proteins with ADP-ribose polymers. The founding member PARP1 is localized predominantly in the nucleus and is activated by binding to DNA lesions. Excessive PARP1 activation following genotoxin treatment causes NAD⁺ depletion and cell death, whereas pharmacological PARP1 inhibition protects cells from genotoxicity. This study investigates whether cellular viability and NAD⁺ metabolism are regulated by tankyrase-1, a PARP member localized predominantly in the cytosol. Using a tetracycline-sensitive promoter to regulate tankyrase-1 expression in Madin–Darby canine kidney (MDCK) cells, we found that a 40-fold induction of tankyrase-1 (from 1500 to 60,000 copies per cell) lowers steady-state NAD⁺ levels but does not affect basal cellular viability. Moreover, the induction confers protection against the oxidative agent H₂O₂ and the alkylating agent MNNG, genotoxins that kill cells by activating PARP1. The cytoprotective effect of tankyrase-1 is not due to enhanced scavenging of oxidants or altered expression of Mcl-1, an anti-apoptotic molecule previously shown to be down-regulated by tankyrase-1 in CHO cells. Instead, tankyrase-1 appears to protect cells by preventing genotoxins from activating PARP1-mediated reactions such as PARP1 automodification and NAD⁺ consumption. Our findings therefore indicate a cytoprotective function of tankyrase-1 mediated through altered NAD⁺ homeostasis and inhibition of PARP1 function.     

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 H₂O₂ 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.     

NAD+ ameliorates endotoxin‐induced acute kidney injury in a sirtuin1–dependent manner via GSK‐3β/Nrf2 signalling pathway

Acute kidney injury (AKI) is a substantial worldwide public health concern with no specific and effective therapies in clinic. NAD⁺ is a pivotal determinant of cellular energy metabolism involved in the progression of AKI; however, its mechanism in kidney injury remains poorly understood. Sirtuin 1 (SIRT1) is an NAD⁺‐dependent deacetylase associated with renal protection and acute stress resistance. In this study, we have investigated the role of NAD⁺ in AKI and the potential mechanism(s) involved in its renoprotective effect. NAD⁺ was notably decreased and negatively correlated with kidney dysfunction in AKI, restoring NAD⁺ with NMN significantly ameliorates LPS‐induced oxidative stress and apoptosis and attenuates renal damage. We also found that the protection of NAD⁺ is associated with SIRT1 expressions and performs in a SIRT1‐dependent manner. Inhibition of SIRT1 blunted the protective effect of NAD⁺ and up‐regulated the activity of glycogen synthase kinase‐3β (GSK‐3β) that was concomitant with mitigated Nrf2 nuclear accumulation, thereby exacerbates AKI. These findings suggest that NAD⁺/SIRT1/GSK‐3β/Nrf2 axis is an important mechanism that can protect against AKI which might be a potential therapeutic target for the treatment of AKI.     

Nicotinamide treatment reduces the levels of histone H3K4 trimethylation in the promoter of the mper1 circadian clock gene and blocks the ability of dexamethasone to induce the acute response

Circadian rhythms, which measure time on a scale of 24 h, are generated by one of the most ubiquitous endogenous mechanisms, the circadian clock. SIRT1, a class III histone deacetylase, and PARP-1, a poly(ADP-ribose) polymerase, are two NAD⁺-dependent enzymes that have been shown to be involved in the regulation of the clock. Here we present evidence that the metabolite nicotinamide, an inhibitor of SIRT1, PARP-1 and mono(ADP-ribosyl) transferases, blocks the ability of dexamethasone to induce the acute response of the circadian clock gene, mper1, while it concomitantly reduces the levels of histone H3 trimethylation of lysine 4 (H3K4me3) in the mper1 promoter. Moreover, application of alternative inhibitors of SIRT1 and ADP-ribosylation did not lead to similar results. Therefore, inhibition of these enzymes does not seem to be the mode by which NAM exerts these effects. These results suggest the presence of a novel mechanism, not previously documented, by which NAM can alter gene expression levels via changes in the histone H3K4 trimethylation state.     

Cdk5 suppression blocks SIRT1 degradation via the ubiquitin-proteasome pathway in Parkinson's disease models

The NAD⁺-dependent protein deacetylase sirtuin 1 (SIRT1), a member of the sirtuin family, may have a neuroprotective effect in multiple neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD) and Amyotrophic lateral sclerosis (ALS). Many studies have suggested that overexpression-induced or resveratrol-treated activation of SIRT1 could significantly ameliorate several neurodegenerative diseases in mouse models. However, the type of SIRT1, protein expression levels and underlying mechanisms remain unclear, especially in PD. In this study, the results demonstrated that SIRT1 knockout markedly worsened the movement function in MPTP-lesioned animal model of PD. SIRT1 expression was found to be markedly decreased not only in environmental factor PD models, neurotoxin MPP⁺-treated primary culture neurons and MPTP-induced mice but also in genetic factor PD models, overexpressed α-synuclein-A30PA53T SH-SY5Y stable cell line and hm²α-SYN-39 transgenic mouse strain. Importantly, the degradation of SIRT1 during MPP⁺ treatment was mediated by the ubiquitin-proteasome pathway. Furthermore, the results indicated that cyclin-dependent kinase 5 (Cdk5) was also involved in the decrease of SIRT1 expression, which could be efficiently blocked by the inhibition of Cdk5. In conclusion, our findings revealed that the Cdk5-dependent ubiquitin-proteasome pathway mediated degradation of SIRT1 plays a vital role in the progression of PD.     

SCD1 regulates the AMPK/SIRT1 pathway and histone acetylation through changes in adenine nucleotide metabolism in skeletal muscle

Stearoyl-CoA desaturase (SCD) is a rate-limiting enzyme that catalyzes the synthesis of monounsaturated fatty acids. It plays an important role in regulating skeletal muscle metabolism. Lack of the SCD1 gene increases the rate of fatty acid β-oxidation through activation of the AMP-activated protein kinase (AMPK) pathway and the upregulation of genes that are related to fatty acid oxidation. The mechanism of AMPK activation under conditions of SCD1 deficiency has been unclear. In the present study, we found that the ablation/inhibition of SCD1 led to AMPK activation in skeletal muscle through an increase in AMP levels whereas muscle-specific SCD1 overexpression decreased both AMPK phosphorylation and the adenosine monophosphate/adenosine triphosphate (AMP/ATP) ratio. Changes in AMPK phosphorylation that were caused by SCD1 down- and upregulation affected NAD⁺ levels following changes in NAD⁺-dependent deacetylase sirtuin-1 (SIRT1) activity and histone 3 (H3K9) acetylation and methylation status. Moreover, mice with muscle-targeted overexpression of SCD1 were more susceptible to high-fat diet-induced lipid accumulation and the development of insulin resistance compared with wild-type mice. These data show that SCD1 is involved in nucleotide (ATP and NAD⁺) metabolism and suggest that the SCD1-dependent regulation of muscle steatosis and insulin sensitivity are mediated by cooperation between AMPK- and SIRT1-regulated pathways. Altogether, the present study reveals a novel mechanism that links SCD1 with the maintenance of metabolic homeostasis and insulin sensitivity in skeletal muscle.     

Biochemical effects of SIRT1 activators

SIRT1 is the closest mammalian homologue of enzymes that extend life in lower organisms. Its role in mammals is incompletely understood, but includes modulation of at least 34 distinct targets through its nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylase activity. Recent experiments using small molecule activators and genetically engineered mice have provided new insight into the role of this enzyme in mammalian biology and helped to highlight some of the potentially relevant targets. The most widely employed activator is resveratrol, a small polyphenol that improves insulin sensitivity and vascular function, boosts endurance, inhibits tumor formation, and ameliorates the early mortality associated with obesity in mice. Many of these effects are consistent with modulation of SIRT1 targets, such as PGC1α and NFκB, however, resveratrol can also activate AMPK, inhibit cyclooxygenases, and influence a variety of other enzymes. A novel activator, SRT1720, as well as various methods to manipulate NAD⁺ metabolism, are emerging as alternative methods to increase SIRT1 activity, and in many cases recapitulate effects of resveratrol. At present, further studies are needed to more directly test the role of SIRT1 in mediating beneficial effects of resveratrol, to evaluate other strategies for SIRT1 activation, and to confirm the specific targets of SIRT1 that are relevant in vivo. These efforts are especially important in light of the fact that SIRT1 activators are entering clinical trials in humans, and “nutraceutical” formulations containing resveratrol are already widely available.     

Inhibition of SIRT1 by a small molecule induces apoptosis in breast cancer cells

Overexpression of SIRT1, a NAD⁺-dependent class III histone deacetylases (HDACs), is implicated in many cancers and therefore could become a promising antitumor target. Here we demonstrate a small molecule SIRT1 inhibitor, ILS-JGB-1741(JGB1741) with potent inhibitory effects on the proliferation of human metastatic breast cancer cells, MDA-MB 231. The molecule has been designed using medicinal chemistry approach based on known SIRT1 inhibitor, sirtinol. The molecule showed a significant inhibition of SIRT1 activity compared to sirtinol. Studies on the antitumor effects of JGB on three different cancer cell lines, K562, HepG2 and MDA-MB 231 showed an IC₅₀ of 1, 10 and 0.5 μM, respectively. Further studies on MDA-MB 231 cells showed a dose-dependent increase in K9 and K382 acetylation of H3 and p53, respectively. Results also demonstrated that JGB1741-induced apoptosis is associated with increase in cytochrome c release, modulation in Bax/Bcl2 ratio and cleavage of PARP. Flowcytometric analysis showed increased percentage of apoptotic cells, decrease in mitochondrial membrane potential and increase in multicaspase activation. In conclusion, the present study indicates the potent apoptotic effects of JGB1741 in MDA-MB 231 cells.