Nuclear Sirtuins and Inflammatory Signaling Pathways
Abstract
The regulation of chronic inflammation has received considerable research attention in recent years because of its contribution to the pathogenesis of chronic diseases such as arthritis, diabetes, metabolic syndrome, and obesity. Thus, strategies that inhibit the inflammatory state may be beneficial in improving the pathophysiology of several inflammation-related disorders. Sirtuins are a family of histone deacetylases that contain seven enzymatic activities in mammals (SIRT1–SIRT7) and function to suppress gene transcription by epigenetic mechanisms. Nuclear sirtuins (SIRT1, 2, 6, and 7) in particular may play an important role in the regulation of inflammatory responses. In this review, we assessed the roles of nuclear sirtuins in inflammatory reactions: SIRT1 has been shown to suppress NF-κB activity, the master regulator of cellular inflammatory response, decrease COX-2 and iNOS production, and increase antioxidant gene expression that suppresses inflammation. SIRT2 activity includes the deacetylation of the p65 subunit of NF-κB and RIP-1, while SIRT6 has been shown to interact with p65/RelA bound to the NF-κB promoter region and repress transcriptional activity. Furthermore, recent studies have shown that the absence of SIRT7 produces an increase in inflammation, illustrating that SIRT7 also functions to decrease inflammation. Given their significant roles in the regulation of chronic inflammation, nuclear sirtuins represent potential therapeutic targets in the control of chronic inflammatory diseases.
Introduction
Inflammation is a biological response involved in the maintenance of homeostasis and is currently receiving attention for its potential role in chronic diseases such as arthritis, diabetes, metabolic syndrome, and obesity. Obesity is linked to a chronic inflammatory response characterized by activation of proinflammatory signaling pathways and abnormal production of cytokines, inducing the release and expression of biological markers of inflammation. The inflammatory changes associated with obesity can be found in both immune and non-immune cells and include the abnormal production of cytokines and chemokines that may further attract and activate immune cells. Hence, a decreased inflammatory state may have beneficial effects on the pathophysiology of obesity and metabolic syndrome.
In this regard, sirtuins may play an important role in reducing inflammation. The family of sirtuins contains seven enzymes in mammals (SIRT1–SIRT7) that share a conserved core catalytic domain but differ in their cellular localization (mitochondrion, cytoplasm, or nucleus) and tissue distribution. Mitochondrial sirtuins (mainly SIRT3, 4, and 5) are well described in the literature to coordinate metabolic pathways involved in stress responses, aging, cardiometabolic diseases, hepatic metabolism, and others. This class of sirtuins seems to actively participate in the mechanisms by which mitochondrial functions are adapted to environmental requirements and metabolic demands. The sirtuins’ role in metabolic pathway signaling is mainly due to their deacetylation capacity, which controls the activities of multiple proteins, consequently affecting enzymatic and protein cascades. The nuclear sirtuins (SIRT1, 2, 6, and 7), on the other hand, among other roles, are known to play an important role in decreasing inflammation. In this context, the present review aimed to assess the role of nuclear sirtuins (SIRT1, 2, 6, and 7) in inflammation.
Molecular Aspects of Inflammation
The inflammatory process presents a very important characteristic, which is endothelial dysfunction that includes the recruitment of adhesion molecules, proinflammatory cytokines, and matrix-degrading enzymes. Acute inflammation starts abruptly and generally resolves rapidly, usually involving mild or strong stimulation of toll-like receptor 4 (TLR4) that, according to Xiao et al., reprograms around 80% of the protein-encoding genome of human blood leukocytes. This shows that the gene programming observed after an acute inflammatory response occurs, emphasizing an epigenetic determination of the inflammation reprogramming. Recognition of TLRs leads to nuclear factor kappa B (NF-κB) translocation to the nucleus, and as a transcription factor, it activates expression of inflammatory molecules such as TNF-α, IL-6, and IL-1β, or by directly activating these molecules without NF-κB participation.
The proinflammatory phase induces gene products such as tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β) and then represses genes that ignited the proinflammatory phase and reprograms many other sets of genes to support the adaptation phase, which lasts much longer than the initiating proinflammatory-phase response before resolving to homeostasis. The signals responsible for the initiation of the acute systemic inflammatory process include subunit p65 (RelA), protein methylases, stress kinases (extracellular signal-regulated kinases (ERK), p38 kinase (p38), c-jun N-terminal kinase (JNK)), and acetyl transferases. The activation of these molecules, among other inflammatory roles via activation of AP-1, leads to insulin resistance through stimulation of the insulin receptor substrate (IRS).
Nuclear factor kappa B (NF-κB) is a master regulator of immune response and inflammation. Activation of NF-κB influences the expression of proinflammatory genes, such as growth factors, chemokines, and cytokines. Deacetylation of the NF-κB subunit p65 leads to a decrease in NF-κB transcription activity by reducing production of cytokines and anti-apoptotic genes. NF-κB is correlated with the synthesis of various cytokines, such as TNF-α, IL-1β, interleukin-6 (IL-6), and interleukin-8 (IL-8), which further amplify the inflammatory status by retro feedback, increasing apoptosis, proliferation, and insulin resistance.
IL-1β is a proinflammatory cytokine that plays an important role in various cellular responses such as apoptosis, inflammation, and proliferation. IL-1β also activates kinases such as mitogen-activated kinase-like protein (MAPKs), serine/threonine kinase (Akt), and SRC proto-oncogene, non-receptor tyrosine kinase (Src), which also lead to increased apoptosis and inflammation.
Another important molecule involved in inflammatory processes is the tumor suppressor p53 kinase (p53), which is stimulated by various stress signals and plays an important role in modulating the cell cycle, apoptosis, and DNA repair. The activation of p53 occurs post-translation, through acetylation and phosphorylation. Acetylation of the lysine residues in p53 increases its binding activity, DNA co-activator recruitment, and cellular senescence.
Macrophage activation and infiltration into resident tissues mediate local inflammation and are a hallmark of metabolic syndrome. Macrophages screen the bloodstream and tissue for signs of damage or infection. Under stimulation, macrophages infiltrate the tissue, perpetuating local inflammation and contributing to the development of insulin resistance and metabolic derangements.
In the context of obesity, the main activators of inflammatory signaling pathways are lipids, saturated fatty acids, and cytokines, which further activate downstream pro-inflammatory molecules and transcription factors. These molecules and pathways are associated with the maintenance of the inflammatory state found in patients with obesity and/or metabolic syndrome. Understanding potential inflammation activators is needed, as they may become therapeutic targets to aid in the treatment of obesity and metabolic syndrome. In this perspective, the sirtuins seem to be important target molecules.
Sirtuins
Sirtuins are proteins classified as histone deacetylases (HDACs), which are molecules that suppress gene transcription by their ability to remove acetyl moieties from the ε-acetamido group on lysine residues within histones. The amino groups of conserved lysine residues present in histone tails can be subjected to reversible acetylation and deacetylation, which plays an important role in regulating gene transcription. These modifications also play an important role in the regulation of DNA damage, genomic instability, proinflammatory gene transcription, and premature aging. This process works through histone acetylation and methylation after DNA damage, which may alter the chromatin status between euchromatin and heterochromatin to recruit DNA repair factors and cofactors to damaged sites.
HDACs also deacetylate non-histone proteins, such as NF-κB, and have the ability to regulate NF-κB-dependent pro-inflammatory gene transcription. Several isoforms of HDACs are identified and grouped into classes. Among them are the sirtuins, HDACs class III members, which use NAD+ as a co-factor. The first member of the sirtuin family was identified in Saccharomyces cerevisiae, titled silent information regulator-2 (Sir2). Sir2 was later found in Drosophila melanogaster and Caenorhabditis elegans, being involved in the regulation of various pathways, including those involved in aging and longevity. In mammals, the first gene of this family was identified as sirtuin 1 (SIRT1). Six other sirtuin genes were subsequently identified in mammals, resulting in seven genes (SIRT1 to SIRT7). These are regulatory genes that influence and respond to various epigenetic factors. These enzymes play an important role in the body’s response to several kinds of stress and toxicity. Sirtuins regulate chronological life expectancy of organisms such as yeast and bacteria and affect biological aspects involved in metabolic diseases and aging in mammals.
The mammalian sirtuin family has seven members, from SIRT1 to SIRT7. Previous studies have shown different subcellular localizations for each sirtuin, with SIRT3, SIRT4, and SIRT5 being mitochondrial proteins, SIRT6 and SIRT7 nuclear proteins, whereas SIRT1 and SIRT2 are both found in the nucleus and the cytoplasm. It is important to highlight that an important role of nuclear sirtuins (SIRT1, 2, 6, and 7) is the control of inflammation.
Nuclear Sirtuins and Inflammation
4.1. SIRT1
SIRT1 is a protein/histone deacetylase with anti-apoptotic, anti-aging, and anti-inflammatory properties. SIRT1 deacetylates histones (H3 and H4) and non-histone proteins, condensing chromatin and silencing gene transcription. SIRT1 acts on co-activators, transcription factors, and signaling molecules such as p53, forkhead box O1 (FOXO), and RelA/p65. It is involved in the improvement of mitochondriogenesis through peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) deacetylation, increased oxidative stress survival response via FOXO1/4, altered apoptosis, altered proliferation mediated by p53 deacetylation, and decreased inflammation via NF-κB suppression.
SIRT1 has been identified as an important regulator of the immune system, with studies showing that SIRT1 can repress inflammation in multiple tissues and macrophages. Artificial overexpression of SIRT1 leads to suppression of the inflammatory response, whereas deletion of SIRT1 results in increased local inflammation. SIRT1 activity in macrophages directly regulates immune response and plays an important therapeutic role in the treatment of chronic inflammatory diseases. The beneficial effect of SIRT1 on metabolic disorders is due in part to its ability to suppress the activity of NF-κB in macrophages. SIRT1 suppresses NF-κB transcription by deacetylating the RelA/p65 subunit of NF-κB at Lys310 and inhibiting NF-κB signaling. SIRT1 interacts with p65, leading to deacetylation at lysine 310, decreasing NF-κB associated transcription. SIRT1 further inhibits NF-κB by interacting with transducin-like enhancer of split 1 (TLE1), a co-repressor of NF-κB. Moreover, SIRT1 deacetylates and inactivates p300/CREB binding protein (p300/CBP), co-activators of NF-κB-dependent inflammatory gene expression.
NF-κB-dependent gene activation is strongly modulated by poly (ADP-ribose) polymerase-1 (PARP-1). SIRT1 interacts with and deacetylates PARP-1, resulting in reduced PARP-1 activity. SIRT1 is capable of suppressing the activity of the PARP-1 gene promoter, leading to a reduction in PARP-1 synthesis. As PARP-1 is required for specific transcriptional activation of NF-κB, SIRT1 could suppress NF-κB signaling by reducing PARP-1.
Moderate overexpression of SIRT1 in mice leads to reduced NF-κB activity, but knockdown of SIRT1 in macrophages increases LPS-stimulated TNF-α secretion. SIRT1-mediated deacetylation of NF-κB inhibits iNOS and cytokine-mediated beta-cell damage in isolated rat islets. SIRT1 also deacetylates and suppresses the transcription activity of activator protein-1 (AP-1), leading to down-regulation of COX-2 gene expression, suggesting the existence of multiple targets of SIRT1 in the regulation of inflammation.
SIRT1 inhibits AP-1 by deacetylating c-Jun and plays a major role in clonal T-cell anergy, a mechanism important for the suppression of self-reactive T-cells. The deacetylation of c-Fos by SIRT1 also contributes to the inhibition of AP-1 and reduces COX-2 expression in macrophages. SIRT1 protects against oxidative stress by up-regulating FOXO3-dependent antioxidant genes (catalase and MnSOD). Induction of ROS-detoxifying enzymes by SIRT1 also suppresses inflammation.
The activity and expression of SIRT1 are also under control of systemic inflammation. Interferon gamma (IFNγ), a pro-inflammatory cytokine, represses the transcription of SIRT1 through class II transactivator. TNFα induces cathepsin B-mediated cleavage and inactivation of SIRT1 in chondrocytes. Mice fed a high-fat diet show induced cleavage of SIRT1 protein in adipose tissue through inflammation-activated caspase-1.
Knockdown of SIRT1 by antisense oligonucleotide (ASO) leads to an increase in apoptosis and of BCL2-associated X protein (Bax), suggesting that down-regulation of SIRT1 is involved in Bax expression and NF-κB activation by I kappa B kinase (IKK) stimulation. This leads to increased phosphorylation of IκBα, activation, and release of NF-κB. SIRT1 inhibition also stimulates p65 acetylation and phosphorylation, correlating with up-regulation of NF-κB-regulated gene products involved in cell proliferation, inflammation, and apoptosis. SIRT1 activators reverse the IL-1β or NA-induced up-regulation of various gene products that mediate matrix degradation, inflammation, and apoptosis, all regulated by NF-κB, but not the ASO against SIRT1. Therefore, SIRT1 and inflammatory signals reciprocally interact at various levels, with SIRT1 being considered a link between inflammation, nutrient, and metabolic dysfunction. Activation of SIRT1 might act as a novel immunomodulatory approach for the treatment of chronic inflammatory diseases via modulation of the NF-κB pathway.
Natural compounds such as resveratrol (found in grapes and other plant species) and caloric restriction have been described as potent SIRT1 activators. These activators induce beneficial effects such as increased thermogenesis and decreased fat accumulation, both of which are consequently associated with downregulation of inflammatory signaling pathways. These findings suggest that SIRT1 is a potential target against inflammation and other disorders, and clinical trials regarding SIRT1 behavior following treatment with such compounds or calorie restriction should be performed.
4.2. SIRT2
Sirtuin 2 (SIRT2), a member of the sirtuin family of proteins, is associated with numerous processes, such as carcinogenesis, infection, cell survival, DNA damage, and cell cycle regulation, but the biological function of SIRT2 in the inflammatory process remains unclear. Studies show that in Sirt2-deficient mice, SIRT2 deletion promotes inflammatory responses by increasing NF-κB acetylation and by reducing the M2-associated anti-inflammatory pathway, showing a protective role for SIRT2 in the development of inflammatory processes.
SIRT2 deacetylates receptor-interacting protein 1 (RIP-1) and the p65 subunit of NF-κB. Cells from SIRT2 knockout mice show hyper-acetylation of p65 concomitantly with increased expression of NF-κB-dependent genes induced by TNF. SIRT2 deacetylates the p65 subunit of NF-κB at lysine 310, resulting in reduced expression of IL-1β, IL-6, matrix metalloproteinase 9 (MMP-9), MMP-13, and monocyte chemoattractant protein 1 (MCP-1). RIP-1 is also involved in inflammatory signaling pathways and was found to be a SIRT2 substrate. SIRT2 deacetylates RIP-1 and stabilizes the RIP-1–RIP-3 protein complex required for TNF-induced cell death in fibroblasts.
Inflammatory factors (LPS, collagen, and TNF-α) reduce the levels of SIRT2, which may account for the decrease in SIRT2 anti-inflammatory functions. SIRT2 is regulated at both transcriptional and post-translational levels depending upon the conditions. SIRT2 is expressed in microglia and LPS-induced inflammation reduces SIRT2 levels in the brain. In microglial cells with SIRT2 knock-down, increased production of IL-6, CD40, CD80, ROS, and NO upon LPS and TNF stimulation was observed. SIRT2 targets NF-κB in microglial cells through p65K310 deacetylation, which seems to prevent activation of microglia upon pro-inflammatory stimulation.
Deficiency of SIRT2 ameliorates NO expression, iNOS, and ROS production by the suppression of LPS-induced activation of NF-κB in macrophages. SIRT2 inhibits inflammation and oxidative stress by reducing the levels of expression of cytokines and ROS, showing that SIRT2 may be a possible therapeutic agent for various disorders, such as skin inflammation. SIRT2 activators have been studied, but little progress has been made. Studies aimed to identify new mechanisms and compounds able to increase SIRT2 expression are urgently needed, as this protein seems to have potential to induce beneficial effects on the whole organism.
4.3. SIRT6
Recent studies have revealed that SIRT6 possesses anti-inflammatory properties. SIRT6 is a histone H3 lysine 9 (H3K9) NAD-dependent deacetylase which modulates telomeric chromatin and gene expression by transferring ADP-ribose from NAD to itself and histones. SIRT6 can also deacetylate H3K56. SIRT6-deficient mice display a degenerative phenotype, leading to death at four to five weeks of age. These mice also show increased expression of NF-κB-dependent genes in a number of tissues.
The anti-inflammatory effects of SIRT6 are a result of its activity as a histone deacetylase, which represses target gene transcription such as NF-κB. SIRT6 modulates NF-κB-dependent genes by its interaction with the RelA subunit; recruitment of SIRT6 to chromatin at the promoters of RelA represses NF-κB target gene expression by deacetylating histone H3K9. SIRT6 and p65/RelA interact at the site of the NF-κB promoter region, deacetylating H3K9 to decrease promoter occupancy by the p65 subunit of NF-κB, while SIRT1 and SIRT2 directly deacetylate p65 and inhibit its transcriptional activity.
In vitro experiments demonstrated that SIRT6 overexpression suppressed NF-κB target gene expression induced by TNF-α. Blocking the NF-κB pathway by SIRT6 in rheumatoid joints reduces both the inflammatory response and tissue destruction. SIRT6 knockdown in human umbilical vein endothelial cells (HUVECs) showed increased expression of proinflammatory cytokines (IL-1β, IL-6, IL-8), multimerin 1 (ECM) remodeling enzymes (MMP-2, MMP-9, and plasminogen activator inhibitor type 1 (PAI-1)), intercellular adhesion molecule 1 (ICAM-1), proangiogenic growth factors VEGF and FGF-2, and the COX-prostaglandin system. SIRT6 knockdown increased the expression of NF-κB, whereas overexpression of SIRT6 decreased NF-κB transcriptional activity. These results show that ablation of SIRT6 in endothelial cells is associated with upregulation of genes involved in inflammation. Thus, SIRT6 may be a potential pharmacological target for inflammatory vascular diseases. Studies aimed to identify SIRT6 activators are needed, especially if the compounds are natural derivatives, as they usually present high efficacy and low toxicity.
4.4. SIRT7
Until recently, SIRT7 was the least studied of the seven mammalian sirtuins. SIRT7 is a vital regulator of rRNA and protein synthesis for maintenance of normal cellular homeostasis. Lysine 18 of histone H3 (H3K18), polymerase (RNA) I polypeptide E, 53 kDa (PAF53), nucleophosmin (NPM1), p53, and GA repeat binding protein, beta 1 (GABP-β1) are targets for the deacetylase activity of SIRT7, which is a key mediator of many cellular activities.
In SIRT7 knockout mice, SIRT7 disruption predisposes the mice to heart hypertrophy together with increasing cardiac inflammation. These mice show increased infiltration of immune cells, with higher levels of pro- and anti-inflammatory cytokine production. These results suggest that SIRT7 is also involved in inflammation. Information regarding the possible involvement of SIRT7 in inflammatory signaling pathways and mechanisms is still lacking in the literature, probably due to the late onset of SIRT7 studies. However, studies connecting SIRT7 with tumorigenesis, stress, and apoptosis may serve as a link for its potential involvement in inflammation, as these are intrinsically connected pathways.
Concluding Remarks
Given the importance of inflammation in chronic diseases, nuclear sirtuins emerge as possible adjuvants against inflammation. They act at the molecular level through the deacetylation of various inflammatory cytokines and transcription factors, decreasing inflammation. The NF-κB pathway appears to be a consensus among the sirtuins’ inflammatory targets. This study may contribute to a better understanding of potential pharmacological NF-κΒ activator 1 targets against inflammatory processes.