Epigenetically rewiring metabolic genes via SIRT6 orchestrates MSC fate determination.

SIRT6 owns versatile types of enzymatic activities as a multitasking protein, including ribosyltransferase and deacetylase. To investigate the epigenetic regulations of SIRT6 on MSC fate determination via histone deacetylation, we used allosteric small molecules specifically controlling its histone 3 deacetylation activities. Results showed that enhanced deacetylation of SIRT6 promoted the ossific lineage commitment of MSC and finally achieved anabolic effects on hard tissues. Mechanistically, H3K9ac and H3K56ac, governed by SIRT6, in MSC orchestrated the transcriptions of crucial metabolic genes, mediating MSC fate determination. Most importantly, our data evidenced that modulating the epigenetic regulations of SIRT6, specifically via enhancing its deacetylation of H3K9ac and H3K56ac, was a promising choice to treat bone loss diseases and promote dentin regeneration. In this study, we revealed the specific roles of SIRT6's histone modification in MSC fate determination. These findings endow us with insights on SIRT6 and the promising therapeutic choices through SIRT6's epigenetic functions for hard tissues regeneration.

Sirtuin 6 ameliorates bleomycin-induced pulmonary fibrosis via activation of lipid catabolism.

Pulmonary fibrosis is a chronic and serious interstitial lung disease with little effective therapies currently. Our incomplete understanding of its pathogenesis remains obstacles in therapeutic developments. Sirtuin 6 (SIRT6) has been shown to mitigate multiple organic fibrosis. However, the involvement of SIRT6-mediated metabolic regulation in pulmonary fibrosis remains unclear. Here, we demonstrated that SIRT6 was predominantly expressed in alveolar epithelial cells in human lung tissues by using a single-cell sequencing database. We showed that SIRT6 protected against bleomycin-induced injury of alveolar epithelial cells in vitro and pulmonary fibrosis of mice in vivo. High-throughput sequencing revealed enriched lipid catabolism in Sirt6 overexpressed lung tissues. Mechanismly, SIRT6 ameliorates bleomycin-induced ectopic lipotoxicity by enhancing lipid degradation, thereby increasing the energy supply and reducing the levels of lipid peroxides. Furthermore, we found that peroxisome proliferator-activated receptor α (PPARα) was essential for SIRT6-mediated lipid catabolism, anti-inflammatory responses, and antifibrotic signaling. Our data suggest that targeting SIRT6-PPARα-mediated lipid catabolism could be a potential therapeutic strategy for diseases complicated with pulmonary fibrosis.

The transcription factor GATA6 accelerates vascular smooth muscle cell senescence-related arterial calcification by counteracting the role of anti-aging factor SIRT6 and impeding DNA damage repair.

Arterial calcification is a hallmark of vascular pathology in the elderly and in individuals with chronic kidney disease (CKD). Vascular smooth muscle cells (VSMCs), after attaining a senescent phenotype, are implicated in the calcifying process. However, the underlying mechanism remains to be elucidated. Here, we reveal an aberrant upregulation of transcriptional factor GATA6 in the calcified aortas of humans, mice with CKD and mice subjected to vitamin D3 injection. Knockdown of GATA6, via recombinant adeno-associated virus carrying GATA6 shRNA, inhibited the development of arterial calcification in mice with CKD. Further gain- and loss-of function experiments in vitro verified the contribution of GATA6 in osteogenic differentiation of VSMCs. Samples of human aorta exhibited a positive relationship between age and GATA6 expression and GATA6 was also elevated in the aortas of old as compared to young mice. Calcified aortas displayed senescent features with VSMCs undergoing premature senescence, blunted by GATA6 downregulation. Notably, abnormal induction of GATA6 in senescent and calcified aortas was rescued in Sirtuin 6 (SIRT6)-transgenic mice, a well-established longevity mouse model. Suppression of GATA6 accounted for the favorable effect of SIRT6 on VSMCs senescence prevention. Mechanistically, SIRT6 inhibited the transcription of GATA6 by deacetylation and increased degradation of transcription factor Nkx2.5. Moreover, GATA6 was induced by DNA damage stress during arterial calcification and subsequently impeded the Ataxia-telangiectasia mutated (ATM)-mediated DNA damage repair process, leading to accelerated VSMCs senescence and osteogenic differentiation. Thus, GATA6 is a novel regulator in VSMCs senescence. Our findings provide novel insight in arterial calcification and a potential new target for intervention.

Activity of DNA Repair Systems in the Cells of Long-Lived Rodents and Bats.

Damages of various origin accumulated in the genomic DNA can lead to the breach of genome stability, and are considered to be one of the main factors involved in cellular senescence. DNA repair systems in mammalian cells ensure effective damage removal and repair of the genome structure, therefore, activity of these systems is expected to be correlated with high maximum lifespan observed in the long-lived mammals. This review discusses current results of the studies focused on determination of the DNA repair system activity and investigation of the properties of its key regulatory proteins in the cells of long-lived rodents and bats. Based on the works discussed in the review, it could be concluded that the long-lived rodents and bats in general demonstrate high efficiency in functioning and regulation of DNA repair systems. Nevertheless, a number of questions around the study of DNA repair in the cells of long-lived rodents and bats remain poorly understood, answers to which could open up new avenues for further research.

Pubertal exposure to Microcystin-LR arrests spermatogonia proliferation by inducing DSB and inhibiting SIRT6 dependent DNA repair in vivo and in vitro.

The reproduction toxicity of pubertal exposure to Microcystin-LR (MC-LR) and the underlying mechanism needs to be further investigated. In the current study, pubertal male ICR mice were intraperitoneally injected with 2 µg/kg MC-LR for four weeks. Pubertal exposure to MC-LR decreased epididymal sperm concentration and blocked spermatogonia proliferation. In-vitro studies found MC-LR inhibited cell proliferation of GC-1 cells and arrested cell cycle in G2/M phase. Mechanistically, MC-LR exposure evoked excessive reactive oxygen species (ROS) and induced DNA double-strand break in GC-1 cells. Besides, MC-LR inhibited DNA repair by reducing PolyADP-ribosylation (PARylation) activity of PARP1. Further study found MC-LR caused proteasomal degradation of SIRT6, a monoADP-ribosylation enzyme which is essential for PARP1 PARylation activity, due to destruction of SIRT6-USP10 interaction. Additionally, MG132 pretreatment alleviated MC-LR-induced SIRT6 degradation and promoted DNA repair, leading to the restoration of cell proliferation inhibition. Correspondingly, N-Acetylcysteine (NAC) pre-treatment mitigated the disturbed SIRT6-USP10 interaction and SIRT6 degradation, causing recovered DNA repair and subsequently restoration of cell proliferation inhibition in MC-LR treated GC-1 cells. Together, pubertal exposure to MC-LR induced spermatogonia cell cycle arrest and sperm count reduction by oxidative DNA damage and simultaneous SIRT6-mediated DNA repair failing. This study reports the effect of pubertal exposure to MC-LR on spermatogenesis and complex mechanism how MC-LR induces spermatogonia cell proliferation inhibition.

Sirtuin 6 protects against hepatic fibrogenesis by suppressing the YAP and TAZ function.

Hepatic fibrosis occurs in response to prolonged tissue injury in the liver, which results in abnormal accumulation of extracellular matrix. Hepatic stellate cells (HSCs) have been suggested to play a major role in liver fibrosis. However, the molecular mechanisms remain incompletely understood. Sirtuin 6 (SIRT6), an NAD+ -dependent deacetylase, has been previously implicated in the regulation of the transforming growth factor ß (TGFß)-SMAD3 pathway that plays a significant role in liver fibrosis. In this work, we aimed to identify other important players during hepatic fibrogenesis, which are modulated by SIRT6. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ or WWTR1), key players in the Hippo pathway, have been implicated in the promotion of hepatic fibrosis. Our data show that HSC-specific Sirt6 knockout mice are more susceptible to high-fat-cholesterol-cholate diet-induced hepatic fibrosis than their wildtype counterparts. Our signaling analyses suggest that in addition to the TGFß-SMAD3 pathway, YAP and TAZ are also highly activated in the SIRT6-deficient HSCs. As it is not clear how SIRT6 might regulate YAP and TAZ, we have decided to elucidate the mechanism underlying the regulation of YAP and TAZ by SIRT6 in HSCs. Overexpression or knockdown of SIRT6 corroborates the role of SIRT6 in the negative regulation of YAP and TAZ. Further biochemical analyses reveal that SIRT6 deacetylates YAP and TAZ and reprograms the composition of the TEA domain transcription factor complex to suppress their downstream target genes, particularly those involved in hepatic fibrosis. In conclusion, our data suggest that SIRT6 plays a critical role in the regulation of the Hippo pathway to protect against hepatic fibrosis.

Sirtuin 6 ameliorates arthritis through modulating cyclic AMP-responsive element binding protein/CCN1/cyclooxygenase 2 pathway in osteoblasts.

INTRODUCTION: CCN1 is an immediate-early gene product pivotal for arthritis progression. We have previously shown that sirtuin 6 (SIRT6) inhibited hypoxia-induced CCN1 expression in osteoblasts. Herein we examined the contribution of cyclic AMP-responsive element binding protein (CREB)/CRE to this suppressive action and the influence of CCN1 on cyclooxygenase (COX) 2 synthesis. MATERIALS AND METHODS: MC3T3-E1 murine osteoblasts were cultured under normoxia (21% oxygen) or hypoxia (2% oxygen). Expressions of CCN1, phospho-CREB (Ser133), COX2 and relevant kinases were assessed by Western blot. SIRT6 was overexpressed in cultured osteoblasts and arthritic joints by a lentiviral-based technique. Activities of CCN1 gene promoter constructs were examined by luciferase reporter assay. Interaction between CREB and CCN1 promoter was assessed by chromatin immunoprecipitation (ChIP). Collagen-induced arthritis (CIA) was established in 20 rats to evaluate the effects of SIRT6 therapy on osteoblastic expressions of phospho-CREB, CCN1 and COX2. RESULTS: SIRT6 suppressed hypoxia-enhanced CCN1 expression and CREB phosphorylation. Attenuation of calcium/calmodulin-dependent protein kinase II (CaMKII) may be responsible for SIRT6-induced CREB inhibition. CRE at - 286 bp upstream of the ATG start codon was essential for CCN1 expression under hypoxia and SIRT6 reduced hypoxia-stimulated CREB/CRE interaction. Forced expression of CREB rescued SIRT6-suppressed CCN1 synthesis. CCN1 induced COX2 expression in osteoblasts. In rat CIA, the therapeutic effect of SIRT6 was accompanied by decreases in osteoblastic expressions of phospho-CREB, CCN1 and COX2. CONCLUSION: Our study indicated that the benefits of SIRT6 to inflammatory arthritis and bone resorption are at least partially derived from its modulation of CREB/CCN1/COX2 pathway in osteoblasts.

SIRT6 regulates obesity-induced oxidative stress via ENDOG/SOD2 signaling in the heart.

The sirtuin 6 (SIRT6) participates in regulating glucose and lipid homeostasis. However, the function of SIRT6 in the process of cardiac pathogenesis caused by obesity-associated lipotoxicity remains to be unveiled. This study was designed to elucidate the role of SIRT6 in the pathogenesis of cardiac injury due to nutrition overload-induced obesity and explore the downstream signaling pathways affecting oxidative stress in the heart. In this study, we used Sirt6 cardiac-specific knockout murine models treated with a high-fat diet (HFD) feeding to explore the function and mechanism of SIRT6 in the heart tissue during HFD-induced obesity. We also took advantage of neonatal cardiomyocytes to study the role and downstream molecules of SIRT6 during HFD-induced injury in vitro, in which intracellular oxidative stress and mitochondrial content were assessed. We observed that during HFD-induced obesity, Sirt6 loss-of-function aggravated cardiac injury including left ventricular hypertrophy and lipid accumulation. Our results evidenced that upon increased fatty acid uptake, SIRT6 positively regulated the expression of endonuclease G (ENDOG), which is a mitochondrial-resident molecule that plays an important role in mitochondrial biogenesis and redox homeostasis. Our results also showed that SIRT6 positively regulated superoxide dismutase 2 (SOD2) expression post-transcriptionally via ENDOG. Our study gives a new sight into SIRT6 beneficial role in mitochondrial biogenesis of cardiomyocytes. Our data also show that SIRT6 is required to reduce intracellular oxidative stress in the heart triggered by high-fat diet-induced obesity, involving the control of ENDOG/SOD2.

SIRT6 ameliorates LPS-induced apoptosis and tight junction injury in ARDS through the ERK1/2 pathway and autophagy.

Sirtuin6 (SIRT6) has been demonstrated to be involved in a range of physiological processes and diseases, while its role in acute respiratory distress syndrome (ARDS) remains unclear. Therefore, this study focused on the role and underlying mechanism of SIRT6 in ARDS with the aim of identifying potential therapeutic targets. In this study, we found that SIRT6 was significantly decreased in lipopolysaccharide (LPS)-induced A549 cells and a murine model. In vitro overexpression of SIRT6 restored the expression of tight junction proteins (ZO-1 and occludin) and alleviated cell apoptosis and inflammation, while knockdown of SIRT6 aggravated the loss of tight junction proteins (ZO-1 and occludin) and promoted cell apoptosis and inflammation in LPS-induced A549 cells. Furthermore, the overexpression of SIRT6 enhanced autophagy and inhibited the ERK1/2 pathway, while the knockdown of SIRT6 inhibited autophagy and activated the ERK1/2 pathway. The autophagy activator rapamycin and the ERK1/2 inhibitor PD98059 rescued the effects of SIRT6 knockdown on tight junction proteins, apoptosis, and inflammation. Mechanistically, SIRT6 deacetylated histone 3 at Lys9 to negatively regulate the ERK1/2 pathway. In vivo, the SIRT6-specific inhibitor OSS_128167 also significantly accelerated LPS-induced loss of tight junction proteins, lung inflammation, and apoptosis. Meanwhile, the SIRT6-specific inhibitor OSS_128167 also activated the ERK1/2 pathway and inhibited lung autophagy. These results suggested that SIRT6 could ameliorate the loss of tight junction proteins, inflammation, and apoptosis in LPS-induced ARDS by inhibiting the ERK1/ 2 pathway and enhancing autophagy, indicating that SIRT6 plays a beneficial role in ARDS and might be a potential therapeutic target for ARDS.

Sirtuin 6 ameliorates alcohol-induced liver injury by reducing endoplasmic reticulum stress in mice.

Alcoholic liver disease (ALD) occurs as a result of chronic and excessive alcohol consumption. It encompasses a wide spectrum of chronic liver abnormalities that range from steatosis to alcoholic hepatitis, progressive fibrosis and cirrhosis. Endoplasmic reticulum (ER) stress induced by ethanol metabolism in hepatocytes has been established as an important contributor to the pathogenesis of ALD. However, whether SIRT6 exerts regulatory effects on ethanol-induced ER stress and contributes to the pathogenesis of ALD is unclear. In this study, we developed and characterized Sirt6 hepatocyte-specific knockout and transgenic mouse models that were treated with chronic-plus-binge ethanol feeding. We observed that hepatic Sirt6 deficiency led to exacerbated ethanol-induced liver injury and aggravated hepatic ER stress. Tauroursodeoxycholic acid (TUDCA) treatment remarkably attenuated ethanol-induced ER stress and ameliorated ALD pathologies caused by Sirt6 ablation. Reciprocally, SIRT6 hepatocyte-specific transgenic mice exhibited reduced ER stress and ameliorated liver injury caused by ethanol exposure. Consistently, knockdown of Sirt6 elevated the expression of ER stress related genes in primary hepatocytes treated with ethanol, whereas overexpression of SIRT6 reduced their expression, indicating SIRT6 regulates ethanol-induced hepatic ER stress in a cell autonomous manner. Collectively, our results suggest that SIRT6 is a positive regulator of ethanol-induced ER stress in the liver and protects against ALD by relieving ER stress.

Sirtuin 6: A potential therapeutic target for cardiovascular diseases.

Cardiovascular diseases (CVDs) are serious diseases endangering human health due to high morbidity and mortality worldwide, and numerous signal molecules are involved in this pathological process. As a member of the Sirtuin family NAD +-dependent deacetylases, indeed, Sirtuin 6 (SIRT6) plays an important role in regulating biological homeostasis, longevity, and various diseases. More importantly, SIRT6 performs as an indispensable role in glucose and lipid metabolism, inflammation and genomic stability for the occurrence and development of various CVDs. Recent advances: among sirtuins, SIRT6 was frequently unveiled thanks for its protective roles against heart failure, cardiovascular remodeling and atherosclerosis, and identified as an essential intervention target of CVDs, bringing SIRT6 into the focus of clinical interest. Herein, we provide an overview of the current molecular mechanism through which SIRT6 regulates CVDs, and we highlight a potential therapeutic target for CVDs.

Endothelial SIRT6 blunts stroke size and neurological deficit by preserving blood-brain barrier integrity: a translational study.

AIMS: Aging is an established risk factor for stroke; genes regulating longevity are implicated in the pathogenesis of ischaemic stroke where to date, therapeutic options remain limited. The blood-brain barrier (BBB) is crucially involved in ischaemia/reperfusion (I/R) brain injury thus representing an attractive target for developing novel therapeutic agents. Given the role of endothelial cells in the BBB, we hypothesized that the endothelial-specific expression of the recently described longevity gene SIRT6 may exhibit protective properties in stroke. METHODS AND RESULTS: SIRT6 endothelial expression was reduced following stroke. Endothelial-specific Sirt6 knockout (eSirt6-/-) mice, as well as animals in which Sirt6 overexpression was post-ischaemically induced, underwent transient middle cerebral artery occlusion (tMCAO). eSirt6-/- animals displayed increased infarct volumes, mortality, and neurological deficit after tMCAO, as compared to control littermates. Conversely, post-ischaemic Sirt6 overexpression decreased infarct size and neurological deficit. Analysis of ischaemic brain sections revealed increased BBB damage and endothelial expression of cleaved caspase-3 in eSIRT6-/- mice as compared to controls. In primary human brain microvascular endothelial cells (HBMVECs), hypoxia/reoxygenation (H/R) reduced SIRT6 expression and SIRT6 silencing impaired the barrier function (transendothelial resistance) similar to what was observed in mice exposed to I/R. Further, SIRT6-silenced HBMVECs exposed to H/R showed reduced viability, increased cleaved caspase-3 expression and reduced activation of the survival pathway Akt. In ischaemic stroke patients, SIRT6 expression was higher in those with short-term neurological improvement as assessed by NIHSS scale and correlated with stroke outcome. CONCLUSION: Endothelial SIRT6 exerts a protective role in ischaemic stroke by blunting I/R-mediated BBB damage and thus, it may represent an interesting novel therapeutic target to be explored in future clinical investigation.

The deacetylase sirtuin 6 protects against kidney fibrosis by epigenetically blocking ß-catenin target gene expression.

Fibrosis is a common pathologic pathway of progressive kidney disease involving complex signaling networks. The deacetylase sirtuin 6 (sirt6) was recently implicated in kidney injury. However, it remains elusive whether and how sirt6 contributes to the regulation of kidney fibrosis. Here, we demonstrate that sirt6 protects against kidney interstitial fibrosis through epigenetic regulation of ß-catenin signaling. Sirt6 is markedly upregulated during fibrogenesis following obstructed nephropathy and kidney ischemia-reperfusion injury. Pharmacological inhibition of sirt6 deacetylase activity aggravates kidney fibrosis in obstructed nephropathy. Consistently, knockdown of sirt6 in mouse kidney proximal tubular epithelial cells aggravates transforming growth factor-ß-induced fibrosis in vitro. Mechanistically, sirt6 deficiency results in augmented expression of the downstream target proteins of ß-catenin signaling. We further show that sirt6 interacts with ß-catenin during transforming growth factor-ß treatment and binds to the promoters of ß-catenin target genes, resulting in the deacetylation of histone H3K56 to prevent the transcription of fibrosis-related genes. Thus, our data reveal the anti-fibrotic function of sirt6 by epigenetically attenuating ß-catenin target gene expression.

SIRT6 deacetylase activity regulates NAMPT activity and NAD(P)(H) pools in cancer cells.

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the NAD+ salvage pathway from nicotinamide. By controlling the biosynthesis of NAD+, NAMPT regulates the activity of NAD+-converting enzymes, such as CD38, poly-ADP-ribose polymerases, and sirtuins (SIRTs). SIRT6 is involved in the regulation of a wide number of metabolic processes. In this study, we investigated the ability of SIRT6 to regulate intracellular NAMPT activity and NAD(P)(H) levels. BxPC-3 cells and MCF-7 cells were engineered to overexpress a catalytically active or a catalytically inactive SIRT6 form or were engineered to silence endogenous SIRT6 expression. In SIRT6-overexpressing cells, NAD(H) levels were up-regulated, as a consequence of NAMPT activation. By immunopurification and incubation with recombinant SIRT6, NAMPT was found to be a direct substrate of SIRT6 deacetylation, with a mechanism that up-regulates NAMPT enzymatic activity. Extracellular NAMPT release was enhanced in SIRT6-silenced cells. Also glucose-6-phosphate dehydrogenase activity and NADPH levels were increased in SIRT6-overexpressing cells. Accordingly, increased SIRT6 levels reduced cancer cell susceptibility to H2O2-induced oxidative stress and to doxorubicin. Our data demonstrate that SIRT6 affects intracellular NAMPT activity, boosts NAD(P)(H) levels, and protects against oxidative stress. The use of SIRT6 inhibitors, together with agents inducing oxidative stress, may represent a promising treatment strategy in cancer.-Sociali, G., Grozio, A., Caffa, I., Schuster, S., Becherini, P., Damonte, P., Sturla, L., Fresia, C., Passalacqua, M., Mazzola, F., Raffaelli, N., Garten, A., Kiess, W., Cea, M., Nencioni, A., Bruzzone, S. SIRT6 deacetylase activity regulates NAMPT activity and NAD(P)(H) pools in cancer cells.

Sirtuin 6 protects human retinal pigment epithelium cells from LPS-induced inflammation and apoptosis partly by regulating autophagy.

Lipopolysaccharides (LPS)-induced retinal inflammation is an important factor in retinal diseases. This study was aimed to investigate the effect of Sirt6 on LPS-induced retinal injury. ARPE-19 cells were incubated with LPS to induce inflammation. The cell viability was determined using CCK-8 assay. The mRNA level and protein expression of corresponding genes was detected using qRT-PCR and western blot, respectively. The production of inflammatory cytokines was measured using ELISA kit. The levels of oxidative stress-related factors were measured using their detection kits. Cell apoptosis was observed using TUNEL assay. The results showed that Sirt6 was downregulated after LPS treatment. Sirt6 strengthened LPS-induced autophagy by promoting the expression of LC3II/I, beclin1 and ATG5. Sirt6 treatment significantly inhibited LPS-induced inflammation, oxidative stress and cell apoptosis, which was then partly abolished by 3 MA. These results suggest Sirt6 to be an important regulator for LPS-induced inflammation, oxidative stress, and apoptosis partly by regulating cell autophagy.

SIRT6 overexpression inhibits cementogenesis by suppressing glucose transporter 1.

Cementum, which shares common features with bone in terms of biochemical composition, is important for the homeostasis of periodontium during periodontitis and orthodontic treatment. Sirtuin 6 (SIRT6), as a member of the sirtuin family, plays key roles in the osteogenic differentiation of bone marrow mesenchymal stem cells. However, the involvement of SIRT6 in cementoblast differentiation and mineralization and the underlying mechanisms remain unknown. In this study, we observed that the expression of SIRT6 increased during cementoblast differentiation initially. Analysis of the gain- and loss-of-function indicated that overexpressing SIRT6 in OCCM-30 cells suppresses cementoblast differentiation and mineralization and downregulating SIRT6 promotes cementogenesis. GLUT1, a glucose transporter necessary in cementogenesis, was inhibited by SIRT6. Overexpressing GLUT1 in SIRT6-overexpressed OCCM-30 cells rescued the inhibitory effect of SIRT6 on cementoblast differentiation and mineralization. Moreover, AMPK was activated after overexpressing SIRT6 and inhibited cementoblast differentiation and mineralization. Downregulating the expression of SIRT6 inhibited AMPK activity. Meanwhile, GLUT1 overexpression significantly decreased AMPK activity. Overall, on one hand, SIRT6 inhibited cementoblast differentiation and mineralization by suppressing GLUT1. On the other hand, SIRT6 inhibited cementoblast differentiation and mineralization by activating the AMPK pathway. GLUT1 overexpression also rescued the increased AMPK pathway activated by SIRT6.

Targeting anti-aging protein sirtuin (Sirt) in the diagnosis of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a severe, incurable, age-associated respiratory disorder that has gained significance because of its unknown etiology and lack of therapeutic approaches. IPF causes maximum damage to the alveolar epithelial cells, thereby leading to lung remodeling and initiating epithelial to mesenchymal transition (EMT). The actual molecular mechanisms underlying IPF still remain unclear, and knowledge about these mechanisms would be helpful in its diagnosis. Sirtuins (Sirt) are class of NAD+-dependent proteins, widely known to exert positive and protective effects on age-related diseases such as diabetes, cancer, and so on, and are also involved in regulating IPF. The sirtuin family comprises of seven members (Sirt1 to Sirt7), out of which Sirt1, Sirt3, Sirt6, and Sirt7 exert positive effects on IPF. Sirt1 is associated with aging and inhibits cellular senescence and fibrosis. Sirt1 is well recognized in controlling pulmonary fibrosis and is also considered as a prime positive mediator of EMT. The expressions of Sirt3 protein tend to decline in IPF patients; hence it is known as an anti-fibrotic protein. Sirt6 indeed has been proven to reduce EMT during IPF. Decreased levels of Sirt7 during IPF regulate lung fibroblasts. Hence, active levels of Sirt1, Sirt3, Sirt6, and Sirt7 can be attractive target models to elucidate a novel potential therapeutic approach for IPF. In this prospect, we have discussed the role of Sirtuins in pulmonary fibrosis by exploring the recent research evidence that highlight the role of sirtuins and also describes their protective effects.

Sirtuin 6 inhibits myofibroblast differentiation via inactivating transforming growth factor-ß1/Smad2 and nuclear factor-κB signaling pathways in human fetal lung fibroblasts.

Fibroblast-to-myofibroblast differentiation, which is characterized by increased expression of α-smooth muscle actin, is known to be involved in the pathogenesis of idiopathic pulmonary fibrosis. Sirtuin 6 (SIRT6), a member of the sirtuin family, has been proved to inhibit epithelial-to-mesenchymal transition during idiopathic pulmonary fibrosis. However, the function of SIRT6 in lung myofibroblast differentiation is still obscure. Transforming growth factor-ß1 (TGF-ß1) is one of the main factors that can powerfully promote myofibroblast differentiation. In the current study, we aimed to explore the role of SIRT6 in the cellular model of fibroblast-to-myofibroblast differentiation induced by TGF-ß1 using human fetal lung fibroblasts (HFL1). We demonstrated that the SIRT6 protein level is upregulated by TGF-ß1 in HFL1 cells. Overexpression of SIRT6 significantly suppresses TGF-ß1-induced myofibroblast differentiation in HFL1 cells. Mechanistically, SIRT6 decreases phosphorylation and nuclear translocation of Smad2 under TGF-ß1 stimulation. Nevertheless, mutant SIRT6 (H133Y) without histone deacetylase activity fails to inhibit phosphorylation and nuclear translocation of Smad2. Meanwhile, SIRT6 interacts with the nuclear factor-κB (NF-κB) subunit p65 and represses TGF-ß1-induced NF-κB-dependent transcriptional activity, which is also dependent on its deacetylase activity. Overexpression of wild-type SIRT6 but not the H133Y mutant inhibits the expression of NF-κB-dependent genes including interleukin (IL)-1ß, IL-6 and matrix metalloproteinase-9 (MMP-9) induced by TGF-ß1, all of which have been demonstrated to promote myofibroblast differentiation. Collectively, our study reveals that SIRT6 prevents TGF-ß1-induced lung myofibroblast differentiation through inhibiting TGF-ß1/Smad2 and NF-κB signaling pathways.

Activation of retinoid X receptor by bexarotene attenuates neuroinflammation via PPARγ/SIRT6/FoxO3a pathway after subarachnoid hemorrhage in rats.

BACKGROUND: Subarachnoid hemorrhage (SAH) is a life-threatening subtype of stroke with high mortality and disabilities. Retinoid X receptor (RXR) has been shown to be neuroprotective against ischemia/reperfusion injury. This study aimed to investigate the effects of the selective RXR agonist bexarotene on neuroinflammation in a rat model of SAH. METHODS: Two hundred male Sprague-Dawley rats were used. The endovascular perforation induced SAH. Bexarotene was administered intraperitoneally at 1 h after SAH induction. To investigate the underlying mechanism, the selective RXR antagonist UVI3003 and RXR siRNA or SIRT6 inhibitor OSS128167 was administered via intracerebroventricular 1 h before SAH induction. Post-SAH assessments including SAH grade, neurological score, brain water content, Western blot, and immunofluorescence were performed. RESULTS: The endogenous RXR and sirtuin 6 (SIRT6) protein levels were increased after SAH. Bexarotene treatment significantly reduced brain edema and improved the short-/long-term neurological deficit after SAH. Mechanistically, bexarotene increased the levels of PPARγ and SIRT6; decreased the expression of phosphorylated FoxO3a (p-FoxO3a), IL-6, IL-1ß, and TNF-a; and inhibited the microglia activation and neutrophils infiltration at 24 h after SAH. Either UVI3003, OSS128167, or RXR siRNA abolished the neuroprotective effects of bexarotene and its regulation on protein levels of PPARγ/SIRT6/p-FoxO3a after SAH. CONCLUSIONS: The activation of RXR by bexarotene attenuated neuroinflammation and improved neurological deficits after SAH. The anti-neuroinflammatory effect was at least partially through regulating PPARγ/SIRT6/FoxO3a pathway. Bexarotene may be a promising therapeutic strategy in the management of SAH patients.

SIRT6/HIF-1α axis promotes papillary thyroid cancer progression by inducing epithelial-mesenchymal transition.

BACKGROUND: In our previous study, we demonstrated that Sirtuin 6 (SIRT6) is upregulated and associated with papillary thyroid cancer (PTC) progression (Qu et al. in Int J Oncol 50(5):1683-92, 2017). This study examined whether SIRT6 promotes epithelial-mesenchymal transition (EMT) of papillary thyroid cancer through hypoxia inducible factor-1α (HIF-1α). METHODS: SIRT6-upregulated TPC-1 and B-CPAP cells were generated by lentivirus. Western blotting, RT-qPCR, immunofluorescence was performed to detect the following EMT associated markers: E-cadherin, Vimentin, Snail, and TWIST. Cell proliferation was detected by CCK8, and cell invasion and migration were detected by transwell and wound healing assays, respectively. HIF-1α expression was further detected by western blotting in both normoxia and hypoxia conditions. A HIF-1α inhibitor was then used to block HIF-1α expression in SIRT6-upregulated PTC cells. The same parameters were then assessed and compared with control HIF-1α cells. RESULTS: E-cadherin was significantly decreased, whereas Vimentin, Snail, and TWIST were increased in SIRT6-upregulated PTC cells. Additionally, SIRT6 promoted the invasion and migration of PTC cells. We found that SIRT6 enhanced HIF-1α stability and synthesis and prolonged the protein half-life. The changes in the EMT associated markers and in the invasion and migration ability were rescued after inhibition of HIF-1α expression. Furthermore, we found that SIRT6 increased PTC resistance to HIF-1α inhibitor-mediated proliferation changes. CONCLUSION: These results confirm that the SIRT6/HIF-1α axis promotes papillary thyroid cancer progression by inducing EMT.