Expression Patterns of ERα, ERß, AR, SIRT1, SIRT2, and SIRT3 in Prostate Cancer Tissue and Normal Prostate Tissue.

BACKGROUND/AIM: The purpose of this study was to examine the expression of estrogen receptor α (ERα) and ß (ERß), androgen receptor (AR), SIRT1, SIRT2 and SIRT3 in prostate cancer (PCa). MATERIALS AND METHODS: From October 2010 to January 2015, 70 patients who had undergone radical prostatectomy following a PCa diagnosis were enrolled in our study. Normal prostate tissue (NPT) and prostate cancer tissues (PCAT) were separated, and the expression of each receptor in each tissue was analyzed with immunochemical staining. Univariate and multivariate analyses were performed to identify factors affecting the development of PCa. RESULTS: ERß and AR were highly expressed in PCAT compared with NPT (p<0.05). SIRT2 was highly expressed in NPT and PCAT (p<0.05). Univariate and multivariate analyses showed that AR and SIRT2 affect PCa development. CONCLUSION: AR is a risk factor for PC, and SIRT2 is associated with a lower incidence of PCa.

AGK2 Alleviates Lipopolysaccharide Induced Neuroinflammation through Regulation of Mitogen-Activated Protein Kinase Phosphatase-1.

Neuroinflammation is associated with the progression of multiple neurological diseases. Many studies show that SIRT2 involves in multiple inflammatory processes. While, the mechanisms remain unclear. The purpose of this study was to explore the effect of SIRT2 inhibitor AGK2 on inflammatory responses and MAPK signaling pathways in LPS activated microglia in vitro and in vivo. The effect of AGK2 on cell viability of BV2 microglial cells was detected by CCK-8 assay. The expression of inflammatory cytokine iNOS was analyzed by western blotting and immunofluorescence. The mRNA expressions of iNOS, TNF-α, and IL-1ß were detected by real-time polymerase chain reaction (RT-PCR). The SIRT2, phospho-P38, P38, phospho-JNK, JNK, phospho-ERK, ERK, α-tubulin, and acetyl-α-tubulin were analyzed by western blotting respectively. The interaction between SIRT2 and MKP-1 was measured by Co-immunoprecipitation (Co-IP) assay. Double immunofluorescent staining was performed to detect the expressions of CD11b and iNOS or SIRT2 in brain tissues. We found that AGK2 could suppress LPS-induced inflammatory cytokines (iNOS, TNF-α, and IL-1ß) expression levels in BV2 microglial cells. Moreover, it could effectively reduce the expression of SIRT2 and increase the acetylation of α-tubulin in LPS activated BV2 microglial cells and LPS induced mice neuroinflammation. In addition, our results showed that AGK2 could reduce the increase of phosphorylation p38, JNK, and ERK after LPS challenge. Co-IP results showed that there was no direct interaction between MKP-1 and SIRT2. However, AGK2 by inhibition of SIRT2 could increase the expression of MKP-1. Furthermore, AGK2 could inhibit the activation of BV2 microglia and expression of iNOS and SIRT2 in LPS treated mice brain tissue. Taken together, our results suggested that AGK2 might alleviate lipopolysaccharide induced neuroinflammation through regulation of mitogen-activated protein kinase phosphatase-1. Graphical abstract.

Anesthetic preconditioning increases sirtuin 2 gene expression in a renal ischemia reperfusion injury model.

BACKGROUND: Renal transplant surgical proceedings are known to elicit periods of hypoxia and consequent blood flow reestablishment triggering ischemia-reperfusion (I-R) injury. Kidney damage induced by I-R injury associates with a higher risk of graft dysfunction and rejection. Anesthetic preconditioning exerts a beneficial effect on I-R injury by reducing oxidative stress, inflammation and apoptosis. However, the degree of renoprotection stimulated by commonly used anesthetics, as well as their mechanisms of action, are largely unknown. Sirtuins are class III histone deacetylases that reduce cellular stress, promote genome stability and regulate senescence. So far, the relationship between sirtuins and anesthetic preconditioning in the context of renal I-R has not been studied. The main objective of the present work was to determine the renal expression of sirtuins after I-R damage in rats under different anesthetic preconditioning treatments. METHODS: Unilateral ischemia was performed via occlusion of the left renal hilum for 45 min and followed by 24 hours of reperfusion. Anesthetic preconditioning schemes (morphine 0.5 mg/kg, fentanyl 10 µg/kg, propofol 7.5 mg/kg, or dexmedetomidine 25 µg/kg) were administered 1 hour before ischemia. Creatinine levels were determined in serum, and expression of kidney injury molecule 1 and sirtuin 1, 2, 3 and 7 in kidney tissue was quantified by RT-PCR. RESULTS: Anesthetic preconditioning with morphine, fentanyl, propofol and dexmedetomidine reduced kidney injury markers after I-R and modulated sirtuin gene expression. Opioids or dexmedetomidine administration before ischemia increased sirtuin 2 expression and correlated with improved renal function. CONCLUSIONS: Anesthetic preconditioning is a promising strategy to prevent I-R injury associated with transplantation. Our results suggest that sirtuin 2 is involved in the protective mechanisms of some commonly used anesthetics against I-R damage.

Rs2015 Polymorphism in miRNA Target Site of Sirtuin2 Gene Is Associated with the Risk of Parkinson's Disease in Chinese Han Population.

Accumulating evidence reveals that the sirtuin family is involved in the pathology of Parkinson's disease (PD). However, the association between the polymorphisms of the sirtuin gene and the risk of PD remains elusive. Here, we investigated the possible association of nine SIRT1 and SIRT2 SNPs with the risk of PD through a clinical case-control study from the Chinese Han population. Our results showed that rs12778366 in the promoter region of SIRT1 and rs2015 in the 3'untranslated region (3'UTR) of the SIRT2 were significantly associated with the risk of PD. Five SNPs related to SIRT1, rs3740051, rs7895833, rs7069102, rs2273773, and rs4746720 and two SNPs related to SIRT2, rs10410544, and rs45592833 did not show an association with PD risk in this study. Moreover, we found that mRNA level of SIRT2 was upregulated, and mRNA level of SIRT1 was downregulated in the peripheral blood of PD patients compared with healthy controls, and we also observed that SNPs rs12778366 and rs2015 influenced the SIRT1 and SIRT2 expression levels, respectively. Further functional assays suggest that rs2015 may affect the expression of SIRT2 by affecting the binding of miR-8061 to the 3'UTR of SIRT2, ultimately contributing to the risk of PD.

Altered levels of sirtuin genes (SIRT1, SIRT2, SIRT3 and SIRT6) and their target genes in adipose tissue from individual with obesity.

INTRODUCTION: Sirtuins regulate energy metabolism and insulin sensitivity through their ability to act as energy sensors and regulators in several metabolic tissues. AIM: To evaluate the expression levels of sirtuin genes SIRT1, SIRT2, SIRT3 and SIRT6 and their target genes (PPAR-α, PGC1-α, NRF1, DGAT1, PPAR-γ and FOXO3a) in subcutaneous adipose tissue collected from individuals with normoweight, overweight and obesity. METHODS: Adipose tissue samples, obtained by lipoaspiration during liposuction surgery, were processed to obtain RNA, which was reverse-transcribed to cDNA. Then, we measured the expression levels of each gene by qPCR. RESULTS: We found differences in the mRNA expression of SIRT1, SIRT2, SIRT3 and SIRT6 and their target genes (PPAR-α, PGC1-α, NRF1, DGAT1, PPAR-γ and FOXO3a) in adipose tissue from overweight or obese subjects when compared to normoweight subjects. All genes analyzed, except SIRT2, showed correlation with BMI. CONCLUSIONS: Our findings in human subcutaneous adipose tissue show that increased body mass index modifies the expression of genes encoding sirtuins and their target genes, which are metabolic regulators of adipose tissue. Therefore, these could be used as biomarkers to predict the ability of adipose tissue to gain mass of adipose tissue.

Sirtuin 2 (Sirt2) Expression Predicts Lymph Node Metastasis and Poor Overall Survival of Patients with Esophageal Squamous Cell Carcinoma.

BACKGROUND: Altered expression of Sirtuin 2 (Sirt2) was associated with cancer development and progression. This study further assessed the association of Sirt2 expression with clinicopathological data and prognosis of patients with esophageal squamous cell carcinoma (ESCC) after postoperative concurrent chemoradiotherapy. METHODS: Tissue specimens from 95 ESCC patients were collected for immunohistochemical analysis of Sirt2 expression, which was used to determine association with patient clinicopathological and survival data. RESULTS: Sirt2 protein was expressed in 53.7% of ESCC tissue specimens but only in 25.3% of normal squamous epithelium (p = 0.000). Sirt2 expression was associated with tumor invasion (p = 0.005), lymph node metastasis (p = 0.003), and advanced clinical stage (p = 0.000), but not with tumor size (p = 0.199), or differentiation (p = 0.177). Sirt2 expression was associated with poor overall and progression-free survival (p = 0.034). The multivariate analysis showed that Sirt2 expression was an independent predictor for overall survival of patients with resected ESCC followed by concurrent chemoradiotherapy (p = 0.048). CONCLUSIONS: Sirt2 protein expression in ESCC tissue specimens was associated with ESCC invasion, lymph node metastasis, and advanced tumor clinical stage, as well as poor overall and poor progression-free survival. Sirt2 expression is an independent prognostic predictor for ESCC patients.

Helicobacter pylori infection increases sirt2 gene expression in gastric epithelial cells of gastritis patients.

BACKGROUND: Helicobacter pylori Infection causes some clinical features of the human stomach such as gastritis, duodenal ulcer, and gastric cancer. It has been shown that Helicobacter pylori infection increases proinflammatory cytokine gene expressions in Gastric Epithelial Cells by activation of NF-kB signaling. Sirt1 and sirt2 as deacetylases play a certain role in the progress of inflammation in arthritis and lung infection by impacting the NF-kB. AIMS: Sirt1 and sirt2 gene expressions in Gastric Epithelial cells of gastritis patients were surveyed with and without Helicobacter pylori infection and rate of prevalence of cagA and hopQ genes in Helicobacter pylori strains were investigated. METHODS: 25 biopsy samples of gastritis patients with Helicobacter pylori infection and 25 biopsy samples of gastritis patients without Helicobacter pylori infection were collected from Tohid Hospital in the city of Sanandaj throughout the year 2016. CDNA was made from total RNA extracted from biopsy samples (Qiagen® Kit). Sirt1 and sirt2 gene expressions were determined using the Corbett machine (Rotor-Gene 6000 Software). CagA and hopQ genes of Helicobacter pylori strains were determined by PCR using specific primers. RESULTS: The sirt2 gene expression was increased in Gastric Epithelial Cells of gastritis patients with Helicobacter pylori infection. No significant relationship was found between sirt1 and sirt2 gene expressions as well as cagA and hopQ as Helicobacter pylori virulence genes. CONCLUSIONS: This study shows the Helicobacter pylori infection duo to sirt2 gene up-expression. There is not a statistically significance relationship between cagA and hopQ Helicobacter pylori genotypes and sirt2 gene up-expression in Gastric Epithelial Cells of gastritis patients.

Reduced gene expression of sirtuins and active AMPK levels in children and adolescents with obesity and insulin resistance.

BACKGROUND: Sirtuins, including SIRT1 and SIRT2, are longevity-associated deacetylase enzymes that modulate metabolic homeostasis in response to the cellular energy state. Adenosine monophosphate activated protein kinase (AMPK) and SIRT1 are interrelated and share several common target pathways. This study aimed to evaluate the SIRT1 and SIRT2 gene expression in peripheral blood mononuclear cells (PBMCs) as well as plasma levels of AMPK, in obese children and adolescents. MATERIALS AND METHODS: Participants included 60 children and adolescents (30 obese and 30 age- and gender-matched control subjects). Real-time polymerase chain reaction (PCR) was used to assess the SIRT1 and SIRT2 gene expression in PBMCs. Serum phospho-AMPK and insulin were measured using enzyme-linked immunosorbent assay (ELISA), and insulin resistance (IR) was calculated by the Homeostasis Model of Assessment of Insulin Resistance (HOMA-IR). Glucose and lipid profile were also measured. RESULTS: SIRT1 gene expression and phospho-AMPK plasma levels were significantly diminished in obese subjects compared to the control group, and both SIRT1 and SIRT2 were significantly lower in obese children with IR compared to those without IR. SIRT1 expression revealed significant negative correlations with body mass index and waist circumference as well as insulin and HOMA-IR and a positive correlation with AMPK. SIRT2 negatively correlated with SIRT1 and positively correlated with high density lipoprotein-cholesterol (HDL-C). CONCLUSION: SIRT1 and SIRT2 expression and AMPK levels decrease in children with obesity and IR. Targeting SIRT1 can be valuable in preventing obesity-associated IR in childhood and adolescence.

NKT-like cells reveal higher than T lymphocytes expression of cellular protective proteins HSP70 and SOD2 and comparably increased expression of SIRT1 in the oldest seniors.

INTRODUCTION: NKT-like cells are "non-classical", "CD1d-independent" NKT cells which represent highly differentiated, conventional T lymphocytes coexpressing several NK (natural killer) associated receptors. They are effector lymphocytes of both innate and adaptive immunity and simultaneously regulatory cells of the adaptive immune system. They reveal large granular lymphocyte morphology and can mediate both MHC-restricted and MHC-unrestricted cytotoxicity, secrete many cytokines and modulate Th1 immune responses. The aim of our study was to analyze the expression of proteins involved in cellular stress response: sirtuin 1 (SIRT1), heat shock protein 70 (HSP70) and manganese superoxide dismutase (SOD2) in NKT-like cells compared to T lymphocytes during ageing. MATERIAL AND METHODS: The study involved three groups of participants: the oldest seniors (n = 25; aged over 85; mean age 88 ± 0.5 ys), the old (n = 30; aged under 85; mean age 76 ± 0.9 ys) and the young (n = 32; mean age 21 ± 0.3 ys). Whole blood samples were analyzed by flow cytometry to assess the NKT-like (CD3+CD56+) and T (CD3+) cell populations. RESULTS: The group of the oldest seniors differed from the other age groups by much higher percentage of both NKT-like cells and T lymphocytes expressing SIRT1, HSP70 and SOD2. The expression of these proteins correlated positively with the age of the participants. Interestingly, the significantly higher expression of the studied protective proteins; i.e. HSP70 and SOD2 was found in CD3+CD56+ cells compared to CD3+ lymphocytes and this phenomenon concerned all the studied age groups. These differences were not significant regarding the expression of SIRT1; however, the same tendency was noticeable. CONCLUSIONS: The analysis of CD3+ and CD3+CD56+ lymphocytes showed the increase in the number of NKT-like cells and decreased number of T cells in the process of ageing. The increased expression of cellular protective proteins SIRT1, HSP70 and SOD2 in NKT-like and T-lymphocytes of the oldest seniors seems to correspond to longevity and the observed correlations may suggest the involvement of these proteins in establishing cellular homeostasis specific for healthy ageing. Furthermore, the higher expression of the protective proteins in NKT-like cells compared to T lymphocytes may indicate their particular role in the interplay between innate and adaptive immunity responses during the process of ageing.

Mutant A53T α-Synuclein Improves Rotarod Performance Before Motor Deficits and Affects Metabolic Pathways.

The protein α-synuclein (α-Syn) interferes with glucose and lipid uptake and also activates innate immune cells. However, it remains unclear whether α-Syn or its familial mutant forms contribute to metabolic alterations and inflammation in synucleinopathies, such as Parkinson's disease (PD). Here, we address this issue in transgenic mice for the mutant A53T human α-Syn (α-SynA53T), a mouse model of synucleinopathies. At 9.5 months of age, mice overexpressing α-SynA53T (homozygous) had a significant reduction in weight, exhibited improved locomotion and did not show major motor deficits compared with control transgenic mice (heterozygous). At 17 months of age, α-SynA53T overexpression promoted general reduction in grip strength and deficient hindlimb reflex and resulted in severe disease and mortality in 50 % of the mice. Analysis of serum metabolites further revealed decreased levels of cholesterol, triglycerides and non-esterified fatty acids (NEFA) in α-SynA53T-overexpressing mice. In fed conditions, these mice also showed a significant decrease in serum insulin without alterations in blood glucose. In addition, assessment of inflammatory gene expression in the brain showed a significant increase in TNF-α mRNA but not of IL-1ß induced by α-SynA53T overexpression. Interestingly, the brain mRNA levels of Sirtuin 2 (Sirt2), a deacetylase involved in both metabolic and inflammatory pathways, were significantly reduced. Our findings highlight the relevance of the mechanisms underlying initial weight loss and hyperactivity as early markers of synucleinopathies. Moreover, we found that changes in blood metabolites and decreased brain Sirt2 gene expression are associated with motor deficits.

High glucose-induced oxidative stress represses sirtuin deacetylase expression and increases histone acetylation leading to neural tube defects.

Aberrant epigenetic modifications are implicated in maternal diabetes-induced neural tube defects (NTDs). Because cellular stress plays a causal role in diabetic embryopathy, we investigated the possible role of the stress-resistant sirtuin (SIRT) family histone deacetylases. Among the seven sirtuins (SIRT1-7), pre-gestational maternal diabetes in vivo or high glucose in vitro significantly reduced the expression of SIRT 2 and SIRT6 in the embryo or neural stem cells, respectively. The down-regulation of SIRT2 and SIRT6 was reversed by superoxide dismutase 1 (SOD1) over-expression in the in vivo mouse model of diabetic embryopathy and the SOD mimetic, tempol and cell permeable SOD, PEGSOD in neural stem cell cultures. 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a superoxide generating agent, mimicked high glucose-suppressed SIRT2 and SIRT6 expression. The acetylation of histone 3 at lysine residues 56 (H3K56), H3K14, H3K9, and H3K27, putative substrates of SIRT2 and SIRT6, was increased by maternal diabetes in vivo or high glucose in vitro, and these increases were blocked by SOD1 over-expression or tempol treatment. SIRT2 or SIRT6 over-expression abrogated high glucose-suppressed SIRT2 or SIRT6 expression, and prevented the increase in acetylation of their histone substrates. The potent sirtuin activator (SRT1720) blocked high glucose-increased histone acetylation and NTD formation, whereas the combination of a pharmacological SIRT2 inhibitor and a pan SIRT inhibitor mimicked the effect of high glucose on increased histone acetylation and NTD induction. Thus, diabetes in vivo or high glucose in vitro suppresses SIRT2 and SIRT6 expression through oxidative stress, and sirtuin down-regulation-induced histone acetylation may be involved in diabetes-induced NTDs. The mechanism underlying pre-gestational diabetes-induced neural tube defects (NTDs) is still elusive. Our study unravels a new epigenetic mechanism in which maternal diabetes-induced oxidative stress represses sirtuin deacetylase 2 (SIRT2) and 6 (SIRT6) expression leading to histone acetylation and gene expression. SIRT down-regulation mediates the teratogenicity of diabetes leading to (NTD) formation. The study provides a mechanistic basis for the development of natural antioxidants and SIRT activators as therapeutics for diabetic embryopathy.

Effect of Resveratrol on Leptin and Sirtuin 2 Expression in the Kidneys in Streptozotocin-induced Diabetic Rats.

OBJECTIVE: To investigate the effect of resveratrol (RSV) on the histopathology and leptin and sirtuin 2 expression levels of the kidneys in streptozotocin (STZ)-induced diabetic rats. STUDY DESIGN: The study was carried out with 33 young, healthy, female Wistar Albino rats. STZ was given (50 mg/kg, intraperitoneal, single dose) to the rats to induce and constitute a diabetes model. After 1 month of STZ, resveratrol (10 mg/kg) was given for 15 days. Then the kidneys were evaluated histopathologically and the leptin and sirtuin 2 expressions were analyzed immunohistochemically. RESULTS: High glucose and fewer weight levels were seen in the STZ-induced diabetes mellitus group. The glucose levels in the RSV-administered diabetic group showed a tendency to decrease but not significantly. Decreased signs of histopathologic kidney damage was seen in the RSV-administered diabetic group, and an increased expression of leptin was seen in the diabetic kidney tissues. There were no significant differences of sirtuin 2 expression levels among the groups. CONCLUSION: It was observed that resveratrol caused changes in the diabetic kidney histology and leptin expression level. Resveratrol may be effective, with its antioxidant and antidiabetic effects, in the prevention of kidney damage caused by long-term hyperglycemia.

Resveratrol Induced Premature Senescence Is Associated with DNA Damage Mediated SIRT1 and SIRT2 Down-Regulation.

The natural polyphenolic compound resveratrol (3,4,5-trihydroxy-trans-stilbene) has broad spectrum health beneficial activities including antioxidant, anti-inflammatory, anti-aging, anti-cancer, cardioprotective, and neuroprotective effects. Remarkably, resveratrol also induces apoptosis and cellular senescence in primary and cancer cells. Resveratrol's anti-aging effects both in vitro and in vivo attributed to activation of a (NAD)-dependent histone deacetylase family member sirtuin-1 (SIRT1) protein. In mammals seven members (SIRT1-7) of sirtuin family have been identified. Among those, SIRT1 is the most extensively studied with perceptive effects on mammalian physiology and suppression of the diseases of aging. Yet no data has specified the role of sirtuins, under conditions where resveratrol treatment induces senescence. Current study was undertaken to investigate the effects of resveratrol in human primary dermal fibroblasts (BJ) and to clarify the role of sirtuin family members in particular SIRT1 and SIRT2 that are known to be involved in cellular stress responses and cell cycle, respectively. Here, we show that resveratrol decreases proliferation of BJ cells in a time and dose dependent manner. In addition the increase in senescence associated ß-galactosidase (SA-ß-gal) activity and methylated H3K9-me indicate the induction of premature senescence. A significant increase in phosphorylation of γ-H2AX, a surrogate of DNA double strand breaks, as well as in levels of p53, p21CIP1 and p16INK4A is also detected. Interestingly, at concentrations where resveratrol induced premature senescence we show a significant decrease in SIRT1 and SIRT2 levels by Western Blot and quantitative RT-PCR analysis. Conversely inhibition of SIRT1 and SIRT2 via siRNA or sirtinol treatment also induced senescence in BJ fibroblasts associated with increased SA-ß-gal activity, γ-H2AX phosphorylation and p53, p21CIP1 and p16INK4A levels. Interestingly DNA damaging agent doxorubicin also induced senescence in BJ fibroblasts associated with decreased SIRT1/2 levels. In conclusion our data reveal that resveratrol induced premature senescence is associated with SIRT1 and SIRT2 down regulation in human dermal fibroblasts. Here we suggest that the concomitant decline in SIRT1/2 expression in response to resveratrol treatment may be a cause for induction of senescence, which is most likely mediated by a regulatory mechanism activated by DNA damage response.

Expression/localization patterns of sirtuins (SIRT1, SIRT2, and SIRT7) during progression of cervical cancer and effects of sirtuin inhibitors on growth of cervical cancer cells.

Sirtuins belong to the family of class III histone deacetylases; its role in neoplasia is controversial as both tumor-suppressive and promoting functions have been reported. There are very few reports available, where expressions of sirtuin isoforms are comprehensively analyzed during neoplasia. Therefore, in the present study, the expression of SIRT1, SIRT2, and SIRT7 during different stages of cervical cancer progression was analyzed. The normal cervical epithelium showed feeble expression of sirtuin isoforms, SIRT1, SIRT2, and SIRT7. A significant increase in SIRT1 expression was noted in the cytoplasm as well as in the nucleus of proliferative layers of cervical epithelium in squamous intraepithelial lesions (SIL); however, in the squamous cell carcinomas (SCC), a heterogeneous pattern of SIRT1 expression varying from low to high was noted. A progressive increase in the expression of both SIRT2 and SIRT7 was noted during cancer progression in the following order: normal < preneoplasia < cancer. Cervical cancer cell lines, HeLa and SiHa, showed higher levels of SIRT1 and SIRT2 in comparison to the immortalized cell counterpart, HaCaT. Specific inhibitors of SIRT1 (Ex527) and SIRT2 (AGK2) impaired the growth of the cervical cancer cells, SiHa, but not of the HaCaT cells. SIRT1 inhibition caused cell death, while SIRT2 inhibition resulted in cell cycle arrest. In conclusion, we report the overexpression of SIRT2 and SIRT7 proteins in cervical cancer and suggest probable application of sirtuin inhibitors as therapeutic targets. Further, a specific increase in the levels of SIRT1 in intraepithelial lesion makes it a promising candidate for identification of preneoplastic changes.

Regulation of the mitogen-activated protein kinase kinase (MEK)-1 by NAD(+)-dependent deacetylases.

Sirtuins are class III deacetylases that regulate many essential processes, including cellular stress, genome stability and metabolism. Although these NAD(+)-dependent deacetylases control adaptive cellular responses, identification of sirtuin-regulated signaling targets remain under-studied. Here, we demonstrate that acetylation of the mitogen-activated protein kinase kinase-1 (MEK1) stimulates its kinase activity, and that acetylated MEK1 is under the regulatory control of the sirtuin family members SIRT1 and SIRT2. Treatment of cells with sirtuin inhibitors, or siRNA knockdown of SIRT1 or SIRT2 proteins, increases MEK1 acetylation and subsequent phosphorylation of the extracellular signal-regulated kinase. Generation of an acetyl-specific MEK1 antibody demonstrates that endogenous acetylated MEK1 is extensively enriched in the nucleus following epidermal growth factor  stimulation. An acetyl-mimic of MEK1 increases inappropriate growth properties, suggesting that acetylation of MEK1 has oncogenic potential.

Changes in microtubule-related proteins and autophagy in long-term vitamin E-deficient mice.

Vitamin E deficiency induces neuronal dysfunction and while oxidative stress is likely to be involved in mediating this process, the detailed mechanisms remain to be elucidated. Previously, we found axonal degeneration in the hippocampal CA1 region in vitamin E-deficient mice of 6 months of age (long-term). However, 3 month-old (short-term) vitamin E-deficient mice did not exhibit axonal degeneration in same region. In order to characterize the mechanisms involved in axonal degeneration in long-term vitamin E-deficient mice, we examined changes in microtubule-related proteins. Long-term vitamin E-deficiency led to significantly increased expression of the phosphorylated form of collapsin response mediator protein (CRMP)-2 compared to short-term deficiency. It is well known that CRMP-2 plays a crucial role in the maintenance of neurite function. Similarly, long-term vitamin E-deficiency significantly decreased the expression of silent mating type information regulation (SIRT)-2 mRNA compared to short-term deficiency. SIRT-2 belongs to a family of class III histone deacetylases (HDACs) and functions in the deacetylation of tubulins. Furthermore, the expression of microtubule-associated protein light chain (MAP-LC)3-2, which is a key autophagy protein was significantly higher in the short-term vitamin E-deficiency than the long-term deficiency. These results indicate that the mechanisms of axonal injury in long-term vitamin E-deficient mice are related to dysfunction in microtubules assembly via alterations in microtubule-related proteins and autophagy.

Aging: filtering out bad mitochondria.

During yeast cytokinesis an aged mother cell gives rise to an immaculate daughter cell. A new study now demonstrates that this rejuvenation encompasses a novel Sir2- and actin-cable-dependent filtering process that prevents feeble mitochondria from entering the daughter cell.

Actin dynamics affect mitochondrial quality control and aging in budding yeast.

Actin cables of budding yeast are bundles of F-actin that extend from the bud tip or neck to the mother cell tip, serve as tracks for bidirectional cargo transport, and undergo continuous movement from buds toward mother cells [1]. This movement, retrograde actin cable flow (RACF), is similar to retrograde actin flow in lamellipodia, growth cones, immunological synapses, dendritic spines, and filopodia [2-5]. In all cases, actin flow is driven by the push of actin polymerization and assembly at the cell cortex, and myosin-driven pulling forces deeper within the cell [6-10]. Therefore, for movement and inheritance from mothers to buds, mitochondria must "swim upstream" against the opposing force of RACF [11]. We find that increasing RACF rates results in increased fitness of mitochondria inherited by buds and that the increase in mitochondrial fitness leads to extended replicative lifespan and increased cellular healthspan. The sirtuin SIR2 is required for normal RACF and mitochondrial fitness, and increasing RACF rates in sir2Δ cells increases mitochondrial fitness and cellular healthspan but does not affect replicative lifespan. These studies support the model that RACF serves as a filter for segregation of fit from less-fit mitochondria during inheritance, which controls cellular lifespan and healthspan. They also support a role for Sir2p in these processes.

Discovery of a potent small molecule SIRT1/2 inhibitor with anticancer effects.

SIRT1 and SIRT2 are deacetylase enzymes that belong to the sirtuin family and are involved in tumorigenesis. In our screen for small molecules inhibiting SIRT1/2 toxoflavin was identified. Toxoflavin potently inhibited SIRT1 activity in in vitro deacetylase assay using purified SIRT1 protein. SIRT2 activity was also inhibited by toxoflavin less potently than SIRT1 in deacetylase assay in vitro. Toxoflavin exhibited growth inhibition of various cancer cell lines including A549 lung cancer cells with a GI(50) of 48 nM. Toxoflavin treatment in A549 cells increased the acetylated form of p53, which is a substrate of SIRT1. The acetylation levels of α-tubulin, a SIRT2 substrate, were also increased by toxoflavin treatment dose-dependently. Several toxoflavin derivatives were synthesized to determine the preliminary structure-activity relationship of toxoflavin. Some of the toxoflavin derivatives showed highly selective inhibition against SIRT1. In conclusion, this study presented toxoflavin as a potent SIRT1/2 inhibitor with anticancer activity.

Differential sirtuin expression patterns in amyotrophic lateral sclerosis (ALS) postmortem tissue: neuroprotective or neurotoxic properties of sirtuins in ALS?

BACKGROUND/AIMS: Sirtuins (SIRT1-7; class III histone deactylases) modulate fundamental mechanisms in age-related neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). We assessed the expression levels of sirtuins in human postmortem ALS and control brain and spinal cord. METHODS AND RESULTS: By quantitative real-time PCR, a significant reduction of SIRT1 and SIRT2 was detected in homogenates of the primary motor cortex (white and gray matter), while there were no differences in spinal cord homogenates. When specifically analyzing mRNA and protein expression in the gray matter (cortical layers I-VI of the precentral gyrus, ventral/dorsal horn of the spinal cord) by in situ hybridization histochemistry and immunohistochemistry, we found increased levels of SIRT1, SIRT2 and SIRT5 in ALS which were significant for SIRT1 and SIRT5 mRNA in the spinal cord. CONCLUSION: Our results indicate a general reduction of SIRT1 and SIRT2 in ALS primary motor cortex, while in situ hybridization histochemistry and immunohistochemistry showed neuron-specific upregulation of SIRT1, SIRT2 and SIRT5, particularly in the spinal cord. Opposed effects have been described for SIRT1 and SIRT2: while SIRT1 activation is mainly associated with neuroprotection, SIRT2 upregulation is toxic to neuronal cells. Novel therapeutic approaches in ALS could therefore target SIRT1 activation or SIRT2 inhibition.