Sirt1 deficiency upregulates glutathione metabolism to prevent hepatocellular carcinoma initiation in mice.

Sirtuin-1 (SIRT1) is involved in various metabolic pathways, including fatty acid synthesis and gluconeogenesis in the liver. However, its role in initiation and progression of liver cancer remains unclear. Studying Sirt1 liver-specific knockout (LKO) mice in combination with diethylnitrosamine (DEN) treatment, we demonstrated that loss of Sirt1 rendered mice resistant to DEN-induced hepatocellular carcinoma (HCC) development. RNA-seq revealed that livers from LKO mice exhibited an enrichment in glutathione metabolism eight months after DEN challenge. Sirt1 deficiency elevated the expression of glutathione-s-transferase family genes by increasing the level of Nrf2, a key regulator of glutathione metabolism. Hence, LKO livers displayed a reductive environment with an increased ratio of GSH to GSSG and an elevated GSH level. Furthermore, using CRISPR knockout techniques, we confirmed that the impairment of HCC formation in LKO mice is mainly dependent on NRF2 signaling. Meanwhile, HCC induced by DEN could be blocked by the administration of N-acetyl cysteine (NAC) when administered one month after DEN challenge. However, NAC treatment starting five months after DEN injection was not able to prevent tumor development. In conclusion, our findings indicate that a reductive environment orchestrated by glutathione metabolism at an early stage can prevent the initiation of HCC.

Intestinal SIRT1 Deficiency-Related Intestinal Inflammation and Dysbiosis Aggravate TNFα-Mediated Renal Dysfunction in Cirrhotic Ascitic Mice.

In advanced cirrhosis, the TNFα-mediated intestinal inflammation and bacteria dysbiosis are involved in the development of inflammation and vasoconstriction-related renal dysfunction. In colitis and acute kidney injury models, activation of SIRT1 attenuates the TNFα-mediated intestinal and renal abnormalities. This study explores the impacts of intestinal SIRT1 deficiency and TNFα-mediated intestinal abnormalities on the development of cirrhosis-related renal dysfunction. Systemic and renal hemodynamics, intestinal dysbiosis [cirrhosis dysbiosis ratio (CDR) as marker of dysbiosis], and direct renal vasoconstrictive response (renal vascular resistance (RVR) and glomerular filtration rate (GFR)) to cumulative doses of TNFα were measured in bile duct ligated (BDL)-cirrhotic ascitic mice. In SIRT1IEC-KO-BDL-ascitic mice, the worsening of intestinal dysbiosis exacerbates intestinal inflammation/barrier dysfunction, the upregulation of the expressions of intestinal/renal TNFα-related pathogenic signals, higher TNFα-induced increase in RVR, and decrease in GFR in perfused kidney. In intestinal SIRT1 knockout groups, the positive correlations were identified between intestinal SIRT1 activity and CDR. Particularly, the negative correlations were identified between CDR and RVR, with the positive correlation between CDR and GFR. In mice with advanced cirrhosis, the expression of intestinal SIRT1 is involved in the linkage between intestinal dysbiosis and vasoconstriction/hypoperfusion-related renal dysfunction through the crosstalk between intestinal/renal TNFα-related pathogenic inflammatory signals.

MAPK pathway and SIRT1 are involved in the down-regulation of secreted osteopontin expression by genistein in metastatic cancer cells.

AIM: The regulation of secreted osteopontin (OPN) expression by genistein and its functional sequel in the metastatic cancer cells (MDA-MB-435 and MDA-MB-231) was ascertained. MAIN METHODS: Western blot and Real-Time PCR were used to analyse the proteins and mRNA transcripts, respectively. Possible transcriptional regulation of secreted OPN was analyzed by chromatin immunoprecipitation assay, bioinformatics analysis, transfection and luciferase reporter assay. The specific siRNAs and constitutive p-ERKs were used to evaluate the role of the MAPK pathway. The functional sequel of genistein in these cells was analyzed by colony formation-, migration- and invasion- assay. KEY FINDINGS: Secreted OPN expression was inhibited (up to ~0.7-fold) by genistein in these cells. Genistein (50 µM) displayed a reduction in the aggressiveness of these cells concerning colony formation rate, migration, and invasion. The p-ERK½ was increased by ~2.5-fold and ~1.5-fold upon 50 µM genistein and 15 µM resveratrol treatments at 24 h, respectively. Knockdown of ERK½ and PD98059, the inhibitor of MEK, promoted secreted OPN expression in vitro in these cells; while, the transfection of the constitutive active ERK2 (L73P and S151D) decreased the secreted OPN expression. Further, silent mating type information regulation 2 homolog 1 (SIRT1) expression in the cells was increased (~1.6-fold) upon genistein treatment (50 µM) likewise with resveratrol (~1.5-fold), an activator for SIRT1. Knockdown of SIRT1 increased OPN mRNA transcripts expression level and secreted OPN protein level in these cells. SIGNIFICANCE: MAPK pathway and SIRT1 activation are involved in the regulation of secreted OPN by genistein in these cells.

Progranulin modulates cartilage-specific gene expression via sirtuin 1-mediated deacetylation of the transcription factors SOX9 and P65.

Progranulin (PGRN) is an autocrine growth factor that exerts crucial roles within cartilage tissue; however, the molecular mechanisms underlying PGRN-mediated cartilage homeostasis remain elusive. In the present study, we investigated the role of PGRN in regulating chondrocyte homeostasis and its therapeutic potential for managing osteoarthritis (OA). We found that PGRN levels are significantly increased in human cartilage in mild OA and that its expression is decreased in the cartilage in severe OA. In vitro, treatment of primary rat chondrocytes with recombinant PGRN significantly enhanced the levels of collagen type II α 1 chain (COL2A1) and aggrecan, and attenuated TNFα-induced up-regulation of matrix metallopeptidase 13 (MMP13) and ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5) in chondrocytes. These effects were abrogated in SIRT1-/- cells, indicating a causative role of SIRT1 in the effects of PGRN on protein expression in chondrocytes. Mechanistically, PGRN increased SIRT1 expression and activity, which reduced the acetylation levels of SRY-box transcription factor (SOX9) and transcription factor P65 (P65) and thereby promoted nuclear translocation of SOX9 and inhibited TNFα-induced P65 nuclear accumulation to maintain chondrocyte homeostasis. In conclusion, our findings reveal a mechanism of action for PGRN that maintains cartilage homeostasis and supports the notion that PGRN up-regulation may be a promising strategy for managing OA.

SIRT1 modulates cell cycle progression by regulating CHK2 acetylation-phosphorylation.

Both the stress-response protein, SIRT1, and the cell cycle checkpoint kinase, CHK2, play critical roles in aging and cancer via the modulation of cellular homeostasis and the maintenance of genomic integrity. However, the underlying mechanism linking the two pathways remains elusive. Here, we show that SIRT1 functions as a modifier of CHK2 in cell cycle control. Specifically, SIRT1 interacts with CHK2 and deacetylates it at lysine 520 residue, which suppresses CHK2 phosphorylation, dimerization, and thus activation. SIRT1 depletion induces CHK2 hyperactivation-mediated cell cycle arrest and subsequent cell death. In vivo, genetic deletion of Chk2 rescues the neonatal lethality of Sirt1-/- mice, consistent with the role of SIRT1 in preventing CHK2 hyperactivation. Together, these results suggest that CHK2 mediates the function of SIRT1 in cell cycle progression, and may provide new insights into modulating cellular homeostasis and maintaining genomic integrity in the prevention of aging and cancer.

The Sirt1 activator resveratrol improved hematopoiesis in pancytopenia mice induced by irradiation.

Bone marrow failure is a disease syndrome with the disability to produce mature blood cells. Pancytopenia is the most common manifestation of bone marrow failure. Sirt1 is important for the function of hematopoietic stem cells, we hypothesized that Sirt1 activation may improve hematopoiesis. The Sirt1 heterozygous and wild type mice were exposed to lethal 6.5 Gy 60Co-γ rays. The survival time and hematopoietic indexes were evaluated. The survival time of Sirt1 deficiency mice was significantly decreased. The numbers of platelets (PLT), reticulocytes (RET) and white blood cells (WBC) were significantly decreased. C57BL/6 mice were exposed to 6.5 Gy 60Co-γ rays then administrated with resveratrol (20 mg/kg/d) or vehicle. Resveratrol increased the survival time and protective against irradiation induced hematopoietic damage. Resveratrol also significantly increased the numbers of PLT, RET and WBC of mice. It also increased the hematopoietic area and karyocytes number. In HEK293T cells, the expression of LKB1 was significantly increased in cytoplasm but not in nuclei when treated with resveratrol (50 µM). These results suggest that Sirt1 deficiency might aggravate bone marrow failure. Resveratrol corrected this hematopoietic defect and LKB1 might involve in the protective effect on bone marrow failure.

Sirt1 ameliorates monosodium urate crystal-induced inflammation by altering macrophage polarization via the PI3K/Akt/STAT6 pathway.

OBJECTIVES: Acute gout is an inflammatory response to MSU crystals. In our previous research, Sirt1 was shown to have an effect in preventing acute gouty inflammation. In the current study, we aimed to investigate the underlying mechanism involving Sirt1 in acute gout. METHODS: The cytological changes and Sirt1 expression in the synovium were observed in patients with acute or intermittent gout. The effect of Sirt1 and its mechanism in gout were studied in macrophages, C57BL/6 mice and Sirt1+/- mice. RESULTS: Sirt1 expression was increased in the peripheral blood mononuclear cells (PBMCs) of patients with acute gout but not in the chronic tophus tissue. The arthritis score and numbers of inflammatory cells in injured paw tissue from murine gout models were upregulated in Sirt1+/- mice compared with wild-type mice. A PCR array of the paw tissue from murine gout models indicated that Sirt1 activation might attenuate MSU-induced inflammation by altering the polarization state of macrophages. Furthermore, in patients with acute gout, the phagocytosis of MSU crystals by a macrophage was found in a smear of the joint fluid and large amounts of macrophages were also found in the synovium. The activation of Sirt1 in gouty mice actually decreased the tendency toward M1 polarization. The inhibition of PI3K/Akt partially blocked the anti-inflammatory effect of Sirt1 and the translocation of STAT6, and phosphorylated STAT6 expression was decreased in RAW 264.7 cells treated with MSU crystals. CONCLUSION: Our studies revealed that Sirt1 ameliorates MSU-induced inflammation by altering macrophage polarization via the PI3K/Akt/STAT6 pathway.

Increase of Hspa1a and Hspa1b genes in the resting B cells of Sirt1 knockout mice.

Sirt1, also known as the longevity gene, is an NAD+-dependent class III histone deacetylase that has been extensively studied in multiple areas of research including cellular metabolism, longevity, cancer, autoimmunity, and immunity. However, little is known about the function of Sirt1 in B cells. This study aimed to investigate the role of Sirt1 in the expression pattern of mRNAs in the resting B cells of mice. CD19+ B cell-specific inducible Sirt1 knockout (KO) mice were divided into tamoxifen-treated Sirt1 KO group (S19T) or control group (S19). mRNAs extracted from resting B cells of both groups were analyzed for differentially expressed genes (DEG) using microarray. DEG analysis showed significant differential expression of 20 genes, of which Hspa1a and Hspa1b showed the highest fold change (FC) in S19T compared with S19 (p value < 0.01 and FC > 3). Further, Kyoto Encyclopedia of Genes and Genomes analysis identified pathways associated with diseases, organismal systems, and antigen processing and presentation. Additionally, the pathways known to involve Hspa1a and Hspa1b were also activated in the S19T group. On the other hand, after in vitro stimulation with lipopolysaccharide, cell viability and IgM production were significantly decreased in Sirt1 KO B cells, while expressions of TNF-α, IL-6, and IL-10 were increased. In summary, our study reveals that Sirt1 may maintain the quiescent state in resting B cells by suppressing the increase of Hspa1a and Hspa1b. This work provides a foundation for further studies on the functional roles of Sirt1 in B cells.

Loss of Sirtuin 1 Alters the Secretome of Breast Cancer Cells by Impairing Lysosomal Integrity.

The NAD+-dependent deacetylase Sirtuin 1 (SIRT1) is down-regulated in triple-negative breast cancer. To determine the mechanistic basis by which reduced SIRT1 expression influences processes related to certain aggressive cancers, we examined the consequences of depleting breast cancer cells of SIRT1. We discovered that reducing SIRT1 levels decreased the expression of one particular subunit of the vacuolar-type H+ ATPase (V-ATPase), which is responsible for proper lysosomal acidification and protein degradation. This impairment in lysosomal function caused a reduction in the number of multi-vesicular bodies (MVBs) targeted for lysosomal degradation and resulted in larger MVBs prior to their fusing with the plasma membrane to release their contents. Collectively, these findings help explain how reduced SIRT1 expression, by disrupting lysosomal function and generating a secretome comprising exosomes with unique cargo and soluble hydrolases that degrade the extracellular matrix, can promote processes that increase breast-cancer-cell survival and invasion.

HSF1/HSP pathway in the hippocampus is involved in SIRT1-mediated caloric restriction-induced neuroprotection after surgery in aged mice.

Postoperative cognitive dysfunction is common in the elderly. Endoplasmic reticulum stress (ER-stress) increases neuronal apoptosis after surgery, and chaperone molecules, such as heat shock proteins (HSPs), help reduce unfolded protein reactions, thereby promoting protein homeostasis. Mammal sirtuin1 (SIRT1)-mediated deacetylation of heat shock factor 1 (HSF1) upregulates HSF1 binding to the HSP70 promoter. Caloric restriction (CR) improves cognition in many neurodegenerative models. In this study, we evaluated whether CR improves impaired learning and memory after surgery by attenuating ER-stress in an SIRT1-dependent manner. Male 18-month-old C57BL/6J mice receiving a 12-week CR or an ad libitum (AL) diet pre-intervention were challenged with tibial open fracture surgery and anesthesia or no treatment. We found a significant protective effect of CR on memory in contextual fear conditioning test after surgery compared with the AL group. CR alleviated ER-stress and neuronal apoptosis in the hippocampus induced by surgery. CR increased HSP70 expression through the HSF1/HSP pathway in a SIRT1-mediated manner, and inhibition of SIRT1 in the hippocampus by lentivirus injection partially reduced the benefits of CR (increased HSP70, deacetylated HSF1, reduced ER-stress, and improved memory). Taken together, our results showed that CR alleviates memory impairment postoperatively via attenuation of ER-stress in the hippocampus in an SIRT1-dependent manner, and the SIRT1/HSF1/HSP70 pathway is involved in this process.

The polyphenol resveratrol promotes skeletal growth in mice through a sirtuin 1-bone morphogenic protein 2 longevity axis.

BACKGROUND AND PURPOSE: The polyphenol resveratrol (RSV) exists in high quantities in certain foods (e.g. grapes and nuts). However, the capacity of RSV to confer physiological health benefits and a biological mechanism through which this might occur remains unclear. EXPERIMENTAL APPROACH: Aged, RSV-treated (300 mg·kg-1 ·day-1 ) and genetically modified [endothelial NOS (eNOS-/- )] female mice were assessed using histomorphometric and µCT analysis. Alongside in vivo analysis, molecular siRNA knockdown and pharmacological manipulation of eNOS, BMP2 and sirtuin 1 (SIRT1) and functional cellular assays in an osteoblast cell line panel, explored the mechanism through which RSV might impact overall bone volume. KEY RESULTS: RSV promoted osteoblast activity and bone growth in vivo. RSV dose-dependently and simultaneously increased alkaline phosphatase (ALP) and eNOS levels. Similarly, NO-donor treatment increased ALP, runt homology transcription factor 2, BMP2 and stimulated bone formation, whilst eNOS-deficient mice displayed a bone loss phenotype. Moreover, RSV-induced increase in ALP and BMP2 expression was blocked in eNOS-/- osteoblasts and by BMP-inhibitor noggin. The longevity-linked SIRT1 enzyme was positively regulated by RSV and SIRT1 deletion reduced eNOS, BMP2 and ALP. Like eNOS deletion, loss of SIRT1 blocked RSV-induced osteoblast activity; however, SIRT1 levels remained unchanged in eNOS-/- mice, indicating RSV activation of SIRT1 stimulates BMP2 release via eNOS. This signalling axis is supported by decreased SIRT1, eNOS and BMP2 confirmed in old versus young bone. CONCLUSIONS AND IMPLICATIONS: These findings suggest a new mechanism of action in bone remodelling and the ageing skeleton, where RSV positively impacts bone homeostasis via SIRT1 activation of BMP2.

[SIRT1 deficiency in CD4+T cells induces acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation].

OBJECTIVE: To study the relationship between acute graft-versus-host disease (aGVHD) and the SIRT1 expression in peripheral blood CD4+T cells from patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT).
 Methods: We collected 40 patients who underwent allo-HSCT from human leukocyte antigen (HLA)-identical sibling donors. SIRT1 expression level in CD4+T cells was measured by real-time PCR and Western blot. Acetylation and phosphorylation of STAT3 in CD4+T cells were detected by Western blot. The binding level between SIRT1 and STAT3 in CD4+T cells was analyzed by co-immunoprecipitation and Western blot. Over-expression of SIRT1 in aGVHD CD4+T cells, as well as STAT3 acetylation and phosphorylation were measured by Western blot. The mRNA levels of RORγt, IL-17A, IL-17F related to Th17 were detected by real-time PCR.
 Results: SIRT1 expression was significantly down-regulated, while STAT3 expression, acetylation and phosphorylation levels were significantly up-regulated in patients with aGVHD compared with patients without aGVHD. The STAT3 acetylation was positively correlated with STAT3 phosphorylation (r=0.69, P<0.01). Less SIRT1-STAT3 complexes were found in CD4+T cells from patients with aGVHD compared with patients without aGVHD. After SIRT1 over-expression in aGVHD CD4+T cells, the STAT3 acetylation and phosphorylation, and the expression of RORγt, IL-17A, and IL-17F related to Th17 were significantly down-regulated (P<0.05).
 Conclusion: SIRT1 deficiency in CD4+T cells plays a crucial role in up-regulation of STAT3 acetylation and phosphorylation, the increase of Th17 related gene expression, and induction of aGVHD after allogeneic hematopoietic stem cell transplantation.

Cervical cancer is addicted to SIRT1 disarming the AIM2 antiviral defense.

Mammalian cells are equipped with antiviral innate immunity. To survive and grow, human papilloma virus (HPV)-infected cervical cancer cells must overcome this host defense system. However, the precise mechanism whereby cervical cancer cells evade the immunity is not fully understood. We noted that Sirtuin 1 (SIRT1) is overexpressed in HPV-infected cervical cancer cells and hypothesized that SIRT1 counteracts antiviral immunity. Here, we found that cervical cancer cells undergo massive death by SIRT1 knockdown, but this effect is reversed by SIRT1 restoration. SIRT1-knocked-down cells showed representative features of pyroptosis, as well as highly expressed absent in melanoma 2 (AIM2) and its downstream genes related to the inflammasome response. Mechanistically, SIRT1 repressed the NF-κB-driven transcription of the AIM2 gene by destabilizing the RELB mRNA. Interestingly, pyroptotic death signaling in SIRT1-knocked-down cells was transmitted to naïve cervical cancer cells, which was mediated by extracellular vesicles carrying AIM2 inflammasome proteins. Furthermore, the growth of cervical cancer xenografts was significantly inhibited by either SIRT1-targeting siRNAs or SIRT1-knockdown-derived extracellular vesicles. Immunohistochemical analyses showed that SIRT1 expression correlated with poor clinical outcomes in cervical cancer. In conclusion, SIRT1 enabled HPV-infected cervical cancer cells to continue growing by nullifying AIM2 inflammasome-mediated immunity. Without SIRT1, cervical cancer cells could no longer survive because of the derepression of the AIM2 inflammasome. SIRT1 could therefore be a target for the effective treatment of cervical cancer.

Myeloid sirtuin1 deficiency aggravates hippocampal inflammation in mice fed high-fat diets.

Chronic low-grade inflammation-induced insulin resistance is associated with neuroinflammation. Myeloid sirtuin1 (SIRT1) deficiency aggravates high-fat diet (HFD)-induced insulin resistance. However, the function of myeloid-specific SIRT1 in the hippocampus of obese mice is largely unknown. To address this question, we fed myeloid SIRT1 knockout (KO) mice a HFD for 40 weeks. We found that HFD-fed SIRT1 KO mice had increased insulin resistance and macrophage infiltration in adipose tissue than wild type (WT) mice. Levels of HFD-induced lipocalin-2 (LCN2) were lower in SIRT1 KO mice than in WT. HFD-induced hippocampal LCN2 expression was lower in HFD-fed SIRT1 KO mice than in WT. Hippocampal acetylation of nuclear factor-κB (NF-κB) and amyloid precursor protein levels were higher in HFD-fed SIRT1 KO mice than in HFD-fed WT mice. Taken together, our results suggest that targeted induction of the anti-inflammatory effects of SIRT1 and LCN2 may help prevent obesity-associated insulin resistance and neuroinflammation.

Cardiomyocyte-specific deletion of Sirt1 gene sensitizes myocardium to ischaemia and reperfusion injury.

Aims: A longevity gene, Sirtuin 1 (SIRT1) and energy sensor AMP-activated protein kinase (AMPK) have common activators such as caloric restriction, oxidative stress, and exercise. The objective of this study is to characterize the role of cardiomyocyte SIRT1 in age-related impaired ischemic AMPK activation and increased susceptibility to ischemic insults. Methods and results: Mice were subjected to ligation of left anterior descending coronary artery for in vivo ischemic models. The glucose and fatty acid oxidation were measured in a working heart perfusion system. The cardiac functions by echocardiography show no difference in young wild-type C57BL/6 J (WT, 4-6 months), aged WT C57BL/6 J (24-26 months), and young inducible cardiomyocyte-specific SIRT1 knockout (icSIRT1 KO) (4-6 months) mice under physiological conditions. However, after 45 mins ischaemia and 24-h reperfusion, the ejection fraction of aged WT and icSIRT1 KO mice was impaired. The aged WT and icSIRT1 KO hearts vs. young WT hearts also show an impaired post-ischemic contractile function in a Langendorff perfusion system. The infarct size of aged WT and icSIRT1 KO hearts was larger than that of young WT hearts. The immunoblotting data demonstrated that aged WT and icSIRT1 KO hearts vs. young WT hearts had impaired phosphorylation of AMPK and downstream acetyl-CoA carboxylase during ischaemia. Intriguingly, AMPK upstream LKB1 is hyper-acetylated in both aged WT and icSIRT1 KO hearts; this could blunt activation of LKB1, leading to an impaired AMPK activation. The working heart perfusion results demonstrated that SIRT1 deficiency significantly impaired substrate metabolism in the hearts; fatty acid oxidation is augmented and glucose oxidation is blunted during ischaemia and reperfusion. Adeno-associated virus (AAV9)-Sirt1 was delivered into the aged hearts via a coronary delivery approach, which significantly rescued the protein level of SIRT1 and the ischemic tolerance of aged hearts. Furthermore, AMPK agonist can rescue the tolerance of aged heart and icSIRT1 KO heart to ischemic insults. Conclusions: Cardiac SIRT1 mediates AMPK activation via LKB1 deacetylation, and AMPK modulates SIRT1 activity via regulation of NAD+ level during ischaemia. SIRT1 and AMPK agonists have therapeutic potential for treatment of aging-related ischemic heart disease.

Metabolic reprogramming of human CD8+ memory T cells through loss of SIRT1.

The expansion of CD8+CD28- T cells, a population of terminally differentiated memory T cells, is one of the most consistent immunological changes in humans during aging. CD8+CD28- T cells are highly cytotoxic, and their frequency is linked to many age-related diseases. As they do not accumulate in mice, many of the molecular mechanisms regulating their fate and function remain unclear. In this paper, we find that human CD8+CD28- T cells, under resting conditions, have an enhanced capacity to use glycolysis, a function linked to decreased expression of the NAD+-dependent protein deacetylase SIRT1. Global gene expression profiling identified the transcription factor FoxO1 as a SIRT1 target involved in transcriptional reprogramming of CD8+CD28- T cells. FoxO1 is proteasomally degraded in SIRT1-deficient CD8+CD28- T cells, and inhibiting its activity in resting CD8+CD28+ T cells enhanced glycolytic capacity and granzyme B production as in CD8+CD28- T cells. These data identify the evolutionarily conserved SIRT1-FoxO1 axis as a regulator of resting CD8+ memory T cell metabolism and activity in humans.

A Possible Reason to Induce Acute Graft-vs.-Host Disease After Hematopoietic Stem Cell Transplantation: Lack of Sirtuin-1 in CD4+ T Cells.

Sirtuin 1 (SIRT1) is a critical suppressor of T cell immunity. However, whether SIRT1 is involved in the progression of acute graft-vs.-host disease (aGVHD) has still remained unclear. PI3K/Akt/mTOR pathway is a crucial element involved in the activation and functions of T cells. Over-activation of PI3K/Akt/mTOR signaling may be related to the occurrence of aGVHD. STAT3 activation requires phosphorylation and acetylation. A recent study showed that STAT3 hyperphosphorylation in CD4+ T cells may be a trigger of aGVHD. The role of the STAT3 acetylation in aGVHD pathogenesis is still unclear. The present study revealed that SIRT1 deficiency as a critical factor is involved in the excessive activation of mTOR pathway and upregulation of STAT3 acetylation and phosphorylation in CD4+ T cells from patients with aGVHD. Exorbitant activation of IL-1ß signaling is the main reason for TAK1-dependent SIRT1 insufficiency. The findings of the present study might provide a new therapeutic target for treating aGVHD.

RNA interference-mediated knockdown of SIRT1 and/or SIRT2 in melanoma: Identification of downstream targets by large-scale proteomics analysis.

Melanoma is the most notorious and fatal of all skin cancers and the existing treatment options have not been proven to effectively manage this neoplasm, especially the metastatic disease. Sirtuin (SIRT) proteins have been shown to be differentially expressed in melanoma. We have shown that SIRTs 1 and 2 were overexpressed in melanoma and inhibition of SIRT1 imparts anti-proliferative responses in human melanoma cells. To elucidate the impact of SIRT 1 and/or 2 in melanoma, we created stable knockdowns of SIRTs 1, 2, and their combination using shRNA mediated RNA interference in A375 human melanoma cells. We found that SIRT1 and SIRT1&2 combination knockdown caused a decreased cellular proliferation in melanoma cells. Further, the knockdown of SIRT 1 and/or 2 resulted in a decreased colony formation in melanoma cells. To explore the downstream targets of SIRTs 1 and/or 2, we employed a label-free quantitative nano-LC-MS/MS proteomics analysis using the stable lines. We found aberrant levels of proteins involved in many vital cellular processes, including cytoskeletal organization, ribosomal activity, oxidative stress response, and angiogenesis. These findings provide clear evidence of cellular systems undergoing alterations in response to sirtuin inhibition, and have unveiled several excellent candidates for future study. SIGNIFICANCE: Melanoma is the deadliest form of skin cancer, due to its aggressive nature, metastatic potential, and a lack of sufficient treatment options for advanced disease. Therefore, detailed investigations into the molecular mechanisms of melanoma growth and progression are needed. In the search for candidate genes to serve as therapeutic targets, the sirtuins show promise as they have been found to be upregulated in melanoma and they regulate a large number of proteins involved in cellular processes known to affect tumor growth, such as DNA damage repair, cell cycle arrest, and apoptosis. In this study, we used a large-scale label-free comparative proteomics system to identify novel protein targets that are affected following knockdown of SIRT1 and/or 2 in A375 metastatic melanoma cell line. Our study offers important insight into the potential downstream targets of SIRTs 1 and/or 2. This may unravel new potential areas of exploration in melanoma research.

SIRT1 Controls Acetaminophen Hepatotoxicity by Modulating Inflammation and Oxidative Stress.

AIMS: Sirtuin 1 (SIRT1) is a key player in liver physiology and a therapeutic target against hepatic inflammation. We evaluated the role of SIRT1 in the proinflammatory context and oxidative stress during acetaminophen (APAP)-mediated hepatotoxicity. RESULTS: SIRT1 protein levels decreased in human and mouse livers following APAP overdose. SIRT1-Tg mice maintained higher levels of SIRT1 on APAP injection than wild-type mice and were protected against hepatotoxicity by modulation of antioxidant systems and restrained inflammatory responses, with decreased oxidative stress, proinflammatory cytokine messenger RNA levels, nuclear factor kappa B (NFκB) signaling, and cell death. Mouse hepatocytes stimulated with conditioned medium of APAP-treated macrophages (APAP-CM) showed decreased SIRT1 levels; an effect mimicked by interleukin (IL)1ß, an activator of NFκB. This negative modulation was abolished by neutralizing IL1ß in APAP-CM or silencing p65-NFκB in hepatocytes. APAP-CM of macrophages from SIRT1-Tg mice failed to downregulate SIRT1 protein levels in hepatocytes. In vivo administration of the NFκB inhibitor BAY 11-7082 preserved SIRT1 levels and protected from APAP-mediated hepatotoxicity. INNOVATION: Our work evidenced the unique role of SIRT1 in APAP hepatoprotection by targeting oxidative stress and inflammation. CONCLUSION: SIRT1 protein levels are downregulated by IL1ß/NFκB signaling in APAP hepatotoxicity, resulting in inflammation and oxidative stress. Thus, maintenance of SIRT1 during APAP overdose by inhibiting NFκB might be clinically relevant. Rebound Track: This work was rejected during standard peer review and rescued by Rebound Peer Review (Antioxid Redox Signal 16:293-296, 2012) with the following serving as open reviewers: Rafael de Cabo, Joaquim Ros, Kalervo Hiltunen, and Neil Kaplowitz. Antioxid. Redox Signal. 28, 1187-1208.

Reduced silent information regulator 1 signaling exacerbates sepsis-induced myocardial injury and mitigates the protective effect of a liver X receptor agonist.

Myocardial injury and dysfunction are critical manifestations of sepsis. Previous studies have reported that liver X receptor (LXR) activation is protective during sepsis. However, whether LXR activation protects against septic heart injury and its underlying mechanisms remain elusive. This study was designed to determine the role of LXR activation in the septic heart with a focus on SIRT1 (silent information regulator 1) signaling. Male cardiac-specific SIRT1 knockout mice (SIRT1-/-) and their wild-type littermates were subjected to sepsis by cecal ligation and puncture (CLP) in the presence or absence of LXR agonist T0901317. The survival rate of mice was recorded during the 7-day period post CLP. Our results demonstrated that SIRT1-/- mice suffered from exacerbated mortality and myocardial injury in comparison with their wild-type littermates. Meanwhile, T0901317 treatment improved mice survival, accompanied by significant ameliorations of myocardial injury and dysfunction in wild-type mice but not in SIRT1-/- mice. Furthermore, the levels of myocardial inflammatory cytokines (TNF-α, IL-6, IL-1ß, MCP-1, MPO and HMGB1), oxidative stress (ROS generation, MDA), endoplasmic-reticulum (ER) stress (protein levels of CHOP, GRP78, GRP94, IRE1α, and ATF6), and cardiac apoptosis following CLP were inhibited by T0901317 treatment in wild-type mice but not in SIRT1-/- mice. Mechanistically, T0901317 enhanced SIRT1 signaling and the subsequent deacetylation and activation of antioxidative FoxO1 and anti-ER stress HSF1, as well as the deacetylation and inhibition of pro-inflammatory NF-ΚB and pro-apoptotic P53, thereby alleviating sepsis-induced myocardial injury and dysfunction. Our data support the promise of LXR activation as an effective strategy for relieving heart septic injury.