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 (ERK). Generation of an acetyl-specific MEK1 antibody demonstrates that endogenous acetylated MEK1 is extensively enriched in the nucleus following epidermal growth factor (EGF) stimulation. An acetyl-mimic of MEK1 increases inappropriate growth properties, suggesting that acetylation of MEK1 has oncogenic potential

Estrogen-dependent dynamic profile of eNOS-DNA associations in prostate cancer

In previous work we have documented the nuclear translocation of endothelial NOS (eNOS) and its participation in combinatorial complexes with Estrogen Receptor Beta (ERβ) and Hypoxia Inducible Factors (HIFs) that determine localized chromatin remodeling in response to estrogen (E2) and hypoxia stimuli, resulting in transcriptional regulation of genes associated with adverse prognosis in prostate cancer (PCa). To explore the role of nuclear eNOS in the acquisition of aggressive phenotype in PCa, we performed ChIP-Sequencing on chromatin-associated eNOS from cells from a primary tumor with poor outcome and from metastatic LNCaP cells. We found that: 1. the eNOS-bound regions (peaks) are widely distributed across the genome encompassing multiple transcription factors binding sites, including Estrogen Response Elements. 2. E2 increased the number of peaks, indicating hormone-dependent eNOS re-localization. 3. Peak distribution was similar with/without E2 with ≈ 55% of them in extragenic DNA regions and an intriguing involvement of the 5′ domain of several miRs deregulated in PCa. Numerous potentially novel eNOS-targeted genes have been identified suggesting that eNOS participates in the regulation of large gene sets. The parallel finding of downregulation of a cluster of miRs, including miR-34a, in PCa cells associated with poor outcome led us to unveil a molecular link between eNOS and SIRT1, an epigenetic regulator of aging and tumorigenicity, negatively regulated by miR-34a and in turn activating eNOS. E2 potentiates miR-34a downregulation thus enhancing SIRT1 expression, depicting a novel eNOS/SIRT1 interplay fine-tuned by E2-activated ER signaling, and suggesting that eNOS may play an important role in aggressive PCa

Dietary Restriction Depends on Nutrient Composition to Extend Chronological Lifespan in Budding Yeast Saccharomyces cerevisiae

10.1371/journal.pone.0064448PLoS ONE85

Sirt1 carboxyl-domain is an ATP-repressible domain that is transferrable to other proteins

Sirt1 is an NAD(+)-dependent protein deacetylase that regulates many physiological functions, including stress resistance, adipogenesis, cell senescence and energy production. Sirt1 can be activated by energy deprivation, but the mechanism is poorly understood. Here, we report that Sirt1 is negatively regulated by ATP, which binds to the C-terminal domain (CTD) of Sirt1. ATP suppresses Sirt1 activity by impairing the CTD's ability to bind to the deacetylase domain as well as its ability to function as the substrate recruitment site. ATP, but not NAD(+), causes a conformational shift to a less compact structure. Mutations that prevent ATP binding increase Sirt1's ability to promote stress resistance and inhibit adipogenesis under high-ATP conditions. Interestingly, the CTD can be attached to other proteins, thereby converting them into energy-regulated proteins. These discoveries provide insight into how extreme energy deprivation can impact Sirt1 activity and underscore the complex nature of Sirt1 structure and regulation

Activation of Peroxisome Proliferator-Activated Receptor Gamma by Rosiglitazone Increases Sirt6 Expression and Ameliorates Hepatic Steatosis in Rats

Sirt6 has been implicated in the regulation of hepatic lipid metabolism and the development of hepatic steatosis. The aim of this study was to address the potential role of Sirt6 in the protective effects of rosiglitazone (RGZ) on hepatic steatosis.) by stomach gavage for 6 weeks. The involvement of Sirt6 in the RGZ's regulation was evaluated by Sirt6 knockdown in AML12 mouse hepatocytes.RGZ treatment ameliorated hepatic lipid accumulation and increased expression of Sirt6, peroxisome proliferator-activated receptor gamma coactivtor-1-α (Ppargc1a/PGC1-α) and Forkhead box O1 (Foxo1) in rat livers. AMP-activated protein kinase (AMPK) phosphorylation was also increased by RGZ, accompanied by alterations in phosphorylation of LKB1. Interestingly, in free fatty acid-treated cells, Sirt6 knockdown increased hepatocyte lipid accumulation measured as increased triglyceride contents (p = 0.035), suggesting that Sirt6 may be beneficial in reducing hepatic fat accumulation. In addition, Sirt6 knockdown abolished the effects of RGZ on hepatocyte fat accumulation, mRNA and protein expression of Ppargc1a/PGC1-α and Foxo1, and phosphorylation levels of LKB1 and AMPK, suggesting that Sirt6 is involved in RGZ-mediated metabolic effects.Our results demonstrate that RGZ significantly decreased hepatic lipid accumulation, and that this process appeared to be mediated by the activation of the Sirt6-AMPK pathway. We propose Sirt6 as a possible therapeutic target for hepatic steatosis

Barnase as a New Therapeutic Agent Triggering Apoptosis in Human Cancer Cells

RNases are currently studied as non-mutagenic alternatives to the harmful DNA-damaging anticancer drugs commonly used in clinical practice. Many mammalian RNases are not potent toxins due to the strong inhibition by ribonuclease inhibitor (RI) presented in the cytoplasm of mammalian cells.In search of new effective anticancer RNases we studied the effects of barnase, a ribonuclease from Bacillus amyloliquefaciens, on human cancer cells. We found that barnase is resistant to RI. In MTT cell viability assay, barnase was cytotoxic to human carcinoma cell lines with half-inhibitory concentrations (IC(50)) ranging from 0.2 to 13 microM and to leukemia cell lines with IC(50) values ranging from 2.4 to 82 microM. Also, we characterized the cytotoxic effects of barnase-based immunoRNase scFv 4D5-dibarnase, which consists of two barnase molecules serially fused to the single-chain variable fragment (scFv) of humanized antibody 4D5 that recognizes the extracellular domain of cancer marker HER2. The scFv 4D5-dibarnase specifically bound to HER2-positive cells and was internalized via receptor-mediated endocytosis. The intracellular localization of internalized scFv 4D5-dibarnase was determined by electronic microscopy. The cytotoxic effect of scFv 4D5-dibarnase on HER2-positive human ovarian carcinoma SKOV-3 cells (IC(50) = 1.8 nM) was three orders of magnitude greater than that of barnase alone. Both barnase and scFv 4D5-dibarnase induced apoptosis in SKOV-3 cells accompanied by internucleosomal chromatin fragmentation, membrane blebbing, the appearance of phosphatidylserine on the outer leaflet of the plasma membrane, and the activation of caspase-3.These results demonstrate that barnase is a potent toxic agent for targeting to cancer cells

HIF-1 and SKN-1 Coordinate the Transcriptional Response to Hydrogen Sulfide in Caenorhabditis elegans

Hydrogen sulfide (H2S) has dramatic physiological effects on animals that are associated with improved survival. C. elegans grown in H2S are long-lived and thermotolerant. To identify mechanisms by which adaptation to H2S effects physiological functions, we have measured transcriptional responses to H2S exposure. Using microarray analysis we observe rapid changes in the abundance of specific mRNAs. The number and magnitude of transcriptional changes increased with the duration of H2S exposure. Functional annotation suggests that genes associated with protein homeostasis are upregulated upon prolonged exposure to H2S. Previous work has shown that the hypoxia-inducible transcription factor, HIF-1, is required for survival in H2S. In fact, we show that hif-1 is required for most, if not all, early transcriptional changes in H2S. Moreover, our data demonstrate that SKN-1, the C. elegans homologue of NRF2, also contributes to H2S-dependent changes in transcription. We show that these results are functionally important, as skn-1 is essential to survive exposure to H2S. Our results suggest a model in which HIF-1 and SKN-1 coordinate a broad transcriptional response to H2S that culminates in a global reorganization of protein homeostasis networks

The activating mutation R201C in GNAS promotes intestinal tumourigenesis in Apc(Min/+) mice through activation of Wnt and ERK1/2 MAPK pathways.

Somatically acquired, activating mutations of GNAS, the gene encoding the stimulatory G-protein Gsalpha subunit, have been identified in kidney, thyroid, pituitary, leydig cell, adrenocortical and, more recently, in colorectal tumours, suggesting that mutations such as R201C may be oncogenic in these tissues. To study the role of GNAS in intestinal tumourigenesis, we placed GNAS R201C under the control of the A33-antigen promoter (Gpa33), which is almost exclusively expressed in the intestines. The GNAS R201C mutation has been shown to result in the constitutive activation of Gsalpha and adenylate cyclase and to lead to the autonomous synthesis of cyclic adenosine monophosphate (cAMP). Gpa33(tm1(GnasR201C)Wtsi/+) mice showed significantly elevated cAMP levels and a compensatory upregulation of cAMP-specific phosphodiesterases in the intestinal epithelium. GNAS R201C alone was not sufficient to induce tumourigenesis by 12 months, but there was a significant increase in adenoma formation when Gpa33(tm1(GnasR201C)Wtsi/+) mice were bred onto an Apc(Min/+) background. GNAS R201C expression was associated with elevated expression of Wnt and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK) pathway target genes, increased phosphorylation of ERK1/2 MAPK and increased immunostaining for the proliferation marker Ki67. Furthermore, the effects of GNAS R201C on the Wnt pathway were additive to the inactivation of Apc. Our data strongly suggest that activating mutations of GNAS cooperate with inactivation of APC and are likely to contribute to colorectal tumourigenesis

SirT3 suppresses hypoxia inducible factor 1α and tumor growth by inhibiting mitochondrial ROS production

It has become increasing clear that alterations in cellular metabolism have a key role in the generation and maintenance of cancer. Some of the metabolic changes can be attributed to the activation of oncogenes or loss of tumor suppressors. Here, we show that the mitochondrial sirtuin, SirT3, acts as a tumor suppressor via its ability to suppress reactive oxygen species (ROS) and regulate hypoxia inducible factor 1α (HIF-1α). Primary mouse embryo fibroblasts (MEFs) or tumor cell lines expressing SirT3 short-hairpin RNA exhibit a greater potential to proliferate, and augmented HIF-1α protein stabilization and transcriptional activity in hypoxic conditions. SirT3 knockdown increases tumorigenesis in xenograft models, and this is abolished by giving mice the anti-oxidant N-acetyl cysteine. Moreover, overexpression of SirT3 inhibits stabilization of HIF-1α protein in hypoxia and attenuates increases in HIF-1α transcriptional activity. Critically, overexpression of SirT3 decreases tumorigenesis in xenografts, even when induction of the sirtuin occurs after tumor initiation. These data suggest that SirT3 acts to suppress the growth of tumors, at least in part through its ability to suppress ROS and HIF-1α

SIRT1 Overexpression Antagonizes Cellular Senescence with Activated ERK/S6k1 Signaling in Human Diploid Fibroblasts

Sir2, a NAD-dependent deacetylase, modulates lifespan in yeasts, worms and flies. The SIRT1, mammalian homologue of Sir2, regulates signaling for favoring survival in stress. But whether SIRT1 has the function to influence cell viability and senescence under non-stressed conditions in human diploid fibroblasts is far from unknown. Our data showed that enforced SIRT1 expression promoted cell proliferation and antagonized cellular senescence with the characteristic features of delayed Senescence-Associated β-galactosidase (SA-β-gal) staining, reduced Senescence-Associated Heterochromatic Foci (SAHF) formation and G1 phase arrest, increased cell growth rate and extended cellular lifespan in human fibroblasts, while dominant-negative SIRT1 allele (H363Y) did not significantly affect cell growth and senescence but displayed a bit decreased lifespan.. Western blot results showed that SIRT1 reduced the expression of p16INK4A and promoted phosphorylation of Rb. Our data also exposed that overexpression of SIRT1 was accompanied by enhanced activation of ERK and S6K1 signaling. These effects were mimicked in both WI38 cells and 2BS cells by concentration-dependent resveratrol, a SIRT1 activator. It was noted that treatment of SIRT1-.transfected cells with Rapamycin, a mTOR inhibitor, reduced the phosphorylation of S6K1 and the expression of Id1, implying that SIRT1-induced phosphorylation of S6K1 may be partly for the decreased expression of p16INK4A and promoted phosphorylation of Rb in 2BS. It was also observed that the expression of SIRT1 and phosphorylation of ERK and S6K1 was declined in senescent 2BS. These findings suggested that SIRT1-promoted cell proliferation and antagonized cellular senescence in human diploid fibroblasts may be, in part, via the activation of ERK/ S6K1 signaling