SUMMARY Hepatic metabolic derangements are key components in the development of fatty liver, insulin resistance, and atherosclerosis. SIRT1, a NAD+-dependent protein deacetylase, is an important regulator of energy homeostasis in response to nutrient availability. Here we demonstrate that hepatic SIRT1 regulates lipid homeostasis by positively regulating PPARα, a nuclear receptor that mediates the adaptive response to fasting and starvation. Hepatocyte-specific deletion of SIRT1 impairs PPARα signaling and decreases fatty acid β-oxidation, whereas overexpression of SIRT1 induces the expression of PPARα targets. SIRT1 interacts with PPARα and is required to activate PPARα co-activator PGC-1α. When challenged with a high-fat diet, liver-specific SIRT1 knockout mice develop hepatic steatosis, hepatic inflammation, and endoplasmic reticulum stress. Taken together, our data indicate that SIRT1 plays a vital role in the regulation of hepatic lipid homeostasis, and that pharmacological activation of SIRT1 may be important for the prevention of obesity-associated metabolic diseases.

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Peroxisome proliferator-activated receptor ␣ (PPAR␣) plays a central role in the cell-specific pleiotropic responses induced by structurally diverse synthetic chemicals designated as peroxisome proliferators. Transcriptional regulation by liganded nuclear receptors involves the participation of cofactors that form multiprotein complexes to achieve cell-and gene-specific transcription. Here we report the identification of such a transcriptionally active PPAR␣-interacting cofactor (PRIC) complex from rat liver nuclear extracts that interacts with full-length PPAR␣ in the presence of ciprofibrate, a synthetic ligand, and leukotriene B 4, a natural ligand. The liganded PPAR␣-PRIC complex enhanced transcription from a peroxisomal enoyl-CoA hydratase͞L-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme gene promoter template that contains peroxisome proliferator response elements. Rat liver PRIC complex comprises some 25 polypeptides, and their identities were established by mass spectrometry and limited sequence analysis. Eighteen of these peptides contain one or more LXXLL motifs necessary for interacting with nuclear receptors. PRIC complex includes known coactivators or coactivator-binding proteins (CBP, SRC-1, PBP, PRIP, PIMT, TRAP100, SUR-2, and PGC-1), other proteins that have not previously been described in association with transcription complexes (CHD5, TOG, and MORF), and a few novel polypeptides designated PRIC300, -285, -215, -177, and -145. We describe the cDNA for PRIC285, which contains five LXXLL motifs. It interacts with PPAR␣ and acts as a coactivator by moderately stimulating PPAR␣-mediated transcription in transfected cells. We conclude that liganded PPAR␣ recruits a distinctive multiprotein complex from rat liver nuclear extracts. The composition of this complex may provide insight into the basis of tissue and species sensitivity to peroxisome proliferators.

8-oxoguanine DNA glycosylase (Oggl) repairs 8-oxo-7,8-dihydroxyguanine (8-oxoG), one of the most abundant DNA adducts caused by oxidative stress. In the mitochondria, Oggl is thought to prevent activation of the intrinsic apoptotic pathway in response to oxidative stress by augmenting DNA repair. However, the predominance of the β-Oggl isoform, which lacks 8-oxoG DNA glycosylase activity, suggests that mitochondrial Oggl functions in a role independent of DNA repair. We report here that overexpression of mitochondria-targeted human α-hOggl (mt-hOggl) in human lung adenocarcinoma cells with some alveolar epithelial cell characteristics (A549 cells) prevents oxidant-induced mitochondrial dysfunction and apoptosis by preserving mitochondrial aconitase. Importantly, mitochondrial α-hOggl mutants lacking 8-oxoG DNA repair activity were as effective as wild-type mt-hOggl in preventing oxidant-induced caspase-9 activation, reductions in mitochondrial aconitase and apoptosis suggesting that the protective effects of mt-hOgg 1 occur independent of DNA repair. Notably, wild-type and mutant mt-hOggl co-precipitate with mitochondrial aconitase. Furthermore, overexpression of mitochondrial aconitase abolishes oxidant-induced apoptosis whereas hOggl silencing using shRNA reduces mitochondrial aconitase and augments apoptosis. These findings suggest a novel mechanism that mt-hOggl acts as a mitochondrial aconitase chaperone protein to prevent oxidant-mediated mitochondrial dysfunction and apoptosis that might be important in the molecular events underlying oxidant-induced toxicity.

Asbestos causes pulmonary toxicity in part by generating reactive oxygen species that cause DNA damage. We previously showed that the mitochondria-regulated (intrinsic) death pathway mediates alveolar epithelial cell (AEC) DNA damage and apoptosis. Because p53 regulates the DNA damage response in part by inducing intrinsic cell death, we determined whether p53-dependent transcriptional activity mediates asbestos-induced AEC mitochondrial dysfunction and apoptosis. We show that inhibitors of p53-dependent transcriptional activation (pifithrin and type 16-E6 protein) block asbestos-induced AEC mitochondrial membrane potential change (⌬⌿m), caspase 9 activation, and apoptosis. We demonstrate that asbestos activates p53 promoter activity, mRNA levels, protein expression, and Bax and p53 mitochondrial translocation. Further, pifithrin, E6, phytic acid, or 0 -A549 cells (cells incapable of mitochondrial reactive oxygen species production) block asbestosinduced p53 activation. Finally, we show that asbestos augments p53 expression in cells at the bronchoalveolar duct junctions of rat lungs and that phytic acid prevents this. These data suggest that p53-dependent transcription pathways mediate asbestos-induced AEC mitochondria-regulated apoptosis. This suggests an important interactive effect between p53 and the mitochondria in the pathogenesis of asbestos-induced pulmonary toxicity that may have broader implications for our understanding of pulmonary fibrosis and lung cancer.

Suchy FJ. Histone H3K4 trimethylation by MLL3 as part of ASCOM complex is critical for NR activation of bile acid transporter genes and is downregulated in cholestasis. Am J Physiol Gastrointest Liver Physiol 300: G771-G781, 2011. First published February 17, 2010 doi:10.1152/ajpgi.00499.2010.-The nuclear receptor Farnesoid x receptor (FXR) is a critical regulator of multiple genes involved in bile acid homeostasis. The coactivators attracted to promoters of FXR target genes and epigenetic modifications that occur after ligand binding to FXR have not been completely defined, and it is unknown whether these processes are disrupted during cholestasis. Using a microarray, we identified decreased expression of mixed lineage leukemia 3 (MLL3), a histone H3 lysine 4 (H3K4) lysine methyl transferase at 1 and 3 days of post-common bile duct ligation (CBDL) in mice. Chromatin immunoprecipitation analysis (ChIP) analysis revealed that H3K4me3 of transporter promoters by MLL3 as part of activating signal cointegrator-2 -containing complex (ASCOM) is essential for activation of bile salt export pump (BSEP), multidrug resistance associated protein 2 (MRP2), and sodium taurocholate cotransporting polypeptide (NTCP) genes by FXR and glucocorticoid receptor (GR). Knockdown of nuclear receptor coactivator 6 (NCOA6) or MLL3/MLL4 mRNAs by small interfering RNA treatment led to a decrease in BSEP and NTCP mRNA levels in hepatoma cells. Human BSEP promoter transactivation by FXR/RXR was enhanced in a dose-dependent fashion by NCOA6 cDNA coexpression and decreased by AdsiNCOA6 infection in HepG2 cells. GST-pull down assays showed that domain 3 and 5 of NCOA6 (LXXLL motifs) interacted with FXR and that the interaction with domain 5 was enhanced by chenodeoxycholic acid. In vivo ChIP assays in HepG2 cells revealed ligand-dependent recruitment of ASCOM complex to FXR element in BSEP and GR element in NTCP promoters, respectively. ChIP analysis demonstrated significantly diminished recruitment of ASCOM complex components and H3K4me3 to Bsep and Mrp2 promoter FXR elements in mouse livers after CBDL. Taken together, these data show that the "H3K4me3" epigenetic mark is essential to activation of BSEP, NTCP, and MRP2 genes by nuclear receptors and is downregulated in cholestasis. hepatoma cells; microarray; epigenetics; gene regulation; activating signal cointegrator-2-containing complex; H3 lysine 4; nuclear receptors; mixed lineage leukemia 3 LIGAND-ACTIVATED NUCLEAR RECEPTORS (NRs), such as Farnesoid x receptor (FXR) and glucocorticoid receptor (GR), initiate transcription by binding to specific DNA regulatory elements in promoters of target genes and recruiting coactivator proteins to modify chromatin structure and induce assembly of the transcription complex containing RNA polymerase II (PolII) (2, 3, 6, 18). The primary coactivators including the p160 coactivators such as TIF2/GRIP-1/SRC-2 and related members of the p300 family bind directly to ligand-activated NRs and recruit an array of secondary coactivators such as p300/cAMP resp...

constitutive androstane receptor N uclear receptors constitute a large superfamily of transcription factors that regulate a diverse array of biological process, including development, differentiation, and neoplasia as well as energy and xenobiotic metabolism (1-3). The binding of specific ligands to nuclear receptors influences the recruitment of initial complex of coactivator proteins with histone acetyltransferase activity necessary for remodeling chromatin (1-3). Docking of several other cofactors results in the formation of a multiprotein coactivator complex, variously called thyroid hormone receptor-associated protein (TRAP)͞vitamin D receptorinteracting protein͞activator-recruited cofactor͞mediator complex, that facilitates interaction with RNA polymerase II and the general basal transcription machinery

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