Genes In Mouse Liver Research Articles

Liver Specific Delivery of siRNA Targeting EGLN Prolyl Hydroxylases Activates Hepatic Erythropoietin Production and Stimulates Erythropoiesis,

Abstract 3161Anemia in chronic kidney disease patients due to impaired renal production of erythropoietin (EPO) and insufficient erythropoiesis is a significant public health problem. One approach to compensate for the low EPO levels in patients with impaired kidney function would be to stimulate the levels of EPO production from non-renal sources. It is well known that during fetal development the liver serves as the primary producer of EPO until the kidney becomes the dominant source after birth. Negative regulation of EPO production is mediated by the EGLN family of prolyl hydroxylases (PHDs1-3) that play an important role in oxygen sensing by targeting the transcription factor hypoxia inducible factor (HIF) for degradation via the proteasome. HIF stabilization and activity is required to mediate EPO gene transcription. Previously it has been shown that liver specific conditional knockout of all three PHDs is required to activate HIF and induce EPO production in liver (Minamishima et al. Science 2010). Here we show that simultaneous siRNA mediated knockdown of all 3 PHD genes in mouse liver using lipid nanoparticles (LNPs) can induce hepatic EPO mRNA activation, elevation of serum EPO levels and stimulation of erythropoiesis. We extend these data by examining siRNA knockdown of different combinations of the 3 PHD genes and demonstrate that PHD2 is the dominant PHD gene regulating hepatic EPO production. In addition, due to the specificity of the LNP delivery system we show that the HIF activation and increase in EPO mRNA occurs specifically in liver. Increases in serum EPO and hematocrit were durable for two weeks and one month after a single intravenous dose of LNP siRNA. Furthermore, PHD siRNA silencing in a 5/6 nephrectomy model successfully elevates hemoglobin levels and corrects anemia. In conclusion, targeting of EGLN prolyl hydroxylase genes with siRNA therapeutics has potential in the treatment of anemia associated with chronic kidney disease and provides liver-specific target gene regulation. Disclosures:Querbes:Alnylam Pharmaceuticals: Employment. Bogorad:Alnylam Pharmaceuticals: Research Funding. Moslehi:Alnylam Pharmaceuticals: Honoraria. Akinc:Alnylam Pharmaceuticals: Employment. Wong:Alnylam Pharmaceuticals: Employment. Zurenko:Alnylam Pharmaceuticals: Employment. Qin:Alnylam Pharmaceuticals: Employment. Hettinger:Alnylam Pharmaceuticals, Inc.: Employment. Kuchimanchi:Alnylam Pharmaceuticals: Employment. Charisse:Alnylam Pharmaceuticals: Employment. Sah:Alnylam Pharmaceuticals, Inc.: Employment. Fitzgerald:Alnylam Pharmaceuticals: Employment. Kotelianski:Alnylam Pharmaceuticals: Employment. Kaelin:Fibrogen: Consultancy, Equity Ownership.

CREB and ChREBP oppositely regulate SIRT1 expression in response to energy availability.

The nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase SIRT1 is a major metabolic regulator activated by energy stresses such as fasting or calorie restriction. SIRT1 activation during fasting not only relies on the increase in the NAD(+)/NADH ratio caused by energy deprivation but also involves an upregulation of SIRT1 mRNA and protein levels in various metabolic tissues. We demonstrate that SIRT1 expression is controlled systemically by the activation of the cyclic AMP response-element-binding protein upon low nutrient availability. Conversely, in the absence of energetic stress, the carbohydrate response-element-binding protein represses the expression of SIRT1. Altogether, these results demonstrate that SIRT1 expression is tightly controlled at the transcriptional level by nutrient availability and further underscore that SIRT1 is a crucial metabolic checkpoint connecting the energetic status with transcriptional programmes.

Overweight induced by chronic risperidone exposure is correlated with overexpression of the SREBP-1c and FAS genes in mouse liver

Weight gain and metabolic disturbances, such as dyslipidemia and hyperglycaemia, are common side effects of most antipsychotic drugs, including risperidone. The aim of this study was to investigate the effects of chronic treatment with risperidone on body weight, fat accumulation, liver weight, and hepatic expression of key genes involved in lipid metabolism in female mice. We also addressed the mechanism of risperidone induction of metabolic side effects by exploring its effect on lipid and cholesterol metabolism in primary cultures of rat hepatocytes. Elevenweeks of treatment with long-acting risperidone (12.5mpk/week) resulted in a significant weight gain associated with an increase of liver and adipose tissue weight. These effects were positively correlated with hepatic mRNA induction of two key genes involved in lipogenesis: sterol regulatory element binding protein-1c (SREBP-1c) and fatty acid synthase (FAS). Furthermore, in line with these in vivo results, risperidone elicited significant inductions of SREBP-1 maturation and FAS mRNA expression in primary cultures of rat hepatocytes associated with an increase of free fatty acid, triacylglycerol, and phospholipid synthesis as assessed by acetate incorporation. The current investigations underscore the usefulness of a mouse model to study the weight gain observed with risperidone treatment in humans. This study shows that risperidone induces similar effects in the liver (in vivo) and in hepatocyte cell cultures (in vitro) on the expression of key genes and/or proteins that control lipid metabolism. This suggests that risperidone could alter lipid metabolism in the liver and induce weight gain in a way that is partly independent of its action on the central nervous system.

Use of Stringent Selection Parameters for the Identification of Possible Selenium-Responsive Marker Genes in Mouse Liver and Gastrocnemius

Selenium is a trace element that, although toxic in higher concentrations, is essential for human and animal health. In this study, we looked at microarray-based gene expression patterns from liver and gastrocnemius tissues in mice fed either a selenium-deficient diet or diets containing sodium selenite, selenomethionine, or a yeast-derived selenium supplement. A p value cutoff of 0.01 was used to identify a select set of selenium-responsive genes that were consistently differentially expressed across three age groups of mice with both ANOVA and t test analyses. A total of 19 gene transcripts were found to be differentially expressed across the three age groups with at least one selenium-deficient/selenium-supplemented diet comparison. Of those 19 genes, 12 had been previously identified as selenoprotein-encoding genes, and four of the genes, Gpx1, Selh, Sep15, and Sepw1, were differentially expressed in both tissues, all three mouse age groups, and all three diet comparisons. Activities associated with non-selenoproteins encoded by selenium-responsive genes included transport and stress response. The selenophosphate synthetase 2 gene Sephs2 in gastrocnemius tissue and the solute carrier gene Slc48a1 in liver tissue, both up-regulated with selenium-deficient diets compared to all three selenium-supplemented diets, are previously overlooked candidates for dietary selenium marker genes.

Zonation of heme synthesis enzymes in mouse liver and their regulation by β-catenin and Ha-ras

Cytochrome P450 (CYP) hemoproteins play an important role in hepatic biotransformation. Recently, β-catenin and Ha-ras signaling have been identified as players controlling transcription of various CYP genes in mouse liver. The aim of the present study was to analyze the role of β-catenin and Ha-ras in the regulation of heme synthesis. Heme synthesis-related gene expression was analyzed in normal liver, in transgenic mice expressing activated β-catenin or Ha-ras, and in hepatomas. Regulation of the aminolevulinate dehydratase promoter was studied in vitro. Elevated expression of mRNAs and proteins involved in heme biosynthesis was linked to β-catenin activation in perivenous hepatocytes, in transgenic hepatocytes, and in hepatocellular tumors. Stimulation of the aminolevulinate dehydratase promoter by β-catenin was independent of the β-catenin/T-cell-specific transcription factor dimer. By contrast, activation of Ha-ras repressed heme synthesis-related gene expression. The present data suggest that β-catenin enhances the expression of both CYPs and heme synthesis-related genes, thus coordinating the availability of CYP apoprotein and its prosthetic group heme. The reciprocal regulation of heme synthesis by β-catenin and Ha-ras-dependent signaling supports our previous hypothesis that antagonistic action of these pathways plays a major role in the control of zonal gene expression in healthy mouse liver and aberrant expression patterns in hepatocellular tumors.

Epigenetic marks of nuclear‐encoded mitochondrial genes by DNA methylation

Mitochondria are semi‐self‐replicating organelles with it's intrinsic mitochondrial DNA (mtDNA). mtDNA is not under epigenetic regulation and, thus, unable to confer tissue or cell type dependent information in mammalian cells. However, of approximately 1,200 proteins in mitochondria, only 13 proteins are encoded by mtDNA. To investigate whether there are differences of epigenetic information of nuclear mitochondrial genes depending on tissues, we studied on tissue‐dependent and differentially methylated regions (T‐DMRs) in the promoters of nuclear mitochondrial genes. We analyzed methylation status of all known mitochondrial genes in mouse liver, brain, and heart. More than 200 mitochondrial genes were identified to have T‐DMRs which are specifically hypomethylated in one of the tissues. The mitochondrial genes with T‐DMRs were related to various mitochondrial functions. We also found that liver‐specific transcription factor binding site were enriched in the genes with liver‐hypomethylated T‐DMR. In conclusion, each tissue examined has inherent DNA methylation pattern of mitochondrial genes, and epigenetic regulation underlies the tissue‐dependent functions of mitochondria.This work was supported by grant from NIBIO.

Intrinsic sex differences in the early growth hormone responsiveness of sex-specific genes in mouse liver.

Sex differences in liver gene expression are dictated by sex differences in circulating GH profiles. Presently, the pituitary hormone dependence of mouse liver gene expression was investigated on a global scale to discover sex-specific early GH response genes that could contribute to sex-specific regulation of downstream GH targets and to ascertain whether intrinsic sex differences characterize hepatic responses to plasma GH stimulation. Global RNA expression analysis identified two distinct classes of sex-specific mouse liver genes: genes subject to positive regulation (class I) and genes subject to negative regulation by pituitary hormones (class II). Genes activated or repressed in hypophysectomized (Hypox) mouse liver within 30-90 min of GH pulse treatment at a physiological dose were identified as putative direct targets of GH action (early response genes). Intrinsic sex differences in the GH responsiveness of a subset of these early response genes were observed. Notably, 45 male-specific genes, including five encoding transcriptional regulators that may mediate downstream sex-specific transcriptional responses, were induced by GH within 30 min in Hypox male but not Hypox female mouse liver. The early GH response genes were enriched in 29 male-specific targets of the transcription factor myocyte enhancer factor 2, whose activation in hepatic stellate cells is associated with liver fibrosis leading to hepatocellular carcinoma, a male-predominant disease. Thus, the rapid activation by GH pulses of certain sex-specific genes is modulated by intrinsic sex-specific factors, which may be associated with prior hormone exposure (epigenetic mechanisms) or genetic factors that are pituitary-independent, and could contribute to sex differences in predisposition to liver cancer or other hepatic patho-physiologies.

Intrinsic Sex Differences in the Early Growth Hormone Responsiveness of Sex-Specific Genes in Mouse Liver

Sex differences in liver gene expression are dictated by sex differences in circulating GH profiles. Presently, the pituitary hormone dependence of mouse liver gene expression was investigated on a global scale to discover sex-specific early GH response genes that could contribute to sex-specific regulation of downstream GH targets and to ascertain whether intrinsic sex differences characterize hepatic responses to plasma GH stimulation. Global RNA expression analysis identified two distinct classes of sex-specific mouse liver genes: genes subject to positive regulation (class I) and genes subject to negative regulation by pituitary hormones (class II). Genes activated or repressed in hypophysectomized (Hypox) mouse liver within 30-90 min of GH pulse treatment at a physiological dose were identified as putative direct targets of GH action (early response genes). Intrinsic sex differences in the GH responsiveness of a subset of these early response genes were observed. Notably, 45 male-specific genes, including five encoding transcriptional regulators that may mediate downstream sex-specific transcriptional responses, were induced by GH within 30 min in Hypox male but not Hypox female mouse liver. The early GH response genes were enriched in 29 male-specific targets of the transcription factor myocyte enhancer factor 2, whose activation in hepatic stellate cells is associated with liver fibrosis leading to hepatocellular carcinoma, a male-predominant disease. Thus, the rapid activation by GH pulses of certain sex-specific genes is modulated by intrinsic sex-specific factors, which may be associated with prior hormone exposure (epigenetic mechanisms) or genetic factors that are pituitary-independent, and could contribute to sex differences in predisposition to liver cancer or other hepatic patho-physiologies.

Discrimination of Tumorigenic Triazole Conazoles from Phenobarbital by Transcriptional Analyses of Mouse Liver Gene Expression

Conazoles are fungicides used to control fungal growth in environmental settings and to treat humans with fungal infections. Mouse hepatotumorigenic conazoles display many of the same hepatic toxicologic responses as the mouse liver carcinogen phenobarbital (PB): constitutive androstane receptor (CAR) activation, hypertrophy, Cyp2b induction, and increased cell proliferation. The goal of this study was to apply transcriptional analyses to hepatic tissues from mice exposed to PB, propiconazole (Pro) or triadimefon (Tri) at tumorigenic exposure levels to reveal similarities and differences in response among these treatments. Mice were administered diets containing PB (850 ppm), Pro (2500 ppm), or Tri (1800 ppm) for 4 and 30 days. Targeted transcriptomic analyses were conducted at the gene level examining differentially expressed genes (DEGs), and subsets of DEGs: cell cycle genes, and transcription factors. Analyses were also conducted on function, pathway and network levels examining Ingenuity Pathway Analysis Tox Lists and Canonical Pathways, and Gene-Go MetaCore dynamic networks and their central hubs. Genes expressed by PB or the two conazoles were also compared with those genes associated with human hepatocellular cancer. The results from these analyses indicated greater differences between PB and the two conazoles than similarities. Significant commonalities between the two conazole treatments were also noted. We posit that the transcriptional profiles of tissues exposed to toxic chemicals inherently contain their mechanisms of toxicity. We conclude that although PB and these 2 conazoles induce mouse liver tumors and exhibit similar toxicological responses, their transcriptional profiles are significantly different and thus their mechanisms of tumorigenic action are likely to differ.

Dose-dependent alterations in gene expression in mouse liver induced by diethylnitrosamine and ethylnitrosourea and determined by quantitative real-time PCR

We examined the dose-dependency of gene expression changes for 51 genes in mouse liver treated with two N-nitroso genotoxic hepatocarcinogens, diethylnitrosamine (DEN) and ethylnitrosourea (ENU) by quantitative real-time PCR (qPCR). DEN (3, 9, 27 and 80 mg/kg bw) or ENU (6, 17, 50 and 150 mg/kg bw) was injected intraperitoneally into groups of five male 9-week-old B6C3F 1 mice and the livers were dissected after 4 h and 28 days. Total RNA from pooled livers was reverse-transcribed to cDNA and the amount of each gene was quantified by qPCR. Results were analyzed by hierarchical and k-means clustering and ingenuity pathway analysis (IPA). The most characteristic result was a similar dose-dependency of gene expression changes with DEN and ENU. Twenty-one genes exhibited a distinct dose-dependent increase in expression at 4 h for both carcinogens [ Bax, Btg2, Ccng1, Cdkn1a, Cyp4a10, Cyp21a1, Fos, Gadd45b, Gdf15, Hmox1, Hspb1, Isg20l1, Jun, Mbd1, Mdm2, Myc, Net1, Plk2, Ppp1r3c, Rcan1 and Tubb2c], although the increase in gene expression due to ENU was generally weaker than that due to DEN. Only Gdf15 showed a dose-dependent increase in expression at 28 days for both carcinogens. The differences between DEN and ENU were in the expression of additional genes (7 for DEN and 8 for ENU). IPA extracted five gene networks: Network-1 included genes related to cancer and cell cycle arrest and associated with Bax, Btg2, Ccng1, Cdkn1a, Gadd45b, Gdf15, Hspb1, Mdm2 and Plk2 and Network-2 was related to DNA replication, recombination, repair and cell death and associated with Cyp21a1, Gdf15, Ppp1r3c, Rcan1 and Tubb2c. The present results show a distinct dose-dependency of gene expression changes induced by DEN and ENU. These changes were associated with cancer, cell cycle arrest, DNA replication, recombination, repair and cell death and were seen not only at 4 h but also, for some, at 28 days after administration.

ChREBP, but not LXRs, is required for the induction of glucose-regulated genes in mouse liver

The transcription factor carbohydrate-responsive element-binding protein (ChREBP) has emerged as a central regulator of lipid synthesis in liver because it is required for glucose-induced expression of the glycolytic enzyme liver-pyruvate kinase (L-PK) and acts in synergy with SREBP to induce lipogenic genes such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). Liver X receptors (LXRs) are also important regulators of the lipogenic pathway, and the recent finding that ChREBP is a direct target of LXRs and that glucose itself can bind and activate LXRs prompted us to study the role of LXRs in the induction of glucose-regulated genes in liver. Using an LXR agonist in wild-type mice, we found that LXR stimulation did not promote ChREBP phosphorylation or nuclear localization in the absence of an increased intrahepatic glucose flux. Furthermore, the induction of ChREBP, L-PK, and ACC by glucose or high-carbohydrate diet was similar in LXRalpha/beta knockout compared with wild-type mice, suggesting that the activation of these genes by glucose occurs by an LXR-independent mechanism. We used fluorescence resonance energy transfer analysis to demonstrate that glucose failed to promote the interaction of LXRalpha/beta with specific cofactors. Finally, siRNA silencing of ChREBP in LXRalpha/beta knockout hepatocytes abrogated glucose-induced expression of L-PK and ACC, further demonstrating the central role of ChREBP in glucose signaling. Taken together, our results demonstrate that glucose is required for ChREBP functional activity and that LXRs are not necessary for the induction of glucose-regulated genes in liver.

Analysis of 2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced Proteome Changes in 5L Rat Hepatoma Cells Reveals Novel Targets of Dioxin Action Including the Mitochondrial Apoptosis Regulator VDAC2

As part of a comprehensive survey of the impact of the environmental pollutant and hepatocarcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the proteome of hepatic cells, we have performed a high resolution two-dimensional gel electrophoresis study on the rat hepatoma cell line 5L. 78 protein species corresponding to 73 different proteins were identified as up- or down-regulated following exposure of the cells to 1 nm TCDD for 8 h. There was an overlap of only nine proteins with those detected as altered by TCDD in our recent study using the non-gel-based isotope-coded protein label method (Sarioglu, H., Brandner, S., Jacobsen, C., Meindl, T., Schmidt, A., Kellermann, J., Lottspeich, F., and Andrae, U. (2006) Quantitative analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced proteome alterations in 5L rat hepatoma cells using isotope-coded protein labels. Proteomics 6, 2407-2421) indicating a strong complementarity of the two approaches. For the majority of the altered proteins, an effect of TCDD on their abundance or posttranslational modifications had not been known before. Several observations suggest that a sizable fraction of the proteins with altered abundance was induced as an adaptive response to TCDD-induced oxidative stress that was demonstrated using the fluorescent probe dihydrorhodamine 123. A prominent group of these proteins comprised various enzymes for which there is evidence that their expression is regulated via the Keap1/Nrf2/antioxidant response element pathway. Other proteins included several involved in the maintenance of mitochondrial energy production and the regulation of the mitochondrial apoptotic pathway. A particularly intriguing finding was the up-regulation of the mitochondrial outer membrane pore protein, voltage-dependent anion channel-selective protein 2 (VDAC2), which was dependent on the presence of a functional aryl hydrocarbon receptor. The regulatability of VDAC2 protein abundance has not been described previously. In view of the recently discovered central role of VDAC2 as an inhibitor of the activation of the proapoptotic protein BAK and the mitochondrial apoptotic pathway, the present data point to a hitherto unrecognized mechanism by which TCDD may affect cellular homeostasis and survival.

Identification of age-dependently expressed genes in mouse liver by differential display–PCR analysis

The aim of this study was to identify genes expressed in an age-dependent manner in mouse ( Mus musculus) liver. To search for age-dependently expressed genes, we used a fluorescence differential display–PCR (FDD–PCR) technique on total RNA extracted from mouse livers collected at seven different developmental stages. All differentially expressed cDNAs detected by FDD–PCR were reamplified, subcloned and sequenced, and six genes were confirmed to show age-dependent expression by quantitative real-time PCR analysis. Nucleotide sequence analyses showed that four of them had high homology with known genes (mitochondrial DNA, cytosolic aldehyde dehydrogenase, cell division cycle 2-like 5 and complement component 8 alpha polypeptide), and two with expressed sequence tags of unknown genes. The FDD–PCR technique was effective for detecting novel age-dependently expressed genes, and also for newly characterizing individual expression patterns of known genes. The age-dependent expression patterns of known genes revealed in this study may provide an opportunity to investigate the unknown physiological roles of the proteins they encode.

Global Analysis of Age-Related Expression Profiles of Mouse Liver Genes: Toward Understanding Age-Related Homeostasis.

For understanding aging as well as various age-related diseases, it is critical to establish the basic regulatory mechanisms underlying age-related homeostatsis. Through systematic analyses of transgenic mice carrying factor IX and protein C genes, we recently discovered the first molecular mechanism of age-related homeostasis, ASE/AIE-mediated age-related regulatory mechanism of gene expression (Kurachi et. al., Science 1999; Zhang et. al., J. Biol. Chem. 2002; J. Thromb. Haemost. 2005). This laid the foundation for further studies toward an integrated understanding of age-related homeostasis. Here we report global analyses of age-related expression profiles of mouse liver genes. For quantification of expression levels of liver genes of mice at 1, 3, 6, 12, 18 and 24 month of age, Affymetrix GeneChip® Mouse Expression Array 430A (MOE430A with 23,643 probes) were used. These microarray analyses detected 9148 probes as qualified meaningful and the data were subjected to extensive analyses by GeneSpring and IPA network analysis softwares. Real time PCR analyses of representative genes were performed for verifying excellent reproducibility of age-related expression profiles determined by microarray analyses. Age-related stages, particularly of puberty and aging, were identified as the major phases for age-related changes in gene expression. Strong relationships between expression level and ontology, indicating that the highest expression gene group, next highest expression group and lowest level expression gene group are of secreted proteins, mitochondoreal proteins and nuclear regulatory protein, respectively. Most importantly, we successfully identified about a dozen unique and fundamental age-related gene expression patterns including those of age-related stable, gradual increase or decrease, and aging-related dramatic increase or decrease types of expression. These findings led us to a new hypothesis that complex and dynamic age-related regulations of genes are produced by a relatively small number of fundamental regulatory mechanisms. These mechanisms may function independently or in various combinations, thus explaining an essential feature of age-related homeostasis.

Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes

Achieving efficient in vivo delivery of siRNA to the appropriate target cell would be a major advance in the use of RNAi in gene function studies and as a therapeutic modality. Hepatocytes, the key parenchymal cells of the liver, are a particularly attractive target cell type for siRNA delivery given their central role in several infectious and metabolic disorders. We have developed a vehicle for the delivery of siRNA to hepatocytes both in vitro and in vivo, which we have named siRNA Dynamic PolyConjugates. Key features of the Dynamic PolyConjugate technology include a membrane-active polymer, the ability to reversibly mask the activity of this polymer until it reaches the acidic environment of endosomes, and the ability to target this modified polymer and its siRNA cargo specifically to hepatocytes in vivo after simple, low-pressure i.v. injection. Using this delivery technology, we demonstrate effective knockdown of two endogenous genes in mouse liver: apolipoprotein B (apoB) and peroxisome proliferator-activated receptor alpha (ppara). Knockdown of apoB resulted in clear phenotypic changes that included a significant reduction in serum cholesterol and increased fat accumulation in the liver, consistent with the known functions of apoB. Knockdown of ppara also resulted in a phenotype consistent with its known function, although with less penetrance than observed in apoB knockdown mice. Analyses of serum liver enzyme and cytokine levels in treated mice indicated that the siRNA Dynamic PolyConjugate was nontoxic and well tolerated.

The Putative Tumor Suppressor Tsc-22 is Downregulated Early in Chemically Induced Hepatocarcinogenesis and may be a Suppressor of Gadd45b

Tsc-22 is a novel tumor suppressor gene that represents a new class of transcription factors that has transcriptional repressor activity. We found Tsc-22 downregulation in livers from B6C3F1 mice following treatment for 2 weeks with carcinogenic doses of the antianxiety drug oxazepam (2500 ppm) or the peroxisome proliferator Wyeth-14,643 (500 ppm) but not with two other carcinogens such as o-nitrotoluene or methyleugenol or three noncarcinogens including p-nitrotoluene, eugenol, or acetaminophen. The expression of Tsc-22 was also repressed in B6C3F1 mouse liver tumors that were induced by several chemicals from 2-year carcinogenicity studies as well as in spontaneous liver tumors. To identify potential Tsc-22 target genes in mouse liver, we transfected small interference RNA (SiRNA) designed to inhibit Tsc-22 into murine liver BNL-CL.2 cells. We selected two potential transcriptional targets of Tsc-22, growth arrest and DNA damage-inducible gene 45 beta (Gadd45b) and leucine zipper, putative tumor suppressor 2 (Lzts2) to test based on our previous complementary DNA microarray studies, showing that expression of these cancer-associated genes was increased when Tsc-22 was repressed. SiRNA treatment of BNL-CL.2 cells with Tsc-22 oligonucleotides but not nonspecific oligonucleotides decreased RNA and protein expression of Tsc-22 by 80-90%, while expression of Gadd45b gene, but not Lzts2, was increased over time after an initial decrease. Treatment of these cells with oxazepam for 48 h also resulted in decreased Tsc-22 and increased Gadd45b expression. These data provide evidence that Tsc-22 is a suppressor of Gadd45b expression, which may contribute to an early antiapoptotic response.

Pharmacodynamic characterization of chemopreventive triterpenoids as exceptionally potent inducers of Nrf2-regulated genes

Synthetic triterpenoids have been developed, which are potent inducers of cytoprotective enzymes and inhibitors of inflammation, greatly improving on the weak activity of naturally occurring triterpenoids. An imidazolide triterpenoid derivative, 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im or TP235), has been previously shown to potently protect against hepatic tumorigenesis, acting in part by inducing cytoprotective genes through Keap1-Nrf2-antioxidant response element (ARE) signaling. In these studies, the pharmacodynamic activity of CDDO-Im is characterized in two distinct lines of ARE reporter mice and by measuring increases in Nqo1 transcript levels as a marker of cytoprotective gene induction. Oral administration of CDDO-Im induces ARE-regulated cytoprotective genes in many tissues in the mouse, including liver, lung, kidney, intestines, brain, heart, thymus, and salivary gland. CDDO-Im induces Nqo1 RNA transcripts in some organs at doses as low as 0.3 mumol/kg body weight (orally). A structure activity evaluation of 15 additional triterpenoids (a) confirmed the importance of Michael acceptor groups on both the A and C rings, (b) showed the requirement for a nitrile group at C-2 of the A ring, and (c) indicated that substituents at C-17 dramatically affected pharmacodynamic action in vivo. In addition to CDDO-Im, other triterpenoids, particularly the methyl ester CDDO-Me (TP155) and the dinitrile TP225, are extremely potent inducers of cytoprotective genes in mouse liver, lung, small intestine mucosa, and cerebral cortex. This pharmacodynamic characterization highlights the chemopreventive promise of several synthetic triterpenoids in multiple target organs.

Methionine flux to transsulfuration is enhanced in the long living Ames dwarf mouse

Long-lived Ames dwarf mice lack growth hormone, prolactin, and thyroid stimulating hormone. Additionally the dwarf mice have enzyme activities and levels that combat oxidative stress more efficiently than those of normal mice. We have shown that methionine metabolism in Ames mice is markedly different than in their wild type littermates. In our previous work we hypothesized that the flux of methionine to the transsulfuration pathway is enhanced in the dwarf mice. The current study was designed to determine whether the flux of methionine to the transsulfuration pathway is increased. We did this by injecting either l-[methyl- 3H]-methionine or l-[ 35S]-methionine into dwarf or normal mice and then determined retained label (in form of S-adenosylmethionine) 45 min later. The amount of retained hepatic 3H and 35S label was significantly reduced in the dwarf mice; at 45 min the specific radioactivity of SAM (pCi/nmol SAM) was 56% lower ( p < 0.05) for 3H-label and 64% lower ( p < 0.005) for 35S-label in dwarf than wild type mice. Retention of 35S was significantly lower in the brain (37%, p < 0.04) and kidney (47%, p < 0.02) of the dwarf compared to wild type mice; there was no statistical difference in retained 3H-label in either brain or kidney. This suggests that both the methyl-moiety and the carbon chain of methionine are lost much faster in the dwarf compared to the wild type mouse, implying that both transmethylation in the liver and transsulfuration in the liver, brain, and kidney are increased in the dwarf mice. As further support, we determined by real-time RT PCR the expression of methionine metabolism genes in livers of mice. Compared to wild type, the Ames dwarf had increased expression of methionine adenosyltransferase 1a (2.3-fold, p = 0.013), glycine N-methyltransferase (3.8-fold, p = 0.023), betaine homocysteine methyltransferase (5.5-fold, p = 0.0006), S-adenosylhomocysteine hydrolase (3.8-fold, p = 0.0005), and cystathionase (2.6-fold; tended to be increased, p = 0.055). Methionine synthase expression was significantly decreased in dwarf compared to wild type (0.48-fold, p = 0.023). These results confirm that the flux of methionine to transsulfuration is enhanced in the Ames dwarf. This, along with data from previous studies support the hypothesis that altered methionine metabolism plays a significant role in the oxidative defense of the dwarf mouse and that the mechanism for the enhanced oxidative defense may be through altered GSH metabolism as a result of the distinctive methionine metabolism.

Circadian Clocks Are Resounding in Peripheral Tissues

Circadian rhythms are prevalent in most organisms. Even the smallest disturbances in the orchestration of circadian gene expression patterns among different tissues can result in functional asynchrony, at the organism level, and may to contribute to a wide range of physiologic disorders. It has been reported that as many as 5%–10% of transcribed genes in peripheral tissues follow a circadian expression pattern. We have conducted a comprehensive study of circadian gene expression on a large dataset representing three different peripheral tissues. The data have been produced in a large-scale microarray experiment covering replicate daily cycles in murine white and brown adipose tissues as well as in liver. We have applied three alternative algorithmic approaches to identify circadian oscillation in time series expression profiles. Analyses of our own data indicate that the expression of at least 7% to 21% of active genes in mouse liver, and in white and brown adipose tissues follow a daily oscillatory pattern. Indeed, analysis of data from other laboratories suggests that the percentage of genes with an oscillatory pattern may approach 50% in the liver. For the rest of the genes, oscillation appears to be obscured by stochastic noise. Our phase classification and computer simulation studies based on multiple datasets indicate no detectable boundary between oscillating and non-oscillating fractions of genes. We conclude that greater attention should be given to the potential influence of circadian mechanisms on any biological pathway related to metabolism and obesity.

Nuclear factor-E2-related factor 2 expression in liver is critical for induction of NAD(P)H:quinone oxidoreductase 1 during cholestasis

Bile duct ligation (BDL) causes hepatocellular oxidative stress and injury. The transcription factor nuclear factor-E2-related factor (Nrf2) induces expression of numerous genes including NAD(P)H:quinone oxidoreductase 1 (Nqo1) during periods of oxidative stress. Therefore, we hypothesized that BDL increases liver expression of mouse antioxidant genes in an Nrf2-dependent manner. BDL or sham surgeries were performed on male C57BL/6, Nrf2-null, and wild-type mice. Livers were collected at 1, 3, and 7 days after surgery for analysis of messenger ribonucleic acid (mRNA) levels of Nrf2-responsive genes as well as Nqo1 protein and activity. BDL increased mRNA expression of multiple Nrf2 genes in mouse liver, compared to sham-operated controls. Follow-up studies investigating protein expression, enzyme activity, and Nrf2 dependency were limited to Nqo1. Nqo1 protein expression and activity in mouse livers was increased 2- to 3-, and 4- to 5-fold at 3 and 7 days after BDL, respectively. Studies also showed that BDL increases Nqol mRNA, protein expression, and enzyme activity in livers from wild-type mice, but not in Nrf2-null mice. In conclusion, expression of Nrf2-dependent genes is increased during cholestasis. These studies also demonstrate that Nqo1 expression and activity in mouse liver are induced via an Nrf2-dependent mechanism.