Growth-factor dependent expression of the translationally controlled tumour protein TCTP is regulated through the PI3-K/Akt/mTORC1 signalling pathway.

Translationally controlled tumour protein TCTP (gene symbol: TPT1) is a highly-conserved, cyto-protective protein implicated in many physiological and disease processes, in particular cancer, where it is associated with poor patient outcomes. To understand the mechanisms underlying the accumulation of high TCTP levels in cancer cells, we studied the signalling pathways that control translation of TCTP mRNA, which contains a 5'-terminal oligopyrimidine tract (5'-TOP). In HT29 colon cancer cells and in HeLa cells, serum increases the expression of TCTP two- and four-fold, respectively, and this is inhibited by rapamycin or mTOR kinase inhibitors. Polysome profiling and mRNA quantification indicate that these effects occur at the level of mRNA translation. Blocking this pathway upstream of mTOR complex 1 (mTORC1) by inhibiting Akt also prevented increases in TCTP levels in both HeLa and HT29 colon cancer cells, whereas knockout of TSC2, a negative regulator of mTORC1, led to derepression of TCTP synthesis under serum starvation. Overexpression of eIF4E enhanced the polysomal association of the TCTP mRNA, although it did not protect its translation from inhibition by rapamycin. Conversely, expression of a constitutively-active mutant of the eIF4E inhibitor 4E-BP1, which is normally inactivated by mTORC1, inhibited TCTP mRNA translation in HEK293 cells. Our results demonstrate that TCTP mRNA translation is regulated by signalling through the PI3-K/Akt/mTORC1 pathway. This explains why TCTP levels are frequently increased in cancers, since mTORC1 signalling is hyperactive in ~80% of tumours.

Altered ceramide acyl chain length and ceramide synthase gene expression in Parkinson's disease.

Genetic studies have provided increasing evidence that ceramide homeostasis plays a role in neurodegenerative diseases including Parkinson's disease (PD). It is known that the relative amounts of different ceramide molecular species, as defined by their fatty acyl chain length, regulate ceramide function in lipid membranes and in signaling pathways. In the present study we used a comprehensive sphingolipidomic case-control approach to determine the effects of PD on ceramide composition in postmortem brain tissue from the anterior cingulate cortex (a region with significant PD pathology) and the occipital cortex (spared in PD), also assessing mRNA expression of the major ceramide synthase genes that regulate ceramide acyl chain composition in the same tissue using quantitative PCR. In PD anterior cingulate cortex but not occipital cortex, total ceramide and sphingomyelin levels were reduced from control levels by 53% (P < 0.001) and 42% (P < 0.001), respectively. Of the 13 ceramide and 15 sphingomyelin molecular lipid species identified and quantified, there was a significant shift in the ceramide acyl chain composition toward shorter acyl chain length in the PD anterior cingulate cortex. This PD-associated change in ceramide acyl chain composition was accompanied by an upregulation of ceramide synthase-1 gene expression, which we consider may represent a response to reduced ceramide levels. These data suggest a significant shift in ceramide function in lipid membranes and signaling pathways occurs in regions with PD pathology. Identifying the regulatory mechanisms precipitating this change may provide novel targets for future therapeutics.

Selective reduction of hydroperoxyeicosatetraenoic acids to their hydroxy derivatives by apolipoprotein D: implications for lipid antioxidant activity and Alzheimer's disease.

ApoD (apolipoprotein D) is up-regulated in AD (Alzheimer's disease) and upon oxidative stress. ApoD inhibits brain lipid peroxidation in vivo, but the mechanism is unknown. Specific methionine residues may inhibit lipid peroxidation by reducing radical-propagating L-OOHs (lipid hydroperoxides) to non-reactive hydroxides via a reaction that generates MetSO (methionine sulfoxide). Since apoD has three conserved methionine residues (Met(49), Met(93) and Met(157)), we generated recombinant proteins with either one or all methionine residues replaced by alanine and assessed their capacity to reduce HpETEs (hydroperoxyeicosatetraenoic acids) to their HETE (hydroxyeicosatetraenoic acid) derivatives. ApoD, apoD(M49-A) and apoD(M157-A) all catalysed the reduction of HpETEs to their corresponding HETEs. Amino acid analysis of HpETE-treated apoD revealed a loss of one third of the methionine residues accompanied by the formation of MetSO. Additional studies using apoD(M93-A) indicated that Met(93) was required for HpETE reduction. We also assessed the impact that apoD MetSO formation has on protein aggregation by Western blotting of HpETE-treated apoD and human brain samples. ApoD methionine oxidation was associated with formation of apoD aggregates that were also detected in the hippocampus of AD patients. In conclusion, conversion of HpETE into HETE is mediated by apoD Met(93), a process that may contribute to apoD antioxidant function.

Proteomic analysis of colon tissue from interleukin-10 gene-deficient mice fed polyunsaturated Fatty acids with comparison to transcriptomic analysis.

Inflammatory bowel disease (IBD) is characterized by intestinal inflammation and is believed to involve complex interactions between genetic, immunological, and environmental factors. We measured changes in the proteome associated with bacterially induced intestinal inflammation in the interleukin 10 gene-deficient (Il10(-/-)) mouse model of IBD, established effects of the dietary polyunsaturated fatty acids (PUFAs) n-3 eicosapentaenoic acid (EPA) and n-6 arachidonic acid (AA) on protein expression (using oleic acid as a control fatty acid), and compared these changes with previously observed transcriptome changes in the same model. Ingenuity pathways analysis of proteomics data showed bacterially induced inflammation was associated with reduced expression of proteins from pathways of metabolism and digestion/absorption/excretion of nutrients/ions, and increased expression of cellular stress and immune response proteins. Both PUFA treatments showed anti-inflammatory activity; EPA appeared to act via the PPARα pathway, whereas AA appeared to increase energy metabolism and cytoskeletal organization and reduce cellular stress responses, possibly enabling a more robust response to inflammation. While there was agreement between proteomic and transcriptomic data with respect to pathways, there was limited concordance between individual gene and protein data, reflecting the importance of having both gene and protein data to better understand complex diseases such as IBD.

Peroxisome proliferator-activated receptor alpha target genes.

The peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPARα serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPARα binds and is activated by numerous fatty acids and fatty acid-derived compounds. PPARα governs biological processes by altering the expression of a large number of target genes. Accordingly, the specific role of PPARα is directly related to the biological function of its target genes. Here, we present an overview of the involvement of PPARα in lipid metabolism and other pathways through a detailed analysis of the different known or putative PPARα target genes. The emphasis is on gene regulation by PPARα in liver although many of the results likely apply to other organs and tissues as well.

Dietary oleic acid as a control fatty acid for polyunsaturated fatty acid intervention studies: a transcriptomics and proteomics investigation using interleukin-10 gene-deficient mice.

Oleic acid (OA) has been used as a control fatty acid in dietary polyunsaturated fatty acid (PUFA) intervention studies due to its lack of effect on eiconasoid biosynthesis. Since the effect of OA as a control fatty acid has not yet been investigated for transcriptomics and proteomics studies, this study aimed to test whether colonic transcriptome and proteome profiles associated with colitis development in mice fed a linoleic acid-rich corn oil-AIN-76A diet (Il10(-/-) compared to C57 mice) where similar to those of OA-fed Il10(-/-) compared to C57 mice (genotype comparison). A close clustering of colonic gene and protein expression profiles between the mice fed the AIN-76A or OA diet was observed. Inflammation-induced regulatory processes associated with cellular and humoral immune responses, cellular stress response and metabolic processes related to energy utilization were identified in Il10(-/-) compared to C57 mice fed either diet. Thus OA was considered as a suitable control unsaturated fatty acid for use in multi-omics PUFA studies. The second aim of this study was to test the effect of an OA-enriched AIN-76A diet compared to a linoleic acid-rich corn oil-AIN-76A diet on colonic transcriptome and proteome changes within Il10(-/-) or C57 mice (diet comparison). Overall, there was a limited concordance observed between measureable transcriptomics and proteomics profiles for genotype and diet comparisons. This underlines the importance and validity of a systems biology approach to understand the effects of diet on gene expression as a function of the genotype.

Diversity of caecal bacteria is altered in interleukin-10 gene-deficient mice before and after colitis onset and when fed polyunsaturated fatty acids.

Interleukin-10 gene-deficient (Il10(-/-)) mice show a hyper-reaction to normal intestinal bacteria and develop spontaneous colitis similar to that of human Crohn's disease when raised under conventional (but not germ-free) conditions. The lack of IL10 protein in these mice leads to changes in intestinal metabolic and signalling processes. The first aim of this study was to identify changes in the bacterial community of the caeca at 7 weeks of age (preclinical colitis) and at 12 weeks of age (when clinical signs of colitis are present), and establish if there were any changes that could be associated with the mouse genotype. We have previously shown that dietary n-3 and n-6 polyunsaturated fatty acids (PUFA) have anti-inflammatory effects and affect colonic gene expression profiles in Il10(-/-) mice; therefore, we also aimed to test the effect of the n-3 PUFA eicosapentaenoic acid (EPA) and the n-6 PUFA arachidonic acid (AA) on the bacterial community of caeca in both Il10(-/-) and C57 mice fed these diets. The lower number of caecal bacteria observed before colitis (7 weeks of age) in Il10(-/-) compared to C57 mice suggests differences in the intestinal bacteria that might be associated with the genotype, and this could contribute to the development of colitis in this mouse model. The number and diversity of caecal bacteria increased after the onset of colitis (12 weeks of age). The increase in caecal Escherichia coli numbers in both inflamed Il10(-/-) and healthy C57 mice might be attributed to the dietary PUFA (especially dietary AA), and thus not be a cause of colitis development. A possible protective effect of E. coli mediated by PUFA supplementation and associated changes in the bacterial environment could be a subject for further investigation to define the mode of action of PUFA in colitis.

Molecular Characterization of the Onset and Progression of Colitis in Inoculated Interleukin-10 Gene-Deficient Mice: A Role for PPARalpha.

The interleukin-10 gene-deficient (Il10(-/-)) mouse is a model of human inflammatory bowel disease and Ppara has been identified as one of the key genes involved in regulation of colitis in the bacterially inoculated Il10(-/-) model. The aims were to (1) characterize colitis onset and progression using a histopathological, transcriptomic, and proteomic approach and (2) investigate links between PPARalpha and IL10 using gene network analysis. Bacterial inoculation resulted in severe colitis in Il10(-/-) mice from 10 to 12 weeks of age. Innate and adaptive immune responses showed differences in gene expression relating to colitis severity. Actin cytoskeleton dynamics, innate immunity, and apoptosis-linked gene and protein expression data suggested a delayed remodeling process in 12-week-old Il10(-/-) mice. Gene expression changes in 12-week-old Il10(-/-) mice were related to PPARalpha signaling likely to control colitis, but how PPARalpha activation might regulate intestinal IL10 production remains to be determined.

Changes in colon gene expression associated with increased colon inflammation in interleukin-10 gene-deficient mice inoculated with Enterococcus species.

BACKGROUND: Inappropriate responses to normal intestinal bacteria may be involved in the development of Inflammatory Bowel Diseases (IBD, e.g. Crohn's Disease (CD), Ulcerative Colitis (UC)) and variations in the host genome may mediate this process. IL-10 gene-deficient (Il10-/-) mice develop CD-like colitis mainly in the colon, in part due to inappropriate responses to normal intestinal bacteria including Enterococcus strains, and have therefore been used as an animal model of CD. Comprehensive characterization of changes in cecum gene expression levels associated with inflammation in the Il10-/- mouse model has recently been reported. Our aim was to characterize changes in colonic gene expression levels in Il10-/- and C57BL/6J (C57; control) mice resulting from oral bacterial inoculation with 12 Enterococcus faecalis and faecium (EF) strains isolated from calves or poultry, complex intestinal flora (CIF) collected from healthy control mice, or a mixture of the two (EF.CIF). We investigated two hypotheses: (1) that oral inoculation of Il10-/- mice would result in greater and more consistent intestinal inflammation than that observed in Il10-/- mice not receiving this inoculation, and (2) that this inflammation would be associated with changes in colon gene expression levels similar to those previously observed in human studies, and these mice would therefore be an appropriate model for human CD. RESULTS: At 12 weeks of age, total RNA extracted from intact colon was hybridized to Agilent 44 k mouse arrays. Differentially expressed genes were identified using linear models for microarray analysis (Bioconductor), and these genes were clustered using GeneSpring GX and Ingenuity Pathways Analysis software. Intestinal inflammation was increased in Il10-/- mice as a result of inoculation, with the strongest effect being in the EF and EF.CIF groups. Genes differentially expressed in Il10-/- mice as a result of EF or EF.CIF inoculation were associated with the following pathways: inflammatory disease (111 genes differentially expressed), immune response (209 genes), antigen presentation (11 genes, particularly major histocompatability complex Class II), fatty acid metabolism (30 genes) and detoxification (31 genes). CONCLUSIONS: Our results suggest that colonic inflammation in Il10-/- mice inoculated with solutions containing Enterococcus strains is associated with gene expression changes similar to those of human IBD, specifically CD, and that with the EF.CIF inoculum in particular this is an appropriate model to investigate food-gene interactions relevant to human CD.

Dietary arachidonic acid-mediated effects on colon inflammation using transcriptome analysis.

Increased levels of n-6 arachidonic acid (AA), a precursor of pro-inflammatory eicosanoids, have been found in the colon mucosa of inflammatory bowel disease patients when compared with healthy subjects. The hypothesis was that dietary AA would aggravate colon inflammation by changing expression of genes in inflammatory signaling pathways. AA-enriched diet was fed to IL10 gene-deficient (Il10-/-) mice, model of a inflammatory bowel disease, and compared with Il10-/- mice fed an oleic acid control diet. Effects of AA on gene expression profiles during colitis were examined using whole genome microarray analysis. Dietary AA decreased the expression levels of some colonic genes in ER stress, complement system, nuclear respiratory factor 2-mediated oxidative stress and positive acute phase response pathways compared with Il10-/- mice fed an oleic acid diet. AA increased the expression levels of fatty acid catabolism genes, but decreased that of lipid synthesis genes during colitis, likely by sterol regulatory element binding transcription factor 1 and target gene regulation. A link has been suggested between AA and reduction of intestinal fibrosis by down-regulating the expression levels of pro-inflammatory and fibrotic marker genes. Contrary to the hypothesis, these findings suggest that dietary AA, in the present experimental conditions, is not pro-inflammatory, reduces ER stress and protects colonocytes from oxidative stress in Il10-/- mice.

Genome-wide analysis of dietary eicosapentaenoic acid- and oleic acid-induced modulation of colon inflammation in interleukin-10 gene-deficient mice.

BACKGROUND/AIMS: Dietary n-3 polyunsaturated fatty acids can reduce inflammation via a range of mechanisms. This study tested the effect of dietary eicosapentaenoic acid (EPA) on intestinal inflammation using interleukin-10 gene-deficient (Il10(-/-)) mice. METHODS: At 35 days of age, 12 weaned Il10(-/-) and 12 C57 mice were randomly assigned to one of two modified AIN-76A diets, supplemented with 3.7% purified ethyl esters of either EPA (n-3) or oleic acid (OA, control). To identify genes relevant to colon inflammation, transcription profiling (microarrays and qRT-PCR) and bioinformatic analyses were used. RESULTS: In this study, dietary EPA reversed the decrease in colon fatty acid beta-oxidation gene expression observed in OA-fed Il10(-/-) compared to C57 mice. Il10(-/-) mice fed the OA diet showed decreased expression of antioxidant enzyme genes, as well as those involved in detoxification of xenobiotics, compared to C57 mice on the same diet. In contrast, dietary EPA increased the expression of these genes in Il10(-/-) mice. CONCLUSIONS: These data indicate that dietary EPA-induced endogenous lipid oxidation which might have a potential anti-inflammatory effect on colon tissue. This is supported by the activation of the Ppara gene that regulates the expression of pro-inflammatory and immunomodulatory genes and proteins.

Nutrigenomics applied to an animal model of Inflammatory Bowel Diseases: transcriptomic analysis of the effects of eicosapentaenoic acid- and arachidonic acid-enriched diets.

In vivo models of Inflammatory Bowel Diseases (IBD) elucidate important mechanisms of chronic inflammation. Complex intestinal responses to food components create a unique "fingerprint" discriminating health from disease. Five-week-old IL10(-/-) and C57BL/6J (C57; control) mice were inoculated orally with complex intestinal microflora (CIF) and/or pure cultures of Enterococcus faecalis and E. faecalis (EF) aiming for more consistent inflammation of the intestinal mucosa. Inoculation treatments were compared to non-inoculated IL10(-/-) and C57 mice, either kept in specific pathogen free (SPF) or conventional conditions (2x5 factorial design). At 12 weeks of age, mice were sacrificed for intestinal histological (HIS) and transcriptomic analysis using limma and Ingenuity Pathway Analysis Software. Colonic HIS was significantly affected (P<0.05) in inoculated IL10(-/-) mice and accounted for approximately 60% of total intestinal HIS. Inoculation showed a strong effect on colonic gene expression, with more than 2000 genes differentially expressed in EF.CIF-inoculated IL10(-/-) mice. Immune response gene expression was altered (P<0.05) in these mice. The second study investigated the effect of arachidonic (AA) and eicosapentaenoic acid (EPA) on colonic HIS and gene expression to test whether EPA, contrary to AA, diminished intestinal inflammation in EF.CIF IL10(-/-) mice (2 x 4 factorial design). AIN-76A (5% corn oil) and AIN-76A (fat-free) +1% corn oil supplemented with either 3.7% oleic acid (OA), AA or EPA were used. IL10(-/-) mice fed EPA- and AA-enriched diets had at least 40% lower colonic HIS (P<0.05) than those fed control diets (AIN-76A and OA diets). The expression of immune response and 'inflammatory disease' genes (down-regulated: TNFalpha, IL6, S100A8, FGF7, PTGS2; up-regulated: PPARalpha, MGLL, MYLK, PPSS23, ABCB4 with EPA and/or AA) was affected in IL10(-/-) mice fed EPA- and AA-enriched diets, compared to those fed AIN-76A diet.