- Email: [email protected]
956 NFAT5 Regulates Intestinal Cell Differentiation by Negatively Regulating AKT
and MGAM contributions to both maltase and glucoamylase assays results. Aim: to examine starch digestion in a series of children with CSID and a population with similar symptoms of recurrent abdominal pain. Material and Methods: Eleven adult control subjects; eleven patients with CSID; and 10 RAP age matched patients in oral breath test using 20mg of UL-13C-substrates and periodic 13CO2 breath enrichment analyses normalized for % of glucose oxidation (CGO%) as previously reported. UL-13C-substrates (G & ST; 20 mg. each, Isotec, Miamisberg, OH) were given in 10 gm unlabeled glucose oligomers (10%) vehicle after an overnight fast and reference sampling on 2 separate days. Serial 13CO2 breath enrichments (every 15 min x 9) were assayed using a 13CO2 infrared spectrophotometer (POCone®, Otsuka Electronics, Tokyo, Japan). A Coefficient of Glucose Oxidation (CGO) was calculated for breath enrichments to adjust for individual differences. Normal adult control subjects were used to define CGO lower reference levels (LL) defined as mean-1 SD (75 ± 19 CGO%, LL 56. Because %CGO were found to be relatively constant in the period of 60-90' after load these values were averaged for each individual. Results: The normal controls had a LL value for starch of 56 %CGO. Eight of ten RAP patients fell below control LL. Within the RAP group (46± 24 %CGO), the LL was of 20% and 1 had < 20%. CSID patients (19 ± 11 %CGO with 6 < 20% and 2 = 20%. Conclusions: Starch digestion by CSID patients was significantly below the 20% %CGO ST/G LL found in RAP patients (p=0.000) and both CSID and RAP populations were fell significantly below the normal LL of 56 %CGO.
952 The Role of Genetic Crohn's Disease Susceptibility Loci in Small Bowel Transplant Outcome Marcel Janse, Rinse K. Weersma, Gerard Dijkstra, Eleonora A. Festen, Debra L. Sudan, David F. Mercer BACKGROUND: Intestinal allograft rejection is a severe complication in small bowel transplantation. Identifying involved pathogenetic mechanisms might make it possible to predict or even prevent rejection and intestinal graft failure. Rejection resembles Crohn's disease (CD) clinically and histopathologically. We investigated the role of CD-associated genetic loci, including NOD2 variants in rejection and graftfailure. METHODS: 99 recipients and 71 donors from the University of Nebraska Medical Center, USA were genotyped for three NOD2 variants and single nucleotide polymorphisms in IBD5, ATG16L1, IRGM, MST1, IL23R, STAT3 and JAK2. Genotypes were related to clinical outcomes rejection, graftfailure and patient-survival using Kaplan-Meier curves and log-rank analysis. RESULTS: Only the MST1 riskallele was more frequent in recipients compared to donors (30,8% and 20,4%, p=0,032). For the other loci, including NOD2, allelic distribution was equal within the two groups. No single locus was significantly associated with rejection, graftfailure or patientsurvival. There is no significant difference in clinical endpoints whether the recipient has a low or high number of risk alleles. Individuals receiving a donorgraft with ≤5 riskalleles in total, have decreased rejection-free survival compared to ≥6 riskalleles (p=0,040), but increased patient three-year survival (mortality: 4/19 and 16/29, p=0,016). CONCLUSIONS: In this relatively large cohort, we could not confirm the association of NOD2, with rejection and graftfailure. Furthermore we found no association of seven other CD-loci with clinical endpoints. However, patients receiving a donorgraft with six or more risk alleles have decreased three-year survival, but -surprisingly- increased rejection-free survival.
955 Associations Between Genetic Variants in the Glucocorticoid Receptor (GR) Gene and Corticosteroid Dependency in Paediatric-Onset Crohn's Disease Alfreda Krupoves, David R. Mack, Emile Levy, Colette Deslandres, Philippe Lambrette, Houda H. Feguery, Jinsong Dong, Vytautas Bucionis, Ernest G. Seidman, Devendra K. Amre Background: Corticosteroids (CS) are the mainstay for the treatment of moderate to severe Crohn's disease (CD) in children. However, CS dependency (~40%) is a significant clinical problem associated with numerous side-effects. Identification of molecular markers that can predict CS response is thus paramount. As the glucocorticoid receptor (GR) is closely related with CS metabolism, we hypothesized that DNA variants in the gene could predict CS dependency in pediatrics-onset CD. Methods: A retrospective cohort study was carried out including patients recruited from two tertiary pediatric gastroenterology clinics in Canada (Montreal and Ottawa). Patients diagnosed with CD prior to age 20 and treated with a first course of CS, were identified and their follow-up information abstracted from the medical charts. Patients were classified as steroid dependent if they experienced clinical relapse during drug reduction or after the end of treatment. Relevant clinical and demographic information was also acquired. Blood or saliva was obtained as a source of DNA. Tag-SNPs (single nucleotide polymorphisms) across the GR gene were genotyped using established methods. Allelic, genotype and haplotype associations between the tag-SNPs and steroid dependency were examined using logistic regression analysis. Odds ratios (OR) and 95% confidence intervals (95% CI) were estimated. Results: A total of 255 CD patients administered a first course of CS were identified. The mean age (±SD) at diagnosis was 12.4(±3.2) yrs. Most patients were male (n=137, 53.7%). A total of 125 (49 %) patients became steroid dependent. All the tag-SNPs were in Hardy-Weinberg Equilibrium (HWE). Of the 12 tagSNPs examined SNPs rs10482682 (allelic OR=1.42, 95% CI: 0.99-2.03, p=0.052; heterozygotes versus common homozygotes: OR=2.23, 95% CI: 1.28-3.87, p=0.004), rs6196 (allelic OR=0.53, 95% CI: 0.31-0.91, p=0.022) and rs2963155 (allelic OR=0.64, 95% CI: 0.420.98, p=0.039) have shown the associations. There was suggestion for association with rs4912911 (allelic OR=0.71, 95% CI: 0.48-1.06, p=0.09; rare homozygotes versus common homozygotes: OR=0.36, 95% CI: 0.14-0.94, p=0.037). Haplotype analysis including the 3 significantly associated markers indicated that two high frequency haplotypes (TTA and CCG) were significantly associated with steroid dependence (p-values of 0.047 and 0.036). When the 4th marker was included, haplotype CCGC was significantly associated with steroid dependence (p=0.004). Conclusion: Our results suggest that DNA variation in the GR gene may be associated with CS dependency in paediatric-onset CD. Further studies are required to replicate these findings in a larger cohort.
953 Tryptophan Hydroxylase Gene Variants and IBS Symptoms Sang-Eun Jun, Kevin C. Cain, Monica Jarrett, Margaret Heitkemper Background: IBS is a common functional gastrointestinal (GI) disorder characterized by recurrent abdominal pain or discomfort, bloating, and altered bowel habits. Serotonin (5HT) is known to play an important role in the regulation of GI motility and sensation. Hence, pharmacological treatments based on the regulation of 5-HT have been developed for IBS patients. Since tryptophan hydroxylase (TPH) is a rate-limiting enzyme in the biosynthesis of 5-HT, TPH variants might be related to disorders involving dysfunction of the 5-HT system. TPH has two isoforms; TPH-1, mostly found in the GI tract, and TPH2, found in the brain. Five single nucleotide polymorphisms (SNPs) from TPH1 gene (rs 4537731, rs684302, rs211105, rs1800532, rs1799913) and one SNP from Tph2 gene (rs4570625) were selected based on literature review. The aim of this study is to assess whether these TPH gene variants were associated with various IBS GI symptoms. Methods: IBS patients (n= 171) who met the Rome-II criteria and gender, ethnicity-matched controls (n = 75) were recruited though public advertisement. For the genotype linkage, we included only Caucasians. DNA was isolated from buffy coat preparations of whole blood and the SNPs were genotyped using custom TaqMan genotyping assay (Applied Biosystems, Foster City, California). IBS patients recorded symptoms in a daily diary for 28 days and filled out the Cognitive Scale for Functional Bowel Disorder (CSFBD). Comparisons between cases and controls were made using Chi-square tests and odds ratios (OR) and 95% confidence intervals (CI) were estimated using logistic regression. Genotypes were modeled assuming a log-additive model which assumes that OR is constant for each additional copy of the minor allele. Results: All SNPs were in Hardy-Weinberg equilibrium. Since there was age differences between groups (IBS 44±14, controls 38±13; p <.002), regressions were adjusted with age. No genotype association was found with IBS in the single locus analysis. However, the minor allele (T) for the rs4570625 was associated with a reduced risk of IBS for each additional copy (OR = 0.67, 95% CI: 0.42, 1.07; p= .088). The four SNPs from TPH 1 gene (rs4537731, rs684302, rs211105 and rs1799913) showed significant (p<.05) association with diarrheal symptoms and urgency. The CC genotype of rs4537731 showed significant association with bloating symptoms (p<.023) and abdominal pain (p<.043). Conclusion: This preliminary analysis suggest that TPH gene variants are related to GI symptoms, especially diarrhea, bloating and abdominal pain. A next step would be to investigate if there is any association between TPH gene polymorphisms and GI motility.
956 NFAT5 Regulates Intestinal Cell Differentiation by Negatively Regulating AKT Qingding Wang, Zheng Guo, Yuning Zhou, Xin Li, Tianyan Gao, Mark Evers Previously, we showed that inhibition of PI3-kinase or overexpression of PTEN enhances intestinal cell differentiation. In addition, NFATc1 and NFATc4, isoforms of the nuclear factor of activated T cells (NFAT) family of transcription factors, regulate PTEN expression and intestinal cell differentiation. NFAT5, a new isoform of NFAT, is a major inducer of osmoprotective genes and has exhibited distinctly different functions compared with NFATc1-c4. The purpose of this study is to investigate: i) the role of NFAT5 in intestinal cell differentiation, and ii) the regulation of Akt phosphorylation by NFAT5. METHODS. Differentiation was induced by treatment of HT29 and Caco-2 human colon cancer cells with sodium butyrate (NaBT) and assayed by measuring intestinal alkaline phosphatase (IAP) activity, an enterocyte differentiation marker. HT29, Caco-2, HCT116 and SW480 cells were transfected with control siRNA, NFAT5 siRNA, or control vector or constructs overexpressing myc-NFAT5 or HA-tagged PH domain leucine-rich repeat protein phosphatase (HA-PHLPP), a novel phosphatase specifically dephosphorylates the hydrophobic motif of Akt (Ser473). PHLPP, protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), p-Akt (Ser473), p-Akt (Thr308), Akt, PTEN and NFAT5 expression was analyzed by Western blot. Immunoprecipitation was performed using an anti-Akt antibody and Akt-PHLPP interaction was determined by Western blotting. NFAT mRNA expression was determined by RT-PCR. RESULTS. Treatment of HT29 cells with NaBT increased NFAT5 mRNA and protein expression. Knockdown of NFAT5 significantly inhibited NaBT-induced IAP activity in HT29 and Caco-2 cells, suggesting a role of NFAT5 in the regulation of intestinal cell differentiation. Knockdown of NFAT5 increased p-Akt (Ser473) level in HT29, Caco-2, HCT116, and SW480 cells, whereas overexpression of NFAT5 decreased p-Akt (Ser473) level in Caco-2 cells. However, knockdown of NFAT5 did not affect expression of PP1, PP2A, p-Akt (Thr308)
954 Carbohydrate Digestion in Congenital Sucrase Isomaltase Deficient and Recurrent Abdominal Pain Children Assesed by 13C- Starch Breath Test Claudia C. Robayo-Torres, Antone R. Opekun, Roberto Quezada-Calvillo, Maricela DiazSotomayor, Susan S. Baker, Buford L. Nichols Background: Starches contribute about half of the food energy needs to the weaned child's diet. Malabsorption of sucrose is associated with abdominal pain, bloating and diarrhea. A genetic disorder called Congenital Sucrase-Isomaltase deficiency (CSID) is suspected when these symptoms follow sugar ingestion and are relieved by a sugar elimination diet. Diagnosis is made upon demonstration of very low or absent sucrase activities in duodenal biopsy enzyme assays with a normal morphology. Treatment is by sucrose elimination diet and oral enzyme supplementation with sacrosidase. Clinical reviews of CSID have suggested that some of these patients also have starch malabsorption. The presence of coexisting starch and sucrose intolerance makes childhood dietary management very difficult and reduces the value of oral sacrosidase supplements. Confirmation of starch intolerance is made difficult because of lack of a specific substrate in mucosal biopsy enzyme assays and the shared SI
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is with rs26538 suggesting that the causal variant lies within the promoter region. REFERENCES 1) Barrett JC.et al. Nat Genet. 2008;40:955-62 2) Travassos LH et al. Nat Immunol. 2009; Nov 8 [Epub ahead of print] 3) Thurston TL et al. Nat Immunol. 2009;10:1215-21 4) Barrett JC et al. Bioinformatics 2005;21:263-5
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or PTEN, suggesting that NFAT5 regulates signaling events that directly control Akt phosphorylation at Ser473 site. Interestingly, knockdown of NFAT5 decreased the interaction between Akt and PHLPP. These results suggest that NFAT5 may regulate Akt (Ser473) phosphorylation through altering PHLPP-mediated inhibition of Akt. CONCLUSIONS. Our results demonstrate a role of NFAT5 in intestinal cell differentiation. Importantly, these data suggest that NFAT5-dependent positive regulation of differentiation is mediated by negatively affecting Akt phosphorylation through the novel regulation of the interaction between PHLPP and Akt.
959 Protease-Activated Receptors (PARS) 1 and 2 Stimulate Interleukin (IL)-8 Expression in HT-29 Cells via ERK/RSK90, Non-Classical NfκB Activation and Histone Acetylation Hongying Wang, France Moreau, Christina L. Hirota, Wallace K. MacNaughton Background: The G-protein-coupled PARs are known to stimulate the production of IL-8, an inflammatory and angiogenic chemokine, in epithelia. Since no mechanism has been described for this phenomenon, our aim was to determine the intracellular signaling pathway by which PARs stimulate IL-8 expression. Methods: HT-29 colonic epithelial cells were treated with the PAR1-activating peptide TFLLR-NH2 or the PAR2-activating peptide SLIGRLNH2 with or without inhibitors of MEK or RSK p90. IL-8 protein was measured in the culture supernatant by ELISA, mRNA levels were measured by RT-PCR and an IL-8 promoterdriven luciferase assay was used to measure promoter activity. Phosphorylation of ERK1/2 and the p65 B subunit was determined by western blot. NFκB transcriptional activity was measured via a κB-driven luciferase assay. Chromatin immunoprecipitation was used to assess binding of transcription factors to the IL-8 promoter. Co-immunoprecipitations were performed to examine interactions between κB subunits and other proteins. siRNA was used to transiently knock-down RSK1 or RSK2. Histone acetlytransferase (HAT) activity was measured using a commercially available kit. For some experiments, cells were stably transfected with HAT-mutated p300 (or empty vector). Results: Both PAR agonists stimulated IL-8 promoter activity, mRNA expression and protein production, which were dependent on ERK1/2. PAR activation also increased NFκB transcriptional activity and binding of the p50 B subunit to the IL-8 promoter, without any change in IκBα phosphorylation or degradation. PAR agonists induced phosphorylation of p65 at ser276 and its appearance in the nucleus, as well as enhanced binding of p300 to the p50 κB subunit. Furthermore, HAT activity toward the histone 3 substrate was also increased following PAR activation, although this was not accompanied by a significant increase in binding of acetylated histone 3 to the IL-8 promoter. PAR-induced phosphorylation of p65 induced nuclear translocation of RSK1 and RSK2, both of which are downstream target kinases from ERK1/2. siRNA knock-down of RSK1 and/or RSK2 reduced PAR-induced p65 phosphorylation, while pharmacological inhibition of RSK significantly blocked IL-8 induction by PAR activation. In cells stably expressing HAT-mutated p300, PAR activation led to significantly lower IL-8 production and promoter activity levels compared to cells transfected with the empty vector. Finally, PAR activation reduced binding of histone deacetylase 2 to p65 in an ERK-independent manner. Conclusions: These studies provide a cellular mechanism for PAR regulation of IL-8 expression in colonic inflammation and tumorigenesis.
957 TNF-α and Notch Signaling Synergistically up-Regulate OLFM4 Expression in Human Intestinal Epithelial Cells Ryuichi Okamoto, Junko Akiyama, Kiichiro Tsuchiya, Tetsuya Nakamura, Mamoru Watanabe Background & Aims: Recent studies in mice have shown that OLFM4 is expressed specifically in intestinal stem cells. However, the precise distribution of OLFM4-expressing cells within normal or inflamed human intestinal mucosa remains unknown. Also, the molecular mechanism regulating its expression within intestinal epithelial cells (IECs) has never been demonstrated. We have previously shown that Notch signaling is constitutively activated in IECs of the inflamed colonic mucosa, and thereby function as a key regulator of lineage-specific gene expression. Present study was planned to clarify the distribution of OLFM4-expressing IECs, and also the underlying mechanism regulating OLFM4 expression in IECs, in inflamed mucosa of human intestine. Methods: Immunostaining was performed to determine the distribution of OLFM4-expressing cells within the human intestinal tissue. Human IECderived cell lines were analyzed by RT-PCR or western blot to examine the expression level of OLFM4 in response to various inflammatory cytokines, including TNF-α, or upon activation of the Notch pathway, which was induced by forced expression of Notch1 intracellular domain (NICD1). Furthermore, a series of promoter assays were performed to analyze the transcriptional regulation of the OLFM4 gene expression within IECs. Results: Immunostaining of normal small intestinal or colonic tissue revealed clear expression of OLFM4 in IECs residing at the lowest part of the crypt, including crypt base columnar cells. However, in inflamed intestinal tissues derived from ulcerative colitis or Crohn's disease patients, increased number of OLFM4-expressing IECs was observed, expanding its distribution to the upper part of the crypt. In LS174T cells, both stimulation by TNF-α and forced intracellular expression of NICD1 significantly up-regulated mRNA and protein expression of OLFM4. Combination of NICD1 expression and TNF-a stimulation had synergistic effect upon upregulation of OLFM-4 mRNA expression, reaching up to 2500 fold increase in LS174T cells. Promoter assays using 5' flanking region of the human OLFM4 gene revealed that both TNF-a and NICD1 up-regulate OLFM4 expression at the transcriptional level, predominantly via the proximal NF-κB binding site. Consistently, TNF-α mediated up-regulation of NFκB-dependent transcriptional activity was significantly enhanced by NICD1 expression in LS174T cells. Conclusion: In the inflamed human intestinal mucosa, TNF-α and activation of Notch signaling synergistically up-regulate expression of OLFM4 in IECs. Such an upregulation of a stem-cell specific gene in IECs might be required to promote the regenerative response of the inflamed mucosa.
960 Disheveled Inhibits ZBP-89 Activation of p21Waf1 and OLFM4, an Intestinal Notch Target Natalia Veniaminova, Milena Saqui-Salces, Juanita L. Merchant BACKGROUND: A number of studies have implicated p21waf1 as a critical molecular switch that toggles intestinal cell fate between proliferation and growth arrest/differentiation. ZBP89 induces p21 in a butyrate-dependent manner and ZBP-89 protein levels increase with growth arrest/differentiation. We recently found by immunolocalization that ZBP-89 expression is elevated in enterochromaffin (EC) cells, which are also negative for βcatenin. This result is consistent with terminal differentiation of ECs and a recent study implicating βcatindependent signaling of ECs (Yadav VK, Cell/08). We therefore tested the hypothesis that βcat-independent signaling modulates ZBP-89 regulation of p21. Since Dishelved (Dvl) proteins interact directly with Wnt receptors and mediate both canonical and non-canonical signaling, Dvl was examined as a candidate ZBP-89 modulator. METHODS: Microarray analysis of tissue from cell specific disruption of ZBP-89 was performed on ZBP-89 floxed mice bred to VillinCre mice (ZBP-89ΔInt). Immunoblot analysis and transfection studies with the p21 and olfactomedin 4 (OLFM4) promoters were performed in mouse STC and human BON neuroendocrine cells. Whole cell extracts prepared from either cell line were used in co-immunoprecipitation experiments to identify protein-protein interactions with Disheveled (Dvl). RESULTS: A microarray analysis of ZBP-89ΔInt revealed that reduced levels of ZBP-89 in the intestine decreased OLFM4, a gene involved in myeloid development recently identified in intestinal Lgr5-progenitors. A recent study also identified OLFM4 in a microarray analysis designed to identify Notch gene targets. Co-immunoprecipitation studies revealed that ZBP-89 preferentially interacted with Dvl2 but not Dvl1 or Dvl3. Treating STC cells with trichostatin A increased the interaction of ZBP-89 with Dvl2. Using the OLFM4 reporter containing the proximal 427 bp of the promoter, we found that ZBP89 induced OLFM4 and that the induction was blocked by Dvl2 in STC cells. A similar result was observed with the p21 promoter. These promoters were not induced in BON cells, which expressed high levels of βcat compared to STC cells, which did not. We identified potential ZBP-89 (GTGGG) consensus-binding sites at -250, -165 and -148 suggesting OLFM4 as a direct ZBP-89 target. CONCLUSION: ZBP-89 induces the basal stem cell marker and putative Notch target gene OLFM4. Dvl2, a component of the Wnt pathway blocks the induction by ZBP-89. We suggest that ZBP-89 participates in intestinal EC cell specification by inducing p21 and OLFM4 to promote growth arrest and cell specification. This event requires restricting βcat-independent Wnt signaling.
958 BACH1 Deficiency Leads to the Suppression of Indomethacin-Induced Apoptosis in the Small Intestine Akihito Harusato, Yuji Naito, Tomohisa Takagi, Shinya Yamada, Katsura Mizushima, Yasuko Hirai, Ryusuke Horie, Ken Inoue, Kohei Fukumoto, Ikuhiro Hirata, Tatsushi Omatsu, Etsuko Kishimoto, Kazuhiko Uchiyama, Osamu Handa, Kazuhiko Igarashi, Toshikazu Yoshikawa BACKGROUND AND AIMS: Recent instrumental development in the diagnosis of small intestine has revealed that non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin, can induce small intestinal mucosal injuries. However, no clinical therapy for those lesions has been established. BTB and CNC homolog 1 (Bach1) is a transcriptional repressor of heme oxygenase-1 (HO-1). Bach1 plays an important role in the feedback regulation of HO-1, which protects cells from various injuries. The authors hypothesized that Bach1 has an important effect on the indomethacin-induced apoptosis in small intestinal mucosal injury by modulating HO-1 expression. METHODS: 8-week-old female C57BL/6 (wild-type) and homozygous Bach1-/- C57BL/6 mice were subjected to this study. Intestinal mucosal injuries induced by a single subcutaneous administration of indomethacin were evaluated macroscopically, histologically, and biochemically. Mucosal protein contents and chemokine mRNA levels were determined by western blotting and real-time PCR, respectively. We also investigated anti-apoptotic property of Bach1 deficiency by fluorescence staining. RESULTS: Indomethacin-induced intestinal injuries were remarkably improved in Bach1-/- mice. The expression of HO-1 mRNA in the intestinal mucosa was significantly increased in both mice after administration of Indomethacin, and it was significantly higher in Bach1-/- mice than in wild-type mice. Administration of indomethacin induced expression of chemokines, such as KC, MCP1 and MIP1-alpha, which was suppressed in Bach1-/- mice. MPO activity levels were also significantly decreased in Bach1-/- mice than in wild-type mice. The result of TUNEL staining in small intestine also showed that the extent of apoptosis was significantly suppressed in Bach1-/- mice compared with that in wild type mice. Western blot analysis showed that the increase in Bak and Bax expression and caspase-3 activation after indomethacin administration was suppressed in Bach1-/- mice. CONCLUSION: Disruption of the Bach1 gene caused inhibition of indomethacin-induced apoptosis indicating that inhibition of Bach1 could be a candidate to treat NSAIDs-induced small intestinal injuries.
961 The Myofibroblast Protein Epimorphin Modulates Hedgehog Signaling in the Colon Anisa Shaker, Shashi Bala, Gowri Kularatna, Marc S. Levin, Deborah C. Rubin In the colon, epithelial Indian hedgehog (Ihh) signals to the mesenchyme, which in turn feeds back to the epithelium to inhibit proliferation and promote differentiation. The precise mechanisms by which Ihh mediates its effects from mesenchyme to epithelium are unclear. Epimorphin (epi) is a mesenchymal/myofibroblast protein that exerts an anti-proliferative effect on overlying normal colon epithelium; epi deletion results in enhanced epithelial
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952 The Role of Genetic Crohn's Disease Susceptibility Loci in Small Bowel Transplant Outcome Marcel Janse, Rinse K. Weersma, Gerard Dijkstra, Eleonora A. Festen, Debra L. Sudan, David F. Mercer BACKGROUND: Intestinal allograft rejection is a severe complication in small bowel transplantation. Identifying involved pathogenetic mechanisms might make it possible to predict or even prevent rejection and intestinal graft failure. Rejection resembles Crohn's disease (CD) clinically and histopathologically. We investigated the role of CD-associated genetic loci, including NOD2 variants in rejection and graftfailure. METHODS: 99 recipients and 71 donors from the University of Nebraska Medical Center, USA were genotyped for three NOD2 variants and single nucleotide polymorphisms in IBD5, ATG16L1, IRGM, MST1, IL23R, STAT3 and JAK2. Genotypes were related to clinical outcomes rejection, graftfailure and patient-survival using Kaplan-Meier curves and log-rank analysis. RESULTS: Only the MST1 riskallele was more frequent in recipients compared to donors (30,8% and 20,4%, p=0,032). For the other loci, including NOD2, allelic distribution was equal within the two groups. No single locus was significantly associated with rejection, graftfailure or patientsurvival. There is no significant difference in clinical endpoints whether the recipient has a low or high number of risk alleles. Individuals receiving a donorgraft with ≤5 riskalleles in total, have decreased rejection-free survival compared to ≥6 riskalleles (p=0,040), but increased patient three-year survival (mortality: 4/19 and 16/29, p=0,016). CONCLUSIONS: In this relatively large cohort, we could not confirm the association of NOD2, with rejection and graftfailure. Furthermore we found no association of seven other CD-loci with clinical endpoints. However, patients receiving a donorgraft with six or more risk alleles have decreased three-year survival, but -surprisingly- increased rejection-free survival.
955 Associations Between Genetic Variants in the Glucocorticoid Receptor (GR) Gene and Corticosteroid Dependency in Paediatric-Onset Crohn's Disease Alfreda Krupoves, David R. Mack, Emile Levy, Colette Deslandres, Philippe Lambrette, Houda H. Feguery, Jinsong Dong, Vytautas Bucionis, Ernest G. Seidman, Devendra K. Amre Background: Corticosteroids (CS) are the mainstay for the treatment of moderate to severe Crohn's disease (CD) in children. However, CS dependency (~40%) is a significant clinical problem associated with numerous side-effects. Identification of molecular markers that can predict CS response is thus paramount. As the glucocorticoid receptor (GR) is closely related with CS metabolism, we hypothesized that DNA variants in the gene could predict CS dependency in pediatrics-onset CD. Methods: A retrospective cohort study was carried out including patients recruited from two tertiary pediatric gastroenterology clinics in Canada (Montreal and Ottawa). Patients diagnosed with CD prior to age 20 and treated with a first course of CS, were identified and their follow-up information abstracted from the medical charts. Patients were classified as steroid dependent if they experienced clinical relapse during drug reduction or after the end of treatment. Relevant clinical and demographic information was also acquired. Blood or saliva was obtained as a source of DNA. Tag-SNPs (single nucleotide polymorphisms) across the GR gene were genotyped using established methods. Allelic, genotype and haplotype associations between the tag-SNPs and steroid dependency were examined using logistic regression analysis. Odds ratios (OR) and 95% confidence intervals (95% CI) were estimated. Results: A total of 255 CD patients administered a first course of CS were identified. The mean age (±SD) at diagnosis was 12.4(±3.2) yrs. Most patients were male (n=137, 53.7%). A total of 125 (49 %) patients became steroid dependent. All the tag-SNPs were in Hardy-Weinberg Equilibrium (HWE). Of the 12 tagSNPs examined SNPs rs10482682 (allelic OR=1.42, 95% CI: 0.99-2.03, p=0.052; heterozygotes versus common homozygotes: OR=2.23, 95% CI: 1.28-3.87, p=0.004), rs6196 (allelic OR=0.53, 95% CI: 0.31-0.91, p=0.022) and rs2963155 (allelic OR=0.64, 95% CI: 0.420.98, p=0.039) have shown the associations. There was suggestion for association with rs4912911 (allelic OR=0.71, 95% CI: 0.48-1.06, p=0.09; rare homozygotes versus common homozygotes: OR=0.36, 95% CI: 0.14-0.94, p=0.037). Haplotype analysis including the 3 significantly associated markers indicated that two high frequency haplotypes (TTA and CCG) were significantly associated with steroid dependence (p-values of 0.047 and 0.036). When the 4th marker was included, haplotype CCGC was significantly associated with steroid dependence (p=0.004). Conclusion: Our results suggest that DNA variation in the GR gene may be associated with CS dependency in paediatric-onset CD. Further studies are required to replicate these findings in a larger cohort.
953 Tryptophan Hydroxylase Gene Variants and IBS Symptoms Sang-Eun Jun, Kevin C. Cain, Monica Jarrett, Margaret Heitkemper Background: IBS is a common functional gastrointestinal (GI) disorder characterized by recurrent abdominal pain or discomfort, bloating, and altered bowel habits. Serotonin (5HT) is known to play an important role in the regulation of GI motility and sensation. Hence, pharmacological treatments based on the regulation of 5-HT have been developed for IBS patients. Since tryptophan hydroxylase (TPH) is a rate-limiting enzyme in the biosynthesis of 5-HT, TPH variants might be related to disorders involving dysfunction of the 5-HT system. TPH has two isoforms; TPH-1, mostly found in the GI tract, and TPH2, found in the brain. Five single nucleotide polymorphisms (SNPs) from TPH1 gene (rs 4537731, rs684302, rs211105, rs1800532, rs1799913) and one SNP from Tph2 gene (rs4570625) were selected based on literature review. The aim of this study is to assess whether these TPH gene variants were associated with various IBS GI symptoms. Methods: IBS patients (n= 171) who met the Rome-II criteria and gender, ethnicity-matched controls (n = 75) were recruited though public advertisement. For the genotype linkage, we included only Caucasians. DNA was isolated from buffy coat preparations of whole blood and the SNPs were genotyped using custom TaqMan genotyping assay (Applied Biosystems, Foster City, California). IBS patients recorded symptoms in a daily diary for 28 days and filled out the Cognitive Scale for Functional Bowel Disorder (CSFBD). Comparisons between cases and controls were made using Chi-square tests and odds ratios (OR) and 95% confidence intervals (CI) were estimated using logistic regression. Genotypes were modeled assuming a log-additive model which assumes that OR is constant for each additional copy of the minor allele. Results: All SNPs were in Hardy-Weinberg equilibrium. Since there was age differences between groups (IBS 44±14, controls 38±13; p <.002), regressions were adjusted with age. No genotype association was found with IBS in the single locus analysis. However, the minor allele (T) for the rs4570625 was associated with a reduced risk of IBS for each additional copy (OR = 0.67, 95% CI: 0.42, 1.07; p= .088). The four SNPs from TPH 1 gene (rs4537731, rs684302, rs211105 and rs1799913) showed significant (p<.05) association with diarrheal symptoms and urgency. The CC genotype of rs4537731 showed significant association with bloating symptoms (p<.023) and abdominal pain (p<.043). Conclusion: This preliminary analysis suggest that TPH gene variants are related to GI symptoms, especially diarrhea, bloating and abdominal pain. A next step would be to investigate if there is any association between TPH gene polymorphisms and GI motility.
956 NFAT5 Regulates Intestinal Cell Differentiation by Negatively Regulating AKT Qingding Wang, Zheng Guo, Yuning Zhou, Xin Li, Tianyan Gao, Mark Evers Previously, we showed that inhibition of PI3-kinase or overexpression of PTEN enhances intestinal cell differentiation. In addition, NFATc1 and NFATc4, isoforms of the nuclear factor of activated T cells (NFAT) family of transcription factors, regulate PTEN expression and intestinal cell differentiation. NFAT5, a new isoform of NFAT, is a major inducer of osmoprotective genes and has exhibited distinctly different functions compared with NFATc1-c4. The purpose of this study is to investigate: i) the role of NFAT5 in intestinal cell differentiation, and ii) the regulation of Akt phosphorylation by NFAT5. METHODS. Differentiation was induced by treatment of HT29 and Caco-2 human colon cancer cells with sodium butyrate (NaBT) and assayed by measuring intestinal alkaline phosphatase (IAP) activity, an enterocyte differentiation marker. HT29, Caco-2, HCT116 and SW480 cells were transfected with control siRNA, NFAT5 siRNA, or control vector or constructs overexpressing myc-NFAT5 or HA-tagged PH domain leucine-rich repeat protein phosphatase (HA-PHLPP), a novel phosphatase specifically dephosphorylates the hydrophobic motif of Akt (Ser473). PHLPP, protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), p-Akt (Ser473), p-Akt (Thr308), Akt, PTEN and NFAT5 expression was analyzed by Western blot. Immunoprecipitation was performed using an anti-Akt antibody and Akt-PHLPP interaction was determined by Western blotting. NFAT mRNA expression was determined by RT-PCR. RESULTS. Treatment of HT29 cells with NaBT increased NFAT5 mRNA and protein expression. Knockdown of NFAT5 significantly inhibited NaBT-induced IAP activity in HT29 and Caco-2 cells, suggesting a role of NFAT5 in the regulation of intestinal cell differentiation. Knockdown of NFAT5 increased p-Akt (Ser473) level in HT29, Caco-2, HCT116, and SW480 cells, whereas overexpression of NFAT5 decreased p-Akt (Ser473) level in Caco-2 cells. However, knockdown of NFAT5 did not affect expression of PP1, PP2A, p-Akt (Thr308)
954 Carbohydrate Digestion in Congenital Sucrase Isomaltase Deficient and Recurrent Abdominal Pain Children Assesed by 13C- Starch Breath Test Claudia C. Robayo-Torres, Antone R. Opekun, Roberto Quezada-Calvillo, Maricela DiazSotomayor, Susan S. Baker, Buford L. Nichols Background: Starches contribute about half of the food energy needs to the weaned child's diet. Malabsorption of sucrose is associated with abdominal pain, bloating and diarrhea. A genetic disorder called Congenital Sucrase-Isomaltase deficiency (CSID) is suspected when these symptoms follow sugar ingestion and are relieved by a sugar elimination diet. Diagnosis is made upon demonstration of very low or absent sucrase activities in duodenal biopsy enzyme assays with a normal morphology. Treatment is by sucrose elimination diet and oral enzyme supplementation with sacrosidase. Clinical reviews of CSID have suggested that some of these patients also have starch malabsorption. The presence of coexisting starch and sucrose intolerance makes childhood dietary management very difficult and reduces the value of oral sacrosidase supplements. Confirmation of starch intolerance is made difficult because of lack of a specific substrate in mucosal biopsy enzyme assays and the shared SI
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is with rs26538 suggesting that the causal variant lies within the promoter region. REFERENCES 1) Barrett JC.et al. Nat Genet. 2008;40:955-62 2) Travassos LH et al. Nat Immunol. 2009; Nov 8 [Epub ahead of print] 3) Thurston TL et al. Nat Immunol. 2009;10:1215-21 4) Barrett JC et al. Bioinformatics 2005;21:263-5
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or PTEN, suggesting that NFAT5 regulates signaling events that directly control Akt phosphorylation at Ser473 site. Interestingly, knockdown of NFAT5 decreased the interaction between Akt and PHLPP. These results suggest that NFAT5 may regulate Akt (Ser473) phosphorylation through altering PHLPP-mediated inhibition of Akt. CONCLUSIONS. Our results demonstrate a role of NFAT5 in intestinal cell differentiation. Importantly, these data suggest that NFAT5-dependent positive regulation of differentiation is mediated by negatively affecting Akt phosphorylation through the novel regulation of the interaction between PHLPP and Akt.
959 Protease-Activated Receptors (PARS) 1 and 2 Stimulate Interleukin (IL)-8 Expression in HT-29 Cells via ERK/RSK90, Non-Classical NfκB Activation and Histone Acetylation Hongying Wang, France Moreau, Christina L. Hirota, Wallace K. MacNaughton Background: The G-protein-coupled PARs are known to stimulate the production of IL-8, an inflammatory and angiogenic chemokine, in epithelia. Since no mechanism has been described for this phenomenon, our aim was to determine the intracellular signaling pathway by which PARs stimulate IL-8 expression. Methods: HT-29 colonic epithelial cells were treated with the PAR1-activating peptide TFLLR-NH2 or the PAR2-activating peptide SLIGRLNH2 with or without inhibitors of MEK or RSK p90. IL-8 protein was measured in the culture supernatant by ELISA, mRNA levels were measured by RT-PCR and an IL-8 promoterdriven luciferase assay was used to measure promoter activity. Phosphorylation of ERK1/2 and the p65 B subunit was determined by western blot. NFκB transcriptional activity was measured via a κB-driven luciferase assay. Chromatin immunoprecipitation was used to assess binding of transcription factors to the IL-8 promoter. Co-immunoprecipitations were performed to examine interactions between κB subunits and other proteins. siRNA was used to transiently knock-down RSK1 or RSK2. Histone acetlytransferase (HAT) activity was measured using a commercially available kit. For some experiments, cells were stably transfected with HAT-mutated p300 (or empty vector). Results: Both PAR agonists stimulated IL-8 promoter activity, mRNA expression and protein production, which were dependent on ERK1/2. PAR activation also increased NFκB transcriptional activity and binding of the p50 B subunit to the IL-8 promoter, without any change in IκBα phosphorylation or degradation. PAR agonists induced phosphorylation of p65 at ser276 and its appearance in the nucleus, as well as enhanced binding of p300 to the p50 κB subunit. Furthermore, HAT activity toward the histone 3 substrate was also increased following PAR activation, although this was not accompanied by a significant increase in binding of acetylated histone 3 to the IL-8 promoter. PAR-induced phosphorylation of p65 induced nuclear translocation of RSK1 and RSK2, both of which are downstream target kinases from ERK1/2. siRNA knock-down of RSK1 and/or RSK2 reduced PAR-induced p65 phosphorylation, while pharmacological inhibition of RSK significantly blocked IL-8 induction by PAR activation. In cells stably expressing HAT-mutated p300, PAR activation led to significantly lower IL-8 production and promoter activity levels compared to cells transfected with the empty vector. Finally, PAR activation reduced binding of histone deacetylase 2 to p65 in an ERK-independent manner. Conclusions: These studies provide a cellular mechanism for PAR regulation of IL-8 expression in colonic inflammation and tumorigenesis.
957 TNF-α and Notch Signaling Synergistically up-Regulate OLFM4 Expression in Human Intestinal Epithelial Cells Ryuichi Okamoto, Junko Akiyama, Kiichiro Tsuchiya, Tetsuya Nakamura, Mamoru Watanabe Background & Aims: Recent studies in mice have shown that OLFM4 is expressed specifically in intestinal stem cells. However, the precise distribution of OLFM4-expressing cells within normal or inflamed human intestinal mucosa remains unknown. Also, the molecular mechanism regulating its expression within intestinal epithelial cells (IECs) has never been demonstrated. We have previously shown that Notch signaling is constitutively activated in IECs of the inflamed colonic mucosa, and thereby function as a key regulator of lineage-specific gene expression. Present study was planned to clarify the distribution of OLFM4-expressing IECs, and also the underlying mechanism regulating OLFM4 expression in IECs, in inflamed mucosa of human intestine. Methods: Immunostaining was performed to determine the distribution of OLFM4-expressing cells within the human intestinal tissue. Human IECderived cell lines were analyzed by RT-PCR or western blot to examine the expression level of OLFM4 in response to various inflammatory cytokines, including TNF-α, or upon activation of the Notch pathway, which was induced by forced expression of Notch1 intracellular domain (NICD1). Furthermore, a series of promoter assays were performed to analyze the transcriptional regulation of the OLFM4 gene expression within IECs. Results: Immunostaining of normal small intestinal or colonic tissue revealed clear expression of OLFM4 in IECs residing at the lowest part of the crypt, including crypt base columnar cells. However, in inflamed intestinal tissues derived from ulcerative colitis or Crohn's disease patients, increased number of OLFM4-expressing IECs was observed, expanding its distribution to the upper part of the crypt. In LS174T cells, both stimulation by TNF-α and forced intracellular expression of NICD1 significantly up-regulated mRNA and protein expression of OLFM4. Combination of NICD1 expression and TNF-a stimulation had synergistic effect upon upregulation of OLFM-4 mRNA expression, reaching up to 2500 fold increase in LS174T cells. Promoter assays using 5' flanking region of the human OLFM4 gene revealed that both TNF-a and NICD1 up-regulate OLFM4 expression at the transcriptional level, predominantly via the proximal NF-κB binding site. Consistently, TNF-α mediated up-regulation of NFκB-dependent transcriptional activity was significantly enhanced by NICD1 expression in LS174T cells. Conclusion: In the inflamed human intestinal mucosa, TNF-α and activation of Notch signaling synergistically up-regulate expression of OLFM4 in IECs. Such an upregulation of a stem-cell specific gene in IECs might be required to promote the regenerative response of the inflamed mucosa.
960 Disheveled Inhibits ZBP-89 Activation of p21Waf1 and OLFM4, an Intestinal Notch Target Natalia Veniaminova, Milena Saqui-Salces, Juanita L. Merchant BACKGROUND: A number of studies have implicated p21waf1 as a critical molecular switch that toggles intestinal cell fate between proliferation and growth arrest/differentiation. ZBP89 induces p21 in a butyrate-dependent manner and ZBP-89 protein levels increase with growth arrest/differentiation. We recently found by immunolocalization that ZBP-89 expression is elevated in enterochromaffin (EC) cells, which are also negative for βcatenin. This result is consistent with terminal differentiation of ECs and a recent study implicating βcatindependent signaling of ECs (Yadav VK, Cell/08). We therefore tested the hypothesis that βcat-independent signaling modulates ZBP-89 regulation of p21. Since Dishelved (Dvl) proteins interact directly with Wnt receptors and mediate both canonical and non-canonical signaling, Dvl was examined as a candidate ZBP-89 modulator. METHODS: Microarray analysis of tissue from cell specific disruption of ZBP-89 was performed on ZBP-89 floxed mice bred to VillinCre mice (ZBP-89ΔInt). Immunoblot analysis and transfection studies with the p21 and olfactomedin 4 (OLFM4) promoters were performed in mouse STC and human BON neuroendocrine cells. Whole cell extracts prepared from either cell line were used in co-immunoprecipitation experiments to identify protein-protein interactions with Disheveled (Dvl). RESULTS: A microarray analysis of ZBP-89ΔInt revealed that reduced levels of ZBP-89 in the intestine decreased OLFM4, a gene involved in myeloid development recently identified in intestinal Lgr5-progenitors. A recent study also identified OLFM4 in a microarray analysis designed to identify Notch gene targets. Co-immunoprecipitation studies revealed that ZBP-89 preferentially interacted with Dvl2 but not Dvl1 or Dvl3. Treating STC cells with trichostatin A increased the interaction of ZBP-89 with Dvl2. Using the OLFM4 reporter containing the proximal 427 bp of the promoter, we found that ZBP89 induced OLFM4 and that the induction was blocked by Dvl2 in STC cells. A similar result was observed with the p21 promoter. These promoters were not induced in BON cells, which expressed high levels of βcat compared to STC cells, which did not. We identified potential ZBP-89 (GTGGG) consensus-binding sites at -250, -165 and -148 suggesting OLFM4 as a direct ZBP-89 target. CONCLUSION: ZBP-89 induces the basal stem cell marker and putative Notch target gene OLFM4. Dvl2, a component of the Wnt pathway blocks the induction by ZBP-89. We suggest that ZBP-89 participates in intestinal EC cell specification by inducing p21 and OLFM4 to promote growth arrest and cell specification. This event requires restricting βcat-independent Wnt signaling.
958 BACH1 Deficiency Leads to the Suppression of Indomethacin-Induced Apoptosis in the Small Intestine Akihito Harusato, Yuji Naito, Tomohisa Takagi, Shinya Yamada, Katsura Mizushima, Yasuko Hirai, Ryusuke Horie, Ken Inoue, Kohei Fukumoto, Ikuhiro Hirata, Tatsushi Omatsu, Etsuko Kishimoto, Kazuhiko Uchiyama, Osamu Handa, Kazuhiko Igarashi, Toshikazu Yoshikawa BACKGROUND AND AIMS: Recent instrumental development in the diagnosis of small intestine has revealed that non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin, can induce small intestinal mucosal injuries. However, no clinical therapy for those lesions has been established. BTB and CNC homolog 1 (Bach1) is a transcriptional repressor of heme oxygenase-1 (HO-1). Bach1 plays an important role in the feedback regulation of HO-1, which protects cells from various injuries. The authors hypothesized that Bach1 has an important effect on the indomethacin-induced apoptosis in small intestinal mucosal injury by modulating HO-1 expression. METHODS: 8-week-old female C57BL/6 (wild-type) and homozygous Bach1-/- C57BL/6 mice were subjected to this study. Intestinal mucosal injuries induced by a single subcutaneous administration of indomethacin were evaluated macroscopically, histologically, and biochemically. Mucosal protein contents and chemokine mRNA levels were determined by western blotting and real-time PCR, respectively. We also investigated anti-apoptotic property of Bach1 deficiency by fluorescence staining. RESULTS: Indomethacin-induced intestinal injuries were remarkably improved in Bach1-/- mice. The expression of HO-1 mRNA in the intestinal mucosa was significantly increased in both mice after administration of Indomethacin, and it was significantly higher in Bach1-/- mice than in wild-type mice. Administration of indomethacin induced expression of chemokines, such as KC, MCP1 and MIP1-alpha, which was suppressed in Bach1-/- mice. MPO activity levels were also significantly decreased in Bach1-/- mice than in wild-type mice. The result of TUNEL staining in small intestine also showed that the extent of apoptosis was significantly suppressed in Bach1-/- mice compared with that in wild type mice. Western blot analysis showed that the increase in Bak and Bax expression and caspase-3 activation after indomethacin administration was suppressed in Bach1-/- mice. CONCLUSION: Disruption of the Bach1 gene caused inhibition of indomethacin-induced apoptosis indicating that inhibition of Bach1 could be a candidate to treat NSAIDs-induced small intestinal injuries.
961 The Myofibroblast Protein Epimorphin Modulates Hedgehog Signaling in the Colon Anisa Shaker, Shashi Bala, Gowri Kularatna, Marc S. Levin, Deborah C. Rubin In the colon, epithelial Indian hedgehog (Ihh) signals to the mesenchyme, which in turn feeds back to the epithelium to inhibit proliferation and promote differentiation. The precise mechanisms by which Ihh mediates its effects from mesenchyme to epithelium are unclear. Epimorphin (epi) is a mesenchymal/myofibroblast protein that exerts an anti-proliferative effect on overlying normal colon epithelium; epi deletion results in enhanced epithelial
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