S1773

Identification of microRNA Expressed in Duodenal and Gastric Mucosa, andModulation By the Duodenal Ulcerogen CysteamineLongchuan Chen, Xiaoming Deng, Tetyana Khomenko, Sandor Szabo

MicroRNAs (miRNAs) are non-coding small RNAs, which are found to regulate gene expres-sion in many tissues/organs in various physiologic processes. Cysteamine induces duodenaland rarely gastric ulcers in patients receiving cysteamine for cystinosis or hepatic damageand animal models. The rat model has been widely used to study the pathogenesis and healingof duodenal ulcers. In this study, we have performed miRNA microarray to complement ourprevious DNA microarray analysis of gene expression in rat duodenal mucosa to identifygene expression changes specifically associated with duodenal ulceration. Goals: To identifymolecular pathways leading to duodenal ulcer formation induced by duodenal ulcerogenslike cysteamine. Methods and Results: Groups of unfasted Sprague-Dawley female rats (180-200g) were given cysteamine-HCl (25mg/100g, once) by gavage. Rats were euthanized byCO2 inhalation 2 hr after cysteamine administration. Proximal duodenal and gastric mucosawas scraped. Total RNA including the miRNA fraction was extracted from mucosal samplesand further processed for Exiqon miRCURY microarray (289 rat miRNAs). Real-time PCRwas performed to confirm some candidate miRNAs. Eight miRNAs were found to have 4-fold or higher difference in expression level between duodenal and gastric mucosa. Amongthem, miR-215 was reported to be upregulated in Barrett's esophagus and is likely a duodenal/small intestinal specific miRNA. Cysteamine changed the expression level of 20 miRNAs induodenal mucosa by over 2-fold; two of these by more than 4-fold. Eleven of them areunpublished novel miRNAs. miR-542-5p was increased more than 8-fold by cysteamine. Insilico analysis shows its target genes include insulin-like growth factor 2 receptor, paired-likehomeodomain transcription factor 3, RCE1 homolog and hypothetical protein LOC152485.Conclusions: 1) We have identified a group of miRNAs differentially expressed in duodenaland gastric mucosa. 2) Cysteamine modulates the expression of several miRNAs which maycontrol wound healing, tissue repair and angiogenesis. 3) Thus, miRNA analysis providesa new way to study gene expression changes in the pathogenesis of duodenal ulceration.

S1774

JunD Represses Importin-α1 Transcription Through Its Proximal PromoterRegion and Regulates Subcellular Localization of RNA-Binding Protein HuR inIntestinal Epithelial CellsJie Chen, Tongtong Zou, Rao N. Jaladanki, Lan Liu, Lan Xiao, Emily Bellavance, TingxiYu, Douglas J. Turner, Jian-Ying Wang

Intestinal epithelium is a rapidly self-renewal tissue in the body and its integrity is highlyregulated. Our recent studies show that RNA-binding protein HuR plays a critical role inregulating in intestinal epithelial cell (IEC) proliferation and that increased cytoplasmic levelsof HuR stabilize mRNAs of p53, nucleophosmin, and ATF-2, thus inhibiting IEC renewal.However, the mechanism underlying HuR trafficking between the nucleus and the cytoplasmremains elusive. JunD is an AP-1 transcription factor and is implicated in negatively regulatingIEC proliferation. Importin-α1 is an adaptor protein that transports bound cargoes throughthe nuclear pore complex and is involved in mediating HuR nuclear import. This studydetermined if the subcellular localization of HuR is regulated by JunD through modulationof importin-α1 expression. Methods: Studies were conducted in human IECs (Caco-2 line).Full-length importin-α1 promoter was cloned, and constructs of wild-type and variousmutated importin-α1-promoter luciferase reporters were generated. Importin-α1 expressionwas examined by measuring its promoter activity and mRNA and protein levels. JunDoverexpression was induced by transient transfection with the JunD expression vector undercontrol of PCMV promoter. Cytoplasmic and nuclear proteins were isolated for detectingHuR subcellular levels. Results: JunD protein levels were increased by ~15-fold when Caco-2 cells were transfected with the JunD expressing vector for 48 and 72 h. Ectopic expressionof JunD repressed importin-α1 gene transcription as indicated by decreased (~70%) importinα1-promoter activity and its mRNA and protein levels, but it did not affect expression ofimportin-β and transportin-1. Studies using deletion- and point-mutations of importin-α1-promoter revealed that JunD repressed importin-α1 transcription through CREB-bindingsites that were located at its proximal promoter region. Reduction of importin-α1 by JunDoverexpression increased cytoplasmic levels of HuR, although it failed to alter levels in totalHuR. Levels of cytoplasmic HuR were ~3-fold the control value after JunD transfection.Furthermore, increased levels of endogenous JunD by depleting cellular polyamines alsoinhibited importin-α1 expression and increased cytoplasmic levels of HuR, which wasassociated with an increase in G1-phase growth arrest. Conclusions: These findings indicatethat JunD represses importin-α1 transcription through its proximal promoter region andthat JunD-mediated inhibition of importin-α1 expression is crucial for the subcellular distri-bution of HuR.

S1775

Reciprocal Expression of Calcium-Sensing Receptor (CaSR) and Ror2 DuringColon Cancer Progression: A New Role for Dkk1Maria Westerhoff, Ivan I. Pacheco, R. John MacLeod, Jerrold R. Turner

Dietary calcium prevents colon cancer progression. The epithelial extracellular CaSR maymediate this effect, but mechanistic details are not well defined. In Vitro, CaSR activationdrives β-catenin degradation in intestinal epithelial cells. This effect is partly-mediated byCaSR-induced secretion of Wnt5a, a ligand for the orphan receptor tyrosine kinase Ror2.In myofibroblasts, CaSR activation drives secretion of Dkk1, a Wnt antagonist. The aim ofthis study was to determine if dysregulated expression of CaSR, Ror2, and Dkk1 mightexplain inappropriate β-catenin activation in colon cancer. METHODS: The ability of Dkk1to inhibit Ror2-induced β-catenin activation was compared in human colon cancer-derivedcell lines HT29 and SW480 which do and do not secrete Dkk1, respectively. The TOPFLASHreporter was used to assess β-catenin activation. CaSR and Ror2 expression were evaluatedin samples of normal colon, dysplasia, and colon cancer from 22 and 15 patients (416 and

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276 tissue microarray cores), respectively, by immunohistochemistry and scored semiquantit-atively from 0 to 3. RESULTS: Transgenic Ror2 expression in SW480 cells enhanced β-catenin activation 3.5±0.2 fold (p<0.05). In contrast, Ror2 expression in HT29 cells inhibitedβ-catenin activation by 62±13% ( p<0.05). This was difference was largely due to Dkk1, sincerecombinant Dkk1 or conditioned medium from CaSR-activated myofibroblasts completelyblocked increased β-catenin activation in Ror2-expressing SW480 cells (p<0.001). To assessthe In Vivo relevance of these observations, CaSR and Ror2 expression was assessed inhuman colon cancers. CaSR was strongly expressed in benign colonocytes (average 2.0±0.2)and was similar in in situ dysplasia (2.3±0.2) and well-differentiated invasive cancer (1.9±0.2),but was detected weakly in poorly-differentiated invasive cancer (1.1±0.2; p<0.01). Con-versely, Ror2 was detected only weakly in benign colonocytes (1.4±0.1), but expression wasprogressively increased in in situ dysplasia (2.0±0.1; p<0.01) and invasive cancer (2.5±0.1;p<0.01). CONCLUSION: These data show that the secreted Wnt antagonist Dkk1 can workas an agonist of Ror2 to inhibit defective Wnt signaling in colon cancer. Together withdecreased CaSR expression and increased Ror2 expression in colon cancer, this suggests thatthe CaSR-Dkk-1-Ror2 signaling pathway is important in preventing oncogenic progression.Together with previous reports that Dkk1 expression is lost in colon cancer, these datasuggest that CaSR-dependent inhibition of carcinogenesis may involve Dkk1-mediated Ror2activation to inhibit inappropriate β-catenin activation.

S1777

Protein Kinase D3 Plays a Pro-Apoptotic Role in Oxidative Stress-InducedApoptosis in Intestinal Epithelial CellsJun Song, Jing Li, B. M. Evers, Dai H. Chung

Protein kinase D (PKD) is a serine/threonine kinase that belongs to the family of calcium/calmodulin-dependent kinases; the PKD family consists of three members, PKD1/PKCµ,PKD2, and PKD3/PKCν. PKD1 was identified as a sensor for oxidative stress that can beactivated by mitochondria-generated and other reactive oxygen species. We have recentlyfound that Rho/PKCδ/PKD1 signaling is an important anti-apoptotic pathway during oxidat-ive stress-induced intestinal cell injury; however, the exact role of PKD3, the most abundantisoform in rat intestinal epithelial cells, during oxidative stress-induced intestinal cellapoptosis is not known. Therefore, the purpose of our study was two-fold: (i) to determinethe exact role of PKD3 during oxidative stress-induced intestinal cell injury, and (ii) toidentify the downstream effectors of PKD3 upon oxidative stress. METHODS. We used ratintestinal epithelial cells (RIE1) for our studies. Stable cell lines of RIE1 cells overexpressingthe wild type PKD3 (RIE1/PKD3WT) or the kinase dead PKD3 (RIE1/PKD3KD) were estab-lished. In order to knockdown the endogenous PKD3, short hairpin PKD3 (shRNA) wasgenerated. Cell death assay was determined using a DNA fragmentation ELISA assay. Westernblotting was performed to assess protein expression. RESULTS. H2O2-induced cell death,as measured by DNA fragmentation, was further increased in RIE1/PKD3WT cells, butdecreased in RIE1/PKD3KD cells and cells transiently transfected with PKD3 shRNA. Consist-ent with DNA fragmentation data, the cleaved caspase-3 and PARP expression was alsoincreased in RIE1/PKD3WT cells, but decreased in RIE1/PKD3KD cells as well as in cellstransfected with PKD3 shRNA. Furthermore, H2O2-induced phosphorylation of p38 andJNK was attenuated in cells transfected with either PKD3 shRNA or RIE1/PKD3KD, butenhanced in RIE1/PKD3WT cells. CONCLUSIONS. Our results demonstrate that the PKD3/p38 or PKD3/JNK pathway plays a pro-apoptotic role in intestinal epithelial cells, in contrastto PKD1. These findings provide potential novel therapeutic targets for the development ofnew strategies to treat inflammatory conditions of the gastrointestinal tract.

S1778

Cigarette Smoke Extract Protects Intestinal- and T-Cells Against Death-Receptor- and Oxidative Stress Induced Apoptosis: An Explanation forDichotomal Effects of Smoking On Ulcerative Colitis and Crohn's Disease?Elise M. van der Logt, Klaas-Nico Faber, Tjasso Blokzijl, Dirk-Jan Slebos, Maikel P.Peppelenbosch, Gerard Dijkstra

Introduction: For long, smoking has been a known risk factor for inflammatory boweldisease, but with a remarkable opposite effect on Crohn's disease (CD) and ulcerative colitis(UC). Where smoking aggravates CD and makes it more resistant to therapy, it actuallyameliorates UC and decreases the need for colectomy. A diminished barrier function of thecolonic epithelial compartment and insufficient activation-induced apoptosis of the T-cellcompartment is recognized in both diseases. We hypothesize that smoke further increasesthe resistance of T-cells to apoptosis in CD patients, whereas such components will providecytoprotection to colonic epithelial cells in UC. Therefore, we studied the effect of cigarettesmoke extract (CSE) on death-receptor- and oxidative stress-induced apoptosis in variousintestinal- and T-cell lines. Methods: Intestinal- (DLD-1, SW480, HCT116, Caco-2) and T-cells (Jurkat, MOLT-4) were exposed to different apoptotic stimuli in presence or absenceof different percentages CSE (v/v). 25 ml culture medium was saturated with smoke from2 standard 3R4F cigarettes (scientific reference) and defined as 100% CSE. Death-receptor-mediated apoptosis was induced by anti-FAS or cytokine mixture (CM; TNF-α + INF-γ +IL-1β). Oxidative stress-mediated apoptosis was induced by the superoxide anion donormenadione. Apoptosis was determined by measuring caspase-3 activity and cleaved PARP,and necrosis by Sytox Green staining. Results: Addition of 0-30% CSE to anti-FAS, CM ormenadione treated cells resulted in a dose-dependent decrease of caspase-3 activity andPARP cleavage in all cell lines investigated. CSE alone did not induce apoptosis, but CSEconcentrations above 50% induced necrosis. CSE inhibitory effects were most pronouncedfor menadione-induced apoptosis followed by CM and anti-FAS. A remarkable observationwas that CSE abolished menadione-induced caspase-3 activity and induced a switch fromapoptosis into necrosis for the highest concentrations of CSE used. In conclusion CSEprotects intestinal- and T-cells against death-receptor- and oxidative stress-induced apoptosis.Increased T-cell resistance against apoptosis is detrimental for CD, while increased colonicepithelial cell resistance against apoptosis, in particular resistancy to oxidative stress, isbeneficial for UC.

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