852 The Tumor Vasculature is a Paracrine Target of Hedgehog in Pancreatic Cancer

AGA Abstracts

healing (decrease from baseline in the Mayo endoscopy sub score >/=1 AND a Mayo score
850 Restoration of Sonic Hedgehog in the Adult Stomach Results in the Recovery of the Gastric Epithelium to the Normal Differentiated State Chang Xiao, Joseph T. Roland, Ki Taek Nam, James R. Goldenring, Yana Zavros Objective: Parietal cell-expressed Sonic Hedgehog (Shh) regulates the function and differentiation of the gastric epithelium. Correlative evidence shows that loss of Shh expression in the adult stomach that is associated with Helicobacter pylori infection results in the disruption of the normal gastric epithelium. We sought to identify whether Shh regulates gastric epithelial cell differentiation in the absence of inflammation. Methods: A mouse model expressing a parietal cell-specific, tamoxifen-inducible deletion of Shh was developed (HKCreERT2;Shhflox/flox). At 4 weeks of age, HKCreERT2;Shhflox/flox mice were treated with either tamoxifen (0.75 mg/day, i.p.) or vehicle for 5 consecutive days. Mice were analyzed 1 and 5 months after the final tamoxifen injection. Epithelial cell proliferation was studied using BrdU labeling 24 hours prior to analysis. Cell lineage changes were studied using markers specific for surface mucous (Ulex europaeus, UEAI), mucous neck (Griffonia simplicifolia, GSII), zymogen (intrinsic factor, IF) and parietal (H+,K+-ATPase) cells. Results: 1 month after the final tamoxifen injection: Compared to the vehicle-treated Control group where H+,K+-ATPase staining co-localized with Shh, Shh immunostaining was absent in the parietal cells of tamoxifen-treated HKCreERT2;Shhflox/flox, verifying the successful deletion of Shh 1 month after the final tamoxifen injection. Compared to Controls, tamoxifen-treated HKCreERT2;Shhflox/flox mice developed foveolar hyperplasia reflected by an expansion of BrdU/UEAI positive cells. Tamoxifen-treated HKCreERT2;Shhflox/flox mice also showed an expansion of GSII/IF co-expressing cells at the base of the gastric gland that indicated a delay in the differentiation of zymogen cells from mucous neck cells. 5 months after the final tamoxifen injection: Immunofluorescence of stomach sections taken from tamoxifentreated HKCreERT2;Shhflox/flox mice 5 months after the final tamoxifen injection showed reexpression of Shh expression within parietal cells. Interestingly, re-expression of Shh within parietal cells of tamoxifen-treated HKCreERT2;Shhflox/flox mice resulted in decreased surface mucous pit cell proliferation down to numbers equivalent to those counted in vehicle-treated Control animals. Conclusion: Loss of Shh in the adult stomach resulted in the development of foveolar hyperplasia and delayed differentiation of the zymogenic cell lineage independent of inflammation. Recovery of the gastric epithelium to the normal differentiated state was correlated with the restoration of parietal cell-expressed Shh.

848 Notch Signaling Induces Expression of the Intestinal Stem Cell Marker Olfactomedin 4 (OLFM4) Kelli L. VanDussen, Åsa Kolterud, Deborah L. Gumucio, Linda C. Samuelson Background/Aims: Notch is a critical pathway regulating proliferation and differentiation in the intestine. Blocking Notch signaling by genetic or pharmacologic means results in decreased epithelial cell proliferation and altered cell fate, suggesting that a stem or progenitor cell is targeted by Notch; however, the identity of this target is unknown. This study sought to identify Notch-responsive cells by analysis of stem and progenitor markers in fetal and adult mouse intestine after Notch inhibition. Methods: Notch signaling was disrupted in two mouse models by treatment with γ-secretase inhibitor (GSI): fetal intestinal cultures treated with 40 μM DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) for 3 days and adult mice treated with 30 μmol/kg DBZ (dibenzazepine) daily for 5 days. Cell lineages were measured by histological analysis and marker gene expression. Stem and progenitor cell marker expression was measured by qRT-PCR, including genes specific for the +4 quiescent stem cell (Bmi1), the active crypt base columnar (CBC) stem cell (Lgr5, Ascl2, Olfm4) and progenitor cells (Msi1, Prom1). The mechanism of Notch regulation of stem cell gene expression was further examined by transient transfection studies in the human colon cancer cell line LS174T. Results: Reduced proliferation and increased secretory cell types, including goblet, Paneth and endocrine cells, were observed in both GSI-treated models, demonstrating effective Notch inhibition. Interestingly, intermediate cells that possess characteristics of both goblet and Paneth cells were observed in the crypts of GSItreated fetal and adult intestine, suggesting that Notch may regulate cell specification and maturation in distinct ways. Analysis of stem/progenitor marker expression showed that disruption of Notch signaling affected the CBC stem cell specific transcript Olfm4, while the other markers were unchanged. Olfm4 was reduced 40-fold in DAPT-treated fetal intestine cultures and 20-fold in DBZ-treated adult mouse intestine, suggesting that Notch activates transcription of this gene. Analysis of an Olfm4-luciferase construct in LS174T cells was consistent with this conclusion, with increased activity observed with constitutive Notch signaling and decreased activity in the presence of GSI or dominant negative mastermind, a key component of the Notch transcriptional complex. Conclusions: Notch signaling targets the intestinal CBC stem cell to induce transcription of the secreted protein olfactomedin 4. In addition, disruption of Notch signaling induces formation of intermediate cells in crypts of fetal and adult mouse intestine.

851 Phylogenomic Analysis of GLI/CI Binding Site Density and Affinity: An Integrative in Silico Approach to Identifying Hedgehog-Regulated Enhancers Aaron M. Udager, Dave S. Parker, Katherine Gurdziel, Mridula Jayaraman, Scott E. Barolo, Deborah L. Gumucio

Hedgehog (Hh) signaling in the mouse intestine is exclusively paracrine (from epithelium to mesenchyme; Kolterud et al., 2009), yet perturbation of Hh pathway activity in the gut has dramatic consequences for intestinal epithelium (Madison et al., 2005). Thus, these epithelial changes are secondary to changes that occur in mesenchymal cells as a result of epithelial Hh signals. Characterization of direct Hh target genes in mesenchymal cells would facilitate the construction of Hh-centric Gene Regulatory Networks (GRN) that, in turn could provide important insight into mechanisms of disease pathogenesis in models of Hh dysregulation (e.g., the potential connection between reduced Hh signaling and IBD; Lees et al., 2009).

Despite intense study in a wide array of species, relatively few direct Hh target genes have been identified In Vivo. Recent data suggest that a precise affinity (either high or low) of Gli/Ci for binding sites in Hh-regulated enhancers is required for a proper response to Hh signaling (unpublished observations; Barolo SE). Since previous computational approaches had focused predominantly on high-affinity sites, we sought to prospectively identify Hh-regulated enhancers in silico by analyzing the phylogenomic distribution of high- and low-affinity Gli/Ci sites. Utilizing available In Vitro DNA binding data for Gli/ Ci, we defined a set of predicted binding sites and cataloged their genomic locations in twelve closely related Drosophila species. A Monte Carlo method was used to model genomewide background clustering and identify Ci-enriched regions (CER) in D. melanogaster. Sequences orthologous to CERs in the other eleven Drosophila species were then collected using either the liftOver tool (genome.ucsc.edu) or custom Perl scripts and pre-existing multiple alignment format (MAF) files. A subset of CERs (conCER) was identified that contains Gli/Ci sites that are highly conserved across all twelve Drosophila species, and interestingly, these sites span a range of predicted binding affinities (ie, high and low). Five previously validated Hh-regulated enhancers contain conCERs, and conCERs are strongly associated with both known and predicted Hh target genes. In addition, many genes not previously known to be Hh targets were linked to conCERs. We evaluated the regulatory potential of a cohort of conCERs In Vivo in transgenic reporter flies and identified previously unrecognized Hh-regulated enhancers for rdx, inv and Mrtf. These data provide proof of principle for an integrative phylogenomic approach to Hh target gene identification in vertebrates, which is now ongoing.

849 A Long-Term, Mesenchyme-Free In Vitro Culture System of Colonic Epithelial Cells Retaining Stem Cell Compartment With Proliferation/Differentiation Properties Shiro Yui, Tetsuya Nakamura, Tomohiro Mizutani, Yasuhiro Nemoto, Ryuichi Okamoto, Kiichiro Tsuchiya, Shizuko Ichinose, Mamoru Watanabe Background & Aims: Having a simple and reproducible method to grow specialized cell types In Vitro is highly desirable in order to study mechanisms by which their proliferation and differentiation are controlled. Recently, several reports showed that primary intestinal epithelial cells containing their stem cell populations could be efficiently maintained In Vitro; however, the long-term and mesenchyme-free culture system of colonic epithelial cells has not been established. This study was undertaken to develop a culture method of pure colonic epithelial cells allowing long-term maintenance of the stem cells In Vitro. Methods: Colonic tissues from normal adult mice at 7 to 9 weeks of age were used to test various conditions for i) the reagent and procedure to isolate the colonic epithelium, ii) the type of matrix in which the cells were embedded, and iii) the use of culture medium and the additional constituents. Cultured cells were morphologically assessed by immunohistochemistry and transmission electron microscopy (TEM). The patterns of gene expression were analyzed by semi-quantitative RT-PCR. Results: After screening a number of combinations of conditions, we have developed a methodology for long-term culture of adult mouse-derived colonic cells in a serum- and mesenchyme-free condition. The cells formed a round cyst-like threedimensional structure having luminal space inside. TEM analysis showed that the cystic structure was composed of monolayer of cells characteristic of epithelium with apicobasal polarity and no mesenchymal cells were present. The epithelial organoid was histologically shown to consist of proliferating cells as well as differentiated cells of multilineage. RT-PCR analysis supported this notion as it showed the expression of Lgr5, a marker gene of the colonic epithelial stem cells In Vivo, in addition to several marker genes for the terminally differentiated cell types. The cells could continuously proliferate with the size of the cystic organoids growing and be repeatedly propagated for months without losing their initial properties. Furthermore, addition of a γ-secretase inhibitor increased the number of goblet cells as well as the expression level of the Muc2 gene, suggesting the cultured cells to retain their physiological capability of differentiating into the secretory lineage by Notch signal inhibition. Conclusions: The techniques that we have developed would be of significant help not only to facilitate the colonic epithelial stem cell biology but also to build a basis for cell replacement therapy for various human colonic diseases.

AGA Abstracts

852 The Tumor Vasculature is a Paracrine Target of Hedgehog in Pancreatic Cancer Yoshiaki Sugiyama, Yusuke Mizukami, Junpei Sasajima, Kazumasa Nakamura, Toru Kawamoto, Kazuya Koizumi, Mikihiro Fujiya, Satoshi Tanno, Hidenori Karasaki, Toru Kono, Daniel C. Chung, Nabeel Bardeesy, Yutaka Kohgo Tumor-derived signals systemically induce a switch in bone marrow (BM) cells that allow the ‘activated' BM-derived cells to function as instigators of tumor growth. The hedgehog (Hh) pathway has been implicated in the development of embryonic blood vessels and pathogenesis of cancer. Smoothened (Smo), one of the receptors in Hh signaling, is a promising molecular target for the treatment of malignancies including pancreatic ductal adenocarcinoma (PDAC), and recent studies have demonstrated an oncogenic function of Hh in stromal cells. We have identified novel molecular mechanisms by which Hh regulates

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tumor angiogenesis through a regulation of BM-derived pro-angiogenic cells (BMPCs). Cyclopamine significantly attenuated the homing of BM-derived cells into the tumor vasculature in human PDAC xenografts, suggesting that Hh signaling may play a role during migration and differentiation of BMPCs. The effect of the Hh ligand Shh in the tumor microenvironment was considered to be cell non-autonomous, and IGF-1 production in the tumor stroma played a key role during the process. In Vitro co-culture experiments demonstrated that KP1N human PDAC cells induced Gli1 and IGF-1 in c-Kit+ BM cultured mononuclear cells utilized as BMPCs, and the induction was significantly attenuated either by cyclopamine or lentiviral shRNA targeting Smo. Tube formation assay with the mouse endothelial line MS1 supports the role of Shh secreted from PDAC cells to induce migration and capillary formation of the BMPCs, and the enhancement of the capillary morphogenesis was blocked by an anti-IGF-1 neutralizing antibody. This “paracrine” effect of Hh seems to be a late event during pancreatic tumorigenesis, as full length Gli2 expression in the neovasculature was detected within PDAC lesions in Pdx1-Cre;LSL-KrasG12D;p53lox/+ mice, but not in precursor PanIN lesions in Pdx1-Cre;LSL-KrasG12D mice. Collectively, Hh derived from cancer cells can have a profound effect on neovascularization through the regulation of BMderived cells during late stages of pancreatic tumorigenesis, and targeting Hh would be a novel therapeutic approach to inhibit tumor angiogenesis.

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Background and aim: Chronic stress exacerbates the morbid symptom of motility dysfunction in inflammatory bowel disease patients. The cellular mechanisms of interactions between psychological and inflammatory stressors to worsen colonic motor dysfunction are not understood. Methods: We employed a 9-day heterotypic chronic stress (HeCS) protocol of randomly distributed daily applications of water avoidance, forced swimming and coldrestraint stressors. Mild inflammation was induced with intraluminal application of 17 mg/ kg TNBS (TNBS 17) on day 2 of chronic stress. Separate groups of rats received TNBS 17 only, HeCS only or no adverse stimuli. Tissues were harvested 8-hours after the last psychological stressor of HeCS or 7-days after TNBS 17 inflammation. Results: TNBS 17 alone modestly suppressed the reactivity of colonic circular smooth muscle strips to ACh (10-6 M to 10-2 M, p<0.05). By contrast, HeCS alone enhanced the reactivity of circular muscle strips to ACh. However, the stressful environment induced by HeCS during TNBS 17 significantly worsened the suppression of reactivity of circular muscle strips to ACh (p<0.05). TNBS 17 or HeCS alone did not increase MPO activity in colonic muscularis externa. However, TNBS 17 during HeCS significantly enhanced the MPO activity (40.3 + 15.2 vs. 8.2 + 4.6 unit/mg of protein, p=0.025). We examined the expression of a panel of 11 cytokines/chemokines of the Th1/Th2 groups in the 4 groups of rats. HeCS alone increased only TNFα, while TNBS 17 alone increased only IL-8 in the muscularis externa. The concurrent applications of HeCS and TNBS 17 significantly enhanced the expressions of IL1β, IL-8, MCP-1, but suppressed the expression of Th2 cytokine IL-4. Other cytokines (IL5, IL-10, IL-12, IFN-γ, and IL-17) were not detected in any group. Next, we investigated the molecular basis of suppression of reactivity to ACh during the concurrent application of HeCS and TNBS 17. The concurrent psychological and inflammatory stimuli significantly suppressed the expression of the G protein Gαq/11, (66 + 14% of the control, p=0.032), without affecting the other cell-signaling proteins of the excitation-contraction coupling (α1C subunit of L-type calcium channels, MLCK, ROK1, MYPT1, PP1c, MLC20, and CPI-17). Conclusion: Concurrent chronic psychological stress significantly worsens colonic motor dysfunction due to mild inflammatory insult by up regulating specific Th1 cytokines and suppressing Th2 cytokines. The suppression of colonic contractility is due to down regulation of the key cell-signaling protein, Gαq/11, of the excitation-contraction coupling in colonic circular smooth muscle cells.

853 Gastric Acid-Mediated Release of Intracellular Calcium Stimulates Sonic Hedgehog Gene Expression Mohamad El Zaatari, Yana Zavros, Arthur Tessier, Meghna Waghray, Stephen I. Lentz, Deborah L. Gumucio, Andrea Todisco, Juanita L. Merchant Introduction: Helicobacter pylori (Hp) infection suppresses acid secretion through its ability to induce a robust pro-inflammatory response that includes secretion of IL-1β. Hp also inhibits Shh gene expression in human subjects. We recently reported that IL-1β inhibits Shh gene expression through its ability to block acid secretion. Since acid stimulates the release of intracellular calcium (Ca2+i), we hypothesized that acid regulates Shh gene expression through its ability to regulate Ca2+-dependent PKC. Methods: We blocked Hh signaling by transgenically overexpressing a secreted form of the Hedgehog interacting protein-1 (sHip-1), a natural inhibitor of hedgehog ligands, from the H,K-ATPase promoter to produce hypochlorhydric mice. Gadolinium, EGTA+BAPTA, PKC-overexpressing adenoviruses, and PKC-inhibitors were used to modulate Ca2+i-release, PKC-activity and Shh expression in primary gastric cultures. Results: sHip-1 mice exhibited lower gastric acidity, reduced somatostatin and increased gastrin gene expression. Shh induced somatostatin gene expression in enriched D cell cultures. Consistent with prior In Vitro evidence that acid induces Shh expression, sHip-1 mice showed lower Shh expression, similar to levels obtained after omeprazole treatment of wild-type mice. We investigated the mechanism of acid-mediated regulation of Shh expression and found that exogenous acid induced parietal cell Ca2+irelease. Gadolinium, a Ca2+-channel blocker, increased Ca2+i and potently induced Shh expression in primary mouse gastric cells. Chelation of Ca2+i with BAPTA plus EGTA reduced Shh expression in gastric organ cultures. Hypothesizing that calcium activation of PKCs regulated Shh gene expression, we showed that overexpression of PKC-α, -β and -δ, but not PKC-ε induced Shh gene expression. In addition, phorbol ester, a direct activator of PKCs, induced a Shh-luciferase promoter construct. Conclusion: Gastric acid induced Shh gene expression through Ca2+i-release and PKC-activation, suggesting that acid-regulated Shh might mediate the regulatory feedback loop through somatostatin.

856 Delayed Gastric Emptying Induced by Chronic Complicated Stress is Improved by Social Attachment in Rats - Involvement of Upregulated Hypothalamic Oxytocin Reji Babygirija, Jun Zheng, Mehmet Bulbul, Kirk A. Ludwig, Toku Takahashi Background: Acute stress delays solid gastric emptying via central corticotrophin-releasing factor (CRF), while centrally released neuropeptide, oxytocin, is believed to be anxiolytic and has stress-attenuating effects. Our recent study showed that 5 consecutive days of repeated restraint stress (chronic single stress) up-regulates hypothalamic oxytocin mRNA expression and restores delayed gastric emptying. In contrast, 7 consecutive days of varying types of stress (chronic complicated stress) fails to up-regulate oxytocin mRNA expression, resulting in maladaptation and delayed gastric emptying. It has been shown that the social interaction of daily life generates a positive environment which continuously activates the system of oxytocin release in both males and females. We investigated the effects of paired housing on gastric emptying during chronic complicated stress in rats. Methods: Adult male Sprague-Dawley rats were either paired or singly housed 1 week prior to the stress loading. Solid gastric emptying was measured after 7 consecutive days of varying types of stress (restraint stress, cold restraint stress, force swimming stress and water avoidance stress). To study whether endogenous oxytocin is involved in restoring the delayed gastric emptying after paired housing, an oxytocin antagonist [d (CH2)5 1.Tyr (Me) 2, Orn8)-oxytocin; 100 ng] was injected intracerebroventricularly (ICV) before the gastric emptying study. CRF and oxytocin mRNA expression in the paraventricular nucleus (PVN) of the hypothalamus was evaluated by real time RT-PCR in singly and paired housed rats after chronic complicated stress loading. Results: In singly housed rats, chronic complicated stress significantly delayed gastric emptying (34.1±4.3%, n=6, P<0.01), compared to that of non-stressed rats (54.3±5.6%, n=6). Delayed gastric emptying observed in singly housed rats induced by chronic complicated stress was significantly improved in paired housed rats (48.6±6.5%, n=8, P<0.05). ICV-administration of oxytocin antagonist attenuated the restored gastric emptying in paired housed rats (39.4±7.1%, n=6) after chronic complicated stress. Increased CRF mRNA expression at the PVN observed in singly housed rats was significantly reduced in paired housed rats, In contrast, oxytocin mRNA expression at the PVN was significantly increased in paired housed rats after chronic complicated stress. Conclusion: These results indicate that social attachment restores delayed gastric emptying following chronic complicated stress, via down-regulating CRF expression and up-regulating oxytocin mRNA expression. This may provide the scientific benefit of social attachment in our daily life.

854 Decreased NR2B Subunit Postsynaptic Levels in the Anterior Cingulated Cortex Impair Long-Term Potentiation, and Suppress Visceral Pain in the Visceral Hypersensitive Rats Zhijun Cao, Shengliang Chen, Xulu Tian, Jin Yan, Jianzhong Mo, Ying Li Anterior cingulated cortex (ACC) modulates visceral pain. We have demonstrated the upregulation of NR2B receptor and Calcium/Calmodulin-dependent Protein Kinase II (CaMKII) in the ACC synapses in visceral hypersensitive (VH) rats. The C-tails of the NR2B bind to CaMKII. Even if the molecular details of these interactions have been addressed, the physiological function of these interactions still remains to be clarified. We hypothesize that synaptic translocation of NR2B receptor induced by CaMKII binding is critical for induction ACC synaptic long term potentiation (LTP) and visceral pain memory. We performed a subcellular fractionation study to prepare synaptic and extrasynaptic NMDARs in normal and VH rats 14 days after induction of colonic anaphylaxis to examine how CaMKII modulate the localization of these different pools of NMDARs. We showed that synaptoneurosome (SS) are enriched for synaptic proteins, NR2B, NR2A, PSD95 and αCaMKII. The Triton-X insoluble fraction (Insol) recovered from SS was to be enriched for the postsynaptic density (PSD) marker protein PSD95, but did not contain the membrane-bound extrasynaptic GABAB receptor. In contrast, the Triton-X soluble fraction (Sol) contained just GABAB receptor. No changed in the protein levels of NR1 and NR2A in whole homogenates (WH), SS or PSD in VH rats were observed. In contrast, increases in NR2B (123±9%, 186±14%, and 210±19 of control), and αCaKMII (127±11%, and 207±15%, 238±11 of control, respectively) were observed. Interestingly, no changes of NR2B and CaMKII were observed in extrasynaptic fraction (Sol) in VH rats. Microinjection of a CaMKII inhibitor, Antennapedia-autocamtide2-related inhibitor peptide II (Ant-AIP-II) that competes with αCaMKII binding to NR2B, into pACC cause a 88% decrease of NB2B level in the PSD (Sol), and a 114% increase of NR2B in extrasynaptic (Insol) fractions. No change was observed in the SS fraction. Western blotting following co-immunoprecipitation shows a reduced NR2B-CaMKII protein complex in PSD. These results indicate that binding to CaMKII represents an important mechanism for postsynaptic anchoring of NR2B receptor. In parallel, theta burst stimulation, which was used to induce LTP, evoked a response of 240±19% of basal in VH rats compared to 107±5% in control, which was 2x longer than that observed in controls. Pretreatment with Ant-AIPII impair the induction of LTP accompanied by a markedly reduction of CRD-induced behavioral pain (visceromotor responses) in VH rats. In conclusion, interaction between NR2B receptor and CaMKII is critical for NR2B stabilization at postsynaptic sites to maintain ACC synaptic plasticity and visceral pain.

857 Aberrant Neuro-Network in Processing of Visceral Placebo Analgesia Among IBS Patients Hsing-Feng Lee, Ching-Liang Lu, Jen-Chuen Hsieh, Tzu-Chen Yeh, Chou-Ming Cheng, Full-Young Chang, Shou-Dong Lee INTRODUCTION: The management of irritable bowel syndrome (IBS) is limited by the lack of efficient treatment and high placebo effect. Large placebo effect was accompanied by a significant decreased neuronal activity in visceral pain matrix upon colorectal balloon distension (CRD) in IBS patients.(Pain 2007; 127:63-72) Similar brain response has been reported in 'healthy volunteers' by our group. (Pain 2009 in press) Aberrant brain processing in visceral pain is seen in IBS patients and differences may exist in the central mechanism

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AGA Abstracts

AGA Abstracts

Cellular Mechanisms of Exacerbation of Colonic Smooth Muscle Dysfunction by Concurrent Psychological and Inflammatory Stressors Xuan-Zheng P. Shi, Sushil K. Sarna