AGA Abstracts Graphical abstract of Deg-AZM Su1096 ROLE OF P21-ACTIVATED KINASE, PAK4, IN ACTIVATION OF NUMEROUS IMPORTANT CELLULAR SIGNALING CASCADES IN PANCREATIC ACINAR CELLS Irene Ramos, Lingaku Lee, Samuel A. Mantey, Tatiana Iordanskaia, Robert T. Jensen Introduction: Present studies show that PAK4 is an important signaling molecule in a number of pancreatic physiological (enzyme/fluid/insulin secretion, development) and patho- physiological processes (pancreatic cancer, pancreatitis). PAK4 is the only member of the Group II p21-activated kinases (PAKs) present in pancreatic acinar cells and is activated by gastrointestinal hormones/neurotransmitters stimulating PLC/cAMP and by various pan- creatic growth factors. However, little is known of the distal mediators of PAK4 activation in cellular signaling cascades in pancreatic acinar cells. Aim: To investigate the role of PAK4 in activating selected pancreatic acinar cellular signaling cascades which mediate a number of pancreatic acinar physiological and pathophysiological responses: activation of MAP Kinases (c-Raf, Mek 1/2, ERK 1/2, p38 and JNK) and GSK3/ß-catenin. Methods: Dispersed rat pancreatic acini were prepared and preincubated with two inhibitors of PAK4, under conditions which did not inhibit other PAKs: PF-3758309 (PF, 0.1 nM. 3 h) and LCH- 7749944 (LCH, 30 μM. 3 h); acini were then incubated with CCK (0.3, 100 nM. 3 min) or TPA (1 μM. 5 min). Results: PF and LCH, decreased CCK (0.3 nM)-induced phosphoryla- tion of Mek1/2 by 21-52%. Both PAK4 inhibitors reduced CCK-induced phosphorylation of Mek1/2 and ERK1/2 by 42-53% and 62-66%, respectively. Inhibition of PAK4 reduced TPA-induced phosphorylation of Mek1/2 and p44/42 (21-46% and 46-65%). Neither activa- tion of c-Raf, p38 or cJNK, induced by the CCK or TPA, were affected by either PAK4 inhibitor. Both CCK and TPA stimulated pS9 GSK3ß and pS675 ß-catenin phosphorylation. PF and LCH inhibited the CCK-induced phosphorylation of pS9 GSK3ß by 50-73% and TPA-induced by 70%. PF and LCH inhibited CCK-induced (82-53%) and TPA-induced phosphorylation of ß-catenin (76-65%). None of the basal levels of activation of the MAP Kinases (c-Raf, Mek 1/2, ERK 1/2, p38 and JNK) or GSK3/ß-catenin were affected by PF or LCH. Conclusion: These results demonstrate that, in pancreatic acinar cells, CCK or TPA activation of Mek1/2, ERK 1/2, GSK3 and ß-catenin are mediated by PAK4; whereas activation of c-Raf, JNK or p38 pathways are independent of PAK4-activation. These results coupled with recent studies showing PAK4 is important in pancreatic fluid/electrolyte/enzyme secretion and acinar cell growth, show that PAK4 plays an important role in different cellular signaling cascades which have been shown to mediate numerous physiological and pathophysiological processes in pancreatic acinar cells. Su1097 TL1A-MEDIATED BACTERIAL TRANSCYTOSIS BY A MLCK SPLICING VARIANT CONTRIBUTES TO INFLAMMATORY BOWEL DISEASE Yu-Chen Pai, David Q. Shih, Stephan R. Targan, Jerrold Turner, Shu Chen Wei, Linda C.H. Yu Abnormal host-microbe interaction and enrichment of mucosa-associated bacteria were reported in inflammatory bowel disease (IBD). Interferon gamma (IFN ) and a novel tumor necrosis factor superfamily member TL1A were implicated in epithelial barrier dysfunction. The distinct timing and mechanisms of bacterial influx through transcellular versus paracellu- lar routes remain poorly understood. The aim of this study was to investigate the relative roles of long myosin light chain kinase (MLCK) splicing variants in barrier defects under proinflammatory stress. Our data showed the presence of intraepithelial bacteria preceding tight junction (TJ) damage in models of chemical-induced enterocolitis in wild-type (WT) mice and spontaneous ileitis in TL1A-transgenic mice. Absence of both transcellular and paracellular barrier defects was seen in long MLCK(-/-) mice given DSS. Neutralizing anti- TL1A reduced the MLCK-activated brush border fanning and bacterial endocytosis, and attenuated mucosal inflammation in WT mice. Bacterial taxa identified within the inflamed epithelial cells included Escherichia, Enterococcus, Staphylococcus, Lactobacillus, Bacillus, and Stenotrophomonas. In vitro studies showed that TL1A elicited bacterial internalization without TJ defects, whereas IFN triggered bacterial endocytosis at low dose and TJ disruption at high dose. TL1A and IFN elevated only the splicing variant MLCK2 but not MLCK1 transcripts. Low-dose TL1A and IFN induced PI3K/Akt/MLCK2-dependent bacterial endo- cytosis, whereas high-dose IFN caused TJ opening via a Src/MLCK1 axis. The phenomenon of bacterial internalization was recapitulated by plasmid transfection of MLCK2 but not MLCK1 to Caco-2 cells with CRISPR/Cas9-based knockout of MLCK gene. Lastly, upregulated expression of TL1A, IFN , and both MLCK variants was confirmed in mucosal biopsy of IBD patients compared to those of non-inflamed controls. In conclusion, divergent pathways of bacterial influx through transcellular and paracellular routes were orchestrated by the S-508 AGA Abstracts activation of distinct MLCK isoforms. Bacterial transcytosis induced by low-grade proinflam- matory cytokine, despite intact TJs, may be an alternative route causing flare-up in IBD. Su1098 FAILURE TO INDUCE OXPHOS IMPAIRS EPITHELIAL MIGRATION AND CRYPT FISSIONING NEEDED FOR ULCER HEALING Ahmed A. Abomhya, Courtney Perry, Tatiana Goretsky, Margarita Avdiushko, Terrence Barrett BACKGROUND/AIM: Pediatric ulcerative colitis (UC) and Crohn’s disease (CD) data show that repressed mitochondrial oxidative phosphorylation (OXPHOS) portends severe clinical outcomes in UC1 (Haberman et al Nat Comm) and Crohn’s disease.2 (Kugrathasan et al Lancet 2017). We posit that repressed mitochondrial respiration is the reason why ulcers DO NOT HEAL IN IBD. Here we examine the impact of repressed mitochondria on ulcer healing in colitis. Mitochondrial transcription factor A (TFAM) is necessary for mitochondrial- encoded gene transcription3 (Gaspari et al., 2004). To examine the role of OXPHOS on ulcer healing, a mouse model of intestinal epithelial cell (IEC)-specific mitochondrial deficiency was generated (VilCre/TFAMfl/fl/mTmG or IEC/TFAM-KO). METHODS: Mitochondrial function was interrogated in WT and IEC/TFAM-KO mice by assessing mitochondrial DNA (mtDNA), mitochondrial complex (Cpx) proteins and mRNA and seahorse analyses in isolated IEC from WT and IEC/TFAM-KO mice. IEC responses to DSS colitis were studied by tracking GFP+ IEC in IEC/TFAM-KO and WT/VilCre//mTmG mice. RESULTS: IEC from WT and IEC/TFAM-KO mice revealed that TFAM-deficient IEC had >30% less mtDNA and oxygen consumption (Seahorse) as well as >50% reduced Cox4 (mitochondria-encoded) mRNA and protein. Staged response to DSS-induced ulceration revealed that TFAM-deficient IEC were capable of proliferation and epithelial mesenchymal transition (EMT) whereas 1) monolayer formation was delayed and 2) new crypt formation/expansion was impaired in IEC/TFAM- KO mice. In particular, we found that crypts undergo fissioning in WT mice whereas crypts in IEC/TFAM-KO mice fail to undergo fissioning in ulcer healing. This deficit was reflected by a transient reversal in IEC proportions from TFAM-deficient (85%) to WT (65%) IEC during the restitution phase of ulcer healing. CONCLUSIONS: These findings reveal the deficit conveyed by severe chronic inflammation on the IEC inability to respond to mucosal ulceration. We posit that severe mucosal inflammation represses IEC mitochondrial respira- tion which impairs IEC migration as well as new crypt formation via fissioning. These data represent a novel observation pertaining to the explanation of why mitochondrial repression has been associated with poor clinical outcomes in IBD, We propose that improved mitochon- drial function should be a treatment goal of new therapeutic agents in IBD. Su1099 INHIBITION OF JAG1/NOTCH1 SIGNALING EXACERBATES INTESTINAL BARRIER DYSFUNCTION BY MODULATING CELL PROLIFERATION AND APOPTOSIS IN MICE WITH COLITIS Tingting Li, Xiao li, Chong Geng, Chunhui Wang Background & aims: The Notch signaling pathway plays a critical role in regulating cell differentiation and proliferation. It has been suggested that Notch activation serves as a protective mechanism in maintenance of intestinal barrier function. However, the exact mechanism remains to be determined. In this study, we aimed to investigate the role and mechanisms of Notch signaling in regulation of intestinal barrier function under inflammatory condition. Methods: In vivo experiments, colitis was induced in C57BL/6 mice by drinking dextran sulfate sodium (DSS)-containing water. For Notch inhibition, mice were administered with LY411575 (a -secretase inhibitor) in a dosage of 10mg/kg by gavage for continuous five days. In vitro experiments, Notch signaling was activated or inhibited by generating a cell line expressing Notch1 intracellular domain (Tet-On NICD1 cells) under the control of doxycycline (DOX, 100 ng/ml) or LY411575, respectively. Transepithelial resistance (TER) value[lx1] was monitored, and cell proliferation, apoptosis, tight junction (TJ) assembly were explored. The expression of ligands and receptors of Notch signaling were analyzed after TNF- exposure in Caco-2 and HT-29 cells. Results: Inhibition of Notch activation by LY411575 treatment in mice resulted in a severe exacerbation of the colitis evidenced by increase in disease activity index (DAI) score and histological examination compared with the vehicle-treated colitis mice. LY411575-treated colitis mice also manifested decrease in proliferation and increase in apoptosis of intestinal epithelial cells (IECs). In addition, immunofluorescence and western blot assays showed that the location and expression of TJ proteins were obviously deteriorated in LY411575-treated colitis mice compared to vehicle-treated colitis mice. In TNF- stimulated Caco-2 and HT-29 cells, ligand of JAG1 and receptor of Notch1 were significantly increased compared to the control group. In Caco-2 and HT-29 cells, LY411575 exposure significantly suppressed cell proliferation and promoted cell apoptosis by CCK-8 and Terminal deoxynucleotidyl transferase mediated dUTP Nick-End Labeling (TUNEL) detections, respectively. In addition, pro-inflammatory cytokines (TNF- and IL-6) were also significantly increased by LY411575 treatment. Fur- thermore, the expression of claudin-3 and claudin-4 were also decreased in LY411575- treated cells compared with the control cells. The expression of Myosin Light Chain Kinase (MLCK) and phosphorylated Myosin Light Chain (p-MLC) were also increased after LY411575 treatment. All these effects could be reversed in Tet-On NICD1 cells. Conclusions: Our results demonstrate that JAG1/Notch1 axis maintain intestinal homeostasis by modulat- ing cell proliferation, apoptosis, and improving tight junctions. This could provide a novel therapeutic target for ulcerative colitis treatment.