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swas examined on HIF1a activation, serotonin release (ELISA), NFkB phosphorylation andexpression of ADORA2A/B (WB). The effect of anHIF1a (FM19G11) inhibitor was quantitatedin this model EC cell system. Results: Transcripts and proteins of HIF1a, ADORA2A/B andTph1 were elevated in Crohn's disease mucosa and isolated Crohn's disease EC cells (2-3.2-fold, p<0.05), (1.5-2.2-fold, p<0.05) and (2.2-3-fold, p<0.05) respectively demonstratingthese cells expressed a “hypoxic” phenotype during active IBD. In KRJ-I cells, 30min and 120min hypoxia stimulated HIF1a protein and transcript (1.5-2, p<0.05), Tph1 and ADORA2Btranscripts and serotonin release (2-10-fold, p<0.05) compared to normoxic conditions.Hypoxia-mediated HIF1a production was reversed by targeting ADORA2B (MRS1754) butnot 2A receptors (SCH442416) while the pan-adenosine agonist (NECA) but not a 2A agonist(GC21680) stimulated HIF1a production and serotonin release. This could be reversed byselectively targeting ADORA2B with MRS1754 and inhibited using FM19G11. Conclusion:In active Crohn's disease, the EC cell expresses a hypoxia-sensitive phenotype with increasedADORA2B expression, Tph1 expression and serotonin synthesis. As these cells also releaseserotonin following ADORA2B activation, we postulate that hypoxia and chemomechanosen-sory events that occur in gut inflammatory conditions drive EC cells to synthesize and secretethis regulatory amine. Furthermore, we speculate that specifically hypoxia and ADORA2Bmaypresent alternatives pathophysiological pathway targets in Crohn's disease therapy.
Three-dimensional Characterization of the Changed Colonic NeuronalNetwork in the Dextran Sodium Sulfate-Induced Murine ColitisYa-Yuan Fu, Pankaj J. Pasricha, Shiue-Cheng Tang
Background & Aims: Impaired innervation in the colitis models has been reported. However,because of the fibrous structure of the enteric neurons, the standard, microtome-based planarmicroscopy cannot provide an integral view to identify the altered 3-dimensional (3D)network. We aimed to apply microtome-free 3D microscopy (Gastroenterology, 139:p1100-1105, 2010) to characterize the murine colitis. Methods: BALB/c mice were subjected todextran sodium sulfate (DSS)-induced ulcerative colitis for 7 days. We applied nuclear andantibody staining (using pan-neuronal marker PGP9.5, glia marker S100b, and sympatheticmarker tyrosine hydroxylase as the targets), optical clearing, and 3D confocal microscopyto characterize the spatial change of the colonic microstructure including the crypts andinnervation. Results: 3D image survey indicated increased ganglia density in the myentericand submucosal plexuses in the diseased colon. High-resolution, stereo projections of theconfocal images helped identify the distortion and disintegration of the honeycomb wall-like connections of neuronal networks in the lamina propria, which otherwise cannot beseen easily by the planar micrographs. Conclusion: The combination of immunostaining,optical clearing, and 3D microscopy provides a useful approach to characterize the spatialchange of the neuronal network in the DSS-induced colitis.
3D projection of the diseased and normal (inset) colonic PGP9.5 staining. Dimensions ofthe projected volume: 520 X 520 X 100 (depth) and 260 X 260 X 100 micrometers for thediseased and normal mucosa, respectively.
TNF Induces Epithelial β-Catenin Cleavage and Chromatin BindingTomas D. Vanagunas, Tatiana Goretsky, Rebecca B. Katzman, Terrence A. Barrett
Background and Aims: Nuclear accumulation of β-catenin is a hallmark of sporadic colorectalcancer (CRC). We previously reported this nuclear β-catenin is cleaved to a 56 kDa fragmentin CRC and can be identified using antibodies specific for epitopes within the armadillorepeat region such as serine 552 phosphorylated β-catenin (pβcat). Nuclear accumulationof cleaved pβcat was also increased in intestinal epithelial cells from ulcerative colitis (UC)and colitis associated cancer (CAC) suggesting this may be a final pathway for β-catenintranscriptional activation. As TNF is abundant in CRC, UC, and CAC tissue, we examined
TNF-induced β-catenin activation in the human colonic epithelial cell line NCM460. β-catenin nuclear localization, cleavage, and chromatin binding were assessed as responses toTNF. Methods and Results: NCM460 cells were stimulated with human recombinant TNFfor 18 hours at three different dosages: 1ng/mL, 5 ng/mL, and 10 ng/mL. As a positivecontrol, cells were treated with 20 mM LiCl. After stimulation, the cells were lysed and proteinseparated into cytosolic, membranous, nuclear soluble, chromatin bound, and cytoskeletalfractions. LiCl increased levels of the 56 kDa fraction of cleaved nuclear p-βcat over control.All three dosages of TNF induced higher levels of the cleaved form of nuclear pβcat overcontrol. The largest increase was found with using 1 ng/mL TNFα stimulation. Importantlythere was a significant increase in 56 kDa cleaved phospho-β-catenin with 1 ng/mL TNFin the chromatin bound fraction relative to control. Conclusions: Human recombinantTNF induces nuclear translocation and cleavage of serine 552 phosphorylated β-catenin inNCM460 cells. As the largest increase was seen in the chromatin bound fraction, these dataimply TNF transcriptionally activates β-catenin in normal colonic epithelial cells. Furtherstudies will be aimed at dissecting the mechanism of this activation. Protein kinase A (PKA)and AKT are likely candidates as previous studies have shown the ability of these two kinasesto phosphorylate β-catenin at serine 552.
Glutamine Enhances Heat Shock Protein Expression in Intestinal EpithelialCells via HSF-1 Activation by PolyaminesYuji Iwashita, Toshio Sakiyama, Mark W. Musch, Mark J. Ropeleski, Hirohito Tsubouchi,Eugene B. Chang
Background and aims Heat shock proteins (Hsps) have a pivotal role in protecting intestinalepithelial cells during physiologic stresses and play an essential role in maintenance ofintestinal homeostasis. The induction of Hsp70 and Hsp25 in intestinal epithelial cellsrequires glutamine, a non-essential amino acid that is rapidly depleted from the body underconditions of stress, e.g. IBD. The mechanisms of glutamine-dependent Hsp induction remainunclear, however. We previously showed that polyamines and their precursor ornithinederived from the metabolism of glutamine, mediate glutamine-dependent induction Hsp70and Hsp25. In this study, we investigate the mechanism by which polyamines enhance Hspexpression in intestinal epithelial cells. Methods: The normal rat small intestinal epithelialcells (IEC-18) were treated with glutamine, ornithine or polyamines (putrescine, spermidineor spermine), alone or in combination with 5 mM α-difluoromethylornithine (DFMO), anirreversible ornithine decarboxylase inhibitor. Cells were heat stressed by incubating themat 42°C for 30 min, and were harvested promptly or after returning to 37°C for 2 h. Theexpression of Hsp70 and Hsp25 was assessed by Western blot analysis and real time RT-PCR. The heat shock transcription factor 1 (HSF-1) localization and DNA binding wereassessed by Western blot analysis and electromobility shift assays respectively. Results: Aspreviously observed, supplementation of ornithine or polyamines restored the heat-inducedexpression of Hsp70 and Hsp25 under conditions of glutamine depletion. In the presenceof glutamine, DFMO significantly decreased the heat stress-induction of Hsp70 and Hsp25.Glutamine, ornithine, polyamines and DFMO did not modify the nuclear localization ofHSF-1. However, DFMO dramatically reduced HSF-1 binding to an oligonucleotide withheat shock elements (HSE) which increased by glutamine. Exogenous polyamines reversedthe inhibitory effect of DFMO on the DNA binding activity. Conclusions: The intestinalepithelial heat shock response is dependent on glutamine which promotes polyamine syn-thesis required for HSF-1 binding to the HSE. The studies provide insight into the essentialrole of glutamine in cell stress.
Effects of Infliximab (IFX) on Intestinal Epithelial Cells: ExploringMechanisms of Mucosal HealingFranco Scaldaferri, Loris Lopetuso, Valentina Petito, Viviana Gerardi, Vincenzo Arena,Egidio Stigliano, Lucia Sparano, Marco Pizzoferrato, Silvia Pecere, Lucrezia Laterza,Alfredo Papa, Valerio Cufino, Giovanni Gasbarrini, Alessandro Sgambato, AntonioGasbarrini
Mucosal healing is one of the biggest issue in the management of IBD, as it seems to beaffected by several categories of drugs, including biologics. Although clinical use of thesedrugs in achieving mucosal healing is a reality, their mechanisms are still not clear. AIMAim of our study is to identify the effect of IFX on intestinal epithelial cells in order toinduce mucosa healing, using In Vitro and In Vivo approaches. METHODS To explore thedirect effects of IFX alone on intestinal epithelial cell proliferation and vitality, an MTT assaywas used on CT26 and Caco2, a murine and human intestinal epithelial cell line, respectively.Cells were incubated in presence of TNF-α (25ng/mL) or IFX (5, 10, 50 μg/ml) and cellvitality was evaluated after 48 hours. To explore the effect of IFX on intestinal epithelialcell migration, a scratch assay, one of the most used In Vitro assay mimicking wound/healingprocess, was used. Briefly, a scratch on a confluent monolayer of CT26 was applied andmigration of cells was measured at regular intervals, in presence of IFX (50 μg/ml) and/orTNF-α(25 ng/ml). Effect of IFX was then explored In Vivo. C57/BL6 mice were exposed for7 days to 2,5 % of DSS and treated with IFX iv (5 mg/Kg) at day 5 or with daily enema ofIFX (300 μg/200 μl) for 3 days starting at day 5. At day 9, mice were sacrificed and histologywas taken. Moreover, human intestinal mucosal biopsies from 2 active UC patients werecultured for 48 h with IFX (10 and 50 ng/ml), and histology was then performed. RESULTSIFX did not significantly affect the proliferation of Caco2 and CT26 at MTT assay. On thecontrary, TNF-α induced a slightly decrease in CT26 proliferation, while not modifyingCaco2 cells proliferation. At scratch assay, IFX with/wo TNF-α was similar to control, whileTNF alone increased epithelial shedding. In Vivo, IFX given as enema ameliorated the severityof colitis in mice, by lowering the disease activity index (DAI) and the loss of body weight,while IFX used iv ameliorated only the DAI, while not modifying weight loss and mortalityat 9 days. At histology no mucosal healing was observed at day 9 in mice, with an effectonly in the reduction of the inflammatory infiltrate in treated mice. Human biopsies exposedto IFX did not show modification in epithelial component, but it was observed a slightdecrease in immune cells infiltration. CONCLUSIONS Taken together our preliminaryobservations suggest that IFX is not acting directly on intestinal epithelial cell component.