- Email: [email protected]
Mechanism of regulation of intestinal epithelial tight junction permeability: Evidence for involvement of occludin phosphorylation
3785 Mechanism Of Regulation Of Intestinal Epithelial Tight Junction Permeabilily: Evidence For Involvement Of Occludin Phosphorylatinn. Thomas Y. Ma, LONG BEACHVA MED Ctr AND UCI-COLLEGEOF MEDICINE, Long Beach, CA; Nell Hoa, All Pedram, UCl-Collegeof Medicine, Irvine, CA; Margaret Merryfield, CSLB, Long Beach, CA The apico-leterallylocatedtight junctions (TJ) act as a structural barrier againstthe paracellular penetration of luminal substances. Previously, it has been proposed that a defectiveintestinal tight junction barrier may play an important pathogenic role in various intestinal diseases including Crohn's disease and NSAID-associated enteropathy. The intracellular processes which modulate intestinal TJ barrier function are poorly understood. The Aim of this study was to determine the involvement of occludin phosphorylation in the regulation of intestinal TJ barrier using an in-vitro model consisting of Caco-2 intestinal epithelial cells grown on permeable inserts. Cytochalasin B (CB), a commonly used TJ disrupting agent, was used to induce an increase in TJ permeability. Methods: Distribution and phosphorylation of occludin was determined by Western blot analysis and double precipitation. Results: 1) CB produced a dose dependentincrease in Caco-2 TJ permeabilityand disassemblyof occludin from their junctional location. 2) CB-induced increase in Caco-2TJ permeabilitywas associatedwith an alteration in detergent (triton X-IO0) solubility of occludin. CB treatment resulted in a rapid translocation of occludin from detergentsoluble to detergentinsoluble fraction. 3) CB-induced occludin translocation was associated with an increase in occludin phosphorytation in the dete~ent insoluble fraction. 4) Probing of the immunoprecipitated oCcludin with specific antibodies for tyrosine and threonine indicated that CB-induced occlodin phosphorylation occurred exclusivelyon tyrosine and not threonine. 5) Pre-treatmentof Caco-2monoleyerswith genistein (a proteintyrosine kinaseinhibitor) preventedCB inducedtyrosine phosphorylationof occludin. 6) Moreover,genisteinalso preventedCB-inducedincreasein Caco-2TJ permeability, suggestingthat CB induced increasein TJ permeabilitywas regulatedby tyrosina phosphoPJiation of occludin. 7) Consistent with these findings, pre-treatment of Caco-2 monoiayer with metabolic inhibitors (2,4-dinitrophenol) also inhibited CB induced occludin phosphorylation and increase in TJ permeability. Conclusion: These results demonstrate that 1) tyrosine phosphorylation of occludin plays an important role in CB induced opening of intestinal epitheliaTJ barrier and 2) occludin tyrosine phosphorylationrepresentsan important potential target in the modulation of intestinal TJ barrier. 3786 Targeted Molecular Inhibition of PLC-y Prevents EGF-Medlated Protutlm el Microtubule (MT) Cytoskeleton and Intestinal Barrier Funoflon (BF). All Banan, Yang Zhang, Jeremy Z. Fields, Ece Mutlu, All Keshavarzian,Rush Univ, Chicago, IL Loss of intestinal BF has beenassociatedwith a variety of oxidative inflammatory Gt disorders including IBD. We recentlyshowedthat EGFprotects the BF of monolayersof human intestinal (Caco-2) cells & the MT cytoskeletonagainst oxidant injury via EGF-receptor(EGF-R)tyrosina kinase. But, the immediate intracellulartransducer responsibleis not known. Becausestudies in non-GI cell models suggest that PLC--f signaling may be activated by EGF-R tyrosine kinases, we surmised that PLC--/activation might be required for EGF-induced prote~lJon. Methods: Caco-2 monolayers were exposed to oxidant (H202) -+ pretreatment with EGF or specific inhibitors of EGFR-tyrosinekinase (e.g,, AG 1478, Tyrphostin 25 or its inactive analog Tyrphostin A1) or of PLC inhibitors (L 108, U-73122 orthe inactive U-73343). In other studies, ceils were stably transfected with a dominant-negativefragment for PLC-yl from the Z region, namely PLCz,to inhibit activation of the PLC--yisoform. Outcome measureswere: monolayer 8F (fluorometry), MT cytoskeletal stability (high resolution laser confocal microscopy), PLC activity (IP3formation), & both PLCz& PLC-'yexpression(immunoblotting) as wall as tubulin assembly & tubulin disassembly(quantitative western blot) (n = 6/group). Results: EGF (via EGF-R) protected against oxidant-induced tubulin disassembly, disruption of microtubute cytoskeleton, & monolayer barrier dysfunction. EGFalso enhancedPLC activity in intact cells; oxidant did not do so. There was a highly significant correlation between EGF protection & PLC activity (p
A-702
or iNOS were examined by RT-PCR. Results: Indomethacin caused hemorrhagic lesions in the small intestine, accompaniedwith an increase in enterobacterialtranslocation and iNOS activity in the mucosa; these all changes were inhibited by pretreatment with dmPGE2.SC560 or rofecoxib by itself did not cause intestinal damage, but the combined administration of these two drugs provokedthe damagewith an incidenceof 100%. SC-560, but not rofecoxib, inhibited PG production and enhancedintestinal hypermotility. The bacterialtranslocation was also increased after SC-560 but not rofecoxib, when examined 6 h after administration. The COX-2 mRNA was up-regulated in the mucosa 6 h after administration of SC-560, and the reduced PGE2contentswere partially restored thereafter. Likewise, SC-560 induced the iNOS mRNA expression in the intestinal mucoss, but the iNOS activity was significantly increased only when rofecoxib was given together with SC-560. Conclusion: These results suggest that the inhibition of both COX-1 and COX-2 is required for NSAID-induced intestinal damage. The COX-1inhibition, despite causing intestinal hypermotility, bacterialtranslocation and iNOS expression, up-regulatesCOX-2expression,and PGs produced by COX-2 may counteract the subsequentevents due to iNOS expression such as overproduction of NO and maintains the mucosal integrity. These sequentialevents related to COX-1 or COX-2 inhibition may explain why intestinal damage occurs only when both COX-f and COX-2 are inhibited. Z7U Tim P,ele el PII(C I ~ In Gel EplUtelial Wound Healing in vitro Margaret M. Lutz, Cecilia J. Song, Jeffrey 8. Matthews, Beth Israel DeaconessMedical Ctr and Harvard Medical Sch, Boston, MA PKCisoforms regulateactin cytoskeletalarchitectureand dynamic remodelingof the basolatsral surface in the human colonic cell line T84. Wound reseating of T84 monolayers has also been used to model in v/vo gut restitution, a process that entails rapid F-actin mobilization and changes in cell polarity. Thus, we postulated that PKC participates in intestinal epithelial wound healing. Methods: Confluent T84 monolayers grown on Transwell supports were woundedby micropipet tips, and fresh medium containing inhibitors or activators of specific PKC isoforms were added. The repair process was analyzedfor 4 hours at 37o and 5% C02 by either time-lapse video microscopy or by capturing initial and final images of multiple wounds. Wound diameters were measuredusing ScanalyticsIP lab spectrum software. Some wounds ware fixed, stained with rhodamine phalloidin and examinedby confocal microscopy. Results: The PKCactivator phorbo112-myristate13 acetate(PMA,IOOnM) dramaticallyaccelerated wound healing. PMA-treatedwounds were 82% smaller after 4 hrs, comparedto control woundsthat were 27% smaller (p
Comparison Of Bacterial And Fluorescent Bead Transcytosis In A Polarised Enterocyts Monolayer Jonathan Samuel White, Margaret Hoper, Rowan Parks, Barry Wd Clements, Thomas Diamond, Queen's Univ of Belfast, Belfast United Kingdom BACKGROUND.The barrier function of the gastrointestinalepitheliumis important in preventing translocation of enteric bacteria and their products to the systemic circulation. This study comparedtransport of an enteric E coil strain and synthetic latex microbeads across a polarisnd enterocyte monolayer. METHODS. Barrier function of caco-2 enterocytes seeded to a twochamber system was examined by measuring the movement of a standard amount of E coil C25 and fluorescent latex beads (diameter 1.5 microns, comparableto enteric bacteria) from the apical to the basal side of the system at various time points after cell seeding. RESULTS. A confluent, polarised cell monoiayer with high electrical resistancewas formed 16 days after seeding. Passage of both E coil and fluorescent beads decreased as the cell monolayer developed,although there was still a significant amount of transcytosis in the mature monolayer= Transcytosis rates for E coil in the mature monolayer were approximetely 28 times higher than for beads. Studies of the temporal course of transcytosis suggestedthat both E coil and beads cross the cell monoiayer by saturabletransport mechanisms. CONCLUSIONS. These results show that there are significant levels of bacterial and bead transcytosis in the mature, electrically-resistant polarised enterocyte monolayer, but that transcytosis rates for E coil C25 are much higher than for latex beadsThis suggeststhat passageof enteric bacteria through enterocytes is not merely a passive process but may involve active mechanisms of uptake and intracelluiar transport. This model may be useful for further eludication of factors regulating uptake and transport of enteric bacteria in enterocytes.
A-702
or iNOS were examined by RT-PCR. Results: Indomethacin caused hemorrhagic lesions in the small intestine, accompaniedwith an increase in enterobacterialtranslocation and iNOS activity in the mucosa; these all changes were inhibited by pretreatment with dmPGE2.SC560 or rofecoxib by itself did not cause intestinal damage, but the combined administration of these two drugs provokedthe damagewith an incidenceof 100%. SC-560, but not rofecoxib, inhibited PG production and enhancedintestinal hypermotility. The bacterialtranslocation was also increased after SC-560 but not rofecoxib, when examined 6 h after administration. The COX-2 mRNA was up-regulated in the mucosa 6 h after administration of SC-560, and the reduced PGE2contentswere partially restored thereafter. Likewise, SC-560 induced the iNOS mRNA expression in the intestinal mucoss, but the iNOS activity was significantly increased only when rofecoxib was given together with SC-560. Conclusion: These results suggest that the inhibition of both COX-1 and COX-2 is required for NSAID-induced intestinal damage. The COX-1inhibition, despite causing intestinal hypermotility, bacterialtranslocation and iNOS expression, up-regulatesCOX-2expression,and PGs produced by COX-2 may counteract the subsequentevents due to iNOS expression such as overproduction of NO and maintains the mucosal integrity. These sequentialevents related to COX-1 or COX-2 inhibition may explain why intestinal damage occurs only when both COX-f and COX-2 are inhibited. Z7U Tim P,ele el PII(C I ~ In Gel EplUtelial Wound Healing in vitro Margaret M. Lutz, Cecilia J. Song, Jeffrey 8. Matthews, Beth Israel DeaconessMedical Ctr and Harvard Medical Sch, Boston, MA PKCisoforms regulateactin cytoskeletalarchitectureand dynamic remodelingof the basolatsral surface in the human colonic cell line T84. Wound reseating of T84 monolayers has also been used to model in v/vo gut restitution, a process that entails rapid F-actin mobilization and changes in cell polarity. Thus, we postulated that PKC participates in intestinal epithelial wound healing. Methods: Confluent T84 monolayers grown on Transwell supports were woundedby micropipet tips, and fresh medium containing inhibitors or activators of specific PKC isoforms were added. The repair process was analyzedfor 4 hours at 37o and 5% C02 by either time-lapse video microscopy or by capturing initial and final images of multiple wounds. Wound diameters were measuredusing ScanalyticsIP lab spectrum software. Some wounds ware fixed, stained with rhodamine phalloidin and examinedby confocal microscopy. Results: The PKCactivator phorbo112-myristate13 acetate(PMA,IOOnM) dramaticallyaccelerated wound healing. PMA-treatedwounds were 82% smaller after 4 hrs, comparedto control woundsthat were 27% smaller (p
Comparison Of Bacterial And Fluorescent Bead Transcytosis In A Polarised Enterocyts Monolayer Jonathan Samuel White, Margaret Hoper, Rowan Parks, Barry Wd Clements, Thomas Diamond, Queen's Univ of Belfast, Belfast United Kingdom BACKGROUND.The barrier function of the gastrointestinalepitheliumis important in preventing translocation of enteric bacteria and their products to the systemic circulation. This study comparedtransport of an enteric E coil strain and synthetic latex microbeads across a polarisnd enterocyte monolayer. METHODS. Barrier function of caco-2 enterocytes seeded to a twochamber system was examined by measuring the movement of a standard amount of E coil C25 and fluorescent latex beads (diameter 1.5 microns, comparableto enteric bacteria) from the apical to the basal side of the system at various time points after cell seeding. RESULTS. A confluent, polarised cell monoiayer with high electrical resistancewas formed 16 days after seeding. Passage of both E coil and fluorescent beads decreased as the cell monolayer developed,although there was still a significant amount of transcytosis in the mature monolayer= Transcytosis rates for E coil in the mature monolayer were approximetely 28 times higher than for beads. Studies of the temporal course of transcytosis suggestedthat both E coil and beads cross the cell monoiayer by saturabletransport mechanisms. CONCLUSIONS. These results show that there are significant levels of bacterial and bead transcytosis in the mature, electrically-resistant polarised enterocyte monolayer, but that transcytosis rates for E coil C25 are much higher than for latex beadsThis suggeststhat passageof enteric bacteria through enterocytes is not merely a passive process but may involve active mechanisms of uptake and intracelluiar transport. This model may be useful for further eludication of factors regulating uptake and transport of enteric bacteria in enterocytes.