Mo1805 Deletion of the KrüPpel-Like Factor 4 Gene in Colonic Epithelium Protects Mice From Dextran...
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Mo1804 Contribution of Intestinal Bacteria and Epithelial Mesenchymal Transition to Intestinal Inflammation and Fibrosis in Enterocyte-Specific Pipk III Deficient Mice Yasuo Horie, Shunsuke Takasuga, Takehiko Sasaki, Hirohide Ohnishi BACKGROUND AND AIM: Genome-wide association studies have provided MTMR3 as a Crohn's-disease-associated gene. Pipk III, which cooperatively functions in phosphatidyl inositol 3,5-bisphosphates [PI(3,5)P2] metabolism with MTMR3, is a kinase that produces PI(3,5)P2 from phophatidyl inositol 3-bisphosphate. We generated enterocyte-specific Pipk III deficient (KO) mice which displayed diarrhea, body weight loss, and pathological findings of epithelial vacuoles, transmural inflammatory cell infiltration, increased fibrosis, and goblet cell depletion, sharing the clinical and histological findings with Crohn's diseae. In this context, we tried to elucidate the mechanisms of intestinal inflammation and fibrosis in Crohn's disease by analyzing those in our KO mice. METHODS: We examined the localization of proteins functioning at apical and basolateral membranes of enterocytes using immunofluo- rescence staining, and the expression of these membranous proteins in intestinal mucosa of both KO and the control mice using western blot analysis. We compared the expression of inflammation- and fibrosis-related genes in intestinal mucosa using RT-PCR. In addition, we monitored the survival of Pipk III KO mice with or without antibiotics to study the relevance of intestinal bacteria in intestinal inflammation of KO mice. Furthermore, we injected fluorescent dextran per anus and observed its mucosal distribution using immuno- fluorescence staining to evaluate how easily intestinal microbiota can invade to mucosa of KO mice. RESULTS: Although western blot analysis did not show any differences in expression of apical and basolateral membranous proteins between KO and the control mice, immunofluo- rescence staining revealed mislocalization of these proteins in KO enterocytes, suggesting impaired intracellular vesicle transport and cellular polarity. We found the increased genes expression of proinflammatory cytokines including TNF-alpha, IL-1beta, and IL-6. Mean- while, the expression of fibrosis-promoting genes such as TGF-beta1 and CTGF, and Snail- 1, which is a transcription factor that promotes epithelial mesenchymal transition (EMT), was increased in KO mice compared to the control mice. Consistent with this result, immunofluorescence staining revealed that KO enterocytes expressed Vimentin protein more intensely than the control. Furthermore, the survival of KO mice treated with antibiotics was prolonged and fluorescent dextran injected per anus was distributed more scattered in KO mice with antibiotics than those without antibiotics. CONCLUSION: Facilitating entry of intestinal bacteria to mucoca based on impaired intracellular vesicle transport and cellular polarity in enterocytes seems to bring about intestinal inflammation in KO mice. Meanwhile, there is a possibility that EMT in KO enterocytes induces intestinal fibrosis. Mo1805 Deletion of the KrüPpel-Like Factor 4 Gene in Colonic Epithelium Protects Mice From Dextran Sulfate Sodium (DSS)-Induced Colitis Amr Ghaleb, Hamed Laroui, Didier Merlin, Vincent W. Yang BACKGROUND: KLF4 is a zinc finger transcription factor normally expressed in the differen- tiated epithelial cells lining the villus border and surface epithelium of the small and large intestine, respectively. KLF4 inhibits cell proliferation by functioning as a cell cycle- checkpoint protein and exhibits tumor suppressor activity in colon cancer. On the other hand, KLF4 is a mediator of proinflammatory signaling in macrophages and its over- expression in the esophageal epithelium activates cytokines, leading to inflammation-medi- ated esophageal squamous cell cancer formation in mice. AIM: To determine whether KLF4 has a proinflammatory activity In Vivo using the DSS-induced colitis model in mice. METHODS: Conditional gene ablation was used to generate C57BL/6 mice lacking Klf4 in their intestinal epithelium by mating floxed Klf4 (Klf4fl/fl) mice with villin-Cre (Vil-Cre) mice. Klf4 mutant mice (Vil-Cre;Klf4fl/fl) and WT (Klf4fl/fl) mice were treated or not with 3% DSS for 6 days and the extent of DSS-induced colonic inflammation was then compared between the groups. WT mice were given or not nanoparticles (NP) loaded Klf4-specific siRNA and concomitantly treated or not with DSS. RESULTS: In untreated mice, the colon of Vil-Cre;Klf4fl/fl mice displayed a distorted architecture and reduction in goblet cell differentiation and maturation. However, deletion of Klf4 did not affect the proliferation rate of colonic epithelial cells. Compared to DSS-treated WT mice, DSS-treated Vil-Cre;Klf4fl/ fl mice had significantly lower clinical scores at day 6 of treatment, as indicated by less weight loss, lower occult blood scores, and longer colon lengths. Histological analysis showed that DSS-treated Vil-Cre;Klf4fl/fl mice had much less epithelial cell loss and lower levels of inflammatory cells infiltrating the colon compared to DSS-treated WT mice. DSS treatment of WT mice induced Klf4 expression in the crypt zone of the colonic epithelium where Klf4 is normally absent. In contrast, DSS-treated Vil-Cre;Klf4fl/fl mice had increased proliferation as indicated by increased Ki67 and BrdU staining. Western blot analysis showed that Klf4 expression was increased in DSS-treated WT mice in a time-dependent manner, peaking at day 3 of treatment. The IkB level was elevated in the colon of DSS-treated Vil-Cre;Klf4fl/fl mice and this was accompanied by a significantly lower inflammatory profile compared to DSS-treated WT mice. Temporary deletion of Klf4 in the colon by siRNA in WT mice also protected from DSS-induced inflammation. CONCLUSIONS: The results show that deletion of Klf4 in mice colon attenuates inflammatory response and increases proliferative/regenerat- ive response following DSS treatment. KLF4 is therefore an important factor that mediates DSS-induced colonic inflammation and could be involved in the pathogenesis and/or propa- gation of inflammatory bowel disease. Mo1806 Gastrokine-1: A Gastric Protein That Protects Mice Against Experimental Colitis Shirley Paski, Thomas M. Nero, F Gary Toback, David L. Boone Background: Gastrokine-1 (GKN1) is an 18-kDa protein expressed predominantly in the gastric antrum where it is secreted into the lumen. A synthetic 21-mer peptide corresponding to the central domain of GKN1 (GKN1-peptide) is mitogenic, motogenic, and cytoprotective S-689 AGA Abstracts in the GI tract of mice exposed to indomethacin. GKN1 peptide is also highly effective at protecting against and treating LPS-induced intestinal hyperpermeability in mice suggesting that GKN1 has effects beyond the stomach. The objective of this study was to evaluate the essential function of GKN1 by phenotyping GKN1 knockout mice under basal conditions and in the setting of experimental colitis. Methods: GKN1-/- mice were developed by ablating the gene in entirety, replacing it with a BAC-based vector expressing the β-galactosidase from the E. coli lacZ gene and neomycin phosphotransferase coding sequences. GKN1+/- embryonic stem cells from C57BL/6 mice were implanted into C57BL/6 embryos to create GKN1+/- mice. To induce colitis, mice received 2% dextran sodium sulfate (DSS) drinking water for 5 days followed by regular acidified water for the remainder of the experiment. Expression of Gkn-1 in mouse tissues was assessed by RT-PCR. Results: GKN1-/- mice develop normally compared to their littermates. GKN1-/- mice demonstrate mild failure to thrive, on average weighing 8% less than their GKN+/+ or +/- littermates by 6 weeks of age. Histology of GKN1-/- mouse stomach, small intestine, and colon is indistinguishable from wild type controls. GKN-1 expression was restricted to the stomach mucosa in mice and was not found in any other part of the small intestine or colon. Challenging GKN1-/- mice with DSS results in more rapid development of severe colitis and increased weight loss compared to wild-type controls. Colon histology of GKN1-/- mice showed increased inflam- mation. Severe colitis is associated with 70% mortality in GKN-/- mice vs 25% wild-type controls. Conclusions: GKN1-/- mice are exquisitely susceptible to DSS colitis with the development of more severe colitis and increased mortality.. These results demonstrate that a naturally occurring stomach protein, GKN1, plays a role in protecting mice against experimental colitis. Further studies are needed to evaluate the efficacy of GKN1 as a novel new therapeutic agent for inflammatory bowel disease. Mo1807 Stable Knockdown of Myosin Vb in CaCo2-Bbe Cells Causes Loss of Apical Microvilli Byron Knowles, Joseph T. Roland, Moorthy Krishnan, Lynne Lapierre, James R. Goldenring, Mitchell Shub Myosin Vb (MYO5B) is positioned at the center of the apical recycling of proteins to the plasma membrane in polarized epithelial cells. MYO5B is an actin based motor that localizes specific Rab small GTPases (Rab8a, Rab10, Rab11, and Rab25) to sub-apical domains. Microvillus Inclusion Disease (MVID) represents a pathophysiologic window into the apical trafficking process, because it arises as a result of inactivating mutations in MYO5B that lead to the loss of apical microvilli in intestinal enterocytes. Understanding how mutations in MYO5B lead to aberrant trafficking in enterocytes will provide novel insights into the fundamental mechanisms governing apical recycling system trafficking and microvilli forma- tion. We hypothesize that MYO5B plays a crucial role in apical trafficking in enterocytes, and MVID patients have defective MYO5B motors that mislocalize Rab small GTPases leading to disruption in apical transport. To test this hypothesis we have established a cellular model of MVID by stably knocking down MYO5B in CaCo2-BBE cells. The cells grown on permeable filters for 15 days were analyzed using immunostaining, Scanning EM, and Transmission EM to examine markers of apical and basolateral polarity, intracellular trafficking, and the establishment of apical microvilli. These studies have demonstrated that we can recapitulate the most important pathological finding of MVID in our cellular model: the loss of apical microvilli. Microvilli were markedly decreased as visualized by either Scanning EM or Transmission EM. These studies have also revealed that apically trafficked brush border proteins, DPPIV and Ezrin, were markedly decreased in the apical membranes of knockdown cells. Additionally, CD10 and transferrin receptor were mislocalized in MYO5B knockdown cells as they are in MVID patients. In addition, MYO5B knockdown caused dispersal of Rab8a and Rab11a-containing vesicles from their usual distribution in the apical region to a more diffuse distribution throughout the cytoplasm. These results suggest that MYO5B in association with Rab11a and Rab8a regulates the establishment and maintenance of the intestinal apical brush border. As a whole, these studies not only provide data on how mutations in MYO5B can lead to MVID, but will also define the modifications in MYO5B- dependent trafficking pathways that lead to alterations in apical trafficking and cell polarity in general. AGA Abstracts
Transcript of Mo1805 Deletion of the KrüPpel-Like Factor 4 Gene in Colonic Epithelium Protects Mice From Dextran...
Mo1804
Contribution of Intestinal Bacteria and Epithelial Mesenchymal Transition toIntestinal Inflammation and Fibrosis in Enterocyte-Specific Pipk III DeficientMiceYasuo Horie, Shunsuke Takasuga, Takehiko Sasaki, Hirohide Ohnishi
BACKGROUND AND AIM: Genome-wide association studies have provided MTMR3 as aCrohn's-disease-associated gene. Pipk III, which cooperatively functions in phosphatidylinositol 3,5-bisphosphates [PI(3,5)P2] metabolism with MTMR3, is a kinase that producesPI(3,5)P2 from phophatidyl inositol 3-bisphosphate. We generated enterocyte-specific PipkIII deficient (KO) mice which displayed diarrhea, body weight loss, and pathological findingsof epithelial vacuoles, transmural inflammatory cell infiltration, increased fibrosis, and gobletcell depletion, sharing the clinical and histological findings with Crohn's diseae. In thiscontext, we tried to elucidate the mechanisms of intestinal inflammation and fibrosis inCrohn's disease by analyzing those in our KOmice. METHODS:We examined the localizationof proteins functioning at apical and basolateral membranes of enterocytes using immunofluo-rescence staining, and the expression of these membranous proteins in intestinal mucosaof both KO and the control mice using western blot analysis. We compared the expressionof inflammation- and fibrosis-related genes in intestinal mucosa using RT-PCR. In addition,we monitored the survival of Pipk III KO mice with or without antibiotics to study therelevance of intestinal bacteria in intestinal inflammation of KO mice. Furthermore, weinjected fluorescent dextran per anus and observed its mucosal distribution using immuno-fluorescence staining to evaluate how easily intestinal microbiota can invade to mucosa of KOmice. RESULTS: Although western blot analysis did not show any differences in expression ofapical and basolateral membranous proteins between KO and the control mice, immunofluo-rescence staining revealed mislocalization of these proteins in KO enterocytes, suggestingimpaired intracellular vesicle transport and cellular polarity. We found the increased genesexpression of proinflammatory cytokines including TNF-alpha, IL-1beta, and IL-6. Mean-while, the expression of fibrosis-promoting genes such as TGF-beta1 and CTGF, and Snail-1, which is a transcription factor that promotes epithelial mesenchymal transition (EMT),was increased in KO mice compared to the control mice. Consistent with this result,immunofluorescence staining revealed that KO enterocytes expressed Vimentin protein moreintensely than the control. Furthermore, the survival of KO mice treated with antibioticswas prolonged and fluorescent dextran injected per anus was distributed more scattered inKO mice with antibiotics than those without antibiotics. CONCLUSION: Facilitating entryof intestinal bacteria to mucoca based on impaired intracellular vesicle transport and cellularpolarity in enterocytes seems to bring about intestinal inflammation in KO mice. Meanwhile,there is a possibility that EMT in KO enterocytes induces intestinal fibrosis.
Mo1805
Deletion of the KrüPpel-Like Factor 4 Gene in Colonic Epithelium ProtectsMice From Dextran Sulfate Sodium (DSS)-Induced ColitisAmr Ghaleb, Hamed Laroui, Didier Merlin, Vincent W. Yang
BACKGROUND: KLF4 is a zinc finger transcription factor normally expressed in the differen-tiated epithelial cells lining the villus border and surface epithelium of the small andlarge intestine, respectively. KLF4 inhibits cell proliferation by functioning as a cell cycle-checkpoint protein and exhibits tumor suppressor activity in colon cancer. On the otherhand, KLF4 is a mediator of proinflammatory signaling in macrophages and its over-expression in the esophageal epithelium activates cytokines, leading to inflammation-medi-ated esophageal squamous cell cancer formation in mice. AIM: To determine whetherKLF4 has a proinflammatory activity In Vivo using the DSS-induced colitis model in mice.METHODS: Conditional gene ablation was used to generate C57BL/6 mice lacking Klf4 intheir intestinal epithelium by mating floxed Klf4 (Klf4fl/fl) mice with villin-Cre (Vil-Cre)mice. Klf4 mutant mice (Vil-Cre;Klf4fl/fl) and WT (Klf4fl/fl) mice were treated or not with3% DSS for 6 days and the extent of DSS-induced colonic inflammation was then comparedbetween the groups. WT mice were given or not nanoparticles (NP) loaded Klf4-specificsiRNA and concomitantly treated or not with DSS. RESULTS: In untreated mice, the colonof Vil-Cre;Klf4fl/fl mice displayed a distorted architecture and reduction in goblet celldifferentiation and maturation. However, deletion of Klf4 did not affect the proliferationrate of colonic epithelial cells. Compared to DSS-treated WTmice, DSS-treated Vil-Cre;Klf4fl/fl mice had significantly lower clinical scores at day 6 of treatment, as indicated by lessweight loss, lower occult blood scores, and longer colon lengths. Histological analysis showedthat DSS-treated Vil-Cre;Klf4fl/fl mice had much less epithelial cell loss and lower levels ofinflammatory cells infiltrating the colon compared to DSS-treated WT mice. DSS treatmentof WT mice induced Klf4 expression in the crypt zone of the colonic epithelium where Klf4is normally absent. In contrast, DSS-treated Vil-Cre;Klf4fl/fl mice had increased proliferationas indicated by increased Ki67 and BrdU staining. Western blot analysis showed that Klf4expression was increased in DSS-treated WT mice in a time-dependent manner, peaking atday 3 of treatment. The IkB level was elevated in the colon of DSS-treated Vil-Cre;Klf4fl/flmice and this was accompanied by a significantly lower inflammatory profile compared toDSS-treated WT mice. Temporary deletion of Klf4 in the colon by siRNA in WT mice alsoprotected from DSS-induced inflammation. CONCLUSIONS: The results show that deletionof Klf4 in mice colon attenuates inflammatory response and increases proliferative/regenerat-ive response following DSS treatment. KLF4 is therefore an important factor that mediatesDSS-induced colonic inflammation and could be involved in the pathogenesis and/or propa-gation of inflammatory bowel disease.
Mo1806
Gastrokine-1: A Gastric Protein That Protects Mice Against ExperimentalColitisShirley Paski, Thomas M. Nero, F Gary Toback, David L. Boone
Background: Gastrokine-1 (GKN1) is an 18-kDa protein expressed predominantly in thegastric antrum where it is secreted into the lumen. A synthetic 21-mer peptide correspondingto the central domain of GKN1 (GKN1-peptide) is mitogenic, motogenic, and cytoprotective
S-689 AGA Abstracts
in the GI tract of mice exposed to indomethacin. GKN1 peptide is also highly effective atprotecting against and treating LPS-induced intestinal hyperpermeability in mice suggestingthat GKN1 has effects beyond the stomach. The objective of this study was to evaluate theessential function of GKN1 by phenotyping GKN1 knockout mice under basal conditionsand in the setting of experimental colitis. Methods: GKN1-/- mice were developed by ablatingthe gene in entirety, replacing it with a BAC-based vector expressing the β-galactosidasefrom the E. coli lacZ gene and neomycin phosphotransferase coding sequences. GKN1+/-embryonic stem cells from C57BL/6 mice were implanted into C57BL/6 embryos to createGKN1+/- mice. To induce colitis, mice received 2% dextran sodium sulfate (DSS) drinkingwater for 5 days followed by regular acidified water for the remainder of the experiment.Expression of Gkn-1 in mouse tissues was assessed by RT-PCR. Results: GKN1-/- micedevelop normally compared to their littermates. GKN1-/- mice demonstrate mild failure tothrive, on average weighing 8% less than their GKN+/+ or +/- littermates by 6 weeks of age.Histology of GKN1-/- mouse stomach, small intestine, and colon is indistinguishable fromwild type controls. GKN-1 expression was restricted to the stomach mucosa in mice andwas not found in any other part of the small intestine or colon. Challenging GKN1-/- micewith DSS results in more rapid development of severe colitis and increased weight losscompared to wild-type controls. Colon histology of GKN1-/- mice showed increased inflam-mation. Severe colitis is associated with 70% mortality in GKN-/- mice vs 25% wild-typecontrols. Conclusions: GKN1-/- mice are exquisitely susceptible to DSS colitis with thedevelopment of more severe colitis and increased mortality.. These results demonstratethat a naturally occurring stomach protein, GKN1, plays a role in protecting mice againstexperimental colitis. Further studies are needed to evaluate the efficacy of GKN1 as a novelnew therapeutic agent for inflammatory bowel disease.
Mo1807
Stable Knockdown of Myosin Vb in CaCo2-Bbe Cells Causes Loss of ApicalMicrovilliByron Knowles, Joseph T. Roland, Moorthy Krishnan, Lynne Lapierre, James R.Goldenring, Mitchell Shub
Myosin Vb (MYO5B) is positioned at the center of the apical recycling of proteins to theplasma membrane in polarized epithelial cells. MYO5B is an actin based motor that localizesspecific Rab small GTPases (Rab8a, Rab10, Rab11, and Rab25) to sub-apical domains.Microvillus Inclusion Disease (MVID) represents a pathophysiologic window into the apicaltrafficking process, because it arises as a result of inactivating mutations in MYO5B thatlead to the loss of apical microvilli in intestinal enterocytes. Understanding how mutationsin MYO5B lead to aberrant trafficking in enterocytes will provide novel insights into thefundamental mechanisms governing apical recycling system trafficking and microvilli forma-tion. We hypothesize that MYO5B plays a crucial role in apical trafficking in enterocytes,and MVID patients have defective MYO5B motors that mislocalize Rab small GTPases leadingto disruption in apical transport. To test this hypothesis we have established a cellular modelof MVID by stably knocking downMYO5B in CaCo2-BBE cells. The cells grown on permeablefilters for 15 days were analyzed using immunostaining, Scanning EM, and TransmissionEM to examine markers of apical and basolateral polarity, intracellular trafficking, and theestablishment of apical microvilli. These studies have demonstrated that we can recapitulatethe most important pathological finding of MVID in our cellular model: the loss of apicalmicrovilli. Microvilli were markedly decreased as visualized by either Scanning EM orTransmission EM. These studies have also revealed that apically trafficked brush borderproteins, DPPIV and Ezrin, were markedly decreased in the apical membranes of knockdowncells. Additionally, CD10 and transferrin receptor were mislocalized in MYO5B knockdowncells as they are in MVID patients. In addition, MYO5B knockdown caused dispersal ofRab8a and Rab11a-containing vesicles from their usual distribution in the apical region toa more diffuse distribution throughout the cytoplasm. These results suggest that MYO5B inassociation with Rab11a and Rab8a regulates the establishment and maintenance of theintestinal apical brush border. As a whole, these studies not only provide data on howmutations in MYO5B can lead to MVID, but will also define the modifications in MYO5B-dependent trafficking pathways that lead to alterations in apical trafficking and cell polarityin general.
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