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582 Dexamethasone Stimulates Tight Junction Sealing in Intestinal Epithelial Cells by up Regulating Claudin-4 Expression via a Pathway Involving MKP-1 and p38
induced a several-fold increase in gastric progenitor cells in the corpus that are marked by lacZ expression driven by a 12.4 kb Villin promoter. Activation of the Hedgehog signaling pathway was markedly lower in corpus of IFNgamma-expressing mice than in controls, which may be due at least in part to a reduction in the pool of parietal cells which expresses Sonic hedgehog. Immunoblot and/or immunostaining for other signaling molecules in IFNgamma mice revealed upregulation of Stat3 and pStat3, Stat1 and pStat1, and Cox2. There was also an increase in the number of SMA+ periglandular myofibroblasts. Analysis of older IFNgamma mice revealed occasional progression to a more disorganized/dysplastic phenotype, which in some areas aberrantly expressed claudin 7, but frank adenocarcinoma was not seen even at 20 months of age. Our findings point to IFNgamma as a key factor driving preneoplastic progression in stomach.
Dexamethasone Stimulates Tight Junction Sealing in Intestinal Epithelial Cells by up Regulating Claudin-4 Expression via a Pathway Involving MKP-1 and p38 Andreas Fischer, Markus Gluth, Ulrich-Frank Pape, Bertram H. Wiedenmann, Daniel C. Baumgart, Franz Theuring Background and aims: Tight junctions within the intestinal epithelium constitute pivotal elements of the intestinal barrier, perturbations of which have been associated with the pathogenesis and progression of inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. We previously demonstrated that glucocorticoid hormones stimulate intestinal tight junction sealing In Vitro by increasing the expression of claudin-4. The aim of this study was to identify the signaling pathways responsible. Methods: Caco-2 monolayers grown on semipermeable filter supports were treated with dexamethasone, the p38 inhibitor SB203580 or the MKP-1 inhibitor triptolide and tight junction function was assessed by measuring transepithelial electrical resistance (TEER). Activation of the claudin-4 promoter was analyzed in Caco-2 cells stably expressing the human claudin-4 promoter fused to a luciferase reporter. In addition, quantitative rt-PCR, Western Blotting and immunofluorescence were employed to analyze changes in the expression, activation and localization of claudin-4, MKP-1 and p38. Results: Treatment of Caco-2 cells with dexamethasone resulted in increased TEERs, increased expression of claudin-4 on the RNA and protein level and activation of the claudin-4 promoter. This was accompanied by decreased phosphorylation of the MAPK p38 as well as increased expression of the MAPK phosphatase MKP-1. Inhibition of p38 by SB203580 was sufficient to mimic the effect of dexamethasone both on TEER and claudin-4 expression as well as claudin-4 promoter activation. Conversely, inhibition of MKP-1 by triptolide prevented the dephosphorylation of p38 by dexamethasone and reversed its effect on TEER, claudin-4 expression and activation of the claudin-4 promoter. Localization of claudin-4 to the tight junction or cell viability was not affected by either of the compounds. Conclusions: Our data provide evidence that glucocorticoid induced up regulation of claudin-4 is mediated by dephosphorylation of p38 resulting from increased expression of the MAPK phosphatase MKP-1. We thereby identified a novel signaling pathway mediating an epithelial specific effect of glucocorticoids on intestinal tight junctions. Given the central role of barrier dysfunction in inflammatory bowel diseases, components of this pathway might represent promising new pharmacological targets for the treatment of these disorders.
585 Overexpression of Interferon-γ Inhibits Gastric Inflammation and Cancer Through Induction of Apoptosis in Immune Cells and Autophagy in Epithelial Cells Shuiping Tu, Michael Quante, Shigeo Takaishi, Govind Bhagat, Guanglin Cui, James G. Fox, Timothy C. Wang Background: Gastric cancer is highly associated with Helicobacter infection. However, accumulating evidence points to the role of the host immune responses in progression to metaplasia, atrophy and dysplasia. Interferon-γ (IFN-γ) is a proinflammatory cytokine that is upregulated in stomachs of humans and mice infected with Helicobacter spp. While IFNγ-/- mice seem to be resistant to gastric metaplasia and IFN-y might induce gastric hyperplasia, IFN-γ may also have a protective effect in terms of Helicobacter infection. Thus, we aimed to clarify the precise role of IFN-γ overexpression on gastric inflammation and carcinogenesis. Methods: To investigate the potential role of IFN-γ in gastric carcinogenesis, we generated a H/K-ATPase/IFN-γ transgene that targets constitutively secreted murine IFN-γ specifically to parietal cells of the stomach. Results: We generated two lines of IFN-γ transgenic mice with overexpression of murine IFN-γ in the gastric mucosa (RT-PCR and ELISA). The presence of increased IFN-γ regulated genes IFN-γ IP-10 and Mig confirmed its bioactivity. During 18 months of observation IFN-γ mice showed no evidence of spontaneous gastritis, atrophy or metaplsia. Overexpression of IFN-γ inhibited IL-1β- and/or H. felis-induced gastric carcinogenesis and increased gastric CD4 T cell apoptosis (TUNEL, FACS), resulting in inhibition of Th1 immune response and a reduction of inflammatory cytokines. Upregulation of Fas expression (qRT-PCR) was detected in gastric immune cells, while expression of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) were decreased in H. felis- infected IFN-γ mice compared to H. felis- infected WT mice. In contrast, Th2 cytokines (IL-4, IL10) remained unchanged. Moreover, overexpression of IFN-γ increased the expression of beclin-1 (qRT-PCR) and induced autophagy (IHC and western blot for LC3) in gastric epithelial cells. Notably, overexpression of IFN-γ inhibited IL-1β- and H. felis-induced cell proliferation (ki67), and Doublecortin and CaM kinase-like-1 (DCAMKL1) positive putative progenitor cells were decreased in H. felis-infected IFN-γ mice compared to H. felis-infected WT mice. The above findings were confirmed in an experiment with 4 weeks of IFN-γ infusion. Conclusion: Overexpression of IFN-γ inhibits the development of gastric inflammation and cancer by induction of apoptosis in immune cells and autophagy in epithelial cells, leading to a suppression of gastric progenitor cell expansion and decreased proliferation in a inflammation induced model of gastric cancer.
583 Cathepsin B Upregulation Mediates Cell Invasion in Esophageal Cancer Gillian L. Allison, Kelsey M. McCowan, Shazia S. Ansari, Claudia D. Andl Introduction: Cathepsin B is overexpressed in tumors of the lung, breast, prostate, colon and stomach. As a cysteine protease, cathepsin B is active in lysosomal vesicles, but its proteolytic activity also facilitates the degradation of the extracellular matrix and aids in tumor cell invasion. We show that cathepsin B upregulation occurs in 70% of esophageal tumors in response to loss of E-cadherin and TGFβ receptor II and is responsible for cell invasion. Methods and Results: We previously established an In Vitro model using immortalized primary human esophageal keratinocytes expressing dominant-negative mutants of Ecad and TβRII (ECdnT). The gene-expression pattern of these cells compared to controls was determined by microarray analysis of invasive and non-invasive tissues after laser-capture microdissection of organotypic reconstruct cultures. Based on the gene-expression analysis cathepsin B was identified as a potential regulator of esophageal cell invasion in ECdnT cells. Cathepsin B expression is upregulated in ECdnT cells in the presence of fibroblasts or when stimulated with fibroblast conditioned media. We confirmed the upregulation of cathepsin B in 70% of patient tumors by immunohistochemistry on tissue microarrays. Mechanistically, we demonstrate that suppression of cathepsin B using chemical inhibitors or shRNA results in diminished cell invasion in Boyden chamber assays in the presence of fibroblast conditioned media and in organotypic cultures. Furthermore, cathepsin B has been demonstrated in infectious disease models to activate TGFβ1, and we show data that cathepsin B expression correlates with high TGFβ1 secretion. Conclusions: Esophageal cell invasion is cathepsin B- dependent. The role of cathepsin B is two-fold, as it is responsible for extracellular matrix degradation as well as for TGFβ1 activation, which in turn results in activation of fibroblasts in the microenvironment. As fibroblast conditioned media regulate the expression of cathepsin B this positive feedback loop enhances tumor progression.
586 Dysregulation of Claudin-7 and β-Catenin by Helicobacter pylori In Vitro and Within Inflamed Gastric Mucosa Lydia E. Wroblewski, Toni Nagy, Rupesh Chaturvedi, M. Blanca Piazuelo, Judith RomeroGallo, Keith T. Wilson, Pelayo Correa, Richard M. Peek Background: Helicobacter pylori induces chronic gastritis, which is the strongest singular risk factor for gastric cancer. Dysregulation of tight junction proteins increases the risk of cancer, and among these proteins are the claudins, a family of 24 closely related integral membrane proteins that mediate tight junction function. Alterations in claudin-7 expression within the context of inflammation lead to barrier dysfunction and carcinogenesis. β-catenin signaling suppresses claudin-7 expression and H. pylori aberrantly activates β-catenin. Therefore, we defined alterations in expression of claudin-7 in response to H. pylori and examined whether β-catenin is involved in H. pylori-induced dysregulation of claudin-7 in gastric epithelium. Methods: Gastric epithelial cells were isolated from human antral biopsies using a dissociation and dispersal technique. Claudin-7 was quantified by flow cytometry using an anti-rabbit claudin-7 antibody; epithelial cell lineage was confirmed using an anti-mouse pan-cytokeratin antibody. Claudin-7 was evaluated in control and H. pylori-infected C57/ BL6 mice (1 week post-inoculation) by immunohistochemistry. In Vitro, expression and localization of claudin-7 in control, or β-catenin-specific siRNA-treated MKN28 gastric epithelial cells was quantified via Western blot and immunocytochemistry. Results: Claudin7 expression was significantly decreased in epithelial cells within H. pylori-infected human gastric mucosa compared with uninfected samples. In uninfected mice, claudin-7 was detected in gastric epithelial cells and strong expression at the lateral membrane was present in a focal pattern. However, similar to colonized human subjects, expression of claudin-7 was decreased in mice infected with H. pylori, with essentially no claudin-7 in the lateral membrane. Concordant with these In Vivo findings, claudin-7 was sharply localized to the cellular margins of uninfected MKN28 gastric epithelial cells; however, co-culture with H. pylori decreased total claudin-7 expression and altered the topography of claudin-7 at the cell membrane. H. pylori induced nuclear translocation and activation of β-catenin in MKN28 cells and treatment with β-catenin-specific siRNA attenuated the ability of H. pylori to decrease claudin-7. Conclusions: Claudin-7 expression is decreased in H. pylori-infected human and rodent gastric epithelium. In Vitro, H. pylori similarly decreases expression of claudin-7 which requires β-catenin activation. These findings suggest that claudin-7 may represent a β-catenin target effector that heightens the risk of carcinogenesis within the context of H. pylori infection.
584 Transgenic Expression of Interferon Gamma in the Gastric Corpus Leads to Inflammation, Progenitor Cell Expansion, and Mucous Metaplasia Li-Jyun Syu, Jennifer Ferris, Xiaotan Qiao, Manas Tetarbe, Juanita L. Merchant, Deborah L. Gumucio, Andrzej Dlugosz Chronic infection with Helicobacter leads to inflammation of the stomach (gastritis) and elaboration of multiple inflammatory cytokines, associated with parietal cell loss, induction of spasmolytic polypeptide-expressing metaplasia (SPEM), intestinal metaplasia, dysplasia, and in some cases, gastric adenocarcinoma. To begin exploring the roles of specific cytokines in neoplastic progression in stomach, we engineered transgenic mice expressing interferon gamma (IFNgamma) under the control of the parietal cell-specific H+/K+ ATPase beta promoter. These IFNgamma mice developed regions of prominent SPEM in the gastric corpus by six weeks of age, with masses of large, foamy mucous cells which stained for the mucous neck cell markers lectin GSII, but not for the goblet cell-specific mucin Muc2 or the intestine-specific transcription factor Cdx2. This widespread gastric metaplasia was accompanied by a reduction or loss of other cell populations, including parietal cells, pit cells, and zymogenic cells, and an increased number of inflammatory cells. Corpus from IFNgamma mice exhibited a striking increase in apoptotic cells (activated caspase 3+) and an expansion of the proliferative (Ki67+) pool of cells. Interestingly, the majority of the apoptotic and proliferating cell populations did not express lineage markers for the major cell types found in the corpus, including the mucous neck/SPEM marker GSII. IFNgamma
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AGA Abstracts
AGA Abstracts
582
Dexamethasone Stimulates Tight Junction Sealing in Intestinal Epithelial Cells by up Regulating Claudin-4 Expression via a Pathway Involving MKP-1 and p38 Andreas Fischer, Markus Gluth, Ulrich-Frank Pape, Bertram H. Wiedenmann, Daniel C. Baumgart, Franz Theuring Background and aims: Tight junctions within the intestinal epithelium constitute pivotal elements of the intestinal barrier, perturbations of which have been associated with the pathogenesis and progression of inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. We previously demonstrated that glucocorticoid hormones stimulate intestinal tight junction sealing In Vitro by increasing the expression of claudin-4. The aim of this study was to identify the signaling pathways responsible. Methods: Caco-2 monolayers grown on semipermeable filter supports were treated with dexamethasone, the p38 inhibitor SB203580 or the MKP-1 inhibitor triptolide and tight junction function was assessed by measuring transepithelial electrical resistance (TEER). Activation of the claudin-4 promoter was analyzed in Caco-2 cells stably expressing the human claudin-4 promoter fused to a luciferase reporter. In addition, quantitative rt-PCR, Western Blotting and immunofluorescence were employed to analyze changes in the expression, activation and localization of claudin-4, MKP-1 and p38. Results: Treatment of Caco-2 cells with dexamethasone resulted in increased TEERs, increased expression of claudin-4 on the RNA and protein level and activation of the claudin-4 promoter. This was accompanied by decreased phosphorylation of the MAPK p38 as well as increased expression of the MAPK phosphatase MKP-1. Inhibition of p38 by SB203580 was sufficient to mimic the effect of dexamethasone both on TEER and claudin-4 expression as well as claudin-4 promoter activation. Conversely, inhibition of MKP-1 by triptolide prevented the dephosphorylation of p38 by dexamethasone and reversed its effect on TEER, claudin-4 expression and activation of the claudin-4 promoter. Localization of claudin-4 to the tight junction or cell viability was not affected by either of the compounds. Conclusions: Our data provide evidence that glucocorticoid induced up regulation of claudin-4 is mediated by dephosphorylation of p38 resulting from increased expression of the MAPK phosphatase MKP-1. We thereby identified a novel signaling pathway mediating an epithelial specific effect of glucocorticoids on intestinal tight junctions. Given the central role of barrier dysfunction in inflammatory bowel diseases, components of this pathway might represent promising new pharmacological targets for the treatment of these disorders.
585 Overexpression of Interferon-γ Inhibits Gastric Inflammation and Cancer Through Induction of Apoptosis in Immune Cells and Autophagy in Epithelial Cells Shuiping Tu, Michael Quante, Shigeo Takaishi, Govind Bhagat, Guanglin Cui, James G. Fox, Timothy C. Wang Background: Gastric cancer is highly associated with Helicobacter infection. However, accumulating evidence points to the role of the host immune responses in progression to metaplasia, atrophy and dysplasia. Interferon-γ (IFN-γ) is a proinflammatory cytokine that is upregulated in stomachs of humans and mice infected with Helicobacter spp. While IFNγ-/- mice seem to be resistant to gastric metaplasia and IFN-y might induce gastric hyperplasia, IFN-γ may also have a protective effect in terms of Helicobacter infection. Thus, we aimed to clarify the precise role of IFN-γ overexpression on gastric inflammation and carcinogenesis. Methods: To investigate the potential role of IFN-γ in gastric carcinogenesis, we generated a H/K-ATPase/IFN-γ transgene that targets constitutively secreted murine IFN-γ specifically to parietal cells of the stomach. Results: We generated two lines of IFN-γ transgenic mice with overexpression of murine IFN-γ in the gastric mucosa (RT-PCR and ELISA). The presence of increased IFN-γ regulated genes IFN-γ IP-10 and Mig confirmed its bioactivity. During 18 months of observation IFN-γ mice showed no evidence of spontaneous gastritis, atrophy or metaplsia. Overexpression of IFN-γ inhibited IL-1β- and/or H. felis-induced gastric carcinogenesis and increased gastric CD4 T cell apoptosis (TUNEL, FACS), resulting in inhibition of Th1 immune response and a reduction of inflammatory cytokines. Upregulation of Fas expression (qRT-PCR) was detected in gastric immune cells, while expression of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) were decreased in H. felis- infected IFN-γ mice compared to H. felis- infected WT mice. In contrast, Th2 cytokines (IL-4, IL10) remained unchanged. Moreover, overexpression of IFN-γ increased the expression of beclin-1 (qRT-PCR) and induced autophagy (IHC and western blot for LC3) in gastric epithelial cells. Notably, overexpression of IFN-γ inhibited IL-1β- and H. felis-induced cell proliferation (ki67), and Doublecortin and CaM kinase-like-1 (DCAMKL1) positive putative progenitor cells were decreased in H. felis-infected IFN-γ mice compared to H. felis-infected WT mice. The above findings were confirmed in an experiment with 4 weeks of IFN-γ infusion. Conclusion: Overexpression of IFN-γ inhibits the development of gastric inflammation and cancer by induction of apoptosis in immune cells and autophagy in epithelial cells, leading to a suppression of gastric progenitor cell expansion and decreased proliferation in a inflammation induced model of gastric cancer.
583 Cathepsin B Upregulation Mediates Cell Invasion in Esophageal Cancer Gillian L. Allison, Kelsey M. McCowan, Shazia S. Ansari, Claudia D. Andl Introduction: Cathepsin B is overexpressed in tumors of the lung, breast, prostate, colon and stomach. As a cysteine protease, cathepsin B is active in lysosomal vesicles, but its proteolytic activity also facilitates the degradation of the extracellular matrix and aids in tumor cell invasion. We show that cathepsin B upregulation occurs in 70% of esophageal tumors in response to loss of E-cadherin and TGFβ receptor II and is responsible for cell invasion. Methods and Results: We previously established an In Vitro model using immortalized primary human esophageal keratinocytes expressing dominant-negative mutants of Ecad and TβRII (ECdnT). The gene-expression pattern of these cells compared to controls was determined by microarray analysis of invasive and non-invasive tissues after laser-capture microdissection of organotypic reconstruct cultures. Based on the gene-expression analysis cathepsin B was identified as a potential regulator of esophageal cell invasion in ECdnT cells. Cathepsin B expression is upregulated in ECdnT cells in the presence of fibroblasts or when stimulated with fibroblast conditioned media. We confirmed the upregulation of cathepsin B in 70% of patient tumors by immunohistochemistry on tissue microarrays. Mechanistically, we demonstrate that suppression of cathepsin B using chemical inhibitors or shRNA results in diminished cell invasion in Boyden chamber assays in the presence of fibroblast conditioned media and in organotypic cultures. Furthermore, cathepsin B has been demonstrated in infectious disease models to activate TGFβ1, and we show data that cathepsin B expression correlates with high TGFβ1 secretion. Conclusions: Esophageal cell invasion is cathepsin B- dependent. The role of cathepsin B is two-fold, as it is responsible for extracellular matrix degradation as well as for TGFβ1 activation, which in turn results in activation of fibroblasts in the microenvironment. As fibroblast conditioned media regulate the expression of cathepsin B this positive feedback loop enhances tumor progression.
586 Dysregulation of Claudin-7 and β-Catenin by Helicobacter pylori In Vitro and Within Inflamed Gastric Mucosa Lydia E. Wroblewski, Toni Nagy, Rupesh Chaturvedi, M. Blanca Piazuelo, Judith RomeroGallo, Keith T. Wilson, Pelayo Correa, Richard M. Peek Background: Helicobacter pylori induces chronic gastritis, which is the strongest singular risk factor for gastric cancer. Dysregulation of tight junction proteins increases the risk of cancer, and among these proteins are the claudins, a family of 24 closely related integral membrane proteins that mediate tight junction function. Alterations in claudin-7 expression within the context of inflammation lead to barrier dysfunction and carcinogenesis. β-catenin signaling suppresses claudin-7 expression and H. pylori aberrantly activates β-catenin. Therefore, we defined alterations in expression of claudin-7 in response to H. pylori and examined whether β-catenin is involved in H. pylori-induced dysregulation of claudin-7 in gastric epithelium. Methods: Gastric epithelial cells were isolated from human antral biopsies using a dissociation and dispersal technique. Claudin-7 was quantified by flow cytometry using an anti-rabbit claudin-7 antibody; epithelial cell lineage was confirmed using an anti-mouse pan-cytokeratin antibody. Claudin-7 was evaluated in control and H. pylori-infected C57/ BL6 mice (1 week post-inoculation) by immunohistochemistry. In Vitro, expression and localization of claudin-7 in control, or β-catenin-specific siRNA-treated MKN28 gastric epithelial cells was quantified via Western blot and immunocytochemistry. Results: Claudin7 expression was significantly decreased in epithelial cells within H. pylori-infected human gastric mucosa compared with uninfected samples. In uninfected mice, claudin-7 was detected in gastric epithelial cells and strong expression at the lateral membrane was present in a focal pattern. However, similar to colonized human subjects, expression of claudin-7 was decreased in mice infected with H. pylori, with essentially no claudin-7 in the lateral membrane. Concordant with these In Vivo findings, claudin-7 was sharply localized to the cellular margins of uninfected MKN28 gastric epithelial cells; however, co-culture with H. pylori decreased total claudin-7 expression and altered the topography of claudin-7 at the cell membrane. H. pylori induced nuclear translocation and activation of β-catenin in MKN28 cells and treatment with β-catenin-specific siRNA attenuated the ability of H. pylori to decrease claudin-7. Conclusions: Claudin-7 expression is decreased in H. pylori-infected human and rodent gastric epithelium. In Vitro, H. pylori similarly decreases expression of claudin-7 which requires β-catenin activation. These findings suggest that claudin-7 may represent a β-catenin target effector that heightens the risk of carcinogenesis within the context of H. pylori infection.
584 Transgenic Expression of Interferon Gamma in the Gastric Corpus Leads to Inflammation, Progenitor Cell Expansion, and Mucous Metaplasia Li-Jyun Syu, Jennifer Ferris, Xiaotan Qiao, Manas Tetarbe, Juanita L. Merchant, Deborah L. Gumucio, Andrzej Dlugosz Chronic infection with Helicobacter leads to inflammation of the stomach (gastritis) and elaboration of multiple inflammatory cytokines, associated with parietal cell loss, induction of spasmolytic polypeptide-expressing metaplasia (SPEM), intestinal metaplasia, dysplasia, and in some cases, gastric adenocarcinoma. To begin exploring the roles of specific cytokines in neoplastic progression in stomach, we engineered transgenic mice expressing interferon gamma (IFNgamma) under the control of the parietal cell-specific H+/K+ ATPase beta promoter. These IFNgamma mice developed regions of prominent SPEM in the gastric corpus by six weeks of age, with masses of large, foamy mucous cells which stained for the mucous neck cell markers lectin GSII, but not for the goblet cell-specific mucin Muc2 or the intestine-specific transcription factor Cdx2. This widespread gastric metaplasia was accompanied by a reduction or loss of other cell populations, including parietal cells, pit cells, and zymogenic cells, and an increased number of inflammatory cells. Corpus from IFNgamma mice exhibited a striking increase in apoptotic cells (activated caspase 3+) and an expansion of the proliferative (Ki67+) pool of cells. Interestingly, the majority of the apoptotic and proliferating cell populations did not express lineage markers for the major cell types found in the corpus, including the mucous neck/SPEM marker GSII. IFNgamma
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AGA Abstracts
AGA Abstracts
582