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Mo2043 Protein Kinase D1 (Pkd1) Activation is an Early Event in Wounded Monolayers of Intestinal Epithelial Cells
Colitis was evaluated by histopathology and epithelial cell proliferation was quantified by BrdU staining. Levels of pro-inflammatory cytokines and chemokines were measured by real-time RT-PCR and ELISA. Expression array profiling of isolated colonic crypts was performed using a mouse whole genome microarray (Agilent). Results: After 3% DSS, mice lacking endogenous GM-CSF compared to WT controls developed more severe colitis with increased mucosal inflammation, proinflammatory chemokine production and mucosal injury with significantly greater mucosal ulceration. During the recovery period, ulcers did not heal in GM-CSF-/- mice, and they became moribund, requiring sacrifice by day 10. In striking contrast, WT mice regenerated the epithelium covering the ulcers by day 12, and survived. To determine the mechanism by which GM-CSF influences repair of the colon epithelium, we found a dose of DSS (1.5%) that caused mucosal ulceration in GM-CSF deficient mice, but was not lethal and allowed recovery of the colon epithelium. The same dose of DSS caused very little to no ulceration of the colon epithelium in WT mice or in GM-CSF-/- mice treated with exogenous murine GM-CSF. Within 1 day of 1.5% DSS there was significantly decreased proliferation of crypt intestinal epithelial cells (IECs) of a similar magnitude in WT and GM-CSF-/- mice. Whereas, IEC proliferation rapidly recovered by 3 days in WT mice, increased IEC proliferation did not occur until day 9 in GM-CSF-/- mice. Consistent with this, microarray analysis of isolated colon crypt epithelium on day 6 of 1.5% DSS treatment revealed down-regulated expression of genes involved in the cell cycle, proliferation, and response to wounding in GM-CSF-/- crypts compared to WT. Conclusion: GM-CSF significantly facilitates epithelial cell proliferation and mucosal ulcer healing in a model of colon injury and inflammation. Supported by NIH grant DK35108.
Identification of a Novel CFTR Interactome in Contributing Diarrhea in Inflammatory Bowel Disease Kavisha Arora, Sunitha Yarlagadda, Aixia Ren, Weiqiang Zhang, Chandrima Sinha, Anjaparavanda P. Naren Background: Inflammatory bowel disease (IBD) is a group of disorders in which the gut suffers from inflammation. Among the few predisposing genetic factors identified in IBD, a cGMP/cAMP-regulated chloride (Cl-) channel CFTR has emerged as one. Our lab and others have reported involvement of CFTR in formation of multiple protein complexes that can regulate the channel function in healthy and diseased states. In our current study, we aim to elucidate a molecular mechanism in the form of a signaling protein complex of CFTR that contributes to disease progression in IBD. Method: (a) Protein-protein interactions were studied using biochemical methods (protein pairwise binding assay, macromolecular complex assembly assay, and co-immunoprecipitation) and In Vitro methods for studying protein interaction in live cell (N-FRETc) in HEK293 cells. (b) Immunohistochemistry was done on mouse colon tissue for studying protein expression and distribution. Real time PCR was performed to monitor relative expression levels of several transcripts under inflammatory conditions. (c) CFTR dependent chloride secretion was monitored using iodide efflux, chloride efflux and short circuit measurements in colonic epithelial cell line HT29CL19A. (d) Dextran sodium sulfate (DSS) induced colitis mouse model was used in Nherf2+/+ and Nherf2-/- C57BL/6 male mice 7-8 weeks of age. Results: (a) Upregulation of iNOS increased CFTR channel function by at least two folds in In Vivo and In Vitro models of inflammation. The phenomenon involves cGMP dependent signaling mechanism. (b) CFTR interacts with iNOS mediated by NHERF2 in a PDZ domain-dependent manner. (c) Diarrhea is attenuated in Nherf2-/- compared to Nherf2+/+ mice in DSS-induced colitis model which simulates IBD. Conclusion: (a) A macromolecular complex of CFTR, NHERF2 and iNOS is integrative to CFTR dependent diarrhea in IBD. This compartmentalization of proteins and consequently of the signaling effectors enables a rapid and fine-tuned regulation of CFTR function. (b) Such signaling modules that are part of pathological profile can be approached as targets for improvement of IBD conditions.
Mo2045 A Novel Vegetable-Derived Probiotics (VDP) Exerts a Therapeutic Effect on DSS Induced Colitis Possibly Mediated by IL-27 Producing CD11c+ Dendritic Cells Yoshikiyo Okada, Yoshikazu Tsuzuki, Toshihide Ueda, Hideaki Hozumi, Shingo Sato, Ryota Hokari, Chie Kurihara, Chikako Watanabe, Kengo Tomita, Shunsuke Komoto, Atsushi Kawaguchi, Shigeaki Nagao, Soichiro Miura BACKGROUND: Probiotics is classified broadly into two categories. One is isolated from dairy products or gut and another is vegetable -derived Probiotics (VDP) isolated from fermented vegetable food such as pickles or a soy-sauce. We recently isolated a novel VDP strain (Lactobacillus buchneri strain s193) from Japanese "Funazushi" (Japanese old-style sushi; salted and fermented with rice and a crusian carp). s193 showed a strong antiinflammatory effect on DSS-induced colitis, but the mechanism by which the strong therapeutic effect occurs remains unknown. In this study, we investigated the effects of s193 on the mRNA expressions of pro- or anti-inflammatory cytokines from CD11c+ cells and master regulators guiding T cell differentiation from CD4+ cells in DSS-induced colitis. MATELIALS AND METHODS: Intragastric administration of s193, a VDP, or L. gasseri (LG; ATCC 33323) isolated from dairy products was performed in 8-week olds-female C57BL/6 mice every day during administration of 5% DSS for 7 days. Colonic inflammation was evaluated by H&E staining. CD11c+ and CD4+ cells were isolated from mesenteric lymph nodes (MLN) and spleen using magnetic bead-based cell separation (MACS). mRNA levels of pro- or antiinflammatory cytokines and master regulators guiding T cell differentiation were determined by real-time PCR. Results: The shortening of colonic length and thickening of colonic wall induced by DSS were significantly attenuated by L. buchneri s193. L. buchneri s193 showed a more drastic anti-inflammatory effect on DSS-induced colitis compared with that of LG. mRNA levels of IL-10, TGF-β and IL-27 in CD11c+ cells isolated from spleen and MLNs of DSS-treated mice were significantly increased by s193 administration compared with DSS alone or LG administration. On the other hand mRNA level of IL-23p19 in CD11c+ cells from MLNs in DSS mice was significantly decreased by s193 administration compared with DSS and LG administration, whereas LG administration rather increased the mRNA levels of IL-12p40 and IL-23p19 in CD11c+ cells from MLN. The increased mRNA levels of RORγt in CD4+ cells from spleen and MLN induced by DSS were significantly attenuated by s193 administration compared with DSS alone or LG administration. Conclusion: Administration of L. buchneri s193 on DSS-colitis demonstrated the high expression of IL-10, TGF-β and IL-27 in CD11c+ cells from spleen and MLN, while these cytokine expressions were not induced by LG treatment. Moreover, mRNA of ROR-γt was significantly decreased by s193 administration compared with DSS alone or LG administration in DSS colitis. These data suggest that L. buchneri s193 may exert a stronger anti-inflammatory effect on DSScolitis than that of LG, possibly through suppression of ROR-γt mediated by high expression of IL-27 and increased secretion of IL-10 and TGF-β from CD11c+ dendritic cells.
Mo2043 Protein Kinase D1 (Pkd1) Activation is an Early Event in Wounded Monolayers of Intestinal Epithelial Cells Steven H. Young, James Sinnett-Smith, Enrique Rozengurt Background: Cell migration and proliferation in the GI mucosa play fundamental roles in homeostatic turnover, restoration of tissue integrity after injury and pathogenesis of intestinal disorders, including IBD and intestinal carcinogenesis. Within minutes of epithelial injury, cells adjacent to a wound extend lamellipodia and migrate over the denuded area to reestablish epithelial continuity, a rapid process known as restitution. Despite its importance, the signaling mechanisms controlling the migration of intestinal epithelial cells remain incompletely understood. Here, we tested the hypothesis that protein kinase D1 (PKD1), the founding member of a new family of protein kinases, functions in the regulation of migration of intestinal epithelial cells. Results: To determine the role of endogenous PKD family (PKD1, PKD2, PKD3) in the migration of intestinal epithelial IEC-6 and IEC-18 cells, cultures of these cells were treated with the selective PKD inhibitor kb NB 142-70, at concentrations (3 μM) that inhibit PKD activity within cells, and then cell migration was assayed by the ability of the monolayers to reconstitute a denuded area of the culture. Treatment with kb NB 142-70 strikingly reduced the ability of IEC-6 and IEC-18 cells to repopulate the denuded area of a wound. PKD inhibition also prevented the polarized morphology of the migrating cells at the leading edge of the wound. Substantiating these results, knockdown of PKD1 protein expression (by 80%) with siRNAs markedly inhibited the migration of IEC-18 cells into the denuded area of the wound. These findings prompted us to test whether PKD1 activation is an early event in wound-induced signaling using immunofluorescence microscopy with an antibody that detects the phosphorylated state of PKD at Ser916, a well established autophosphorylation site. Wounding induced a dramatic and selective PKD1 activation in the cells located at the edge of the wound rather than in the whole cell monolayer. An increase in the phosphorylated state of Ser916 was detected as early as 30 s after wounding and reached a maximum after 3 min. The increase in Ser916 phosphorylation was prevented by treatment with inhibitors of PKD family activity (either CRT0066101 or kb NB 142-70) prior to wounding. Identical results were obtained with monolayers of IEC-6 cells. These results were corroborated by using In Vitro kinase assays in which PKD1 catalytic activity in response to wounding by multiple parallel scrapes was determined after immunoprecipitation. Conclusion: Our results support the hypothesis that PKD1 plays a key role in mediating cell migration in untransformed intestinal epithelial cells and demonstrate that PKD1 activation is one of the early events initiated by wounding monolayers of intestinal epithelial cells.
Mo2046 Sulforaphane Protects Small Intestinal Mucosa From Indomethacin-Induced Injury in Mice via Dual Beneficial Mechanisms In Vivo Junya Sato, Atsushi Fukumoto, Yuhei Suzuki, Akinori Yanaka Background and Aims: Recent studies have revealed that daily use of NSAIDs frequently causes injuries in small intestinal mucosa. However, effective drugs to prevent NSAIDsinduced small intestinal injury have not been well defined until recently. It has been suggested that both the oxidative stress and the overgrowth of luminal enterobacteria play a significant role in pathogenesis of NSAIDs-induced small intestinal injury. Sulforaphane (SFN), an isothiocyanate compound rich in broccoli sprouts, shows potent antioxidant activity by upregulating nrf2-dependent antioxidant enzymes (McMahon M, Cancer Res 2001). Our recent study showed that SFN protects small intestinal epithelial cells from aspirin-induced injury by up-regulating hemoxygenase-1 (HO-1) In Vitro (DDW2010, W1356). Our study also showed that SFN possesses anti-bacterial activity against H. pylori (Yanaka A, CPR 2:353360, 2009). Based on these backgrounds, we hypothesized that SFN may have dual beneficial effects on small intestine during NSAIDs treatment. In the present study, we examined if SFN protects small intestinal mucosa from NSAIDs-induced injury via stimulating nrf2dependent up-regulation of antioxidant enzyme activity and/or and inhibiting overgrowth of enterobacteria using indomethacin (IND)-treated mice In Vivo. Methods: IND-induced small intestinal mucosal injury was induced in mice, as described previously (Miura N, Biol Pharm Bull 2007), using male ddY mice (7-wks old). Sulforaphane glucosinolate (SGS),
Mo2044 GM-CSF Facilitates Repair of Injured Colon Mucosa in Mice In Vivo by Promoting Crypt Epithelial Cell Proliferation Laia Egea, Steve Shenouda, Martin F. Kagnoff Background and aims: Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) is a hematopoietic growth factor that targets cells of the lymphocyte/myeloid linage. Earlier studies found that GM-CSF administered to patients with Crohn's disease had a clinically beneficial effect thought to reflect its known activity on populations of myeloid cells and host innate immunity. Using a murine colon infection model that causes mucosal inflammation, but little epithelial injury, we found that endogenous GM-CSF had a significant and non-redundant role in regulating colon mucosal inflammation (Cell Host & Microbe 7, 151163, 2010). The aim of this study was to probe the role of endogenous GM-CSF in an injury model of colitis that damages colon epithelium. Methods: Genetically mutant GMCSF deficient mice (GM-CSF-/-) and control wild type (WT) C57BL/6 mice were administered 3% or 1.5% of dextran sulfate sodium (DSS) for 6 days followed by a recovery period.
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AGA Abstracts
AGA Abstracts
Mo2042
Identification of a Novel CFTR Interactome in Contributing Diarrhea in Inflammatory Bowel Disease Kavisha Arora, Sunitha Yarlagadda, Aixia Ren, Weiqiang Zhang, Chandrima Sinha, Anjaparavanda P. Naren Background: Inflammatory bowel disease (IBD) is a group of disorders in which the gut suffers from inflammation. Among the few predisposing genetic factors identified in IBD, a cGMP/cAMP-regulated chloride (Cl-) channel CFTR has emerged as one. Our lab and others have reported involvement of CFTR in formation of multiple protein complexes that can regulate the channel function in healthy and diseased states. In our current study, we aim to elucidate a molecular mechanism in the form of a signaling protein complex of CFTR that contributes to disease progression in IBD. Method: (a) Protein-protein interactions were studied using biochemical methods (protein pairwise binding assay, macromolecular complex assembly assay, and co-immunoprecipitation) and In Vitro methods for studying protein interaction in live cell (N-FRETc) in HEK293 cells. (b) Immunohistochemistry was done on mouse colon tissue for studying protein expression and distribution. Real time PCR was performed to monitor relative expression levels of several transcripts under inflammatory conditions. (c) CFTR dependent chloride secretion was monitored using iodide efflux, chloride efflux and short circuit measurements in colonic epithelial cell line HT29CL19A. (d) Dextran sodium sulfate (DSS) induced colitis mouse model was used in Nherf2+/+ and Nherf2-/- C57BL/6 male mice 7-8 weeks of age. Results: (a) Upregulation of iNOS increased CFTR channel function by at least two folds in In Vivo and In Vitro models of inflammation. The phenomenon involves cGMP dependent signaling mechanism. (b) CFTR interacts with iNOS mediated by NHERF2 in a PDZ domain-dependent manner. (c) Diarrhea is attenuated in Nherf2-/- compared to Nherf2+/+ mice in DSS-induced colitis model which simulates IBD. Conclusion: (a) A macromolecular complex of CFTR, NHERF2 and iNOS is integrative to CFTR dependent diarrhea in IBD. This compartmentalization of proteins and consequently of the signaling effectors enables a rapid and fine-tuned regulation of CFTR function. (b) Such signaling modules that are part of pathological profile can be approached as targets for improvement of IBD conditions.
Mo2045 A Novel Vegetable-Derived Probiotics (VDP) Exerts a Therapeutic Effect on DSS Induced Colitis Possibly Mediated by IL-27 Producing CD11c+ Dendritic Cells Yoshikiyo Okada, Yoshikazu Tsuzuki, Toshihide Ueda, Hideaki Hozumi, Shingo Sato, Ryota Hokari, Chie Kurihara, Chikako Watanabe, Kengo Tomita, Shunsuke Komoto, Atsushi Kawaguchi, Shigeaki Nagao, Soichiro Miura BACKGROUND: Probiotics is classified broadly into two categories. One is isolated from dairy products or gut and another is vegetable -derived Probiotics (VDP) isolated from fermented vegetable food such as pickles or a soy-sauce. We recently isolated a novel VDP strain (Lactobacillus buchneri strain s193) from Japanese "Funazushi" (Japanese old-style sushi; salted and fermented with rice and a crusian carp). s193 showed a strong antiinflammatory effect on DSS-induced colitis, but the mechanism by which the strong therapeutic effect occurs remains unknown. In this study, we investigated the effects of s193 on the mRNA expressions of pro- or anti-inflammatory cytokines from CD11c+ cells and master regulators guiding T cell differentiation from CD4+ cells in DSS-induced colitis. MATELIALS AND METHODS: Intragastric administration of s193, a VDP, or L. gasseri (LG; ATCC 33323) isolated from dairy products was performed in 8-week olds-female C57BL/6 mice every day during administration of 5% DSS for 7 days. Colonic inflammation was evaluated by H&E staining. CD11c+ and CD4+ cells were isolated from mesenteric lymph nodes (MLN) and spleen using magnetic bead-based cell separation (MACS). mRNA levels of pro- or antiinflammatory cytokines and master regulators guiding T cell differentiation were determined by real-time PCR. Results: The shortening of colonic length and thickening of colonic wall induced by DSS were significantly attenuated by L. buchneri s193. L. buchneri s193 showed a more drastic anti-inflammatory effect on DSS-induced colitis compared with that of LG. mRNA levels of IL-10, TGF-β and IL-27 in CD11c+ cells isolated from spleen and MLNs of DSS-treated mice were significantly increased by s193 administration compared with DSS alone or LG administration. On the other hand mRNA level of IL-23p19 in CD11c+ cells from MLNs in DSS mice was significantly decreased by s193 administration compared with DSS and LG administration, whereas LG administration rather increased the mRNA levels of IL-12p40 and IL-23p19 in CD11c+ cells from MLN. The increased mRNA levels of RORγt in CD4+ cells from spleen and MLN induced by DSS were significantly attenuated by s193 administration compared with DSS alone or LG administration. Conclusion: Administration of L. buchneri s193 on DSS-colitis demonstrated the high expression of IL-10, TGF-β and IL-27 in CD11c+ cells from spleen and MLN, while these cytokine expressions were not induced by LG treatment. Moreover, mRNA of ROR-γt was significantly decreased by s193 administration compared with DSS alone or LG administration in DSS colitis. These data suggest that L. buchneri s193 may exert a stronger anti-inflammatory effect on DSScolitis than that of LG, possibly through suppression of ROR-γt mediated by high expression of IL-27 and increased secretion of IL-10 and TGF-β from CD11c+ dendritic cells.
Mo2043 Protein Kinase D1 (Pkd1) Activation is an Early Event in Wounded Monolayers of Intestinal Epithelial Cells Steven H. Young, James Sinnett-Smith, Enrique Rozengurt Background: Cell migration and proliferation in the GI mucosa play fundamental roles in homeostatic turnover, restoration of tissue integrity after injury and pathogenesis of intestinal disorders, including IBD and intestinal carcinogenesis. Within minutes of epithelial injury, cells adjacent to a wound extend lamellipodia and migrate over the denuded area to reestablish epithelial continuity, a rapid process known as restitution. Despite its importance, the signaling mechanisms controlling the migration of intestinal epithelial cells remain incompletely understood. Here, we tested the hypothesis that protein kinase D1 (PKD1), the founding member of a new family of protein kinases, functions in the regulation of migration of intestinal epithelial cells. Results: To determine the role of endogenous PKD family (PKD1, PKD2, PKD3) in the migration of intestinal epithelial IEC-6 and IEC-18 cells, cultures of these cells were treated with the selective PKD inhibitor kb NB 142-70, at concentrations (3 μM) that inhibit PKD activity within cells, and then cell migration was assayed by the ability of the monolayers to reconstitute a denuded area of the culture. Treatment with kb NB 142-70 strikingly reduced the ability of IEC-6 and IEC-18 cells to repopulate the denuded area of a wound. PKD inhibition also prevented the polarized morphology of the migrating cells at the leading edge of the wound. Substantiating these results, knockdown of PKD1 protein expression (by 80%) with siRNAs markedly inhibited the migration of IEC-18 cells into the denuded area of the wound. These findings prompted us to test whether PKD1 activation is an early event in wound-induced signaling using immunofluorescence microscopy with an antibody that detects the phosphorylated state of PKD at Ser916, a well established autophosphorylation site. Wounding induced a dramatic and selective PKD1 activation in the cells located at the edge of the wound rather than in the whole cell monolayer. An increase in the phosphorylated state of Ser916 was detected as early as 30 s after wounding and reached a maximum after 3 min. The increase in Ser916 phosphorylation was prevented by treatment with inhibitors of PKD family activity (either CRT0066101 or kb NB 142-70) prior to wounding. Identical results were obtained with monolayers of IEC-6 cells. These results were corroborated by using In Vitro kinase assays in which PKD1 catalytic activity in response to wounding by multiple parallel scrapes was determined after immunoprecipitation. Conclusion: Our results support the hypothesis that PKD1 plays a key role in mediating cell migration in untransformed intestinal epithelial cells and demonstrate that PKD1 activation is one of the early events initiated by wounding monolayers of intestinal epithelial cells.
Mo2046 Sulforaphane Protects Small Intestinal Mucosa From Indomethacin-Induced Injury in Mice via Dual Beneficial Mechanisms In Vivo Junya Sato, Atsushi Fukumoto, Yuhei Suzuki, Akinori Yanaka Background and Aims: Recent studies have revealed that daily use of NSAIDs frequently causes injuries in small intestinal mucosa. However, effective drugs to prevent NSAIDsinduced small intestinal injury have not been well defined until recently. It has been suggested that both the oxidative stress and the overgrowth of luminal enterobacteria play a significant role in pathogenesis of NSAIDs-induced small intestinal injury. Sulforaphane (SFN), an isothiocyanate compound rich in broccoli sprouts, shows potent antioxidant activity by upregulating nrf2-dependent antioxidant enzymes (McMahon M, Cancer Res 2001). Our recent study showed that SFN protects small intestinal epithelial cells from aspirin-induced injury by up-regulating hemoxygenase-1 (HO-1) In Vitro (DDW2010, W1356). Our study also showed that SFN possesses anti-bacterial activity against H. pylori (Yanaka A, CPR 2:353360, 2009). Based on these backgrounds, we hypothesized that SFN may have dual beneficial effects on small intestine during NSAIDs treatment. In the present study, we examined if SFN protects small intestinal mucosa from NSAIDs-induced injury via stimulating nrf2dependent up-regulation of antioxidant enzyme activity and/or and inhibiting overgrowth of enterobacteria using indomethacin (IND)-treated mice In Vivo. Methods: IND-induced small intestinal mucosal injury was induced in mice, as described previously (Miura N, Biol Pharm Bull 2007), using male ddY mice (7-wks old). Sulforaphane glucosinolate (SGS),
Mo2044 GM-CSF Facilitates Repair of Injured Colon Mucosa in Mice In Vivo by Promoting Crypt Epithelial Cell Proliferation Laia Egea, Steve Shenouda, Martin F. Kagnoff Background and aims: Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) is a hematopoietic growth factor that targets cells of the lymphocyte/myeloid linage. Earlier studies found that GM-CSF administered to patients with Crohn's disease had a clinically beneficial effect thought to reflect its known activity on populations of myeloid cells and host innate immunity. Using a murine colon infection model that causes mucosal inflammation, but little epithelial injury, we found that endogenous GM-CSF had a significant and non-redundant role in regulating colon mucosal inflammation (Cell Host & Microbe 7, 151163, 2010). The aim of this study was to probe the role of endogenous GM-CSF in an injury model of colitis that damages colon epithelium. Methods: Genetically mutant GMCSF deficient mice (GM-CSF-/-) and control wild type (WT) C57BL/6 mice were administered 3% or 1.5% of dextran sulfate sodium (DSS) for 6 days followed by a recovery period.
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AGA Abstracts
AGA Abstracts
Mo2042