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405 Intestinal Epithelial Deletion of Sphingosine Kinase 1 Suppresses Crypt Regeneration Following Radiation Injury
were analysed for overall 96 hours, in a 24 hour interval. Additionally, we generated organoids from SurviviniΔIEC and control mice. Survivin deletion was performed by addition of tamoxifen. Specimens were investigated by quantitative PCR, immunohistochemistry and western blot analysis. Results: Time course analysis of gene deletion of survivin in IECs revealed an initial pulse of apoptosis at the crypt bottom 48 hours after tamoxifen injection. This did not led to death of SurviviniΔIEC animals. Histological analysis at later time points revealed aberrant chromosome segregation in IECs and the formation of multinucleated cells starting from the crypt bottom. Importantly, these cells did not show signs of cell death but of DNA damage, as confirmed by phopho-H2AX and phospho-p53 positive nuclei. Quantitative PCR showed elevated gene expression levels of p21 and ddit3 (dna-damageinducible-transcript 3) in SurviviniΔIEC mice, suggesting mitotic catastrophe in IEC depleted of survivin. Conclusions: Although survivin belongs to the IAP family, we could not see extensive apoptosis after survivin deletion in IECs. Our data demonstrates that the loss of survivin initially leads to the death of apoptosis prone IECs, followed by the formation of multinucleated cells with signs of DNA damage in apoptosis reluctant IECs. This leads to an alternative cell death pathway, known as mitotic catastrophe.
injury. Sphk1 in the intestinal epithelial cells might protect adult stem cell population from radiation injury.
A Self-Reinforcing Pathway of Protective Mucosal Immunity Mediated by Epithelial CD1d Torsten Olszak, Joana F. Neves, Marie C. Dowds, Kristi Baker, Jonathan Glickman, Nicholas Davidson, Christian Jobin, Stephan Brand, Werner Muller, Koichiro Wada, Kazufumi Katayama, Atsushi Nakajima, Hiroyuki Mizuguchi, Kunito Kawasaki, Kazuhiro Nagata, Stefan Schreiber, Arthur Kaser, Sebastian Zeissig, Richard S. Blumberg Background & Aim: The mechanisms by which homeostasis at mucosal surfaces is maintained is of central importance to the pathogenesis of inflammatory bowel disease (IBD). Critical to these processes is the intestinal epithelial cell (IEC), which regulates the responses of immune cells to environmental factors. CD1d presents lipid antigens to natural killer T (NKT) cells, which are critical for the pathogenesis of ulcerative colitis (UC), a major form of IBD. Since CD1d cross-linking on model IEC lines results in the production of barrier protective cytokines, we aimed to characterize the role of epithelial CD1d in IBD and its contribution to intestinal inflammation. Methods: Villin(V)-CreERT2xIl10fl/fl mice (IL10ΔIEC), V-CreERT2xCd1d1fl/fl (CD1dΔIEC), and V-CreERT2xMttpfl/fl (MTPΔIEC) were generated to allow for tamoxifen-induced, IEC-specific deletion of IL-10, CD1d, and microsomal triglyceride transfer protein (MTP), a lipid transfer protein critical for CD1d function. Oxazolone-induced colitis was investigated in the presence or absence of anti-CD1d- or anti-IL-10R blockade as well as using adenoviruses which express heat-shock protein 110 (HSP110). RNA and protein expression by IECs were assessed using microarrays, qPCR, ELISA, Western blot and immunohistochemistry. Results: We show that oxazolone colitis leads to rapid mortality and morbidity of MTPΔIEC, CD1dΔIEC and IL-10ΔIEC mice compared to wildtype littermates. Bone marrow chimera experiments with HSP110-deficient mice demonstrated that epithelial deficiency of HSP110 phenocopied observations made for epithelial deficiency of MTP, CD1d, and IL-10. Mechanistically, we demonstrate that CD1d, HSP110, and IL-10 participate in a self-reinforcing epithelial pathway, which converges on, and is mediated by, STAT3. As such, STAT3 silencing inhibited constitutive expression of epithelial CD1d, HSP110, and IL-10 and prevented HSP110-induced upregulation of IL-10 and CD1d. In accordance with these findings, adenoviral reconstitution of HSP110 in vivo restored epithelial IL-10 expression and protected from inflammation-associated morbidity and mortality in the oxazolone model in an IL-10 receptor-dependent manner. Conclusion: These results indicate a critical role for epithelial MTP, CD1d, HSP110 and IL-10 in a selfreinforcing pathway, which governs mucosal homeostasis and protects from intestinal inflammation.
404 AMPK Positively Regulates Neurotensin Secretion Through Inhibition of mTORC1 Signaling Jun Song, Jing Li, Heidi L. Weiss, Tianyan Gao, Courtney M. Townsend, B. Mark Evers Neurotensin (NT) is a gut peptide produced and stored in N cells of the distal small bowel. NT has been implicated in the regulation of gastrointestinal functions, including the stimulation of intestinal fat absorption, inhibition of gastric acid secretion and gastroduodenal motility. AMPK and mTORC1 are two important signaling pathways regulating energy balance. Previously, we have shown that mTORC1 inhibits NT gene expression and peptide secretion through feedback inhibition of ERK1/2. We also found that AMPK activation increases NT secretion. The purpose of the present study was to investigate whether NT secretion is regulated by a mechanistic crosstalk between AMPK and mTORC1 signaling. METHODS. i) The human endocrine cell lines, BON (pancreatic carcinoid) and QGP-1 (pancreatic somatostatinoma), were used. AMPK was activated by Aicar (1 mM) or 2-DG (10 mM) or inhibited by compound C (CC) (10 μM). To confirm findings obtained in cell lines, experiments were performed using a murine primary intestinal crypt model. The isolated crypts were cultured in serum-free bronchial epithelial growth medium, which enriches for neuroendocrine (NE) cells and inhibits fibroblast growth. In addition, mice were injected with saline (vehicle) or Aicar and blood samples collected for measurement of plasma NT. ii) Expression of TSC2, an mTORC1 negative regulator activated by AMPK phosphorylation, was decreased by RNA interference (RNAi). iii) To detect the involvement of ERK1/2, cells were treated with PD98059 (10 μM), an MEK inhibitor. RESULTS. NT mRNA expression was increased in BON cells treated with Aicar in a time-dependent fashion (0, 3 and 24 h). Enriched NT-positive cells were noted in isolated primary intestinal crypts by immunofluorescent microscopic analysis; 2-DG treatment for 3 h increased NT secretion from primary intestinal crypts. Plasma NT was also increased in mice injected with Aicar (500 mg/kg body weight) for 1 h compared to saline injection (n=5 mice per group). Phosphorylation of S6K1 was decreased by Aicar treatment for 3 h which was reversed by CC. Consistently, RNAi-mediated inhibition of TSC2 expression decreased Aicar-stimulated NT release. Furthermore, Aicar-stimulated NT release was attenuated by PD98059 treatment. Aicar treatment resulted in ERK1/2 translocation from cytosol to the nucleus, which was inhibited by CC. CONCLUSIONS. AMPK plays a positive role in the regulation of NT secretion through inhibition of mTORC1. These findings suggest that AMPK and mTORC1 are important signaling pathways in the control of NT gene expression and peptide release in response to low or high energy status in endocrine cells.
407 Mitochondrial Unfolded Protein Responses Control Epithelial Stem Cell Proliferation in the Intestine Emanuel Berger, Detian Yuan, Nadine Waldschmitt, Eva Rath, Michael Allgäuer, Ori Staszewski, Mark V. Boekschoten, Michael Müller, Marco Prinz, Achim Weber, Markus Gerhard, Klaus-Peter Janssen, Mathias Heikenwälder, Dirk Haller Background and aim: Heat shock protein 60 (HSP60), a mitochondrial unfolded protein response (mtUPR)-associated chaperone, is highly expressed in intestinal epithelial cells (IEC) of mouse models of chronic inflammation and in patients with inflammatory bowel disease (IBD). This study investigates the role of HSP60 in epithelial homeostasis using novel tissue-specific knockout mouse models. Methods and Results: Generation of IEC-specific Hsp60 knockout mice (Hsp60flox/floxXVillinCre) antagonized embryonic development at day E11.5 and induced embryonic lethality. Postnatal induction of HSP60 deficiency (Hsp60flox/ flox XVillinCreERT2) caused massive aberrations in the villus-crypt architecture of the small intestine associated with symptoms of severe wasting and mortality within one week. Sporadic failure of Cre-mediated Hsp60 deletion resulted in the generation of highly proliferative HSP60-positive escaper cells expressing the stem cell marker olfactomedin 4 (OLFM4). At the macroscopic level, the colonic tissue was free of any pathology, despite an increased infiltration of macrophages. Independent of hyperproliferative crypt foci, HSP60-deficient crypts in colon and small intestine revealed hallmarks of mtUPR, associated with an abrogated expression of Ki67, Lgr5 and Olfm4, indicating a significant loss of epithelial stemness. Consistently, tamoxifen-induced deletion of Hsp60 ex vivo reduced growth of small intestinal crypt organoids. Microarray analysis of HSP60-deficient colonic epithelium identified 2,512 differentially expressed genes (q<0.01), including a set of distinct C/EBP-homologous protein (CHOP) target genes. Comparative analysis with the transcriptional profile of epithelial cellspecific CHOP transgenic (ChopIEC Tg/Tg) mice identified 165 shared genes, emphasizing on metabolic processes. Mitochondria in the HSP60-deficient epithelium showed altered morphology, as well as decreased expression of functional markers like the mitochondrial encoded cytochrome c oxidase I (mtCoxI) subunit located in the respiratory chain. Conclusion: Tissue-specific deletion of Hsp60 disrupts epithelial cell homeostasis in both the pre- and postnatal gut, independent of an inflammation-associated pathology. Hsp60 deletion in the intestinal epithelium triggers mtUPR and alterations in mitochondrial function, leading to an impaired proliferative response and a loss of stemness.
405 Intestinal Epithelial Deletion of Sphingosine Kinase 1 Suppresses Crypt Regeneration Following Radiation Injury Jung Wan Choe, Beom Jae Lee, Moon Kyung Joo, Hyo jung Kim, Jong-Jae Park, Jae Seon Kim, Young-Tae Bak, Dae Sik Yang Background: Genotoxins such as γ-irradiation lead to major increases in crypt apoptosis and the loss of crypt stem cells. Sphingosine kinase 1(Sphk1) is a key enzyme in the sphingolipid pathway to produce the shingolipid metabolite, sphingosine-1-phosphate. Sphk1 is overexpressed in many solids tumors, including prostate, stomach, kidney and oral squamous cancer. Overexpressed Sphk1 in these cancers is associated with radiation resistance. We investigated the role of intestinal epithelial Sphk1 on crypt regeneration and the expression of stem cell markers after radiation injury Methods: The Sphk1 was conditionally deleted from intestinal epithelial cells in mice by crossing Sphk1 flox/flox mice with villin-cre transgenic mice to generate Villin-cre Sphk1 flox/flox (Sphk1 KO mice). Sphk1 flox/flox mice were used for control. Mice were irradiated at a single dose of 12 Gy. To examine Sphk1/S1P pathway during radiation injury, Sphk1/2, S1PR1-3 were assessed by qPCR at 0hr, 24hr, 84hr and 120 hr after 12 Gy whole body radiation (WBR). IEC apoptosis in the small bowel was assessed by Tunnel and H&E staining. To determine crypt survival and regeneration, microcolony assay was done at 84hrs after 12 Gy WBR. Quantitative RT-PCR was performed with specific primers for mice Lgr5, Bmi1, Msi1, Hopx, Oflm4, Defa5, Pla2v5, lysozyme, MMP-7, cyclin D1 and c-myc at each time point. Immunohistochemistry for DCAMKL-1 was done at 120hrs after irradiation. Results: Expression of Sphk1 m RNA began to increase at after irradiation and peak level was observed at 120hrs after 12 Gy WBR. Sphk1/Sphk2 ratio was significantly increased at this time point. In Sphk1 KO mice, epithelial cell apoptosis in the crypt of small intestine was significantly increased in both H&E staining and Tunnel activity. In microcolony assay, crypt survival and regeneration was significantly decreased 2 fold in Sphk1 KO mice compared with control(p<0.05). Quantitative RT-PCR revealed significantly reduced stem cell markers (Lgr5, Olfm4, Bmi1, Msi1) and paneth cell markers (Defa5, Lysozyme, MMP-7) in Sphk1 KO mice compared with control mice at 120hr after irradiation. Conclusion These data suggest that epithelial Sphk1 plays an important role in the intestinal epithelial cell regeneration after radiation
408 Epithelial Derived MMP9 Exhibits Tumor Suppressive Role in Colitis Associated Cancer Noopur Bhatnagar, Lewins Walter, Hamed Laroui, Pallavi Garg Background and Aims: Individuals with Inflammatory Bowel Disease (IBD) have increased risk of developing colorectal cancer (CRC). Colitis-associated cancer (CAC) a subtype of CRC is unique as it develops to adenocarcinoma via ‘inflammation-dysplasia-carcinoma' sequence compared to ‘adenoma-carcinoma sequence' of CRC. Matrix metalloproteinases
S-87
AGA Abstracts
AGA Abstracts
406
injury. Sphk1 in the intestinal epithelial cells might protect adult stem cell population from radiation injury.
A Self-Reinforcing Pathway of Protective Mucosal Immunity Mediated by Epithelial CD1d Torsten Olszak, Joana F. Neves, Marie C. Dowds, Kristi Baker, Jonathan Glickman, Nicholas Davidson, Christian Jobin, Stephan Brand, Werner Muller, Koichiro Wada, Kazufumi Katayama, Atsushi Nakajima, Hiroyuki Mizuguchi, Kunito Kawasaki, Kazuhiro Nagata, Stefan Schreiber, Arthur Kaser, Sebastian Zeissig, Richard S. Blumberg Background & Aim: The mechanisms by which homeostasis at mucosal surfaces is maintained is of central importance to the pathogenesis of inflammatory bowel disease (IBD). Critical to these processes is the intestinal epithelial cell (IEC), which regulates the responses of immune cells to environmental factors. CD1d presents lipid antigens to natural killer T (NKT) cells, which are critical for the pathogenesis of ulcerative colitis (UC), a major form of IBD. Since CD1d cross-linking on model IEC lines results in the production of barrier protective cytokines, we aimed to characterize the role of epithelial CD1d in IBD and its contribution to intestinal inflammation. Methods: Villin(V)-CreERT2xIl10fl/fl mice (IL10ΔIEC), V-CreERT2xCd1d1fl/fl (CD1dΔIEC), and V-CreERT2xMttpfl/fl (MTPΔIEC) were generated to allow for tamoxifen-induced, IEC-specific deletion of IL-10, CD1d, and microsomal triglyceride transfer protein (MTP), a lipid transfer protein critical for CD1d function. Oxazolone-induced colitis was investigated in the presence or absence of anti-CD1d- or anti-IL-10R blockade as well as using adenoviruses which express heat-shock protein 110 (HSP110). RNA and protein expression by IECs were assessed using microarrays, qPCR, ELISA, Western blot and immunohistochemistry. Results: We show that oxazolone colitis leads to rapid mortality and morbidity of MTPΔIEC, CD1dΔIEC and IL-10ΔIEC mice compared to wildtype littermates. Bone marrow chimera experiments with HSP110-deficient mice demonstrated that epithelial deficiency of HSP110 phenocopied observations made for epithelial deficiency of MTP, CD1d, and IL-10. Mechanistically, we demonstrate that CD1d, HSP110, and IL-10 participate in a self-reinforcing epithelial pathway, which converges on, and is mediated by, STAT3. As such, STAT3 silencing inhibited constitutive expression of epithelial CD1d, HSP110, and IL-10 and prevented HSP110-induced upregulation of IL-10 and CD1d. In accordance with these findings, adenoviral reconstitution of HSP110 in vivo restored epithelial IL-10 expression and protected from inflammation-associated morbidity and mortality in the oxazolone model in an IL-10 receptor-dependent manner. Conclusion: These results indicate a critical role for epithelial MTP, CD1d, HSP110 and IL-10 in a selfreinforcing pathway, which governs mucosal homeostasis and protects from intestinal inflammation.
404 AMPK Positively Regulates Neurotensin Secretion Through Inhibition of mTORC1 Signaling Jun Song, Jing Li, Heidi L. Weiss, Tianyan Gao, Courtney M. Townsend, B. Mark Evers Neurotensin (NT) is a gut peptide produced and stored in N cells of the distal small bowel. NT has been implicated in the regulation of gastrointestinal functions, including the stimulation of intestinal fat absorption, inhibition of gastric acid secretion and gastroduodenal motility. AMPK and mTORC1 are two important signaling pathways regulating energy balance. Previously, we have shown that mTORC1 inhibits NT gene expression and peptide secretion through feedback inhibition of ERK1/2. We also found that AMPK activation increases NT secretion. The purpose of the present study was to investigate whether NT secretion is regulated by a mechanistic crosstalk between AMPK and mTORC1 signaling. METHODS. i) The human endocrine cell lines, BON (pancreatic carcinoid) and QGP-1 (pancreatic somatostatinoma), were used. AMPK was activated by Aicar (1 mM) or 2-DG (10 mM) or inhibited by compound C (CC) (10 μM). To confirm findings obtained in cell lines, experiments were performed using a murine primary intestinal crypt model. The isolated crypts were cultured in serum-free bronchial epithelial growth medium, which enriches for neuroendocrine (NE) cells and inhibits fibroblast growth. In addition, mice were injected with saline (vehicle) or Aicar and blood samples collected for measurement of plasma NT. ii) Expression of TSC2, an mTORC1 negative regulator activated by AMPK phosphorylation, was decreased by RNA interference (RNAi). iii) To detect the involvement of ERK1/2, cells were treated with PD98059 (10 μM), an MEK inhibitor. RESULTS. NT mRNA expression was increased in BON cells treated with Aicar in a time-dependent fashion (0, 3 and 24 h). Enriched NT-positive cells were noted in isolated primary intestinal crypts by immunofluorescent microscopic analysis; 2-DG treatment for 3 h increased NT secretion from primary intestinal crypts. Plasma NT was also increased in mice injected with Aicar (500 mg/kg body weight) for 1 h compared to saline injection (n=5 mice per group). Phosphorylation of S6K1 was decreased by Aicar treatment for 3 h which was reversed by CC. Consistently, RNAi-mediated inhibition of TSC2 expression decreased Aicar-stimulated NT release. Furthermore, Aicar-stimulated NT release was attenuated by PD98059 treatment. Aicar treatment resulted in ERK1/2 translocation from cytosol to the nucleus, which was inhibited by CC. CONCLUSIONS. AMPK plays a positive role in the regulation of NT secretion through inhibition of mTORC1. These findings suggest that AMPK and mTORC1 are important signaling pathways in the control of NT gene expression and peptide release in response to low or high energy status in endocrine cells.
407 Mitochondrial Unfolded Protein Responses Control Epithelial Stem Cell Proliferation in the Intestine Emanuel Berger, Detian Yuan, Nadine Waldschmitt, Eva Rath, Michael Allgäuer, Ori Staszewski, Mark V. Boekschoten, Michael Müller, Marco Prinz, Achim Weber, Markus Gerhard, Klaus-Peter Janssen, Mathias Heikenwälder, Dirk Haller Background and aim: Heat shock protein 60 (HSP60), a mitochondrial unfolded protein response (mtUPR)-associated chaperone, is highly expressed in intestinal epithelial cells (IEC) of mouse models of chronic inflammation and in patients with inflammatory bowel disease (IBD). This study investigates the role of HSP60 in epithelial homeostasis using novel tissue-specific knockout mouse models. Methods and Results: Generation of IEC-specific Hsp60 knockout mice (Hsp60flox/floxXVillinCre) antagonized embryonic development at day E11.5 and induced embryonic lethality. Postnatal induction of HSP60 deficiency (Hsp60flox/ flox XVillinCreERT2) caused massive aberrations in the villus-crypt architecture of the small intestine associated with symptoms of severe wasting and mortality within one week. Sporadic failure of Cre-mediated Hsp60 deletion resulted in the generation of highly proliferative HSP60-positive escaper cells expressing the stem cell marker olfactomedin 4 (OLFM4). At the macroscopic level, the colonic tissue was free of any pathology, despite an increased infiltration of macrophages. Independent of hyperproliferative crypt foci, HSP60-deficient crypts in colon and small intestine revealed hallmarks of mtUPR, associated with an abrogated expression of Ki67, Lgr5 and Olfm4, indicating a significant loss of epithelial stemness. Consistently, tamoxifen-induced deletion of Hsp60 ex vivo reduced growth of small intestinal crypt organoids. Microarray analysis of HSP60-deficient colonic epithelium identified 2,512 differentially expressed genes (q<0.01), including a set of distinct C/EBP-homologous protein (CHOP) target genes. Comparative analysis with the transcriptional profile of epithelial cellspecific CHOP transgenic (ChopIEC Tg/Tg) mice identified 165 shared genes, emphasizing on metabolic processes. Mitochondria in the HSP60-deficient epithelium showed altered morphology, as well as decreased expression of functional markers like the mitochondrial encoded cytochrome c oxidase I (mtCoxI) subunit located in the respiratory chain. Conclusion: Tissue-specific deletion of Hsp60 disrupts epithelial cell homeostasis in both the pre- and postnatal gut, independent of an inflammation-associated pathology. Hsp60 deletion in the intestinal epithelium triggers mtUPR and alterations in mitochondrial function, leading to an impaired proliferative response and a loss of stemness.
405 Intestinal Epithelial Deletion of Sphingosine Kinase 1 Suppresses Crypt Regeneration Following Radiation Injury Jung Wan Choe, Beom Jae Lee, Moon Kyung Joo, Hyo jung Kim, Jong-Jae Park, Jae Seon Kim, Young-Tae Bak, Dae Sik Yang Background: Genotoxins such as γ-irradiation lead to major increases in crypt apoptosis and the loss of crypt stem cells. Sphingosine kinase 1(Sphk1) is a key enzyme in the sphingolipid pathway to produce the shingolipid metabolite, sphingosine-1-phosphate. Sphk1 is overexpressed in many solids tumors, including prostate, stomach, kidney and oral squamous cancer. Overexpressed Sphk1 in these cancers is associated with radiation resistance. We investigated the role of intestinal epithelial Sphk1 on crypt regeneration and the expression of stem cell markers after radiation injury Methods: The Sphk1 was conditionally deleted from intestinal epithelial cells in mice by crossing Sphk1 flox/flox mice with villin-cre transgenic mice to generate Villin-cre Sphk1 flox/flox (Sphk1 KO mice). Sphk1 flox/flox mice were used for control. Mice were irradiated at a single dose of 12 Gy. To examine Sphk1/S1P pathway during radiation injury, Sphk1/2, S1PR1-3 were assessed by qPCR at 0hr, 24hr, 84hr and 120 hr after 12 Gy whole body radiation (WBR). IEC apoptosis in the small bowel was assessed by Tunnel and H&E staining. To determine crypt survival and regeneration, microcolony assay was done at 84hrs after 12 Gy WBR. Quantitative RT-PCR was performed with specific primers for mice Lgr5, Bmi1, Msi1, Hopx, Oflm4, Defa5, Pla2v5, lysozyme, MMP-7, cyclin D1 and c-myc at each time point. Immunohistochemistry for DCAMKL-1 was done at 120hrs after irradiation. Results: Expression of Sphk1 m RNA began to increase at after irradiation and peak level was observed at 120hrs after 12 Gy WBR. Sphk1/Sphk2 ratio was significantly increased at this time point. In Sphk1 KO mice, epithelial cell apoptosis in the crypt of small intestine was significantly increased in both H&E staining and Tunnel activity. In microcolony assay, crypt survival and regeneration was significantly decreased 2 fold in Sphk1 KO mice compared with control(p<0.05). Quantitative RT-PCR revealed significantly reduced stem cell markers (Lgr5, Olfm4, Bmi1, Msi1) and paneth cell markers (Defa5, Lysozyme, MMP-7) in Sphk1 KO mice compared with control mice at 120hr after irradiation. Conclusion These data suggest that epithelial Sphk1 plays an important role in the intestinal epithelial cell regeneration after radiation
408 Epithelial Derived MMP9 Exhibits Tumor Suppressive Role in Colitis Associated Cancer Noopur Bhatnagar, Lewins Walter, Hamed Laroui, Pallavi Garg Background and Aims: Individuals with Inflammatory Bowel Disease (IBD) have increased risk of developing colorectal cancer (CRC). Colitis-associated cancer (CAC) a subtype of CRC is unique as it develops to adenocarcinoma via ‘inflammation-dysplasia-carcinoma' sequence compared to ‘adenoma-carcinoma sequence' of CRC. Matrix metalloproteinases
S-87
AGA Abstracts
AGA Abstracts
406