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135 MTG16 Plays a Critical Role in Intestinal Stem Cell Differentiation and Injury Responses
132 Intestinal Epithelial Tissue-Specific miR-222 Overexpression Inhibits Mucosal Growth Hee Kyoung Chung, Rao N. Jaladanki, Lan Xiao, Douglas J. Turner, Peixin Yang, JianYing Wang Posttranscriptional processes, particularly altered mRNA stability and translation, play an important role in the maintenance of gut epithelial integrity, and they are governed predominantly by microRNAs (miRNAs) through interaction with cis-elements located at the 3'untranslated regions of target mRNAs. Our previous in vitro cell biology studies demonstrate that normal intestinal epithelial cells (IECs) express miRNA-222 (miR-222) and that ectopic overexpression of a miR-222 precursor represses cyclin-dependent kinase 4 (CDK4) translation, thus inhibiting IEC proliferation. Since these studies were conducted in cultured IECs, the exact biological function of miR-222 in the intestinal epithelium remains unknown. In this study, we developed miR-222-transgenic (miR222-tg) mice that specifically overexpress miR-222 in the intestinal epithelium and further examined its effect on intestinal mucosal growth. Methods: The intestinal epithelial tissue-specific A33-promoter was chosen to generate miR222-tg mice in this study. The cDNA encoding primary-miR222 was cloned to IRESGFP vector, and the final DNA construct (A33-miR222-GFP-NLS) was used for microinjection to embryos. The levels of miR-222 in gut mucosa and other tissues were measured by quantitative real-time PCR analysis and GFP immunofluorescence assay, while histological features of intestine were examined by H&E staining. Mucosal growth was measured by BrdU incorporation and immunohistochemistry staining. Results: Two founders were generated after microinjection and their germ line transmissions were checked by genotyping. One founder showed the specific expression of miR222/GFP in small intestinal mucosa only, and other showed broader spectrum expression in mucosa of the small intestine, caecum and colon. Levels of miR-222 in small intestinal mucosa in both lines of miR222-tg mice were increased by >2.5-fold, although its expression was unchanged in gastric mucosa, liver, and lung. Interestingly, miR222-tg mice exhibited a significant inhibition of intestinal mucosal growth as indicated by deceased levels of DNA synthesis and cell proliferation and by shrinkage of crypt area and villi. Levels of cell proliferation-associated protein PCNA in the mucosa was also decreased in miR222-tg mouse. Expression of CDK4 in small intestinal mucosa was inhibited by locally increasing miR-222 level in miR222-tg mice, similar to the pattern of our previous findings in vitro. In addition, miR-222-tg mice also showed irregular and destructive features in crypt area. Conclusions: These results indicate that 1) miR-222 functions as a negative regulator in normal intestinal mucosal growth and 2) miR-222 inhibits small intestinal mucosal renewal at least partially by repressing CDK4 expression.
134 The Impact of the Pentadecapeptide BPC 157 on the Healing of Ileorectal Anastomosis in Rats Following the Subtotal Colectomy Robert Klicek, Tihomir Grgic, Gorana Aralica, Leonardo Patrlj, Masa Hrelec Patrlj, Dora Grgic, Jelena Suran, Sven Seiwerth, Predrag Sikiric Aim. The pentadecapeptide BPC 157 has already been proved effective at healing ileoileal and jejunoileal anastomosis at short bowel syndrome (SurgToday, 2007 ; Dig Dis Sci, 2009). In this study we wanted to assess whether it is effective at healing ileorectal anastomosis. Another goal would be to see whether BPC 157 recovers animals` state following the subtotal colectomy. Methods. 60 male Wistar Albino rats under deep anesthesia underwent subtotal colectomy with ileorectal anastomosis formation. The animals were randomly assigned into two groups; half of them were in control group that was treated with saline (5mL/kg, i.p.), while the other received BPC 157 (10 μg/kg). The treatment was once daily, the first dose was applied immediately after the operation and the animals received the last dose 24h before they were sacrificed. The animals were sacrificed 7 and 14 days following the surgery and the biomechanical, functional, macroscopic and microscopic assessment were performed. Results. In BPC 157 treated group mortality was llower significantly, the same was with body weight loss. The ileorectal anastomosis could sustain less volume of water at control animals before leakage started. During all experimental period control animals had diarrhea, while in BPC 157 treated animals the stool was formed from 3rd postoperative day on. Conclusions. Accordingly to these results BPC 157 could present a novel therapeutic agent which affects the healing of ileorectal anastomosis and reduces adverse effects of subtotal colectomy.
133 Myosin Phosphatase Is a Critical Regulator of Epithelial Homeostasis and Survival Whose Expression Is Downregulated in IBD Weiqi He, Juanmin Zha, Minsheng Zhu, Jerrold R. Turner BACKGROUND & AIMS: Intestinal barrier loss has been implicated in inflammatory bowel disease (IBD). Increased phosphorylation of myosin regulatory light chain (MLC) mediated by MLC kinase (MLCK) is responsible for tumor necrosis factor (TNF)-induced barrier loss. While the contributions of MLCK activation to pathogenesis of human and experimental IBD have been the subject of intense scrutiny, myosin phosphatase, which opposes MLCK action, has not been studied. Myosin phosphatase is composed of a catalytic subunit (PP1cδ) and a regulatory subunit (MYPT1). MYPT1 confers specificity and restrains activity of PP1cδ, but can be inhibited by Rho-associated kinase (ROCK), thereby providing an additional means of managing MLC phosphorylation. The aim of this study was to determine the potential contributions of MYPT1 to barrier regulation in disease. METHODS: MYPT1 and PP1cδ expression were analyzed in CD4CD45RBhi adoptive transfer colitis. MYPT1f/f mice were crossed to villin-Cre and villin-CreERT2 mice. RESULTS: Intestinal epithelial expression of both MYPT1 and PP1cδ expression were downregulated in immune-mediated experimental IBD. To assess the significance of reduced epithelial MYPT1 expression in disease, MYPT1fl/ fl /villin-Cre mice were generated. However, MYPT1fl/fl/villin-Cre embryos were lost prior to intestinal organogenesis, indicating an essential role of MYPT1 in early development. In
135 MTG16 Plays a Critical Role in Intestinal Stem Cell Differentiation and Injury Responses Shenika Poindexter, Rupesh Chaturvedi, Xi Chen, Pauline K. Lund, Mukul K. Mittal, Wei Ning, Bobak Parang, Vishruth K. Reddy, Kshipra Singh, J. Joshua Smith, Amanda Williams, Keith T. Wilson, Christopher S. Williams Myeloid translocation genes (MTGs) are transcriptional co-repressors that complex with NCoR, HDACs, and mSin3A and have been implicated in a variety of biological processes including malignancy, development, differentiation and stem cell function. Our previously published studies have shown that Mtg16 -/- mice were more sensitive to chemically induced colonic injury than wild-type mice. Based on these observations and because little is known about the function of Mtg16 in the small intestine (SI), we hypothesized that MTG16
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contrast, MYPT1fl/fl/villin-CreERT2 mice were born in expected Mendelian proportions. At 6 weeks of age, MYPT1fl/fl/villin-CreERT2 and MYPT1fl/fl mice received daily tamoxifen injections. By the fifth injection, MYPT1fl/fl/villin-CreERT2, but not MYPT1fl/fl, mice developed rectal bleeding, weight loss, and lethargy. Histologic analyses demonstrated severe epithelial damage, including small intestinal villous loss and colonic ulceration, and dense mixed inflammatory infiltrates, indicating a critical homeostatic function of MYPT1. Further examination showed apical (brush border) membrane rounding, thickening and increased density of the perijunctional actomyosin ring, and retraction of epithelial lateral membranes. To separate primary epithelial effects of MYPT1 loss from those secondary to the mucosal immune response, intestinal stem cells were isolated from MYPT1fl/fl/villin-CreERT2 mice prior to tamoxifen treatment and grown as enteroids. In vitro treatment of MYPT1fl/fl/villinCreERT2 enteroids with tamoxifen resulted in epithelial cell death, while villin-CreERT2 enteroids were unaffected. Thus, MYPT1 loss activates cell-intrinsic epithelial death. CONCLUSIONS: These data demonstrate that MYPT1, and likely PP1cδ, are critical determinants of epithelial homeostasis. Further studies are required to determine whether the mechanisms that lead to epithelial death in vitro and severe enterocolitis in vivo following MYPT1 knockout.
as a transcription factor, Signal Transducer and Activator of Transcription 3 (STAT3) resides in the mitochondria of cells where phosphorylation at serine 727 (S727), but not tyrosine 705 (Y705), is required for optimal ETC activity and protects against stress-induced mitochondrial dysfunction. In this study, we explored whether PHB protects against mitochondrial dysfunction and apoptosis induced by pro-inflammatory cytokines via mitochondrial pS727-Stat3. Methods: Mitochondrial dysfunction, as indicated by increased mitochondrial reactive oxygen species (ROS) production, decreased ATP concentration, and reduced activity of ETC complexes, was induced in Caco2-BBE and IEC-6 intestinal epithelial cell lines by treatment with tumor necrosis factor α (TNFα), a pro-inflammatory cytokine involved in IBD that promotes cellular injury via mitochondrial damage. The effect of PHB overexpression on mitochondrial dysfunction and apoptosis was determined during TNFα treatment. The interaction of PHB with STAT3 was assessed by co-immunoprecipitation and immunofluorescent staining. To determine whether mitochondrial STAT3 is necessary for the protective effect of PHB on mitochondrial function, cells overexpressing PHB were transfected with a mitochondrial targeted pS727-STAT3 dominant negative (MLS-S727-STAT3dn) during TNFα treatment. Results: PHB overexpression protects against TNFα-induced mitochondrial dysfunction and apoptosis in cultured IECs. This protection was eliminated by expression of MLS-S727-STAT3dn. PHB co-immunoprecipitates and co-localizes with pS727-STAT3, but not pY705-STAT3, in the mitochondria of cultured IECs and in vivo in colonic epithelium from wild-type mice. PHB/pS727-STAT3 interaction is decreased in IECs during TNFαinduced mitochondrial stress due at least in part to decreased PHB expression. PHB overexpression maintains PHB/pS727-STAT3 interaction during TNFα treatment. Conclusions: Our data demonstrate a potential mechanistic link between PHB and mitochondrial STAT3 in the intestinal epithelium to prevent mitochondrial dysfunction, which is thought to participate in the pathogenesis of IBD. Reduced levels of PHB during IBD may be an underlying factor promoting mitochondrial dysfunction and apoptosis of the intestinal epithelium.
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regulates stem cell differentiation and injury responses in the SI. Baseline characterization of wildtype and Mtg16-/- SI revealed significantly decreased number of goblet cells (11.6 vs 14.9 PAS+ cells/crypt-villus, p=0.014), indicating a previously unrecognized role for Mtg16 in gut lineage allocation. Furthermore, Mtg16-/- mice have increased proliferation (6.6 vs 4.5 phospho-Histone H3+ cells/crypt-villus, p=0.011) but no differences in apoptosis (0.4 vs 0.5 TUNEL+ cells/cypt-villus, p=0.51). To assess intestinal injury responses, wildtype and Mtg16-/- mice were exposed to 12-Gy whole body radiation (WBR). Mtg16-/- mice had increased crypt viability (92.8% vs 77.9%, p<0.0001), and decreased apoptosis (1.4 vs 2.3 TUNEL+ cells/ crypt villus, p=0.001). To decipher the mechanism underlying this phenomenon, we looked for DNA damage in these cells by immunofluorescence staining for phospho-γH2AX foci, an early marker for DNA damage. We found fewer γH2AX+ foci in Mtg16-/- SI. Flow cytometry analysis using antibody to γH2AX confirmed decreased DNA damage in irradiated Mtg16-/- mice (15.1% vs 32.8%, p=0.003), indicating that loss of MTG16 impairs radiosensitivity. Since high doses of radiation are known to induce endothelial damage, we established enteroids from non-irradiated and irradiated wildtype and Mtg16-/mice to focus on the role of MTG16 in epithelial cell differentiation and response to radiation induced injury. Enteroids isolated from Mtg16-/- mice had higher plating efficiencies than those isolated from wildtype mice (43.3% vs 36.3%, p<0.05) before and after 12 Gy WBR (1.7% vs 0.7%, p<0.0001). We also found decreased efficiency in isolated crypts forming branched enteroids from non-irradiated Mtg16-/- mice (3.9% vs 23.9%, p<0.0001). In sum, our data provide evidence that loss of MTG16 impacts intestinal stem cell differentiation, and promotes radio-resistance. This might be due to shifting cellular response away from DNA damage-induced apoptosis and DNA repair after injury. These studies may have important implications for MTG16 being a diagnostic marker for radiation injury response and could lead to improved treatment modalities for patients receiving radiotherapy treatments.
real-time PCR with a set of primers within the MUC1, MUC2, and MUC13 promoters (ChiP). Results: Mice with conditional ablation of the Prep1 gene from the intestinal epithelium were viable. However, transgenic animals appeared smaller than wt, with a defective development of both the small and large intestine. VillinCRE/Prep1flox/flox mice showed an altered Goblet cell maturation and differentiation, resulting in MUC2 (soluble mucin) overproduction and MUC1/MUC13 (membrane-bound mucins) overexpression. Mutant mice were protected from DSS-induced colitis, as assessed by body weight loss, colitis activity Index, and histological scores. This correlated with reduced intestinal permeability (p<0,001), and bacterial penetration (p<0,05) within the inner mucus layer. Finally, Prep1 was find to be up-regulated in the intestinal epithelium of IBD patients, when compared to controls, and ChiP assay revealed that Prep1 modulate the transcription of the human MUC2, MUC1 and MUC13 genes, by directly binding to their promoters and acting as a repressor. Conclusion: Our findings demonstrate an essential role for Prep1 in intestinal development, epithelial homeostasis and maintenance of the mucosal barrier. Its manipulation, could indeed represent a novel therapeutic approach for the treatment of IBD, possibly restoring the mucin barrier. 138 ErbB4 Activation Protects Paneth Cells and Ameliorates Experimental Necrotizing Enterocolitis Steven J. McElroy, Shannon L. Castle, Jessica K. Bernard, Dana Almohazey, Catherine J. Hunter, Mark R. Frey Breastfed infants have a lower risk of developing necrotizing enterocolitis (NEC), a devastating intestinal disorder which affects up to 10% of premature infants and has a mortality of ~30%. While the protective factors in milk are not yet well-characterized, promising candidate molecules include growth factor ligands for ErbB receptor tyrosine kinases. Neuregulin-4 (NRG4) is an ErbB4-specific ligand that promotes cell survival in adult colonocytes; however, its role in the developing small intestine is uncharacterized. A common feature of NEC and other intestinal inflammatory diseases is Paneth cell (PC) loss; these cells provide antimicrobial peptides and also support the Lgr5+ intestinal stem cells, and are thus necessary for intestinal homeostasis and defense. In this study, we tested whether NRG4-ErbB4 signaling is protective in experimental NEC, and asked if it protects PCs during inflammation. METHODS: NRG4 was tested in an intestinal injury model which mimics human NEC-associated PC loss and intestinal infection. P14-16 C57Bl/6 mice were given i.p. injections of dithizone to ablate PCs, followed by Klebsiella pneumoniae gavage, to trigger NEC-like injury. Some mice received NRG4 (250μg/kg, i.p.) along with the dithizone. Effects of ErbB4 signaling on the small intestinal epithelium were tested in vitro with ileal enteroid cultures grown from ErbB4flox/ flox (functionally wild-type) and ErbB4flox/flox;Villin-Cre (intestinal epithelial-specific ErbB4 knockout) mice. Presence of the NRG4-ErbB4 signaling axis in developing human intestine was tested by immunoblot and immunohistochemistry on human milk and tissue samples collected from infants undergoing bowel resection. RESULTS: In the Dithizone/Klebsiella (DK) NEC model, ErbB4 activation by NRG4 administration reduced intestinal injury (66% decrease in histological damage score, p<0.05), and prevented DK-induced Paneth cell loss (mean PCs/crypt: control 2.80 +/- 0.26, DK 1.35 +/- 0.44, DK + NRG4 3.00 +/- 0.25). In vitro, cultured ErbB4-null ileal epithelial enteroids displayed a dysmorphic phenotype with fewer crypt buds than wild type, and had reduced RNA expression for PC (Lyz1, 57% reduction, p<0.05) and intestinal stem cell (Lgr5, 75% reduction, p<0.01) markers. Furthermore, ErbB4-null enteroids were highly sensitive to TNF, losing all crypt architecture after exposure. The potential clinical relevance of these findings was demonstrated by the observation that NRG4 and its receptor ErbB4 were detectable in human breast milk and developing human intestine, respectively. CONCLUSIONS: ErbB4 supports Paneth cells, promotes normal morphogenesis of the intestinal epithelium, and protects the intestinal epithelium from inflammatory insult. NRG4-ErbB4 signaling may contribute to the protective effects of breastfeeding, and represents a novel target pathway for therapeutic intervention in NEC.
136 PKD1 Functions Upstream of PKA in a Novel Pathway That Links GPCR Activation to β-Catenin Phosphorylation At Ser552 in Intestinal Epithelial Cells James Sinnett-Smith, Yang Ni, Steven H. Young, Enrique Rozengurt Background: Protein kinase D1 (PKD1) signaling stimulates intestinal epithelial cell proliferation, as revealed by using epithelial cells in culture and a novel PKD1 transgenic mouse model (J Biol Chem, 286, 511-520, 2011). Given that β-catenin phosphorylation at Ser552 promotes its dissociation from cell-cell contacts and stimulates its transcriptional activity, we examined whether GPCR/PKD1 activation stimulates β-catenin phosphorylation at this regulatory site. Results: Stimulation of intestinal epithelial IEC-18 cells with angiotensin II (ANG II), a mitogenic agonist that activates GPCRs endogenously expressed by these cells, induced a striking increase in the phosphorylation of β-catenin at Ser552 in IEC-18 cells which persisted for at least 24 h. In contrast, stimulation of IEC-18 with EGF induced transient Ser552 phosphorylation that declined to near baseline levels within 2 h. ANGIIinduced phosphorylation of β-catenin at Ser552 was completely prevented by cell exposure to PKD family inhibitors kb NB 142-70 or CRT006610 as well as by siRNA-mediated knockdown of PKD1 expression. These results implied that PKD1 plays a critical role in mediating GPCR-induced β-catenin phosphorylation at Ser552. Given that Ser552 does not reside within a PKD family consensus amino acid sequence, we hypothesized that PKD1 regulates β-catenin phosphorylation at this residue through other kinases implicated in Ser552 phosphorylation, including PKA and Akt. ANGII is known to induce transient Akt activation in IEC-18 cells but stimulates sustained β-catenin phosphorylation at Ser552, implying that GPCR activation induces phosphorylation at Ser552 through an Akt-independent pathway. In contrast, treatment of IEC-18 cells with H-89, an inhibitor of PKA, prevented β-catenin phosphorylation at Ser552 in response to ANGII in a dose-dependent manner, without impairing PKD1 activation, as scored by autophosphorylation at Ser916. These results suggested that PKA lies downstream of PKD1 in a novel pathway. In line with this hypothesis, stimulation with the adenylyl cyclase activator forskolin (FSK) and the phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) to increase cAMP/PKA, potently induced sustained β-catenin phosphorylation at Ser552 but did not induce PKD1 activation. Accordingly, the response to FSK+IBMX, in contrast to ANGII, was not blocked by kb NB 142-70 but abolished by H-89, an inhibitor of PKA but not PKD1. Using transgenic mice that express elevated PKD1 protein in intestinal epithelial cells, we found that over-expression of PKD1 was associated with enhanced β-catenin phosphorylation at Ser552, suggesting that the PKD1/ β-catenin pathway functions in vivo. Conclusion: Collectively, these results imply that PKD1 functions upstream of PKA in a novel pathway that links GPCRs to β-catenin phosphorylation at Ser552 in intestinal epithelial cells.
139 ATOH1 Transcriptional Targets in the Adult Intestine Yuan-Hung Lo, Maxime M. Mahe, Eunah Chung, Sunghee Oh, Joo-Seop Park, Noah F. Shroyer Background and Aims: Notch signaling has a critical function controlling the fate of intestinal progenitors by differentially regulating two opposing basic helix-loop-helix (bHLH) transcription factors, Atoh1 (atonal homolog1) and HES1 (hairy/enhancer of split1). Previous studies showed progenitors that escape Notch activation express high levels of Atoh1 and commit to a secretory lineage fate. These results indicated Atoh1 is a critical gatekeeper for the program of Notch-mediated differentiation and maturation of intestinal epithelial cells. Although some transcription factors downstream of Atoh1, such SPDEF and Gfi1, have been identified to specify differentiation and maturation of specific cell types, the bona fide targets of Atoh1 at the genome wide level in the adult intestine are still largely unknown. Here we aim to identify Atoh1 transcriptional signature in both distal small intestine and colon. Methods: Mice with an Atoh1-GFP fusion protein inserted into the Atoh1 locus (Atoh1GFP) were used in this study. In order to generate Atoh1 signature in adult intestines, the distal small intestinal crypts or colon crypts were isolated from 10~12 weeks old adult mice followed by H3K27ac, H3K27me3 as well as Atoh1 ChIP-sequencing. In parallel, RNAsequencing was performed on Atoh1 deficient crypts and normal crypts. Finally, the transcriptional profile of Atoh1 expressing cells in the crypts were generated by cell sorting GFP+ cells from Atoh1GFP intestines followed by RNA-sequencing. In order to confirm the potential direct targets of Atoh1 generated from our ChIP-sequencing and RNA-sequencing, ChIPqPCR was performed on both mouse colon crypts and human colon cancer cell lines, which transiently overexpress flag-tagged Atoh1. Results: In this study, we identified Atoh1 targets in the crypts of both distal small intestine and colon. As expected, Atoh1 shares a conserved DNA binding motif 5'-NCANNTG-3' of bHLH transcription factors. A refined matrix for Atoh1 DNA binding was defined by de novo motif analysis. Furthermore, DNA binding motifs for several other intestine-enriched transcription factors (e.g., SPDEF, GATA4, CDX2) were found within Atoh1-bound chromatin. Moreover, our ChIP-sequencing data showed Atoh1 binds to the promoter/enhancer regions marked by opened-chromatin marker H3K27ac, such as the promoter/enhancer of SPDEF and Gfi1, which are suggested to be
137 Conditional Deletion of Prep1 in the Intestinal Epithelium Alters Epithelial Homeostasis, Intestinal Development, and Controls Colitis Susceptibility Silvia D'Alessio, Carlotta Tacconi, Carmen Correale, Alessandro Gandelli, Marco Genua, Stefania Vetrano, Silvio Danese Background: Balanced and dynamic interactions among mucus layers, intestinal epithelial cells, and microbiota, are essential for the maintenance of the intestinal mucosal homeostasis. The disruption of this balance leads to a defective mucus barrier with increased permeability that results in intestinal inflammation. The homeodomain transcription factor, Prep1, is expressed in the post-mitotic differentiated intestinal epithelial cells and is essential in embryonic development. Here we report for the first time the involvement of Prep1 in intestinal epithelial homeostasis, and its functional role in experimental colitis. Methods: Mice lacking Prep1 in intestinal epithelium were generated by mating Prep1flox/flox mice with VillinCRE animals. Goblet cells staining was carried out by Alcian Blue and Periodic Acid-Schiff (PAS) staining, while mucins were quantified by real-time PCR and immunohistochemistry. The dextran sulfate sodium (DSS) model of colitis was used in VillinCRE/Prep1flox/ flox and wild-type (wt) mice. Moreover, Prep1 expression was evaluated in the intestinal epithelium of surgical specimens from normal individuals and patients with IBD, by Western Blot. Cross-linked, sonicated chromatin from human intestinal epithelial cells, was then immunoprecipitated with an anti-Prep1 antibody and the precipitated DNA analyzed by
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134 The Impact of the Pentadecapeptide BPC 157 on the Healing of Ileorectal Anastomosis in Rats Following the Subtotal Colectomy Robert Klicek, Tihomir Grgic, Gorana Aralica, Leonardo Patrlj, Masa Hrelec Patrlj, Dora Grgic, Jelena Suran, Sven Seiwerth, Predrag Sikiric Aim. The pentadecapeptide BPC 157 has already been proved effective at healing ileoileal and jejunoileal anastomosis at short bowel syndrome (SurgToday, 2007 ; Dig Dis Sci, 2009). In this study we wanted to assess whether it is effective at healing ileorectal anastomosis. Another goal would be to see whether BPC 157 recovers animals` state following the subtotal colectomy. Methods. 60 male Wistar Albino rats under deep anesthesia underwent subtotal colectomy with ileorectal anastomosis formation. The animals were randomly assigned into two groups; half of them were in control group that was treated with saline (5mL/kg, i.p.), while the other received BPC 157 (10 μg/kg). The treatment was once daily, the first dose was applied immediately after the operation and the animals received the last dose 24h before they were sacrificed. The animals were sacrificed 7 and 14 days following the surgery and the biomechanical, functional, macroscopic and microscopic assessment were performed. Results. In BPC 157 treated group mortality was llower significantly, the same was with body weight loss. The ileorectal anastomosis could sustain less volume of water at control animals before leakage started. During all experimental period control animals had diarrhea, while in BPC 157 treated animals the stool was formed from 3rd postoperative day on. Conclusions. Accordingly to these results BPC 157 could present a novel therapeutic agent which affects the healing of ileorectal anastomosis and reduces adverse effects of subtotal colectomy.
133 Myosin Phosphatase Is a Critical Regulator of Epithelial Homeostasis and Survival Whose Expression Is Downregulated in IBD Weiqi He, Juanmin Zha, Minsheng Zhu, Jerrold R. Turner BACKGROUND & AIMS: Intestinal barrier loss has been implicated in inflammatory bowel disease (IBD). Increased phosphorylation of myosin regulatory light chain (MLC) mediated by MLC kinase (MLCK) is responsible for tumor necrosis factor (TNF)-induced barrier loss. While the contributions of MLCK activation to pathogenesis of human and experimental IBD have been the subject of intense scrutiny, myosin phosphatase, which opposes MLCK action, has not been studied. Myosin phosphatase is composed of a catalytic subunit (PP1cδ) and a regulatory subunit (MYPT1). MYPT1 confers specificity and restrains activity of PP1cδ, but can be inhibited by Rho-associated kinase (ROCK), thereby providing an additional means of managing MLC phosphorylation. The aim of this study was to determine the potential contributions of MYPT1 to barrier regulation in disease. METHODS: MYPT1 and PP1cδ expression were analyzed in CD4CD45RBhi adoptive transfer colitis. MYPT1f/f mice were crossed to villin-Cre and villin-CreERT2 mice. RESULTS: Intestinal epithelial expression of both MYPT1 and PP1cδ expression were downregulated in immune-mediated experimental IBD. To assess the significance of reduced epithelial MYPT1 expression in disease, MYPT1fl/ fl /villin-Cre mice were generated. However, MYPT1fl/fl/villin-Cre embryos were lost prior to intestinal organogenesis, indicating an essential role of MYPT1 in early development. In
135 MTG16 Plays a Critical Role in Intestinal Stem Cell Differentiation and Injury Responses Shenika Poindexter, Rupesh Chaturvedi, Xi Chen, Pauline K. Lund, Mukul K. Mittal, Wei Ning, Bobak Parang, Vishruth K. Reddy, Kshipra Singh, J. Joshua Smith, Amanda Williams, Keith T. Wilson, Christopher S. Williams Myeloid translocation genes (MTGs) are transcriptional co-repressors that complex with NCoR, HDACs, and mSin3A and have been implicated in a variety of biological processes including malignancy, development, differentiation and stem cell function. Our previously published studies have shown that Mtg16 -/- mice were more sensitive to chemically induced colonic injury than wild-type mice. Based on these observations and because little is known about the function of Mtg16 in the small intestine (SI), we hypothesized that MTG16
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contrast, MYPT1fl/fl/villin-CreERT2 mice were born in expected Mendelian proportions. At 6 weeks of age, MYPT1fl/fl/villin-CreERT2 and MYPT1fl/fl mice received daily tamoxifen injections. By the fifth injection, MYPT1fl/fl/villin-CreERT2, but not MYPT1fl/fl, mice developed rectal bleeding, weight loss, and lethargy. Histologic analyses demonstrated severe epithelial damage, including small intestinal villous loss and colonic ulceration, and dense mixed inflammatory infiltrates, indicating a critical homeostatic function of MYPT1. Further examination showed apical (brush border) membrane rounding, thickening and increased density of the perijunctional actomyosin ring, and retraction of epithelial lateral membranes. To separate primary epithelial effects of MYPT1 loss from those secondary to the mucosal immune response, intestinal stem cells were isolated from MYPT1fl/fl/villin-CreERT2 mice prior to tamoxifen treatment and grown as enteroids. In vitro treatment of MYPT1fl/fl/villinCreERT2 enteroids with tamoxifen resulted in epithelial cell death, while villin-CreERT2 enteroids were unaffected. Thus, MYPT1 loss activates cell-intrinsic epithelial death. CONCLUSIONS: These data demonstrate that MYPT1, and likely PP1cδ, are critical determinants of epithelial homeostasis. Further studies are required to determine whether the mechanisms that lead to epithelial death in vitro and severe enterocolitis in vivo following MYPT1 knockout.
as a transcription factor, Signal Transducer and Activator of Transcription 3 (STAT3) resides in the mitochondria of cells where phosphorylation at serine 727 (S727), but not tyrosine 705 (Y705), is required for optimal ETC activity and protects against stress-induced mitochondrial dysfunction. In this study, we explored whether PHB protects against mitochondrial dysfunction and apoptosis induced by pro-inflammatory cytokines via mitochondrial pS727-Stat3. Methods: Mitochondrial dysfunction, as indicated by increased mitochondrial reactive oxygen species (ROS) production, decreased ATP concentration, and reduced activity of ETC complexes, was induced in Caco2-BBE and IEC-6 intestinal epithelial cell lines by treatment with tumor necrosis factor α (TNFα), a pro-inflammatory cytokine involved in IBD that promotes cellular injury via mitochondrial damage. The effect of PHB overexpression on mitochondrial dysfunction and apoptosis was determined during TNFα treatment. The interaction of PHB with STAT3 was assessed by co-immunoprecipitation and immunofluorescent staining. To determine whether mitochondrial STAT3 is necessary for the protective effect of PHB on mitochondrial function, cells overexpressing PHB were transfected with a mitochondrial targeted pS727-STAT3 dominant negative (MLS-S727-STAT3dn) during TNFα treatment. Results: PHB overexpression protects against TNFα-induced mitochondrial dysfunction and apoptosis in cultured IECs. This protection was eliminated by expression of MLS-S727-STAT3dn. PHB co-immunoprecipitates and co-localizes with pS727-STAT3, but not pY705-STAT3, in the mitochondria of cultured IECs and in vivo in colonic epithelium from wild-type mice. PHB/pS727-STAT3 interaction is decreased in IECs during TNFαinduced mitochondrial stress due at least in part to decreased PHB expression. PHB overexpression maintains PHB/pS727-STAT3 interaction during TNFα treatment. Conclusions: Our data demonstrate a potential mechanistic link between PHB and mitochondrial STAT3 in the intestinal epithelium to prevent mitochondrial dysfunction, which is thought to participate in the pathogenesis of IBD. Reduced levels of PHB during IBD may be an underlying factor promoting mitochondrial dysfunction and apoptosis of the intestinal epithelium.
AGA Abstracts
regulates stem cell differentiation and injury responses in the SI. Baseline characterization of wildtype and Mtg16-/- SI revealed significantly decreased number of goblet cells (11.6 vs 14.9 PAS+ cells/crypt-villus, p=0.014), indicating a previously unrecognized role for Mtg16 in gut lineage allocation. Furthermore, Mtg16-/- mice have increased proliferation (6.6 vs 4.5 phospho-Histone H3+ cells/crypt-villus, p=0.011) but no differences in apoptosis (0.4 vs 0.5 TUNEL+ cells/cypt-villus, p=0.51). To assess intestinal injury responses, wildtype and Mtg16-/- mice were exposed to 12-Gy whole body radiation (WBR). Mtg16-/- mice had increased crypt viability (92.8% vs 77.9%, p<0.0001), and decreased apoptosis (1.4 vs 2.3 TUNEL+ cells/ crypt villus, p=0.001). To decipher the mechanism underlying this phenomenon, we looked for DNA damage in these cells by immunofluorescence staining for phospho-γH2AX foci, an early marker for DNA damage. We found fewer γH2AX+ foci in Mtg16-/- SI. Flow cytometry analysis using antibody to γH2AX confirmed decreased DNA damage in irradiated Mtg16-/- mice (15.1% vs 32.8%, p=0.003), indicating that loss of MTG16 impairs radiosensitivity. Since high doses of radiation are known to induce endothelial damage, we established enteroids from non-irradiated and irradiated wildtype and Mtg16-/mice to focus on the role of MTG16 in epithelial cell differentiation and response to radiation induced injury. Enteroids isolated from Mtg16-/- mice had higher plating efficiencies than those isolated from wildtype mice (43.3% vs 36.3%, p<0.05) before and after 12 Gy WBR (1.7% vs 0.7%, p<0.0001). We also found decreased efficiency in isolated crypts forming branched enteroids from non-irradiated Mtg16-/- mice (3.9% vs 23.9%, p<0.0001). In sum, our data provide evidence that loss of MTG16 impacts intestinal stem cell differentiation, and promotes radio-resistance. This might be due to shifting cellular response away from DNA damage-induced apoptosis and DNA repair after injury. These studies may have important implications for MTG16 being a diagnostic marker for radiation injury response and could lead to improved treatment modalities for patients receiving radiotherapy treatments.
real-time PCR with a set of primers within the MUC1, MUC2, and MUC13 promoters (ChiP). Results: Mice with conditional ablation of the Prep1 gene from the intestinal epithelium were viable. However, transgenic animals appeared smaller than wt, with a defective development of both the small and large intestine. VillinCRE/Prep1flox/flox mice showed an altered Goblet cell maturation and differentiation, resulting in MUC2 (soluble mucin) overproduction and MUC1/MUC13 (membrane-bound mucins) overexpression. Mutant mice were protected from DSS-induced colitis, as assessed by body weight loss, colitis activity Index, and histological scores. This correlated with reduced intestinal permeability (p<0,001), and bacterial penetration (p<0,05) within the inner mucus layer. Finally, Prep1 was find to be up-regulated in the intestinal epithelium of IBD patients, when compared to controls, and ChiP assay revealed that Prep1 modulate the transcription of the human MUC2, MUC1 and MUC13 genes, by directly binding to their promoters and acting as a repressor. Conclusion: Our findings demonstrate an essential role for Prep1 in intestinal development, epithelial homeostasis and maintenance of the mucosal barrier. Its manipulation, could indeed represent a novel therapeutic approach for the treatment of IBD, possibly restoring the mucin barrier. 138 ErbB4 Activation Protects Paneth Cells and Ameliorates Experimental Necrotizing Enterocolitis Steven J. McElroy, Shannon L. Castle, Jessica K. Bernard, Dana Almohazey, Catherine J. Hunter, Mark R. Frey Breastfed infants have a lower risk of developing necrotizing enterocolitis (NEC), a devastating intestinal disorder which affects up to 10% of premature infants and has a mortality of ~30%. While the protective factors in milk are not yet well-characterized, promising candidate molecules include growth factor ligands for ErbB receptor tyrosine kinases. Neuregulin-4 (NRG4) is an ErbB4-specific ligand that promotes cell survival in adult colonocytes; however, its role in the developing small intestine is uncharacterized. A common feature of NEC and other intestinal inflammatory diseases is Paneth cell (PC) loss; these cells provide antimicrobial peptides and also support the Lgr5+ intestinal stem cells, and are thus necessary for intestinal homeostasis and defense. In this study, we tested whether NRG4-ErbB4 signaling is protective in experimental NEC, and asked if it protects PCs during inflammation. METHODS: NRG4 was tested in an intestinal injury model which mimics human NEC-associated PC loss and intestinal infection. P14-16 C57Bl/6 mice were given i.p. injections of dithizone to ablate PCs, followed by Klebsiella pneumoniae gavage, to trigger NEC-like injury. Some mice received NRG4 (250μg/kg, i.p.) along with the dithizone. Effects of ErbB4 signaling on the small intestinal epithelium were tested in vitro with ileal enteroid cultures grown from ErbB4flox/ flox (functionally wild-type) and ErbB4flox/flox;Villin-Cre (intestinal epithelial-specific ErbB4 knockout) mice. Presence of the NRG4-ErbB4 signaling axis in developing human intestine was tested by immunoblot and immunohistochemistry on human milk and tissue samples collected from infants undergoing bowel resection. RESULTS: In the Dithizone/Klebsiella (DK) NEC model, ErbB4 activation by NRG4 administration reduced intestinal injury (66% decrease in histological damage score, p<0.05), and prevented DK-induced Paneth cell loss (mean PCs/crypt: control 2.80 +/- 0.26, DK 1.35 +/- 0.44, DK + NRG4 3.00 +/- 0.25). In vitro, cultured ErbB4-null ileal epithelial enteroids displayed a dysmorphic phenotype with fewer crypt buds than wild type, and had reduced RNA expression for PC (Lyz1, 57% reduction, p<0.05) and intestinal stem cell (Lgr5, 75% reduction, p<0.01) markers. Furthermore, ErbB4-null enteroids were highly sensitive to TNF, losing all crypt architecture after exposure. The potential clinical relevance of these findings was demonstrated by the observation that NRG4 and its receptor ErbB4 were detectable in human breast milk and developing human intestine, respectively. CONCLUSIONS: ErbB4 supports Paneth cells, promotes normal morphogenesis of the intestinal epithelium, and protects the intestinal epithelium from inflammatory insult. NRG4-ErbB4 signaling may contribute to the protective effects of breastfeeding, and represents a novel target pathway for therapeutic intervention in NEC.
136 PKD1 Functions Upstream of PKA in a Novel Pathway That Links GPCR Activation to β-Catenin Phosphorylation At Ser552 in Intestinal Epithelial Cells James Sinnett-Smith, Yang Ni, Steven H. Young, Enrique Rozengurt Background: Protein kinase D1 (PKD1) signaling stimulates intestinal epithelial cell proliferation, as revealed by using epithelial cells in culture and a novel PKD1 transgenic mouse model (J Biol Chem, 286, 511-520, 2011). Given that β-catenin phosphorylation at Ser552 promotes its dissociation from cell-cell contacts and stimulates its transcriptional activity, we examined whether GPCR/PKD1 activation stimulates β-catenin phosphorylation at this regulatory site. Results: Stimulation of intestinal epithelial IEC-18 cells with angiotensin II (ANG II), a mitogenic agonist that activates GPCRs endogenously expressed by these cells, induced a striking increase in the phosphorylation of β-catenin at Ser552 in IEC-18 cells which persisted for at least 24 h. In contrast, stimulation of IEC-18 with EGF induced transient Ser552 phosphorylation that declined to near baseline levels within 2 h. ANGIIinduced phosphorylation of β-catenin at Ser552 was completely prevented by cell exposure to PKD family inhibitors kb NB 142-70 or CRT006610 as well as by siRNA-mediated knockdown of PKD1 expression. These results implied that PKD1 plays a critical role in mediating GPCR-induced β-catenin phosphorylation at Ser552. Given that Ser552 does not reside within a PKD family consensus amino acid sequence, we hypothesized that PKD1 regulates β-catenin phosphorylation at this residue through other kinases implicated in Ser552 phosphorylation, including PKA and Akt. ANGII is known to induce transient Akt activation in IEC-18 cells but stimulates sustained β-catenin phosphorylation at Ser552, implying that GPCR activation induces phosphorylation at Ser552 through an Akt-independent pathway. In contrast, treatment of IEC-18 cells with H-89, an inhibitor of PKA, prevented β-catenin phosphorylation at Ser552 in response to ANGII in a dose-dependent manner, without impairing PKD1 activation, as scored by autophosphorylation at Ser916. These results suggested that PKA lies downstream of PKD1 in a novel pathway. In line with this hypothesis, stimulation with the adenylyl cyclase activator forskolin (FSK) and the phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) to increase cAMP/PKA, potently induced sustained β-catenin phosphorylation at Ser552 but did not induce PKD1 activation. Accordingly, the response to FSK+IBMX, in contrast to ANGII, was not blocked by kb NB 142-70 but abolished by H-89, an inhibitor of PKA but not PKD1. Using transgenic mice that express elevated PKD1 protein in intestinal epithelial cells, we found that over-expression of PKD1 was associated with enhanced β-catenin phosphorylation at Ser552, suggesting that the PKD1/ β-catenin pathway functions in vivo. Conclusion: Collectively, these results imply that PKD1 functions upstream of PKA in a novel pathway that links GPCRs to β-catenin phosphorylation at Ser552 in intestinal epithelial cells.
139 ATOH1 Transcriptional Targets in the Adult Intestine Yuan-Hung Lo, Maxime M. Mahe, Eunah Chung, Sunghee Oh, Joo-Seop Park, Noah F. Shroyer Background and Aims: Notch signaling has a critical function controlling the fate of intestinal progenitors by differentially regulating two opposing basic helix-loop-helix (bHLH) transcription factors, Atoh1 (atonal homolog1) and HES1 (hairy/enhancer of split1). Previous studies showed progenitors that escape Notch activation express high levels of Atoh1 and commit to a secretory lineage fate. These results indicated Atoh1 is a critical gatekeeper for the program of Notch-mediated differentiation and maturation of intestinal epithelial cells. Although some transcription factors downstream of Atoh1, such SPDEF and Gfi1, have been identified to specify differentiation and maturation of specific cell types, the bona fide targets of Atoh1 at the genome wide level in the adult intestine are still largely unknown. Here we aim to identify Atoh1 transcriptional signature in both distal small intestine and colon. Methods: Mice with an Atoh1-GFP fusion protein inserted into the Atoh1 locus (Atoh1GFP) were used in this study. In order to generate Atoh1 signature in adult intestines, the distal small intestinal crypts or colon crypts were isolated from 10~12 weeks old adult mice followed by H3K27ac, H3K27me3 as well as Atoh1 ChIP-sequencing. In parallel, RNAsequencing was performed on Atoh1 deficient crypts and normal crypts. Finally, the transcriptional profile of Atoh1 expressing cells in the crypts were generated by cell sorting GFP+ cells from Atoh1GFP intestines followed by RNA-sequencing. In order to confirm the potential direct targets of Atoh1 generated from our ChIP-sequencing and RNA-sequencing, ChIPqPCR was performed on both mouse colon crypts and human colon cancer cell lines, which transiently overexpress flag-tagged Atoh1. Results: In this study, we identified Atoh1 targets in the crypts of both distal small intestine and colon. As expected, Atoh1 shares a conserved DNA binding motif 5'-NCANNTG-3' of bHLH transcription factors. A refined matrix for Atoh1 DNA binding was defined by de novo motif analysis. Furthermore, DNA binding motifs for several other intestine-enriched transcription factors (e.g., SPDEF, GATA4, CDX2) were found within Atoh1-bound chromatin. Moreover, our ChIP-sequencing data showed Atoh1 binds to the promoter/enhancer regions marked by opened-chromatin marker H3K27ac, such as the promoter/enhancer of SPDEF and Gfi1, which are suggested to be
137 Conditional Deletion of Prep1 in the Intestinal Epithelium Alters Epithelial Homeostasis, Intestinal Development, and Controls Colitis Susceptibility Silvia D'Alessio, Carlotta Tacconi, Carmen Correale, Alessandro Gandelli, Marco Genua, Stefania Vetrano, Silvio Danese Background: Balanced and dynamic interactions among mucus layers, intestinal epithelial cells, and microbiota, are essential for the maintenance of the intestinal mucosal homeostasis. The disruption of this balance leads to a defective mucus barrier with increased permeability that results in intestinal inflammation. The homeodomain transcription factor, Prep1, is expressed in the post-mitotic differentiated intestinal epithelial cells and is essential in embryonic development. Here we report for the first time the involvement of Prep1 in intestinal epithelial homeostasis, and its functional role in experimental colitis. Methods: Mice lacking Prep1 in intestinal epithelium were generated by mating Prep1flox/flox mice with VillinCRE animals. Goblet cells staining was carried out by Alcian Blue and Periodic Acid-Schiff (PAS) staining, while mucins were quantified by real-time PCR and immunohistochemistry. The dextran sulfate sodium (DSS) model of colitis was used in VillinCRE/Prep1flox/ flox and wild-type (wt) mice. Moreover, Prep1 expression was evaluated in the intestinal epithelium of surgical specimens from normal individuals and patients with IBD, by Western Blot. Cross-linked, sonicated chromatin from human intestinal epithelial cells, was then immunoprecipitated with an anti-Prep1 antibody and the precipitated DNA analyzed by
AGA Abstracts
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