subepithelial follicules showed tight correlation with the severity of inflammation (r = 0.9). The number of EGFR and HGFR positive subepithelial cells was found to be significantly elevated in severe (21.6±2.1%/21.3±1.9%), moderate (14.3±1.7%/14.6±1.6%) and mild (7.2±1.6%/7.4±1.3%) inflammation compared to healthy colon mucosa (2.6±1.4%/ 2.4±1.03%) (p<0.005). IGFR1 immunoreactive cells were only found in a trace number in the subepithelial layer in all cases. Low number of CDX2 and CK positive subepithelial cells were also found in ILFs, and their increase in number was in correlation (r=0.8) with the severity of inflammation. RT-PCR validated the immunohistochemical results. CONCLUSION: EGFR/HGFR positive subepithelial cells showing numerical correlation with the activity of inflammation in ulcerative colitis may indicate their involvement in regenerative mucosal processes. The presence of CDX2 or CK positive subepithelial cells suggest that the mesenchymal-epithelial transition may be located to ILFs.
M1711
In response to various stressful conditions, rapid changes in gene expression patterns in intestinal epithelial cells (IECs) control cell division and survival, thereby preserving the epithelial homeostasis. c-Myc is a nuclear transcriptional factor and plays an important role in the regulation of gut mucosal growth and repair after injury. Besides transcriptional regulation, c-Myc expression is also tightly controlled at the translational level, but its exact mechanism remains elusive. The RNA-binding protein CUG-BP1 binds to GC-rich sequences of mRNAs and regulates translation of its target transcripts. Stress granules (SGs) are cytoplasmic aggregates of stalled translational preinitiation complexes during stress and have emerged as important players in the posttranscriptional regulation of gene expression. The current study determines if CUG-BP1 regulates c-Myc translation by enhancing c-Myc mRNA translocation into SGs, thus modulating IEC growth. Methods: Studies were conducted in IEC-6 cells, derived from rat intestinal crypts. CUG-BP1 binding to c-Myc mRNA was examined by biotin RNA pull-down assays and ribonucleoprotein immunoprecipitation analysis. c-Myc translation was measured by polyribosomal profile analysis and chimeric cMyc ARE luciferease reporter gene activity. Co-localization of c-Myc mRNA with SGs was examined by in situ hybridization and immunofluorescence staining. Functions of CUGBP1 were investigated by its gene silencing and overexpression. Results: Ectopic CUG-BP1 overexpression increased [CUG-BP1/c-Myc mRNA] complex, repressed c-Myc translation (by ~51%), decreased c-Myc protein level (by ~60%), and inhibited IEC proliferation (by ~35%). In contrast, CUG-BP1 silencing reduced CUG-BP1 association with the c-Myc mRNA, promoted c-Myc translation, and increased c-Myc protein level (by ~5-fold), thus stimulating IEC growth. Interestingly, increased CUG-BP1 also enhanced c-Myc mRNA translocation into SGs, whereas CUG-BP1 silencing reduced c-Myc mRNA levels in SGs. Furthermore, increased levels of endogenous CUG-BP1 protein by depletion of cellular polyamines also enhanced the recruitment of c-Myc mRNA into SGs and repressed c-Myc translation, which was associated with a reduction in IEC growth. CUG-BP1 silencing in polyamine-deficient cells not only prevented increased levels of c-Myc mRNA in SGs but also promoted c-Myc translation, leading to an increase in c-Myc protein and cell proliferation. Conclusion: These results indicate that CUG-BP1 represses c-Myc translation by increasing accumulation of cMyc mRNA in SGs, thus inhibiting intestinal epithelial cell growth.
M1709 Analysis of Proliferative/Apoptotic Ratio, Mitotic Index and Apoptotic Index in Histologically Intact Children Colonic Biopsy Samples and Colorectal Adenoma-Dysplasia-Carcinoma Sequence in Adults Katalin Leiszter, Ferenc Sipos, Orsolya Galamb, Tibor Krenács, Sándor Spisák, Gábor Veres, András Kiss, Barnabas Wichmann, Kinga Tóth, Gabor Valcz, Alexandra Kalmar, Ottó Jász, Béla Molnár, Zsolt Tulassay Background: The reformation of colonic epithelium takes 3-5 days. The imbalance of epithelial proliferation and apoptosis may result in age-related gastrointestinal alterations like sporadic colorectal cancer whom incidence increases over age of 40. Aims: Our aims were the determination and comparison of proliferative/apoptotic ratio (PAR), mitotic index (MI) and apoptotic index (AI) in healthy samples from children and in colorectal adenoma-dysplasiacarcinoma sequence (ADCS). Materials and methods: Colonic biopsies from 10 healthy adults and 14 healthy children, 10 colorectal adenomas, 10 colorectal cancers were collected, fixed in formalin and embedded in paraffin. The proliferating cell specific Ki-67 monoclonal antibody was used and labelled with rodamin. Apoptotic cells were detected with TUNEL method using green fluorescent labeled nucleotides (FITC); Hoechst nuclear co-staining was done. After digital scanning slides were analysed with digital microscope; one-way ANOVA test was performed for data analysis of paired sample groups. Results: The PAR and MI were significantly higher in histologically intact children colonic samples (PAR=3,52±2,49; MI=0,34±0,07) compared to healthy adults (PAR=0,89±0,23; MI=0,15±0,06) (p<0,05), and they showed continuous increase with the colorectal ADCS. The PAR and MI were significantly higher in histologically intact children colonic samples and colorectal cancer samples (PAR= 9,83±7,72; MI=0,42±0,11) compared to healthy adult samples (p<0,05). The highest PAR and MI were found in colorectal cancer samples. The AI was lower in healthy children samples (0,13±0,06) compared to histologically intact adult colonic samples (0,17±0,05) and showed continuous decrease with the colorectal ADCS. The lowest AI was found in colorectal cancer samples (0,06±0,04). Conclusions: The epithelial cell proliferation increased and the apoptosis decreased in histologically intact children colonic samples compared to histologically intact adult colonic samples, but in contrast to colorectal cancer it can be a well-regulated, well-balanced regenerative process. In adults the given steps of colorectal ADCS may be characterised by using PAR, MI and AI.
M1712 The Role of α-Gustducin in the Effect of Bitter Taste Receptor Agonists on Ghrelin Secretion and Food Intake Sara Janssen, Pieter-Jan Verhulst, Jan F. Tack, Inge Depoortere Background: Circulating ghrelin levels constitute a hunger signal to the hypothalamus. The nutritional status influences ghrelin secretion from specialized endocrine cells but the factors involved in chemosensation of the ghrelin cell are not known. It is known that specific G proteins, α-gustducin and α-transducin, mediate bitter and sweet gustatory signals in the intestine. This study aimed to investigate whether bitter taste receptor agonists (T2R) can affect ghrelin secretion via α-gustducin. Methods: The co-localization between ghrelin and α-gustducin or α-transducin was investigated by double labeling immunofluorescence on sections of the mouse corpus. Fasted wild-type (α-gust+/+) and α-gustducin knockout (αgust-/-) mice were gavaged with a mixture of T2R agonists (denatonium benzoate (10mM), phenylthiocarbamide (10mM), 6-propyl-2-thiouracil (5mM), quinine (1,5mM), D-[-]salicin (5mM)). Blood samples were taken at 0 and 40 min after gavage and plasma ghrelin levels were determined by radioimmunoassay. Food intake after oral administration of T2R agonists was followed and mRNA expression of NPY, AgRP, ghrelin and POMC in the hypothalamus was measured by real time-PCR. Results: α-gustducin IR cells are located in brush cells and endocrine cells but only the latter co-localized to some extent with ghrelin. However, the majority of α-transducin IR cells co-localized with ghrelin. Oral gavage of T2R agonists increased plasma octanoyl ghrelin levels by 112±30% in α-gust+/+ mice. In α-gust-/- mice the effect was significantly lower (71±16%). Plasma total ghrelin levels were increased to a similar extent in both genotypes (α-gust+/+: 56±6%, α-gust-/- : 52±8%). The most effective T2R agonist to stimulate octanoyl ghrelin was phenylthiocarbamide (182%), the least effective was salicin (27%). Food intake was increased from 1.35±0.09 to 1.62±0.06 g/h during the first 30 min after gavage of T2R agonists in α-gust+/+ mice but not in α-gust-/- mice (from 1.28±0.10 to 1.19±0.05 g/h). Simultaneously, the mRNA expression of AgRP was significantly increased by 49% in the hypothalamus of α-gust+/+, but not of α-gust-/- mice. NPY expression tended to be increased (13%) in α-gust+/+ mice but was decreased (-22%) in α-gust-/- mice. The expression of POMC and ghrelin mRNA was not affected in both genotypes. Conclusion: The increase in plasma octanoyl ghrelin levels after gavage of bitter taste receptor agonists is partially mediated via α-gustducin but may also involve α-transducin. The accompanied temporary increase in food intake and the higher expression of orexigenic peptides in the hypothalamus occurs via α-gustducin and probably involves octanoyl ghrelin.
M1710 Vitamins B Deficiency During Gestation and Suckling Periods Induces Growth Retardation Related With Atrophic Gatritis and Altered Ghrelin Expression in the Rat Pup Carine Pourie, Gregory Pourie, Jean-Luc Daval, Catherine Tomasetto, Violette Koziel, Laurent Peyrin Biroulet, Marie Christine Rio, Jean-Louis Gueant, Bernard Beck Methyl donor deficiency (MDD) during pregnancy induces intrauterine growth retardation, a characteristic at risk of obesity and metabolic syndrome, according to the fetal programming hypothesis. Whether the gastric endocrine and exocrine secretions are involved in fetal programming is poorly documented. We showed recently that the growth retardation of MDD rat pups was related to an altered expression of grhelin in the pit region of oxyntic glands and to a subsequent decreased release in plasma. We aimed to further evaluate the morphological and functional alteration of the stomach of MDD pups. We studied the gastric oxyntic mucosa cellular organization in weanling rat pups of dams deprived of choline, folate and B12 during gestation and suckling periods, by using specific markers and approaches (Periodic Acid Schiff, bromodeoxyuridine, homocysteine, TUNEL, immunostaining, RT-PCR…). Deficient pups were smaller and weighed less (p<0.001) than control pups. MDD induced an increase in plasma and tissue homocysteine and a decrease of plasma folate and vitamin B12. At weaning, their gastric oxyntic mucosa was characterized by loss of cell polarity, anarchic cell migration, abnormal progenitor differentiation, apoptosis, and signs of surface layer erosion. Moreover, pups had atrophic glands with signs of surface layer erosion and inflammation as illustrated by homocysteine, TFF1, mucins, Cox-2 and TIMP1 increased expression. In MDD rats, the zymogenic cells producing intrinsic factor were less numerous in the base of the oxyntic glands whereas a very strong staining appeared in the pit region of the gland. The same pit region was affected by changes in cell interactions due to loss of beta-catenin and E-cadherin expression. Taken together, our results indicated that beside its deleterious effect on grhelin expression, the MDD induced gastric mucosal inflammation and cell surface layer erosion. TIMP 1, Cox-2 and TFF 1 were activated whereas cell junctions were modified in order to influence rapid cell migration, healing and restitution of mucosa integrity. The remodelling of gastric cellular organisation led also to altered expression of intrinsic factor, the key protein for vitamin B12 absorption.
M1713 Mechanisms of Crosstalk Between Intestinal EC and L Cells: the Roles of Serotonin and Glucagon-Like Peptide 1 in Regulating Neuroendocrine Cell Function Alexander D. Kazberouk, Francesco Giovinazzo, Andrew Timberlake, Betty De Smet, Roswitha Pfragner, Irvin Modlin, Mark Kidd Background: Serotonin (5-HT)-secreting enterochromaffin (EC) cells and GLP-1 secreting L cells are chemosensory enteroendocrine cells that colocalize in the duodenum and terminal ileum of the GI tract. As both cell types play key roles in regulating the response of the GI tract to the contents of the lumen (i.e. motility, islet secretion), we hypothesized that they interacted at a secretory product/receptor level in much the same fashion as gastrin G cells/ histamine ECL cells in the regulation of acid secretion. We therefore evaluated the receptor
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AGA Abstracts
AGA Abstracts
RNA-Binding Protein Cug-Bp1 Represses c-MYC Translation by Recruiting Its mRNA Into Stress Granules Inhibiting Intestinal Epithelial Cell Growth Lan Liu, Jaladanki N. Rao, Tongtong Zou, Lan Xiao, Tingxi Yu, Douglas J. Turner, Myriam Gorospe, Jian-Ying Wang