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Su1743 Estrogen Receptors Regulate Intestinal Epithelial Cell Proliferation and the Underlying Mechanisms
debate. Since in addition to enzymatic inhibition, change in COX-2 mRNA stability through inhibiting COX-2 N-glycosylation can also lead to COX-2 inhibition, we hypothesized that the addition of GS-HCl can reduce the dose of NSAID required for authentic action of NSAID because previously we reported that GS-HCl led to inhibition of COX-2 N-glycosylation and COX-2 protein turnover (Jang et al, JBC, 2007). Stimulation of IEC-6 with indomethacin resulted in significantly increased expressions of COX-2, iNOS, ICAM-1, VCAM-1, IL-8, and IL-1 as well as increased apoptosis, whereas the co-treatment of 5mM GS-HCl and indomethacin significantly decreased expressions of these inflammatory mediators as well as attenuated apoptosis relevant to significant inhibition of COX-2 N-glycosylation. Indomethacin-induced gastric ulcers were significantly attenuated in animal group co-treated with GS-HCl and lowered dose of indomethacin under the improved efficacy of collageninduced arthritis than high doses of indomethacin alone group. Our study provided the first insight that GS-HCl contributed to COX-2 weakening action through N-glycosylation inhibition, leading to novel strategy that combination of GS-HCl and lowered dosing of NSAID secured lesser GI toxicity under the similar COX inhibiting action. Su1743
Su1740
Estrogen Receptors Regulate Intestinal Epithelial Cell Proliferation and the Underlying Mechanisms Hui Dong, Chang-Whan Kim, Jeong Wook Kim, Jimmy Y. Chow, Biguang Tuo
Mucin Side Chain Sugar Residues Expression in Helicobacter pylori, NSAID, and Idiopathic Peptic Ulcers Yaron Niv, Doron Boltin, Marisa Halpern, Miriam Cohen, Zohar Levi, Alex Vilkin, Sara Morgenstern, Samuel B. Ho
Background and Aims: We recently reported that estrogen and estrogen receptors (ERs) play an important role in human intestinal mucosal protection and epithelial cell homeostasis; however, the underlying mechanisms are largely unknown. We therefore sought to investigate the cellular and molecular mechanisms by which estrogen and ERs protect human intestine and regulate epithelial cell proliferation. Methods: SCBN cell, an intestinal epithelial crypt cell line, and CHO cells stably expressing Na+/Ca2+ exchanger (NCX1-CHO) were used. 17b-estradiol (E2, 1-10 nM) was used as a ERs agonist. Gramicidin (1 ug/ml) or ouabain (1 uM) was used to raise intracellular Na+ concentrations and stimulate the Ca2+ entry mode of NCX1. RT-PCR and Western blot analysis were used to detect expression of ERs, NCX1 and CFTR. Cytosolic free Ca2+ concentration ([Ca2+]cyt) in single cells was measured with a digital cell imaging system and XTT assay was applied to study cell proliferation. Results: The expression of ER subtypes (ERa and ERb) was detected by RT-PCR and Western blot analysis in SCBN cells. E2 at 1-10 nM significantly suppressed cell proliferation for 24 and 48 h (p<0.001, n=10); however, progesterone at 1-10 nM did not significantly affect cell proliferation. E2-induced suppression of cell proliferation was reversed by CFTRinh172 (10 uM). The expression of CFTR and NCX1 were confirmed in SCBN cells. To further elucidate the mechanisms underlying E2-induced suppression of cell proliferation, NCX1CHO cells were used. After intracellular Na+ concentrations were raised by gramicidin (1 ug/ml) or ouabain (1 uM) to stimulate the Ca2+ entry mode of NCX1, [Ca2+]cyt was increased in NCX1-CHO cells but not in control CHO cells (n=50 cells for each group, p<0.001). Similarly, either gramicidin or ouabain increased proliferation of NCX1-CHO cells (n=5, p<0.05), but not control CHO cells (n=5, p>0.05). Interestingly, gramicidininduced proliferation of NCX1-CHO cells was significantly suppressed by E2 (n=5, p<0.05). Conclusions: ERs are functionally expressed in intestinal epithelial cells to regulate cell proliferation. Our findings not only reveal the novel roles for CFTR and NCX1 in regulating intestinal epithelial cell proliferation but also suggest these molecules may be involved in ERs-suppressed epithelial cell proliferation that is associated with intestinal inflammation and carcinogenesis.
Introduction: Idiopathic peptic ulcer is rising in the Western world, and may have a different etiology and pathogenesis than H, pylori or NSAID. The main protective factor of the gastric mucosa is the mucus unstirred layer. Change in mucin expression may lead to ulcer formation. Aim: To determine the expression pattern of mucin's type 2 backbone structure and sialic acid residues in H. pylori, NSAID, and idiopathic-gastric ulcers. Methods: We randomly selected 92 patients with H. pylori (Group 1, n=30), NSAID (Group 2, n=18), combined H. pylori and NSAID associated gastric ulcers (Group 3, n=24), and patients with idiopathic gastric ulcers (Group 4, n=20). ECA (specific for Neu5Acα2-6gal/GalNAc) and SNA (specific for Galβ1-4GlcNAc, type 2 backbone structure exposed after removal of sialic acid), lectin staining performed on sections of the mucosa from the ulcer margins. Inflammation score was assessed according to the Sidney system. Results: Bleeding and mortality rates were significantly higher in patients with idiopathic ulcer. The expression of sialic acid residues stained by SNA lectin was higher in these patients, Group 4, than in Group 1, reaching significance in the foveola (P < 0.0001, and P = 0.004, respectively). EMA staining intensity was significantly higher in the foveola than in the glands, 13.49±3.08 versus 5.71±4.72 (P < 0.0001), with no difference between Group 1 and Group 4. Conclusion: This observation might be important, since increased number of sialic acid residues may decrease the protection efficiency against acid, pepsin, toxic material or bacterial adhesion. Su1741 Klotho, an Aging Suppressor, and Fibroblast Growth Factor 23 is Involved in Gastric Ulcer Healing Tetsuya Tanigawa, Toshio Watanabe, Yuji Nadatani, Koji Otani, Hirohisa Machida, Hirotoshi Okazaki, Hirokazu Yamagami, Kenji Watanabe, Kazunari Tominaga, Yasuhiro Fujiwara, Tetsuo Arakawa
Su1744
Background and Aim: The klotho gene was originally identified as an aging suppressor gene, which extends lifespan when overexpressed and accelerates the development of aginglike phenotypes when disrupted in mice. Klotho protein is involved in modulation of mineral metabolism and aging, and recent researches revealed that Klotho functions as a humoral factor with pleiotropic activities. Klotho protein forms a complex with fibroblast growth factor (FGF) receptors and functions as an obligate co-receptor for FGF23. In this study, we investigated whether Klotho and FGF23 is involved in the mechanism of gastric ulcer healing. Materials and Methods: Experimental gastric ulcers were induced in C57BL/6J mice by focal serosal application of 60% acetic acid for 30 sec. Mouse recombinant Klotho (10 and 20 μg/kg body weight/day) or vehicle (PBS) was administered intraperitoneally to the mice with ulcers from day 0 (3 days after inducing ulceration) to day 7. On day 4 and 7, the stomachs were removed and the ulcer size was measured; the ulcer size was expressed in the form of an ulcer index (the product of maximum length and minimum length of an ulcer). Expression of mRNA for FGF23 and Klotho in normal gastric tissue and ulcer tissue was determined by real-time quantitative RT-PCR. Results: Expression of mRNA for FGF23 was almost undetectable in normal gastric mucosa and it was markedly induced in ulcer tissue. Expression of mRNA for Klotho in gastric epithelial cells at the ulcer margin was reduced by 90% compared with normal gastric tissue. Expression of mRNA for Klotho was hardly detectable in ulcer bed. Administration of recombinant Klotho accelerated gastric ulcer healing [ulcer index: 8.25 ± 2.46 mm2 in vehicle-treated group vs 3.81 ± 0.66 mm2 in Klotho (10 μg/kg body weight/day)-treated group and 4.38 ± 1.09 mm2 in Klotho (20 μg/kg body weight/day)-treated group]. Conclusions: Our findings suggest that Klotho promotes gastric ulcer healing.
Some Water-Soluble Vitamins are Likely to Be Deficient at the Time of Peptic Ulcer Development: Time-Course Changes in Blood Concentrations of WaterSoluble Vitamins Wataru Sato, Kazumasa Miyake, Hiroyuki Nagoya, Yasuhiro Kodaka, Tomotaka Shindo, Nobue Ueki, Masafumi Kusunoki, Tetsuro Kawagoe, Seiji Futagami, Katya Gudis, Choitsu Sakamoto [Background and aim] Unlike fat-soluble vitamins (V), water-soluble V have a metabolic feature. Water-soluble V are relatively easy to become depleted because they are unstable, excreted readily in urine, and difficult to be stored in the body. Lack of water-soluble V which have an antioxidant action can be one of the causative factors of the onset of peptic ulcer (PU), though the relationship between PU development and such V is not clear yet. Of the water-soluble V B6 and V B12 and folic acid are involved in the metabolism of homocysteine which can cause arteriosclerosis and phlebothrombosis. In other words, homocysteine concentrations in blood may be an indicator of such vitamin deficiency and arteriosclerosis. Therefore, in this study, lack of water-soluble V in PU development was evaluated by measuring blood concentrations of water-soluble V and homocysteine in patients with hemorrhagic PU. [Methods] This prospective study included the patients who needed hospitalization for the treatment of PU. Infusion using a vitamin preparation was not performed during hospitalization. Fasting blood concentrations of water-soluble V (V B1,V B2, V B6, V B12, V C, and folic acid) and homocysteine were measured at 3 time points (at the time of admission and hospital discharge and 3 months after discharge). Of the 20 consecutive patients who met the inclusion criteria, 10 patients who completed measurements at 3 time points were analyzed. All of these 10 patients were male. [Results] Patients were 3 with gastric ulcer and 7 with duodenal ulcer, with an average age of 58.4±15.1 years, and with an average hospitalization period of 13.7±6.1 days. Scarring of ulcers and biopsy results confirmed no malignant findings in all 10 patients at 3 months after hospital discharge. Mean blood concentrations of V B1, V B2, V B6, and V C at the time of admission were at the lower limit of normal. Time-specific effects had been confirmed by one-way repeatedmeasures analysis of variance in mean concentrations of V B1, V B2 and V B6 (P< 0.05), but not V B12, V C, folic acid and homocysteine, Mean V B2 concentration at the time of hospital discharge was significantly higher compared with at the time of admission (P<0.05). Furthermore, mean concentrations of V B1, V B2, and V B6 significantly increased 3 months after discharge compared with at the time of admission (P<0.05). Moreover, mean concentrations of V B1 and V B6 significantly increased 3 months after discharge compared with at the time of discharge (P<0.05). [Conclusion] Since V B1, V B2, and V B6 appear
Su1742 NSAID-Sparing Effect of Glucosamine Hydrochloride Through Preventing COX-2 N-Glycosylation Secures Lesser GI Toxicity Under the Warranty of NSAID Action Hua Hong, Yoon Jae Kim, Eun-Hee Kim, Young-Min Han, Ki-Baik Hahm Though non-steroidal antiinflammatory drug (NSAID) has been prescribed for pain alleviation as well as potent anti-inflammation popularly, the notorious GI toxicity limits its general usage, after which “Coxib” has been invented to guarantee GI safety. Though glucosamine (GS), an agent composed of an amino monosaccharide, has been widely used for the alternative regimen of arthritis, their clinical implication in this matter has been under
S-493
AGA Abstracts
AGA Abstracts
esophageal epithelium. Design Gene expression microarray was used to survey gene expression in the esophagus at three critical stages, specification, metaplasia and maturation. The esophagi were isolated from wild-type, Nrf2-/-, Keap1-/-, or Nrf2-/-;Keap1-/- embryos or young adults. Array data were statistically analyzed for differentially expressed genes and pathways. Histochemical and immunohistochemical staining were used to verify potential involvement of the Wnt pathway, PPARβ/δ and the PI3K/Akt pathway in the development of esophageal epithelium. Results Dynamic gene expression pattern accompanied the morphological changes of the developing esophagus at the critical stages. Particularly, the Nrf2/ Keap1 pathway had a baseline activity in the metaplasia phase and was further activated in the maturation phase. The Wnt pathway was active early and became inactive later in the metaplasia stage. In addition, global transcript profiling of the esophagus isolated from Keap1-/- mice showed increased expression of Nrf2 downstream targets and genes involved in keratinization. Microarray data also suggested that esophageal hyperkeratosis in the Keap1-/- mice was due to activation of PPARβ/δ and the PI3K/Akt pathway. Conclusions Morphological changes of the esophageal epithelium are associated with dynamic changes in gene expression. The activity of Nrf2/Keap1 pathway is required for the maturation of the esophagus.
Su1740
Estrogen Receptors Regulate Intestinal Epithelial Cell Proliferation and the Underlying Mechanisms Hui Dong, Chang-Whan Kim, Jeong Wook Kim, Jimmy Y. Chow, Biguang Tuo
Mucin Side Chain Sugar Residues Expression in Helicobacter pylori, NSAID, and Idiopathic Peptic Ulcers Yaron Niv, Doron Boltin, Marisa Halpern, Miriam Cohen, Zohar Levi, Alex Vilkin, Sara Morgenstern, Samuel B. Ho
Background and Aims: We recently reported that estrogen and estrogen receptors (ERs) play an important role in human intestinal mucosal protection and epithelial cell homeostasis; however, the underlying mechanisms are largely unknown. We therefore sought to investigate the cellular and molecular mechanisms by which estrogen and ERs protect human intestine and regulate epithelial cell proliferation. Methods: SCBN cell, an intestinal epithelial crypt cell line, and CHO cells stably expressing Na+/Ca2+ exchanger (NCX1-CHO) were used. 17b-estradiol (E2, 1-10 nM) was used as a ERs agonist. Gramicidin (1 ug/ml) or ouabain (1 uM) was used to raise intracellular Na+ concentrations and stimulate the Ca2+ entry mode of NCX1. RT-PCR and Western blot analysis were used to detect expression of ERs, NCX1 and CFTR. Cytosolic free Ca2+ concentration ([Ca2+]cyt) in single cells was measured with a digital cell imaging system and XTT assay was applied to study cell proliferation. Results: The expression of ER subtypes (ERa and ERb) was detected by RT-PCR and Western blot analysis in SCBN cells. E2 at 1-10 nM significantly suppressed cell proliferation for 24 and 48 h (p<0.001, n=10); however, progesterone at 1-10 nM did not significantly affect cell proliferation. E2-induced suppression of cell proliferation was reversed by CFTRinh172 (10 uM). The expression of CFTR and NCX1 were confirmed in SCBN cells. To further elucidate the mechanisms underlying E2-induced suppression of cell proliferation, NCX1CHO cells were used. After intracellular Na+ concentrations were raised by gramicidin (1 ug/ml) or ouabain (1 uM) to stimulate the Ca2+ entry mode of NCX1, [Ca2+]cyt was increased in NCX1-CHO cells but not in control CHO cells (n=50 cells for each group, p<0.001). Similarly, either gramicidin or ouabain increased proliferation of NCX1-CHO cells (n=5, p<0.05), but not control CHO cells (n=5, p>0.05). Interestingly, gramicidininduced proliferation of NCX1-CHO cells was significantly suppressed by E2 (n=5, p<0.05). Conclusions: ERs are functionally expressed in intestinal epithelial cells to regulate cell proliferation. Our findings not only reveal the novel roles for CFTR and NCX1 in regulating intestinal epithelial cell proliferation but also suggest these molecules may be involved in ERs-suppressed epithelial cell proliferation that is associated with intestinal inflammation and carcinogenesis.
Introduction: Idiopathic peptic ulcer is rising in the Western world, and may have a different etiology and pathogenesis than H, pylori or NSAID. The main protective factor of the gastric mucosa is the mucus unstirred layer. Change in mucin expression may lead to ulcer formation. Aim: To determine the expression pattern of mucin's type 2 backbone structure and sialic acid residues in H. pylori, NSAID, and idiopathic-gastric ulcers. Methods: We randomly selected 92 patients with H. pylori (Group 1, n=30), NSAID (Group 2, n=18), combined H. pylori and NSAID associated gastric ulcers (Group 3, n=24), and patients with idiopathic gastric ulcers (Group 4, n=20). ECA (specific for Neu5Acα2-6gal/GalNAc) and SNA (specific for Galβ1-4GlcNAc, type 2 backbone structure exposed after removal of sialic acid), lectin staining performed on sections of the mucosa from the ulcer margins. Inflammation score was assessed according to the Sidney system. Results: Bleeding and mortality rates were significantly higher in patients with idiopathic ulcer. The expression of sialic acid residues stained by SNA lectin was higher in these patients, Group 4, than in Group 1, reaching significance in the foveola (P < 0.0001, and P = 0.004, respectively). EMA staining intensity was significantly higher in the foveola than in the glands, 13.49±3.08 versus 5.71±4.72 (P < 0.0001), with no difference between Group 1 and Group 4. Conclusion: This observation might be important, since increased number of sialic acid residues may decrease the protection efficiency against acid, pepsin, toxic material or bacterial adhesion. Su1741 Klotho, an Aging Suppressor, and Fibroblast Growth Factor 23 is Involved in Gastric Ulcer Healing Tetsuya Tanigawa, Toshio Watanabe, Yuji Nadatani, Koji Otani, Hirohisa Machida, Hirotoshi Okazaki, Hirokazu Yamagami, Kenji Watanabe, Kazunari Tominaga, Yasuhiro Fujiwara, Tetsuo Arakawa
Su1744
Background and Aim: The klotho gene was originally identified as an aging suppressor gene, which extends lifespan when overexpressed and accelerates the development of aginglike phenotypes when disrupted in mice. Klotho protein is involved in modulation of mineral metabolism and aging, and recent researches revealed that Klotho functions as a humoral factor with pleiotropic activities. Klotho protein forms a complex with fibroblast growth factor (FGF) receptors and functions as an obligate co-receptor for FGF23. In this study, we investigated whether Klotho and FGF23 is involved in the mechanism of gastric ulcer healing. Materials and Methods: Experimental gastric ulcers were induced in C57BL/6J mice by focal serosal application of 60% acetic acid for 30 sec. Mouse recombinant Klotho (10 and 20 μg/kg body weight/day) or vehicle (PBS) was administered intraperitoneally to the mice with ulcers from day 0 (3 days after inducing ulceration) to day 7. On day 4 and 7, the stomachs were removed and the ulcer size was measured; the ulcer size was expressed in the form of an ulcer index (the product of maximum length and minimum length of an ulcer). Expression of mRNA for FGF23 and Klotho in normal gastric tissue and ulcer tissue was determined by real-time quantitative RT-PCR. Results: Expression of mRNA for FGF23 was almost undetectable in normal gastric mucosa and it was markedly induced in ulcer tissue. Expression of mRNA for Klotho in gastric epithelial cells at the ulcer margin was reduced by 90% compared with normal gastric tissue. Expression of mRNA for Klotho was hardly detectable in ulcer bed. Administration of recombinant Klotho accelerated gastric ulcer healing [ulcer index: 8.25 ± 2.46 mm2 in vehicle-treated group vs 3.81 ± 0.66 mm2 in Klotho (10 μg/kg body weight/day)-treated group and 4.38 ± 1.09 mm2 in Klotho (20 μg/kg body weight/day)-treated group]. Conclusions: Our findings suggest that Klotho promotes gastric ulcer healing.
Some Water-Soluble Vitamins are Likely to Be Deficient at the Time of Peptic Ulcer Development: Time-Course Changes in Blood Concentrations of WaterSoluble Vitamins Wataru Sato, Kazumasa Miyake, Hiroyuki Nagoya, Yasuhiro Kodaka, Tomotaka Shindo, Nobue Ueki, Masafumi Kusunoki, Tetsuro Kawagoe, Seiji Futagami, Katya Gudis, Choitsu Sakamoto [Background and aim] Unlike fat-soluble vitamins (V), water-soluble V have a metabolic feature. Water-soluble V are relatively easy to become depleted because they are unstable, excreted readily in urine, and difficult to be stored in the body. Lack of water-soluble V which have an antioxidant action can be one of the causative factors of the onset of peptic ulcer (PU), though the relationship between PU development and such V is not clear yet. Of the water-soluble V B6 and V B12 and folic acid are involved in the metabolism of homocysteine which can cause arteriosclerosis and phlebothrombosis. In other words, homocysteine concentrations in blood may be an indicator of such vitamin deficiency and arteriosclerosis. Therefore, in this study, lack of water-soluble V in PU development was evaluated by measuring blood concentrations of water-soluble V and homocysteine in patients with hemorrhagic PU. [Methods] This prospective study included the patients who needed hospitalization for the treatment of PU. Infusion using a vitamin preparation was not performed during hospitalization. Fasting blood concentrations of water-soluble V (V B1,V B2, V B6, V B12, V C, and folic acid) and homocysteine were measured at 3 time points (at the time of admission and hospital discharge and 3 months after discharge). Of the 20 consecutive patients who met the inclusion criteria, 10 patients who completed measurements at 3 time points were analyzed. All of these 10 patients were male. [Results] Patients were 3 with gastric ulcer and 7 with duodenal ulcer, with an average age of 58.4±15.1 years, and with an average hospitalization period of 13.7±6.1 days. Scarring of ulcers and biopsy results confirmed no malignant findings in all 10 patients at 3 months after hospital discharge. Mean blood concentrations of V B1, V B2, V B6, and V C at the time of admission were at the lower limit of normal. Time-specific effects had been confirmed by one-way repeatedmeasures analysis of variance in mean concentrations of V B1, V B2 and V B6 (P< 0.05), but not V B12, V C, folic acid and homocysteine, Mean V B2 concentration at the time of hospital discharge was significantly higher compared with at the time of admission (P<0.05). Furthermore, mean concentrations of V B1, V B2, and V B6 significantly increased 3 months after discharge compared with at the time of admission (P<0.05). Moreover, mean concentrations of V B1 and V B6 significantly increased 3 months after discharge compared with at the time of discharge (P<0.05). [Conclusion] Since V B1, V B2, and V B6 appear
Su1742 NSAID-Sparing Effect of Glucosamine Hydrochloride Through Preventing COX-2 N-Glycosylation Secures Lesser GI Toxicity Under the Warranty of NSAID Action Hua Hong, Yoon Jae Kim, Eun-Hee Kim, Young-Min Han, Ki-Baik Hahm Though non-steroidal antiinflammatory drug (NSAID) has been prescribed for pain alleviation as well as potent anti-inflammation popularly, the notorious GI toxicity limits its general usage, after which “Coxib” has been invented to guarantee GI safety. Though glucosamine (GS), an agent composed of an amino monosaccharide, has been widely used for the alternative regimen of arthritis, their clinical implication in this matter has been under
S-493
AGA Abstracts
AGA Abstracts
esophageal epithelium. Design Gene expression microarray was used to survey gene expression in the esophagus at three critical stages, specification, metaplasia and maturation. The esophagi were isolated from wild-type, Nrf2-/-, Keap1-/-, or Nrf2-/-;Keap1-/- embryos or young adults. Array data were statistically analyzed for differentially expressed genes and pathways. Histochemical and immunohistochemical staining were used to verify potential involvement of the Wnt pathway, PPARβ/δ and the PI3K/Akt pathway in the development of esophageal epithelium. Results Dynamic gene expression pattern accompanied the morphological changes of the developing esophagus at the critical stages. Particularly, the Nrf2/ Keap1 pathway had a baseline activity in the metaplasia phase and was further activated in the maturation phase. The Wnt pathway was active early and became inactive later in the metaplasia stage. In addition, global transcript profiling of the esophagus isolated from Keap1-/- mice showed increased expression of Nrf2 downstream targets and genes involved in keratinization. Microarray data also suggested that esophageal hyperkeratosis in the Keap1-/- mice was due to activation of PPARβ/δ and the PI3K/Akt pathway. Conclusions Morphological changes of the esophageal epithelium are associated with dynamic changes in gene expression. The activity of Nrf2/Keap1 pathway is required for the maturation of the esophagus.