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The Colonic Surface Goblet Cells Secrete Mucus Important for Proper Protective Mucus Layer Formation
AGA Abstracts
lymphocyte count compared to vehicle-receiving controls (0.21 ± 0.08 x 103 cells/µL vs. 1.15 ± 0.71 x 103 cells/µL, respectively, p < 0.05). CyTOF analysis of peripheral blood leucocytes revealed both diminished CD8+ T and B cells upon treatment. This finding was further confirmed by the increased apoptosis upon A-1211212 in splenocytes and lamina propria mononuclear cells compared with controls. Upon treatment, increased apoptosis was accompanied by a decrease in Tnf (0.76 ± 0.55 vs 0.99 ± 0.60), Il1β (0.71± 0.55 vs 1.23 ± 1.00), Ifnγ (0.78 ± 0.27 vs 1.23 ± 1.00) and Il6 (1.38 ± 1.12 vs 2.17 ± 2.09) compared with controls. A-1211212 positively altered the colonic mucosa at a macroscopic and microscopic level and ameliorated intestinal inflammation as shown by colonoscopy and histology. Discussion Pro-apoptotic A-1211212 was efficacious in diminishing accumulated lymphocytes and ameliorating colitis in the IL-10-/- animal model of spontaneous colitis. Regulating inappropriate survival of autoreactive lymphocytes by A-1211212 may provide a new therapeutic strategy in IBD.
Tu2020 CRITICAL ROLE OF USP9X IN INITIAL STEPS OF VMP1-MEDIATED AUTOPHAGY Maria I. Vaccaro, Daniel Grasso, Felipe Renna The intracellular activation of zymogens is involved in acute pancreatitis pathogenesis and the eventual pancreas self-digestion. Most of the cases of acute pancreatitis are self-limited, suggesting the importance of the adaptive response mechanism of pancreatic acinar cells. VMP1 is an autophagy-related protein which is able to induce autophagy even in nonstarved condition. Moreover, VMP1 is induced by acute pancreatitis and mediates zymophagy, the selective autophagic degradation of zymogen granules. Here we demonstrated that the ubiquitin protease USP9x interacts with VMP1 during zymophagy in acute pancreatitis. The objective of this work is to know the role of VMP1-USP9x interaction in the initial steps of autophagosome formation. We demonstrate by immunofluorescence that USP9x responds as early as 10 min after autophagy induction, with a relocation pattern without changing its expression by western blot. At basal conditions, USP9x is distributed over the whole cytoplasm. However, upon autophagy induction, it quickly moves to a perinuclear area compatible with ERES/ERGIC compartment, considered as an autophagosome formation site. USP9x is esential for autophagosome formation since shRNA-mediated depletion of USP9x inhibits autophagy, evaluated by LC3 recruitment. USP9x interacts with VMP1 and colocalizes with VMP1 in the ERES/ERGIC compartment, suggesting its implication in the initiation mechanism of autophagosome formation. We also demonstrate that USP9x deubiquitinates BECN1, which is another VMP1 interactor essential for autophagosome formation. We show the ubiquitination of BECN1 during autophagy and its accumulation when it is co-expressed with a ubiquitin molecule without chain formation capability. Moreover, shRNA-mediated depletion of USP9x increases the general ubiquitin signal in the cytoplasm and inhibits autophagosome formation, confirming the critical role of VMP1, BECN1 and USP9x in the autophagosome formation. In conclusion, We demonstrated for the first time that the deubiquitin protease USP9x is a novel autophagy-related protein involved in initial steps of autophagosome formation. USP9x relocates to the ERES/ERGIC compartment interacting with VMP1 and BECN1 in response to autophagy induction. Hierarchically, USP9x acts downstream of VMP1, since VMP1 expression induces USP9x relocation during autophagosome formation. Finally, we suggest that USP9x is a new player in the molecular mechanism of zymophagy during acute pancreatitis, being part in the initial steps of VMP1-mediated autophagy.
Tu2021 THE ROLE OF GΑQ/GΑ11 SIGNALING IN A RAT INTESTINAL EPITHELIAL CELL Hirosato Mashima, Noboru Watanabe, Masanari Sekine, Takeharu Asano, Takeshi Uehara, Shunsuke Urayoshi, Kenichi Yamanaka, Satohiro Matsumoto, Noriyoshi Sagihara, Shinichi Asabe, Hiroyuki Miyatani, Hirohide Ohnishi Background & Aims: Intestinal homeostasis and coordinated action of digestion, absorption and excretion are tightly regulated by a number of gastrointestinal hormones. Most of them exert their actions through G-protein coupled receptors. We have shown recently that the lack of Gαq/Gα11 signaling impaired Paneth cell maturation, induced its differentiation toward goblet cell, and affected the regeneration of colonic mucosa in experimental colitis (Watanabe N. et al. Cell Mol Gastroenterol Hepatol 2(6); 767-782, 2016). Enterocytes and enteroendocrine cells seemed to be not affected in Int-Gq/11 double knock-out intestine. In this study, we examined whether the signaling through Gαq/Gα11 did not play a role in enterocytes using an intestinal epithelial cell line. Methods: We used a rat IEC6 intestinal epithelial cell line. We over-expressed Gαq and/or Gα11 protein using a retrovirus system and puroduced the following cells; IEC6-cont, IEC6-Gq, IEC6-G11, IEC6-Gq/11. We knocked-down the expression of Gαq/Gα11 using siRNA and prepared the cells; IEC6-sicont, IEC6-G∆q, IEC6-G∆11, IEC6-G∆q/11. We evaluated the proliferation of these cells with BrdU incorporation assay and/or counting the cell numbers. We examined the effect on differentiation by qPCR using the markers of enterocytes, goblet cells, Paneth cells and enteroendocrine cells. We also evaluated the changes in the Wnt/β-catenin and Notch signaling. Results: Proliferation was decreased in IEC6-Gq and IEC6-G11 and severely inhibited in IEC6-Gq/11 cells. In contrast, proliferation was enhanced in IEC6-G∆q, IEC6G∆11, and IEC6-G∆q/11 cells. Relative expression of Muc2 was elevated in IEC6-G∆q, IEC6-G∆11, and IEC6-G∆q/11 cells, suggesting that loss of Gαq/Gα11 signaling induced the differentiation of IEC6 cells toward goblet cells. Evaluation of the changes in Wnt/βcatenin and Notch signaling is under way. Conclusions: The signaling through Gαq/Gα11 may play a role in the preservation of enterocytes. The loss of Gαq/Gα11-mediated signaling induced the differentiation of IEC6 cells toward goblet cells.
Tu2019 THE COLONIC SURFACE GOBLET CELLS SECRETE MUCUS IMPORTANT FOR PROPER PROTECTIVE MUCUS LAYER FORMATION Elisabeth E. Nyström, Beatriz Martínez-Abad, Liisa Arike, George Birchenough, Malin E V. Johansson The colonic epithelium is covered by mucus keeping the microbiota at bay, thus limiting direct host-bacterial contact (1). Breaches in the mucus barrier will allow bacterial penetration and prolonged exposure leads to inflammation, as seen in mouse models and ulcerative colitis patients (2). Good mucus protection is achieved by constant renewal, a balance between mucus secretion and removal by distal propulsion. The secreted mucus expands to form the inner mucus layer which is proteolytically processed to looser outer mucus which facilitates smooth removal. Mucus is produced by goblet cells which differ in function upon location along the crypt-surface axis. There is a constant basal mucus secretion, but the upper crypt goblet cells, containing large amounts of stored mucus in their apical granulae, can also respond to acute danger. This massive mucus release is a response to bacterial components sensed by the recently identified sentinel goblet cell (3). Goblet cell differentiation is tightly controlled and goblet cells maturation is at least in part regulated by the transcription factor SPDEF. We have now used the Spdef deficient mouse model to study goblet cell impairment and its role in mucus protection. In this model the mucus is thinner and less protective. The animals develop colitis over time in addition to being more susceptible to induced colitis. The most pronounced effect is in the goblet cells located in the surface epithelium. The surface goblet cells are different from the upper crypt goblet cells as they rapidly produce and secrete mucus and by this contribute to the maintenance of the protective mucus system (4). Using live explants we here show the role and importance of the mucus secreted from these cells in forming a functional mucus layer. Proteomic analysis of the collected mucus revealed alterations of a few proteins. Goblet cell specific proteomic and transcriptomic analyses were also performed on goblet cells isolated by FACS from wild type and Spdef -/- RedMUC2 transgenic mice. These data reveal specific signatures of mucus biosynthesis which has the largest impact on fast producing cells, as the surface goblet cells. This data support our previous observations that goblet cells associated with different functions are present at various locations in the epithelium. All of these goblet cells contribute in a coordinated way to the protection of the intestinal epithelium. This study indicates a unique role for the surface goblet cells in maintaining good protection of the epithelium. Reference List 1. M. E. Johansson et al., Proc. Natl. Acad. Sci. U. S. A 105, 15064 (2008). 2. M. E. Johansson et al., Gut 63, 281 (2014). 3. G. M. Birchenough, E. E. Nystrom, M. E. Johansson, G. C. Hansson, Science 352, 1535 (2016). 4. M. E. Johansson, PLoS. ONE. 7, e41009 (2012).
AGA Abstracts
Tu2022 THE UBIQUITIN HYBRID GENE UBA52 REGULATES CELL CYCLE AND UBIQUITINATION OF RIBOSOME IN COLON CANCER Masanori Kobayashi, Shigeru Oshima, Chiaki Maeyashiki, Yoichi Nibe, Kana Otsubo, Mamoru Watanabe [Background & Aims]The biogenesis of ribosomes is a tightly regulated activity and it is linked to other fundamental cellular processes. Many studies have shown that ribosome dysfunction is associated with developmental defects and increased risk of cancer, including colon cancer. Therefore, some anticancer drugs targeting ribosome have been developed. Recent studies have shown that regulatory 40S ribosomal ubiquitination is an important phase of translational control. In addition, translation reactions terminate with ub-dependent removal of defective nascent chain. UBA52, one of the four ubiquitin-coding genes, comprise a single ubiquitin fused at the C-terminus to ribosomal protein (RP) L40. The increased expression of UBA52 was reported in human colorectal adenoma. Nevertheless, the association between ubiquitin-dependent regulation of translation and the ubiquitin-ribosomal hybrid gene UBA52 remains unclear. We aimed to analyze the functions of UBA52 and to identify novel therapeutic candidate pathway of colon cancer. [Methods]We analyzed the
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lymphocyte count compared to vehicle-receiving controls (0.21 ± 0.08 x 103 cells/µL vs. 1.15 ± 0.71 x 103 cells/µL, respectively, p < 0.05). CyTOF analysis of peripheral blood leucocytes revealed both diminished CD8+ T and B cells upon treatment. This finding was further confirmed by the increased apoptosis upon A-1211212 in splenocytes and lamina propria mononuclear cells compared with controls. Upon treatment, increased apoptosis was accompanied by a decrease in Tnf (0.76 ± 0.55 vs 0.99 ± 0.60), Il1β (0.71± 0.55 vs 1.23 ± 1.00), Ifnγ (0.78 ± 0.27 vs 1.23 ± 1.00) and Il6 (1.38 ± 1.12 vs 2.17 ± 2.09) compared with controls. A-1211212 positively altered the colonic mucosa at a macroscopic and microscopic level and ameliorated intestinal inflammation as shown by colonoscopy and histology. Discussion Pro-apoptotic A-1211212 was efficacious in diminishing accumulated lymphocytes and ameliorating colitis in the IL-10-/- animal model of spontaneous colitis. Regulating inappropriate survival of autoreactive lymphocytes by A-1211212 may provide a new therapeutic strategy in IBD.
Tu2020 CRITICAL ROLE OF USP9X IN INITIAL STEPS OF VMP1-MEDIATED AUTOPHAGY Maria I. Vaccaro, Daniel Grasso, Felipe Renna The intracellular activation of zymogens is involved in acute pancreatitis pathogenesis and the eventual pancreas self-digestion. Most of the cases of acute pancreatitis are self-limited, suggesting the importance of the adaptive response mechanism of pancreatic acinar cells. VMP1 is an autophagy-related protein which is able to induce autophagy even in nonstarved condition. Moreover, VMP1 is induced by acute pancreatitis and mediates zymophagy, the selective autophagic degradation of zymogen granules. Here we demonstrated that the ubiquitin protease USP9x interacts with VMP1 during zymophagy in acute pancreatitis. The objective of this work is to know the role of VMP1-USP9x interaction in the initial steps of autophagosome formation. We demonstrate by immunofluorescence that USP9x responds as early as 10 min after autophagy induction, with a relocation pattern without changing its expression by western blot. At basal conditions, USP9x is distributed over the whole cytoplasm. However, upon autophagy induction, it quickly moves to a perinuclear area compatible with ERES/ERGIC compartment, considered as an autophagosome formation site. USP9x is esential for autophagosome formation since shRNA-mediated depletion of USP9x inhibits autophagy, evaluated by LC3 recruitment. USP9x interacts with VMP1 and colocalizes with VMP1 in the ERES/ERGIC compartment, suggesting its implication in the initiation mechanism of autophagosome formation. We also demonstrate that USP9x deubiquitinates BECN1, which is another VMP1 interactor essential for autophagosome formation. We show the ubiquitination of BECN1 during autophagy and its accumulation when it is co-expressed with a ubiquitin molecule without chain formation capability. Moreover, shRNA-mediated depletion of USP9x increases the general ubiquitin signal in the cytoplasm and inhibits autophagosome formation, confirming the critical role of VMP1, BECN1 and USP9x in the autophagosome formation. In conclusion, We demonstrated for the first time that the deubiquitin protease USP9x is a novel autophagy-related protein involved in initial steps of autophagosome formation. USP9x relocates to the ERES/ERGIC compartment interacting with VMP1 and BECN1 in response to autophagy induction. Hierarchically, USP9x acts downstream of VMP1, since VMP1 expression induces USP9x relocation during autophagosome formation. Finally, we suggest that USP9x is a new player in the molecular mechanism of zymophagy during acute pancreatitis, being part in the initial steps of VMP1-mediated autophagy.
Tu2021 THE ROLE OF GΑQ/GΑ11 SIGNALING IN A RAT INTESTINAL EPITHELIAL CELL Hirosato Mashima, Noboru Watanabe, Masanari Sekine, Takeharu Asano, Takeshi Uehara, Shunsuke Urayoshi, Kenichi Yamanaka, Satohiro Matsumoto, Noriyoshi Sagihara, Shinichi Asabe, Hiroyuki Miyatani, Hirohide Ohnishi Background & Aims: Intestinal homeostasis and coordinated action of digestion, absorption and excretion are tightly regulated by a number of gastrointestinal hormones. Most of them exert their actions through G-protein coupled receptors. We have shown recently that the lack of Gαq/Gα11 signaling impaired Paneth cell maturation, induced its differentiation toward goblet cell, and affected the regeneration of colonic mucosa in experimental colitis (Watanabe N. et al. Cell Mol Gastroenterol Hepatol 2(6); 767-782, 2016). Enterocytes and enteroendocrine cells seemed to be not affected in Int-Gq/11 double knock-out intestine. In this study, we examined whether the signaling through Gαq/Gα11 did not play a role in enterocytes using an intestinal epithelial cell line. Methods: We used a rat IEC6 intestinal epithelial cell line. We over-expressed Gαq and/or Gα11 protein using a retrovirus system and puroduced the following cells; IEC6-cont, IEC6-Gq, IEC6-G11, IEC6-Gq/11. We knocked-down the expression of Gαq/Gα11 using siRNA and prepared the cells; IEC6-sicont, IEC6-G∆q, IEC6-G∆11, IEC6-G∆q/11. We evaluated the proliferation of these cells with BrdU incorporation assay and/or counting the cell numbers. We examined the effect on differentiation by qPCR using the markers of enterocytes, goblet cells, Paneth cells and enteroendocrine cells. We also evaluated the changes in the Wnt/β-catenin and Notch signaling. Results: Proliferation was decreased in IEC6-Gq and IEC6-G11 and severely inhibited in IEC6-Gq/11 cells. In contrast, proliferation was enhanced in IEC6-G∆q, IEC6G∆11, and IEC6-G∆q/11 cells. Relative expression of Muc2 was elevated in IEC6-G∆q, IEC6-G∆11, and IEC6-G∆q/11 cells, suggesting that loss of Gαq/Gα11 signaling induced the differentiation of IEC6 cells toward goblet cells. Evaluation of the changes in Wnt/βcatenin and Notch signaling is under way. Conclusions: The signaling through Gαq/Gα11 may play a role in the preservation of enterocytes. The loss of Gαq/Gα11-mediated signaling induced the differentiation of IEC6 cells toward goblet cells.
Tu2019 THE COLONIC SURFACE GOBLET CELLS SECRETE MUCUS IMPORTANT FOR PROPER PROTECTIVE MUCUS LAYER FORMATION Elisabeth E. Nyström, Beatriz Martínez-Abad, Liisa Arike, George Birchenough, Malin E V. Johansson The colonic epithelium is covered by mucus keeping the microbiota at bay, thus limiting direct host-bacterial contact (1). Breaches in the mucus barrier will allow bacterial penetration and prolonged exposure leads to inflammation, as seen in mouse models and ulcerative colitis patients (2). Good mucus protection is achieved by constant renewal, a balance between mucus secretion and removal by distal propulsion. The secreted mucus expands to form the inner mucus layer which is proteolytically processed to looser outer mucus which facilitates smooth removal. Mucus is produced by goblet cells which differ in function upon location along the crypt-surface axis. There is a constant basal mucus secretion, but the upper crypt goblet cells, containing large amounts of stored mucus in their apical granulae, can also respond to acute danger. This massive mucus release is a response to bacterial components sensed by the recently identified sentinel goblet cell (3). Goblet cell differentiation is tightly controlled and goblet cells maturation is at least in part regulated by the transcription factor SPDEF. We have now used the Spdef deficient mouse model to study goblet cell impairment and its role in mucus protection. In this model the mucus is thinner and less protective. The animals develop colitis over time in addition to being more susceptible to induced colitis. The most pronounced effect is in the goblet cells located in the surface epithelium. The surface goblet cells are different from the upper crypt goblet cells as they rapidly produce and secrete mucus and by this contribute to the maintenance of the protective mucus system (4). Using live explants we here show the role and importance of the mucus secreted from these cells in forming a functional mucus layer. Proteomic analysis of the collected mucus revealed alterations of a few proteins. Goblet cell specific proteomic and transcriptomic analyses were also performed on goblet cells isolated by FACS from wild type and Spdef -/- RedMUC2 transgenic mice. These data reveal specific signatures of mucus biosynthesis which has the largest impact on fast producing cells, as the surface goblet cells. This data support our previous observations that goblet cells associated with different functions are present at various locations in the epithelium. All of these goblet cells contribute in a coordinated way to the protection of the intestinal epithelium. This study indicates a unique role for the surface goblet cells in maintaining good protection of the epithelium. Reference List 1. M. E. Johansson et al., Proc. Natl. Acad. Sci. U. S. A 105, 15064 (2008). 2. M. E. Johansson et al., Gut 63, 281 (2014). 3. G. M. Birchenough, E. E. Nystrom, M. E. Johansson, G. C. Hansson, Science 352, 1535 (2016). 4. M. E. Johansson, PLoS. ONE. 7, e41009 (2012).
AGA Abstracts
Tu2022 THE UBIQUITIN HYBRID GENE UBA52 REGULATES CELL CYCLE AND UBIQUITINATION OF RIBOSOME IN COLON CANCER Masanori Kobayashi, Shigeru Oshima, Chiaki Maeyashiki, Yoichi Nibe, Kana Otsubo, Mamoru Watanabe [Background & Aims]The biogenesis of ribosomes is a tightly regulated activity and it is linked to other fundamental cellular processes. Many studies have shown that ribosome dysfunction is associated with developmental defects and increased risk of cancer, including colon cancer. Therefore, some anticancer drugs targeting ribosome have been developed. Recent studies have shown that regulatory 40S ribosomal ubiquitination is an important phase of translational control. In addition, translation reactions terminate with ub-dependent removal of defective nascent chain. UBA52, one of the four ubiquitin-coding genes, comprise a single ubiquitin fused at the C-terminus to ribosomal protein (RP) L40. The increased expression of UBA52 was reported in human colorectal adenoma. Nevertheless, the association between ubiquitin-dependent regulation of translation and the ubiquitin-ribosomal hybrid gene UBA52 remains unclear. We aimed to analyze the functions of UBA52 and to identify novel therapeutic candidate pathway of colon cancer. [Methods]We analyzed the
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