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Pandey A, Shen C, Feng S, Enosi Tuipulotu D, Ngo C, Liu C, Kurera M, Mathur A, Venkataraman S, Zhang J, Talaulikar D, Song R, Wong JJL, Teoh N, Kaakoush NO, Man SM. Ku70 senses cytosolic DNA and assembles a tumor-suppressive signalosome. SCIENCE ADVANCES 2024; 10:eadh3409. [PMID: 38277448 PMCID: PMC10816715 DOI: 10.1126/sciadv.adh3409] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/25/2023] [Accepted: 12/26/2023] [Indexed: 01/28/2024]
Abstract
The innate immune response contributes to the development or attenuation of acute and chronic diseases, including cancer. Microbial DNA and mislocalized DNA from damaged host cells can activate different host responses that shape disease outcomes. Here, we show that mice and humans lacking a single allele of the DNA repair protein Ku70 had increased susceptibility to the development of intestinal cancer. Mechanistically, Ku70 translocates from the nucleus into the cytoplasm where it binds to cytosolic DNA and interacts with the GTPase Ras and the kinase Raf, forming a tripartite protein complex and docking at Rab5+Rab7+ early-late endosomes. This Ku70-Ras-Raf signalosome activates the MEK-ERK pathways, leading to impaired activation of cell cycle proteins Cdc25A and CDK1, reducing cell proliferation and tumorigenesis. We also identified the domains of Ku70, Ras, and Raf involved in activating the Ku70 signaling pathway. Therapeutics targeting components of the Ku70 signalosome could improve the treatment outcomes in cancer.
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Affiliation(s)
- Abhimanu Pandey
- Division of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia
| | - Cheng Shen
- Division of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia
| | - Shouya Feng
- Division of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia
| | - Daniel Enosi Tuipulotu
- Division of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia
| | - Chinh Ngo
- Division of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia
| | - Cheng Liu
- Conjoint Gastroenterology Laboratory, QIMR Berghofer Medical Research Institute, Herston, Australia
- School of Medicine, University of Queensland, Herston, Australia
- Mater Pathology, Mater Hospital, South Brisbane, Australia
| | - Melan Kurera
- Division of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia
| | - Anukriti Mathur
- Division of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia
| | - Shweta Venkataraman
- Division of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia
| | - Jing Zhang
- Division of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia
| | - Dipti Talaulikar
- Division of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia
- Haematology Translational Research Unit, ACT Pathology, Canberra Health Services, Canberra, Australian Capital Territory, Australia
- Department of Human Genomics, ACT Pathology, Canberra, Australian Capital Territory, Australia
- School of Medicine and Psychology, College of Health and Medicine, The Australian National University, Canberra, Australia
| | - Renhua Song
- Epigenetics and RNA Biology Program Centenary Institute, The University of Sydney, Camperdown 2050, Australia
- Faculty of Medicine and Health, The University of Sydney, Camperdown 2050, Australia
| | - Justin J.-L. Wong
- Epigenetics and RNA Biology Program Centenary Institute, The University of Sydney, Camperdown 2050, Australia
- Faculty of Medicine and Health, The University of Sydney, Camperdown 2050, Australia
| | - Narci Teoh
- Gastroenterology and Hepatology Unit, The Australian National University Medical School at The Canberra Hospital, The Australian National University, Canberra, Australia
| | - Nadeem O. Kaakoush
- School of Biomedical Sciences, University of New South Wales, Sydney, NSW, Australia
| | - Si Ming Man
- Division of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia
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Ji W, Liu W, Huo Y, Hu C, Zhang Y. Banxia Xiexin decoction ameliorates dextran sulfate sodium (DSS)-induced ulcerative colitis via inhibiting serine-threonine protein kinase (Akt)/mitogen-activated protein kinase (MAPK) signaling pathway. Biotechnol Appl Biochem 2023; 70:1530-1542. [PMID: 36806191 DOI: 10.1002/bab.2451] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2022] [Revised: 02/02/2023] [Accepted: 02/11/2023] [Indexed: 02/19/2023]
Abstract
Banxia Xiexin decoction (BXD), a traditional Chinese medicine, was widely used in treating ulcerative colitis (UC). However, the active components of BXD and its mechanism in UC remain elusive. Therefore, we used network pharmacology in vivo experiments, molecular docking, and surface plasmon resonance strategy (SPR) to uncover BXD's potential mechanism. A UC rat model was established by orally administering 7% dextran sulfate sodium (DSS) in drinking water, BXD and palmatine were orally administered for 7 days. Network pharmacology was used to investigate the main bioactive components and crucial targets of BXD in treating UC. Molecular docking was used to investigate interactions between components and crucial targets, verifying the results by SPR. By network pharmacology predicting, 20 active components and 44 candidate anti-UC targets of BXD were identified, and the crucial proteins were screeded from PPI network, including extracellular regulated protein kinases (ERK), AKT1, and tumor necrosis factor-α (TNF). In addition, some key active components (palmatine, sexangularetin, and skullcapflavone II) were screened out from the active components-targets network. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and in vivo experiments showed that protein-serine-threonine kinase (Akt)/MAPK pathway was involved in BXD treatment for UC; BXD and palmatine significantly ameliorated the severity of DSS-induced UC in rats. Our study might assist in further investigation of the active components in Chinese medicine.
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Affiliation(s)
- Wanli Ji
- School of Pharmacy, Shanghai University of Medicine and Health Sciences, Shanghai, China
| | - Wangzhenzu Liu
- School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Yan Huo
- Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Cheng Hu
- Science and Technology Experiment Center, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Yifan Zhang
- School of Pharmacy, Shanghai University of Medicine and Health Sciences, Shanghai, China
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Wang S, Pu J, Li X, Yan Z, Li C, Zheng Y, Luo Z, Cui L. UBE2W Improves the Experimental Colitis by Inhibiting the NF-κB Signaling Pathway. Dig Dis Sci 2022; 67:5529-5539. [PMID: 35314916 DOI: 10.1007/s10620-022-07453-4] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/14/2021] [Accepted: 02/16/2022] [Indexed: 01/05/2023]
Abstract
BACKGROUND The NF-κB signaling cascade regulates immune response and is often dysregulated in tumor development. UBE2W is a novel type I ubiquitin-conjugating enzyme (E2) whose biological function is still unclear. AIMS This study was designed to investigate whether UBE2W regulates NF-κB signaling pathway and is involved in the progression of experimental colitis. METHODS At the cellular level, the effect of UBE2W on NF-κB transcriptional activity was measured using a dual-luciferase reporter assay. The influence of UBE2W on NF-κB pathway activation and the entry of p65 into the nucleus were determined by Western blot and immunofluorescence analyses, respectively. Moreover, the colitis model was established by administering 2.5% dextran sulfate sodium (DSS)/water to UBE2W overexpression, UBE2W-knockdown and control mice. Body weight, stool consistency, colon length and clinical severity were examined. Expression of pro-inflammatory cytokines and phosphorylation of p65 and IκB in the colon tissue were measured by qRT-PCR and Western blot, respectively. RESULTS UBE2W inhibited TNFα-induced NF-κB transcription activity, attenuated IκB and p65 phosphorylation, downregulated TNFα and IL-8 expression and blocked the entry of p65 into the nucleus. In the DSS-induced colitis model, UBE2W-knockdown mice had increased weight loss, more serious diarrhea and mucosal injures compared with the control mice. Moreover, phosphorylation of IκB and p65 and the expression of pro-inflammatory mediators such as TNFα, IL-6 were significantly increased in UBE2W knockdown mice. However, these changes were completely reversed in UBE2W overexpression mice. CONCLUSIONS The overexpression of UBE2W ameliorates the severity of DSS-induced colitis, which may be mediated by inhibiting the expression of pro-inflammatory mediators and activation of the NF-κB signaling pathway. These findings provide evidence that UBE2W might have potential therapeutic implications in IBD.
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Affiliation(s)
- Shaoxin Wang
- Department of Gastroenterology, The First Medical Center of Chinese PLA General Hospital, 28 Fuxing Road, Beijing, 100853, China
| | - Jiang Pu
- Department of Gastroenterology, The First Medical Center of Chinese PLA General Hospital, 28 Fuxing Road, Beijing, 100853, China
| | - Xiaowei Li
- Department of Gastroenterology, The First Medical Center of Chinese PLA General Hospital, 28 Fuxing Road, Beijing, 100853, China
| | - Zhihui Yan
- Department of Gastroenterology, The First Medical Center of Chinese PLA General Hospital, 28 Fuxing Road, Beijing, 100853, China
| | - Chao Li
- Department of Gastroenterology, The First Medical Center of Chinese PLA General Hospital, 28 Fuxing Road, Beijing, 100853, China
| | - Yan Zheng
- Department of Gastroenterology, The First Medical Center of Chinese PLA General Hospital, 28 Fuxing Road, Beijing, 100853, China
| | - Zhe Luo
- Department of Gastroenterology, The First Medical Center of Chinese PLA General Hospital, 28 Fuxing Road, Beijing, 100853, China
| | - Lihong Cui
- Department of Gastroenterology, The First Medical Center of Chinese PLA General Hospital, 28 Fuxing Road, Beijing, 100853, China.
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Henry C, Bassignani A, Berland M, Langella O, Sokol H, Juste C. Modern Metaproteomics: A Unique Tool to Characterize the Active Microbiome in Health and Diseases, and Pave the Road towards New Biomarkers—Example of Crohn’s Disease and Ulcerative Colitis Flare-Ups. Cells 2022; 11:cells11081340. [PMID: 35456018 PMCID: PMC9028112 DOI: 10.3390/cells11081340] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2022] [Revised: 04/06/2022] [Accepted: 04/11/2022] [Indexed: 11/16/2022] Open
Abstract
Thanks to the latest developments in mass spectrometry, software and standards, metaproteomics is emerging as the vital complement of metagenomics, to make headway in understanding the actual functioning of living and active microbial communities. Modern metaproteomics offers new possibilities in the area of clinical diagnosis. This is illustrated here, for the still highly challenging diagnosis of intestinal bowel diseases (IBDs). Using bottom-up proteomics, we analyzed the gut metaproteomes of the same twenty faecal specimens processed either fresh or after a two-month freezing period. We focused on metaproteomes of microbial cell envelopes since it is an outstanding way of capturing host and host–microbe interaction signals. The protein profiles of pairs of fresh and frozen-thawed samples were closely related, making feasible deferred analysis in a distant diagnosis centre. The taxonomic and functional landscape of microbes in diverse IBD phenotypes—active ulcerative colitis, or active Crohn’s disease either with ileo-colonic or exclusive colonic localization—differed from each other and from the controls. Based on their specific peptides, we could identify proteins that were either strictly overrepresented or underrepresented in all samples of one clinical group compared to all samples of another group, paving the road for promising additional diagnostic tool for IBDs.
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Affiliation(s)
- Céline Henry
- PAPPSO, Micalis Institute, INRAE, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France; (C.H.); (A.B.)
- Micalis Institute, INRAE, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France;
| | - Ariane Bassignani
- PAPPSO, Micalis Institute, INRAE, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France; (C.H.); (A.B.)
- Micalis Institute, INRAE, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France;
- MGP, INRAE, Université Paris-Saclay, 78350 Jouy-en-Josas, France;
| | - Magali Berland
- MGP, INRAE, Université Paris-Saclay, 78350 Jouy-en-Josas, France;
| | - Olivier Langella
- PAPPSO, GQE-Le Moulon, AgroParisTech, CNRS, INRAE, Université Paris-Saclay, 91190 Gif-sur-Yvette, France;
| | - Harry Sokol
- Micalis Institute, INRAE, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France;
- Gastroenterology Department, INSERM, Centre de Recherche Saint-Antoine, CRSA, AP-HP, Saint Antoine Hospital, Sorbonne Université, 75012 Paris, France
- Paris Centre for Microbiome Medicine (PaCeMM) FHU, Service de Gastro-Entérologie et Nutrition, Hôpital Saint-Antoine, Direction de la Recherche Clinique et de l’Innovation (DRCI) de l’AP-HP, CEDEX 12, 75571 Paris, France
| | - Catherine Juste
- Micalis Institute, INRAE, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France;
- Correspondence: ; Tel.: +33-67-72-82-035
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5
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Ruiz Castro PA, Yepiskoposyan H, Gubian S, Calvino-Martin F, Kogel U, Renggli K, Peitsch MC, Hoeng J, Talikka M. Systems biology approach highlights mechanistic differences between Crohn's disease and ulcerative colitis. Sci Rep 2021; 11:11519. [PMID: 34075172 PMCID: PMC8169754 DOI: 10.1038/s41598-021-91124-3] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2021] [Accepted: 05/21/2021] [Indexed: 12/11/2022] Open
Abstract
The molecular mechanisms of IBD have been the subject of intensive exploration. We, therefore, assembled the available information into a suite of causal biological network models, which offer comprehensive visualization of the processes underlying IBD. Scientific text was curated by using Biological Expression Language (BEL) and compiled with OpenBEL 3.0.0. Network properties were analysed by Cytoscape. Network perturbation amplitudes were computed to score the network models with transcriptomic data from public data repositories. The IBD network model suite consists of three independent models that represent signalling pathways that contribute to IBD. In the “intestinal permeability” model, programmed cell death factors were downregulated in CD and upregulated in UC. In the “inflammation” model, PPARG, IL6, and IFN-associated pathways were prominent regulatory factors in both diseases. In the “wound healing” model, factors promoting wound healing were upregulated in CD and downregulated in UC. Scoring of publicly available transcriptomic datasets onto these network models demonstrated that the IBD models capture the perturbation in each dataset accurately. The IBD network model suite can provide better mechanistic insights of the transcriptional changes in IBD and constitutes a valuable tool in personalized medicine to further understand individual drug responses in IBD.
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Affiliation(s)
- Pedro A Ruiz Castro
- Philip Morris International R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, 2000, Neuchâtel, Switzerland.
| | - Hasmik Yepiskoposyan
- Philip Morris International R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, 2000, Neuchâtel, Switzerland.
| | - Sylvain Gubian
- Philip Morris International R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, 2000, Neuchâtel, Switzerland
| | - Florian Calvino-Martin
- Philip Morris International R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, 2000, Neuchâtel, Switzerland
| | - Ulrike Kogel
- Philip Morris International R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, 2000, Neuchâtel, Switzerland
| | - Kasper Renggli
- Philip Morris International R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, 2000, Neuchâtel, Switzerland
| | - Manuel C Peitsch
- Philip Morris International R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, 2000, Neuchâtel, Switzerland
| | - Julia Hoeng
- Philip Morris International R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, 2000, Neuchâtel, Switzerland
| | - Marja Talikka
- Philip Morris International R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, 2000, Neuchâtel, Switzerland.
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Sprouty2 limits intestinal tuft and goblet cell numbers through GSK3β-mediated restriction of epithelial IL-33. Nat Commun 2021; 12:836. [PMID: 33547321 PMCID: PMC7864916 DOI: 10.1038/s41467-021-21113-7] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2019] [Accepted: 01/12/2021] [Indexed: 02/07/2023] Open
Abstract
Dynamic regulation of intestinal cell differentiation is crucial for both homeostasis and the response to injury or inflammation. Sprouty2, an intracellular signaling regulator, controls pathways including PI3K and MAPKs that are implicated in differentiation and are dysregulated in inflammatory bowel disease. Here, we ask whether Sprouty2 controls secretory cell differentiation and the response to colitis. We report that colonic epithelial Sprouty2 deletion leads to expanded tuft and goblet cell populations. Sprouty2 loss induces PI3K/Akt signaling, leading to GSK3β inhibition and epithelial interleukin (IL)-33 expression. In vivo, this results in increased stromal IL-13+ cells. IL-13 in turn induces tuft and goblet cell expansion in vitro and in vivo. Sprouty2 is downregulated by acute inflammation; this appears to be a protective response, as VillinCre;Sprouty2F/F mice are resistant to DSS colitis. In contrast, Sprouty2 is elevated in chronic colitis and in colons of inflammatory bowel disease patients, suggesting that this protective epithelial-stromal signaling mechanism is lost in disease. Dynamic regulation of colonic secretory cell numbers is a critical component of the response to intestinal injury and inflammation. Here, the authors show that loss of the intracellular signalling regulator Sprouty2 in the intestinal epithelial cells is a protective response to injury that leads to increased secretory cell numbers, thus limiting colitis severity.
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7
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Zhou C, Tan Y, Wang Y, Liao F, Wang Q, Li J, Peng S, Peng X, Zou Y. PM 2.5-inducible long non-coding RNA (NONHSAT247851.1) is a positive regulator of inflammation through its interaction with raf-1 in HUVECs. ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 2020; 196:110476. [PMID: 32278143 DOI: 10.1016/j.ecoenv.2020.110476] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/01/2019] [Revised: 03/08/2020] [Accepted: 03/11/2020] [Indexed: 06/11/2023]
Abstract
Several studies have demonstrated that PM2.5 inhalation is associated with an increased risk of cerebrovascular disease (CVD), in which inflammation plays an important role. The mechanisms of this disease are not fully understood to date. Long non-coding RNAs (lncRNAs) are involved in many pathophysiological processes, such as immune responses; however, their functions associated with inflammation are largely unexplored. High-throughput sequencing assay and obtained numerous lncRNAs that altered the expression in response to PM2.5 treatment in HUVECs. NONHSAT247851.1 was also identified, which was significantly up-regulated to control the expression of immune response genes. Mechanistically, the results indicated that NONHSAT247851.1 knockdown reduced the expression of IL1β. In study, we investigated NONHSAT247851.1 as a promoter in regulating immune response genes via binding with raf-1 to regulate the phosphorylation level of p65 protein in HUVECs. The data collected suggests that NONHSAT247851.1 regulates inflammation via interaction with raf-1 to control the inflammatory expression in PM2.5 exposure.
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Affiliation(s)
- CaiLan Zhou
- School of Public Health, Guangxi Medical University, Nanning, 530021, China
| | - Yi Tan
- State Environmental Protection Key Laboratory of Environmental Pollution Health Risk Assessment, South China Institute of Environmental Sciences, Ministry of Ecology and Environment, Guangzhou, 510535, China
| | - YuYu Wang
- State Environmental Protection Key Laboratory of Environmental Pollution Health Risk Assessment, South China Institute of Environmental Sciences, Ministry of Ecology and Environment, Guangzhou, 510535, China
| | - FangPing Liao
- School of Public Health, Guangxi Medical University, Nanning, 530021, China
| | - QiuLing Wang
- School of Public Health, Guangxi Medical University, Nanning, 530021, China
| | - JingLin Li
- School of Public Health, Guangxi Medical University, Nanning, 530021, China
| | - SuJuan Peng
- School of Public Health, Wuhan University of Science and Technology, Wuhan, 430081, China
| | - XiaoWu Peng
- School of Public Health, Guangxi Medical University, Nanning, 530021, China; State Environmental Protection Key Laboratory of Environmental Pollution Health Risk Assessment, South China Institute of Environmental Sciences, Ministry of Ecology and Environment, Guangzhou, 510535, China.
| | - YunFeng Zou
- School of Public Health, Guangxi Medical University, Nanning, 530021, China.
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8
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Dubé PE, Liu CY, Girish N, Washington MK, Polk DB. Pharmacological activation of epidermal growth factor receptor signaling inhibits colitis-associated cancer in mice. Sci Rep 2018; 8:9119. [PMID: 29904166 PMCID: PMC6002410 DOI: 10.1038/s41598-018-27353-w] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2018] [Accepted: 05/30/2018] [Indexed: 12/15/2022] Open
Abstract
Current treatments for inflammatory bowel disease (IBD) target the overactive immune response of the intestinal mucosa. However, epidermal growth factor (EGF), an activating ligand of the EGF receptor (EGFR), has been shown to induce disease remission through direct targeting of intestinal mucosal healing. Despite promising preclinical and clinical results, this EGFR-activating therapy has not progressed, in part due to the potential for carcinogenesis associated with long-term use and the increased risk of colitis-associated cancer (CAC) in IBD. Here we tested whether pharmacological modulation of EGFR altered outcomes of CAC in the murine azoxymethane/dextran sulfate sodium model. We found that administering EGF during the period of maximum colitis severity ("early"), coincident with the initiation and early promotion of tumors, improved outcomes of colitis and reduced tumor size. In contrast, daily EGF administration beginning ~2 months after tumor initiation ("late") increased tumor size. Administration of the EGFR kinase inhibitor gefitinib increased the tumor size when the drug was given early and decreased the tumor size when the drug was administered late. EGF administration not only reduced colonic cytokine and chemokine expression during injury, but also baseline chemokine expression in homeostasis. These results suggest that EGFR activation during acute bouts of colitis may reduce the long-term burden of CAC.
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Affiliation(s)
- Philip E Dubé
- Division of Pediatric Gastroenterology, Hepatology, and Nutrition, Children's Hospital Los Angeles, Los Angeles, CA, USA
- Taconic Biosciences, Hudson, NY, USA
| | - Cambrian Y Liu
- Division of Pediatric Gastroenterology, Hepatology, and Nutrition, Children's Hospital Los Angeles, Los Angeles, CA, USA
| | - Nandini Girish
- Division of Pediatric Gastroenterology, Hepatology, and Nutrition, Children's Hospital Los Angeles, Los Angeles, CA, USA
| | - M Kay Washington
- Department of Pathology, Vanderbilt University Medical Center, Nashville, TN, USA
| | - D Brent Polk
- Division of Pediatric Gastroenterology, Hepatology, and Nutrition, Children's Hospital Los Angeles, Los Angeles, CA, USA.
- Department of Biochemistry and Molecular Medicine, Keck School of Medicine of University of Southern California, Los Angeles, CA, USA.
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9
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Zhang W, Zhu B, Xu J, Liu Y, Qiu E, Li Z, Li Z, He Y, Zhou H, Bai Y, Zhi F. Bacteroides fragilis Protects Against Antibiotic-Associated Diarrhea in Rats by Modulating Intestinal Defenses. Front Immunol 2018; 9:1040. [PMID: 29868005 PMCID: PMC5954023 DOI: 10.3389/fimmu.2018.01040] [Citation(s) in RCA: 77] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2018] [Accepted: 04/26/2018] [Indexed: 01/03/2023] Open
Abstract
Antibiotic-associated diarrhea (AAD) is iatrogenic diarrhea characterized by disruption of the gut microbiota. Probiotics are routinely used to treat AAD in clinical practice; however, the effectiveness and mechanisms by which probiotics alleviate symptoms remain poorly understood. We previously isolated a non-toxic Bacteroides fragilis strain ZY-312, which has been verified to be beneficial in certain infection disorders. However, the precise role of this commensal bacterium in AAD is unknown. In this study, we successfully established an AAD rat model by exposing rats to appropriate antibiotics. These rats developed diarrhea symptoms and showed alterations in their intestinal microbiota, including overgrowth of some pathogenic bacteria. In addition, gastrointestinal barrier defects, indicated by compromised aquaporin expression, aberrant tight junction proteins, and decreased abundance of mucus-filled goblet cells, were also detected in ADD rats compared with control animals. Of note, oral treatment with B. fragilis strain ZY-312 ameliorated AAD-related diarrhea symptoms by increasing the abundance of specific commensal microbiota. Interestingly, we demonstrated that these changes were coincident with the restoration of intestinal barrier function and enterocyte regeneration in AAD rats. In summary, we identified a potential probiotic therapeutic strategy for AAD and identified the vital roles of B. fragilis strain ZY-312 in modulating the colonic bacterial community and participating in microbiota-mediated epithelial cell proliferation and differentiation.
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Affiliation(s)
- Wendi Zhang
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Institute of Gastroenterology of Guangdong Province, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Bo Zhu
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Institute of Gastroenterology of Guangdong Province, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Jiahui Xu
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Institute of Gastroenterology of Guangdong Province, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Yangyang Liu
- Guangzhou ZhiYi Biotechnology Co. Ltd., Guangzhou, China
| | - Enqi Qiu
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Institute of Gastroenterology of Guangdong Province, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Zhijun Li
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Institute of Gastroenterology of Guangdong Province, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Zhengchao Li
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Institute of Gastroenterology of Guangdong Province, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Yan He
- State Key Laboratory of Organ Failure Research, Division of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - Hongwei Zhou
- State Key Laboratory of Organ Failure Research, Division of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - Yang Bai
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Institute of Gastroenterology of Guangdong Province, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Fachao Zhi
- Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Institute of Gastroenterology of Guangdong Province, Nanfang Hospital, Southern Medical University, Guangzhou, China
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10
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Esculentoside A exerts anti-inflammatory activity in microglial cells. Int Immunopharmacol 2017; 51:148-157. [DOI: 10.1016/j.intimp.2017.08.014] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2017] [Revised: 08/07/2017] [Accepted: 08/16/2017] [Indexed: 01/09/2023]
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Dorard C, Vucak G, Baccarini M. Deciphering the RAS/ERK pathway in vivo. Biochem Soc Trans 2017; 45:27-36. [PMID: 28202657 DOI: 10.1042/bst20160135] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2016] [Revised: 11/03/2016] [Accepted: 11/07/2016] [Indexed: 12/19/2022]
Abstract
The RAS/ERK pathway has been intensely studied for about three decades, not least because of its role in human pathologies. ERK activation is observed in the majority of human cancers; in about one-third of them, it is driven by mutational activation of pathway components. The pathway is arguably one of the best targets for molecule-based pharmacological intervention, and several small-molecule inhibitors are in clinical use. Genetically engineered mouse models have greatly contributed to our understanding of signaling pathways in development, tissue homeostasis, and disease. In the specific case of the RAS/ERK pathway, they have revealed unique biological roles of structurally and functionally similar proteins, new kinase-independent effectors, and unsuspected relationships with other cascades. This short review summarizes the contribution of mouse models to our current understanding of the pathway.
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Affiliation(s)
- Coralie Dorard
- Max F. Perutz Laboratories, Center for Molecular Biology, University of Vienna, Vienna 1030, Austria
| | - Georg Vucak
- Max F. Perutz Laboratories, Center for Molecular Biology, University of Vienna, Vienna 1030, Austria
| | - Manuela Baccarini
- Max F. Perutz Laboratories, Center for Molecular Biology, University of Vienna, Vienna 1030, Austria
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12
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Khajah MA, Ananthalakshmi KV, Edafiogho I. Anti-Inflammatory Properties of the Enaminone E121 in the Dextran Sulfate Sodium (DSS) Colitis Model. PLoS One 2016; 11:e0168567. [PMID: 27997590 PMCID: PMC5173236 DOI: 10.1371/journal.pone.0168567] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2016] [Accepted: 12/03/2016] [Indexed: 02/07/2023] Open
Abstract
BACKGROUND Enaminones are synthetic compounds with an established role in the prevention of various forms of seizures. Recent evidence suggests potent anti-tussive, bronchodilation and anti-inflammatory properties. Pre-treatment with particularly E121 compound resulted in a decrease in leukocyte recruitment in the ovalbumin induced-model of asthma, immune cell proliferation and cytokine release in vitro. We hypothesize that E121 might serve as a therapeutic potential in intestinal inflammation through modulating immune cell functions. METHODS Colitis was induced by daily dextran sulfate sodium (DSS) administration for 5 days, and its severity was determined by gross and histological assessments. The plasma level of various cytokines was measured using flow cytometry-based assay. The colonic expression/ phosphorylation level of various molecules was determined by immunofluorescence and western blotting. The effects of E121 treatment on in vitro neutrophil chemotaxis (under-agarose assay), superoxide release (luminol oxidation assay) and apoptosis (annexin V/7AAD) were also determined. RESULTS DSS-induced colitis in mice was significantly reduced by daily E121 treatment (30-100 mg/kg) at gross and histological levels. This effect was due to modulated plasma levels of interleukin (IL-2) and colonic expression levels of various signaling molecules and proteins involved in apoptosis. In vitro neutrophil survival, chemotaxis, and superoxide release were also reduced by E121 treatment. CONCLUSION Our results indicate important anti-inflammatory actions of E121 in the pathogenesis of IBD.
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Affiliation(s)
| | | | - Ivan Edafiogho
- Department of Pharmaceutical Sciences, University of Saint Joseph School of Pharmacy, Hartford, Connecticut, United States of America
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13
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A Novel Role of Spred2 in the Colonic Epithelial Cell Homeostasis and Inflammation. Sci Rep 2016; 6:37531. [PMID: 27869219 PMCID: PMC5116627 DOI: 10.1038/srep37531] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2016] [Accepted: 10/27/2016] [Indexed: 12/13/2022] Open
Abstract
Rapid and adequate mucosal healing is important for a remission of ulcerative colitis (UC) patients. Here, we examined whether Spred2, a member of the Sprouty-related EVH1-domain-containing proteins that inhibit the Ras/Raf/ERK pathway, plays a role in colonic mucosal homeostasis and inflammation by using Spred2 knockout (KO) mice. We first detected increased epithelial cell proliferation and cadherin 1 expression in the colon of naïve Spred2 KO mice compared to wild-type mice. Interestingly, Spred2 KO mice were resistant to dextran sulfate sodium (DSS)-induced acute colitis as indicated by lower levels of body weight loss and disease activity index. Histologically, epithelial cell injury and inflammation were milder in the colonic mucosa of Spred2 KO mice on day 3 and almost undetectable by day 8. Experiments with bone chimeric mice indicated that Spred2-deficiency in non-hematopoietic cells was responsible for the reduced sensitivity to DSS. Finally, Spred2 KO mice developed significantly fewer tumors in response to azoxymethane plus DSS. Taken together, our results demonstrate, for the first time, that Spred2 plays an important role in the regulation of colonic epithelial cell proliferation and inflammation by potentially down-regulating the activation of ERK. Thus, Spred2 may be a new therapeutic target for the treatment of UC.
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14
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Kuo WT, Lee TC, Yu LCH. Eritoran Suppresses Colon Cancer by Altering a Functional Balance in Toll-like Receptors That Bind Lipopolysaccharide. Cancer Res 2016; 76:4684-95. [PMID: 27328732 DOI: 10.1158/0008-5472.can-16-0172] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2016] [Accepted: 06/05/2016] [Indexed: 11/16/2022]
Abstract
Colorectal carcinogenesis is affected by overexpression of the lipopolysaccharide (LPS) receptors CD14 and TLR4, which antagonize each other by affecting epithelial cell proliferation and apoptosis. Eritoran is an investigational drug for sepsis treatment that resembles the lipid A moiety of LPS and therefore acts as a TLR4 inhibitor. In the present study, we explored the potential therapeutic uses and mechanisms of action of eritoran in reducing colon cancer progression. Eritoran administration via intracolonic, intragastric, or intravenous routes significantly reduced tumor burden in a chemically induced mouse model of colorectal carcinoma. Decreased proliferation and increased apoptosis were observed in mouse tumor cells after eritoran treatment. In vitro cultures of mouse primary tumor spheroids and human cancer cell lines displayed increased cell proliferation and cell-cycle progression following LPS challenge. This effect was inhibited by eritoran and by silencing CD14 or TLR4. In contrast, apoptosis induced by eritoran was eliminated by silencing CD14 or protein kinase Cζ (PKCζ) but not TLR4. Lastly, LPS and eritoran caused hyperphosphorylation of PKCζ in a CD14-dependent and TLR4-independent manner. Blocking PKCζ activation by a Src kinase inhibitor and a PKCζ-pseudosubstrate prevented eritoran-induced apoptosis. In summary, our work offers a preclinical proof of concept for the exploration of eritoran as a clinical treatment, with a mechanistic rationale to reposition this drug to improve the management of colorectal cancer. Cancer Res; 76(16); 4684-95. ©2016 AACR.
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Affiliation(s)
- Wei-Ting Kuo
- Graduate Institute of Physiology, National Taiwan University College of Medicine, Taipei, Taiwan
| | - Tsung-Chun Lee
- Graduate Institute of Physiology, National Taiwan University College of Medicine, Taipei, Taiwan. Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
| | - Linda Chia-Hui Yu
- Graduate Institute of Physiology, National Taiwan University College of Medicine, Taipei, Taiwan.
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15
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Chen T, Zheng F, Tao J, Tan S, Zeng L, Peng X, Wu B. Insulin-Like Growth Factor-1 Contributes to Mucosal Repair by β-Arrestin2-Mediated Extracellular Signal-Related Kinase Signaling in Experimental Colitis. THE AMERICAN JOURNAL OF PATHOLOGY 2016; 185:2441-53. [PMID: 26362717 DOI: 10.1016/j.ajpath.2015.05.020] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/05/2015] [Revised: 05/19/2015] [Accepted: 05/28/2015] [Indexed: 12/12/2022]
Abstract
Insulin-like growth factor-1 (IGF-1) possesses the ability to attenuate intestinal damage and promote mucosal repair of colitis. β-Arrestins, as the scaffolding proteins of G protein-coupled receptors or non-G protein-coupled receptors signaling, can be involved in IGF-1-mediated signaling pathways. However, the interaction of IGF-1 and β-arrestin2 in the mucosal repair of experimental colitis remains unexplored. Ulcerative colitis was induced in β-arrestin2 wild-type mice and β-arrestin2 knockout littermates by using 3% dextran sulfate sodium for 5 days, followed by regular water consumption for 1, 2, 3, and 4 weeks to analyze the mucosal repair from experimental colitis. Disease activity index and histologic score analyses were performed. Apoptosis and proliferation were assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and Ki-67 staining, respectively. The expressions of β-arrestin2, phospho (p)-IGF-1R, and p-extracellular signal-regulated kinase (ERK)1/2 were examined. Furthermore, β-arrestin2 was overexpressed or altered in HCT116 cells by transfection before IGF-1 treatment in vitro. IGF-1 and β-arrestin2 expression was up-regulated in the repairing phase of experimental colitis. Targeted deletion of β-arrestin2 delayed the repair of colitis by inhibiting cell proliferation without affecting the levels of IGF-1 and p-IGF-1R. The β-arrestin2/ERK signaling pathway was involved in IGF-1-mediated mucosal repair through promoting epithelial cell and goblet cell regeneration from experimental colitis. These results indicate that IGF-1 contributes to the mucosal repair by β-arrestin2-mediated ERK signaling in experimental colitis.
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Affiliation(s)
- Tingting Chen
- Department of Gastroenterology, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China
| | - Fengping Zheng
- Department of Gastroenterology, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China
| | - Jin Tao
- Department of Gastroenterology, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China
| | - Siwei Tan
- Department of Gastroenterology, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China
| | - Lixian Zeng
- Department of Gastroenterology, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China
| | - Xiaojie Peng
- Department of Gastroenterology, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China
| | - Bin Wu
- Department of Gastroenterology, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China.
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16
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Lappas M. RAF1 is increased in labouring myometrium and modulates inflammation-induced pro-labour mediators. Reproduction 2016; 151:411-20. [DOI: 10.1530/rep-15-0607] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2015] [Accepted: 01/25/2016] [Indexed: 01/25/2023]
Abstract
Inflammation plays a central role in the terminal process of human labour and delivery, including myometrial contractions. RAF1 proto-oncogene serine/threonine-protein kinase (RAF1) can activate ERK (official gene symbolMAPK1) and/or nuclear factor-kappa B (NF-κB) to regulate genes involved in inflammation. There are, however, no studies on the role of RAF1 in the processes of human labour and delivery. Thus, the aims of this study were to determine the effect of i) human labour and pro-inflammatory cytokines interleukin 1 beta (IL1B) and tumour necrosis factor (TNF) alpha on RAF1 protein expression in myometrium and ii) siRNA knockdown ofRAF1on pro-inflammatory and pro-labour mediators in human myometrial primary cells. Term labour was associated with an increase in RAF1 protein expression. Furthermore, RAF1 protein expression was increased in myometrial cells treated with IL1B and TNF, two likely factors contributing to preterm birth. Knockdown ofRAF1by siRNA in primary myometrial cells significantly decreased IL1B- and TNF-inducedIL1A, IL1B, IL6,(C-X-C motif) ligand 8 (CXCL8)and chemokine (C-C motif) ligand 2 (CCL2) mRNA abundance and IL6, IL8 and CCL2; prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA levels and prostaglandin PGF2αrelease; and NF-κB activation. Furthermore,RAF1knockdown was associated with decreased activation of ERK in the presence of IL1B but not TNF. Concordantly, the ERK inhibitor U0126 significantly decreased IL1B-inducedIL6,CXCL8,CCL2andPTGS2mRNA abundance; IL6, CXCL8, CCL2 and PGF2αrelease; and NF-κB activation. In conclusion, IL1B induces the expression and secretion of pro-labour mediators through the RAF1–MAPK1–NF-κB signalling pathway. TNF, on the other hand, regulates pro-labour mediators through the RAF1–NF-κB signalling pathway via an MAPK1-independent mechanism.
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17
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Wen YA, Li X, Goretsky T, Weiss HL, Barrett TA, Gao T. Loss of PHLPP protects against colitis by inhibiting intestinal epithelial cell apoptosis. Biochim Biophys Acta Mol Basis Dis 2015; 1852:2013-23. [PMID: 26187040 DOI: 10.1016/j.bbadis.2015.07.012] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2015] [Revised: 07/10/2015] [Accepted: 07/13/2015] [Indexed: 12/12/2022]
Abstract
A common feature of inflammatory bowel disease (IBD) is the loss of intestinal epithelial barrier function due to excessive apoptosis of intestinal epithelial cells (IECs). However, the molecular mechanism underlying increased IEC apoptosis remains unclear. Here, we investigated the role of PHLPP, a novel family of protein phosphatases, in regulating inflammation-induced IEC apoptosis in mouse models of colitis. Both Phlpp1 and Phlpp2 genes were deleted in mice. Compared with wild-type mice, PHLPP double knockout (DKO) mice were protected from colitis induced by DSS as demonstrated by lower histopathological scores, and this reduced susceptibility to colitis was associated with decreased apoptosis and increased Akt activity in IECs in vivo. In addition, epithelial organoids derived from PHLPP DKO mice were more resistant to inflammation-induced apoptosis while inhibition of Akt activity abolished the protective effect of PHLPP-loss. Furthermore, we found that PHLPP expression was significantly reduced in IECs following the induction of colitis by DSS and in human IBD patient samples. This inflammation-induced downregulation of PHLPP was partially blocked by treating cells with a proteasome inhibitor. Taken together, our results indicated that proteasome-mediated degradation of PHLPP at the onset of inflammation plays an important role in protecting IEC injury by inhibiting apoptosis.
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Affiliation(s)
- Yang-An Wen
- Markey Cancer Center, University of Kentucky, Lexington, KY 40536-0509, USA
| | - Xin Li
- Markey Cancer Center, University of Kentucky, Lexington, KY 40536-0509, USA
| | - Tatiana Goretsky
- Division of Digestive Diseases and Nutrition, Department of Internal Medicine, University of Kentucky, Lexington, KY 40536-0509, USA
| | - Heidi L Weiss
- Markey Cancer Center, University of Kentucky, Lexington, KY 40536-0509, USA
| | - Terrence A Barrett
- Division of Digestive Diseases and Nutrition, Department of Internal Medicine, University of Kentucky, Lexington, KY 40536-0509, USA
| | - Tianyan Gao
- Markey Cancer Center, University of Kentucky, Lexington, KY 40536-0509, USA; Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536-0509, USA.
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18
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Kuo WT, Lee TC, Yang HY, Chen CY, Au YC, Lu YZ, Wu LL, Wei SC, Ni YH, Lin BR, Chen Y, Tsai YH, Kung JT, Sheu F, Lin LW, Yu LCH. LPS receptor subunits have antagonistic roles in epithelial apoptosis and colonic carcinogenesis. Cell Death Differ 2015; 22:1590-604. [PMID: 25633197 DOI: 10.1038/cdd.2014.240] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2014] [Revised: 11/24/2014] [Accepted: 12/22/2014] [Indexed: 12/15/2022] Open
Abstract
Colorectal carcinoma (CRC) is characterized by unlimited proliferation and suppression of apoptosis, selective advantages for tumor survival, and chemoresistance. Lipopolysaccharide (LPS) signaling is involved in both epithelial homeostasis and tumorigenesis, but the relative roles had by LPS receptor subunits CD14 and Toll-like receptor 4 (TLR4) are poorly understood. Our study showed that normal human colonocytes were CD14(+)TLR4(-), whereas cancerous tissues were CD14(+)TLR4(+), by immunofluorescent staining. Using a chemical-induced CRC model, increased epithelial apoptosis and decreased tumor multiplicity and sizes were observed in TLR4-mutant mice compared with wild-type (WT) mice with CD14(+)TLR4(+) colonocytes. WT mice intracolonically administered a TLR4 antagonist displayed tumor reduction associated with enhanced apoptosis in cancerous tissues. Mucosa-associated LPS content was elevated in response to CRC induction. Epithelial apoptosis induced by LPS hypersensitivity in TLR4-mutant mice was prevented by intracolonic administration of neutralizing anti-CD14. Moreover, LPS-induced apoptosis was observed in primary colonic organoid cultures derived from TLR4 mutant but not WT murine crypts. Gene silencing of TLR4 increased cell apoptosis in WT organoids, whereas knockdown of CD14 ablated cell death in TLR4-mutant organoids. In vitro studies showed that LPS challenge caused apoptosis in Caco-2 cells (CD14(+)TLR4(-)) in a CD14-, phosphatidylcholine-specific phospholipase C-, sphingomyelinase-, and protein kinase C-ζ-dependent manner. Conversely, expression of functional but not mutant TLR4 (Asp299Gly, Thr399Ile, and Pro714His) rescued cells from LPS/CD14-induced apoptosis. In summary, CD14-mediated lipid signaling induced epithelial apoptosis, whereas TLR4 antagonistically promoted cell survival and cancer development. Our findings indicate that dysfunction in the CD14/TLR4 antagonism may contribute to normal epithelial transition to carcinogenesis, and provide novel strategies for intervention against colorectal cancer.
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Affiliation(s)
- W-T Kuo
- Graduate Institute of Physiology, National Taiwan University College of Medicine, Taipei, Taiwan
| | - T-C Lee
- Graduate Institute of Physiology, National Taiwan University College of Medicine, Taipei, Taiwan.,Department of Internal Medicine, National Taiwan University College of Medicine, Taipei, Taiwan
| | - H-Y Yang
- Graduate Institute of Physiology, National Taiwan University College of Medicine, Taipei, Taiwan
| | - C-Y Chen
- Graduate Institute of Physiology, National Taiwan University College of Medicine, Taipei, Taiwan
| | - Y-C Au
- Graduate Institute of Physiology, National Taiwan University College of Medicine, Taipei, Taiwan
| | - Y-Z Lu
- Graduate Institute of Physiology, National Taiwan University College of Medicine, Taipei, Taiwan
| | - L-L Wu
- Graduate Institute of Physiology, National Taiwan University College of Medicine, Taipei, Taiwan
| | - S-C Wei
- Department of Internal Medicine, National Taiwan University College of Medicine, Taipei, Taiwan
| | - Y-H Ni
- Department of Pediatrics, National Taiwan University College of Medicine, Taipei, Taiwan
| | - B-R Lin
- Department of Surgery, National Taiwan University College of Medicine and Hospital, Taipei, Taiwan
| | - Y Chen
- Department of Surgery, Far Eastern Memorial Hospital, New Taipei, Taiwan.,Department of Chemical Engineering and Material Science, Yuan-Ze University, Tao-Yuan, Taiwan
| | - Y-H Tsai
- Department of Surgery, Far Eastern Memorial Hospital, New Taipei, Taiwan.,Department of Chemical Engineering and Material Science, Yuan-Ze University, Tao-Yuan, Taiwan
| | - J T Kung
- Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan
| | - F Sheu
- Department of Horticulture, National Taiwan University, Taipei, Taiwan
| | - L-W Lin
- Department of Pathology, National Taiwan University Hospital, Yunlin Branch, Yunlin, Taiwan
| | - L C-H Yu
- Graduate Institute of Physiology, National Taiwan University College of Medicine, Taipei, Taiwan
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19
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Williams CS, Bradley AM, Chaturvedi R, Singh K, Piazuelo MB, Chen X, McDonough EM, Schwartz DA, Brown CT, Allaman MM, Coburn LA, Horst SN, Beaulieu DB, Choksi YA, Washington MK, Williams AD, Fisher MA, Zinkel SS, Peek RM, Wilson KT, Hiebert SW. MTG16 contributes to colonic epithelial integrity in experimental colitis. Gut 2013; 62:1446-55. [PMID: 22833394 PMCID: PMC3663894 DOI: 10.1136/gutjnl-2011-301439] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
OBJECTIVE The myeloid translocation genes (MTGs) are transcriptional corepressors with both Mtg8(-/-) and Mtgr1(-/-) mice showing developmental and/or differentiation defects in the intestine. We sought to determine the role of MTG16 in intestinal integrity. METHODS Baseline and stress induced colonic phenotypes were examined in Mtg16(-/-) mice. To unmask phenotypes, we treated Mtg16(-/-) mice with dextran sodium sulphate (DSS) or infected them with Citrobacter rodentium and the colons were examined for ulceration and for changes in proliferation, apoptosis and inflammation. RESULTS Mtg16(-/-) mice have altered immune subsets, suggesting priming towards Th1 responses. Mtg16(-/-) mice developed increased weight loss, diarrhoea, mortality and histological colitis and there were increased innate (Gr1(+), F4/80(+), CD11c(+) and MHCII(+); CD11c(+)) and Th1 adaptive (CD4) immune cells in Mtg16(-/-) colons after DSS treatment. Additionally, there was increased apoptosis and a compensatory increased proliferation in Mtg16(-/-) colons. Compared with wild-type mice, Mtg16(-/-) mice exhibited increased colonic CD4;IFN-γ cells in vehicle-treated and DSS-treated mice. Adoptive transfer of wild-type marrow into Mtg16(-/-) recipients did not rescue the Mtg16(-/-) injury phenotype. Isolated colonic epithelial cells from DSS-treated Mtg16(-/-) mice exhibited increased KC (Cxcl1) mRNA expression when compared with wild-type mice. Mtg16(-/-) mice infected with C rodentium had more severe colitis and greater bacterial colonisation. Last, MTG16 mRNA levels were reduced in human ulcerative colitis versus normal colon tissues. CONCLUSIONS These observations indicate that MTG16 is critical for colonocyte survival and regeneration in response to intestinal injury and provide evidence that this transcriptional corepressor regulates inflammatory recruitment in response to injury.
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Affiliation(s)
- Christopher S Williams
- Department of Medicine, Division of Gastroenterology, Vanderbilt University, Nashville, Tennessee, USA,Department of Cancer Biology, Vanderbilt University, Nashville, Tennessee, USA,Vanderbilt Ingram Cancer Center, Nashville, Tennessee, USA,Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee, USA
| | - Amber M Bradley
- Department of Medicine, Division of Gastroenterology, Vanderbilt University, Nashville, Tennessee, USA
| | - Rupesh Chaturvedi
- Department of Medicine, Division of Gastroenterology, Vanderbilt University, Nashville, Tennessee, USA,Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee, USA
| | - Kshipra Singh
- Department of Medicine, Division of Gastroenterology, Vanderbilt University, Nashville, Tennessee, USA,Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee, USA
| | - Maria B Piazuelo
- Department of Medicine, Division of Gastroenterology, Vanderbilt University, Nashville, Tennessee, USA
| | - Xi Chen
- Department of Biostatistics, Vanderbilt University, Nashville, Tennessee, USA
| | - Elizabeth M McDonough
- Department of Pediatrics, Division of Pediatric Gastroenterology, Vanderbilt University, Nashville, Tennessee, USA
| | - David A Schwartz
- Department of Medicine, Division of Gastroenterology, Vanderbilt University, Nashville, Tennessee, USA
| | - Caroline T Brown
- Department of Medicine, Division of Gastroenterology, Vanderbilt University, Nashville, Tennessee, USA
| | - Margaret M Allaman
- Department of Medicine, Division of Gastroenterology, Vanderbilt University, Nashville, Tennessee, USA
| | - Lori A Coburn
- Department of Medicine, Division of Gastroenterology, Vanderbilt University, Nashville, Tennessee, USA,Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee, USA
| | - Sara N Horst
- Department of Medicine, Division of Gastroenterology, Vanderbilt University, Nashville, Tennessee, USA,Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee, USA
| | - Dawn B Beaulieu
- Department of Medicine, Division of Gastroenterology, Vanderbilt University, Nashville, Tennessee, USA
| | - Yash A Choksi
- Department of Medicine, Division of Gastroenterology, Vanderbilt University, Nashville, Tennessee, USA
| | | | - Amanda D Williams
- Department of Medicine, Division of Gastroenterology, Vanderbilt University, Nashville, Tennessee, USA
| | - Melissa A Fisher
- Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, USA
| | - Sandra S Zinkel
- Department of Cancer Biology, Vanderbilt University, Nashville, Tennessee, USA,Vanderbilt Ingram Cancer Center, Nashville, Tennessee, USA
| | - Richard M Peek
- Department of Medicine, Division of Gastroenterology, Vanderbilt University, Nashville, Tennessee, USA,Vanderbilt Ingram Cancer Center, Nashville, Tennessee, USA,Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee, USA
| | - Keith T Wilson
- Department of Medicine, Division of Gastroenterology, Vanderbilt University, Nashville, Tennessee, USA,Department of Cancer Biology, Vanderbilt University, Nashville, Tennessee, USA,Vanderbilt Ingram Cancer Center, Nashville, Tennessee, USA,Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee, USA
| | - Scott W Hiebert
- Vanderbilt Ingram Cancer Center, Nashville, Tennessee, USA,Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, USA
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20
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Ziogas DC, Gras-Miralles B, Mustafa S, Geiger BM, Najarian RM, Nagel JM, Flier SN, Popov Y, Tseng YH, Kokkotou E. Anti-melanin-concentrating hormone treatment attenuates chronic experimental colitis and fibrosis. Am J Physiol Gastrointest Liver Physiol 2013; 304:G876-84. [PMID: 23538494 PMCID: PMC3652072 DOI: 10.1152/ajpgi.00305.2012] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Fibrosis represents a major complication of several chronic diseases, including inflammatory bowel disease (IBD). Treatment of IBD remains a clinical challenge despite several recent therapeutic advances. Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide shown to regulate appetite and energy balance. However, accumulating evidence suggests that MCH has additional biological effects, including modulation of inflammation. In the present study, we examined the efficacy of an MCH-blocking antibody in treating established, dextran sodium sulfate-induced experimental colitis. Histological and molecular analysis of mouse tissues revealed that mice receiving anti-MCH had accelerated mucosal restitution and lower colonic expression of several proinflammatory cytokines, as well as fibrogenic genes, including COL1A1. In parallel, they spared collagen deposits seen in the untreated mice, suggesting attenuated fibrosis. These findings raised the possibility of perhaps direct effects of MCH on myofibroblasts. Indeed, in biopsies from patients with IBD, we demonstrate expression of the MCH receptor MCHR1 in α-smooth muscle actin(+) subepithelial cells. CCD-18Co cells, a primary human colonic myofibroblast cell line, were also positive for MCHR1. In these cells, MCH acted as a profibrotic modulator by potentiating the effects of IGF-1 and TGF-β on proliferation and collagen production. Thus, by virtue of combined anti-inflammatory and anti-fibrotic effects, blocking MCH might represent a compelling approach for treating IBD.
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Affiliation(s)
| | | | | | | | | | | | | | - Yury Popov
- 1Beth Israel Deaconess Medical Center and
| | - Yu-Hua Tseng
- 2Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts
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21
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Rosen MJ, Chaturvedi R, Washington MK, Kuhnhein LA, Moore PD, Coggeshall SS, McDonough EM, Weitkamp JH, Singh AB, Coburn LA, Williams CS, Yan F, Van Kaer L, Peebles RS, Wilson KT. STAT6 deficiency ameliorates severity of oxazolone colitis by decreasing expression of claudin-2 and Th2-inducing cytokines. THE JOURNAL OF IMMUNOLOGY 2013; 190:1849-58. [PMID: 23303670 DOI: 10.4049/jimmunol.1201373] [Citation(s) in RCA: 66] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Patients suffering from ulcerative colitis (UC) exhibit chronic colonic inflammation caused by a dysregulated mucosal immune response and epithelial barrier disruption. Th2 cytokines, including IL-13, have been implicated in the pathogenesis of UC. IL-13 induces phosphorylation of STAT6, and we previously demonstrated increased epithelial p-STAT6 in children with UC. In this study, we investigated the role of STAT6 in oxazolone colitis, a murine model of UC, by inducing colitis in STAT6-deficient (STAT6(-/-)) and wild type (WT) mice. We observed increased epithelial cell, T cell, macrophage, and NKT cell STAT6 phosphorylation, as well as increased p-STAT6(+) IL-13-producing NKT cells, in colitic WT mice. Colitis was attenuated in STAT6(-/-) mice, with improvements in weight, colon length, and histopathology. There was decreased induction of the pore-forming tight junction protein claudin-2 in STAT6(-/-) mice. Similarly, short hairpin RNA STAT6 knockdown reduced claudin-2 induction and transepithelial resistance decrease in IL-13-treated human T84 cells. Tissue expression of IL-13, IFN-γ, IL-17, and IL-10 mRNA was similarly induced in WT and STAT6(-/-) colitic mice; however, we observed increased mRNA expression for the Th2-inducing cytokines IL-33 and thymic stromal lymphopoietin in WT mice with colitis, which was abrogated in STAT6(-/-) mice. Mesenteric lymph node cells from STAT6(-/-) mice with colitis exhibited reduced secretion of IL-4, IL-5, IL-13, and IFN-γ. IL-33 augmented mesenteric lymph node cell secretion of IL-5, IL-13, IL-6, and IFN-γ. These data implicate STAT6 in the pathogenesis of colitis in vivo with important roles in altering epithelial barrier function and regulating Th2-inducing cytokine production.
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Affiliation(s)
- Michael J Rosen
- Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
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Rose WA, Sakamoto K, Leifer CA. Multifunctional role of dextran sulfate sodium for in vivo modeling of intestinal diseases. BMC Immunol 2012; 13:41. [PMID: 22853702 PMCID: PMC3488029 DOI: 10.1186/1471-2172-13-41] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2012] [Accepted: 07/06/2012] [Indexed: 12/15/2022] Open
Abstract
Background Inflammatory bowel diseases (IBDs) are chronic, relapsing disorders that affect the gastrointestinal tract of millions of people and continue to increase in incidence each year. While several factors have been associated with development of IBDs, the exact etiology is unknown. Research using animal models of IBDs is beginning to provide insights into how the different factors contribute to disease development. Oral administration of dextran sulfate sodium (DSS) to mice induces a reproducible experimental colitis that models several intestinal lesions associated with IBDs. The murine DSS colitis model can also be adapted to quantify intestinal repair following injury. Understanding the mechanistic basis behind intestinal repair is critical to development of new therapeutics for IBDs because of their chronic relapsing nature. Results The murine DSS colitis model was adapted to provide a system enabling the quantification of severe intestinal injury with impaired wound healing or mild intestinal injury with rapid restoration of mucosal integrity, by altering DSS concentrations and including a recovery phase. We showed that through a novel format for presentation of the clinical disease data, the temporal progression of intestinal lesions can be quantified on an individual mouse basis. Additionally, parameters for quantification of DSS-induced alterations in epithelial cell populations are included to provide insights into mechanisms underlying the development of these lesions. For example, the use of the two different model systems showed that toll-like receptor 9, a nucleic acid-sensing pattern recognition receptor, is important for protection only following mild intestinal damage and suggests that this model is superior for identifying proteins necessary for intestinal repair. Conclusions We showed that using a murine DSS-induced experimental colitis model system, and presenting data in a longitudinal manner on a per mouse basis, enhanced the usefulness of this model, and provided novel insights into the role of an innate immune receptor in intestinal repair. By elucidating the mechanistic basis of intestinal injury and repair, we can begin to understand the etiology of IBDs, enabling development of novel therapeutics or prophylactics.
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Affiliation(s)
- William A Rose
- Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
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ErbB2 and ErbB3 regulate recovery from dextran sulfate sodium-induced colitis by promoting mouse colon epithelial cell survival. J Transl Med 2012; 92:437-50. [PMID: 22157714 PMCID: PMC3289719 DOI: 10.1038/labinvest.2011.192] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
ErbB2 and ErbB3 receptor tyrosine kinases are key regulators of proliferation, migration, differentiation and cell survival; however, their roles in gastrointestinal biology remain poorly defined. We hypothesized that ErbB2 and ErbB3 promote colon epithelial cell survival in the context of the wound-healing response following colitis. In this study, mice bearing intestinal epithelial-specific deletion of ErbB2 or ErbB3 were treated with dextran sulfate sodium (DSS). Colon sections were examined for injury, cytokine expression, epithelial cell proliferation and apoptosis. Deletion of epithelial ErbB2 did not affect the extent of intestinal injury in response to DSS, whereas deletion of ErbB3 slightly increased injury. However, the roles of both receptors were more apparent during recovery from DSS colitis, in which ErbB2 or ErbB3 epithelial deletion resulted in greater inflammation and crypt damage during the early reparative period. Moreover, loss of ErbB3 prevented normal epithelial regeneration in the long term, with damage persisting for at least 6 weeks following a single round of DSS. Delayed recovery in mice with epithelial deletion of ErbB2 or ErbB3 was associated with increased colonic expression of tumor necrosis factor alpha and increased epithelial apoptosis. Furthermore, epithelial ErbB3 deletion increased apoptosis at baseline and during DSS injury. Additionally, epithelial cell hyperproliferation during recovery was exacerbated by deletion of either ErbB2 or ErbB3. These results suggest that ErbB2 and ErbB3 have important cytoprotective and reparative roles in the colonic epithelium following injury, by promoting colon epithelial cell survival.
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24
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Yu LCH, Wang JT, Wei SC, Ni YH. Host-microbial interactions and regulation of intestinal epithelial barrier function: From physiology to pathology. World J Gastrointest Pathophysiol 2012; 3:27-43. [PMID: 22368784 PMCID: PMC3284523 DOI: 10.4291/wjgp.v3.i1.27] [Citation(s) in RCA: 175] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/11/2011] [Revised: 10/04/2011] [Accepted: 02/08/2012] [Indexed: 02/06/2023] Open
Abstract
The gastrointestinal tract is the largest reservoir of commensal bacteria in the human body, providing nutrients and space for the survival of microbes while concurrently operating mucosal barriers to confine the microbial population. The epithelial cells linked by tight junctions not only physically separate the microbiota from the lamina propria, but also secrete proinflammatory cytokines and reactive oxygen species in response to pathogen invasion and metabolic stress and serve as a sentinel to the underlying immune cells. Accumulating evidence indicates that commensal bacteria are involved in various physiological functions in the gut and microbial imbalances (dysbiosis) may cause pathology. Commensal bacteria are involved in the regulation of intestinal epithelial cell turnover, promotion of epithelial restitution and reorganization of tight junctions, all of which are pivotal for fortifying barrier function. Recent studies indicate that aberrant bacterial lipopolysaccharide-mediated signaling in gut mucosa may be involved in the pathogenesis of chronic inflammation and carcinogenesis. Our perception of enteric commensals has now changed from one of opportunistic pathogens to active participants in maintaining intestinal homeostasis. This review attempts to explain the dynamic interaction between the intestinal epithelium and commensal bacteria in disease and health status.
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25
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Chang F, Lacey MR, Bouljihad M, Höner Zu Bentrup K, Fortgang IS. Tumor necrosis factor receptor 1 functions as a tumor suppressor. Am J Physiol Gastrointest Liver Physiol 2012; 302:G195-206. [PMID: 22052015 PMCID: PMC3341116 DOI: 10.1152/ajpgi.00209.2011] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/31/2011] [Accepted: 11/02/2011] [Indexed: 01/31/2023]
Abstract
Tumor necrosis factor (TNF) is a key player in inflammatory bowel disease and has been variably associated with carcinogenesis, but details of the cross talk between inflammatory and tumorigenic pathways remain incompletely understood. It has been shown that, in C57BL/6 mice, signaling via TNF receptor 1 (TNFR1) is protective from injury and inflammation in experimental colitis. Therefore, we hypothesized that loss of TNFR1 signaling would confer increased risk of developing colitis-associated carcinoma. Using three models of murine tumorigenesis based on repeated bouts of inflammation or systemic tumor initiator, we sought to determine the roles of TNF and TNFR1 with regard to neoplastic transformation in the colon in wild-type (WT), TNFR1 knockout (R1KO), and TNF knockout (TNFKO) mice. We found R1KO animals to have more severe disease, as defined by weight loss, hematochezia, and histology. TNFKO mice demonstrated less weight loss but were consistently smaller, and rates and duration of hematochezia were comparable to WT mice. Histological inflammation scores were higher and neoplastic lesions occurred more frequently and earlier in R1KO mice. Apoptosis is not affected in R1KO mice although epithelial proliferation following injury is more ardent even before tumorigenesis is apparent. Lastly, there is earlier and more intense expression of activated β-catenin in these mice, implying a connection between TNFR1 and Wnt signaling. Taken together, these findings show that in the context of colitis-associated carcinogenesis TNFR1 functions as a tumor suppressor, exerting this effect not via apoptosis but by modulating activation of β-catenin and controlling epithelial proliferation.
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Affiliation(s)
- Fengqi Chang
- Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Tulane University School of Medicine, New Orleans, LA 70112, USA
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26
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Rosen MJ, Frey MR, Washington MK, Chaturvedi R, Kuhnhein LA, Matta P, Revetta FL, Wilson KT, Polk DB. STAT6 activation in ulcerative colitis: a new target for prevention of IL-13-induced colon epithelial cell dysfunction. Inflamm Bowel Dis 2011; 17:2224-34. [PMID: 21308881 PMCID: PMC3120916 DOI: 10.1002/ibd.21628] [Citation(s) in RCA: 87] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/01/2010] [Accepted: 12/08/2010] [Indexed: 12/12/2022]
Abstract
BACKGROUND Interleukin 13 (IL-13) is upregulated in ulcerative colitis (UC) and increases colon epithelial permeability by inducing apoptosis and expression of the pore-forming tight junction protein claudin-2. IL-13 induces activation of signal transducer and activator of transcription 6 (STAT6). However, the STAT6 phosphorylation status in patients with UC is unknown, as is the effect of STAT6 inhibition on colonic epithelium exposed to IL-13. The study aims were to determine if mucosal STAT6 phosphorylation is increased in patients with UC, and if STAT6 inhibition attenuates IL-13-induced colon epithelial cell dysfunction. METHODS Immunohistochemical staining for phosphorylated (p) STAT6 was performed on colonic tissue from newly diagnosed pediatric subjects with UC (early UC) or Crohn's disease (CD), colectomy tissue from adults with UC (advanced UC), and controls. Colon HT-29 and T84 cells were transfected with STAT6 small interfering RNA (siRNA), or treated with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor that inhibits STAT6, prior to IL-13 treatment. RESULTS The median score for epithelial pSTAT6 was 0 in control subjects, 2 in early UC (versus control P = 0.019), 4 in advanced UC (P = 0.003), and 0 in CD (P = 0.4). Cell transfection with STAT6 siRNA prevented IL-13-induced apoptosis and claudin-2 expression. SAHA inhibited IL-13-induced STAT6 phosphorylation, apoptosis, and claudin-2 expression, and mitigated IL-13-induced reductions in transepithelial resistance. CONCLUSIONS UC is associated with increased colonic epithelial STAT6 phosphorylation, and STAT6 inhibition prevents IL-13-induced apoptosis and barrier disruption. These data identify STAT6 as a novel target for UC treatment and support further study of SAHA as a therapeutic agent.
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Affiliation(s)
- Michael J Rosen
- Department of Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition, Vanderbilt University School of Medicine, Nashville, TN, USA.
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Koch S, Nusrat A. The life and death of epithelia during inflammation: lessons learned from the gut. ANNUAL REVIEW OF PATHOLOGY 2011; 7:35-60. [PMID: 21838548 DOI: 10.1146/annurev-pathol-011811-120905] [Citation(s) in RCA: 107] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Epithelial cells form protective barriers that physically separate an organism from the outside world. Rather than being merely static, impregnable shields, epithelia are highly dynamic structures that can adjust their proliferation, differentiation, and death in response to intrinsic and extrinsic signals. The advantages as well as pitfalls of this flexibility are highlighted in inflammatory disorders such as inflammatory bowel diseases and psoriasis, which are characterized by a chronically dysregulated homeostasis of the epithelium. In recent years, it has become increasingly apparent that epithelial cells communicate with their surroundings through converging, integrated signaling cascades and that even minor alterations in these pathways can have dramatic pathologic consequences. In this review, we discuss how inflammatory cytokines and other signaling molecules, directly or through cross talk, regulate epithelial homeostasis in the intestine, and we highlight parallels and differences in a few other organs.
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Affiliation(s)
- Stefan Koch
- Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
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28
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Koboziev I, Karlsson F, Zhang S, Grisham MB. Pharmacological intervention studies using mouse models of the inflammatory bowel diseases: translating preclinical data into new drug therapies. Inflamm Bowel Dis 2011; 17:1229-45. [PMID: 21312318 PMCID: PMC3075372 DOI: 10.1002/ibd.21557] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/28/2010] [Accepted: 10/04/2010] [Indexed: 12/14/2022]
Abstract
Most therapeutic agents used in clinical practice today were originally developed and tested in animal models so that drug toxicity and safety, dose-responses, and efficacy could be determined. Retrospective analyses of preclinical intervention studies using animal models of different diseases demonstrate that only a small percentage of the interventions reporting promising effects translate to clinical efficacy. The failure to translate therapeutic efficacy from bench to bedside may be due, in part, to shortcomings in the design of the clinical studies; however, it is becoming clear that much of the problem resides within the preclinical studies. One potential strategy for improving our ability to identify new therapeutics that may have a reasonable chance of success in clinical trials is to identify the most immunologically-relevant mouse models of IBD and pharmacologic strategies that most closely mimic the clinical situation. This review presents a critical evaluation of the different mouse models and pharmacological approaches that may be used in intervention studies as well as discuss emerging issues related to study design and data interpretation of preclinical studies.
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Affiliation(s)
- Iurii Koboziev
- Immunology and Inflammation Research Group LSU Health Sciences Center Shreveport, LA 71130
,Department of Molecular and Cellular Physiology LSU Health Sciences Center Shreveport, LA 71130
| | - Fridrik Karlsson
- Immunology and Inflammation Research Group LSU Health Sciences Center Shreveport, LA 71130
,Department of Molecular and Cellular Physiology LSU Health Sciences Center Shreveport, LA 71130
| | - Songlin Zhang
- Immunology and Inflammation Research Group LSU Health Sciences Center Shreveport, LA 71130
,Department of Pathology LSU Health Sciences Center Shreveport, LA 71130
| | - Matthew B. Grisham
- Immunology and Inflammation Research Group LSU Health Sciences Center Shreveport, LA 71130
,Department of Molecular and Cellular Physiology LSU Health Sciences Center Shreveport, LA 71130
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29
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Guo X, Roberts MR, Becker SM, Podd B, Zhang Y, Chua SC, Myers MG, Duggal P, Houpt ER, Petri WA. Leptin signaling in intestinal epithelium mediates resistance to enteric infection by Entamoeba histolytica. Mucosal Immunol 2011; 4:294-303. [PMID: 21124310 PMCID: PMC3079783 DOI: 10.1038/mi.2010.76] [Citation(s) in RCA: 91] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
Leptin is an adipocytokine that links nutrition to immunity. Previous observation that a genetic polymorphism in the leptin receptor affected susceptibility to Entamoeba histolytica infection led to the hypothesis that leptin signaling has a protective role during intestinal amebic infection. In this study we show that mice lacking the functional leptin receptor developed devastating mucosal destruction after E. histolytica infection. Bone marrow chimera experiments demonstrated that leptin receptor expressed on hematopoietic cells was not sufficient to confer resistance. Similarly, peripheral knockout of the leptin receptor rendered animals susceptible, indicating that central expression of the leptin receptor was not sufficient to confer protection. The site of leptin action was localized to the gut via an intestinal epithelium-specific deletion of the leptin receptor, which rendered mice susceptible to infection and mucosal destruction by the parasite. Mutation of tyrosine 985 or 1138 in the intracellular domain of the leptin receptor, which mediates signaling through the SH2-containing tyrosine phosphatase/extracellular signal-regulated kinase (SHP2/ERK) and signal transducer and activator of transcription 3 (STAT3) pathways, respectively, demonstrated that both were important for mucosal protection. We conclude that leptin-mediated resistance to amebiasis is via its actions on intestinal epithelium rather than hematopoietic cells or the brain, and requires leptin receptor signaling through both the STAT3 and SHP2/ERK pathways.
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Affiliation(s)
- Xiaoti Guo
- Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, Virginia
| | - Margo R. Roberts
- Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, Virginia
| | - Stephen M. Becker
- Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, Virginia
| | - Bradley Podd
- Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, Virginia
| | - Yiying Zhang
- Department of Pediatrics, Division of Molecular Genetics & Naomi Berrie Diabetes Center Columbia University Medical Center, New York, New York
| | - Streamson C. Chua
- Departments of Medicine and Neuroscience, Albert Einstein College of Medicine, Bronx, New York
| | - Martin G. Myers
- Departments of Internal Medicine & Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan
| | - Priya Duggal
- Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland
| | - Eric R. Houpt
- Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, Virginia.,Corresponding Authors: William A. Petri, Jr., Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, VA, 434 924 5621 (direct), 434 924 0075 (fax) , *Eric R. Houpt, Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, VA, 434 243 9326 (direct), 434 924 0075 (fax)
| | - William A. Petri
- Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, Virginia
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30
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Singh K, Chaturvedi R, Barry DP, Coburn LA, Asim M, Lewis ND, Piazuelo MB, Washington MK, Vitek MP, Wilson KT. The apolipoprotein E-mimetic peptide COG112 inhibits NF-kappaB signaling, proinflammatory cytokine expression, and disease activity in murine models of colitis. J Biol Chem 2011; 286:3839-50. [PMID: 21115487 PMCID: PMC3030385 DOI: 10.1074/jbc.m110.176719] [Citation(s) in RCA: 63] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2010] [Indexed: 12/12/2022] Open
Abstract
Inflammatory bowel disease (IBD), consisting of Crohn's disease and ulcerative colitis, is a source of substantial morbidity and remains difficult to treat. New strategies for beneficial anti-inflammatory therapies would be highly desirable. Apolipoprotein (apo) E has immunomodulatory effects and synthetically derived apoE-mimetic peptides are beneficial in models of sepsis and neuroinflammation. We have reported that the antennapedia-linked apoE-mimetic peptide COG112 inhibits the inflammatory response to the colitis-inducing pathogen Citrobacter rodentium in vitro by inhibiting NF-κB activation. We now determined the effect of COG112 in mouse models of colitis. Using C. rodentium as an infection model, and dextran sulfate sodium (DSS) as an injury model, mice were treated with COG112 by intraperitoneal injection. With C. rodentium, COG112 improved the clinical parameters of survival, body weight, colon weight, and histologic injury. With DSS, COG112 ameliorated the loss of body weight, reduction in colon length, and histologic injury, whether administered concurrently with induction of colitis, during induction plus recovery, or only during the recovery phase of disease. In both colitis models, COG112 inhibited colon tissue inducible nitric-oxide synthase (iNOS), KC, TNF-α, IFN-γ, and IL-17 mRNA expression, and reduced nuclear translocation of NF-κB, as determined by immunoblot and immunofluorescence confocal microscopy. IκB kinase (IKK) activity was also reduced, which is necessary for activation of the canonical NF-κB pathway. Isolated colonic epithelial cells exhibited marked attenuation of expression of iNOS and the CXC chemokines KC and MIP-2. These studies indicate that apoE-mimetic peptides such as COG112 are novel potential therapies for IBD.
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Affiliation(s)
- Kshipra Singh
- From the Departments of Medicine, Division of Gastroenterology
- the Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee 37212, and
| | - Rupesh Chaturvedi
- From the Departments of Medicine, Division of Gastroenterology
- the Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee 37212, and
| | - Daniel P. Barry
- From the Departments of Medicine, Division of Gastroenterology
| | - Lori A. Coburn
- the Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee 37212, and
| | - Mohammad Asim
- From the Departments of Medicine, Division of Gastroenterology
- the Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee 37212, and
| | - Nuruddeen D. Lewis
- From the Departments of Medicine, Division of Gastroenterology
- Cancer Biology, Vanderbilt University Medical Center, Nashville, Tennessee 37232
| | | | | | | | - Keith T. Wilson
- From the Departments of Medicine, Division of Gastroenterology
- Cancer Biology, Vanderbilt University Medical Center, Nashville, Tennessee 37232
- the Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee 37212, and
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31
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Wimmer R, Baccarini M. Partner exchange: protein-protein interactions in the Raf pathway. Trends Biochem Sci 2010; 35:660-8. [PMID: 20621483 DOI: 10.1016/j.tibs.2010.06.001] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2010] [Revised: 05/27/2010] [Accepted: 06/07/2010] [Indexed: 01/07/2023]
Abstract
The three-tiered Raf-MEK-ERK kinase module is activated downstream of Ras and has been traditionally linked to cellular proliferation. Mammals have three Raf, two Mek and two Erk genes. Recently, the analysis of protein-protein interactions in the pathway has begun to provide a rationale for the redundancy within each tier. New results show that the MEK-ERK-activating unit consists of Raf hetero- and homodimers; downstream of Raf, MEK1-MEK2 heterodimers and ERK dimers are required for temporal and spatial pathway regulation. Finally, C-Raf mediates pathway crosstalk downstream of Ras by directly binding to and inhibiting kinases engaged in other signaling cascades. Given the roles of these interactions in tumorigenesis, their study will provide new opportunities for molecule-based therapies that target the pathway.
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Affiliation(s)
- Reiner Wimmer
- University of Vienna, Center for Molecular Biology, Max F. Perutz Laboratories, Doktor-Bohr-Gasse 9, A-1030 Vienna, Austria
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32
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Xia XM, Wang FY, Xu WA, Wang ZK, Liu J, Lu YK, Jin XX, Lu H, Shen YZ. CXCR4 antagonist AMD3100 attenuates colonic damage in mice with experimental colitis. World J Gastroenterol 2010; 16:2873-80. [PMID: 20556832 PMCID: PMC2887582 DOI: 10.3748/wjg.v16.i23.2873] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the effects of the chemokine stromal cell-derived factor-1 (CXCL12) receptor (CXCR4) antagonist AMD3100 on colonic inflammation and epithelial barrier in dextran sulfate sodium (DSS)-induced colitis in mice.
METHODS: Experimental colitis was induced by administration of 5% DSS for 7 d, and assays performed on intestinal segments from the ileocecal valve to the anus. Colonic morphology was examined by hematoxylin and eosin staining. Colonic cytokines were determined by enzyme-linked immunosorbent assay. Myeloperoxidase (MPO) activity (indicator of inflammatory infiltration) was observed spectrophotometrically. Gut permeability was assessed by mucosal-to-serosal clearance of fluorescein isothiocyanate-conjugated dextran 4000 (FD4) in everted gut sacs. The apoptosis of colonic epithelium was assessed by Hoechst-33342 staining. To further elucidate the role of CXCR4 in colonic inflammation, we also investigated the effect of AMD3100 on migration and cytokine production of isolated peripheral blood mononuclear cells (PBMCs).
RESULTS: DSS-induced colitis was characterized by morphologic changes, as well as increased colonic cytokines, inflammatory infiltration, epithelial apoptosis, and intestinal permeability in mice. In AMD3100-treated mice, epithelial destruction, inflammatory infiltration, and submucosal edema were markedly reduced; colonic tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interferon-γ (IFN-γ) levels, as well as MPO activity were significantly decreased. Increased intestinal permeability in DSS-treated mice was significantly reduced by AMD3100. The number of apoptotic cells in colitis mice was markedly increased after DSS administration, and decreased when treated with the CXCR4 antagonist AMD3100. In pre-activated PBMCs, CXCL12 stimulation significantly increased the migration of PBMCs, and was inhibited by AMD3100. Moderately increased TNF-α, IL-6, and IFN-γ from CXCL12-treated PBMCs were also reduced by AMD3100.
CONCLUSION: The CXCR4 antagonist AMD3100 exerts therapeutic effects on experimental colitis by inhibiting colonic inflammation and enhancing epithelial barrier integrity.
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33
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Stadnyk AW. When expression is not enough: the evolving thoughts on Fas/FasL and epithelial cell apoptosis. Inflamm Bowel Dis 2010; 16:1061-2. [PMID: 20027648 DOI: 10.1002/ibd.21197] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/09/2022]
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34
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Yan F, Polk DB. Disruption of NF-kappaB signalling by ancient microbial molecules: novel therapies of the future? Gut 2010; 59:421-6. [PMID: 20332512 PMCID: PMC3036986 DOI: 10.1136/gut.2009.179614] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Affiliation(s)
- Fang Yan
- Department of Pediatrics, Monroe Carrel Jr Children’s Hospital at Vanderbilt, Vanderbilt University School of Medicine, Nashville, Tennessee, USA, Digestive Diseases Research Center, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
| | - D Brent Polk
- Department of Pediatrics, Monroe Carrel Jr Children’s Hospital at Vanderbilt, Vanderbilt University School of Medicine, Nashville, Tennessee, USA, Digestive Diseases Research Center, Vanderbilt University School of Medicine, Nashville, Tennessee, USA, Department of Cell & Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
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35
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Vongsa RA, Zimmerman NP, Dwinell MB. CCR6 regulation of the actin cytoskeleton orchestrates human beta defensin-2- and CCL20-mediated restitution of colonic epithelial cells. J Biol Chem 2009; 284:10034-45. [PMID: 19233848 DOI: 10.1074/jbc.m805289200] [Citation(s) in RCA: 63] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Intestinal inflammation is exacerbated by defects in the epithelial barrier and subsequent infiltration of microbes and toxins into the underlying mucosa. Production of chemokines and antimicrobial peptides by an intact epithelium provide the first line of defense against invading organisms. In addition to its antimicrobial actions, human beta defensin-2 (HBD2) may also stimulate the migration of dendritic cells through binding the chemokine receptor CCR6. As human colonic epithelium expresses CCR6, we investigated the potential of HBD2 to stimulate intestinal epithelial migration. Using polarized human intestinal Caco2 and T84 cells and non-transformed IEC6 cells, HBD2 was equipotent to CCL20 in stimulating migration. Neutralizing antibodies confirmed HBD2 and CCL20 engagement to CCR6 were sufficient to induce epithelial cell migration. Consistent with restitution, motogenic concentrations of HBD2 and CCL20 did not induce proliferation. Stimulation with those CCR6 ligands leads to calcium mobilization and elevated active RhoA, phosphorylated myosin light chain, and F-actin accumulation. HBD2 and CCL20 were unable to stimulate migration in the presence of either Rho-kinase or phosphoinositide 3-kinase inhibitors or an intracellular calcium chelator. Together, these data indicate that the canonical wound healing regulatory pathway, along with calcium mobilization, regulates CCR6-directed epithelial cell migration. These findings expand the mechanistic role for chemokines and HBD2 in mucosal inflammation to include immunocyte trafficking and killing of microbes with the concomitant activation of restitutive migration and barrier repair.
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Affiliation(s)
- Rebecca A Vongsa
- Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA
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36
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Edelblum KL, Goettel JA, Koyama T, McElroy SJ, Yan F, Polk DB. TNFR1 promotes tumor necrosis factor-mediated mouse colon epithelial cell survival through RAF activation of NF-kappaB. J Biol Chem 2008; 283:29485-94. [PMID: 18713739 PMCID: PMC2570867 DOI: 10.1074/jbc.m801269200] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Tumor necrosis factor (TNF) is a therapeutic target in the treatment of inflammatory bowel disease; however, the exact role of TNF signaling in the colon epithelium remains unclear. We demonstrate that TNF activation of TNF receptor (R)1 stimulates both pro- and anti-apoptotic signaling pathways in the colon epithelium; however, TNFR1 protects against colon epithelial cell apoptosis following TNF exposure. To investigate anti-apoptotic signaling pathways downstream of TNFR1, we generated an intestinal epithelium-specific Raf knock-out mouse and identified Raf kinase as a key regulator of colon epithelial cell survival in response to TNF. Surprisingly, Raf promotes NF-kappaB p65 phosphorylation, independent of MEK signaling, to support cell survival. Taken together, these data demonstrate a novel pathway in which Raf promotes colon epithelial cell survival through NF-kappaB downstream of TNFR1 activation. Thus, further understanding of colon epithelial cell-specific TNFR signaling may result in the identification of new targets for inflammatory bowel disease treatment and define novel mediators of colitis-associated cancer.
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Affiliation(s)
- Karen L Edelblum
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0696, USA
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