Oihane Erice1, Patricia Munoz-Garrido1,2, Javier Vaquero3,4, Maria J Perugorria1,2,5, Maite G Fernandez-Barrena2,6, Elena Saez6, Alvaro Santos-Laso1, Ander Arbelaiz1, Raul Jimenez-Agüero1, Joaquin Fernandez-Irigoyen7, Enrique Santamaria7, Verónica Torrano8,9, Arkaitz Carracedo5,8,9,10, Meenakshisundaram Ananthanarayanan11, Marco Marzioni12, Jesus Prieto2,6, Ulrich Beuers13, Ronald P Oude Elferink13, Nicholas F LaRusso14, Luis Bujanda1,2, Jose J G Marin2,3, Jesus M Banales1,2,5. 1. Department of Liver and Gastrointestinal Diseases, Biodonostia Research Institute-Donostia University Hospital, University of the Basque Country (UPV/EHU), San Sebastian, Spain. 2. National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd), Carlos III National Institute of Health, Madrid, Spain. 3. Experimental Hepatology and Drug Targeting (HEVEFARM), Biomedical Research Institute of Salamanca (IBSAL), University of Salamanca, Salamanca, Spain. 4. Sorbonne Universités, UPMC Université Paris 06, INSERM, Saint-Antoine Research Center, Paris, and Fondation ARC, Villejuif, France. 5. IKERBASQUE, Basque Foundation for Science, Bilbao, Spain. 6. Division of Hepatology, CIMA of the University of Navarra, Pamplona, Spain. 7. Proteored-ISCIII, Proteomics Unit, Navarrabiomed, Navarra Health Department, Public University of Navarra, Navarra Institute for Health Research (IdiSNA), Pamplona, Spain. 8. CIC bioGUNE, Bizkaia Technology Park, Derio, Bizkaia, Spain. 9. CIBERONC. 10. Biochemistry and Molecular Biology Department, University of the Basque Country (UPV/EHU), Bilbao, Spain. 11. Liver Center, Yale University School of Medicine, New Haven, CT. 12. Department of Gastroenterology, Università Politecnica delle Marche, Ancona, Italy. 13. Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands. 14. Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN.
Abstract
Primary biliary cholangitis (PBC) is a chronic cholestatic liver disease associated with autoimmune phenomena targeting intrahepatic bile duct cells (cholangiocytes). Although its etiopathogenesis remains obscure, development of antimitochondrial autoantibodies against pyruvate dehydrogenase complex E2 is a common feature. MicroRNA (miR) dysregulation occurs in liver and immune cells of PBC patients, but its functional relevance is largely unknown. We previously reported that miR-506 is overexpressed in PBC cholangiocytes and directly targets both Cl- / HCO3- anion exchanger 2 and type III inositol 1,4,5-trisphosphate receptor, leading to cholestasis. Here, the regulation of miR-506 gene expression and its role in cholangiocyte pathophysiology and immune activation was studied. Several proinflammatory cytokines overexpressed in PBC livers (such as interleukin-8 [IL8], IL12, IL17, IL18, and tumor necrosis factor alpha) stimulated miR-506 promoter activity in human cholangiocytes, as revealed by luciferase reporter assays. Experimental overexpression of miR-506 in cholangiocytes dysregulated the cell proteomic profile (by mass spectrometry), affecting proteins involved in different biological processes including mitochondrial metabolism. In cholangiocytes, miR-506 (1) induced dedifferentiation with down-regulation of biliary and epithelial markers together with up-regulation of mesenchymal, proinflammatory, and profibrotic markers; (2) impaired cell proliferation and adhesion; (3) increased oxidative and endoplasmic reticulum stress; (4) caused DNA damage; and (5) sensitized to caspase-3-dependent apoptosis induced by cytotoxic bile acids. These events were also associated with impaired energy metabolism in mitochondria (proton leak and less adenosine triphosphate production) and pyruvate dehydrogenase complex E2 overexpression. Coculture of miR-506 overexpressing cholangiocytes with PBC immunocytes induced activation and proliferation of PBC immunocytes. CONCLUSION: Different proinflammatory cytokines enhance the expression of miR-506 in biliary epithelial cells; miR-506 induces PBC-like features in cholangiocytes and promotes immune activation, representing a potential therapeutic target for PBC patients. (Hepatology 2018;67:1420-1440).
Primary biliary cholangitis (PBC) is a chronic cholestatic liver disease associated with autoimmune phenomena targeting intrahepatic bile duct cells (cholangiocytes). Although its etiopathogenesis remains obscure, development of antimitochondrial autoantibodies against pyruvate dehydrogenase complex E2 is a common feature. MicroRNA (miR) dysregulation occurs in liver and immune cells of PBCpatients, but its functional relevance is largely unknown. We previously reported that miR-506 is overexpressed in PBC cholangiocytes and directly targets both Cl- / HCO3- anion exchanger 2 and type III inositol 1,4,5-trisphosphate receptor, leading to cholestasis. Here, the regulation of miR-506 gene expression and its role in cholangiocyte pathophysiology and immune activation was studied. Several proinflammatory cytokines overexpressed in PBC livers (such as interleukin-8 [IL8], IL12, IL17, IL18, and tumor necrosis factor alpha) stimulated miR-506 promoter activity in human cholangiocytes, as revealed by luciferase reporter assays. Experimental overexpression of miR-506 in cholangiocytes dysregulated the cell proteomic profile (by mass spectrometry), affecting proteins involved in different biological processes including mitochondrial metabolism. In cholangiocytes, miR-506 (1) induced dedifferentiation with down-regulation of biliary and epithelial markers together with up-regulation of mesenchymal, proinflammatory, and profibrotic markers; (2) impaired cell proliferation and adhesion; (3) increased oxidative and endoplasmic reticulum stress; (4) caused DNA damage; and (5) sensitized to caspase-3-dependent apoptosis induced by cytotoxic bile acids. These events were also associated with impaired energy metabolism in mitochondria (proton leak and less adenosine triphosphate production) and pyruvate dehydrogenase complex E2 overexpression. Coculture of miR-506 overexpressing cholangiocytes with PBC immunocytes induced activation and proliferation of PBC immunocytes. CONCLUSION: Different proinflammatory cytokines enhance the expression of miR-506 in biliary epithelial cells; miR-506 induces PBC-like features in cholangiocytes and promotes immune activation, representing a potential therapeutic target for PBCpatients. (Hepatology 2018;67:1420-1440).
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