Post-translational regulation of the type III inositol 1,4,5-trisphosphate receptor by miRNA-506.
The type III isoform of the inositol 1,4,5-trisphosphate receptor (InsP3R3) is apically localized and triggers Ca(2+) waves and secretion in a number of polarized epithelia. However, nothing is known about epigenetic regulation of this InsP3R isoform. We investigated miRNA regulation of InsP3R3 in primary bile duct epithelia (cholangiocytes) and in the H69 cholangiocyte cell line, because the role of InsP3R3 in cholangiocyte Ca(2+) signaling and secretion is well established and because loss of InsP3R3 from cholangiocytes is responsible for the impairment in bile secretion that occurs in a number of liver diseases. Analysis of the 3'-UTR of human InsP3R3 mRNA revealed two highly conserved binding sites for miR-506. Transfection of miR-506 mimics into cell lines expressing InsP3R3-3'UTR-luciferase led to decreased reporter activity, whereas co-transfection with miR-506 inhibitors led to enhanced activity. Reporter activity was abrogated in isolated mutant proximal or distal miR-506 constructs in miR-506-transfected HEK293 cells. InsP3R3 protein levels were decreased by miR-506 mimics and increased by inhibitors, and InsP3R3 expression was markedly decreased in H69 cells stably transfected with miR-506 relative to control cells. miR-506-H69 cells exhibited a fibrotic signature. In situ hybridization revealed elevated miR-506 expression in vivo in human-diseased cholangiocytes. Histamine-induced, InsP3-mediated Ca(2+) signals were decreased by 50% in stable miR-506 cells compared with controls. Finally, InsP3R3-mediated fluid secretion was significantly decreased in isolated bile duct units transfected with miR-506, relative to control IBDU. Together, these data identify miR-506 as a regulator of InsP3R3 expression and InsP3R3-mediated Ca(2+) signaling and secretion.
From the Departments of Medicine and ananth.meena@yale.edu.
Meenakshisundaram
M
Ananthanarayanan
the Department of Liver and Gastrointestinal Diseases, Biodonostia Research Institute, Donostia University Hospital, University of Basque Country (UPV/EHU), CIBERehd, IKERBASQUE, AECC, 20014 San Sebastian, Spain, and the Division of Gene Therapy and Hepatology, CIMA of the University of Navarra, Ciberehd, 31009 Pamplona, Spain.
Jesus M
JM
Banales
From the Departments of Medicine and.
Mateus T
MT
Guerra
From the Departments of Medicine and.
Carlo
C
Spirli
the Department of Liver and Gastrointestinal Diseases, Biodonostia Research Institute, Donostia University Hospital, University of Basque Country (UPV/EHU), CIBERehd, IKERBASQUE, AECC, 20014 San Sebastian, Spain, and.
Patricia
P
Munoz-Garrido
Pathology, Section of Digestive Diseases and the Liver Center, Yale University School of Medicine, New Haven, Connecticut 06520.
Kisha
K
Mitchell-Richards
From the Departments of Medicine and.
Denisse
D
Tafur
the Division of Gene Therapy and Hepatology, CIMA of the University of Navarra, Ciberehd, 31009 Pamplona, Spain.
Elena
E
Saez
From the Departments of Medicine and.
Michael H
MH
Nathanson
D020413:3' Untranslated Regions; D001483:Base Sequence; D001652:Bile Ducts / Q000378:metabolism / Q000473:pathology; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002460:Cell Line; D044127:Epigenesis, Genetic; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000235:genetics* / Q000378:metabolism; D008099:Liver / Q000378:metabolism / Q000473:pathology; D008105:Liver Cirrhosis, Biliary / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding
D020413:3' Untranslated Regions; D053496:Inositol 1,4,5-Trisphosphate Receptors; C561508:MIRN506 microRNA, human; D035683:MicroRNAs; D008156:Luciferases; D002118:Calcium
Calcium; Cholangiocytes; Epithelial Cell; IP3 Receptor Type III; IP3 Receptor Type III H69 Cells; Inositol 1,4,5-Trisphosphate (IP3); MicroRNA (miRNA); Secretion; miR-506