Post-translational Regulation of the Type III Inositol 1,4,5-Trisphosphate Receptor by miRNA-506

Background: Inositol 1,4,5-trisphosphate receptor (InsP(3)R3) is critical to secretion in a number of epithelia and its expression is lost in secretory disorders. Results: miR-506 down-regulates InsP(3)R3 expression and impairs Ca2+ signaling and secretion. Conclusion: Post-translational regulation of InsP(3)R3 expression by miR-506 might contribute to disease phenotype. Significance: Restoring InsP(3)R3 expression by use of anti-miR-506 therapy might be beneficial in a variety of secretory disorders. The type III isoform of the inositol 1,4,5-trisphosphate receptor (InsP(3)R3) is apically localized and triggers Ca2+ 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 InsP(3)R3 in primary bile duct epithelia (cholangiocytes) and in the H69 cholangiocyte cell line, because the role of InsP(3)R3 in cholangiocyte Ca2+ signaling and secretion is well established and because loss of InsP(3)R3 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 InsP(3)R3 mRNA revealed two highly conserved binding sites for miR-506. Transfection of miR-506 mimics into cell lines expressing InsP(3)R3-3UTR-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. InsP(3)R3 protein levels were decreased by miR-506 mimics and increased by inhibitors, and InsP(3)R3 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, InsP(3)-mediated Ca2+ signals were decreased by 50% in stable miR-506 cells compared with controls. Finally, InsP(3)R3-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 InsP(3)R3 expression and InsP(3)R3-mediated Ca2+ signaling and secretion.