Dysregulated activation of Hippo-YAP1 signaling induces oxidative stress and aberrant development of intrahepatic biliary cells in biliary atresia

Abstract

The canonical Hippo-YAP1 signaling pathway is crucial for liver development and regeneration but its role in repair and regeneration of intrahepatic bile duct in biliary atresia (BA) remains largely unknown. YAP1 expression in the liver tissues of patients with BA and rhesus rotavirus (RRV) induced experimental BA mouse model were examined using quantitative reverse transcriptase-PCR (qRT-PCR) and double immunofluorescence. Mouse EpCAM-expressing cells derived liver organoids were generated and treated with Hippo-YAP1 pathway activators (Xmu-mp-1 and TRULI) or an inhibitor (Peptide17). Morphological, immunofluorescence, RNA-seq, and bioinformatic analyses were performed. Oxidative stress in human intrahepatic biliary epithelial cells (HiBECs) transfected with a constitutively active YAP1 (YAPS127A) plasmid was assessed using qRT-PCR and fluorescence-activated cell sorting analysis. PRDX1 expression in BA and experimental BA mouse model livers was examined by double immunofluorescence. The mRNA expression and nuclear localization of YAP1 in EpCAM-expressing bile duct cells were increased in the livers of BA and experimental BA mouse model. Aberrant development of intrahepatic organoids, differential expression of oxidative stress response genes Sod3 and Prdx1, enrichment of oxidative stress, and mitochondrial reactive oxidative stress (mito-ROS)-associated gene sets were observed in organoids treated with Hippo-YAP1 activators, whereas organoid development was unaffected by the addition of the Hippo-YAP1 inhibitor. Transfection with constitutively active YAP1 led to the downregulation of PRDX1 and oxidative stress in HiBECs. Additionally, reduced PRDX1 expression was also observed in the bile duct of human BA and experimental BA mouse livers. In conclusion, dysregulated activation of Hippo-YAP1 signaling induces oxidative stress and impairs the development of intrahepatic biliary organoids, which indicates therapeutic strategies targeting Hippo-YAP1 signaling may offer potential to improve biliary repair and regeneration in patients with BA.

Keywords: Biliary atresia; Biliary repair and regeneration; Organoids; Reactive oxygen species; YAP1.