Abstract
Biliary atresia is a neonatal liver disease with extrahepatic bile duct obstruction and progressive liver fibrosis. The etiology and pathogenesis of the disease are unknown. We previously identified a plant toxin, biliatresone, responsible for biliary atresia in naturally-occurring animal models, that causes cholangiocyte destruction in in-vitro models. Decreases in reduced glutathione (GSH) mimic the effects of biliatresone, and agents that replenish cellular GSH ameliorate the effects of the toxin. The goals of this study were to define signaling pathways downstream of biliatresone that lead to cholangiocyte destruction and to determine their relationship to GSH. Using cholangiocyte culture and 3D cholangiocyte spheroid cultures, we found that biliatresone and decreases in GSH upregulated RhoU/Wrch1, a Wnt signaling family member, which then mediated an increase in Hey2 in the NOTCH signaling pathway, causing downregulation of the transcription factor Sox17. When these genes were up- or down-regulated, the biliatresone effect on spheroids was phenocopied, resulting in lumen obstruction. Biopsies of patients with biliary atresia demonstrated increased RhoU/Wrch1 and Hey2 expression in cholangiocytes. We present a novel pathway of cholangiocyte injury in a model of biliary atresia, which is relevant to human BA and may suggest potential future therapeutics.
Highlights
Biliary Atresia (BA) is a pediatric liver disease which occurs in neonates and is characterized by extrahepatic bile duct obstruction and progressive liver fibrosis
In mouse cholangiocytes cultured in 3D as spheroids, biliatresone treatment resulted in loss of lumens, cholangiocyte polarity changes including microtubule destabilization, and increased monolayer permeability; in bile duct explants from neonatal pups, biliatresone treatment led to lumen obstruction and sub epithelial fibrosis[12,13,14]
We found that biliatresone decreased mRNA levels of the transcription factor Sox[17] and that Sox[17] silencing phenocopied the effects of biliatresone[14]
Summary
Biliary Atresia (BA) is a pediatric liver disease which occurs in neonates and is characterized by extrahepatic bile duct obstruction and progressive liver fibrosis. Biliatresone binds strongly to reduced glutathione (GSH)[12,15] It causes decreases in intracellular levels of GSH; artificial reductions in GSH through treatment with DL-buthionine sulfoximine (BSO) mimic the phenotype of biliatresone treatment. In Sox[17] haploinsufficient mice, a BA-like phenotype with hypoplastic gallbladder epithelia detached cells from the luminal wall, leading to bile duct stenosis or atresia[22]. In this work we identified three genes as key players in biliatresone-mediated cholangiocyte injury: RhoU/ Wrch[1], Hey[2] and Sox[17]. Understanding the cellular mechanisms underlying biliatresone-induced cholangiocyte injury, may provide valuable insight into BA in humans
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