Protein Quality Control is a Risk Factor and Therapeutic Target in Toxin-Induced Biliary Atresia

Abstract

BACKGROUND and AIMS Extra-hepatic biliary atresia (BA) is a pediatric liver disease with no approved medical therapy. Recent studies using human samples and experimental modeling suggest that glutathione redox metabolism and heterogeneity play a role in disease pathogenesis. We sought to dissect the mechanistic basis of liver redox variation and explore how other stress responses affect cholangiocyte injury in BA.METHODS We performed quantitative in situ hepatic glutathione redox mapping in zebrafish larvae carrying targeted mutations in glutathione metabolism genes and correlated these findings with sensitivity to the plant-derived BA-linked toxin biliatresone. We also determined whether genetic disruption of HSP90 protein quality control pathway genes implicated in human BA altered biliatresone toxicity in zebrafish and human cholangiocytes. An in vivo screen of a known drug library was performed to identify novel modifiers of cholangiocyte injury in the zebrafish experimental BA model with subsequent validation.RESULTS Glutathione metabolism gene mutations caused regionally distinct changes in the redox potential of cholangiocytes that differentially sensitized them to biliatresone. Disruption of human BA-implicated HSP90 pathway genes sensitized zebrafish and human cholangiocytes to biliatresone-induced injury independent of glutathione. Phosphodiesterase-5 inhibitors (PDE5i) and other cGMP signaling activators worked synergistically with the glutathione precursor N- acetylcysteine (NAC) in preventing biliatresone-induced injury in zebrafish and human cholangiocytes. PDE5i enhanced proteasomal degradation and required intact HSP90 chaperone.CONCLUSION Regional variation in glutathione metabolism underlies sensitivity to the biliary toxin biliatresone, and mirrors recently reported BA risk stratification linked to glutathione metabolism gene expression. Human BA can be causatively linked to genetic modulation of protein quality control. Combined treatment with NAC and cGMP signaling enhancers warrants further investigation as therapy for BA.Background and Context Biliary atresia (BA) is an obstructive fibrosing cholangiopathy that is the leading indication for liver transplantation in the pediatric population. There are no known treatments to prevent progressive liver injury after surgical restoration of bile flow.New Findings The authors identify factors that affect susceptibility of cholangiocytes to oxidative injury using a toxin-induced BA model. This information is used to validate genetic risk factors for human BA and identified PDE5i as a potential treatment for biliary atresia, either on its own or in combination with the anti-oxidant N-acetyl-cysteine.Limitations The work done in animal and cell culture models needs further study in human tissue-derived models and a larger cohort of BA patients.Impact The findings from this study provide a rationale for identifying new genetic risk factors that predispose to BA and for an interventional study to prevent progressive liver injury in this enigmatic disease.Short Summary This study uses zebrafish and human cell culture models to identify novel injury mechanisms, genetic risk factors and new therapies for the pediatric liver disease biliary atresia.Competing Interest StatementThe authors have declared no competing interest.Abbreviations17-AAG7-N-allylamino-17-demethoxygeldanamycin;BAbiliary atresia;bilbiliatresone;8-Br-cGMP8-Bromoguanosine 3’,5’-cyclic monophosphate;EHCextrahepatic cholangiocytes;Gclglutamate cysteine ligase;GGAgeranylgeranyl acetate;gsrglutathione reductase;GSHreduced glutathione;GSSGoxidized glutathione;IHCintrahepatic cholangiocytes;NACN-acetylcysteine;PDE5iPhosphodiesterase-5 inhibitors;PQCprotein quality control;WTwild-typeView Full Text