181. Real-Time Monitoring of Transcription Factor Activity Using In Vitro and In Vivo Models of Cholestasis

Abstract

Biliary obstruction results in cholestasis characterised by fibrosis, progressive cirrhosis and cholangiocyte hyperplasia leading to liver failure. The underlying molecular mechanisms remain unclear but likely involve deregulation of bi-potent progenitor cells. Transfection of cell lines with vectors containing serial transcription factor (TF) binding sequences upstream of a minimal promoter driving luciferase expression have been widely used to study TF activity in vitro. We have expanded on this technology, both in vitro and in vivo, to enable quantification of bioluminescence output in living cell cultures and animals. We have developed a library of lentiviral vectors expressing either firefly luciferase or a secreted NanoLuc luciferase conditionally activated by a range of TFs. Human HepaRG cells can be cultured as bi-potent progenitors capable of differentiating into cholangiocytes or hepatocytes and act as a valuable tool to understand cholangiocyte hyperplasia. We have evaluated TF activity in living HepaRG cultures subjected to pro-cholestatic agonists by measuring NanoLuc activity in conditioned medium. For in vivo experiments, high-titer VSV-G pseudotyped lentiviral preps containing TF activated reporter cassettes were administered by intravascular injection to P0 neonatal mice. This resulted in liver-restricted transduction and lifelong tolerance of the transgene. After establishing a bioimaging baseline, adult mice injected with either the NF-κB or the Smad2/3 reporter constructs, were subjected to partial bile duct ligation (pBDL) and serial bioimaging thereafter. Responses were observed in NF-kb (p = 0.048) and Smad2/3 (p = 0.009) reporter mice post-BDL. These tools have allowed us to evaluate and compare the temporal activity of candidate TFs during cholestatic insult in living cells and living mice. We envisage that this study could result in the elucidation of novel therapeutic targets.