Dysregulated Cholangiocyte Autophagy Contributes to Progression of Polycystic Liver Disease

ANATOLIY I. MASYUK; TATYANA V. MASYUK; MARIA LORENZO PISARELLO; BING Q. HUANG; NICHOLAS F. LARUSSO

Congreso; Liver Meeting - American Association for Study of Liver Diseases; 2016

BACKGROUND: Polycystic Liver Disease (PLD) is a group of genetic disorders characterized by fluid-filled, hepatic cysts arising from cholangiocytes by unclear mechanisms, with lim- ited treatment and significant morbidity. AIM: Given the emerg- ing role of autophagy in the pathogenesis of many diseases, here we tested the hypothesis that dysregulated cholangiocyte autophagy occurs in PLD and contributes to disease progres- sion. METHODS: We employed NextGen Sequencing (NGS) and Functional Pathway Cluster Analysis (FPCA), transmission electron microscopy (TEM), immunofluorescence confocal microscopy, and western blotting to assess the autophagy path- way. We utilized a 3-D cholangiocyte culture model to study cystogenesis in vitro, and the PCK rat model of PLD to study disease progression in vivo. RESULTS: By quantitative TEM, we observed that the number of organelles involved in autoph- agy (i.e., autophagosomes, lysosomes and autolysosomes) in cholangiocytes of animal models and humans with PLD are all increased ~ 3-4-fold compared to appropriate controls. Further analysis of the transcriptome of cultured human and rodent healthy and PLD cholangiocytes by NGS and FPCA revealed that 25 of 30 autophagy-related genes were activated in PLD cholangiocytes; moreover, among 41 affected intracellular pathways, the lysosomal pathway was the second most up-ulated. By confocal microscopy of healthy and PLD livers and by western blot of normal and PLD cultured cholangiocytes, we found a ~2-fold increase in expression of four autopha- gy-related proteins (i.e., ATG5, ATG6, ATG7 and ATG8). In cultured PLD cholangiocytes, autophagic flux was increased ~ 2-fold compared to normal cholangiocytes as assessed by western blots of an autophagosome marker, LC3-II, and an autophagy receptor, p62, as well as by the number of LC3-GFP puncta in the presence and absence of Bafilomycin A1. The level of autophagy assessed by LC3-GFP puncta was increased ~3-fold in PLD cholangiocytes treated with forskolin and db-cAMP. Pharmacologic inhibition of autophagy with Bafilo- mycin A1 and hydroxychloroquine decreased cholangiocyte proliferation and cyst growth ~2-fold in 3-D culture. Finally, hydroxychloroquine reduced hepatic cystogenesis by 25% in the PCK rat. CONCLUSION: Our data support the hypothesis that autophagy is dysregulated in PLD cholangiocytes, likely in a cAMP-dependent manner, contributes to hepatic cystogenesis and represents a novel target for PLD treatment.