Tight junction stabilization prevents HepaRG cell death in drug-induced intrahepatic cholestasis

Abstract

Entacapone (ENT), a catechol-O-methyltransferase inhibitor, causes liver injury by inducing bile canaliculi (BC) dilation through inhibition of the myosin light kinase pathway. Loss of tight junctions (TJs) induces hepatocyte depolarization, which causes bile secretory failure, leading to liver damage. To understand the influence of TJ structural changes as a consequence of BC dynamics, we compared the datasets of time-lapse and immunofluorescence images for TJ protein ZO-1 in hepatocytes cultured with ENT, forskolin (FOR), ENT/FOR, and those cultured without any drugs. Retrospective analysis revealed that the drastic change in BC behaviors caused TJ disruption and apoptosis in cells cultured with ENT. Exposure to FOR or sodium taurocholate facilitated TJ formation in the cells cultured with ENT and suppressed BC dynamic changes, leading to the inhibition of TJ disruption and apoptosis. Our findings clarify that hepatocyte TJ stabilization protects against cell death induced by BC disruption.

Quantitative understanding of HepaRG cells during drug‐induced intrahepatic cholestasis through changes in bile canaliculi dynamics

“…Exposure of cells to TOL and ENT, which are known to induce hepatic cholestasis, accelerates BC dilation by relaxing actomyosin via the Rho kinase/myosin light kinase pathway 10,11 . Rapid BC dilation causes BC disruption, resulting in TJ disruption and apoptosis 14 5 and 6), and maintained TJ integrity in BCs.…”

“…Time‐lapse images of HepaRG cells were captured from triplicate cultures in 6‐well plates using an image analyzer (Eclipse Ti2; Nikon) with a 10× objective lens and phase‐contrast images (5.3 × 5.3 mm) at 16 bits in gray scale with 1.88 pixels/μm 2 resolution, every 20 min for 48 h. Following time‐lapse observation, fluorescence staining of ZO‐1 protein (Abcam), F‐actin, and cell nuclei of HepaRG cells was conducted according to a previously described method 14 . Briefly, HepaRG cells washed with PBS were fixed with 2% paraformaldehyde for 15 min at 4°C, and permeabilized by incubation for 30 min in 0.05% Triton X‐100 at 4°C.…”

“…Exposure time, t , was determined from the moment following the addition of ENT, TOL, or FOR. To understand the changes in BC dynamics following exposure to drugs, the exposure time and concentration of drugs were determined based on the cytotoxicity data and BC dynamics in published reports 10,14 …”

“…Meanwhile, the Rho small GTPase family regulates the assembly and disassembly of the actin cytoskeleton, 9 and drugs that induce intrahepatic cholestasis have been found to alter BC dynamics via the Rho kinase/myosin light kinase pathway 10,11 . The actin cytoskeleton and adapter proteins, such as zonula occludens‐1 (ZO‐1), interact to form tight junctions (TJs), 12,13 while certain drugs may cause the interaction between TJs and the actin cytoskeleton to become unbalanced, thereby disrupting TJ integrity 14 . Thus, an investigation of BC dynamics may provide important information regarding changes in cellular fate.…”

“…More specifically, exposure to TOL and ENT causes BC dilation via the Rho kinase/myosin light kinase pathway 10,11 . A previous retrospective study analyzed time‐lapse and immunofluorescence images of the TJ protein, ZO‐1, in BCs cultured with or without ENT and forskolin (FOR), a drug that facilitates TJ formation, to better understand the effects of TJ structural changes due to altered BC dynamics 14 . Moreover, we previously found that exposure to ENT and FOR suppresses changes in BC dynamics, resulting in inhibition of TJ disruption and apoptosis.…”