Aberrant Activation of Atypical Protein Kinase C in Carbon Tetrachloride–Induced Oxidative Stress Provokes a Disturbance of Cell Polarity and Sealing of Bile Canalicular Lumen

Abstract

Polarized hepatocytes contain tight junctions (TJs), which are among the most important junctions for sealing the bile canalicular lumen from the sinusoidal space. Alterations in TJs are implicated in chronic cholestatic liver diseases, such as primary biliary cirrhosis and primary sclerosing cholangitis, which have lipid peroxidation marker elevations or antioxidant vitamin decreases. However, the effect of oxidative stress on hepatocyte polarity or liver morphology is unknown. We found that carbon tetrachloride (CCl4)-induced oxidative stress resulted in disassembly of TJs. Ultrastructural analysis revealed disruption in TJs, Golgi morphology, and expansion of the bile canalicular lumen size in CCl4-treated hepatocytes. The Par complex [Par-3-atypical protein kinase C (aPKC) and Par-6 ternary complex] regulates TJs and lumen formation, and the Par-3-aPKC complex formation was inhibited by CCl4 treatment. Moreover, the antioxidant compound vitamin E prohibited a CCl4-induced disturbance in TJs and Par-3-aPKC complex formation. aPKC phosphorylates Par-3 and down-regulates its own affinity with Par-3. Importantly, aPKC kinase activity and Par-3 phosphorylation were significantly increased in CCl4-treated rat livers. These results indicate that the Par-3-aPKC complex plays a crucial role in the maintenance of hepatocyte polarity and sealing of the bile canalicular lumen. Our findings suggest that bile canalicular lumen expansion might explain the presence of cholestasis in patients with primary biliary cirrhosis and primary sclerosing cholangitis.

Low-dose acetaminophen induces early disruption of cell-cell tight junctions in human hepatic cells and mouse liver

“…This suggests that APAP toxicity, via oxidative stress, may involve a PKC-dependent signalling pathway 62 . PKC involvement is also observed in other cell types, including bronchial epithelial cells 63 , and in vivo following carbon tetrachloride (CCl 4 )-induced oxidative stress in rat liver 50 . PKC signaling is also shown to play an important role in Clostridium difficile toxin A-mediated damage on TJ structure and functions in human colonic cells 53 .…”

“…1e ). Interestingly, recent work shows that CCl 4 -induced oxidative stress in rats, resulted in disassembly of TJ and depolarization of hepatocytes, through disruption of atypical-PKC complex formation, evident after only 2 hours of CCl 4 -leading to liver cholestasis and cirrhosis 50 . Indeed, given atypical-PKC regulates TJ assembly and epithelial cell polarity, it is feasible that APAP-induced oxidative stress may also lead to dysregulation of TJ-mediated cell-signaling and membrane trafficking 51 , particularly at a high-dose of APAP, resulting in cell death.…”

α‐Tocopherol promotes HaCaT keratinocyte wound repair through the regulation of polarity proteins leading to the polarized cell migration

“…The commercially available antibodies were as follows: anti‐annexin II (Clone 5) mouse monoclonal antibodies (mAbs) (BD Biosciences, San Jose, CA); anti‐aPKCζ (C20) rabbit polyclonal antibody (pAb), anti‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH; 6C5) mouse mAb, and mouse–rabbit–rat control IgG (Santa Cruz Biotechnology, Santa Cruz, CA); anti‐Par‐3 rabbit pAb (Merck Millipore, Darmstadt, Germany); anti‐BrdU mouse mAb (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan); anti‐phospho‐aPKCζ T560 rabbit mAb (Abcam Ltd., Cambridge, UK); anti‐phospho‐Akt S473 (#4060S); and anti‐Akt (#9272) rabbit mAbs, and anti‐β‐actin rabbit pAb (#4967) (Cell Signaling Technology, Beverly, MA); horseradish peroxidase‐conjugated secondary Ab (GE Healthcare Bio‐Sciences, Piscataway, NJ). The phospho‐specific mouse mAb against S653 of human lethal (2) giant larvae (Llgl2) was produced against a phospho‐peptide [KSLRES(P)FRKLR] as described previously .…”

“…IP‐kinase assay was performed as described previously . The cells were harvested and lysed in IP lysis buffer (20 mM Tris–HCl, pH 7.4, 0.25 M sucrose, 1.2 mM EDTA, 20 mM β‐mercaptoethanol, 150 mM NaCl, 1 mM NaF, 1 mM Na 4 P 2 O 7 , 0.5% Triton X‐100, 0.5% Nonidet P‐40, 0.001% aprotinin, and 1 mM PMSF).…”

Structural and functional hepatocyte polarity and liver disease

“…A recent study has shown that induction in oxidative stress by administration of carbon tetrachloride disrupted Par-3 – alpha-PKC complex formation resulting in disassembly of TJ and depolarization of hepatocytes. These changes led to cholestasis and cirrhosis [147] . The mobilization of fibrogenic cells has been proposed to involve conversion of polarized hepatocytes and biliary epithelial cells into mesenchymal cells with concomitant loss of epithelial polarization and acquisition of a mobile phenotype, but this is controversial [148,149] .…”