Yosuke Horikoshi

The cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion molecule (JAM)

The establishment and maintenance of cellular polarity are critical for the development of multicellular organisms. PAR (partitioning‐defective) proteins were identified in Caenorhabditis elegans as determinants of asymmetric cell division and polarized cell growth. Recently, vertebrate orthologues of two of these proteins, ASIP/PAR‐3 and PAR‐6, were found to form a signalling complex with the small GTPases Cdc42/Rac1 and with atypical protein kinase C (PKC). Here we show that ASIP/PAR‐3 associates with the tight‐junction‐associated protein junctional adhesion molecule (JAM) in vitro and in vivo. No binding was observed with claudin‐1, ‐4 or ‐5. In fibroblasts and CHO cells overexpressing JAM, endogenous ASIP is recruited to JAM at sites of cell–cell contact. Over expression of truncated JAM lacking the extracellular part disrupts ASIP/PAR‐3 localization at intercellular junctions and delays ASIP/PAR‐3 recruitment to newly formed cell junctions. During junction formation, JAM appears early in primordial forms of junctions. Our data suggest that the ASIP/PAR‐3–aPKC complex is tethered to tight junctions via its association with JAM, indicating a potential role for JAM in the generation of cell polarity in epithelial cells.

Mammalian Lgl Forms a Protein Complex with PAR-6 and aPKC Independently of PAR-3 to Regulate Epithelial Cell Polarity

BACKGROUND Epithelial cells have apicobasal polarity and an asymmetric junctional complex that provides the bases for development and tissue maintenance. In both vertebrates and invertebrates, the evolutionarily conserved protein complex, PAR-6/aPKC/PAR-3, localizes to the subapical region and plays critical roles in the establishment of a junctional complex and cell polarity. In Drosophila, another set of proteins called tumor suppressors, such as Lgl, which localize separately to the basolateral membrane domain but genetically interact with the subapical proteins, also contribute to the establishment of cell polarity. However, how physically separated proteins interact remains to be clarified. RESULTS We show that mammalian Lgl competes for PAR-3 in forming an independent complex with PAR-6/aPKC. During cell polarization, mLgl initially colocalizes with PAR-6/aPKC at the cell-cell contact region and is phosphorylated by aPKC, followed by segregation from apical PAR-6/aPKC to the basolateral membrane after cells are polarized. Overexpression studies establish that increased amounts of the mLgl/PAR-6/aPKC complex suppress the formation of epithelial junctions; this contrasts with the previous observation that the complex containing PAR-3 promotes it. CONCLUSIONS These results indicate that PAR-6/aPKC selectively interacts with either mLgl or PAR-3 under the control of aPKC activity to regulate epithelial cell polarity.

PAR‐6 regulates aPKC activity in a novel way and mediates cell‐cell contact‐induced formation of the epithelial junctional complex

Background PAR‐6, aPKC and PAR‐3 are polarity proteins that co‐operate in the establishment of cell polarity in Caenorhabditis elegans and Drosophila embryos. We have recently shown that mammalian aPKC is required for the formation of the epithelia‐specific cell‐cell junctional structure. We have also revealed that a mammalian PAR‐6 forms a ternary complex with aPKC and ASIP/PAR‐3, and localizes at the most apical end of the junctional complex in epithelial cells.

Interaction between PAR-3 and the aPKC–PAR-6 complex is indispensable for apical domain development of epithelial cells

The evolutionarily conserved polarity proteins PAR-3, atypical protein kinase C (aPKC) and PAR-6 critically regulate the apical membrane development required for epithelial organ development. However, the molecular mechanisms underlying their roles remain to be clarified. We demonstrate that PAR-3 knockdown in MDCK cells retards apical protein delivery to the plasma membrane, and eventually leads to mislocalized apical domain formation at intercellular regions in both two-dimensional and three-dimensional culture systems. The defects in PAR-3 knockdown cells are efficiently rescued by wild-type PAR-3, but not by a point mutant (S827/829A) that lacks the ability to interact with aPKC, indicating that formation of the PAR-3–aPKC–PAR-6 complex is essential for apical membrane development. This is in sharp contrast with tight junction maturation, which does not necessarily depend on the aPKC–PAR-3 interaction, and indicates that the two fundamental processes essential for epithelial polarity are differentially regulated by these polarity proteins. Importantly, highly depolarized cells accumulate aPKC and PAR-6, but not PAR-3, on apical protein-containing vacuoles, which become targeted to PAR-3-positive primordial cell-cell contact sites during the initial stage of the repolarization process. Therefore, formation of the PAR-3–aPKC–PAR-6 complex might be required for targeting of not only the aPKC–PAR-6 complex but also of apical protein carrier vesicles to primordial junction structures.

Lgl mediates apical domain disassembly by suppressing the PAR-3-aPKC-PAR-6 complex to orient apical membrane polarity

The basolateral tumor suppressor protein Lgl is important for the regulation of epithelial cell polarity and tissue morphology. Recent studies have shown a physical and functional interaction of Lgl with another polarity-regulating protein machinery, the apical PAR-3-aPKC-PAR-6 complex, in epithelial cells. However, the mechanism of Lgl-mediated regulation of epithelial cell polarity remains obscure. By an siRNA method, we here show that endogenous Lgl is required for the disassembly of apical membrane domains in depolarizing MDCK cells induced by Ca2+ depletion. Importantly, this Lgl function is mediated by the suppression of the apical PAR-3-aPKC-PAR-6 complex activity. Analysis using 2D- or 3D-cultured cells in collagen gel suggests the importance of this suppressive regulation of Lgl on the collagen-mediated re-establishment of apical membrane domains and lumen formation. These results indicate that basolateral Lgl plays a crucial role in the disassembly of apical membrane domains to induce the orientation of apical membrane polarity, which is mediated by the suppression of apical PAR-3-aPKC-PAR-6 complex activity.

KIBRA Suppresses Apical Exocytosis through Inhibition of aPKC Kinase Activity in Epithelial Cells

Epithelial cells possess apical-basolateral polarity and form tight junctions (TJs) at the apical-lateral border, separating apical and basolateral membrane domains. The PAR3-aPKC-PAR6 complex plays a central role in TJ formation and apical domain development during tissue morphogenesis. Inactivation and overactivation of aPKC kinase activity disrupts membrane polarity. The mechanism that suppresses active aPKC is unknown. KIBRA, an upstream regulator of the Hippo pathway, regulates tissue size in Drosophila and can bind to aPKC. However, the relationship between KIBRA and the PAR3-aPKC-PAR6 complex remains unknown. We report that KIBRA binds to the PAR3-aPKC-PAR6 complex and localizes at TJs and apical domains in epithelial tissues and cells. The knockdown of KIBRA causes expansion of the apical domain in MDCK three-dimensional cysts and suppresses the formation of apical-containing vacuoles through enhanced de novo apical exocytosis. These phenotypes are restored by inhibition of aPKC. In addition, KIBRA directly inhibits the kinase activity of aPKC in vitro. These results strongly support the notion that KIBRA regulates epithelial cell polarity by suppressing apical exocytosis through direct inhibition of aPKC kinase activity in the PAR3-aPKC-PAR6 complex.

Carbon tetrachloride-induced hepatic injury through formation of oxidized diacylglycerol and activation of the PKC/NF-κB pathway.

Protein kinase C (PKC) participates in signal transduction, and its overactivation is involved in various types of cell injury. PKC depends on diacylglycerol (DAG) for its activation in vivo We have previously reported that DAG peroxides (DAG-O(O)H) activate PKC in vitro more strongly than unoxidized DAG, suggesting that DAG-O(O)H, if generated in vivo under oxidative stress, would act as an aberrant signal transducer. The present study examined whether DAG-O(O)H are formed in carbon tetrachloride (CCl4)-induced acute rat liver injury in association with activation of the PKC/nuclear factor (NF)-κB pathway. A single subcutaneous injection of CCl4 resulted in a marked increase in hepatic DAG-O(O)H content. At the molecular level, immunohistochemistry and subcellular fractionation combined with immunoblotting localized PKCα, βI, βII and δ isoforms to cell membranes, while immunoblotting showed phosphorylation of the p65 subunit of NF-κB, and immunoprecipitation using isoform-specific anti-PKC antibodies revealed specific association of PKCα and p65. In addition, expression of tumor necrosis factor α (TNFα) and neutrophil invasion increased in the CCl4-treated rats. Furthermore, we demonstrated that Vitamin E, one of the most important natural antioxidants that suppresses peroxidation of membrane lipids, significantly inhibited the CCl4-induced increase in hepatic DAG-O(O)H content and TNFα expression as well as phosphorylation of PKCα and p65. These data demonstrate for the first time that DAG-O(O)H are generated in the process of CCl4-induced liver injury, resulting in activation of the PKC/NF-κB pathway and TNFα-mediated aggravation of liver injury.

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

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.

Compound 48/80, a mast cell degranulator, causes oxidative damage by enhancing vitamin C synthesis via reduced glutathione depletion and lipid peroxidation through neutrophil infiltration in rat livers

In this study, we examined whether compound 48/80 (C48/80), a mast cell degranulator, causes hepatic oxidative damage in rats. Serum and liver biochemical parameters were determined 0.5, 3 or 6 h after a single treatment with C48/80 (0.75 mg/kg). Serum histamine and serotonin levels increased 0.5 h after C48/80 treatment but diminished thereafter. Increases in serum vitamin C (VC) and transaminases and hepatic hydrogen peroxide, lipid peroxide, and myeloperoxidase levels and a decrease in hepatic reduced glutathione level occurred 0.5 h after C48/80 treatment and further proceeded at 3 h, but these changes diminished at 6 h. Serum lipid peroxide and hepatic VC levels increased 3 h after C48/80 treatment. Hepatic glycogen level decreased 0.5 h after C48/80 treatment and further decreased at 3 h. Pre-administered ketotifen diminished all these changes found at 3 h after treatment, while pre-administered NPC 14686 diminished these changes except changes in serum histamine and serotonin levels. Hepatocellular apoptosis observed at 3 h after C48/80 treatment was attenuated by pre-administered ketotifen and NPC 14686. These results indicate that C48/80 causes oxidative damage by enhancing VC synthesis via reduced glutathione depletion-dependent glycogenolysis and lipid peroxidation through neutrophil infiltration following mast cell degranulation in rat livers.

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

In many developed countries including Japan, how to care the bedridden elderly people with chronic wounds such as decubitus becomes one of the most concerned issues. Although antioxidant micronutrients including vitamin E, especially α-tocopherol (α-Toc), are reported to shorten a period of wound closure, the promoting effect of α-Toc on wound healing independent of its antioxidant activity remains to be fully elucidated. The aim of this study was to examine whether α-Toc affects wound-mediated HaCaT keratinocyte polarization process including the recruitment of polarity regulating proteins, leading to wound repair independently of its antioxidant activity. We investigated the effects of α-Toc and other antioxidants such as Trolox, a cell-permeable α-Toc analog on the migration, proliferation, and cell polarization of HaCaT keratinocytes after wounding. We analyzed the localization and complex formation of polarity proteins, partitioning defective 3 (Par3), and atypical protein kinase C (aPKC), and aPKC activity by immunohistochemistry, immunoprecipitation analyses, and in vitro kinase assays, respectively. α-Toc but not other antioxidants enhanced the wound closure and cell polarization in HaCaT keratinocytes after wounding. α-Toc regulated the localization and complex formation of Par3 and aPKC during wound healing. Knockdown of aPKC or Par3 abrogated α-Toc-mediated promotion of the wound closure and cell polarization in HaCaT keratinocytes. Furthermore, aPKC kinase activity was significantly increased in α-Toc-treated cells through activation of phosphatidylinositol 3-kinase/Akt signaling pathway. These results suggest that α-Toc promotes HaCaT keratinocyte wound repair by regulating the aPKC kinase activity and the formation of aPKC-Par3 complex.