Hypoxia increases transepithelial electrical conductance and reduces occludin at the plasma membrane in alveolar epithelial cells via PKC-ζ and PP2A pathway.
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ABSTRACT:
SUBMITTER: Caraballo JC
PROVIDER: S-EPMC3075095 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Inhaled particulate matter (PM) causes lung inflammation and epithelial dysfunction. However, the direct effect of PM on alveolar epithelial barrier integrity is not well understood. Our aim is to determine whether PM exposure affects the alveolar epithelial cells (AEC) transepithelial electrical conductance (Gt) and tight junction (TJ) proteins.Human AEC (A549) and primary rat AEC were exposed to PM of <10 µm in size (PM(10) ) and diesel exhaust particles (DEP), using titanium dioxide (TiO(2) ) as a control for particle size effects. Gt and permeability to fluorescein isothiocyanate-dextran (FITC-Dextran) were measured to assess barrier integrity. TJ integrity was evaluated by analysing penetration of Lanthanum nitrate (La(3+) ) under transmission electron microscopy. Surface proteins were labelled with biotin and analysed by western blot. Immunofluorescence was performed to assess colocalization of TJ proteins including occludin and zonula occludens-1 (ZO-1). PM induced dissociation of occludin-ZO-1 was evaluated by co-immunoprecipitation.PM(10) and DEP increased Gt and disrupted TJ after 3 h of treatment. PM(10) and DEP induced occludin internalization from the plasma membrane into endosomal compartments and dissociation of occludin from ZO-1. Overexpression of antioxidant enzymes manganese superoxide dismutase (MnSOD) and catalase, prevented PM-induced Gt increase, occludin reduction from the plasma membrane and its dissociation from ZO-1.PM induces alveolar epithelial dysfunction in part via occludin reduction at the plasma membrane and ZO-1 dissociation in AEC. Furthermore, these effects are prevented by overexpression of two different antioxidant enzymes.
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Project description:Occludin, a tight junction protein, has been reported to regulate barrier function - particularly the leak pathway for larger solutes - in epithelia. Therefore, we aimed to precisely define its role in macromolecule passage at single cell-cell junctions. A combination of varying occludin expression by transient and stable knockdown including systematic seeding strategies was employed to achieve a broad and defined pattern of variance in occludin expression over epithelia. This variance model enabled us to examine occludin function in the leak pathway using global and local analysis, i.e. to analyze macromolecule flux across epithelia and macromolecule passage at single-cell level. Macromolecular flux was found not to correlate with occludin expression in intestinal epithelial cells. In fact, by spatially resolving macromolecular permeation sites using a recently developed method we uncovered leaky cell junctions at the edge of Transwells resulting in increased passage. This demonstrates that rare leaks can determine net flux of macromolecules across epithelia while the vast majority of cellular junctions do not contribute significantly. Hence, concomitant local analysis of macromolecule passage across epithelial barriers is indispensable for interpretation of global flux data. By combining this new approach with cell culture models of the leak pathway, we can present evidence that lack of occludin is not sufficient to stimulate the epithelial leak pathway.
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Project description:PKC eta is expressed predominantly in the epithelial tissues; however, its role in the regulation of epithelial tight junctions (TJs) is unknown. We present evidence that PKC eta phosphorylates occludin on threonine residues (T403 and T404) and plays a crucial role in the assembly and/or maintenance of TJs in Caco-2 and MDCK cell monolayers. Inhibition of PKC eta by specific pseudo substrate inhibitor or knockdown of PKC eta by specific shRNA disrupts the junctional distribution of occludin and ZO-1 and compromises the epithelial barrier function. Expression of dominant negative, PKC eta(K394R) disrupts the TJ and barrier function, whereas wild-type PKC eta and constitutively active PKC eta(A161E) enhance the TJ integrity. Inhibition and knockdown of PKC eta or expression of PKC eta(K394R) induce dephosphorylation of occludin on threonine residues, whereas active PKC eta elevates occludin phosphorylation. PKC eta directly interacts with the C-terminal domain of occludin and phosphorylates it on highly conserved T403 and T404. T403/404A mutations result in the loss of occludin's ability to localize at the TJs, whereas T403/404D mutations attenuates the PKC eta inhibitor-mediated redistribution of occludin from the intercellular junctions. These results reveal an important mechanism of epithelial TJ regulation by PKC eta.
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Project description:Doxorubicin (Dox) is a chemotherapeutic drug used to treat a wide range of cancers, but its clinical application is limited due to its cardiotoxicity. Protein kinase C-ζ (PKC-ζ) is a serine/threonine kinase belonging to atypical protein kinase C (PKC) subfamily, and is activated by its phosphorylation. We and others have reported that PKC-ζ induced cardiac hypertrophy by activating the inflammatory signaling pathway. This study focused on whether PKC-ζ played an important role in Dox-induced cardiotoxicity. We found that PKC-ζ phosphorylation was increased by Dox treatment in vivo and in vitro. PKC-ζ overexpression exacerbated Dox-induced cardiotoxicity. Conversely, knockdown of PKC-ζ by siRNA relieved Dox-induced cardiotoxicity. Similar results were observed when PKC-ζ enzyme activity was inhibited by its pseudosubstrate inhibitor, Myristoylated. PKC-ζ interacted with β-catenin and inhibited Wnt/β-catenin signaling pathway. Activation of Wnt/β-catenin signaling by LiCl protected against Dox-induced cardiotoxicity. The Wnt/β-catenin inhibitor XAV-939 aggravated Dox-caused decline of β-catenin and cardiomyocyte apoptosis and mitochondrial damage. Moreover, activation of Wnt/β-catenin suppressed aggravation of Dox-induced cardiotoxicity due to PKC-ζ overexpression. Taken together, our study revealed that inhibition of PKC-ζ activity was a potential cardioprotective approach to preventing Dox-induced cardiac injury.
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