Ethanol selectively impairs clathrin-mediated internalization in polarized hepatic cells.
Although alcoholic liver disease is clinically well-described, the molecular basis for alcohol-induced hepatotoxicity is not well understood. Previously, we determined that the clathrin-mediated internalization of asialoglycoprotein receptor was impaired in ethanol-treated WIF-B cells whereas the internalization of a glycophosphatidylinositol-anchored protein thought to be endocytosed via a caveolae/raft-mediated pathway was not changed suggesting that clathrin-mediated endocytosis is selectively impaired by ethanol. To test this possibility, we examined the internalization of a panel of proteins and compounds internalized by different mechanisms in control and ethanol-treated WIF-B cells. We determined that the internalization of markers known to be internalized via clathrin-mediated mechanisms was impaired. In contrast, the internalization of markers for caveolae/raft-mediated endocytosis, fluid phase internalization or non-vesicle-mediated uptake was not impaired in ethanol-treated cells. We further determined that clathrin heavy chain accumulated at the basolateral surface in small puncta in ethanol-treated cells while there was decreased dynamin-2 membrane association. Interestingly, the internalization of resident apical proteins that lack any known internalization signals was also disrupted by ethanol suggesting that these proteins are internalized via clathrin-mediated mechanisms. This conclusion is consistent with our findings that dominant negative dynamin-2 overexpression impaired internalization of known clathrin markers and single spanning apical residents, but not of markers of fluid phase or raft-mediated internalization. Together these results indicate that ethanol exposure selectively impairs hepatic clathrin-mediated internalization by preventing vesicle fission from the plasma membrane.
Fernandez
David J
DJ
Department of Biology, The Catholic University of America, Washington, DC 20064, USA.
McVicker
Benita L
BL
Tuma
Dean J
DJ
Tuma
Pamela L
PL
D021941:Caveolae / Q000187:drug effects* / Q000378:metabolism; D002460:Cell Line; D016764:Cell Polarity / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism / Q000502:physiology*; D033922:Clathrin Heavy Chains / Q000737:chemistry / Q000494:pharmacology; D004347:Drug Interactions; D004705:Endocytosis / Q000187:drug effects* / Q000502:physiology; D000431:Ethanol / Q000494:pharmacology*; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000166:cytology; D021962:Membrane Microdomains / Q000187:drug effects* / Q000378:metabolism; D021381:Protein Transport; D055293:Receptors, Urokinase Plasminogen Activator / Q000378:metabolism
D002966:Clathrin; D055293:Receptors, Urokinase Plasminogen Activator; D033922:Clathrin Heavy Chains; D000431:Ethanol