title: Phosphorylation dynamics of radixin in hypoxia-induced hepatocyte injury.
abstract: The most prominent ezrin-radixin-moesin protein in hepatocytes is radixin, which is localized primarily at the canalicular microvilli and appears to be important in regulation of cell polarity and in localizing the multidrug resistance-associated protein 2 (Mrp-2) function. Our aim was to investigate how hypoxia affects radixin distribution and Mrp-2 function. We created wild-type and mutant constructs (in adenoviral vectors), which were expressed in WIF-B cells. The cellular distribution of Mrp-2 and radixin was visualized by fluorescence microscopy, and a 5-chloromethylfluorescein diacetate (CMFDA) assay was used to measure Mrp-2 function. Under usual conditions, cells infected with wild-type radixin, nonphosphorylatable radixin-T564A, and radixin-T564D (active phospho-mimicking mutant) were found to be heavily expressed in canalicular membrane compartment vacuoles, typically colocalizing with Mrp-2. In contrast, after hypoxia for 24 h, both endogenous and overexpressed wild-type radixin and the radixin-T564A mutant were found to be translocated to the cytoplasmic space. However, distribution of the radixin-T564D mutant, which mimics constant phosphorylation, was remarkably different, being associated with canalicular membranes even in hypoxic conditions. This dominant-active construct also prevented dissociation of radixin from the plasma membrane. Hypoxia also led to Mrp-2 mislocalization and caused Mrp-2 to be dissociated from radixin; the radixin phospho-mimicking mutant (T564D) abrogated this effect of hypoxia. Finally, hypoxia diminished the secretory response (measured using the CMFDA assay) in WIF-B cells, and the dominant-active construct (radixin-T567D) rescued this phenotype. Taken collectively, these findings suggest that radixin regulates Mrp-2 localization and function in hepatocytes and is important in hypoxic liver injury.
authors.affiliation: Division of Gastrointestinal and Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles, California;
authors.forename: Jo
authors.initials: J
authors.lastname: Suda
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina.
authors.forename: Don C
authors.initials: DC
authors.lastname: Rockey
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina karvar@musc.edu.
authors.forename: Serhan
authors.initials: S
authors.lastname: Karvar
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000818:Animals; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D003593:Cytoplasm / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D005838:Genotype; D022781:Hepatocytes / Q000378:metabolism* / Q000473:pathology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010641:Phenotype; D010766:Phosphorylation; D021381:Protein Transport; D034622:RNA Interference; D051381:Rats; D014162:Transfection
chemical_list: D018528:ATP-Binding Cassette Transporters; C423013:Abcc2 protein, rat; D003598:Cytoskeletal Proteins; C497444:Hif1a protein, rat; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008565:Membrane Proteins; C071776:radixin
keywords: bile transporter; cytoskeleton; ischemia; liver; reperfusion