Suda Jo, Rockey Don C, Karvar Serhan
Division of Gastrointestinal and Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles, California;
Division of Gastroenterology and Hepatology, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina.
Am J Physiol Gastrointest Liver Physiol. 2015 Feb 15;308(4):G313-24. doi: 10.1152/ajpgi.00369.2014. Epub 2014 Dec 11.
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.
肝细胞中最主要的埃兹蛋白-根蛋白-膜突蛋白是根蛋白，它主要定位于胆小管微绒毛，似乎在细胞极性调节以及多药耐药相关蛋白2（Mrp-2）功能定位中发挥重要作用。我们的目的是研究缺氧如何影响根蛋白分布和Mrp-2功能。我们构建了野生型和突变体构建体（腺病毒载体形式），并在WIF-B细胞中表达。通过荧光显微镜观察Mrp-2和根蛋白的细胞分布，并用5-氯甲基荧光素二乙酸酯（CMFDA）试验检测Mrp-2功能。在正常条件下，感染野生型根蛋白、非磷酸化根蛋白-T564A和根蛋白-T564D（活性磷酸化模拟突变体）的细胞在胆小管膜区室泡中大量表达，通常与Mrp-2共定位。相比之下，缺氧24小时后，内源性和过表达的野生型根蛋白以及根蛋白-T564A突变体均转移至细胞质空间。然而，模拟持续磷酸化的根蛋白-T564D突变体的分布明显不同，即使在缺氧条件下也与胆小管膜相关。这种显性活性构建体还可防止根蛋白从质膜解离。缺氧还导致Mrp-2定位错误，并使Mrp-2与根蛋白解离；根蛋白磷酸化模拟突变体（T564D）可消除缺氧的这种作用。最后，缺氧减弱了WIF-B细胞中的分泌反应（用CMFDA试验测定），而显性活性构建体（根蛋白-T567D）挽救了这种表型。总体而言，这些发现表明根蛋白调节肝细胞中Mrp-2的定位和功能，在缺氧性肝损伤中起重要作用。
