Radixin is required to maintain apical canalicular membrane structure and function in rat hepatocytes
Radixin is required to maintain apical canalicular membrane structure and function in rat hepatocytes
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DOI：
10.1053/j.gastro.2006.06.013
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发表时间：
2006-09-01
期刊：
影响因子：
29.4
通讯作者：
Boyer, James L.
中科院分区：
文献类型：
--
作者：
Wang, Wei;Soroka, Carol J.;Boyer, James L.
背景与目的：Ezrin-radixin-moesin蛋白是细胞质膜和肌动蛋白丝之间的交联蛋白。Radixin是肝细胞中主要的ezrin-radixin-moesin蛋白，据报道可选择性地将多药耐药相关蛋白2与顶小管膜结合。然而，这是否是其主要功能仍有待确定。研究方法：腺病毒介导的短干扰RNA（siRNA）下调radixin在胶原β-培养的大鼠肝细胞的表达和形态和功能的变化进行了定量分析。结果如下：在对照培养物中，广泛的胆小管网络与适当定位的顶端和基底侧转运蛋白，提供功能性分泌的荧光亲胆物质进入胆小管腔。与暴露于编码乱序siRNA的腺病毒的对照细胞相比，siRNA诱导的对radixin的抑制与小管膜结构的显著减少相关，如通过微分干涉对比显微镜和F-肌动蛋白染色所观察到的。间接免疫荧光显示，顶端转运蛋白（多药耐药相关蛋白2，胆盐输出泵，和多药耐药蛋白1）从他们的正常位置在顶端膜解离，并发现主要与Rab 11含内体。基底外侧膜转运蛋白，有机阴离子转运多肽2（Oatp 2）的本地化，没有受到影响。与这种顶端转运体的错位一致，谷胱甘肽-甲基荧光素和胆酰甘氨酰氨基-荧光素的胆汁排泄在根蛋白缺陷细胞中显著降低，但在对照siRNA细胞中不降低。结论：Radixin对于维持小管膜转运蛋白的极化靶向和/或保留是必不可少的，并且是肝细胞顶膜的整体结构和功能的关键决定因素。
Background & Aims: Ezrin-radixin-moesin proteins are cross-linkers between the plasma membrane and actin filaments. Radixin, the dominant ezrin-radixin-moesin protein in hepatocytes, has been reported to selectively tether multidrug-resistance-associated protein 2 to the apical canalicular membrane. However, it remains to be determined if this is its primary function. Methods: An adenovirus-mediated short interfering RNA (siRNA) was used to down-regulate radixin expression in collagen sandwich-cultured rat hepatocytes and morphologic and functional changes were characterized quantitatively. Results: In control cultures, an extensive bile canalicular network developed with properly localized apical and basolateral transporters that provided for functional excretion of fluorescent cholephiles into the bile canalicular lumina. siRNA-induced suppression of radixin was associated with a marked reduction in the canalicular membrane structure as observed by differential interference contrast microscopy and F-actin staining, in contrast to control cells exposed to adenovirus encoding scrambled siRNA. Indirect immunofluorescence showed that apical transporters (multidrug-resistance-associated protein 2, bile salt export pump, and multidrug-resistance protein 1) dissociated from their normal location at the apical membrane and were found largely associated with Rab11-containing endosomes. Localization of the basolateral membrane transporter, organic anion transporting polypeptide 2 (Oatp2), was not affected. Consistent with this dislocation of apical transporters, the biliary excretion of glutathione-methylfluorescein and cholylglycylamido-fluorescein was decreased significantly in the radixin-deficient cells, but not in the control siRNA cells. Conclusions: Radixin is essential for maintaining the polarized targeting and/or retaining of canalicular membrane transporters and is a critical determinant of the overall structure and function of the apical membrane of hepatocytes.