Protein kinase C-dependent distribution of the multidrug resistance protein 2 from the canalicular to the basolateral membrane in human HepG2 cells
Protein kinase C-dependent distribution of the multidrug resistance protein 2 from the canalicular to the basolateral membrane in human HepG2 cells
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DOI：
10.1053/jhep.2001.25959
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发表时间：
2001-08-01
期刊：
影响因子：
13.5
通讯作者：
Häussinger, D
中科院分区：
文献类型：
--
作者：
Kubitz, R;Huth, C;Häussinger, D
肝胆转运蛋白的亚细胞定位直接影响胆汁形成的速率，例如，缀合物输出泵多药耐药蛋白2（MRP 2）通过从肝中小管膜取回和插入肝中小管膜而在短期规模上受到调节。本研究报道了蛋白激酶C对人肝母细胞瘤HepG 2细胞中MRP 2定位和活性的影响。免疫细胞化学和Western blot检测到MRP 2在HepG 2细胞中的表达。使用荧光MRP 2底物通过共聚焦激光扫描显微镜评估功能活性。在未经处理的HepG 2细胞中，MRP 2几乎完全定位于顶膜。用佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）处理HepG 2细胞导致位于顶部的MRP 2迅速减少，并且在4小时内超过90%的假小管丢失。这是伴随着减少假小管分泌的MRP 2底物谷胱甘肽-甲基荧光素。有趣的是，PMA处理（1-100 nmol/L）导致在30分钟内在基底外侧膜上出现免疫反应性MRP 2。这是通过其与MRP 1，人二肽基肽酶IV（DPPIV），和转染的大鼠Ntcp共定位。PMA对MRP 2定位的影响对蛋白激酶C（PKC）抑制剂Go 6850敏感，但对PD 098059对MEK的抑制不敏感。基底外侧MRP 2的外观没有抑制放线菌酮或破坏微管或微丝。PMA（100 nmol）诱导大鼠肝脏胆汁淤积，Mrp 2与Ntcp共定位于基底膜的某些区域。这些数据表明，由于PKC激活，小管MRP 2重新靶向基底外侧膜可能代表了一种可能导致胆汁淤积的新机制。
The subcellular localization of hepatobiliary transport proteins directly affects the rate of bile formation, e.g., the conjugate export pump multidrug resistance protein 2 (MRP2) is regulated on a short-term scale by retrieval from and insertion into the canalicular membrane in the liver. This study reports on the effects of protein kinase C on MRP2 localization and activity in human hepatoblastoma HepG2 cells. MRP2 was detected in HepG2 cells by immunocytochemistry and Western blot analysis. Functional activity was assessed by confocal laser scanning microscopy using fluorescent MRP2 substrates. In untreated HepG2 cells MRP2 was almost exclusively localized at the apical membrane. Treatment of HepG2 cells with phorbol-12-myristate-13-acetate (PMA) resulted in a rapid decrease of apically localized MRP2 and a loss of more than 90% of pseudocanaliculi within 4 hours. This was accompanied by a reduced pseudocanalicular secretion of the MRP2 substrate glutathione-methylfluorescein. Interestingly, PMA treatment (1-100 nmol/L) led to the appearance of immunoreactive MRP2 at the basolateral membrane within 30 minutes. This was shown by its colocalization with MRP1, human dipeptidylpeptidase IV (DPPIV), and transfected rat Ntcp. The effects of PMA on MRP2 localization were sensitive to the protein kinase C (PKC) inhibitor Go6850 but insensitive to inhibition of MEK by PD098059. Basolateral MRP2-appearance was not inhibited by cycloheximide or by disruption of microtubules or microfilaments. In rat livers cholestasis was induced by PMA (100 nmol) and Mrp2 was detected at the basolateral membrane in some areas, colocalizing with Ntcp. The data suggest that retargeting of canalicular MRP2 to the basolateral membrane due to PKC activation may represent a novel mechanism that may contribute to cholestasis.