Department of Membrane Enzymology, Bijvoet Center and Institute of Biomembranes, Utrecht University, the Netherlands.
Hepatology. 2010 Jun;51(6):2049-60. doi: 10.1002/hep.23586.
Mutations in ATP8B1 cause familial intrahepatic cholestasis type 1, a spectrum of disorders characterized by intrahepatic cholestasis, reduced growth, deafness, and diarrhea. ATP8B1 belongs to the P(4) P-type adenosine triphosphatase (ATPase) family of putative aminophospholipid translocases, and loss of aminophospholipid asymmetry in the canalicular membranes of ATP8B1-deficient liver cells has been proposed as the primary cause of impaired bile salt excretion. To explore the origin of the hepatic and extrahepatic symptoms associated with ATP8B1 deficiency, we investigated the impact of ATP8B1 depletion on the domain-specific aminophospholipid translocase activities and polarized organization of polarized epithelial Caco-2 cells. Caco-2 cells were stably transfected with short hairpin RNA constructs to block ATP8B1 expression. Aminophospholipid translocase activity was assessed using spin-labeled phospholipids. The polarized organization of these cells was determined by pulse-chase analysis, cell-fractionation, immunocytochemistry, and transmission electron microscopy. ATP8B1 was abundantly expressed in the apical membrane of Caco-2 cells, and its expression was markedly induced during differentiation and polarization. Blocking ATP8B1 expression by RNA interference (RNAi) affected neither aminophospholipid transport nor the asymmetrical distribution of aminophospholipids across the apical bilayer. Nonetheless, ATP8B1-depleted Caco-2 cells displayed profound perturbations in apical membrane organization, including a disorganized apical actin cytoskeleton, a loss in microvilli, and a posttranscriptional defect in apical protein expression.
Our findings point to a critical role of ATP8B1 in apical membrane organization that is unrelated to its presumed aminophospholipid translocase activity, yet potentially relevant for the development of cholestasis and the manifestation of extrahepatic features associated with ATP8B1 deficiency.
ATP8B1 基因突变可导致 1 型家族性肝内胆汁淤积症，这是一组以肝内胆汁淤积、生长迟缓、耳聋和腹泻为特征的疾病。ATP8B1 属于 P(4) P 型三磷酸腺苷（ATPase）家族的假定氨磷脂转运体，据推测 ATP8B1 缺陷肝细胞的胆小管膜中氨磷脂不对称性的缺失是胆汁盐排泄受损的主要原因。为了探究与 ATP8B1 缺乏相关的肝内和肝外症状的起源，我们研究了 ATP8B1 耗竭对域特异性氨磷脂转运体活性和极化上皮 Caco-2 细胞极化组织的影响。用短发夹 RNA 构建体稳定转染 Caco-2 细胞以阻断 ATP8B1 表达。用自旋标记磷脂评估氨磷脂转运体活性。通过脉冲追踪分析、细胞分级分离、免疫细胞化学和透射电子显微镜来确定这些细胞的极化组织。ATP8B1 在 Caco-2 细胞的顶膜中大量表达，其表达在分化和极化过程中明显增加。用 RNA 干扰（RNAi）阻断 ATP8B1 表达既不影响氨磷脂转运，也不影响氨磷脂在顶双层的不对称分布。尽管如此，ATP8B1 耗竭的 Caco-2 细胞的顶膜组织出现严重紊乱，包括顶 actin 细胞骨架紊乱、微绒毛缺失以及顶蛋白表达的转录后缺陷。
我们的研究结果表明，ATP8B1 在顶膜组织中起着关键作用，与假定的氨磷脂转运体活性无关，但可能与胆汁淤积的发生和与 ATP8B1 缺乏相关的肝外特征的表现有关。
