Kagawa Tatehiro, Orii Reiko, Hirose Shunji, Arase Yoshitaka, Shiraishi Koichi, Mizutani Akiko, Tsukamoto Hidekazu, Mine Tetsuya
Division of Gastroenterology, Department of Internal Medicine, Tokai University School of Medicine, Shimokasuya 143, Isehara, Kanagawa, 259-1193, Japan,
J Gastroenterol. 2014 May;49(5):890-9. doi: 10.1007/s00535-013-0833-y. Epub 2013 May 31.
Ursodeoxycholic acid (UDCA) partly exerts choleretic effects by modifying the function of the bile salt export pump (Bsep, ABCB11). UDCA induces insertion of Bsep into the canalicular membrane of hepatocytes; however, underlying mechanisms remain unknown. We aimed to elucidate molecular mechanisms behind UDCA-induced Bsep activation.
We established MDCK II cells stably expressing both Bsep and Na(+)-taurocholate cotransporting polypeptide, and investigated the effect of UDCA on activity and protein expression of Bsep using these cells. We performed inhibitor study to know the molecules involved in UDCA-induced Bsep activation, and also tested the influence of UDCA on Bsep having a disease-associated mutation.
UDCA activated Bsep in a dose-dependent manner. UDCA did not affect Bsep protein expression in whole cell lysates but increased its apical surface expression by extending the half-life from 2.4 to 5.0 h. This effect was specific to Bsep because UDCA did not affect other apical and basolateral proteins, and was independent of protein kinase A, adenylate cyclase, p38(MAPK), phosphatidylinositide 3-kinase, Ca(2+), and microtubules. NorUDCA activated Bsep similar to UDCA; however, cholic acid, taurocholic acid, and tauroUDCA had no effect. UDCA significantly increased the activity of Bsep with a benign recurrent intrahepatic cholestasis 2 mutation (A570T) but did not affect Bsep with a progressive familial intrahepatic cholestasis 2 mutation (G982R or D482G).
We demonstrated that UDCA stabilizes Bsep protein in the apical membrane and increases its activity in MDCK II cells, presumably by retarding the endocytotic process.
熊去氧胆酸（UDCA）部分通过改变胆盐输出泵（Bsep，ABCB11）的功能发挥利胆作用。UDCA诱导Bsep插入肝细胞的胆小管膜；然而，其潜在机制仍不清楚。我们旨在阐明UDCA诱导Bsep激活背后的分子机制。
我们建立了稳定表达Bsep和牛磺胆酸钠共转运多肽的MDCK II细胞，并使用这些细胞研究UDCA对Bsep活性和蛋白表达的影响。我们进行了抑制剂研究以了解参与UDCA诱导Bsep激活的分子，还测试了UDCA对具有疾病相关突变的Bsep的影响。
UDCA以剂量依赖性方式激活Bsep。UDCA不影响全细胞裂解物中Bsep蛋白的表达，但通过将半衰期从2.4小时延长至5.0小时增加了其顶端表面表达。这种作用对Bsep具有特异性，因为UDCA不影响其他顶端和基底外侧蛋白，并且独立于蛋白激酶A、腺苷酸环化酶、p38（MAPK）、磷脂酰肌醇3激酶、Ca（2+）和微管。NorUDCA与UDCA类似地激活Bsep；然而，胆酸、牛磺胆酸和牛磺UDCA没有作用。UDCA显著增加了具有良性复发性肝内胆汁淤积2突变（A570T）的Bsep的活性，但不影响具有进行性家族性肝内胆汁淤积2突变（G982R或D482G）的Bsep。
我们证明UDCA稳定顶端膜中的Bsep蛋白并增加其在MDCK II细胞中的活性，可能是通过延缓内吞过程实现的。
