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Abstract
The polycystic kidney (PCK) rat is an animal model of Caroli’s disease as well as autosomal recessive polycystic kidney disease (ARPKD). The signaling pathways involving the mammalian target of rapamycin (mTOR) are aberrantly activated in ARPKD. This study investigated the effects of inhibitors for the cell signaling pathways including mTOR on cholangiocyte proliferation of the PCK rat. Cultured PCK cholangiocytes were treated with rapamycin and everolimus [inhibitors of mTOR complex 1 (mTOC1)], LY294002 [an inhibitor of phosphatidylinositol 3-kinase (PI3K)] and NVP-BEZ235 (an inhibitor of PI3K and mTORC1/2), and the cell proliferative activity was determined in relation to autophagy and apoptosis. The expression of phosphorylated (p)-mTOR, p-Akt, and PI3K was increased in PCK cholangiocytes compared to normal cholangiocytes. All inhibitors significantly inhibited the cell proliferative activity of PCK cholangiocytes, where NVP-BEZ235 had the most prominent effect. NVP-BEZ235, but not rapamycin and everolimus, further inhibited biliary cyst formation in the three-dimensional cell culture system. Rapamycin and everolimus induced apoptosis in PCK cholangiocytes, whereas NVP-BEZ235 inhibited cholangiocyte apoptosis. Notably, the autophagic response was significantly induced following the treatment with NVP-BEZ235, but not rapamycin and everolimus. Inhibition of autophagy using siRNA against protein-light chain3 and 3-methyladenine significantly increased the cell proliferative activity of PCK cholangiocytes treated with NVP-BEZ235. In vivo, treatment of the PCK rat with NVP-BEZ235 attenuated cystic dilatation of the intrahepatic bile ducts, whereas renal cyst development was unaffected. These results suggest that the aberrant activation of the PI3K/mTOR pathway is involved in cystic proliferation of cholangiocytes of the PCK rat, and inhibition of the pathway can reduce cholangiocyte proliferation via the mechanism involving apoptosis and/or autophagy.
Highlights
Caroli’s disease is characterized by the progressive, multiple cystic dilatation of intrahepatic bile ducts, and is frequently associated with portal fibrosis corresponding to congenital hepatic fibrosis [1]
Western blot analysis showed that the expression p-mammalian target of rapamycin (mTOR) (Ser2448), p-mTOR (Ser2481), p-Akt (Ser473), p-S6, phosphatidylinositol 3-kinase (PI3K) p100a, and PI3K p85 was increased in cultured polycystic kidney (PCK) cholangiocytes compared to normal cholangiocytes (Figure 1B and 1C), where the expression p-mTOR (Ser2448) was indicative of the activation of mTOR complex 1 (mTORC1), and p-mTOR (Ser2481) and p-Akt (Ser473) were indicative of the activation of mTORC2
This study demonstrated that the PI3K/Akt/mTOR pathway was activated in PCK cholangiocytes
Summary
Caroli’s disease is characterized by the progressive, multiple cystic dilatation of intrahepatic bile ducts, and is frequently associated with portal fibrosis corresponding to congenital hepatic fibrosis [1]. It belongs to a group of congenital hepatorenal fibrocystic syndrome, and is a hepatic manifestation of autosomal recessive polycystic kidney disease (ARPKD) [2]. The polycystic kidney (PCK) rat is an established animal model of Caroli’s disease with congenital hepatic fibrosis as well as ARPKD [3]. The mTORC2 has been suggested that it lies downstream of phosphatidylinositol 3-kinase (PI3K) signaling and phosphorylates
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