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Rifampicin induces clathrin-dependent endocytosis and ubiquitin–proteasome degradation of MRP2 via oxidative stress-activated PKC-ERK/JNK/p38 and PI3K signaling pathways in HepG2 cells

Authors: Bao-yan Xu1, Xu-dong Tang2, Jing Chen2, Hong-bo Wu2, Wen-sheng Chen2, Lei Chen2
1 Department of Infectious Disease, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing 400038, China
2 Institute of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing 400038, China
Correspondence to: Lei Chen: 13883895566@163.com,
DOI: 10.1038/s41401-019-0266-0
Received: 18 October 2018
Accepted: 30 May 2019
Advance online: 17 July 2019

Abstract

It was reported that antituberculosis medicines could induce liver damage via oxidative stress. In this study, we investigated the effects of rifampicin (RFP) on the membrane expression of multidrug resistance-associated protein 2 (MRP2) and the relationship between oxidative stress and RFP-induced endocytosis of MRP2 in HepG2 cells. We found that RFP (12.5–50 μM) dose-dependently decreased the expression and membrane localization of MRP2 in HepG2 cells without changing the messenger RNA level. RFP (50 μM) induced oxidative stress responses that further activated the PKC-ERK/JNK/p38 (protein kinase C-extracellular signal-regulated kinase/c-JUN N-terminal kinase/p38) and PI3K (phosphoinositide 3-kinase) signaling pathways in HepG2 cells. Pretreatment with glutathione reduced ethyl ester (2 mM) not only reversed the changes in oxidative stress indicators and signaling molecules but also diminished RFP-induced reduction in green fluorescence intensity of MRP2. We conducted co-immunoprecipitation assays and revealed that a direct interaction existed among MRP2, clathrin, and adaptor protein 2 (AP2) in HepG2 cells, and their expression was clearly affected by the changes in intracellular redox levels. Knockdown of clathrin or AP2 with small interfering RNA attenuated RFP-induced decreases of membrane and total MRP2. We further demonstrated that RFP markedly increased the ubiquitin–proteasome degradation of MRP2 in HepG2 cells, which was mediated by the E3 ubiquitin ligase GP78, but not HRD1 or TEB4. In conclusion, this study demonstrates that RFP-induced oxidative stress activates the PKC-ERK/JNK/p38 and PI3K signaling pathways that leads to clathrin-dependent endocytosis and ubiquitination of MRP2 in HepG2 cells, which provides new insight into the mechanism of RFP-induced cholestasis.
Keywords: rifampicin; cholestasis; MRP2; oxidative stress; PKC-ERK/JNK/p38; PI3K; endocytosis; cholestasis; HepG2 cells

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