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 signalregulated 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.