Department of Infectious Disease, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, China.
Institute of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, China.
Acta Pharmacol Sin. 2020 Jan;41(1):56-64. doi: 10.1038/s41401-019-0266-0. Epub 2019 Jul 17.
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.
据报道，抗结核药物可通过氧化应激诱导肝损伤。在这项研究中，我们研究了利福平（RFP）对多药耐药相关蛋白 2（MRP2）膜表达的影响，以及氧化应激与 RFP 诱导 HepG2 细胞内吞 MRP2 之间的关系。我们发现，RFP（12.5-50 μM）剂量依赖性地降低了 HepG2 细胞中 MRP2 的表达和膜定位，而不改变信使 RNA 水平。RFP（50 μM）诱导氧化应激反应，进一步激活了 HepG2 细胞中的蛋白激酶 C-细胞外信号调节激酶/ c-JUN N 末端激酶/ p38（蛋白激酶 C-细胞外信号调节激酶/ c-JUN N 末端激酶/ p38）和磷脂酰肌醇 3-激酶（PI3K）信号通路。用谷胱甘肽还原乙基酯（2 mM）预处理不仅逆转了氧化应激指标和信号分子的变化，而且减少了 RFP 诱导的 MRP2 绿色荧光强度的降低。我们进行了共免疫沉淀测定，结果表明，MRP2、网格蛋白和衔接蛋白 2（AP2）之间在 HepG2 细胞中存在直接相互作用，其表达明显受到细胞内氧化还原水平变化的影响。用小干扰 RNA 敲低网格蛋白或 AP2 可减弱 RFP 诱导的膜和总 MRP2 的减少。我们进一步证明，RFP 显著增加了 HepG2 细胞中 MRP2 的泛素蛋白酶体降解，这是由 E3 泛素连接酶 GP78 介导的，但不是 HRD1 或 TEB4。总之，本研究表明，RFP 诱导的氧化应激激活了蛋白激酶 C-细胞外信号调节激酶/ c-JUN N 末端激酶/ p38 和 PI3K 信号通路，导致 HepG2 细胞中网格蛋白依赖性内吞和 MRP2 的泛素化，为 RFP 诱导的胆汁淤积机制提供了新的见解。
