TNFα Induces Multidrug Resistance-Associated Protein 4 Expression through p38-E2F1-Nrf2 Signaling in Obstructive Cholestasis

Purpose: To explore the molecular mechanism of the upregulation of multidrug resistance-associated protein 4 (MRP4) in cholestasis. Materials and Methods: The mRNA and protein levels of MRP4 in liver samples from cholestatic patients were determined by quantitative real-time PCR and Western blot. In human hepatoma HepG2 cells, electrophoretic mobility shift assay (EMSA) was used to determine the affinity of nuclear factor-E2-related factor (Nrf2) binding to MRP4 promoter. Dual-luciferase reporter assay was used to detect the binding of tumor necrosis factor alpha (TNF alpha) to the promotor of E2F1. The bile duct ligation mouse models were established using male C57BL/6 mice. Results: The mRNA and protein levels of MRP4 were significantly increased in cholestatic patients. TNF alpha treatment induced the expression of MRP4 and Nrf2 and enhanced cell nuclear extract binding activity to MRP4 promoter, as demonstrated by EMSA. Nrf2 knockdown reduced MRP4 mRNA levels in both HepG2 and Hep-3B cells. In addition, TNF alpha increased Rb phosphorylation and expression of MRP4 and Nrf2 and activated E2F1 and phosphorylated p38 in HepG2 and Hep-3B cells. These effects were markedly inhibited by pretreatment with E2F1 siRNA. Dual-luciferase reporter assay validated that TNF alpha induces the transcription of E2F1. Furthermore, the expression of MRP4, Nrf2, E2F1, and p-p38 proteins was improved with treatment of TNF alpha in a mouse model of cholestasis. E2F1 siRNA lentivirus or SB 203580 (p38 inhibitor) inhibited these positive effects. Conclusion: Our findings indicated that TNF alpha induces hepatic MRP4 expression through activation of the p38-E2F1-Nrf2 signaling pathway in human obstructive cholestasis.