Yonsei Med J.  2019 Nov;60(11):1045-1053. 10.3349/ymj.2019.60.11.1045.

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

• Lian W
• Liu X
• Chen W

• KMID: 2460221
• DOI: http://doi.org/10.3349/ymj.2019.60.11.1045

Abstract

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 α (TNFα) 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. TNFα 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, TNFα 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 TNFα induces the transcription of E2F1. Furthermore, the expression of MRP4, Nrf2, E2F1, and p-p38 proteins was improved with treatment of TNFα in a mouse model of cholestasis. E2F1 siRNA lentivirus or SB 203580 (p38 inhibitor) inhibited these positive effects.
CONCLUSION
Our findings indicated that TNFα induces hepatic MRP4 expression through activation of the p38-E2F1-Nrf2 signaling pathway in human obstructive cholestasis.

Keyword

Obstructive cholestasis; multidrug resistance-associated protein 4; Nrf-2; TNFα

MeSH Terms

Animals
Bile Ducts
Blotting, Western
Carcinoma, Hepatocellular
Cholestasis*
Electrophoretic Mobility Shift Assay
Hep G2 Cells
Humans
Lentivirus
Ligation
Liver
Male
Mice
Multidrug Resistance-Associated Proteins*
Phosphorylation
Real-Time Polymerase Chain Reaction
RNA, Messenger
RNA, Small Interfering
Tumor Necrosis Factor-alpha
Up-Regulation
Multidrug Resistance-Associated Proteins
RNA, Messenger
RNA, Small Interfering
Tumor Necrosis Factor-alpha