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

Figure

• 

  Fig. 1 Expression of MRP4 and plasma levels of TNFα were increased in liver samples of patients with obstructive cholestasis. (A) The expression of MRP4 protein and mRNA was detected by Western blot and qRT-PCR analysis. Representative blots and corresponding densitometry are shown (n=14). (B) Plasma levels of TNFα, IL-6, and IL-1β in patients with obstructive cholestasis were detected using ELISA kits (n=14). *p<0.05, vs. controls. C, control liver tissues samples; CH, cholestasis liver samples, MRP4, multidrug resistance-associated protein 4; Nrf2, nuclear factor-E2-related factor; TNFα, tumor necrosis factor α.

• 

  Fig. 2 TNFα-induced Nrf2 expression and binding activity to Nrf2 response element in MRP4 promoter. The influence of TNFα on Nrf2 expression in HepG2 cells (A) and Hep-3B cells (B) was detected by qRT-PCR and Western blot analysis. (C) HepG2 cells were serum-starved overnight before TNFα treatment. Nrf2 binding activity to the Nrf2 response element in MRP4/ABCC4 promoter after TNFα treatment was detected by EMSA analysis. (D) HepG2 cells and Hep-3B cells were transfected with Nrf2 siRNA upon treatment with 50 ng/mL of TNFα. Transcription of Nrf2 and MRP4 in HepG2 and Hep-3B cells was detected by qRT-PCR. (E) ROS levels in cells were determined using Reactive Oxygen Species Assay kits. NAC was used to eliminate ROS. The intensity of ROS fluorescence was measured by flow cytometry at 488 nm. (F) The mRNA levels of MRP4 were detected by qRT-PCR. *p<0.05, vs. controls; †p<0.05, vs. TNFα. MRP4, multidrug resistance-associated protein 4; Nrf2, nuclear factor-E2-related factor; TNFα, tumor necrosis factor α; ROS, reactive oxygen species.

• 

  Fig. 3 The p38-Rb-E2F1 pathway mediates TNFα-induced MRP4 expression. (A) The influence of TNFα on p-p38 and E2F1 in HepG2 cells and Hep-3B cells was detected by Western blot analysis. (B) The interaction between E2F1 3′-UTR and TNFα was detected by luciferase activity assay. Wild-type or mutated E2F1 3′-UTR containing binding sites for TNFα was co-transfected with pre-TNFα or pre-DMSO negative control in HEK293T cells. (C and D) The expression of p-p38, p38, E2F1, p-Rb, MRP4, and Nrf2 protein levels in both HepG2 cells and Hep-3B cells was detected by Western blot and their corresponding densitometry values after TNFα and siRNA treatment were quantified. *p<0.05, vs. controls; †p<0.05, vs. TNFα. MRP4, multidrug resistance-associated protein 4; Nrf2, nuclear factor-E2-related factor; TNFα, tumor necrosis factor α.

• 

  Fig. 4 The p38-Rb-E2F1 pathway mediates TNFα-induced MRP4/ABCC4 expression in mouse models of cholestasis. (A) Plasma levels of TNFα, IL-6, and IL-1β in BDL mice were detected by ELISA after BDL operation. (B) Serum levels of ALT and AST in mice were detected using an automatic biochemical meter. (C and D) Representative Western blots for MRP4, Nrf2, p-p38, and E2F1 and their corresponding densitometry values after E2F1 siRNA lentiviral, SB 203580 pretreatment and TNFα injection (% of control group). *p<0.05, vs. controls. †p<0.05, vs. TNFα. MRP4, multidrug resistance-associated protein 4; Nrf2, nuclear factor-E2-related factor; TNFα, tumor necrosis factor α; ALT, alanine aminotransferase; AST, aspartate transaminase; BDL, bile duct ligation.

• 

  Fig. 5 Pathway of TNFα-induced expression of MRP4. MRP4, multidrug resistance-associated protein 4; Nrf2, nuclear factor-E2-related factor; TNFα, tumor necrosis factor α.

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