role of TNFα in promoting hepatic MRP4 expression via the p38-Rb-E2F1 pathway in human obstructive cholestasis

• Published in: Biochemical and biophysical research communications 2016
• Main Authors: Lian, Wei, Xiaocong Liu, Long Yang, Liangjun Zhang, Xinchan Feng, Wensheng Chen
• Format: Article
• Language: English
• Subjects: animal models; bile secretion; cholestasis; human cell lines; Human obstructive cholestasis; humans; Lentivirus; liver; messenger RNA; MRP4/ABCC4; multiple drug resistance; mutation; Nrf-2; patients; phosphorylation; proteins; quantitative polymerase chain reaction; reverse transcriptase polymerase chain reaction; signal transduction; small interfering RNA; TNFÎ; toxic substances; tumor necrosis factor-alpha; Western blotting
• ISSN: 0006-291X; 1090-2104

Cholestasis includes a spectrum of hepatobiliary diseases of diverse etiologies that are characterized by impaired hepatocellular secretion of bile, resulting in liver damage. The upregulation of multidrug resistance associated protein 4 (MRP4/ABCC4) has been shown to play an important role in an adaptive response that may protect the liver from the accumulation of toxic compounds, but the molecular mechanism of this up-regulation remains elusive. In this study, we demonstrated that TNFα regulated MRP4 expression in vivo and in vitro. The expression of MRP4/ABCC4 mRNA and protein were significantly increased 3.4- and 3.8- fold, respectively in cholestatic patients, while elevated plasma TNFα levels (6.3-fold) were also observed by qRT-PCR and western blot analyses. In vitro, TNFα treatment induced the expression of MRP4/ABCC4 and nuclear factor-E2-related factor (Nrf2) in a dose- and time-dependent manner and enhanced cell nuclear extract (Nrf2) binding activity to the MRP4/ABCC4 promoter, as demonstrated by EMSA in HepG2 cells. In addition, TNFα increased Rb phosphorylation and expression of MRP4 and Nrf2, activated E2F1 and phosphorylated p38 in a time-dependent manner in HepG2 cells, but these effects were markedly inhibited by pretreatment with E2F1 siRNA. The dual-luciferase reporter assay validated that TNFα directly bound the 3′-untranslated region (UTR) of E2F1, which could be abolished by mutation of the E2F1. Furthermore, the expression of MRP4, Nrf2, E2F1 and p-p38 proteins was improved with treatment of TNFα in a mouse model of cholestasis, either E2F1 siRNA lentivirus or SB 203580 (p38 inhibitor) could inhibit these positive effects. Our findings indicated that TNFα induced hepatic MRP4/ABCC4 expression through activation of the p38-Rb-E2F1 signaling pathway, accompanied by an increase in Nrf2 expression in human obstructive cholestasis.