AP-1 Inhibition by SR 11302 Protects Human Hepatoma HepG2 Cells from Bile Acid-Induced Cytotoxicity by Restoring the NOS-3 Expression

Sandra González-Rubio,Clara I Linares,Patricia Aguilar-Melero,Manuel De La Mata,Gustavo Ferrín,José L Montero-Álvarez,Manuel Rodríguez-Perálvarez

doi:10.1371/journal.pone.0160525

Abstract

The harmful effects of bile acid accumulation occurring during cholestatic liver diseases have been associated with oxidative stress increase and endothelial nitric oxide synthase (NOS-3) expression decrease in liver cells. We have previously reported that glycochenodeoxycholic acid (GCDCA) down-regulates gene expression by increasing SP1 binding to the NOS-3 promoter in an oxidative stress dependent manner. In the present study, we aimed to investigate the role of transcription factor (TF) AP-1 on the NOS-3 deregulation during GCDCA-induced cholestasis. The cytotoxic response to GCDCA was characterized by 1) the increased expression and activation of TFs cJun and c-Fos; 2) a higher binding capability of these at position -666 of the NOS-3 promoter; 3) a decrease of the transcriptional activity of the promoter and the expression and activity of NOS-3; and 4) the expression increase of cyclin D1. Specific inhibition of AP-1 by the retinoid SR 11302 counteracted the cytotoxic effects induced by GCDCA while promoting NOS-3 expression recovery and cyclin D1 reduction. NOS activity inhibition by L-NAME inhibited the protective effect of SR 11302. Inducible NOS isoform was no detected in this experimental model of cholestasis. Our data provide direct evidence for the involvement of AP-1 in the NOS-3 expression regulation during cholestasis and define a critical role for NOS-3 in regulating the expression of cyclin D1 during the cell damage induced by bile acids. AP-1 appears as a potential therapeutic target in cholestatic liver diseases given its role as a transcriptional repressor of NOS-3.

Highlights

Bile acids (BAs) are physiological detergents synthesized from cholesterol in the liver, which are required for absorption and transport of dietary fats and lipid-soluble vitamins, and disposal of toxic metabolites, drugs and xenobiotics
Bioinformatic analysis of the pNOS-3 sequence identified candidate TF binding sites (TFBS) involved in the modulation of the NOS-3 expression
When we later investigated the effect of AP-1 inhibition in Glycochenodeoxycholic acid (GCDCA)-treated pGL4-NOS3 cells, we found that all assayed TF inhibitors (TFIs), curcumin, quercetin and the specific AP-1 inhibitor SR

Summary

Introduction

Bile acids (BAs) are physiological detergents synthesized from cholesterol in the liver, which are required for absorption and transport of dietary fats and lipid-soluble vitamins, and disposal of toxic metabolites, drugs and xenobiotics. Cholestasis may produce accumulation of toxic BAs in liver leading to hepatocellular apoptosis, which represents a key cellular mechanism for BA-mediated liver injury and progression of CLD towards fibrosis, cirrhosis and chronic liver failure [1] [2] [3]. Glycochenodeoxycholic acid (GCDCA) is the major human hydrophobic BA [4] that has been held responsible for cholestasis-associated liver injury [5] [6]. By contrast, another physiological constituent of human bile, ursodeoxycholic acid (UDCA), has been demonstrated to exert anticholestatic effects in several cholestatic disorders and is the treatment of choice in primary biliary cirrhosis, which represents the leading cause of small-duct biliary diseases. UDCA is frequently combined or replaced by other therapeutics agents such as corticosteroids, immunosuppressant and other drugs [6] [7]

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