Down Regulation of a Matrix Degrading Cysteine Protease Cathepsin L, by Acetaldehyde: Role of C/EBPα

Riyaz A Mir,Shyam S Chauhan,Christian Schönbach

doi:10.1371/journal.pone.0020768

Abstract

BackgroundThe imbalance between extra cellular matrix (ECM) synthesis and degradation is critical aspect of various hepatic pathologies including alcohol induced liver fibrosis. This study was carried out to investigate the effect of acetaldehyde on expression of an extra cellular matrix degrading protease cathepsin L (CTSL) in HepG2 cells.Methodology and ResultsWe measured the enzymatic activity, protein and, mRNA levels of CTSL in acetaldehyde treated and untreated cells. The binding of CAAT enhancer binding protein α (C/EBP α) to CTSL promoter and its key role in the transcription from this promoter and conferring responsiveness to acetaldehyde was established by site directed mutagenesis, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) assays and siRNA technology. Acetaldehyde treatment significantly decreased CTSL activity and protein levels in HepG2 cells. A similar decrease in the mRNA levels and promoter activity was also observed. This decrease by acetaldehyde was attributed to the fall in the liver enriched transcription factor C/EBP α levels and it's binding to the CTSL promoter. Mutagenesis of C/EBP α binding motifs revealed the key role of this factor in CTSL transcription as well as conferring responsiveness to acetaldehyde. The siRNA mediated silencing of the C/EBP α expression mimicked the effect of acetaldehyde on CTSL levels and its promoter activity. It also abolished the responsiveness of this promoter to acetaldehyde.ConclusionAcetaldehyde down regulates the C/EBP α mediated CTSL expression in hepatic cell lines. The decreased expression of CTSL may at least in part contribute to ECM deposition in liver which is a hallmark of alcoholic liver fibrosis.

Highlights

Alcohol induced hepatic fibrosis is preceded by the accumulation of extra cellular matrix (ECM) proteins in the liver
The decreased expression of cathepsin L (CTSL) may at least in part contribute to ECM deposition in liver which is a hallmark of alcoholic liver fibrosis
To ascertain that the observed decrease in CTSL was not due the direct inhibition of the enzyme by acetaldehyde we quantiated this protease by Western blotting and a representative blot is presented in (Figure 2C). human CTSL (hCTSL) is synthesized as a 42 kDa pre-proenzyme which is processed to a 34 kDa proenzyme and to a 26 kDa enzymatically active form

Summary

Introduction

Alcohol induced hepatic fibrosis is preceded by the accumulation of ECM proteins in the liver. The accumulation of these proteins which are predominantly synthesized by the activated hepatic stellate cells is governed by the rate of their synthesis and degradation. While the effect of alcohol or its oxidative metabolite acetaldehyde on the levels of various ECM degrading enzymes except CTSL have been extensively studied in stellate cells [7]. The imbalance between extra cellular matrix (ECM) synthesis and degradation is critical aspect of various hepatic pathologies including alcohol induced liver fibrosis. This study was carried out to investigate the effect of acetaldehyde on expression of an extra cellular matrix degrading protease cathepsin L (CTSL) in HepG2 cells

Methods

Results

Conclusion