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

BACKGROUND: 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. METHODOLOGY AND RESULTS: We 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. CONCLUSION: Acetaldehyde 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

Mutagenesis of C/EBP α binding motifs abolishes CTSL promoter activity and its responsiveness to acetaldehyde.

<p>The two C/EBPα binding motifs were mutated individually or in combination and the resulting constructs were transfected in HepG2 cells. The mutated motifs have been marked by <b>X</b>. The luciferase activity of these constructs was assayed after culturing the transfected cells in the presence or absence of acetaldehyde for 24 hours. Values are mean ± S.D from three independent experiments. Values significantly different from untreated controls have been marked by *.</p

Silencing of C/EBP α expression simulate the effect of acetaldehyde.

<p>(A). Simulation of the effect of acetaldehyde treatment on CTSL expression by Silencing of C/EBP α expression. HepG2 cells transfected with 2.0 or 4.0 µg of C/EBP α si RNA expression vectors ( pU6-C/EBPα-1+pU6-C/EBPα-2 ) or 4.0 µg of scrambled Si RNA expression vector(pU6-SC) were cultured in the presence or absence of 5 um acetaldehyde as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020768#pone-0020768-g003" target="_blank">figure 3</a>. After 24 hours the cells were lysed and levels of C/EBP α, CTSL, lamin B and α tubulin were detected by western blotting using specific antibodies. The cell lysates prepared from untransfected, untreated or treated with acetaldehyde cells were also subjected to western blotting using the above mentioned antibodies and served as control. Each blot shown is a representative of at least three separate experiments. (B). Silencing of C/EBP α expression simulates the effect of acetaldehyde treatment on CTSL promoter activity. HepG2 cells co transfected with 1.0 µg of pRB-1.75 and 4.0 µg of C/EBP α si RNA expression vectors ( pU6-C/EBPα-1+pU6-C/EBPα-2 ) or scrambled Si RNA expression vector(pU6-SC) were cultured in the presence or absence of 5 µM acetaldehyde as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020768#pone-0020768-g003" target="_blank">figure 3</a>. After 24 hours the cells were lysed and luciferase activity was assayed. Each transfection was done in triplicates and results are expressed as mean ± SD from three independent experiments. Values significantly different from control has been marked by *.</p

Acetaldehyde treatment decreases CTSL and C/EBP α level in human hepatic stellate cells.

<p>Cell lysates of acetaldehyde treated or untreated hepatic stellate cells were resolved on SDS-PAGE and subjected to western bloting using specific antibodies for CTSL, C/EBPα, C/EBPβ or C/EBPδ as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020768#s2" target="_blank">Materials and Methods</a> section. Simultaneously western blot for α-Tubulin was performed and used for normalization for equal loading.</p

List of oligo nucleotides used for various purposes in the present study.

<p> <b>Mutated nucleotides are shown in lower case and bold face.</b></p

Reduction of CTSL mRNA in acetaldehyde treated HepG2 cells.

<p>Total cellular RNA isolated from acetaldehyde treated or untreated cells was reverse transcribed and subjected to real time PCR using sybergreen and gene specific primers for (A) CTSL (B) cathepsin B or (C) cystatin C. Similarly real time PCR for 18 S rRNA was performed and used as an internal control. Cycle threshold (Ct) values were calculated for each PCR and relative fold change was calculated using 2−^{ΔΔ Ct} method. Each set of observation was compared to the other set using a paired two-tailed t-test, assuming unequal variances among the sample means. A p value of ≤0.05 was considered to be statistically significant. Values are mean ± SD from three independent experiments performed in triplicates. Values significantly different from control has been marked by *. (D) Analysis of PCR products. After the completion of real time PCR amplified products were resolved on agarose gel to confirm the amplification of a single product. Representative gel for each target is given in the figure.</p

Effect of acetaldehyde on CTSL promoter activity and identification of acetaldehyde response element.

<p>(A). CTSL promoter activity in acetaldehyde treated and untreated cells. HepG2 cells were transfected with CTSL promoter reporter construct (pRB1.75) or pGL3C (control) vector. After 24 hours the transfected cells were treated with 5.0 µM acetaldehyde or vehicle solution (PBS) for another 24 hours and then the cells were lysed followed by assaying luciferase activity in the cell lysate. (B). Identification of acetaldehyde response elements in CTSL promoter by deletion analysis. HepG2 cells transfected with various CTSL promoter reporter deletion constructs were cultured in the presence or absence of acetaldehyde as described above and luciferase activity was assayed. All constructs were cotransfected with the pRLnull vector and the renilla luciferase activity was used to normalize for the transfection efficiency. Each transfection was done in triplicates and results are expressed as mean ± SD from three independent experiments. Values significantly different from control has been marked by *.</p

Growth rates of HepG2 cells in the presence and absence of acetaldehyde.

<p>10⁵ cells were plated in each well of a six well plate and allowed to grow in the presence or absence of 5.0 µM acetaldehyde. After various time points the cells were trypsinized and counted. Values are mean ±SD of three independent experiments performed in triplicate.</p

Reduction in the activity and levels of CTSL by acetaldehyde treatment in HepG2 cells.

<p>Cathepsin L+cathepsin B activity was assayed in the cell lysates spectroflourimetrically as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020768#s2" target="_blank">materials and methods</a>. The specific activity of CTSL was measured by including a specific cathepsin B inhibitor (CA-074) in the assay mixture and used to calculate cathepsin B activity, by subtracting it from the total enzymatic activity of L+B. (A) Effect of acetaldehyde treatment on CTSL activity (B) Effect of acetaldehyde treatment on cathepsin B activity. Values are mean ± S.D from at least three independent experiments performed in triplicate. Values significantly different from untreated controls have been marked by *(P≤0.05). (C) Effect of acetaldehyde treatment on CTSL levels. CTSL levels in acetaldehyde treated and untreated cells were detected by western blotting using a monoclonal antibody. Simultaneously western blot for α Tubulin was performed. Representative blots are shown in figure. (D) Densitometric quantitation of CTSL levels in acetaldehyde treated and untreated cells. The three specific bands of CTSL (42 kDa, 34 kDa and 26 kDa) representing prepro, pro and mature forms of the protease were quantitated densitometrically and the values thus obtained for each form were added. The values obtained for α-tubulin were used as an internal control for equal loading. Values are mean ± S.D from three independent experiments. Values significantly different from untreated controls have been marked by *(P≤0.05).</p