Hepatitis C Virus Infection Increases c-Jun N-Terminal Kinase (JNK) Phosphorylation and Accentuates Hepatocyte Lipoapoptosis

Abstract

BACKGROUND Hepatitis C virus (HCV) infection and metabolic diseases including nonalcoholic steatohepatitis (NASH) exhibit a complex interplay. Although free fatty acid-mediated apoptosis is a prominent feature of NASH, the impact of HCV infection on hepatocyte lipotoxicity has remained largely unexplored. The study aimed at identifying whether infection by HCV affected the apoptotic pathway in hepatocytes during fatty acid assault. MATERIAL AND METHODS OR6 cells, which are derived from human hepatocellular carcinoma Huh-7 cells and harbor a full-length HCV RNA genome replication system, were treated with palmitate. Apoptosis was examined by 4',6-diamidino-2-phenylindole staining. Activation and expression of JNK, Bim, cIAP-1, and Mcl-1 were examined by immunoblotting. mRNA expression of CHOP, a major player in endoplasmic reticulum stress-mediated apoptosis, was assessed by real-time PCR. RESULTS Palmitate-induced hepatocyte apoptosis was significantly enhanced in OR6 cells compared to cured cells, in which the HCV genome had been eradicated by treatment with interferon-α. Although basal expression of CHOP mRNA was enhanced in OR6 cells compared to cured cells, it was similarly upregulated in both cell lines following palmitate treatment. Notably, palmitate-induced JNK phosphorylation was accentuated in OR6 cells compared to cured cells. Inhibition of JNK with SP600125 attenuated palmitate-induced apoptosis. Palmitate-mediated upregulation of BH3-only protein Bim, which acts downstream of JNK, was also enhanced in OR6 cells compared to cured cells. In contrast, Mcl-1 and cIAP-1 were equally reduced in OR6 cells and cured cells following palmitate treatment. CONCLUSIONS These findings suggest that during lipoapoptosis, HCV infection may enhance hepatocyte toxicity by increasing JNK phosphorylation.

Figure 1

Induction of hepatocyte lipoapoptosis in HCV replicon cells. (A) Upper panel: OR6 cells and Huh-7 cells were treated with palmitate for 24 hours. Nuclear staining with DAPI and fluorescence microscopy was used to assess apoptotic cells. Palmitate-induced hepatocyte apoptosis was enhanced in OR6 cells, * p<0.05. Data are expressed as mean ±SE. Lower panel: Images of DAPI staining overlaid with bright field view. Each image is representative of at least three independently taken photographs. Arrows indicate apoptotic cells. Original magnification was 400×. (B) Cleaved and uncleaved PARP were examined by immunoblotting after 24 hours of palmitate treatment in OR6 cells and cured cells. (C) Huh-7 cells were treated with IFN-α for two weeks. Cells were then treated by palmitate for 24 hours and lipoapoptosis was examined by staining cells with DAPI. Data are expressed as mean ±SE. For apoptosis measurements, we first confirmed the homogeneity of the data within experimental conditions using the Chi-squared test. The dose-dependent increase in apoptosis among palmitate-treated cells was then assessed by the Cochran-Armitage test. Finally, statistical differences between OR6 cells and cured cells, as well as differences between IFN-α treated cells and untreated cells under each experimental condition were determined by Fisher’s exact test.

Figure 2

HCV infection induces JNK phosphorylation. (A) JNK phosphorylation was examined by immunoblotting after treatment with palmitate for eight hours. (B) Expression of GSK was examined by immunoblotting after treatment with palmitate for eight hours. (C) Left panel: OR6 cells were treated with JNK inhibitor SP600125, and lipoapoptosis was examined by staining cells with DAPI after treatment for 24 hours, * p<0.05. Data are expressed as mean ±SE. Right panel: images of DAPI staining overlaid with bright field view. Lower panel: images of DAPI staining overlaid with bright field view. Each image is representative of at least three independently taken photographs. Arrows indicate apoptotic cells. Original magnification was 400×.

Figure 3

Expression of CHOP and Bim in OR6 cells increases following palmitate treatment. OR6 cells and cured cells were treated with palmitate for eight hours. (A) Bim expression was examined by immunoblotting. Data are expressed as mean ±SE. (B) CHOP expression was assessed by RT-PCR. Differences between groups were compared using ANOVA followed by a post hoc test (Bonferroni’s method), or a two-way ANOVA followed by Bonferroni’s method. A p value <0.05 was considered statistically significant.

Figure 4

Expression of anti-apoptotic proteins Mcl-1 and cIAP-1 in both OR6 cells and cured cells decreases following palmitate treatment. OR6 cells and Huh-7 cells were treated with palmitate for eight hours. Expression of Mcl-1 and cIAP-1 was examined by immunoblotting. Homogeneity of the data within experimental conditions was confirmed by the Chi-squared test. The dose-dependent increase in apoptosis among palmitate-treated cells was then assessed by the Cochran-Armitage test. Finally, statistical differences between JNK-treated and untreated cells under each experimental condition were determined by Fisher’s exact test.