Critical role of PA28gamma in hepatitis C virus-associated steatogenesis and hepatocarcinogenesis

Abstract

Hepatitis C virus (HCV) is a major cause of chronic liver disease that frequently leads to steatosis, cirrhosis, and eventually hepatocellular carcinoma (HCC). HCV core protein is not only a component of viral particles but also a multifunctional protein because liver steatosis and HCC are developed in HCV core gene-transgenic (CoreTg) mice. Proteasome activator PA28gamma/REGgamma regulates host and viral proteins such as nuclear hormone receptors and HCV core protein. Here we show that a knockout of the PA28gamma gene induces the accumulation of HCV core protein in the nucleus of hepatocytes of CoreTg mice and disrupts development of both hepatic steatosis and HCC. Furthermore, the genes related to fatty acid biosynthesis and srebp-1c promoter activity were up-regulated by HCV core protein in the cell line and the mouse liver in a PA28gamma-dependent manner. Heterodimer composed of liver X receptor alpha (LXRalpha) and retinoid X receptor alpha (RXRalpha) is known to up-regulate srebp-1c promoter activity. Our data also show that HCV core protein enhances the binding of LXRalpha/RXRalpha to LXR-response element in the presence but not the absence of PA28gamma. These findings suggest that PA28gamma plays a crucial role in the development of liver pathology induced by HCV infection.

Fig. 1.

Preparation and characterization of PA28γ-knockout HCV core-transgenic mice. (A) The structures of the wild-type and mutated PA28γ genes and the transgene encoding the HCV core protein under the control of the HBV X promoter were investigated. Positions corresponding to the screening primers and sizes of PCR products are shown. PCR products of the HCV core gene as well as wild-type and mutated PA28γ alleles were amplified from the genomic DNAs of PA28γ^+/+, PA28γ^+/+CoreTg, PA28γ^−/−, and PA28γ^−/−CoreTg mice. (B) Body weights of PA28γ^+/+, PA28γ^+/+CoreTg, PA28γ^−/−CoreTg, and PA28γ^−/− mice at the age of 6 months. (C) HCV core protein levels in the livers of PA28γ^+/+CoreTg and PA28γ^−/−CoreTg mice were determined by ELISA (mean ± SD, n = 10). (D) Localization of HCV core protein in the liver. Liver sections of PA28γ^+/+, PA28γ^+/+CoreTg, and PA28γ^−/−CoreTg mice at the age of 2 months were stained with anti-HCV core antibody.

Fig. 2.

Accumulation of lipid droplets by expression of HCV core protein. (A) Liver sections of the mice at the age of 6 months were stained with hematoxylin/eosin (HE). (B) (Upper) Liver sections of PA28γ^+/+CoreTg and PA28γ^−/−CoreTg mice at the age of 6 months were stained with oil red O. (Lower) The area occupied by lipid droplets of PA28γ^+/+ (white), PA28γ^+/+CoreTg (gray), PA28γ^−/−CoreTg (black), and PA28γ^−/− (dark gray) mice was calculated by Image-Pro software (MediaCybernetics, Silver Spring, MD) (mean ± SD, n = 10).

Fig. 3.

Transcription of genes regulating lipid biosynthesis in the mouse liver. (A) Total RNA was prepared from the livers of 2-month-old mice; and the transcription of genes encoding SREBP-1a, SREBP-1c, and SREBP-2 was determined by real-time PCR. (B) The transcription of genes encoding SREBP-1c, fatty acid synthase, acetyl-CoA carboxylase, stearoyl-CoA desaturase, HMG-CoA synthase, and HMG-CoA reductase of 6-month-old mice was measured by real-time PCR. The transcription of the genes was normalized with that of hypoxanthine phosphoribosyltransferase, and the values are expressed as relative activity (n = 5; ∗, P < 0.05; ∗∗, P < 0.01). The transcription of each gene in PA28γ^+/+, PA28γ^+/+CoreTg, PA28γ^−/−CoreTg, and PA28γ^−/− mice is indicated by white, gray, black, and dark gray bars, respectively.

Fig. 4.

Activation of the srebp-1c promoter by HCV core protein. (A) FLAG-LXRα and HA-RXRα were expressed in 293T cells together with or without HCV core protein. Ligands for LXRα and RXRα dissolved in ethanol [Ligands (+)] or ethanol alone [Ligands (−)] were added to the culture supernatant at 24 h posttransfection. Cells were harvested at 48 h posttransfection, and nuclear extracts were mixed with the reaction buffer for EMSA in the presence or absence of antibody (100 ng) against HA, FLAG, HCV core or PA28γ, or nonlabeled LXRE probe (Competitor). (Left) The resulting mixtures were subjected to PAGE and blotted with horseradish peroxidase/streptavidin. The mobility shift of the LXRE probe and its supershift are indicated by a gray and black arrow, respectively. (Right) Expression of HCV core, HA-RXRα, FLAG-LXRα, and PA28γ in cells was detected by immunoblotting. (B) Effects of ligands for RXRα, 9-cis-retinoic acid (9cisRA), and for LXRα, 22(R)-hydroxylcholesterol (22RHC), on the activation of the srebp-1c promoter in 293T cells expressing RXRα, LXRα, and/or HCV core protein. Ligands were added into the medium at 24 h posttransfection at a concentration of 5 μM, and the cells were harvested after 24 h of incubation.

Fig. 5.

PA28γ is required for HCV core-dependent activation of the srebp-1c promoter. (A) Effect of PA28γ knockdown on the LXRα/RXRα–DNA complex. FLAG-LXRα and HA-RXRα were expressed in FLC4 (control) or PA28γ-knockdown (PA28γ KD) cells together with or without HCV core protein. Cells were harvested at 48 h posttransfection, and nuclear extracts were mixed with the reaction buffer for EMSA. (Upper) The resulting mixtures were subjected to PAGE and blotted with horseradish peroxidase-streptavidin. The mobility shift of the LXRE probe is indicated by an arrow. (Lower) Expression of HCV core, HA-RXRα, FLAG-LXRα, and PA28γ in cells was detected by immunoblotting. (B) Effect of PA28γ knockout on the LXRα/RXRα–DNA complex in the mouse liver. (Upper) Nuclear extracts were prepared from the livers of 2-month-old PA28γ^−/−, PA28γ^+/+CoreTg, PA28γ^−/−CoreTg, and PA28γ^+/+ mice and subjected to EMSA. The mobility shift of the LXRE probe is indicated by an arrow. (Lower) The expression of HCV core, PA28γ, and β-actin in the livers of the mice was detected by immunoblotting. (C) Effect of HCV core protein on srebp-1 promoter activity in PA28γ-knockout fibroblasts. A plasmid encoding firefly luciferase under the control of the srebp-1c promoter was transfected into MEFs prepared from PA28γ^+/+ (Left) or PA28γ^−/− (Right) mice together with a plasmid encoding a Renilla luciferase. An empty plasmid or plasmids encoding mouse RXRα or LXRα were also cotransfected into the cells together with (gray bars) or without (white bars) a plasmid encoding HCV core protein. Luciferase activity under the control of the srebp-1c promoter was determined, and it is expressed as the fold increase in relative luciferase activity after standardization with the activity of Renilla luciferase.