Volume 132, Issue 5, Pages 1955-1967 (May 2007) Hepatitis B Virus X Protein Induces Hepatic Steatosis Via Transcriptional Activation of SREBP1 and PPARγ  Kook Hwan Kim, Hye–Jun Shin, Kyeongjin Kim, Hyun Mi Choi, Sang Hoon Rhee, Hyung–Bae Moon, Hyeong Hoe Kim, Ung Suk Yang, Dae–Yeul Yu, Jaehun Cheong  Gastroenterology  Volume 132, Issue 5, Pages 1955-1967 (May 2007) DOI: 10.1053/j.gastro.2007.03.039 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 The effect of HBx on hepatic lipid accumulation. (A) Macroscopic analyses of livers. Hepatomegaly and pale coloring was observed in 11-week-old HBx-transgenic mice. (B) Liver weight in HBx-transgenic mice and wild-type mice. The liver weight/body weight ratio was analyzed in wild-type and HBx-transgenic mice at 11 weeks. Values represent mean ± SD (n = 10 mice/group). *P < .05 compared with wild-type mice. (C) H&E staining in liver tissue of 11-week-old HBx-transgenic mice and wild-type mice. The droplets are indicative of neutral lipid accumulation. (D) Oil-Red O staining of histologic sections of liver taken from 24-week-old HBx-transgenic mice. The red staining indicates neutral lipid. (E) The establishment of stable cell lines constitutively expressing HBx. The expression level of HBx mRNA and protein in HepG2-HBx stable cells was analyzed by RT-PCR and Western blotting, respectively. Oil-Red O staining reveals the increased hepatocellular lipids in HepG2-HBx stable cells. Gastroenterology 2007 132, 1955-1967DOI: (10.1053/j.gastro.2007.03.039) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 The effect of HBx on SREBP1 and PPARα gene expression. (A) The expression of SREBP1 protein in HepG2-HBx stable cells. Western blotting and RT-PCR were performed on cell extracts of HepG2-HBx stable cells using anti-SREBP1 antibody and a specific primer. The equivalence of mRNA and protein loading in the lanes was confirmed by the expression of β-actin and actin, respectively. Data shown are 1 experiment representative of 3 independent experiments. (B) The mRNA and protein expression of SREBP1 in Chang liver cells transiently transfected with HBx expression vector. Cells were transfected with 2 μg of HBx expression plasmid and empty vector. After 48 hours of transfection, RT-PCR and Western blotting were performed for investigating expression of the indicated mRNA and protein, respectively. Data from RT-PCR were quantified and normalized relative to the β-actin mRNA level using ImageJ version 1.35d (National Institutes of Health Image). Data for Western blotting are representative of 5 independent experiments. Data for RT-PCR are representative of 4 independent experiments. Values represent mean ± SD (n = 4). *P < .01 compared with mock transfectants. (C) The effect on the mRNA and protein expression of PPARα in HBx-transfected Chang liver cells. Data for Western blotting and RT-PCR are representative of 4 independent experiments. Values represent mean ± SD (n = 4). (D) The mRNA and protein expression of SREBP1 and PPARα in 11-week-old HBx-transgenic mice and wild-type mice. Data are representative of at least 3 independent experiments performed in duplicate. Gastroenterology 2007 132, 1955-1967DOI: (10.1053/j.gastro.2007.03.039) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 SREBP1 transcriptional activity and lipogenic gene expression in Chang liver cells expressing HBx. (A) The effect of HBx on SREBP1-increased FAS promoter activity. Cells were transfected with 0.2 μg of each indicated expression plasmid, along with 0.2 μg of FAS reporter plasmid as indicated. After 48 hours of transfection, cell lysates were obtained and luciferase activity was measured. The amount of DNA in each transfection was kept constant by adding an appropriate amount of the pcDNA3.1/HisC vector. Relative luciferase activity was quantified relative to the level of luciferase activity from cells transfected with FAS reporter plasmid only. Luciferase activity was normalized for transfection efficiency by corresponding β-galactosidase activity. Data shown are the mean ± SD (n = 7). *P < .005 compared with SREBP1a transfectants. **P < .001 compared with SREBP1c transfectants. (B) The mRNA expression of FAS in SREBP1a/HBx-cotransfected Chang liver cells. RT-PCR was performed with specific primers. Data shown are 1 experiment representative of 4 independent experiments. Values represent mean ± SD (n = 4). ***P < .05 compared with SREBP1c transfectants. (C) The interaction between HBx and HA-SREBP1c by co-immunoprecipitation assay. Chang liver cells were transfected with HBx plasmid or empty vector, along with HA-SREBP1c plasmid. After 48 hours of transfection, cell lysates were obtained and immunoprecipitated with HA antibody followed by Western blotting using HBx antibody. (D) The interaction between HBx and GST-SREBP1c by in vivo GST pull-down assay. Chang liver cells were transfected with vectors for GST or GST-SREBP1c proteins, along with HBx expression vector. After 48 hours of transfection, cell lysates were obtained and immobilized onto glutathione-Sepharose 4B beads followed by Western blotting using HBx antibody and GST-antibody. (E) Colocalization of HBx and SREBP1c in nuclear foci. Both GFP-HBx and RFP-SREBP1c plasmids were cotransfected into Chang liver cells. After 24 hours of transfection, cells were counterstained with Hoechst to label nuclei and cell imaging was assessed by fluorescence microscopy. Gastroenterology 2007 132, 1955-1967DOI: (10.1053/j.gastro.2007.03.039) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 The requirement for activation of the PI3K/AKT pathway in HBx-mediated SREBP1 activation. (A) The increased phosphorylation of AKT by HBx protein. The phosphorylated form of AKT in Chang liver cells expressing HBx protein was determined by Western blotting. Data were quantified and normalized relative to the AKT band level using ImageJ version 1.35d (National Institutes of Health Image). Data shown are representative of 4 independent experiments. Values represent mean ± SD (n = 4). *P < .01 compared with mock transfectants. (B) The decreased mRNA and protein expression of PTEN in HBx-transgenic mice at 11 weeks. Data are representative of at least 3 independent experiments performed in duplicate. (C) The PI3K inhibitor, LY294002, abolishes HBx-mediated SREBP1 gene expression. Chang liver cells were transfected with 2 μg of expression plasmids for HBx protein. After 24 hours of transfection, cells were treated with 25 μmol/L LY294002 or vehicle for 24 hours. Then RT-PCR (left panel) and Western blotting (right panel) were performed with specific primers and antibodies, respectively. Data for RT-PCR are representative of 4 independent experiments. Values represent mean ± SD (n = 4). **P < .01 compared with mock transfectants. ***P < .01 compared with HBx transfectants without LY294002. (D) LY294002 alleviates HBx-induced SREBP1 transcriptional activity. Chang liver cells were transfected with 0.2 μg of each indicated expression plasmid, along with 0.2 μg of FAS reporter plasmid. After 24 hours of transfection, cells were treated with LY294002 or vehicle for 24 hours. Cells were harvested and luciferase activity was measured. (E) LY294002 blocks HBx-mediated increase of FAS mRNA. Chang liver cells were transfected with 2 μg of HBx expression vector. After 24 hours of transfection, cells were treated with LY294002 or vehicle for 24 hours. Total RNA was extracted from cells and analyzed for RT-PCR. Data are representative of 4 independent experiments. Values represent mean ± SD (n = 4). **P < .01 compared with mock transfectants. ***P < .01 compared with HBx transfectants without LY294002. Gastroenterology 2007 132, 1955-1967DOI: (10.1053/j.gastro.2007.03.039) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 The effect of HBx on the expression and activity of C/EBPα and PPARγ. (A) The up-regulation of PPARγ gene expression in HepG2-HBx stable cells. Cell lysates were prepared and analyzed by RT-PCR and Western blotting as indicated. Data shown are 1 experiment representative of 3 independent experiments. (B) The increase of PPARγ gene expression in HBx-transfected Chang liver cells. RT-PCR and Western blotting were performed as indicated. Data for Western blotting are representative of 4 independent experiments. Data for RT-PCR are representative of 3 or 4 independent experiments. Values represent mean ± SD (n = 3–4). *P < .01 compared with mock transfectants. (C) The increased mRNA and protein expression of PPARγ gene in HBx-transgenic mice at 11 weeks. Data are representative of at least 3 independent experiments performed in duplicate. (D) The enhanced expression of C/EBPα gene in HepG2-HBx stable cells and HBx-transgenic mice. Western blotting and RT-PCR were performed in HepG2-HBx stable cells and 11-week-old HBx transgenic mice, respectively. Data are representative of at least 2 or 3 independent experiments. (E) HBx enhances C/EBPα-induced activation of PPARγ promoter. HepG2 cells were transfected with indicated expression plasmid. After 48 hours of transfection, luciferase activity was determined. Data shown are the mean ± SD (n = 4). *P < .01 compared with C/EBPα transfectants. (F) The effect of HBx on PPARγ transcriptional activity. Chang liver cells were transfected with indicated expression plasmids. After 24 hours of transfection, cells were treated with 1 μmol/L rosiglitazone or vehicle for 24 hours. After 48 hours of transfection, luciferase activity was determined. Data shown are the mean ± SD (n = 4). *P < .05 and **P < .01 compared with PPARγ1 transfectants without/with rosiglitazone, respectively. ***P < .01 and ****P < .05 compared with PPARγ2 transfectants without/with rosiglitazone, respectively. Gastroenterology 2007 132, 1955-1967DOI: (10.1053/j.gastro.2007.03.039) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 The effect of HBx on the expression of adipogenic and lipogenic genes. (A) The enhanced expression of adipogenic and lipogenic genes in Chang liver cells expressing HBx protein. Data are representative of 4 independent experiments. Values represent mean ± SD (n = 4). *P < .05 compared with mock transfectants. **P < .01 compared with mock transfectants. (B) The increased expression of adipogenic/lipogenic genes in 11-week-old HBx-transgenic mice. Data are representative of at least 3 independent experiments performed in duplicate. Gastroenterology 2007 132, 1955-1967DOI: (10.1053/j.gastro.2007.03.039) Copyright © 2007 AGA Institute Terms and Conditions

Figure 7 Mechanisms by which HBx induces hepatic steatosis. HBx may lead to increased AKT activity and inhibition of PTEN expression, resulting in activation of SREBP1, which up-regulates hepatic lipid synthesis. The enhancement of C/EBPα expression and activity by HBx may lead to increased PPARγ activity, thereby inducing the expression of hepatic adipogenic and lipogenic genes. Gastroenterology 2007 132, 1955-1967DOI: (10.1053/j.gastro.2007.03.039) Copyright © 2007 AGA Institute Terms and Conditions