HBV (hepatitis B virus) is a primary cause of chronic liver disease, which frequently results in hepatitis, cirrhosis and ultimately HCC (hepatocellular carcinoma). Recently, we showed that HBx (HBV protein X) expression induces lipid accumulation in hepatic cells mediated by the induction of SREBP1 (sterol-regulatory-element-binding protein 1), a key regulator of lipogenic genes in the liver. However, the molecular mechanisms by which HBx increases SREBP1 expression and transactivation remain to be clearly elucidated. In the present study, we demonstrated that HBx interacts with LXRα (liver X receptor α) and enhances the binding of LXRα to LXRE (LXR-response element), thereby resulting in the up-regulation of SREBP1 and FAS (fatty acid synthase) in the presence or absence of the LXR agonist T0901317 in the hepatic cells and HBx-transgenic mice. Furthermore, HBx also augments the ability to recruit ASC2 (activating signal co-integrator 2), a transcriptional co-activator that controls liver lipid metabolic pathways, to the LXRE with LXRα. These studies place LXRα in a key position within the HBx-induced lipogenic pathways, and suggest a molecular mechanism through which HBV infection can stimulate the SREBP1-mediated control of hepatic lipid accumulation.
- activating signal co-integrator 2 (ASC2)
- fatty acid synthase (FAS)
- hepatic steatosis
- hepatitis B virus (HBV)
- sterol-regulatory-element-binding protein (SREBP)
The human liver infected with HBV (hepatitis B virus) and HCV (hepatitis C virus) can develop chronic hepatitis, cirrhosis and, in certain instances, HCC (hepatocellular carcinoma) [1–3]. In addition, CHB (chronic viral hepatitis B) and CHC (chronic viral hepatitis C) are both associated frequently with hepatic steatosis; the frequency of steatosis in CHB ranges from 27 to 51%, whereas in CHC, it is between 31 and 72% [4–8]. Among the four proteins that originate from the HBV genome, including polymerase, surface, core and HBx (HBV protein X), the 154-amino-acid HBx is a multifunctional regulator which modulates a variety of host processes via interaction with the virus and host factors . It has been reported previously that the expression of HBx induces the expression of lipid-synthesis-related genes in transgenic mice [10,11]. These reports indicate that HBx performs a crucial function in the development of a variety of liver failure types, resulting from the derangement of hepatic metabolism. However, the precise functions of HBV proteins in the accumulation of fatty acid remain to be elucidated, primarily because of a lack of systems sufficient for the investigation of HBV pathogenesis.
The LXRs (liver X receptors), LXRα (NR1H3) and LXRβ (NR1H2), are members of the nuclear hormone receptor superfamily of transcription factors. LXRα is abundantly expressed within the liver and is also detected in adipose tissue, intestines, kidneys and macrophages, whereas the expression of LXRβ is detectable in the majority of tissues [12,13]. LXRs have been defined as sterol sensors, as they can be activated by cholesterol-derived oxysterols, including 24(S),25-epoxycholesterol, 24(S)-hydroxycholesterol and (22R)-hydroxycholesterol . Several other synthetic LXR agonists, including the synthetic T0901317 (TO1317), have also been previously described . In the liver, LXR is involved in the transcriptional control of cholesterol 7-α-hydroxylase 1 (CYP7A1), encoding a critical enzyme in the conversion of cholesterol into bile acids [14,16]. LXR has also been demonstrated to control genes that encode proteins associated with de novo lipogenesis, including SREBP1c (sterol-regulatory-element-binding protein 1c), a transcription factor which regulates the expression of a variety of lipogenic genes, including genes that encode ACC1 (acetyl-CoA carboxylase 1) and FAS (fatty acid synthase) [15–19]. Furthermore, LXRs have been shown to directly influence the transcription of genes that encode FAS , CETP (cholesterol ester transfer protein) , LPL (lipoprotein lipase)  and SCD1 (stearoyl-CoA desaturase 1) [23,24].
Recent studies using a cDNA microarray have suggested that SREBP1 was induced in patients with chronic hepatitis B , and we determined that cellular lipid accumulation by HBx is modulated via the up-regulation of SREBP1 and PPARγ (peroxisome-proliferator-activated receptor γ) with the other lipogenic-associated proteins, FAS, ACC1 and SCD1. However, the manner in which HBx activates SREBP1 expression remains to be clearly demonstrated. It has been determined that de novo lipogenesis induced by SREBP1 is reminiscent of that induced by the activation of LXRs with an agonist.
In the present study, we explored the effects of HBx on LXRα-mediated SREBP1 induction using a combination of molecular, cellular and animal models. This mechanism for the regulation of SREBP1 and FAS expression by LXRs bears significant implications for the development of LXR agonists as modulators of HBx-induced lipid accumulation. Furthermore, the present study was designed to determine a possible role of ASC2 (activating signal co-integrator 2), a co-activator that controls liver lipid metabolism, in the transcriptional activation of LXRα and its agonists. To achieve these goals, we demonstrated that the expression of lipogenesis-associated genes was enhanced by HBx and LXR agonist in cultured liver cells. This may clarify the molecular mechanisms by which HBV infection induces SREBP1 and FAS expression.
MATERIALS AND METHODS
Plasmids, reagents and antibodies
pCMX/hLXRα and LXRE (LXR-response element)–tk (thymidine kinase)–Luc (luciferase) were kindly donated by Dr David J. Mangelsdorf (Department of Pharmacology, University of Texas Southwestern Medical Centre, Dallas, TX, U.S.A.) . pGL4.10/hSREBP1c-Luc  and pGL2B/rFAS-Luc (−1594/+65)  were generously donated by Dr A. Bennett (School of Biomedical Sciences, Medical School, Queen's Medical Centre, Nottingham, U.K.) and Dr Timothy F. Osborne (Department of Molecular Biology and Biochemistry, University of California Irvine, Irvine, CA, U.S.A.) respectively. HBV 1.2-mer wild-type and HBx-null mutant vectors were kindly provided by Dr W.S. Ryu (Yonsei University, Seoul, Republic of Korea) . pcDNA3/HA/hLXRα, pM/hLXRα (Gal4/hLXRα) or pVP16/hLXRα full-length and deletion constructs were constructed via the insertion of PCR fragments of ORFs (open reading frames) into EcoRV/XhoI- and BamHI/HindIII-digested HA (haemagglutinin) epitope-tagged pcDNA3 vector (Invitrogen) (pcDNA/HA), pM (Gal4) and pVP16 vectors (Clontech). pcDNA3/GST/HBx, pM/HBx (Gal4/HBx) and pVP16/HBx were generated using the restriction enzymes EcoRI/XhoI or EcoRI/XbaI. pcDNA3/RFP or pcDNA3/GFP were prepared via the insertion of the PCR ORF fragment into KpnI/EcoRI-digested pcDNA3 from pDsRed-Expression C1 (Clontech) and pEGFP-C1 vector. pcDNA3/RFP/hLXRα or pcDNA3/GFP/HBx were subcloned via the insertion of PCR fragments into EcoRI/XhoI-digested RFP (red fluorescent protein) or GFP (green fluorescent protein) epitope-tagged pcDNA3 vector. pSG5/HBx, pcDNA3/HA/HBx, pcDNA3/ASC2, pcDNA3/ASC2 (DN2) and pCMX/Gal4N/ASC2 were described previously [11,30].
The LXR agonist, T0901317, was acquired from Cayman. The transfection reagents PolyFect, SuperFect and JetPEI were purchased from Qiagen and Polyplus Transfection. All other reagents were purchased from Sigma. Antibodies against SREBP1 (SC-13551), GST (glutathione transferase) (SC-138) or p300 (sc-584) were purchased from Santa Cruz Biotechnology and antibodies against β-actin (A2066), FAS (610962), LXRα (ab3585), HBx (MAB8419), HA (1 867 423) and ASC2 (BL1874) were obtained from Sigma, BD Biosciences, Abcam, Chemicon, Roche and Bethyl Laboratories respectively.
Chang liver (A.T.C.C. CCL 13), HepG2, Huh7 and Hep3B cells were obtained from the American Type Culture Collection (A.T.C.C.), Manassas, VA, U.S.A. Chang liver, HepG2, Huh7 and Hep3B cells were maintained in DMEM (Dulbecco's modified Eagle's medium)/10% (v/v) FBS (fetal bovine serum) (Gibco BRL) at 37 °C in a humid atmosphere of 5% CO2.
Transient transfection, luciferase assay and stable transfection
Transient transfections of Chang liver, HepG2, Huh7 and HepG3B cells were conducted using PolyFect, SuperFect or JetPEI reagent in 24-well culture plates with the indicated reporter plasmids and co-transfected with mammalian expression vectors. The total quantities of expression vectors were maintained at constant levels via the addition of empty vectors. The cells were lysed in cell culture lysis buffer (Promega), and relative luciferase activities were determined using luciferin (BD Biosciences). Stable HepG2 transfectant cells with HA and HA–HBx were described previously  and were maintained in DMEM/10% (v/v) FBS containing 200 μg/ml G418.
The wild-type or HBx homozygous transgenic mice (11 weeks old, approx. 20 g each) were kindly provided by Dr D.Y. Yu (Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea) . These mice received daily intraperitoneal injections of either vehicle (control) or 50 mg/kg T0901317 for 5 days. T0901317 was dissolved in DMSO and diluted (5:1) with PBS before injection. The mice were killed, and the liver tissues were isolated. The principles of laboratory animal care were followed and the study was approved by the local ethics committee.
RNA isolation, RT (reverse transcription)–PCR and quantitative real-time PCR
Total RNA from Chang liver cells was prepared using TRIzol® (Invitrogen) in accordance with the manufacturer's recommendations. The cDNA was synthesized from 3 μg of total RNA with MMLV (Moloney murine leukaemia virus) reverse transcriptase (Promega) using a random hexamer at 37 °C for 1 h. A 1/25th aliquot of the cDNA was subjected to PCR amplification using gene-specific primers (Table 1). Real-time PCR was performed with an SYBR Green I LightCycler-based real-time PCR assay (Roche Applied Science). The reaction mixtures were prepared using LightCycler Fast Start DNA master mixture for SYBR Green I, 0.5 μM of each primer and 4 mM MgCl2. All PCR conditions and primers were optimized to produce a single product of the correct base pair size.
RNA interference and transfection
For the siRNA (small interfering RNA)-mediated down-regulation of LXRα, LXRα-specific siRNA (siLXRα) and negative-control siRNA were purchased from Bioneer. Chang liver cells were transfected with either siLXRα or with a negative-control siRNA sequence using HiPerFect transfection reagent (Qiagen).
In vivo interaction assays
Chang liver cells grown in DMEM supplemented with 10% (v/v) FBS were plated 1 day before transfection. In brief, 3 μg of each plasmid DNA was transfected into the Chang liver cells using PolyFect. At 48 h after transfection, the cells were solubilized with 300 μl of RIPA buffer (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P40 and 0.25% sodium deoxycholate) containing protease inhibitor cocktail (Roche), 1 mM sodium fluoride, 1 mM sodium orthovanadate and 1 mM PMSF. The cleared lysates were then mixed with 40 μl of glutathione–Sepharose beads (GE Healthcare) and rotated overnight at 4 °C. The bound proteins were eluted in 15 mM GSH, separated via SDS/PAGE (12% gels), and then transferred on to PVDF membranes (Millipore). The membranes were probed with anti-HA antibody (Roche) and developed via ECL® (enhanced chemiluminescence) (GE Healthcare), in accordance with the manufacturer's instructions.
ChIP (chromatin immunoprecipitation) assay
ChIP assays were conducted as described by the manufacturer (Upstate Biotechnology) with some modifications. Cross-linking between transcription factors and chromatin was achieved via the addition of formaldehyde (1% final concentration) for 10 min at 37 °C, and then halted via the addition of 125 mM glycine for 5 min at room temperature (25 °C). Chromatin solutions were sonicated and incubated with anti-HBx (Chemicon), anti-p300 or anti-ASC2 antibody, or with control IgG, then rotated overnight at 4 °C. The immune complexes were collected with Protein A– or G–Sepharose slurry (Invitrogen) and salmon sperm DNA for 4 h with rotational washing and then incubated overnight at 65 °C for reverse cross-linking. Chromatin DNA was purified and subjected to PCR analysis. In order to amplify the SREBP1c promoter regions containing LXRE, the following primer set was utilized: 5′-CTCAGGGTGCCAGCGAACCAG-3′ (forward) and 5′-GGGTTACTAGCGGACGTCCGCC-3′ (reverse). After amplification, the PCR products were resolved on a 1.5% agarose gel and visualized via ethidium bromide staining.
Fluorescence microscopy in living cells
Fluorescence microscopy was conducted on Chang liver cells transfected with pcDNA3/RFP/hLXRα and pcDNA3/GFP/HBx constructs. Following transfection, the cells were incubated for 48 h. Before imaging, the cells were counterstained with Hoechst dye for 10 min at 37 °C to stain the nuclei, and were visualized with a Zeiss Axiovert 200M fluorescence microscope.
For immunoprecipitation, the cells were plated and permitted to adhere overnight. The cells were lysed via the addition of RIPA buffer and were incubated for 10 min on ice, then scraped into microcentrifuge tubes. After 15 min of high-speed centrifugation, an aliquot of the lysates was removed for Western blotting, and the remainder was immunoprecipitated overnight with 1.5 μg of anti-HA antibody (Roche) and 40 μl of Protein G–Sepharose (50% suspension). The lysates and immunoprecipitates were then separated via SDS/PAGE (12% gels) and transferred on to PVDF membranes for blotting. The proteins were detected using horseradish-peroxidase-conjugated secondary antibodies and visualized via ECL®.
Oil Red O staining
Oil Red O staining was conducted in accordance with the procedure described previously , with minor modifications. In brief, cells were washed twice in PBS and fixed for 1 h with 10% (w/v) formaldehyde in PBS. After two washes in 60% isopropyl alcohol, the cells were stained overnight in freshly diluted Oil Red O solution (6 vol. of Oil Red O stock solution to 4 vol. of water; Oil Red O stock solution is 0.5% Oil Red O in isopropyl alcohol). The stain was then removed, and the cells were washed twice in water. The number of Oil Red O-positive cells was counted under a bright-field microscope in ten replicates of ten fields and averaged. Absorbance of eluted Oil Red O by adding 100% propan-2-ol at 500 nm was then measured in a spectrophotometer.
Statistical analyses were conducted via unpaired or paired Student's t tests as appropriate. All data are expressed as means±S.D. P<0.05 was considered to be significant.
HBx induces the transcriptional activity of LXRα in hepatic cells
Recently, we determined that HBx overexpression induces lipid accumulation in Chang liver, HepG2 cells and HBx-transgenic mice by inducing the expression and transcriptional activity of SREBP1 . In addition, the LXR agonist treatment induces the expression of genes associated with fatty acid biosynthesis and augments hepatic triacylglycerol levels in animal models . In order to determine whether HBx affects the transcriptional activity of LXRα, we co-transfected a reporter plasmid harbouring multimerized LXR-binding sites with the expression plasmid of LXRα and HBx . As shown in Figure 1(A), HBx profoundly augmented the activation of reporter gene expression by LXRα in the presence or absence of a synthetic LXR agonist T0901317 in a variety of hepatic cell lines. We also noted that HBx enhanced the transcriptional activity of LXRα in the presence of the endogenous LXR ligands, (22R)-hydroxycholesterol (Figure 1B) and 25-hydroxycholesterol (results not shown) in Chang liver cells [2,32]. In addition, we noted that HBx overexpression augmented the transcriptional activation of LXRα more significantly than was observed with other common co-activators, including CBP [CREB (cAMP-response-element-binding protein)-binding protein], p300 and SRC-1 (steroid receptor co-activator 1) (Figure 1C).
HBx may modulate LXRα activity directly, by binding to LXRα, or indirectly, via the generation of endogenous ligands or post-transcriptional modifications. In order to assess these possibilities, we tested the effects on the activity of a Gal4–hLXRα (human LXRα) fusion protein by HBx. As is shown in Figure 1(D), the activation of the Gal4–hLXRα chimaera was enhanced by HBx overexpression in a dose-dependent manner. Taken together, the results of these studies indicate that HBx activates LXRα via transcriptional mechanisms.
HBx promotes SREBP1 and FAS gene expression through LXRα activation
Furthermore, HBx was shown to augment the promoter activity of SREBP1c and FAS in the presence or absence of T0901317, as seen in our analysis of the luciferase activity compared with basal levels (Figure 2A). Also, it can be expected that the increased SREBP1 and FAS activity by HBx was accompanied by increases in the levels of hepatic mRNA. As shown in Figures 2(B) and 2(C), the mRNA induction in the RT–PCR (Figure 2B) and quantitative real-time PCR (Figure 2C) was confirmed for SREBP1 and FAS expression by HBx. In addition, T0901317 treatment resulted in further elevations of these genes, particularly in the presence of HBx.
In an effort to determine whether LXRs perform a function in SREBP1 and FAS gene expression by HBx, we attempted to knock down LXRα expression using siLXRα. Chang liver cells were co-transfected with HBx construct and/or siLXRα. As is shown in Figure 2(D), siLXRα-transfected cells did not affect an increase in SREBP1 and FAS promoter activity, even in the presence of HBx. We confirmed the siRNA-mediated knockdown of LXRα expression using Western blotting with the antibody specific for LXRα (Figure 2D, lower panel). As shown in Figures 2(E) and 2(F), the induction of SREBP1 and FAS mRNA in the RT–PCR and quantitative real-time PCR was also not induced by HBx in the siLXRα-transfected cells. These results indicate clearly that LXRα is a crucial transcription factor that mediates the HBx-induced expression of the SREBP1 and FAS genes.
HBx enhances the expression of SREBP1 and FAS protein induced by the LXR agonist
In an effort to determine whether HBx augments the levels of SREBP1 and FAS protein in hepatic cells, HepG2 cells stably expressing HBx were prepared via transfection with the expression plasmid of HA-tagged HBx, and the cell extract was subjected to Western blot assay  using antibodies against SREBP1 and FAS. As shown in Figure 3(A), HBx expression augmented the induction of SREBP1 and FAS protein. In addition, T0901317 treatments increased the levels of these proteins, particularly in the presence of HBx. For the protein expression analysis on the tissue level, the wild-type and HBx-transgenic mice were treated for 5 days with daily single intraperitoneal doses of T0901317 (50 mg/kg) or DMSO (vehicle). In the liver of HBx-transgenic mice (Figure 3B), increases in the levels of SREBP1 and FAS proteins were elevated after T0901317 treatment. These results showed that lipogenic protein expression induced by HBx required the activation of LXR.
The HBV replicon increases the transactivation of LXRα in an HBx expression-dependent manner
We noted that the expression level of HBx induced by a strong promoter in the transfection system is guaranteed to be greater than the physiological level expressed during chronic HBV infection [29,33]. In order to address this, we assessed the effects of modest levels of HBx expressed from a HBV replicon, a greater-than-genome-length construct (1.2-mer), on the transactivation of LXRα. As shown in Figure 4(A), the transfection of the wild-type HBV 1.2-mer (WT) in HBV-negative liver cell lines, Chang liver cells and HBV-infected liver cell lines, Hep3B, resulted in a significant increase in hLXRα-dependent transcription, whereas the transfection of the HBx-null mutant (X−) of HBV 1.2-mer, which was constructed by inserting a stop codon after the first and second AUG of the HBx ORF by site-directed mutagenesis , did not increase its transactivation sufficiently compared with what was observed in the wild-type. Furthermore, the etopically expressed HBx could recover the transactivation of the HBx-null mutant (X−) of HBV 1.2-mer with LXRα in a dose-dependent fashion. The reporter assay results gained using the native promoter of SREBP1c and FAS supported further the notion that HBx was responsible for the activation of LXRα in hepatic cells, as is shown in Figure 4(B). On the basis of these data, we concluded that the HBx expressed from a replicating HBV genome confers on hepatocytes the ability to functionally activate LXRα upon the expression of the target gene.
LXRα interacts physically with HBx
We hypothesized that the physical interactions between LXRα and HBx might augment the enhancement of LXRα activity. In an effort to determine whether LXRα interacts with HBx on the cellular level, we employed several approaches. First, we exploited the mammalian two-hybrid system in which full-length LXRα and HBx were fused to Gal4 or VP16 (virus protein 16) respectively. As the consequence of a direct interaction between LXRα and HBx, the increased transactivity of a reconstituted Gal4–VP16 protein was monitored by the luciferase reporter plasmid under the command of a multimerized Gal4-response element. In particular, it has been demonstrated previously that the interaction between LXRα and HBx was increased in the presence of T0901317 (Figure 5A). We then assessed the co-immunoprecipitation of HBx and LXRα. Transiently expressed HBx associated with LXRα via anti-HA antibody immunoprecipitation, followed by Western blotting with antibody against HBx (Figure 5B).
In addition, we evaluated the physical association of LXRα and HBx using an in vivo GST pull-down assay. The Chang liver cells were transfected with constructs that encode the GST-fused HBx and HA-tagged hLXRα. The results of in vivo GST pull-down indicated that LXRα binds only to GST–HBx, and not to the GST protein (Figure 5C). In order to determine whether HBx forms a transcriptional protein complex with LXRα on the LXREs within the promoter region of the endogenous LXR target gene, SREBP1c, we conducted ChIP assays. As is shown in Figure 5(D), HBx was detected in a transcriptional complex to the LXRE of the SREBP1c promoter. No PCR products were detected when control IgG was utilized in immunoprecipitation. The p300 co-activator was employed as the positive control . Finally, in order to confirm these findings further, we next assessed the cellular localization of LXRα and HBx via fluorescence microscopy in living cells. The RFP- and GFP-fusion proteins of LXRα and HBx were expressed in Chang liver cells, demonstrating that LXRα and HBx were co-localized in the nucleus, as shown in Figure 5(E). This result was also verified in the presence of the LXR agonist T0901317 (results not shown).
Collectively, these findings indicate that HBx was presented with LXRα on the LXRE, including another co-activator, such as p300, to the SREBP1c promoter, resulting in the transactivation of SREBP1c transcription.
Mapping of the LXRα interaction domain with HBx
In order to map the LXRα domains that mediate the interaction between LXRα and HBx, we constructed encoding truncated forms of LXRα  fused by the Gal4, VP16 or HA proteins (Figure 6A). A series of LXRα deletions fused to the VP16 activation domain were co-transfected into Chang liver cells with full-length HBx fused to the Gal4 DBD (DNA-binding domain). The co-expression of VP16–hLXRα (full) and VP16–hLXRα (D3) with Gal4–HBx augmented Gal4–tk–Luc-dependent transactivation, whereas other VP16–hLXRα deletion mutants did not (Figure 6B). The results expressing Gal4–hLXRα (full) and Gal4–hLXRα (D3) showed similar effects as those observed using VP16–hLXRα constructs (results not shown).
In an effort to confirm further the identification of the LXRα-interaction domain with HBx, we conducted an in vivo GST pull-down assay exploiting truncated forms of HA-tagged LXRα proteins. Chang liver cells were transfected with the construct that encodes the HA-fused LXRα full or deletion mutants with GST-fused HBx. The results indicated that HBx interacted with the full-length and D3 mutants harbouring the LBD (ligand-binding domain), but not with the D1 and D2 deletion mutants that harboured amino acids 1–161, which are known to accommodate the AF1 (activation function domain 1) and DBDs (Figure 6C).
ASC2 potentiates LXRα-mediated transactivation with HBx
Previous results have shown that LXRs bind to co-repressors and repress transcription in cases in which LXR is not occupied by an agonist , whereas LXRs bind to co-activators to augment gene activation in the presence of an agonist . This prompted us to attempt to determine whether the interaction between the LBD of LXRα and HBx induces the intracellular levels of secreted co-repressors and recruited co-activators within the LXRα target gene promoter. The transient transfection of co-repressors including HDAC1 (histone deacetylase 1), NCoR (nuclear receptor co-repressor), SHP (short heterodimer partner) and SMRT (silencing mediator for retinoic acid receptor and thyroid-hormone receptor) down-regulated the transactivation of the SREBP1c promoter; however, the overexpression of HBx induced a slight recovery of the effects of co-repressors in the absence of an agonist (results not shown). Furthermore, we assumed that HBx could enhance the recruitment of co-activators which perform a function in the regulation of lipid accumulation. We noted that co-activators, including CBP, p300, SRC-1, PGC (PPARγ coactivator)-1α, PGC-1β and ASC2, up-regulated the transcriptional activity of LXRα-mediated SREBP1c in the presence or absence of HBx. Among many co-activators, we focused on the effects of ASC2 on HBx-mediated LXRα activation. ASC2 has been identified as a physiologically important transcriptional co-activator of LXRs in vivo with regard to cholesterol and lipogenic metabolism in the liver [36,37]. We also reported previously that HBx potentiates the co-activator function of ASC2 via direct molecular association . Initially, the co-transfection of intact ASC2 with LXRα and HBx further stimulated transcription from the LXRE reporter construct (Figure 7A) and the native promoters of SREBP1c and FAS (Figure 7B). By way of contrast, as shown in Figure 7(C), the overexpression of the truncated ASC2 construct (DN2: amino acids 1429–1511) that encodes the LXRα-interacting region with ASC2  diminished HBx-induced transactivation by activated LXRα.
We observed similar results using the mammalian two-hybrid assay. The interaction between Gal4-fused ASC2 and VP16-fused LXRα was enhanced by HBx (Figure 7D). The reverse attempt using Gal4–hLXRα and VP16–ASC2 also showed effects similar to those observed in previous studies (results not shown). Finally, we confirmed the recruitment of ASC2 on the SREBP1c promoter via ChIP assays. In the HBx-transfected cells, the binding of ASC2 to the SREBP1c promoter was increased in the absence or presence of T0901317 as compared with the mock-transfected cells (Figure 7E). These results show that HBx performs an important function in the formation of an active transcriptional complex of LXRα and ASC2 co-operatively for significant lipogenic gene expression in the occurrence of HBV infection.
HBx regulates the gene expression of lipogenesis transcription factors and enzymes with LXRα activation
We postulated that the association of LXRα, HBx and co-activators including ASC2 and p300 may augment the expression of lipogenic enzymes and the associated transcription factors. In order to assess this, we evaluated the expression of key lipogenic genes in response to HBx transfection with the LXR agonist T0901317. Using RT–PCR (Figure 8A) and quantitative real-time PCR (Figure 8B) analysis, we determined that the mRNA levels of lipogenic enzymes, including ACC1 and SCD1, were enhanced after treatment with T0901317 and were increased significantly in the presence of HBx. Also, the results of previous studies showed that HBx elevates the gene expression and transcriptional activity of PPARγ, regulating lipid synthesis, transport and storage [38,39]. On the basis of this finding, it was shown that PPARγ expression was increased by T0901317 treatment and HBx. Furthermore, the expression of the CD36 gene, which performs a critical function in fatty acid uptake and is a novel target gene of PPARγ , was increased (Figures 8A and 8B). Conversely, no significant differences were observed in the expression of the lipid β-oxidation regulatory genes, PGC1α and ACO (aconitase) (Figure 8A) [41,42].
We also assessed HBx-stimulated lipogenesis in the presence of T0901317, as determined by an increase in Oil Red O staining in HepG2 cells (Figure 8C) and Chang liver cells (results not shown). We determined that T0901317-activated LXRs promoted lipogenesis to a slight degree in the presence of HBx. Oil Red O-positive cells were counted in each cell, and triacylglycerol accumulation was also measured by spectrophotometric determination (Figure 8C). Collectively, these results indicated that HBx stimulated LXRα-induced lipogenesis via the augmentation of the accumulation of cytosolic lipids by lipogenic gene expression.
HBV infects >350 million individuals worldwide, and then induces liver diseases, including hepatic inflammation, cirrhosis, and ultimately HCC . Among the four proteins that originate from the HBV genome (polymerase, surface, core and HBx), HBx is associated with HBV-related pathogenesis in the liver . We reported previously that HBx induces hepatic steatosis via the transcriptional activation of SREBP1 and PPARγ using human cell lines and HBx-transgenic mice . Moreover, it was shown that HBx induces lipid accumulation via an induced increase in the expression of hepatic adipogenic and lipogenic genes. However, the specific functions of HBx, which include potential mechanisms of the transcriptional regulation of SREBP1, remain to be clearly delineated.
In the present study, we have demonstrated that HBx increases the expression of the SREBP1c and FAS genes through a distinct transcriptional regulation occurring via an LXRE in the SREBP1c and FAS promoter, thereby suggesting that the HBx-mediated regulation of lipogenesis in hepatocytes involves direct targeting to LXRα. In addition to their ability to modulate cholesterol metabolism , LXRs are also crucial regulators of hepatic lipogenesis. The treatment of mice with synthetic LXR agonists increases triacylglycerol levels in the liver, and effects that pose a significant obstacle to the development of these compounds as human therapeutics [15,20]. The lipogenic activity of LXRs results in the up-regulation of hepatic lipogenic regulators, including SREBP1c [18,45], FAS , ACC1 and SCD1 [13,24,46]. Although HBx did not augment the expression of the LXRα gene, we present evidence that places HBx within the LXRα regulatory pathway and HBV-induced hepatic steatosis. Our results also show that HBx is capable of interacting with LXRα and the region of LXRα mediating the interaction between LXRα and HBx harbours the previously described LBD.
We also sought to determine whether the interaction of LXRα LBD with HBx retains the ability to release co-repressors or to recruit co-activators. It was demonstrated that HBx also mediates the interaction between LXRα and ASC2. In the present study, we focused on ASC2 as a bona fide co-activator of LXRs in the presence of HBx; however, we also assessed the activities of other transcriptional co-activators, including CBP, p300, SRC-1, PGC-1α and PGC-1β. These co-activators also augmented the transcriptional activity of LXRα with HBx on the SREBP1c promoter, and, unexpectedly, PGC-1α significantly amplified the LXRα-mediated transcriptional activation of the SREBP1c promoter in the presence of HBx. In the present study, it was proposed that PGC-1α functions as a potent co-activator of LXRs . PGC-1α functions as a transcriptional co-activator for multiple metabolic pathways in the liver, including fatty acid oxidation, gluconeogenesis and mitochondrial respiration [48,49]. Although the physiological relevance of LXR co-activation by PGC-1α remains to be elucidated, our findings may suggest the potential role of PGC-1α in lipid metabolism, which is associated with HBV infection. For instance, previous studies have demonstrated the association of the PGC1α gene locus with obesity and Type 2 diabetes [47,50,51]. However, considering our observations that PGC-1α activates the LXRα regulatory pathway via multiple mechanisms, including co-operation with HBx as well as the transcriptional activation of the SREBP1c gene, the mechanisms will hopefully be unveiled in additional studies.
The initial stages of NASH (non-alcoholic steatohepatitis) are characterized by steatosis or fatty liver with no detectable inflammation. Furthermore, in advanced stages of NASH, a growing body of evidence suggests that increased liver triacylglycerols exert increased oxidative stress in animals and humans [52–56]. We also noted that hepatic inflammation has been observed in 11-week-old HBx-transgenic mice . The activation of inflammatory signalling pathways and releases of inflammatory mediators are fundamental to the diverse immune functions of macrophages. By way of contrast with the role of HBx in the mediation of the inflammatory pathways, LXRs reciprocally repress a set of inflammatory genes, including iNOS (inducible nitric oxide synthase), COX (cyclo-oxygenase)-2, IL (interleukin)-6, IL-1β and MMP-9 (matrix metalloproteinase 9) in macrophages [57,58]. Participation in both metabolic and inflammatory control is a common feature of the LXR signalling pathways. However, it remains difficult at this point to predict the detrimental effects of the activation of inflammatory pathways by chronic HBV infection in liver, compared with the relative benefits of these pathways by LXRα activation in the macrophages.
In conclusion, we have determined that HBx augments the hepatic LXRα transcriptional activity that is robustly induced in response to conditions that dictate increased SREBP1 and FAS expression. HBx performs a crucial function as an LXRα-interacting protein which co-activates selective targets of the LXRα co-activation circuitry in cases of HBV infection. Collectively, these findings also demonstrate a specific mechanism by which LXRα transcription is activated in response to agonist stimuli. Notably, we found that HBx functions as the link between LXRα and a subset of its target transcription co-activators to ameliorate the hepatic lipogenic pathway ability facilitated by HBV infection.
This work was supported by a grant of the Korea Healthcare Technology, Ministry of Health, Welfare and Family Affairs, Republic of Korea [grant number A080907].
We thank Dr D. J. Mangelsdorf, Dr A. Bennett, Dr T. F. Osborne, Dr W. S. Ryu and Dr D. Y. Yu for the constructs and mice, as noted in the text.
Abbreviations: ACC1, acetyl-CoA carboxylase 1; ASC2, activating signal co-integrator 2; CBP, CREB (cAMP-response-element-binding protein)-binding protein; CHB, chronic viral hepatitis B; CHC, chronic viral hepatitis C; ChIP, chromatin immunoprecipitation; DBD, DNA-binding domain; DMEM, Dulbecco's modified Eagle's medium; FAS, fatty acid synthase; FBS, fetal bovine serum; GFP, green fluorescent protein; GST, glutathione transferase; HA, haemagglutinin; HBV, hepatitis B virus; HBx, HBV protein X; HCC, hepatocellular carcinoma; IL, interleukin; LBD, ligand-binding domain; Luc, luciferase; LXR, liver X receptor; hLXRα, human LXRα; LXRE, LXR-response element; NASH, non-alcoholic steatohepatitis; ORF, open reading frame; PPARγ, peroxisome-proliferator-activated receptor γ; PGC, PPARγ co-activator; RFP, red fluorescent protein; RT, reverse transcription; SCD1, stearoyl-CoA desaturase 1; siRNA, small interfering RNA; siLXRα, LXRα-specific siRNA; SRC-1, steroid receptor co-activator 1; SREBP1, sterol-regulatory-element-binding protein 1; tk, thymidine kinase; VP16, virus protein 16
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