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Originally published In Press as doi:10.1074/jbc.M403176200 on April 29, 2004

J. Biol. Chem., Vol. 279, Issue 27, 28106-28112, July 2, 2004
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The Hepatitis B Virus X Protein Inhibits Secretion of Apolipoprotein B by Enhancing the Expression of N-Acetylglucosaminyltransferase III*

Sung-Koo Kang{ddagger}, Tae-Wook Chung{ddagger}, Ji-Young Lee{ddagger}, Young-Choon Lee§, Richard E. Morton¶, and Cheorl-Ho Kim{ddagger}||

From the {ddagger}National Research Laboratory for Glycobiology, and Department of Biochemistry and Molecular Biology, College of Oriental Medicine, Dongguk University, Kyungju, Kyungbuk 780-714, Korea, §Faculty of Biotechnology, Dong-A University, Saha-gu, Busan 604-714, Korea, and Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195

Received for publication, March 22, 2004 , and in revised form, April 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The X protein of hepatitis B virus (HBx) plays a major role on hepatocellular carcinoma (HCC). Apolipoprotein B (apoB) in the liver is an important glycoprotein for transportation of very low density lipoproteins and low density lipoproteins. Although lipid accumulation in the liver is known as one of the factors for the HCC, the relationship between HBx and apoB during the HCC development is poorly understood. To better understand the biological significance of HBx in HCC, liver Chang cells that specifically express HBx were established and characterized. In this study we demonstrate that overexpression of HBx significantly up-regulates the expression of UDP-N-acetylglucosamine:{beta}-D-mannoside-1,4-N-acetylglucosaminyltransferase-III (GnT-III), an enzyme that functions as a bisecting-N-acetylglucosamine (GlcNAc) transferase in apoB, and increases GnT-III promoter activity in a chloramphenicol acetyltransferase assay. GnT-III expression levels of HBx-transfected cells appeared to be higher than that of hepatocarcinoma cells as well as GnT-III-transfected cells, indicating that HBx may has a strong GnT-III promotor-enhancing activity. Intracellular levels of apoBs, which contained the increased bisecting GlcNAc, were accumulated in HBx-transfected liver cells. These cells as well as GnT-III-transfected liver cells revealed the inhibition of apoB secretion and the increased accumulation of intracellular triglyceride and cholesterol compared with vector-transfected cells. Moreover, overexpression of GnT-III and HBx in liver cells was shown to down-regulate the transcriptional level of microsomal triglyceride transfer protein, which regulates the assembly and secretion of apoB. Therefore, our study strongly suggested that the HBx increase in intracellular accumulation of aberrantly glycosylated apoB resulted in inhibition of secretion of apoB as well as intracellular lipid accumulation by elevating the expression of GnT-III.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In Western countries 75–90% of hepatocellular carcinomas (HCCs)1 are associated with chronic liver diseases (1). Hepatitis B virus is a major causative agent of acute and chronic hepatitis in humans (2) and is closely associated with the incidence of human liver cancer. Among the four proteins that originate from the hepatitis B virus genome, HBx protein is a 17-kDa multifunctional regulatory protein and has been detected with high frequency in liver cells from patients with chronic hepatitis, cirrhosis, and liver cancer (3). In our previous study HBx has an inhibitory effect on the p53-mediated transcription of the 3'-inositol phosphatase and tensin homologue deleted on chromosome 10, which is associated with tumor suppression (4). Therefore, HBx is thought to be associated with the development of HCC. However, the precise function of HBx in the tumorigenic transformation of liver cells remains unclear.

The liver is the major organism for both the production of plasma lipoproteins and their uptake from plasma and catabolism (5). The production of apolipoprotein B (apoB, a 500-kDa protein)-containing lipoproteins by the liver is required for the assembly and secretion of very low density lipoproteins and low density lipoproteins (6–10). The assembly of apoB with lipid to form a secretion-competent particle is a complex process (11, 12). It is widely accepted that hepatic lipid availability is obligatory for apoB-containing lipoprotein assembly within the liver. This finding has been supported by studies demonstrating the necessity of triglyceride (12, 13) and phospholipid (14). The microsomal triglyceride transfer protein (MTP) also plays a key role in apoB secretion by catalyzing the transfer of lipids to the nascent apoB molecule as it is co-translationally translocated across the endoplasmic reticulum membrane (15, 16).

GnT-III catalyzes the attachment of a GlcNAc residue to mannose in the {beta}(1–4) configuration in the region of N-glycans and forms a bisecting GlcNAc (17), as shown in Scheme 1. Recent investigations revealed that the bisecting GlcNAc residue, a product of GnT-III activity, correlated with a number of biological events including the suppression of metastasis of mouse melanoma cells (18) and has been reported to be significantly elevated in the serum of human subjects with hepatomas, liver cirrhosis, as well as in HCC (19–25). Therefore, GnT-III also could be a factor for the development of HCC.



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  		SCHEME 1.
A bisecting GlcNAc chain in N-glycans in biosynthesized by GnT-III. GnT-III catalyzes the attachment of {beta}-1,4-GlcNAc to the {beta}-D-mannoside of the tri-mannose core structure of an N-glycan.

 
ApoB is a glycoprotein and contains high mannose N-glycans or biantennary-type oligosaccharides in the case of human low density lipoproteins (26, 27). Some investigators show that the glycosylation of proteins and lipids are associated with development, differentiation, and carcinogenesis (28–30). Recently, aberrant glycosylation as the direct result of the formation of bisecting-GlcNAc by GnT-III has been shown to disrupt the function of apoB, leading to the generation of fatty liver (31, 32). On the basis of the findings reported herein, we provide evidence of the molecular mechanism underlying the inhibition of apoB secretion and intracellular accumulation of triglyceride and cholesterol in vitro model by HBx/GnT-III expression.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Transfection—Chang cells (ATCC number CCL-13), a human liver cell line, were maintained using Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C in a humidified 5% CO2 incubator. The cells were used for stable transfection with HBx and GnT-III cDNA using LipofectAMINE reagent following the manufacturer's instructions. The 465-bp and 1.6-kb cDNAs encoding the open reading frames for HBx and GnT-III were inserted into pcDNA3 expression vector at the HindIII/KpnI and BamHI/EcoRI sites, respectively. Transfected cells were then selected by cell culture medium containing 600 µg/ml G418 sulfate.

Reverse Transcriptase (RT)-PCR—Total RNA from parental Chang cells and various transfected cells was prepared using TRIZOL (Invitrogen), and the cDNAs were synthesized by reverse transcriptase with an oligo(dT) adaptor primer from the RNA LA PCR kit (Takara, Japan). To specifically detect the expression of endogenous human GnT-III and MTP, PCR was performed with the selective primers for human GnT-III and MTP in a PCR cycler. The primers used in this study were designed to detect mRNA for: human GnT-III, 5'-ACTTCTTCAAGACCCTGTCCTATGT-3' (sense) and 5'-GAGCCGTTGGCCCC CTCAGGCTTCT-3' (antisense); MTP, 5'-TGCTGTCAGCATCTGG CGACCCT-3' (sense) and 5'-TCAAAACCATCCGCTGGAAGTACTAT-3' (antisense); {beta}-actin. 5'-CAAGAGATGGCCACGGCTGCT-3' (sense) and 5'-TCCTTCTGCATCCTGTCGGCA-3' (antisense). The sizes of products that were yielded by the PCR using these primers were expected to be 345, 500, and 247 bp for GnT-III, MTP, and {beta}-actin, respectively.

Northern Blot Analysis—To detect mRNA levels of HBx, GnT-III, and MTP, total RNA was prepared using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. For Northern blot analysis, 10 µg of total RNA was separated by electrophoresis in a 1% formaldehyde agarose gel. After electrophoresis the gel was blotted on Hybond C membrane (Amersham Biosciences). RNA was fixed to the membrane by cross-linking for 3 min using ultraviolet (UV). Hybridization was performed in Expresshyb (Clontech) to a random prime-labeled probe (Clontech) that encompassed the partial HBx, GnT-III, and MTP genes.

Production GnT-III Antibody—Aglycosyl recombinant N-acetylglucosaminyltransferase-III protein deficient in the first 23 amino acids was expressed in Escherichia coli and purified by DEAE-Sephacel chromatography (Amersham Biosciences) followed by Sephacryl S-200 gel (Amersham Biosciences) filtration, and finally, preparative gel electrophoresis (33). The procedure employed for the production of monoclonal antibodies was based on the protocol described by Harlow and Lane (34). Balb/c mice were immunized by an intraperitoneal injection of aglycosyl recombinant N-acetylglucosaminyltransferase-III and mixed with Freund's complete adjuvant. Spleen cells from the mice were fused with the murine myeloma cell line SP2/0-Ag14. The monoclonal antibody-producing hybridomas were cloned by the limiting dilution technique and propagated by injecting them into mice. Subsequently, ascitic fluids were harvested and processed by protein G-Sepharose 4B chromatography to obtain purified monoclonal antibody as described previously (35).

Western Blot and Lectin Blot Analysis—Liver Chang cells were lysed in radioimmune precipitation assay buffer containing 150 mM NaCl, 20 mM Tris (pH 7.5), 1% Triton X-100, 2 mM EDTA, 10% (v/v) glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mM Na3VO4, 20 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 100 kallikrein inhibitor units of aprotinin per ml. Cell lysates were clarified by centrifugation at 14,000 x g for 10 min at 4 °C. Protein (25 µg) was separated on SDS-PAGE gels and then transferred to nitrocellulose membrane. After blocking nonspecific binding sites, the membranes were incubated with specific antibodies, anti-GnT-III, anti-glyceraldehyde-3-phosphate dehydrogenase (Chemicon), anti-HBx (Koma Biotechnology, Korea), and anti-apoB (Calbiochem). After washing the membranes with phosphate-buffered saline three times, they were further incubated with horseradish peroxidase-conjugated antibody. For detection of bisecting-GlcNAc residues, the membrane was incubated with 2 µg/ml biotinylated erythroagglutinating phytohemagglutinin (E-PHA), Roche Applied Science). Lectins were detected by using horseradish peroxidase-conjugated lectins (Seikagakukougyo, Kyoto, Japan). Immunoblots were revealed by autoradiography using the enhanced chemiluminescence detection kit (Amersham Biosciences).

Immunoprecipitation—The cell lysate (0.5 mg/ml) was precleared with 50 µl of protein A-Sepharose beads at 4 °C for 1 h and clarified by centrifugation at 14,000 rpm for 10 min. The precleared lysate was incubated with an anti-apoB antibody for 1 h, then 50 µl of protein A-Sepharose beads were added, and the mixture was incubated for 1 h. After extensive washing with radioimmune precipitation assay buffer, the immunoprecipitated apoB were eluted from beads with 50 µl of SDS sample buffer and subjected to 6% SDS-PAGE under reducing conditions. Western blot and lectin blot were performed with anti-apoB and E-PHA, respectively, as described above.

Construction of Plasmids and Transfections of Chang Cells with GnT-III Promoter-Chloramphenicol Acetyltransferase (CAT) Gene Fusion Vector—pSV0-CAT, which expresses chloramphenicol acetyltransferase (CAT), was from the laboratory of Molecular Glycobiology, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejon, Korea. pGNT-CAT plasmid was constructed by ligating DNA fragment ranging from –1 to –1058 bp of GnT-III upstream region of promoter in pSV0-CAT. Cells were co-transfected with 10 µg of pGNT-CAT plasmid and different concentration of pCDNA-HBx gene by a LipofectAMINE-based transfection method. Transfected cells were cultured at 37 °C in 3% CO2 for 24 h followed by 5% CO2 for 24 h and used for the CAT assay.

CAT Assay—The procedure previously developed by Bullock and Gorman (36) was employed. Transfected cells from 1 plate were harvested, resuspended in 100 µl of 0.25 M Tris-HCl (pH 7.8), and subjected to 3 cycles of freezing in a dry ice-ethanol bath and thawing in 37 °C incubator. The cell debris was removed by centrifugation, and the supernatant was saved in a clean tube. Total cell lysates were assayed for CAT activity were mixed with 4 µl of [14C]chloramphenicol (54 mCi/mmol), 70 µl of 1.0 M Tris-HCl (pH 7.8), 20 µl of 4 mM acetyl-CoA, and distilled water to a final volume of 150 µl. After incubation at 37 °C for 1 h, chloramphenicol was redissolved in 30 µl of ethyl acetate and applied to a TLC plate (Merck). The plate was incubated for 15 min in chloroform:methanol (95:5) and air-dried. After autoradiography, the radioactive spots on TLC plate were shown.

Measurement of Triglyceride and Cholesterol in Liver Cells—To determine triglyceride and cholesterol content in vector and GnT-III- and HBx-transfected cells, Chang cells were starved for 24 h and then incubated with Dulbecco's modified Eagle's medium supplemented with 1 µM concentrations of all-trans-retinoic acids (t-RAs) for 48 h. Triglyceride and cholesterol mass content from the cells was measured enzymatically (Sigma).

Statistics—Results are expressed as the mean ± S.D. and averages of three to five experiments. Means were compared by t tests to determine statistical significance. A p value of <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
HBx Enhances the Expression of GnT-III by the Promoter Activity of the GnT-III Gene—Chang cells were transfected with the HBx cDNA using pcDNA expression vector. The expression of both HBx mRNA and protein was verified before the investigation of HBx-induced effects. The expression of HBx mRNA was confirmed by Northern blot analysis, and the RNA controls of the corresponding blots are shown in Fig. 1A. Furthermore, the expression of HBx protein was confirmed by Western blot analysis using monoclonal anti-HBx antibodies (Fig. 1B).



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  		FIG. 1.
Expression level of HBx mRNA and proteins in HBx-transfected liver cells. A, twenty µg of total RNA extracted from Chang, Chang-pcDNA, and Chang-HBx-transfected cells were electrophoresed on 1% agarose gel containing 2.2 M formaldehyde and then analyzed by Northern blot hybridization using 32P-labeled HBx cDNA. B, proteins (25 µg) extracted from these cells were subjected to 15% SDS-polyacrylamide gel electrophoresis. Expression of HBx (17 kDa) was analyzed by Western blot using a specific antibody to HBx. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

 
Although we were searching for some factors that induced GnT-III, we found that HBx protein enhanced the mRNA and protein expression of GnT-III in the HBx-transfected cells, Chang-HBx. To elucidate whether the endogenous GnT-III gene is activated in the Chang-HBx, the GnT-III mRNA and protein levels were measured by Northern blot (Fig. 2A), RT-PCR (Fig. 2B), and Western blot analysis (Fig. 2C). As shown by these data, the expression of GnT-III gene and protein was significantly elevated in Chang-HBx cells compared with Chang and Chang-pcDAN3 cells.



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  		FIG. 2.
Detection of mRNA and protein for GnT-III in HBx-transfected liver cells. A, 20 µg of total RNAs extracted from HBx-transfected Chang cells were electrophoresed on a 1.0% agarose gel containing 2.2 M formaldehyde and then analyzed by Northern blot hybridization using 32P-labeled GnT-III cDNA (upper panel). {beta}-Actin indicates that equal amounts of RNAs were loaded in each lane (lower panel). B, one µg of total RNA from each cell was subjected to RT-PCR (upper panel). {beta}-Actin mRNA expression was also examined as a control (lower panel). C, 25 µg of proteins extracted from Chang cells, Chang-pcDNA3, and Chang-HBx cells were subjected to 10% SDS-polyacrylamide gel electrophoresis. Expression of GnT-III (68 kDa) was analyzed by Western blot using a specific antibody to GnT-III (upper panel). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) indicates that equal amounts of proteins were loaded in each lane (lower panel).

 
Here, we postulate whether HBx may enhance the promoter activity of the GnT-III gene in the endogenous system. In a previous study we isolated and characterized the 5'-flanking region of GnT-III gene from human placental genome library (37). A schematic diagram of the GnT-III promoter from –1058 to –1 is shown in Fig. 3A. Putative promoter elements are based on sequence comparison to known motifs: GRE, half-palindromic glucocorticoid-response element (TGTCCT), recognition sites for CREB (CGTGACGA), AP-2 (GGCCTGGGGA), and SP1 (GGGCGG). The EMBL data library accession number is L48489 [GenBank] . To examine the effect of HBx on the promoter activity of human GnT-III gene, the plasmid pGNT-CAT containing GnT-III promoter in front of the CAT reporter gene was co-transfected in human normal liver Chang cells with an increasing amount of HBx expression vector (pcDNA3-HBx) (see "Experimental Procedures"). Indeed, as shown in Fig. 3B, the CAT activity of the GnT-III promoter was gradually elevated up to 5-fold by increasing the HBx expression vector, although detailed characterization of the promoter activity is limited due to its comprehensive properties of the GnT-III promoter region (38, 39). Therefore, these results showed that the HBx protein transactivated GnT-III gene transcriptionally and translationally.



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  		FIG. 3.
HBx transactivates GnT-III promoter. A, schematic diagram of GnT-III promoter. The nucleotide sequence from –1058 to –1 is shown. Putative promoter elements are based on sequence comparison to known motifs: half-palindromic glucocorticoid-response element (GRE) (TGTCCT) and recognition sites for CREB (CGTGACGA), AP-2 (GGCCTGGGGA), and SP1 (GGGCGG). B, 10 µg of pGNT-CAT were cotransfected into Chang cells with different amounts of HBx expression vector (pcDNA3-HBx) as indicated. Transfected cells from one plate were harvested, resuspended in 100 µl of 0.25 M Tris-HCl (pH 7.8), and subjected to 3 cycles of freezing in a dry ice-ethanol bath and thawing in 37 °C incubator. The cell debris was removed by centrifugation, and the supernatant was saved in a clean tube. Portions of cell extracts assayed for CAT activity were mixed with 4 µl of [14C]chloramphenicol (54 mCi/mmol), 70 µl of 1 M Tris-HCl (pH 7.8), 20 µl of 4 mM acetyl-CoA, and distilled water to a final volume of 150 µl. After incubation at 37 °C for 1 h, chloramphenicol was redissolved in 30 µl of ethyl acetate and applied to a TLC plate. The plate was incubated for 15 min in chloroform:methanol (95:5) and was air-dried before autoradiography.

 
Comparison of the Levels of HBx and GnT-III mRNA and Protein in Normal Cells, Hepatocarcinoma Cells, and Our Transfected Cells—To compare the expression levels of HBx and GnT-III among the several cell lines, GnT-III cDNA was transfected into liver Chang cells, and RT-PCR and Western blot analysis were carried out using normal liver Chang cells, the hepatocarcinoma cell line (HepG2), and our transfected cells as described under "Experimental Procedures." As shown in Fig. 5, we could not detect the HBx level in HepG2 cells by RT-PCR (Fig. 4A) and Western blot (Fig. 4B) because HepG2 is well known as hepatocarcinoma cell line, which is not integrated with hepatitis B virus genome. The level of GnT-III expression in HepG2 cells was similar to that of GnT-III-transfected cells but appeared to be lower than that in HBx-transfected cells. Moreover, in densitometry analysis (Fig. 4C) based on protein levels, GnT-III levels in HBx-transfected cells was increased up to 2-fold over that in GnT-III-transfected cells.



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  		FIG. 5.
Characterization of immunoprecipitated apoB from HBx-transfected cells. Immunoprecipitation analysis in liver Chang cells was performed as described under "Experimental Procedures." Immunoprecipitated apoB from pcDNA- and HBx-transfected cells were subjected to 6% SDS-PAGE followed by Western blot (A) and lectin blot (B) with anti-apoB antibody and E-PHA, respectively. On the lectin blot the membrane was incubated with E-PHA in the presence or absence of 100 mM GalNAc. These results were reproducible in three independent experiments for each cell line.

 



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  		FIG. 4.
Comparison of the levels of HBx and GnT-III mRNA and protein in normal cells, hepatocarcinoma cells, and our transfected cells. A, 1 µg of total RNA from each cell was subjected to RT-PCR for HBx (upper panel) and GnT-III (middle panel). {beta}-Actin mRNA expression was also examined as a control (lower panel). B, proteins (25 µg) extracted from normal Chang, HepG2, Chang-GnT-III, and Chang-HBx cells were subjected to 10 and 12% SDS-polyacrylamide gel electrophoresis. Expressions of HBx (upper panel) and GnT-III (middle panel) were analyzed by Western blot using a specific antibody to HBx and GnT-III. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) indicates that equal amounts of proteins were loaded in each lane (lower panel). C, protein bands were quantitated by densitometry.

 
Accumulation of ApoB with Increased Bisecting-GlcNAc in HBx-transfected Cells—To investigate the increase of accumulation of intracellular apoB in HBx-transfected liver cells, immunoprecipitation analysis was performed using cell lysates with anti-apoB antibody as described under "Experimental Procedures." As shown in Fig. 5A, compared with pcDNA transfected cells as control, HBx-transfected cells contained higher levels of intact apoB bands, 150- and 50-kDa apoB species.

Next, to determine whether these apoB species are aberrantly glycosylated, lectin blot analysis were performed using E-PHA lectins, which are known to react preferentially with bisecting-GlcNAc (40). In Fig. 5B, immunoprecipitated apoB species showed increased bisecting-GlcNAc, which is a consistent result with Western blot analysis in HBx-transfected cells. However, although an ~80-kDa protein reacted with E-PHA, this band appeared to be nonspecific because it could not detect in Western blot analysis (Fig. 5A). Furthermore, the reactivity of immunoprecipitated apoB to E-PHA was blocked in the presence of an authentic inhibitor, GalNAc (41). Therefore, these results indicated that HBx-transfected cells significantly increased the intracellular accumulation of apoB species, which contained increased bisecting GlcNAc.

Enhanced Expression of GnT-III by HBx Decreases the Secretion of ApoB and Increases Accumulation of Cellular Triglyceride and Cholesterol Contents—Based on the finding that the expression level of GnT-III in HBx-transfected cell was higher than that in GnT-III-transfected cell as shown in Fig. 4, we hypothesized that the aberrant glycosylation of apoB by HBx-induced GnT-III expression may be involved in apoB secretion; the apoB protein level was measured by Western blot from culture media in the control vector, GnT-III, and HBx-transfected cells because apoB plays an important role for delivery of triglyceride from liver to peripheral tissue. Cells were treated with t-RA for 2 days in serum-free media because secretion of apoB induced by t-RA was increased in dose and time-dependent manner (data not shown). The same result was observed in treatment with oleic acid, and there was no apoptotic fragmentation in the cell (data not shown). The secretion of apoB was significantly decreased by 40 and 95% in GnT-III- and HBx-transfected cells, respectively, compared with in vector control (Fig. 6A), indicating that GnT-III expression level is an important factor for the inhibition of apoB secretion. This finding suggested that aberrant glycosylation of apoB mediated by enhanced expression of HBx-induced GnT-III inhibits the secretion of apoB and increases accumulation of intracellular apoB.



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  		FIG. 6.
GnT-III expression levels, inhibition of apoB secretion from culture media, and changes in cellular triglyceride and cholesterol contents in GnT-III- and HBx-transfected liver cells. A, after being permanently transfected with HBx and GnT-III cDNAs, Chang cells were incubated with 1 µM t-RA in serum-free media for 2 days. Proteins (25 µg) from media were subjected to 6% SDS-PAGE. Secretion of apoB from the media was analyzed by Western blot and bands were quantitated by densitometry. B, changes in cellular triglyceride (TG) and cholesterol mass were measured in Chang cells using enzymatic reagents. Values are the means for three to five experiments.

 
As a result of the Western blot experiment of inhibition of apoB secretion in HBx- and GnT-III-transfected cells, as shown in Fig. 6B, we postulated that cellular triglyceride and cholesterol mass may be increased in the HBx- and GnT-III-transfected cells. To test this, we measured the accumulation of triglyceride and cholesterol in the cytosolic fraction in Chang cells transfected with HBx and GnT-III. As expected, in Fig. 6C cellular triglyceride levels in GnT-III- and HBx-transfected cell were higher than vector-transfected cells up to 25% (p < 0.05) and 85% (p < 0.01), respectively. Cellular cholesterol levels in GnT-III- and HBx-transfected cells were slightly increased by 21% (p < 0.05) and 35% (p < 0.01), respectively. These results clearly indicated that increased accumulation of intracellular apoBs caused an accumulation of triglyceride and cholesterol by enhanced expression of HBx-induced GnT-III.

Expression Levels of MTP mRNA—Because MTP has been shown to play a critical role for apoB assembly and secretion, to determine whether MTP expression may be affected in the GnT-III- and HBx-transfected liver cells, MTP mRNA levels were measured by RT-PCR and Northern blot analysis (Fig. 7, A and B). The expression of MTP was significantly decreased in GnT-III- and HBx-transfected cells compared with pcDNA-transfected cells after treatment of t-RA for 48 h in serum-free media. When the cells were treated with t-RA, however, there was no difference in apoB gene expression by RT-PCR (data not shown). This result suggested that the relationship between HBx and GnT-III may regulate MTP expression for apoB assembly and secretion.



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  		FIG. 7.
Expression level of MTP mRNA in GnT-III and HBx-transfected liver cells. A, 1 µg of total RNA from each cell was subjected to RT-PCR (upper panel) as described under "Experimental Procedures." {beta}-Actin mRNA expression was also examined as a control (lower panel). B, 20 µg of total RNAs extracted from vector and GnT-III- and HBx-transfected Chang cells were electrophoresed on a 1.0% agarose gel containing 2.2 M formaldehyde and then analyzed by Northern blot hybridization using 32P-labeled PCR product of MTP gene (upper panel). {beta}-Actin indicates that equal amounts of RNAs were loaded in each lane (lower panel).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here, we provide evidence of the relationship between HBx and GnT-III responsible for inhibition of apoB secretion in vitro model. Many investigators have used HepG2 cell for studying apoB secretion. However, the HepG2 cell line is already a HCC, and increase of the expression of GnT-III gene was reported in human hepatocarcinoma tissues, fetal liver tissues, and hepatoma cell lines (42). In this study we have chosen a liver Chang cell line that is originally derived from normal liver tissue but is subsequently established via HeLa cell contamination.

Miyoshi et al. (43) observe that during hepatocarcinogenesis, GnT-III messenger RNA levels were increased in LEC rats, and Ishibashi et al. (21) also reported that GnT-III activity in human serum, and liver and hepatoma tissues were increased in liver cirrhosis and hepatoma patients. We observed that HBx increases GnT-III expression by transcription as well as translation levels. In the previous study we found that the promoter region of GnT-III has seven AP-2 sites by sequence homology search (37). Elevation of GnT-III gene expression by HBx may be modulated by AP-2 activation since HBx has been shown to activate promoters through several transcription factors such as AP-1, AP-2, NF-{kappa}B, CREB, and ATF-2 (45–48), although HBx does not bind directly to DNA (49). We also showed that GnT-III expression levels in HBx-transfected cells is higher than that in hepatocarcinoma HepG2 cells as well as GnT-III-transfected cells, indicating that HBx induces GnT-III expression with strong GnT-III promotor activity. Therefore, this finding supports several studies which show that GnT-III expression may be involved in the development of HCC (23, 25). Unfortunately, this study did not determine the precise promotor region involved in the activation for GnT-III expression by HBx because of the limitation of the comprehensive properties of the GnT-III promoter region (38, 39), but this is the first report that HBx transactivates GnT-III expression.

The hepatic production of apoB-containing lipoproteins is regulated largely at posttranscriptional levels, with nascent apoB molecules secreted or degraded intracellularly (11). Based on our findings that intracellular accumulation of aberrantly glycosylated apoB species, which exhibit strong reactivities to E-PHA, is increased and low molecular weight immunoreactive apoB species (especially 150 and 50 kDa) are detected in HBx-transfected cells, the present study supports our previous results (31) and those of Ihara et al. (32), who report that overexpression of GnT-III in transgenic hepatocytes induced aberrant glycosylation of apoB and disrupted apoB secretion. They also showed that the 130- and 50-kDa apoB species were immunoprecipitated and detected with lectin blot analysis. In addition, apoB mRNA levels in liver Chang cells are not affected by HBx transfection by RT-PCR (data not shown), consistent with the concept that, under most conditions of altered apoB secretion from HepG2 cells, apoB mRNA levels remain unchanged (13, 51, 52).

Some investigators suggest that hepatic triglyceride accumulation has a greater influence on apoB secretion (53) and cholesteryl ester is the major lipid species stimulating very low density lipoprotein secretion (54). However, in our study, even though there is intracellular accumulation of triglyceride and cholesterol in GnT-III- as well as HBx-transfected liver Chang cells, inhibition of apoB secretion occurs in these cells. Therefore, intracellular accumulation of apoB caused by the ability of HBx to induce GnT-III expression is the major determinant for the inhibition of apoB secretion and intracellular lipid accumulation.

With regard to the association of apoB with tumorigenesis, lipid accumulation in the liver resulted in the development of dysplasia and carcinoma of the liver in mice expressing aberrantly truncated apoB (55). Our study also revealed that an increase of the accumulation of triglyceride and cholesterol in liver cells is detected in GnT-III- as well as HBx-transfected cells, which is consistent with previous studies by Lee et al. (31) and Ihara et al. (32), which show that lipid accumulation in the liver in GnT-III transgenic mice leads to the generation of liver abnormality. Thus, intracellular accumulation of triglyceride and cholesterol in liver cells caused by HBx-induced GnT-III expression may give us a new insight for HBx-mediated HCC development.

In addition, MTP, an intraluminal protein in the endoplasmic reticulum plays an essential role in regulating the assembly and secretion of apoB containing lipoproteins (44, 50, 56). It is interesting to note that HBx- and GnT-III-transfected cells showed down-expression of MTP mRNA but not in vector-transfected cell. Because cells were treated with t-RA in our experiment, MTP expression levels by RT-PCR and secretion of apoB by Western blot were increased in dose- and time-dependent manners (data not shown). However, these data questioned whether down-regulation of MTP transcriptional levels by overexpression of GnT-III and HBx may result from the accumulation of triglyceride and cholesterol in liver cells primarily or secondarily. The contribution of down-regulation of MTP expression by GnT-III and HBx remains to be clarified.

The availability of transgenic mice aberrantly expressing the human GnT-III in the liver may help not only to elucidate the role of protein/lipid glycosylation in the development and pathological change of the liver but also to develop therapeutic agents for human diseases caused by glycosylation abnormality. We are currently investigating whether hepatocytes from these transgenic mice show an altered sensitivity to viral infection or abnormal receptor-ligand interactions as the result of aberrant glycosylation of cell surface protein and lipids. In conclusion, HBx induced GnT-III expression may disrupt lipid metabolism by abnormal glycosylation of apoB and may target MTP for assembly and secretion of apoB in the liver cells.


   FOOTNOTES
 
* This work was supported by National Research Laboratory Program, Ministry of Science and Technology, South Korea Grant M10203000024-02J0000-01300 (to C.-H. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: NRLG and Dept. of Biochemistry and Molecular Biology, Dongguk University, Sukjang-Dong 707, Kyungju City, Kyungbuk 780-714, Korea. Tel.: 82-54-770-2663; Fax: 82-54-770-2281; E-mail: chkimbio{at}dongguk.ac.kr.

1 The abbreviations used are: HCC, hepatocellular carcinoma; HBx, X protein of hepatitis B virus; apoB, apolipoprotein B; GnT-III, UDP-N-acetylglucosamine:{beta}-D-mannoside-1,4-N-acetylglucosaminyltransferase-III; MTP, microsomal triglyceride transfer protein; RT, reverse transcriptase; CAT, chloramphenicol acetyltransferase; t-RA, all-transretinoic acid; CREB, cAMP-response element-binding protein; E-PHA, erythroagglutinating phytohemagglutinin. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Colombo, M. (1998) Hepatogastroenterology 45, 1221–1225
  2. Tiollais, P., Charnay, P., and Vyas G. N. (1981) Science 213, 406–411[Abstract/Free Full Text]
  3. Kim, C., Koike, K., Saito, I., Miyamura, T., and Jay, G. (1991) Nature 351, 317–320[CrossRef][Medline] [Order article via Infotrieve]
  4. Chung, T. W., Lee, Y. C., Ko, J. H., and Kim, C. H. (2003) Cancer Res. 63, 3453–3458[Abstract/Free Full Text]
  5. Davis, R. A., and Hui, T. Y. (2001) Arterioscler. Thromb. Vasc. Biol. 21, 887–898[Abstract/Free Full Text]
  6. Davis, R. A. (1999) Biochim. Biophys. Acta 1441, 1–31[Medline] [Order article via Infotrieve]
  7. Olofsson, S. O., Asp, L., and Boren, J. (1999) Curr. Opin. Lipidol. 10, 341–346[CrossRef][Medline] [Order article via Infotrieve]
  8. Davidson, N. O., and Shelness, G. S. (2000) Annu. Rev. Nutr. 20, 169–193[CrossRef][Medline] [Order article via Infotrieve]
  9. Fisher, E. A., and Ginsberg, H. N. (2002) J. Biol. Chem. 277, 17377–17380[Free Full Text]
  10. Yao, Z., and McLeod, R. S. (1994) Biochim. Biophys. Acta 1212, 152–166[Medline] [Order article via Infotrieve]
  11. Yao, Z., Tran, K., and McLeod, R. S. (1997) J. Lipid Res. 38, 1937–1953[Abstract]
  12. Ginsberg, H. N. (1997) Clin. Exp. Pharmacol. Physiol. 24, 29–32
  13. Pullinger, C. R., North, J. D., Teng, B-B., Rifici, V. A., Ronhild de Brito, A. E., and Scott, J. (1989) J. Lipid Res. 30, 1065–1077[Abstract]
  14. Yao, Z., and Vance, D. E. (1988) J. Biol. Chem. 263, 2998–3004[Abstract/Free Full Text]
  15. Jamil, H., Dickson, J. K., Jr., Chu, C., Lago, M. W., Rinehart, J. K., Biller, S. A., Gregg, R. E., and Wetterau, J. R. (1995) J. Biol. Chem. 270, 6549–6554[Abstract/Free Full Text]
  16. Wetterau, J. R., Gregg, R. E., Harrity, T. W., Arbeeny, C., Cap, M., Connolly, F., Chu, C.-H., George, R. J., Gordon, D. A., Jamil, H., Jolibois, K. G., Kunselman, L. K., Lan, S.-J., Maccagnan, T. J., Ricci, B., Yan, M., Young, D., Chen, Y., Fryszman, O. M., Logan, J. V. M., Musial, C. L., Poss, M. A., Robl, J. A., Simpkins, L. M., Slusarchyk, W. A., Sulsky, R., Taunk, P., Magnin, D. A., Tino, J. A., Lawrence, R. M., Dickson, J. K., Jr., and Biller, S. A. (1998) Science 282, 751–754[Abstract/Free Full Text]
  17. Nishikawa, A., Fujii, S., Sugiyama, T., Hayashi, N., and Taniguchi, N. (1988) Biochem. Biophys. Res. Commun. 152, 107–112[CrossRef][Medline] [Order article via Infotrieve]
  18. Pascale, R., Narasimhan, S., and Rajalakshmi, S. (1989) Carcinogenesis 10, 961–964[Medline] [Order article via Infotrieve]
  19. Ohno, M., Nishikawa, A., Koketsu, M., Taga, H., Endo, Y., Hada, T., Higashino, K., and Taniguchi, N. (1992) Int. J. Cancer 51, 315–317[Medline] [Order article via Infotrieve]
  20. Taniguchi, N., Miyoshi, E., Ko, J. H., Ikeda, Y., and Ihara, Y. (1999) Biochim. Biophys. Acta 455, 287–300
  21. Ishibashi, K., Nishikawa, A., Hayashi, N., Kasahara, A., Sato, N., Fujii, S., Kamada, T., and Taniguchi, N. (1989) Clin. Chim. Acta 185, 325–332[CrossRef][Medline] [Order article via Infotrieve]
  22. Yoshimura, M., Nishikawa, A., Ihara, Y., Taniguchi, S., and Taniguchi, N. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8754–8758[Abstract/Free Full Text]
  23. Mori, S, Aoyagi, Y., Yanagi, M., Suzuki, Y., and Asakura, H. (1998) J. Gastroenterol. Hepatol. 13, 610–619[Medline] [Order article via Infotrieve]
  24. Nishikawa, A., Ihara, Y., Hatakeyama, M., and Taniguchi, N. (1992) J. Biol. Chem. 267, 18199–18204[Abstract/Free Full Text]
  25. Narasimhan, S., Schachter, H., and Rajalakshmi, S. (1988) J. Biol. Chem. 263, 1273–1281[Abstract/Free Full Text]
  26. Swaminathan, N., and Aladjem, F. (1976) Biochemistry 15, 1516–1522[CrossRef][Medline] [Order article via Infotrieve]
  27. Taniguchi, T., Ishikawa, Y., Tsunemitsu, M., and Fukuzaki, H. (1989) Arch. Biochem. Biophys. 273, 197–205[CrossRef][Medline] [Order article via Infotrieve]
  28. Hakomori, S. (1989) Adv. Cancer Res. 52, 257–331[Medline] [Order article via Infotrieve]
  29. Rademacher, T. W., Parekh, R. B., and Dwek, R. A. (1988) Annu. Rev. Biochem. 57, 785–838[CrossRef][Medline] [Order article via Infotrieve]
  30. Yamashita, K, Hitoi, A., Taniguchi, N., Yokosawa, N., Tsukada, Y., and Kobata, A. (1983) Cancer Res. 43, 5059–5063[Abstract/Free Full Text]
  31. Lee, J. W., Song, E. Y., Chung, T. W., Kang, S. K., Kim, K. S., Chung, T. H., Yeom, Y. I., and Kim, C. H. (2004) Arch. Biochem. Biophys. 426, 18–31[CrossRef][Medline] [Order article via Infotrieve]
  32. Ihara, Y., Yoshimura, M., Miyoshi, E., Nishikawa, A., Sultan, A. S., Toyosawa, S., Ohnishi, A., Suzuki, M., Yamamura, K., and Taniguchi, N. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2526–2530[Abstract/Free Full Text]
  33. Song, E. Y., Kim, K. A., Kim, Y. D., Kwon, D. H., Lee, H. S., Kim, C. H., Chung, T. W., and Byun, S. M. (2000) Biotechnol. Lett. 22, 201–204[CrossRef]
  34. Harlow, E. D., and Lane, P. (1988) Antibodies: A Laboratory Manual, pp. 139–244, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  35. Song, E. Y., Kim, K. S., Kim, K. A., Kim, Y. D., Kwon, D. H., Byun, S. M., Kim, H. J., Chung, T. W., Choe, Y. K., Chung, T. W., and Kim, C. H. (2003) Glycoconj. J. 9, l415–1421
  36. Bullock C., and Gorman, C. (2000) Methods Enzymol. 326, 202–221[Medline] [Order article via Infotrieve]
  37. Kim, Y. J., Kim, K. S., Ko, J. H., Lee, Y. C., Chung, T. W., Choe, I. S., and Kim, C. H. (1996) Gene (Amst.) 170, 281–283[CrossRef][Medline] [Order article via Infotrieve]
  38. Koyama, N., Miyoshi, E., Ihara, Y., Kang, R., Nishikawa, A., and Taniguchi, N. (1996) Eur. J. Biochem. 238, 853–861[Medline] [Order article via Infotrieve]
  39. Kang, B. S., Kim, Y. J., Shim, J. K., Song, E. Y., Park, Y. G., Lee, Y. C., Nam, K. S., Kim, J. K., Lee, T. K., Chung, T. W., and Kim, C. H. (1998) J. Biochem. Mol. Biol. 31, 578–584
  40. Yamashita, K., Hitoi, A., and Kobata, A. (1983) J. Biol. Chem. 258, 14753–14755[Abstract/Free Full Text]
  41. Serafini-Cessi, F., Franceschi, C., and Sperti, S. (1979) Biochem. J. 183, 381–388[Medline] [Order article via Infotrieve]
  42. Song, E. Y., Lee, Y. C., Park, Y. G., Chung, T. W., and Kim, C. H. (2001) Cancer Investig. 19, 797–805
  43. Miyoshi, E., Nishikawa, A., Ihara, Y., Gu, J., Sugiyama, T., Hayashi, N., Fusamoto, H., Kamada, T., and Taniguchi, N. (1993) Cancer Res. 53, 3899–3902[Abstract/Free Full Text]
  44. Gordon, D. A., Jamil, H., Gregg, R. E., Olofsson, S.-O., and Boren, J. (1996) J. Biol. Chem. 271, 33047–33053[Abstract/Free Full Text]
  45. Imagawa, M., Chiu, R., and Karin, M. (1987) Cell 51, 251–260[CrossRef][Medline] [Order article via Infotrieve]
  46. Hyman, S. E., Comb, M., Pearlberg, J., and Goodman, H. M. (1989) Mol. Cell. Biol. 9, 321–324[Abstract/Free Full Text]
  47. Kannan, P., Buettner, R., Chiao, P. J., Yim, S. O., Sarkiss, M., and Tainsky, M. A. (1994) Genes Dev. 8, 1258–1269[Abstract/Free Full Text]
  48. Caselmann, W. H., and Koshy, R. (1998) Hepatitis B Virus: Molecular Mechanisms in Disease and Novel Strategies for Therapy, pp 161–181, Imperial College Press, London
  49. Haviv, I., Shamay, M., Doitsh, G., and Shaul, Y. (1998) Mol. Cell. Biol. 18, 1562–1569[Abstract/Free Full Text]
  50. Jamil, H., Gordon, D. A., Eustice, D. C., Broods, C. M., Dickson, J. J. K., Chen, Y., Ricci, B., Chu, C.-H., Harrity, T. W., Ciosek, J. C. P., Biller, S. A., Gregg, R. E., and Wetterau, J. R. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11991–11995[Abstract/Free Full Text]
  51. Wilcox, L. J., Barrett, P. H. R., Newton, R. S., and Huff, M. W. (1999) Arterioscler. Thromb. Vasc. Biol. 19, 939–949[Abstract/Free Full Text]
  52. Wilcox, L. J., Barrett, P. H. R., and Huff, M. W. (1999) J. Lipid Res. 40, 1078–1089[Abstract/Free Full Text]
  53. Benoist, F., and Grand-Perret, T. (1996) Arterioscler. Thromb. Vasc. Biol. 16, 1229–1235[Abstract/Free Full Text]
  54. Avramoglu, R. K., Cianflone, K., and Sniderman, A. D. (1995) J. Lipid Res. 36, 2513–2528[Abstract]
  55. Yamanaka, S., Balestra, M. E., Ferrell, L. D., Fan, J., Arnold, K. S., Taylor, S., Taylor, J. M., and Innerarity, T. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8483–8487[Abstract/Free Full Text]
  56. Hussain, M. M., Shi, J., and Dreizen, P. (2003) J. Lipid Res. 44, 22–32[Abstract/Free Full Text]

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