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• W4235187828 abstract "Cyclin D1 is frequently overexpressed in hepatocellular carcinoma (HCC) exhibiting increased malignant phenotypes. It has also been known that the hepatitis Bx (HBx) protein is strongly associated with HCC development and progression. Although overexpression of both proteins is related to HCC, the relationship between the two has not been well studied. Here we show that HBx up-regulates cyclin D1 and that this process is mediated by the NF-κB2(p52)/BCL-3 complex. Our experiments indicate that HBx up-regulates BCL-3 in the mRNA level, which subsequently results in the up-regulation of the NF-κB2(p52)/BCL-3 complex in the nucleus. Moreover, impaired HBx-mediated BCL-3 up-regulation by small interfering RNA for BCL-3 reduced HBx-mediated cyclin D1 up-regulation. Down-regulation of the HBx protein level by p53 also reduced HBx-mediated cyclin D1 up-regulation. From these results, we conclude that the up-regulation of cyclin D1 by HBx is mediated by the up-regulation of NF-κB2(p52)/BCL-3 in the nucleus. This HBx-mediated-cyclin D1 up-regulation might play an important role in the HBx-mediated HCC development and progression. Cyclin D1 is frequently overexpressed in hepatocellular carcinoma (HCC) exhibiting increased malignant phenotypes. It has also been known that the hepatitis Bx (HBx) protein is strongly associated with HCC development and progression. Although overexpression of both proteins is related to HCC, the relationship between the two has not been well studied. Here we show that HBx up-regulates cyclin D1 and that this process is mediated by the NF-κB2(p52)/BCL-3 complex. Our experiments indicate that HBx up-regulates BCL-3 in the mRNA level, which subsequently results in the up-regulation of the NF-κB2(p52)/BCL-3 complex in the nucleus. Moreover, impaired HBx-mediated BCL-3 up-regulation by small interfering RNA for BCL-3 reduced HBx-mediated cyclin D1 up-regulation. Down-regulation of the HBx protein level by p53 also reduced HBx-mediated cyclin D1 up-regulation. From these results, we conclude that the up-regulation of cyclin D1 by HBx is mediated by the up-regulation of NF-κB2(p52)/BCL-3 in the nucleus. This HBx-mediated-cyclin D1 up-regulation might play an important role in the HBx-mediated HCC development and progression. Hepatitis B virus (HBV) 3The abbreviations used are: HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HBx, hepatitis Bx; NF-κB, nuclear factor κB; CDKs, cyclin-dependent kinases; BCL-3, B-cell lymphoma protein-3; HDAC1, histone deacetylase 1; DAPI, 4′, 6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate-conjugated; siRNA, small interfering RNA; HA, hemagglutinin; mAb, monoclonal antibody; pAb, polyclonal antibody; RT, reverse transcription; PBS, phosphate-buffered saline; CMV, cytomegalovirus; IKK, IκB kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CREB, cAMP-response element-binding protein. 3The abbreviations used are: HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HBx, hepatitis Bx; NF-κB, nuclear factor κB; CDKs, cyclin-dependent kinases; BCL-3, B-cell lymphoma protein-3; HDAC1, histone deacetylase 1; DAPI, 4′, 6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate-conjugated; siRNA, small interfering RNA; HA, hemagglutinin; mAb, monoclonal antibody; pAb, polyclonal antibody; RT, reverse transcription; PBS, phosphate-buffered saline; CMV, cytomegalovirus; IKK, IκB kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CREB, cAMP-response element-binding protein. is a member of the hepadnavirus family by which 350 million people have been infected worldwide (1Zuckerman A.J. Br. Med. J. 1999; 318: 1213Crossref PubMed Google Scholar). In 5-10% of the patients, the initial infection progresses to a lifelong chronic hepatitis B infection, which is a frequent precursor to cirrhosis and hepatocellular carcinoma (HCC) (2Feitelson M.A. J. Cell. Physiol. 1999; 181: 188-202Crossref PubMed Scopus (98) Google Scholar). HBV has a partially double-stranded DNA gene encoding four unspliced overlapping messages that terminate at a common polyadenylation signal (3Seeger C. Mason W.S. Microbiol. Mol. Biol. Rev. 2000; 64: 51-68Crossref PubMed Scopus (1216) Google Scholar). Each messenger RNA encodes the core protein and S proteins (small S, middle S, and large S proteins) as the viral structure proteins, HBV polymerase for gene replication and hepatitis Bx (HBx), which induces various signal transduction (3Seeger C. Mason W.S. Microbiol. Mol. Biol. Rev. 2000; 64: 51-68Crossref PubMed Scopus (1216) Google Scholar). These HBV proteins interact with many host factors where the interactions are associated with viral replication and the virus-mediated pathogenesis (4Hu J. Seeger C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1060-1064Crossref PubMed Scopus (294) Google Scholar, 5Park S.G. Jung G. J. Virol. 2001; 75: 6962-6968Crossref PubMed Scopus (63) Google Scholar, 6Kim K.H. Seong B.L. EMBO J. 2003; 22: 2104-2116Crossref PubMed Scopus (115) Google Scholar, 7Tanaka Y. Kanai F. Kawakami T. Tateishi K. Ijichi H. Kawabe T. Arakawa Y. Nishimura T. Shirakata Y. Koike K. Omata M. Biochem. Biophys. Res. Commun. 2004; 318: 461-469Crossref PubMed Scopus (84) Google Scholar, 8Park S.G. Lee S.M. Jung G. J. Biol. Chem. 2003; 278: 39851-39857Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The HBx protein is a small protein, composed of 154 amino acids, which is necessary for establishing productive infection in vivo (9Zoulim F. Saputelli J. Seeger C. J. Virol. 1994; 68: 2026-2030Crossref PubMed Google Scholar). HBx expression is known to be strongly associated with HCC development and progression as animal model experiments show that transgenic mice expressing the HBx protein are susceptible to HCC development (10Kim C.M. Koike K. Saito I. Miyamura T. Jay G. Nature. 1991; 351: 317-320Crossref PubMed Scopus (1053) Google Scholar, 11Slagle B.L. Lee T.H. Medina D. Finegold M.J. Butel J.S. Mol. Carcinog. 1996; 15: 261-269Crossref PubMed Scopus (163) Google Scholar). HBx can trigger changes in a variety of intracellular signal transduction (12Rabe C. Cheng B. Caselmann W.H. Dig. Dis. 2001; 19: 279-287Crossref PubMed Scopus (27) Google Scholar). Among them, nuclear factor-κB (NF-κB) activation is one of the most important events due to its close links with tumorigenesis (13Lin A. Karin M. Semin. Cancer Biol. 2003; 13: 107-114Crossref PubMed Scopus (347) Google Scholar, 14Baldwin A.S. J. Clin. Investig. 2001; 107: 241-246Crossref PubMed Scopus (1199) Google Scholar). The transcription factor NF-κB is a key regulator in oncogenesis, which, by promoting proliferation and inhibiting apoptosis, tips the balance between proliferation and apoptosis toward malignant growth in tumor cells (13Lin A. Karin M. Semin. Cancer Biol. 2003; 13: 107-114Crossref PubMed Scopus (347) Google Scholar). NF-κB is a class of dimeric transcription factors that belong to the Rel family including RelA (p65), RelB, c-Rel, NF-κB1 (p50), and NF-κB2 (p52) (15Ghosh S. Karin M. Cell. 2002; 109: S81-S96Abstract Full Text Full Text PDF PubMed Scopus (3290) Google Scholar). It has been reported that different stimuli can induce the activation of different NF-κB complexes (16Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3361) Google Scholar). This selective activation is a key to the regulation of gene expression since the recognition sequences of different NF-κB complexes differ from each other (17Ghosh G. Huang D.B. Huxford T. Curr. Opin. Struct. Biol. 2004; 14: 21-27Crossref PubMed Scopus (37) Google Scholar). Previous studies have suggested a link between HBx and cyclin D1 where cyclin D1 is shown to be up-regulated by HBx (18Klein A. Guhl E. Tzeng Y.J. Fuhrhop J. Levrero M. Graessmann M. Graessmann A. Oncogene. 2003; 22: 2910-2919Crossref PubMed Scopus (34) Google Scholar). Overexpression of cyclin D1 is involved in tumor development and growth (19Fu M. Wang C. Li Z. Sakamaki T. Pestell R.G. Endocrinology. 2004; 145: 5439-5447Crossref PubMed Scopus (815) Google Scholar) as well as in HCC. Although amplification of the cyclin D1 gene is responsible for only a proportion of the cases (20Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4971) Google Scholar), other mechanisms remain to be clarified (21Ozturk M. Semin. Liver Dis. 1999; 19: 235-242Crossref PubMed Scopus (156) Google Scholar). Generally, cyclin D1 induces the progression of cell cycle from the G1 to S phase by the activation of cyclin-dependent kinases (CDK), namely CDK4 and CDK6 (19Fu M. Wang C. Li Z. Sakamaki T. Pestell R.G. Endocrinology. 2004; 145: 5439-5447Crossref PubMed Scopus (815) Google Scholar, 22Attwooll C. Lazzerini Denchi E. Helin K. EMBO J. 2004; 23: 4709-4716Crossref PubMed Scopus (418) Google Scholar). These kinases phosphorylate retinoblastoma, and the hyperphosphorylation of retinoblastoma promotes the release of the E2F family of transcriptional factors, which in turn promotes entry into the S phase via activation of key target genes (22Attwooll C. Lazzerini Denchi E. Helin K. EMBO J. 2004; 23: 4709-4716Crossref PubMed Scopus (418) Google Scholar). Cyclin D1 is upregulated by the NF-κB2(p52)/B-cell lymphoma protein-3 (BCL-3) complex (23Westerheide S.D. Mayo M.W. Anest V. Hanson J.L. Baldwin Jr., A.S. Mol. Cell. Biol. 2001; 21: 8428-8436Crossref PubMed Scopus (153) Google Scholar, 24Rocha S. Martin A.M. Meek D.W. Perkins N.D. Mol. Cell. Biol. 2003; 23: 4713-4727Crossref PubMed Scopus (202) Google Scholar) where the nuclear localization of BCL-3 is dependent on its polyubiquitination in the activation condition (25Massoumi R. Chmielarska K. Hennecke K. Pfeifer A. Fassler R. Cell. 2006; 125: 665-677Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar). In this study, we present the HBx-mediated NF-κB2(p52)/BCL-3 activation and its effect on the gene expression of cyclin D1. Our experiments show that NF-κB2(p52)/BCL-3 complex activation by HBx is mediated by the up-regulation and nuclear localization of BCL-3, which eventually induces the up-regulation of cyclin D1. This HBx-mediated up-regulation of cyclin D1 might play a role in HBx-mediated HCC development and progression. Plasmids—The construction of pCMV-HA/HBx has been described previously (26Park S.G. Ryu H.M. Lim S.O. Kim Y.I. Hwang S.B. Jung G. Gastroenterology. 2005; 128: 2042-2053Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). pCMV-Myc/p53 was constructed by inserting the p53 gene fragment into the EcoRI site of pCMV-Myc (Clontech); the fragment was generated by PCR using HepG2 cell cDNA. pCMV-HA/RelA, pCMV-HA/RelB, pCMV-HA/NF-κB1(p50), and pCMV-HA/NF-κB2(p52) plasmids were constructed in the same manner. pCMV-HA/HDAC1 and pCMV-HA/BCL-3 constructs were also generated by inserting histone deacetylase 1 (HDAC1) and BCL-3 gene fragments, PCR-generated from HepG2 cell cDNA, into the EcoRI site of pCMV-HA, respectively (Clontech). Construction of p973CD1LUC, which contains the cyclin D1 promoter from -973 to +137, and p29CD1LUC, which contains the cyclin D1 promoter from -29 to +137, was described previously (27Park Y.G. Park S. Lim S.O. Lee M.S. Ryu C.K. Kim I. ChoChung Y.S. Biochem. Biophys. Res. Commun. 2001; 281: 1213-1219Crossref PubMed Scopus (28) Google Scholar). A p66CD1LUC construct containing the cyclin D1 promoter from -66 to +137, and p66CD1LUCΔκB, which contains the cyclin D1 promoter from -66 to +137 with κB site mutation, also followed previous description (28Joyce D. Bouzahzah B. Fu M. Albanese C. D'Amico M. Steer J. Klein J.U. Lee R.J. Segall J.E. Westwick J.K. Der C.J. Pestell R.G. J. Biol. Chem. 1999; 274: 25245-25249Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). pSV-Basic-β-galactosidase was purchased from Promega (Madison, WI). Antibodies and Reagents—The following antibodies were purchased from the listed manufacturers: anti-HA mouse monoclonal antibody (mAb, Sigma), anti-Myc mouse mAb (BD Biosciences), anti-p53 mouse mAb (BD Biosciences), anti-BCL-3 rabbit pAb, anti-cyclin D1 mouse mAb, and anti-HDAC1 mouse mAb (Upstate Biotechnology, Charlottesville, VA), anti-NF-κB2(p52) rabbit pAb (Santa Cruz Biotechnology, Santa Cruz, CA). The anti-β-actin mouse mAb, anti-mouse horseradish peroxidase, anti-rabbit horseradish peroxidase, anti-mouse fluorescein isothiocyanate (FITC), and anti-rabbit FITC were purchased from Sigma, and antimouse-rhodamine and anti-goat FITC were purchased from Chemicon and Santa Cruz Biotechnology, respectively. The anti-HBx rabbit pAb was a gift from Dr. Yun (Ewha Women’s University). 4′, 6-Diamidino-2-phenylindole (DAPI) was purchased from Merck (Darmstadt, Germany). The small interfering RNAs (siRNAs) specific for BCL-3 and green fluorescent protein were synthesized by Samchully Pharm. Co. (Seoul, Korea). Cells and Transfections—Chang liver cells were cultured in minimum essential medium (Invitrogen) supplemented with 10% fetal bovine serum (JBI, Daegu, Korea). HepG2.2.15 cells (HBV-producing cells) (29Sells M.A. Chen M.L. Acs G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1005-1009Crossref PubMed Scopus (997) Google Scholar) and HepG2 cells (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum (JBI). The cells were seeded in 12-well plates at a density of 0.3 × 106 cells/well for immunoblot analysis or reporter assay. The cells were transfected using the FuGENE 6 transfection reagent (Roche Applied Science, Mannheim, Germany) following the manufacturer’s instructions. Cells were transfected with 0.2-0.8 μg of pCMV-HA/HBx for the expression of HBx. In transfections, the total DNA amount was normalized with backbone DNA, such as pCMV-HA, pCMV-Myc, and pRc/CMV. Total protein extracts were prepared with lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholic acid, 0.5% SDS, and 1× protease inhibitor mix (Sigma) in 1× phosphate-buffered saline (PBS)) after 48 h of transfection. For siRNA transfection, the cells were transfected using Lipofectamine™ 2000 transfection reagent (Invitrogen), as instructed by the manufacturer. Construction of pNF-κB/luciferase was as described previously (26Park S.G. Ryu H.M. Lim S.O. Kim Y.I. Hwang S.B. Jung G. Gastroenterology. 2005; 128: 2042-2053Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Construction of pCMV-IKKβ/DN, which is a dominant negative form of IKKβ, was as described previously (30Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1068) Google Scholar). RT-PCR Analysis—Two days after transfection, total RNA was extracted with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was generated by reverse transcription (RT) followed by amplification using 2.5 units of Taq polymerase (Qbio, Seoul, Korea), a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific primer set and a BCL-3-(5′-GGCGCTTTACTACCCCGGAGCCTTA-3′ and 5′-GTTTCTTGGCACTCGGTGTTCACTG-3′) or cyclin D1-(5′-AGCTCCTGTGCTGCGAAGTGGAAAC-3′ and 5′-AGTGTTCAATGAAATCGTGCGGGGT-3′) specific primer set. PCR was performed using a Gene Amp PCR system 2400 (PerkinElmer Life Sciences) with a 5-min initial denaturation at 95 °C and 20-30 cycles of 50 s at 95 °C, 50 s at 60 °C, 50 s at 72 °C; these cycles were followed by a 7-min extension at 72 °C. The PCR fragments were separated by running on a 1% agarose gel. Luciferase Reporter Assay—Cells were transfected using Fugene6 with 650 ng of promoter reporter plasmids, 650 ng of pSV-Basic-β-galactosidase, and the appropriate DNAs. After transfection (48 h), cell extracts were prepared and assayed for luciferase activity according to the instructions in the luciferase assay system (Promega, Madison, WI). Transfection efficiency was normalized by β-galactosidase activity. Fluorescent Microscopy Analysis—Chang liver and HepG2 cells were seeded on poly-l-lysine-coated coverslips at a density of 0.05 × 106 cells/coverslip and were either transfected or treated. The low transfection efficiency may be caused by low seeding density. Slides were washed with 1× PBS, fixed with ice-cold methanol for 10 min, and blocked with 1% bovine serum albumin in 1× PBS for 1 h. The slides were then probed for 90 min at room temperature with appropriate antibodies. After probing, the slides were washed three times with 1× PBS for 10 min/wash. Incubation followed for 30 min at room temperature with either FITC-conjugated secondary antibody (Sigma) or rhodamine-conjugated secondary antibody (Chemicon). Nuclei were stained using DAPI. The slides were washed, mounted with the mounting medium (Sigma), and secured with rubber cement. Micrographs were acquired at a magnification of ×400 with a BX51 system equipped with DP70 (Olympus). The initial attempt resulted in inconclusive detection of endogenous protein localization. Therefore, cells were transfected with expression plasmids for analyzing the localization of each protein by immunofluorescent microscopy analysis. In addition, fluorescence signal intensities of proteins stained with either FITC or rhodamine do not correlate with their expression levels because the exposure time was optimized for clearer visualization of the stained protein. Immunoprecipitation—Preparation of nuclear extract has been described previously (26Park S.G. Ryu H.M. Lim S.O. Kim Y.I. Hwang S.B. Jung G. Gastroenterology. 2005; 128: 2042-2053Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). For immunoprecipitation by anti-NF-κB2(p52) antibodies, the nuclear extracts were diluted with dilution buffer at a final concentration of 20 mm HEPES, pH 7.9, 5% glycerol, 100 mm NaCl, 0.5% Nonidet P-40, and 1× protease inhibitor mix. This diluted nuclear extract was mixed with anti-NF-κB2(p52) antibodies for 2 h at 4°C. Then protein A-CL4B beads were added and further mixed for 2 h at 4°C. After the mixing, the beads were washed sequentially twice with ice-cold 1× PBS containing 0.5% Nonidet P-40 and twice with ice-cold 1× PBS. The bound proteins were eluted with 1× SDS sample buffer (10 mm Tris, pH 6.8, 5% glycerol, 2% SDS, 5% β-mercaptoethanol, and 0.05% bromphenol blue). Then eluted proteins were separated by 7.5% polyacrylamide gel electrophoresis and immunoblotted with anti-BCL-3 antibody. Data Analysis—Statistical analyses were performed using Student’s t test. p value of <0.01 was regarded as significant. Cyclin D1 and BCL-3 Are Up-regulated by HBx—It has been known that the NF-κB2(p52) homodimer associates with BCL-3 or HDAC1 to function as a transcriptional activator or inactivator, respectively (24Rocha S. Martin A.M. Meek D.W. Perkins N.D. Mol. Cell. Biol. 2003; 23: 4713-4727Crossref PubMed Scopus (202) Google Scholar). In this study, we observed whether HBx has an effect on the formation of the individual complexes. First, in Chang liver cells, changes in the expression level of endogenous proteins such as BCL-3, HDAC1, and NF-κB2(p52) due to HBx were detected by immunoblot analyses. Endogenous protein levels of HDAC1 and NF-κB2(p52) remained unchanged, whereas the levels of BCL-3 and cyclin D1 were up-regulated by HBx expression (Fig. 1A). The level of proteins was compared between HepG2 cells and HepG2.2.15 cells, which have 1.2× HBV genome for the production of the HBV particle. Endogenous protein levels of BCL-3 and cyclin D1 dramatically increased in HepG2.2.15 cells when compared with HepG2 cells (Fig. 1B). NF-κB2(p52) slightly increased. HBV might be responsible for this up-regulation of proteins in the HepG2.2.15 cells; in particular, long time exposure to HBV seems to be critical for the up-regulation of NF-κB2(p52) since the protein level is not increased in Chang liver cells transfected with pCMV-HA/HBx for 2 days. In general, the unprocessed form of NF-κB2(p100) is converted to the processed form of NF-κB2(p52) by a certain stimulus such as lymphotoxin-α. However, in both Chang liver cells and HepG2 cells, the unprocessed form of NF-κB2(p100) was not detected by our immunoblot analysis, the exact reason for which remains unclear. To determine the point of regulation, we looked at the mRNA level of BCL-3 and cyclin D1 in Chang liver cells. HBx expression up-regulates mRNA levels of BCL-3 and cyclin D1. (Fig. 1C). In addition, mRNA levels are also up-regulated in HepG2.2.15 cells when compared with those in HepG2 cells (Fig. 1D). This result indicates that HBx up-regulates BCL-3 and cyclin D1 at the mRNA level. HBx Up-regulates the NFκB2/BCL-3 Complex in the Nucleus—The effect of HBx on the localization of NF-κB2(p52), HDAC1, and BCL-3 was studied by immunofluorescent microscopy analysis. The localization of NF-κB2(p52) and HDAC1 was constant (Fig. 2, A and B), whereas the localization of BCL-3 was affected by HBx (Fig. 2C) in Chang liver cells. BCL-3 was translocated into the nucleus by HBx in Chang liver cells. This finding indicates that HBx affects the BCL-3 localization as well as the protein level. However, we were unable to see direct interaction between HBx and BCL-3, which means that HBx may indirectly affect BCL-3 protein localization (data not shown). Immunoprecipitation data show that the formation of the NF-κB2(p52)/BCL-3 complex in the nucleus increases due to HBx expression in Chang liver cells (Fig. 2D). Since IKK-β/DN inhibits HBx-mediated BCL-3 mRNA upregulation (Fig. 2E), the increased complex formation in the nucleus might be due to up-regulated endogenous BCL-3 by HBx dependent on IKK-β-mediated NF-κB activation (Fig. 2E). In addition, the level of NF-κB2(p52)/HDAC1 complex in nucleus is decreased, whereas the level of NF-κB2(p52)/BCL-3 complex in nucleus is increased in the HepG2.2.15 cells when compared with the HepG2 cells (Fig. 2F). From these data, it can be inferred that hepatitis B virus up-regulates the NF-κB2(p52)/BCL-3 complex formation in the nucleus through induction of BCL-3 nuclear localization and up-regulation of BCL-3 by HBx. During this process, HDAC1 might be discharged from NF-κB2(p52). HBx Activates Cyclin D1 Promoter through κB Site in Chang Liver Cells—Previous reports have shown that the NF-κB2(p52)/BCL-3 complex activates the cyclin D1 promoter through the κB site (23Westerheide S.D. Mayo M.W. Anest V. Hanson J.L. Baldwin Jr., A.S. Mol. Cell. Biol. 2001; 21: 8428-8436Crossref PubMed Scopus (153) Google Scholar, 24Rocha S. Martin A.M. Meek D.W. Perkins N.D. Mol. Cell. Biol. 2003; 23: 4713-4727Crossref PubMed Scopus (202) Google Scholar). As our results indicate that the NF-κB2(p52)/BCL-3 complex in the nucleus is increased by HBx, we tested whether HBx would activate the cyclin D1 promoter through the κB site on the cyclin D1 promoter. A reporter plasmid containing the cyclin D1 promoter was used. To analyze the role of the κB site on HBx-mediated cyclin D1 promoter activation, we used a minimal promoter (from -66 to +137) containing one κB site or a mutated κB site. In our experiments, the cyclin D1 promoter activation by HBx in Chang liver cells transfected with p973CD1LUC is higher than that in cells transfected with p66CD1LUC. This is thought to be caused by the existence of three κB sites in p973CD1LUC. The results exhibit that HBx activates the cyclin D1 promoter (Fig. 3A), and this activation is inhibited by the mutation of the κB site (Fig. 3B). This cyclin D1 promoter activation and inhibition by the mutated κB site also occur in HepG2.2.15 cells (Fig. 3, C and D). Cyclin D1 Up-regulation by HBx Is Mediated by NFκB2(p52) and BCL-3—In a previous study, the cyclin D1 promoter was shown to be activated by the NF-κB2(p52)/BCL-3 complex but inactivated by the NF-κB2(p52)/HDAC1 complex (24Rocha S. Martin A.M. Meek D.W. Perkins N.D. Mol. Cell. Biol. 2003; 23: 4713-4727Crossref PubMed Scopus (202) Google Scholar). Our aforementioned data indicate that HBx up-regulates the NF-κB2(p52)/BCL-3 complex in the nucleus and activates the cyclin D1 promoter through the κB site. Therefore, we proceeded to test whether NF-κB2(p52) and BCL-3 would play a role in the activation of the cyclin D1 promoter. The effect of overexpressed NF-κB2(p52) and BCL-3 on the cyclin D1 promoter activity was analyzed in Chang liver cells. Among four different NF-κB proteins, only NF-κB2(p52) overexpression activates the cyclin D1 promoter (Fig. 4, A and B). In addition, HBx-mediated cyclin D1 promoter activation is more enhanced by NF-κB2(p52) overexpression (Fig. 4C) or BCL-3 overexpression (Fig. 4D). Meanwhile, treatment with siRNA specific for BCL3 resulted in impaired HBx-induced cyclin D1 promoter activation (Fig. 4E). Moreover, when the HBx-mediated BCL-3 up-regulation was impaired by BCL-3 siRNA, up-regulation of endogenous cyclin D1 induced by HBx was reduced (Fig. 4F). This indicates that HBx increases the cyclin D1 level through up-regulation of BCL-3 protein. p53 Inhibits HBx-mediated Cyclin D1 Up-regulation in Chang Liver Cells—Overexpression of p53 down-regulates the HBx protein level by facilitating HBx degradation. 4S. G. Park, C. Chung, Y. M. Park, and G. Jung, unpublished results. Fig. 5A shows that high expression level of p53 induces down-regulation of HBx protein. Along with this, HBx-mediated up-regulation of endogenous BCL-3 and cyclin D1 was impaired by p53. However, levels of endogenous HDAC1 and NF-κB2(p52) remain constant. In addition, we also observed that the overexpression of p53 inhibits HBx-mediated cyclin D1 promoter activation (Fig. 5, B and C), which might be caused by down-regulated HBx. Our data indicate that the level of HBx affects the NF-κB2(p52)/BCL-3-mediated cyclin D1 up-regulation. HBx protein is known as a tumorigenic protein involved in HCC development and progression (31Murakami S. J. Gastroenterol. 2001; 36: 651-660Crossref PubMed Scopus (288) Google Scholar). Many reports have suggested that HBx activates a variety of signaling pathways including NF-κB activation, which is also related to HCC development and progression. In our study to reveal the detailed mechanism of the role of HBx in HCC, we used an expression system derived from the CMV promoter, which mimics the HBx-overexpressing cells frequently observed both in cancerous and in uncancerous regions of liver tissues (32Su Q. Schroder C.H. Hofmann W.J. Otto G. Pichlmayr R. Bannasch P. Hepatology. 1998; 27: 1109-1120Crossref PubMed Scopus (192) Google Scholar). In vitro analyses using HBx-overexpressing cell lines have shown that the overexpression of HBx increases its mobility associated with HCC progression. HBx down-regulates E-cadherin (33Lara-Pezzi E. Roche S. Andrisani O.M. Sanchez-Madrid F. Lopez-Cabrera M. Oncogene. 2001; 20: 3323-3331Crossref PubMed Scopus (81) Google Scholar, 34Lee J.O. Kwun H.J. Jung J.K. Choi K.H. Min do S. Jang K.L. Oncogene. 2005; 24: 6617-6625Crossref PubMed Scopus (163) Google Scholar) and up-regulates MMPs (35Chung T.W. Lee Y.C. Kim C.H. FASEB J. 2004; 18: 1123-1125Crossref PubMed Scopus (355) Google Scholar, 36Lara-Pezzi E. Gomez-Gaviro M.V. Galvez B.G. Mira E. Iniguez M.A. Fresno M. Martinez A.C. Arroyo A.G. Lopez-Cabrera M. J. Clin. Investig. 2002; 110: 1831-1838Crossref PubMed Scopus (173) Google Scholar), thereby enhancing the cell mobility and eventually increasing the malignant phenotype of HCC. Overexpression of cyclin D1 is frequently observed in cancer and is suggested as a good prognostic marker (19Fu M. Wang C. Li Z. Sakamaki T. Pestell R.G. Endocrinology. 2004; 145: 5439-5447Crossref PubMed Scopus (815) Google Scholar). Additionally, cyclin D1 overexpression is also involved in the progression of HCC (37Sato Y. Itoh F. Hareyama M. Satoh M. Hinoda Y. Seto M. Ueda R. Imai K. J. Gastroenterol. 1999; 34: 486-493Crossref PubMed Scopus (42) Google Scholar). Recent data suggest that HBx-mediated cyclin D1 up-regulation in mammary gland cells can induce tumors as well despite the differences between hepatocyte and mammary gland cells (18Klein A. Guhl E. Tzeng Y.J. Fuhrhop J. Levrero M. Graessmann M. Graessmann A. Oncogene. 2003; 22: 2910-2919Crossref PubMed Scopus (34) Google Scholar). In this report, we show that HBx up-regulates cyclin D1 in Chang liver cells (immortalized hepatocyte cell line) and HepG2.2.15 cells (HBV-producing cell line derived from hepatoblastoma cell line). That is, HBx can up-regulate cyclin D1 in hepatocyte and hepatoma cells. It has been well established that HBx activates NF-κB, which leads to the induction of many genes (26Park S.G. Ryu H.M. Lim S.O. Kim Y.I. Hwang S.B. Jung G. Gastroenterology. 2005; 128: 2042-2053Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 38Park S.G. Lee T. Kang H.Y. Park K. Cho K.H. Jung G. FEBS Lett. 2006; 580: 822-830Crossref PubMed Scopus (49) Google Scholar, 39Su F. Schneider R.J. J. Virol. 1996; 70: 4558-4566Crossref PubMed Google Scholar, 40Lee S.H. Park S.G. Lim S.O. Jung G. Biochim. Biophys. Acta. 2005; 1741: 75-84Crossref PubMed Scopus (12) Google Scholar). However, no previous study has identified the specific NF-κB complexes and target genes activated by HBx among the individual NF-κB complexes with different DNA recognition sequences (17Ghosh G. Huang D.B. Huxford T. Curr. Opin. Struct. Biol. 2004; 14: 21-27Crossref PubMed Scopus (37) Google Scholar). Our study shows that HBx activates the NF-κB2(p52)/BCL-3 complex through up-regulation and nuclear translocation of BCL-3. Our data indicate that general IKK-β-dependent NF-κB activation by HBx induces BCL-3 mRNA up-regulation and that BCL-3 nuclear translocation is induced by HBx. As a result, the level of NF-κB2(p52)/BCL-3 complex is increased in the nucleus, leading to the up-regulation of cyclin D1. Generally, NF-κB proteins form a homo- or heteodimer complex functioning as a transcription factor. However, a homodimer complex of NF-κB1(p50) and NF-κB2(p52) can form complexes with other proteins such as CREB-binding protein/p300, HDAC1, and BCL-3 (24Rocha S. Martin A.M. Meek D.W. Perkins N.D. Mol. Cell. Biol. 2003; 23: 4713-4727Crossref PubMed Scopus (202) Google Scholar, 41Zhong H. May M.J. Jimi E. Ghosh S. Mol. Cell. 2002; 9: 625-636Abstract Full Text Full Text PDF PubMed Scopus (816) Google Scholar, 42Nolan G.P. Fujita T. Bhatia K. Huppi C. Liou H.C. Scott M.L. Baltimore D. Mol. Cell. Biol. 1993; 13: 3557-3566Crossref PubMed Google Scholar). In the absence of stress, the NF-κB2(p52) homodimer binds to BCL-3 or HDAC1, which determines the function of the complex to be either a transcriptional activator or a repressor (24Rocha S. Martin A.M. Meek D.W. Perkins N.D. Mol. Cell. Biol. 2003; 23: 4713-4727Crossref PubMed Scopus (202) Google Scholar). In our experiments, the cyclin D1 promoter is activated by the overexpression of NF-κB2(p52) and BCL-3 but repressed by the overexpression of HDAC1, which means that the NF-κB2(p52)/BCL-3 complex activates the cyclin D1 promoter. Although overexpression of either NF-κB2(p52) or BCL-3 with HBx expression intensifies the HBx-mediated cyclin D1 promoter activation, HDAC1 overexpression inhibits the HBx-mediated cyclin D1 promoter activation. In conclusion, HBx-mediated BCL-3 up-regulation induces complex formation of NF-κB2(p52)/BCL-3, which triggers the discharge of HDAC1 from NF-κB2(p52). Moreover, the nuclear localization of BCL-3 is induced by HBx. Therefore, the up-regulation and nuclear localization of BCL-3 by HBx can increase the NF-κB2(p52)/BCL-3 complex in the nucleus, leading to the up-regulation of cyclin D1 through activation of the cyclin D1 promoter (Fig. 6). Recent reports provide a clue on the nuclear localization of BCL-3 during activation: polyubiquitination of BCL-3. In our results, overexpression of BCL-3 alone can activate cyclin D1 promotor, whereas the activation is synergistically enhanced by HBx expression. This indicates that the nuclear localization of BCL-3 by HBx plays a role in HBx-mediated cyclin D1 up-regulation. Whether the suggested modification of BCL-3 is responsible for the translocation requires further investigation. In this study, we identified a detailed mechanism of HBx-mediated cyclin D1 up-regulation. (i) HBx up-regulates BCL-3 at the mRNA level, which is mediated by IKK-β-dependent NF-κB activation, (ii) HBx facilitates the nuclear localization of BCL-3, and (iii) this consequentially up-regulates the NF-κB2(p52)/BCL-3 complex and down-regulates the NF-κB2(p52)/HDAC1 complex in the nucleus. In conclusion, HBx activates the NF-κB2(p52)/BCL-3 complex, and its specific targets, such as cyclin D1, are up-regulated. Our study is a crucial progress in further understanding the mechanisms of HBV-mediated HCC development and progression. We thank K. H. Kim, J. Cheong, and Y. D. Yoo for the critical reading of the manuscript. We thank Y. K. Park, R. Pestell, J. B. Kim, Y. J. Bang, and Tularik, Inc. for providing the plasmids. We also thank Y. Yun for the anti-HBx antibody." @default.
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• W4235187828 title "Up-regulation of Cyclin D1 by HBx Is Mediated by NF-κB2/BCL3 Complex through κB Site of Cyclin D1 Promoter" @default.
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