HBx Protein Induces Expression Of MIG And Increases Migration Of Leukocytes Through Activation Of NF-κB

Objective1. To investigate the effect of HBV on the MIG expression.2. To investigate the molecular mechanism of MIG upregulation induced by HBx.3. To investigate whether HBx-induced MIG could increase migration of peripheral bloodlymphocytes.Methods一. HBV induces MIG expression in hepatocytes.1. The plasmid pBlue-HBV containing 1.3-fold HBV genome were transfected into HepG2cells, and the protein levels of MIG in the supernatants were measured 48 h after thetransfectioin by ELISA.2. Next, we transfected a series of plasmids containing individual seven viral genes of HBV(pCMV-S, pCMV-preS1, pCMV-preS2, pCMV-HBe, pCMV-HBc, pCMV-HBx orpCMV-HBp) or control plasmids (pCMV-tag2) into HepG2 cells, respectively.3. To determine whether protein encoded by HBV induces MIG gene transcription, aconstruct containing the sequence from -983 to +33 (relative to the transcriptional start site)of 5'-flanking region of the human MIG gene was co-tranfected with individual genes ofHBV or control plasmids, respectively, into HepG2 cells. 4. We next investigated the dose-dependence of HBx on MIG promoter activity in HepG2cells. HepG2 cells were co-transfected with the MIG promoter reporter plasmids andincreased concentration of pCMV-HBx plasmids, and then the luciferase activity wasmeasured.二. The molecular mechanism of HBx-induced MIG expression.1. The reporter constructs containing serial 5' deletions of the MIG promoter wereco-transfected with pCMV-HBx, respectively, into HepG2 cells, and luciferase activity wasmeasured 48 h after the transfection.2. To investigate the role of the two NF-κB binding sites on MIG activation, these twoNF-κB sites (NF-κB1, and NF-κB2) and a C/eBP site near the NF-κB2 were mutated bymultiple rounds of PCR.3. To determine the roles of NF-κB in the HBx-induced activation of MIG promoter,pCMV-HBx and (-983/+33)MIG were co-transfected into HepG2 cells and then the cellswere treated with MG-132 (an NF-κB inhibitor) at different concentrations.4. NF-κB is composed of homo- and heterodimers of Rel family members, typicallyp65:p50 heterodimers. To investigate the effect of p50 and p65 on the transcriptionalactivation of MIG, HepG2 cells were co-transfected with plasmids expressing p65 or p50and the reporter plasmids (-495/+33)MIG or the mutant plasmids NF-κB2 Mut, and MIGpromoter activities were measured by luciferase reporter assay. We examined the effect ofHBx protein on the translocation of p65 from cytosol to nucleus by transfection of HepG2cells with pCMV-HBx. At 0, 24, and 48 h post-transfection, protein levels of p65 in thecytosol and nucleus fractions were analyzed by Western blot using anti-p65 antibody.5. To determine whether NF-κB binds to the MIG promoter after transfection withpCMV-HBx, EMSA was performed using a biotin-labeled NF-κB consensusoligonucleotides in the MIG promoter (-149 to -127) as a probe. HepG2 cells weretransfected with control plasmids or pCMV-HBx, and the nuclear extracts were incubated with the biotin-labeled probe at room temperature. To determine the specificity of NF-κBbinding activity, nuclear extracts were incubated with the labeled NF-κB consensusoligonucleotides in the presence of either an unlabeled wild type NF-κB binding probe or amutated probe. We confirmed the authenticity of the NF-κB band using supershift assay byincubation of nuclear extracts with antibody against p50, p65, p52, c-Rel or RelB. We nextperformed a ChIP assay to determine whether NF-κB binds to the MIG promoter in vivo.Chromatin fragments were prepared from HepG2 cells transfected with pCMV-HBx andimmunoprecipitated with antibodies against either the p50 or the p65 subunit. A 156-bpDNA fragment containing the NF-κB binding site in MIG promoter was amplified by PCRusing specific primers.三. The effect of HBx-induced MIG on the migration of PBLs.1. To assess the functionality of the induced MIG protein, we performed migration assayusing activated PBLs. HepG2 cells were transfected with pCMV-HBx or pCMV-Tag2, andchemotatic activity of supernatants were determined.2. To determine whether NF-κB was involved in the migration effect, pCMV-HBx wastransfected into HepG2 cells and then the cells were treated with MG-132 (an NF-κBinhibitor).3. To investigate the effect of p50 and p65 subunits of NF-κB in the migration effect,HepG2 cells was transfected with pCMV-p50 or pCMV-p65, or were co-transfected withpCMV-p50 and pCMV-p65.4. To assess the functionality of the induced MIG protein, HepG2 cells were transfectedwith plasmids expressing HBx, p50, or p65, with or without treatment anti-MIG antibody,and chemotatic activity of supernatants were determined.Results一. HBV induces MIG expression in hepatocytes.1. It was found that MIG protein levels significantly increased in cells transfected with pBlue-HBV compared with the cells transfected with control plasmids.2. It was found that MIG protein levels increased in cells transfected with pCMV-HBx,while the rest of the constructs for viral proteins (S, preS1, preS2, HBe, HBc, P) had nosignificant effects on MIG protein expression.3. MIG promoter activity was significantly activated by HBx protein.4. It was found that the levels of luciferase activity increased as the concentration ofplasmids pCMV-HBx increased, indicating that the activation of MIG promoter by HBxprotein was concentration-dependent.二. The molecular mechanism of HBx-induced MIG expression.1. Deletion from nt -983 to -185 did not affect HBx-induced luciferase activity. Furtherdeletion to nt -129 significantly decreased HBx-induced luciferase activity. These dataindicated that the sequence between nt -185 and -129 was critical for the activation of MIGpromoter by HBx protein.2. Analysis of the promoter of MIG gene revealed that there were two NF-κB binding siteson these regions, mutation of the NF-κBl and C/eBP binding sites did not significantlyaffect HBx-induced promoter activity, while mutation of NF-κB2 significantly abolishedHBx-induced MIG activation.3. Luciferase activity assay revealed that the MIG promoter activity was gradually inhibitedas the concentrations of MG-132 were increased, indicating that NF-κB was required forthe HBx-induced activation of MIG promoter.4. Luciferase activity assays showed that the transcriptional activity of MIG was elevated inthe presence of p50 or p65 protein when compared to that of control. The transcriptionalactivity of MIG reached the highest level when both p50 and p65 protein were present.However, the mutation of NF-κB2 binding site of MIG promoter abolished the effect ofp50 and/or p65 protein on the activation of the MIG promoter.Results clearly revealed that the protein levels of p65 decreased in cytosol and increased innucleus as the time of transfection increased in cells transfected with pCMV-HBx. However, the protein levels of p65 were relatively unchanged in both cytosol and nucleusas the time of transfection increased in cells transfected with pCMV-Tag2.5. The DNA binding activity of NF-κB was significantly increased in the cells transfectedwith pCMV-HBx, compared with the HepG2 cells transfected with control plasmids, thewild type NF-κB consensus oligonucleotides, but not the mutated oligonucleotidessignificantly abrogated NF-κB complexes. Incubation of the nuclear extracts with theantibody either the p50 or the p65 supershifted the specific retardation signal, suggestingthat the band represented a p65/p50 heterodimer. We also examined the effect of MG-132on HBx-induced NF-κB DNA binding activity. The DNA binding activity of NF-κB wassignificantly inhibited by the treatment with MG-132. PCR products were only producedfrom DNA isolated from cells transfected with pCMV-HBx in the presence of anti-p50 oranti-p65 antibody, but they were not detected in the presence of control plasmids andcontrol antibody.三. The effect of HBx-induced MIG on the migration of PBLs.1. After cells were transfected with pCMV-HBx, the chemotatic activity increased 5.19 fold,compared with cells transfected with control plasmid pCMV-Tag2.2. Treatment with NF-κB inhibitor MG-132 after transfection with pCMV-HBxsignificantly decreased the chemotactic activity.3. When cells were transfected with pCMV-p50 and pCMV-p65, the chemotatic activityincreased 3.2 and 4.0 fold, respectively, compared with control. Moreover, when cells wereco-transfected with pCMV-p50 and pCMV-p65, the chemotatic activity increased 6.9 fold.4. In response to supernatants from cells transfected with plasmids expressing HBx, p50, orp65, the migratory activity of PBLs was substantially increased. Interestingly, this activitywas significantly decreased upon the addition of neutralizing anti-MIG antibody to thesupernatants of transfected HepG2 cells, whereas a control antibody had little effect onmigration. Conclusion1. HBV induces MIG gene expression in hepatocellular carcinoma cells.2. HBx induces translocation of NF-κB subunits into the nucleus, which binds to theNF-κB2 binding element of MIG promoter and enhances MIG promoter activity.3. HBx-induced MIG increases migration of peripheral blood lymphocytes.