NF-κB Activation Induced By MHBs^t167/HBx And Its Possible Mechanisms During Renal Epithelial Cell HK-2 Injure

BackgroudHepatitis B virus (HBV) infection is common in china, which can causehepatitis B virus associated glomerulonephritis (HBVGN). Clinical feature ofHBVGN is characterized by nephritic syndrome and its major morphologicalchange is atypical membranous glomerulonephritis.The pathogenesis of HBVGN has not been clearly defined, but three majortheories may be included: immune complex deposition, specific immunologicaleffect damage and direct viral-induced injure. The first two theories have beenwildly accepted. Some studies observed that there were HBV antigens in renaltissue, HBV-DNA and RNA in glomerular and tubular epithelial cells, and eventhe complete viral particles or resembling viral particles in renal tissue. Moreover,the existence of HBV-DNA was closely related with the duration of proteinuria. Itsuggests that direct viral-induced pathological alterations might be involved inHBVGN, but it is still lack of direct evidences for its pahtogenetic mechanism.Nuclear transcription factor-kappa B (NF-κB) is an important pleiotropictranscription factor, which can regulate a lot of gene expression and participate inmany signal transductions. In quiescent cells, NF-κB is sequestered in the cytoplasm through its interaction with the inhibitor of nuclear factor kappa B(IκB). It can be stimulated by various factors, then the inhibitory units may bedegraded and NF-κB transfers from cytoplasm to nucleus, where it binds tospecial promoters (κB-binding sites) and initiates gene transcription. It may beinvolved in the processes of inflammation, proliferation, extracellular matrixcrosslinking and cell apoptosis. Excessive NF-κB activation is closely relatedwith human renal diseases, and may evoke the renal inflammatory damage.In hepatic studies, the C-terminally truncated middle size surface proteins(MHBst) and HBx proteins are shown to act as a transcriptional transactivator andcan stimulate some transcription factors (such as NF-κB and AP-1). Although themechanisms about NF-κB activation induced by MHBst/HBx have not been fullyunderstood, some studies show that it may be related with protein kinase C (PKC)activation and Ras/Raf/MAPK pathway.Previously, we have firstly reported that there was nuclear translocation ofNF-κB in HBVGN renal tissue, especially in renal tubular epithelial cells.Whether is NF-κB activation also induced by MHBst/HBx in renal cells or not?How about its possible mechanism? What effect does the NF-κB activation haveon renal cells? Those studies have not been reported in any literatures and mayprovide laboratory evidences for HBV direct viral-induced renal injure inHBVGN.Objective1, Investigate whether NF-κB activation was related with MHBst¹⁶⁷/HBx, and itsrelationship with renal tubular epithelial cellular apoptosis.2, Study the possible mechanisms that MHBst¹⁶⁷/HBx induce NF-κB activationin renal tubular epithelial cells.Methods 1, Construct the pcDNA3.1(+)MHBst¹⁶⁷ and pcDNA3.1(+)HBx vectors byamplifying specific MHBst¹⁶⁷ and HBx fragments from p1.2II plasmid (HBV,adr serotype) with PCR method.2, After human renal proximal tubular epithelial cells HK-2 were transientlytransfected by MHBst¹⁶⁷ and/or HBx, the NF-κB activation level was detectedby immunocytochemistry, Western blot and EMSA. In addition, a specificNF-κB inhibitor pyrrolidine dithiocarbamate (PDTC, 30μM) was used asexperimental control.3, After MHBst¹⁶⁷ or/and HBx transfection (with or without PDTC treatment),HK-2 cellsíapoptosis index was assessed by Hochest33258 stainning and itsrelationship with NF-κB activation was analyzed by Pearson correlationanalysis.4, Primarily study the mechanism about NF-κB activation induced byMHBst¹⁶⁷/HBx in HK-2 cells:(1) The reporter plasmid pGL3-p-(κB)5 contained five repeatedκB-binding sitewas constructed and theκB-dependent transcription (including self-regulationof NF-κB) was detected by dual luciferase reporter assay (DLR).(2) After HK-2 cells transfected with MHBst¹⁶⁷ or/and HBx (with or withoutspecific PKC inhibitor G? 6983 treatment), PKC/Raf/ERK pathway and itsrelationship with NF-κB activation were analyzed by PKC kinase activityassay, immunoprecipitation and Western blot.Results1, The eukaryotic expression vectors pcDNA3.1(+)MHBst¹⁶⁷ andpcDNA3.1(+)HBx were constructed successfully. The target genes wererespectively identical to the published HBV genome sequences AF052576.1and AY123424.1. 2, Increase of NF-κB translocation and phospho-IκBαprotein expression inHK-2 cells with MHBst¹⁶⁷ or/and HBx transfection.(1) Immunocytochemisty results: The nuclear NF-κBp65 positive ratio ofMHBst¹⁶⁷, HBx and MHBst¹⁶⁷+HBx transfected groups were 12.58%±0.543%, 22%±0.477% and 30.76%±0.49% respectively, which were allmore significantly increased than pcDNA3.1(+) transfected group (P<0.05).Moreover, MHBst¹⁶⁷+HBx co-transfected group was increased moresignificantly than MHBst¹⁶⁷ or HBx transfected alone (P<0.05) and thenuclear NF-κBp65 positive ratios were inhibited after PDTC treatment(P<0.05).(2) Western blot results:a) Compared with pcDNA3.1(+) group, NF-κBp65 level in nucleus ofMHBst¹⁶⁷, HBx and MHBst¹⁶⁷+HBx transfected groups were significantlyincreased to 1.8-fold, 1.9-fold and 2.1-fold respectively (P<0.05), and theNF-κBp65 and NF-κBp50 expression in cytosol were also significantlyincreased to 1.9².9 folds (P<0.05); furthermore, the cytosolic andnuclear NF-κB proteins were increased more markedly in theMHBst¹⁶⁷+HBx co-transfected group than MHBst¹⁶⁷ or HBx transfectedgroups (P<0.05), and PDTC also could inhibit the increased NF-κBproteins expression (P<0.05).b) In cellular total extracts, there was no significant difference of IκBαprotein expression in all transfection groups (P>0.05) and IκBαproteinswere increased slightly after PDTC treatment; in comparison with emptyvector transfected group, phospho-IκBαprotein of MHBst¹⁶⁷, HBx andMHBst¹⁶⁷+HBx transfected groups were increased to 1.44-fold, 1.52-foldand 2.03-fold (P<0.05), the MHBst¹⁶⁷+HBx co-transfected group was increased more markedly than MHBst¹⁶⁷ or HBx transfected groups(P<0.05), and the expression of phosphor-IκBαprotein could be inhibitedby PDTC (P<0.05); the same results were shown that total NF-κBp65and NF-κBp50 proteins of MHBst¹⁶⁷ or/and HBx transfected groups werealso increased than that of the pcDNA3.1(+) transfected group (P<0.05),the MHBst¹⁶⁷+HBx co-transfected group was increased more obviouslythan MHBst¹⁶⁷ or HBx transfected groups (P<0.05) and all groups couldbe inhibited by PDTC (P<0.05).3, Enhancement ofκB-specific DNA binding ability with MHBst¹⁶⁷ or/andHBx transfection in HK-2 cells. The nuclearκB-specific DNA-proteincomplexes of MHBst¹⁶⁷, HBx and MHBst¹⁶⁷+HBx transfected groups weresignificantly increased (P<0.05), which were 1.7-fold, 3.4-fold and 5.9-foldrespectively compared with pcDNA3.1(+) transfected group, and theMHBst¹⁶⁷+HBx co-transfected group was significantly higher than MHBst¹⁶⁷ orHBx transfected alone (P<0.05). PDTC also could reduce theκB-specificDNA-protein complex in all transfected groups (P<0.05).4, Increased apoptosis index in HK-2 cells with MHBst¹⁶⁷ or/and HBxtransfection. Compared with pcDNA3.1(+) transfected (3.17%), the apoptoticindex of MHBst¹⁶⁷, HBx and MHBst¹⁶⁷+HBx transfected groups weresignificantly increased after MHBst¹⁶⁷(8.18%) or HBx (10.23%) transfectedalone, and MHBst¹⁶⁷+HBx co-transfected (13.98%) (P<0.05). The apoptosisindex of MHBst¹⁶⁷+HBx co-transfected group was significantly higher than thatof MHBst¹⁶⁷ or HBx transfected alone (P<0.05). After treated with PDTC, theapoptosis index of all transfected groups was significantly decreased (P<0.05).A correlation statistic analysis showed that the apoptosis index of HK-2 cellswas consistent with the nucleus NF-κBp65 protein level (r=0.841, P<0.05) andtheκB-specific binding ability (r=0.894, P<0.05). 5, Increase ofκB-controlled genes transcription after MHBst¹⁶⁷/HBxtransfection. The luciferace activity in MHBst¹⁶⁷ or/and HBx plasmidtransfected groups were increased significantly more than that in equal amountof pcDNA3.1(+) transfected group (P<0.05), and the MHBst¹⁶⁷+HBxco-transfected group increased more highly than the MHBst¹⁶⁷ or HBxtransfected alone (P<0.05); meanwhile, the DLR results showed there was adose-dependent manner to MHBst¹⁶⁷ or/and HBx (P<0.05), but not atime-dependent manner included (P>0.05). In addition, the transcriptionprocess could also be inhibited by PDTC treatment (P<0.05).6, The effect of MHBst¹⁶⁷/HBx on the PKC/Raf/ERK pathway in HK-2 cells.(1) In comparison with empty vector transfection, the PKC kinase activity wasslightly increased after MHBst¹⁶⁷ or/and HBx transfection (P<0.05), andcould be inhibited by G? 6983 (P<0.05).(2) There were no c-raf-1-ERK1/2 complexes formulation in HK-2 cells afterMHBst¹⁶⁷ or/and HBx transfection.(3) After MHBst¹⁶⁷ or/and HBx transfection (with or without G? 6983treatment), the Raf-1 protein in all groups was negative, which wasconsistent with the immunoprecipitation results. The expression of ERK2protein in all transfected groups was relatively uniform, without significantdifference (P>0.05). After MHBst¹⁶⁷ or/and HBx transfected, theexpression of NF-κBp65 and phospho-ERK proteins were increased incomparison with empty vector transfected group (P<0.05), and theMHBst¹⁶⁷+HBx co-transfected group increased more markedly than theMHBst¹⁶⁷ or HBx transfected alone (P<0.05). After G? 6983 treatment theexpression of NF-κBp65 and phospho-ERK proteins were decreased.Conclusions1, After human renal proximal tubular epithelial cells HK-2 were transfectedwith MHBst¹⁶⁷ or/and HBx, NF-κB protein was activated, which exhibited as the increase of phospho-IκB protein, NF-κB protein nuclear translocation,nuclearκB-site binding ability andκB-controlled genes transcription. Thelevel of NF-κB activation was closely related with HK-2 apoptosis and PDTCcould effectively inhibit those processes.2, There might also be a synergistic effect of MHBst¹⁶⁷ and HBx in the processof NF-κB activation and tubular cells apoptosis.3, In HK-2 cells, the mechanisms about NF-κB activation induced byMHBst¹⁶⁷/HBx might be as the followings:(1) Through the increase of cellular PKC kinase activity, the phospho-ERKprotein level (independent of Raf pathway) and the degeneration of IκBprotein, NF-κB was activated and translocated to nucleus.(2) On the other hand, after NF-κB protein activated by MHBst¹⁶⁷/HBx,NF-κB protein could also be transactivated and up-regulated by itself.