The hepatitis B virus X protein activates nuclear factor of activated T cells (NF-AT) by a cyclosporin A-sensitive pathway

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The hepatitis B virus X protein activates nuclear factor of activated T cells (NF-AT) by a cyclosporin A-sensitive pathway
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  The EMBO Journal  Vol.17 No.23 pp.7066–7077, 1998 The hepatitis B virus X protein activates nuclearfactor of activated T cells (NF-AT) by a cyclosporinA-sensitive pathway Enrique Lara-Pezzi, Angel Luis Armesilla 1 ,Pedro L.Majano 2 , Juan Miguel Redondo 1 and Manuel Lo´ pez-Cabrera 3 Unidades de Biologı´a Molecular y  2 Hepatologı´a, Hospital de laPrincesa, Universidad Auto´noma de Madrid, 28006 Madrid and 1 Centro de Biologı´a Molecular, Consejo Superior de InvestigacionesCientı´ficas (CSIC), Cantoblanco, 28049 Madrid, Spain 3 Corresponding authore-mail: mlcabrera/princesa@hup.es The X gene product of the human hepatitis B virus(HBx) is a transcriptional activator of various viraland cellular genes. We recently have determined thatthe production of tumor necrosis factor- α   (TNF- α  )by HBV-infected hepatocytes is transcriptionally up-regulated by HBx, involving nuclear factor of activatedT cells (NF-AT)-dependent activation of the TNF- α  gene promoter. Here we show that HBx activates NF-AT by a cyclosporin A-sensitive mechanism involvingdephosphorylation and nuclear translocation of thetranscription factor. Luciferase gene expression assaysdemonstrated that HBx transactivates transcriptionthrough NF-AT-binding sites and activates a Gal4–NF-AT chimeric protein. DNA–protein interaction assaysrevealed that HBx induces the formation of NF-AT-containing DNA-binding complexes. Immunofluores-cence analysis demonstrated that HBx induces thenuclear translocation of NF-AT, which can be blockedby the immunosuppressive drug cyclosporin A. Fur-thermore, immunoblot analysis showed that the HBx-induced activation and translocation of NF-AT areassociated with its dephosphorylation. Thus, HBx mayplay a relevant role in the intrahepatic inflammatoryprocesses by inducing locally the expression of cyto-kines that are regulated by NF-AT. Keywords : cyclosporin A/HBx/hepatitis B virus/NF-AT/ transcription Introduction The hepatitis B virus (HBV) is a hepatotrophic viruscomposed of a partially double-stranded circular DNAgenome that causes acute and chronic hepatic injury.Persistent HBV infection is strongly associated with thedevelopment of hepatocellular carcinoma (Ganem andVarmus, 1987). Four genes, S/preS, C/preC, P and X, areencoded by the viral genome (Tiollais  et al ., 1985). TheX gene encodes a 17 kDa protein, termed HBx, that hasbeenshowntofunctionasatranscriptionaltransactivatorof a variety of viral and cellular promoter/enhancer elements(reviewed in Yen, 1996).HBx does not bind directly to DNA, but it is ableto transactivate transcription through multiple  cis -acting 7066  © Oxford University Press elements including AP-1, AP-2, ATF/CREB, NF- κ  B,C/EBP and Egr-1-binding sites (Maguire  et al ., 1991;Kekule´  et al ., 1993; Yoo  et al ., 1996). However, the exactmechanism of transactivation still remains unresolved. Ithas been determined that HBx interacts in the nucleuswith components of the basal transcription machinery,including RPB5, a subunit of all three mammalian RNApolymerases, and several transcription factors (Maguire et al ., 1991; Cheong  et al ., 1995; Qadri  et al ., 1995; Haviv et al ., 1996, 1998; Lin  et al ., 1997). Thus, HBx may exertits effect by mimicking the cellular coactivator function(Haviv  et al ., 1996). Another proposed mechanism forHBx activity involves the activation of signal transductionpathways such as the Ras/Raf/ERK and MEKK-1/JNKcascades, leading to induction of AP-1, NF- κ  B and prob-ably other transcription factors (Benn and Schneider, 1994;Natoli  et al ., 1994b; Doria  et al ., 1995; Benn  et al ., 1996;Su and Schneider, 1996; Klein and Schneider, 1997).Whether protein kinase C (PKC) is involved in the signaltransduction pathways activated by HBx is less clear(Cross  et al ., 1993; Kekule´  et al ., 1993; Benn andSchneider, 1994; Murakami  et al ., 1994; Natoli  et al .,1994a; Chirillo  et al ., 1996). HBx has been found to bedistributed in the cytoplasm, but also to some extent inthe nucleus of transfected cells (Doria  et al ., 1995; Haviv et al ., 1998). Thus, HBx may have a dual function, onerelated to its cytoplasmic localization, that can mediatethe activation of signal transduction pathways, and anothernuclear, that may account for the interaction with transcrip-tion factors and components of the transcription apparatusto enhance the binding or activity of these proteins (Doria et al ., 1995).Although there is emerging evidence of the involvementof HBx in hepatocarcinogenesis (Koike, 1995), very littleis known about the role that this viral protein plays in theintrahepatic inflammatory processes. In this context, wepreviously have reported the production of the pro-inflammatory cytokine tumor necrosis factor- α  (TNF- α )by hepatocytes from patients chronically infected by HBV.We also demonstrated that transient or stable transfectionof the hepatoma cell line HepG2 with either the wholeHBV genome or HBx expression vectors resulted inTNF- α  production (Gonza´lez-Amaro  et al ., 1994). Inaddition, it has been reported that the gene encodinghuman interleukin 8 (IL-8) is also transactivated by HBx(Mahe´  et al ., 1991). We have shown recently that HBx-induced TNF- α  production by hepatocytes is regulated atthe transcriptional level involving the activation of nuclearfactor of activated T cells (NF-AT) (Lara-Pezzi  et al .,1998).The expression of TNF- α , IL-8 and other cytokine-encoding genes is regulated in a co-ordinate manner bythe transcription factor NF-AT in cells of the immunesystem (Okamoto  et al ., 1994; Tsai  et al ., 1996; Rao  et al .,  Activation of NF-AT by HBx 1997). Therefore, NF-AT is required for initiating andcontrolling effective immune and inflammatory responses.NF-AT is a family of transcription factors that includes atleast four structurally related proteins; NF-AT1 (previouslynamed NF-ATp), NF-ATc, NF-AT3 and NF-AT4. Multipleisoforms and species-specific variants of these proteinshave also been identified. Although NF-AT proteins arenot expressed exclusively by cells of the immune system(reviewed in Rao  et al ., 1997), little information isavailable regarding the expression and function of NF-AT-related proteins outside the immune system (Rao, 1994;Rao  et al ., 1997).The activity of NF-AT proteins is tightly regulated bythe calcium/calmodulin-dependent phosphatase calci-neurin (Sigal and Dumont, 1992; Jain  et al ., 1993; Crabtreeand Clipstone, 1994; Cantrell, 1996; Loh  et al ., 1996), aprimary target for inhibition by the immunosuppressivedrugs cyclosporin A (CsA) and FK506 (Schreiber andCrabtree, 1992; Shaw  et al ., 1995). Calcineurin controlsthe translocation of NF-AT proteins from the cytoplasmto the nucleus of activated cells by interacting with anN-terminal regulatory domain conserved in all the mem-bers of the NF-AT family (Luo  et al ., 1996b). NF-ATproteins are able to bind cooperatively with transcriptionfactors of the AP-1 family to form composite NF-AT:AP-1 sites (Jain  et al ., 1993), which are found in the regulatoryelements of many genes that are transcribed inducibly bycells of the immune system (Boise  et al ., 1993; Cockeril et al ., 1995; Jain  et al ., 1995; Rooney  et al ., 1995).In this report, we demonstrate that HBx transactivatestranscription through NF-AT-binding sites in liver-derivedChang (CHL) cells, and that a chimeric Gal4–NF-ATprotein, containing the N-terminal transactivation domainof NF-AT1 fused to the Gal4 DNA-binding domain (DBD)is activated by HBx. We also show that HBx induces, inCHL cells, NF-AT-containing protein complexes that bindindependently of AP-1 to an NF-AT site of the murineIL-4 promoter. Furthermore, we demonstrate that HBxtriggers dephosphorylation and nuclear translocation of NF-AT by a CsA-sensitive mechanism. Results Transactivation of NF-AT-dependent transcription by HBx  To analyze whether HBx was able to transactivate trans-cription through NF-AT-binding sites, CHL cells weretransiently co-transfected with the HBx expression vectorpSV-X along with a luciferase reporter plasmid driven bythree tandem copies of the distal NF-AT site of the IL-2enhancer (pNF-AT-Luc), which has been reported to bindNF-AT cooperatively with transcription factors of theAP-1 family (Boise  et al ., 1993; Northrop  et al ., 1993;Jain  et al ., 1995). As shown in Figure 1A, the expressionof HBx induced the NF-AT-dependent transcription 4- to6-fold. Moreover, in the presence of either the mitogenphorbol 12-myristate 13-acetate (PMA) or the calciumionophore A23187, HBx further induced (11- to 14-fold)NF-AT transcriptional activity, leading to luciferaseexpression levels similar to those obtained by co-stimula-tion with PMA plus the calcium ionophore, which providesa full stimulus for NF-AT-dependent transcription (Rao et al ., 1997). In contrast, PMA or calcium ionophore alone 7067 exerted a weakeffect on the inductionof the transcriptionalactivity of the NF-AT enhancer, thus suggesting that HBxmay compensate both the mitogenic signals induced byPMA and the calcium signals induced by calcium iono-phore in the activation of NF-AT sites. Strikingly, HBxfurther increased the induction of the NF-AT enhancerby co-stimulation with PMA plus calcium ionophore(Figure 1B). A dominant-negative version of NF-AT,which lacks the DNA-binding domain and the C-terminaltransactivation domain (Northrop  et al ., 1994), was ableto prevent the induction by HBx, either alone or in thepresence of PMA and/or calcium ionophore (Figure 1Aand 1B). In addition, CsA, a pharmacological inhibitor of thephosphatasecalcineurinthatcontrolsNF-ATactivation,alsoblockedtheinductionbyHBxaswellasthesynergisticinduction by HBx and PMA plus calcium ionophore(Figure 1C), suggesting that activation of NF-AT proteinswas mediating the induction of the NF-AT enhancerelement by HBx.The synergism of HBx and PMA plus the calciumionophore was not due merely to an increase in HBxexpression levels, since these agents did not induce signi-ficantly the activity of the promoter/enhancer elementsdriving the expression of HBx within the plasmid pSV-X(data not shown). Therefore, distinct mechanisms mightbe mediating the synergistic activation of the NF-AT-dependent transcription by HBx. One such mechanismcould be a further increase by HBx of the PMA-inducedtranscriptional activity of AP-1, which binds cooperativelywith NF-AT to the NF-AT enhancer element. To addressthis issue, CHL cells were co-transfected with pSV-Xvector and the reporter plasmid pAP-1-Luc, containingfour AP-1-binding sites, and the transfected cells wereeither left untreated or stimulated with PMA or PMA pluscalcium ionophore. As shown in Figure 1D, HBx furtheractivated the AP-1-dependent transcription induced byPMA (not shown) and by PMA plus calcium ionophore.However, the activation of AP-1 transcriptional activityby HBx, either alone or in the presence of PMA orPMA plus calcium ionophore, was not blocked by CsA(Figure 1D, and data not shown). Thus, the synergisticinduction of the NF-AT enhancer element by HBx andPMA plus calcium ionophore may be due, at least in part,to a stronger activation of the AP-1 component of thiscomposite NF-AT:AP-1 site.To substantiate further that HBx was able to inducetranscription through the NF-AT enhancer element viaactivation of NF-AT proteins, CHL cells were co-transfected with a cDNA encoding full-length NF-ATcalong with the pNF-AT-Luc reporter plasmid, either inthe presence or the absence of HBx. Although NF-ATchad a weak effect on the luciferase activity, probablydue to its cytoplasmic localization after transfection,when co-transfected with HBx it potently enhanced NF-AT-Luc expression in a dose-dependent manner (Figure2A). In addition, the HBx-mediated induction in thepresence of NF-ATc was blocked by the dominant-negative mutant of NF-ATc (Figure 2A). Similarfunctional results were obtained using clones of CHLcells stably transfected with HBx (CMX), in which NF-ATc enhanced HBx-mediated induction of the NF-ATelement   6-fold (Figure 2B).  E.Lara-Pezzi  et al  . Fig. 1.  HBx transactivates a multimeric NF-AT-containing plasmid. ( A ) CHL cells were co-transfected with 0.2  µ g of the reporter plasmid pNF-AT-Luc along with 5  µ g of either pSV-X or the control plasmid pSV-hygro. Transfected cells were either left untreated or stimulated with PMA orcalcium ionophore (Io). Treatment with PMA plus calcium ionophore was used as control for full stimulus of NF-AT-dependent transcription.( B ) HBx synergizes with PMA plus calcium ionophore in the activation of the NF-AT enhancer element. CHL cells were co-transfected as describedabove, and either left untreated or stimulated with PMA and calcium ionophore (PMA   Io). To demonstrate the specificity of the activation of thereporter gene expression, 2  µ g of the plasmid pSH102C ∆ 418, which encodes a dominant-negative mutant of NF-AT, were included in thetransfection experiments. ( C ) Cyclosporin A blocks the activation of the NF-AT enhancer element by HBx. CHL cells were co-transfected asdescribed above and, where indicated, treated with CsA (200 ng/ml) and/or PMA plus calcium ionophore. ( D ) HBx synergizes with PMA pluscalcium ionophore in the activation of a multimeric AP-1-containing plasmid in a CsA-independent manner. CHL cells were co-transfected with0.2  µ g of the reporter plasmid pAP-1-Luc along with 5  µ g of pSV-X or pSV-hygro. Cells were either left untreated or treated with CsA and/or PMAplus calcium ionophore. The luciferase activities are represented as fold induction over the expression of pNF-AT-Luc or pAP-1-Luc in the absenceof any stimuli. The values shown in (A), (B) and (C) represent the mean fold-induction (  SD) of at least three independent experiments. The valuesshown in (D) are representative of three experiments. HBx targets the transactivation domain of NF-AT  To confirm the role of NF-AT proteins in HBx-mediatedtranscriptional activation of the NF-AT enhancer element,a Gal4-derived reporter system was employed, which inmammalian cells responds only to artificial activators. Tocarry out these studies, the chimeric vector, pGal4-NF-AT1(1–415), encoding the Gal4 DBD fused to the trans-activation domain of NF-AT1, or the parental vectorpRSV-Gal4-DBD (Luo  et al ., 1996a) were co-transfectedinto CHL cells along with the luciferase reporter plasmidpGal4-Luc, either in the presence or the absence of theHBx expression vector pSV-X. As expected, HBx did 7068 not stimulate transcription when co-transfected with thecontrol plasmid encoding the Gal4 DBD (Figure 3A).However, transcription of the pGal4-Luc reporter plasmidwas induced up to 16-fold by HBx when co-transfectedwith Gal4–NF-AT1 expression vector (Figure 3A). Sim-ilarly, in CMX cells, HBx induced Gal4-Luc expression~18-fold, when compared with control CMO cells, usingthree differentamounts ofGal4–NF-AT1 expressionvector(Figure 3B). Taken together, these results strongly indicatethat HBx is able to activate NF-AT either by directprotein–protein interaction or by mimicking the signalsinvolved in the activation of these proteins.  Activation of NF-AT by HBx Fig. 2.  HBx synergizes with transfected full-length NF-ATc-encodingcDNA. ( A ) CHL cells were co-transfected with 0.2  µ g of pNF-AT-Luc, 5  µ g of pSV-X or pSV-hygro, and increasing amounts of pNF-ATcwt. To keep the amount of plasmid DNA in each transfection pointconstant, the empty vector pBJ5 and 5  µ g of the carrier plasmidpGEM7 were used. ( B ) Empty vector cells (CMO) and HBxexpression cells (CMX) cells were co-transfected with 0.2  µ g of pNF-AT-Luc, 0.2  µ g of the expression plasmid pNF-ATcwt or the emptyvector pBJ5. The dominant-negative NF-AT-encoding plasmidpSH102C ∆ 418 (2  µ g) was included in the transfection experiments todemonstrate the specificity of the activation. The luciferase activitiesare represented as fold induction over the expression of pNF-AT-Lucin the absence of any stimuli. Results shown are representative of fourexperiments. Three different clones of CMO and CMX cells wereemployed in the experiments. HBx induces NF-AT DNA-binding activity  To examine whether HBx was able to induce the formationof NF-AT-containing DNA–protein complexes, electro-phoretic mobility shift assays (EMSAs) were performedusing a  32 P-labeled oligonucleotide that contained the 7069 Fig. 3.  HBx activates a Gal4–NF-AT1 chimeric protein containing thetransactivation domain of NF-AT1. ( A ) CHL cells were co-transfectedwith 1  µ g of the reporter plasmid pGal4-Luc along with 5  µ g of eitherthe expression vector encoding the fusion protein Gal4–NF-AT1(1–415) or the parental empty vector pRSV-Gal4-DBD, and increasingamounts of pSV-X. To keep the total amount of DNA constant,pSV-hygro was used. ( B ) CMO and CMX cells were co-transfectedwith 1  µ g of pGal4-Luc and increasing amounts of the expressionvector pGal4-NF-AT1(1–415). To keep the amount of DNA constant,the parental vector pRSV-Gal4-DBD was used. The luciferaseactivities are represented as fold-induction over the values obtained inCHL cells or CMO cells co-transfected with pGal4-Luc and pRSV-Gal4-DBD, and are representative of three independent experiments.The results obtained with the stable transfectants were verified byusing different clones of CMO and CMX cells. NF-AT-binding site of the mouse IL-4 promoter, whichcan bind NF-AT independently of AP-1 (Rooney  et al .,1994, 1995), and nuclear extracts from CHL cells stablytransfected witheither CMX or CMO. Three major specificcomplexes were resolved using nuclear extracts fromHBx-expressing cells that were not detectable in CMOcontrol cells (Figure 4A). These inducible complexesresulted from specific DNA binding since their formation  E.Lara-Pezzi  et al  . Fig. 4.  HBx induces the formation of NF-AT-containing DNA-bindingcomplexes. ( A ) A 2  µ g aliquot of protein from CMO or CMX nuclearextracts was incubated with a  32 P-labeled probe containing the NF-AT-binding site of the murine IL-4 promoter. For competition, a 130-foldmolar excess of the homologous NF-AT oligonucleotide or anoligonucleotide containing an SP-1-binding site were used. The threespecific complexes formed in the presence of HBx are indicated.( B ) CMO and CMX cells were stimulated with PMA (10 ng/ml) pluscalcium ionophore (1  µ M) for 16 h and the complexes formedcompared with those obtained in the absence of stimuli. ( C ) TheHBx-induced complexes are competed by the homologous NF-AToligonucleotide (40-fold excess) but not by a mutated NF-AToligonucleotide (40- and 130-fold) or an AP-1 oligonucleotide(130-fold excess). ( D ) Prior to adding the IL-4-NF-AT probe, 0.5  µ l of either pre-immune antiserum (P. I.) or antibodies against differentmembers of the NF- κ  B, NF-AT or AP-1 families were included in thebinding reaction. Supershifted bands induced by the anti-NF-ATcmonoclonal antibody and by the anti-NF-AT1 antiserum (672) areindicated by arrows. 7070 was blocked by an excess of unlabeled IL4-NFAT oligo-nucleotide, but not by an unrelated oligonucleotide con-taining an SP-1-binding site. Three DNA–proteincomplexes, with electrophoretic mobility identical to thoseinduced by HBx, were resolved using nuclear extractsfromCMOcellstreatedwithPMApluscalciumionophore,which were used as a positive control for NF-AT activation(Figure 4B). It is noteworthy that PMA plus calciumionophore treatment was a stronger stimulus than HBxfor the formation of these complexes. In addition, HBxexpression did not appear to enhance further the bindingactivity of these proteins induced by PMA plus calciumionophore (Figure 4B). To characterize these HBx-inducedcomplexes, an IL4-NFAT oligonucleotide bearingmutations in the core NF-AT-binding site was included inthe competition assays. As shown in Figure 4C, twodifferent amounts of the mutated IL4-NFAT oligonucleo-tide had no detectable effect on the generation of theinducible complexes, whereas similar amounts of the wild-type oligonucleotide completely abolished their formation.As an additional control, an oligonucleotide including anAP-1 consensus site failed to compete the specific bandsgenerated with the NF-AT probe, suggesting that thesecomplexes contained NF-AT-related proteins that bind tothe oligonucleotide independently of the Fos/Jun familyproteins.To identify further the IL4-NFAT-binding factorsinduced by HBx, specific antibodies for NF- κ  B, NF-ATand Fos/Jun proteins were added to the binding reactions.As shown in Figure 4D, neither a pre-immune antiserumnor antisera against the NF- κ  B family members p50,Rel A and p52 (not shown) affected any of the DNA–protein complexes. In contrast, addition of a monoclonalantibody specific for NF-ATc prevented the formation of complex 1 and generated a supershifted band. An anti-serum against NF-AT, which recognizes NF-AT1 andprobably other members of the family (see Materials andmethods), reduced complex 2 and prevented the formationof complex 3. On the other hand, an antiserum specificfor NF-AT1 (672) significantly reduced complex 3, abol-ished the formation of complex 2 and generated a super-shifted band. Taken together, these results suggest thatcomplex 1 is composed mainly by NF-ATc, and thatcomplexes 2 and 3 contain NF-AT1. These results alsoindicate that the inducible bands do not contain detectableamounts of NF- κ  B-related proteins, in agreement withprevious observations which demonstrated that NF- κ  Bfamily members bind with very low affinity to theNF-AT site of the mouse IL-4 promoter (Casolaro  et al .,1995). Consistent with the competition assays, the HBx-induced complexes were not affected by antibodies recog-nizing various members of the Fos and Jun families. HBx triggers nuclear translocation and dephosphorylation of NF-AT  To investigate whether the activation of NF-AT-dependenttranscription and NF-AT binding by HBx involved thenuclear translocation of NF-AT proteins, the cellulardistribution of a transiently expressed HA-tagged NF-ATcprotein (Northrop  et al ., 1994) was analyzed by indirectimmunofluorescence staining in clones of CHL cells eitherexpressing or not expressing HBx. As summarized inTable I, ~40% of the stable CMX cells displayed a nuclear
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