Transforming growth factor beta 1 stimulates expression of the Epstein-Barr virus BZLF1 immediate-early gene product ZEBRA by an indirect mechanism which requires the MAPK kinase pathway.
Disruption of Epstein-Barr virus (EBV) latency is mediated by ZEBRA, the protein product of the immediate-early EBV gene, BZLF1. In vitro, phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC), induces reactivation of EBV. However, the physiological stimuli responsible for the disruption of viral latency are not well characterized. Transforming growth factor beta 1 (TGF-beta1) has also been shown to trigger the reactivation of EBV in Burkitt lymphoma cell lines; however, the effect of TGF-beta1 on ZEBRA expression has not been reported. To further understand this phenomenon, we have investigated the effect of TGF-beta1 on ZEBRA expression. Our results indicate that the treatment of different EBV-positive Burkitt's lymphoma cell lines with TGF-beta1 induces a time-dependent activation of BZLF1 transcription with a corresponding increase in the production of the protein ZEBRA. TGF-beta1 has been shown to exert its effects through a wide range of intracellular routes; in the present study, we have explored these pathways. Transient expression of Smad proteins on their own had no effect on ZEBRA expression. A specific inhibitor of p38 mitogen-activated protein kinase (MAPK), SB203580, did not affect TGF-beta1-induced ZEBRA expression, whereas treatment with the MAPK/ERK kinase inhibitors, PD98059 and U0126, dramatically decreased this induction. This suggests that TGF-beta1 effect on BZLF1 expression requires the MAPK pathway. However, in Raji and B95-8 cells additional routes can be used, as (i) the inhibition of ZEBRA induction by PD98059 or U0126 was incomplete, whereas these inhibitors completely abolished PMA-induced ZEBRA expression, (ii) TGF-beta1 induction of ZEBRA expression occurs in PKC-depleted cells, (iii) in Raji and in B95-8 cells, the effect of TGF-beta1 and PMA are additive. Transient transfection of the EBV-negative B-cell line DG75 with a BZLF1 promoter-fusion construct (Zp-CAT) showed that under conditions where the BZLF1 promoter is activated by PMA treatment, TGF-beta1 had no significant effect on the expression of the chloramphenicol acetyltransferase gene. Furthermore, TGF-beta1 induction of BZLF1 transcripts is dependent on de novo protein synthesis, which suggests that TGF-beta1 induces BZLF1 expression by an indirect mechanism.
Fahmi
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Laboratoire de Pharmacologie Expérimentale et Clinique, INSERM EPI 99-32, Institut de Génétique Moléculaire, 75010 Paris, France.
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D002070:Butadienes / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral / Q000187:drug effects*; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D017874:Immediate-Early Proteins / Q000096:biosynthesis / Q000235:genetics*; D007700:Kinetics; D020935:MAP Kinase Signaling System; D009570:Nitriles / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011725:Pyridines / Q000494:pharmacology; D012333:RNA, Messenger; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D051906:Smad7 Protein; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics*; D016212:Transforming Growth Factor beta / Q000378:metabolism / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D014764:Viral Proteins
C063676:BZLF1 protein, Herpesvirus 4, Human; D002070:Butadienes; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007093:Imidazoles; D017874:Immediate-Early Proteins; D009570:Nitriles; D011725:Pyridines; D012333:RNA, Messenger; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; C497806:SMAD7 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D051906:Smad7 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; C113580:U 0126; D014764:Viral Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D013755:Tetradecanoylphorbol Acetate; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one