12-O-tetradecanoylphorbol-13-acetate induces Epstein-Barr virus reactivation via NF-kappa B and AP-1 as regulated by protein kinase C and mitogen-activated protein kinase

Signaling pathway components mediating Epstein-Barr virus (EBV) reactivatio n by 12-O-tetradecanoylphorbol-13-acetate (TPA) were characterized in terms of induction and modification of specific transacting factors. The consequ ences of protein kinase C (PKC) activation by TPA in inhibiting inducible n itric oxide synthase (iNOS) mRNA expression were analyzed in the EBV-infect ed gastric epithelial cell line GT38. Spontaneous expression of the EBV BZL F1 gene product ZEBRA became undetectable upon long-term culturing of GT38 cells, while iNOS mRNA expression increased. In such cells the PKC inhibito rs 1-(5-isoquinolinesulphonyl)-2,5-dimethylpiperazine (H7) and staurosporin e inhibited TPA-induced expression of BZLF1 and BRLF1 and reversed TPA-medi ated inhibition of iNOS gene expression. The mitogen-activated protein kina se inhibitor PD98059 inhibited TPA-induced BZLF1 expression. Electrophoreti c mobility shift assays demonstrated that transcription factors NF-kappaB a nd AP-1 were also activated by TPA in a time-dependent manner. The TPA-indu ced NF-kappaB activation was inhibited by prior treatment of the cells with the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC). TPA-induced BZ LF1 expression was also inhibited by the treatment with PDTC. Northern blot analyses characterized changes in levels of the c-jun and junB expressions of the AP-1 family. These results show that TPA induces EBV reactivation v ia NF-kappaB and AP-1 and that PKC is an important mediator in regulating g ene expression leading to EBV reactivation after TPA treatment of GT38 cell s. (C) 2001 Academic Press.