Mitogen stimulation of T-cells increases c-Fos and c-Jun protein levels, AP-1 binding and AP-1 transcriptional activity.

We have analysed the effect of mitogenic lectins on c-Fos and c-Jun protein levels as well as on activator protein-1 (AP-1) binding and enhancer activity in Jurkat T-cells. Both c-Fos and c-Jun protein levels were increased after Con A and PHA stimulation. Since T-cell stimulation increases both intracellular Ca2+ and cAMP levels and activates protein kinase C (PKC), the possible involvement of these intracellular messengers in c-Fos and c-Jun induction was tested. PMA, which directly activates PKC, mimicked the effect of the lectins on c-Fos and c-Jun, but elevation of either intracellular Ca2+ or cAMP levels had little or no effect. The mitogen-induced increase of c-Fos and c-Jun immunoreactivity was inhibited by H-7, a kinase inhibitor with relatively high specificity for PKC, and less efficiently by H-8, a structurally related kinase inhibitor less active on PKC, but more active on cyclic nucleotide-dependent kinases. Con A stimulation was found to increase both binding of AP-1 to the AP-1 consensus sequence, TRE, and AP-1 enhancer activity, in Jurkat cells. PMA was also found to increase the AP-1 enhancer activity, whereas elevation of Ca2+ or cAMP had only minor effects. We conclude that stimulation with mitogenic lectins is sufficient to increase both c-Fos and c-Jun protein levels, AP-1 binding and AP-1 enhancer activity in Jurkat cells and that they act via mechanisms that could involve the activation of PKC.     

Activator protein-1 mediates induced but not basal epidermal growth factor receptor gene expression

BACKGROUND: The epidermal growth factor receptor (EGFR) is expressed at different levels in many cell types and found overexpressed in many cancers. EGFR expression is increased or decreased in response to extracellular stimuli. We examined the effect of increased c-Jun expression on EGFR promoter activity. MATERIALS AND METHODS: We used DNAse I foot-printing analysis to determine the binding of activator protein 1 (AP-1) to the promoter region. We also used cotransfection experiments and western blotting analysis to determine the effect of AP-1 family members on EGFR expression. RESULTS: AP-1 was able to bind to at least seven sites in the EGFR promoter region. Cotransfection of MCF-7 cells with a c-Jun expression vector and the EGFR promoter reporter resulted in a 7-fold increase in promoter activity. JunB, but not c-fos, also enhanced the EGFR promoter activity. An A-Fos-dominant negative shown to inhibit Jun-dependent transactivation was able to prevent c-Jun induction of the promoter activity, but only slightly decreased the basal activity of the promoter. Furthermore, the A-Fos dominant negative was able to inhibit phorbol ester induction of the EGFR promoter. Examination of EGFR expression of MCF-7 stable cell lines that overexpress c-Jun revealed an increase in EGFR expression. Additionally, a cisplatin-resistant cell line, A2780/CP70, which has an increase in AP-1 activity compared with the parental cell line, A2780, was found to have an increase in EGFR level. CONCLUSIONS: These results indicate that AP-1 can act to increase the expression of EGFR and may play a role in upregulation of EGFR in cancer cells.