ENHANCED AP-1 AND NF-KAPPA-B ACTIVITIES AND STABILITY OF INTERLEUKIN-8 (IL-8) TRANSCRIPTS ARE IMPLICATED IN IL-8 MESSENGER-RNA SUPERINDUCTION IN LUNG EPITHELIAL H292 CELLS

Inhibition of protein synthesis may result in superinduction of short- lived transcripts and has been attributed variably to stabilization of transcripts and/or increased gene transcription. Little is known abou t the kinetics of these processes and relevant transcriptional element s have not been identified. In this study, we describe superinduction of interleukin 8 (IL-8) mRNA, an important inflammatory mediator, in l ung epithelial-like H292 cells and identify the underlying molecular m echanisms and their kinetics. Cycloheximide (CHI, 10 mu g/ml), an inhi bitor of protein synthesis, maximally increased IL-8 mRNA levels 30-fo ld in H292 cells. Tumour necrosis factor alpha (TNF-alpha), which indu ced IL-8 mRNA 3-fold, synergized with CHI causing a 150-fold increase at 6 h. CHI early on increased the stability of IL-8 mRNA (from 40 min in cells cultured with medium to more than 4h with CHI). CHI also inc reased transcription as shown by transfection with IL-8 promoter const ructs. Truncated and mutated constructs identified NF-kappa B and AP-1 binding sites as primary cis-acting elements in IL-8 gene transcripti on and IL-8 mRNA superinduction. Electrophoretic mobility shift assays indicated that CHI increased NF-kappa B and prolonged AP-1 DNA-bindin g activities and that the synergism of TNF-alpha and CHI on IL-8 mRNA expression was paralleled by a further increase of AP-1 DNA-binding ac tivity. This synergism was still noticed when 4h elapsed between the a ddition of CHI and that of TNF-alpha. Taken together, our results indi cate that CHI interferes with both post-transcriptional and transcript ional repressive mechanisms of IL-8 mRNA expression.