P38 AND EXTRACELLULAR SIGNAL-REGULATED KINASE MITOGEN-ACTIVATED PROTEIN-KINASE PATHWAYS ARE REQUIRED FOR NUCLEAR FACTOR KAPPA-B P65 TRANSACTIVATION MEDIATED BY TUMOR-NECROSIS-FACTOR

Interleukin-6 (IL-6) is a pleiotropic cytokine, which is involved in i nflammatory and immune responses, acute phase reactions, and hematopoi esis. In the mouse fibrosarcoma cell line L929, the nuclear factor (NF )-kappa B plays a crucial role in IL-6 gene expression mediated by tum or necrosis factor (TNF). The levels of the activated factor do not, h owever, correlate with the variations of IL-6 gene transcription; ther efore, other factors and/or regulatory mechanisms presumably modulate the levels of IL-6 mRNA production, Upon analysis of various deletion and point-mutated variants of the human IL-6 gene promoter coupled to a reporter gene, we screened for possible cooperating transcription fa ctors. Even the smallest deletion variant, containing almost exclusive ly a NF-kappa B-responsive sequence preceding the IL-6 minimal promote r, as well as a recombinant construction containing multiple kappa B-m otifs, could still be stimulated with TNF. We observed that the p38 mi togen-activated protein kinase (MAPK) inhibitor SB203580 was able to r epress TNF-stimulated expression of the IL-6 gene, as well as of a kap pa B-dependent reporter gene construct, without affecting the levels o f NF-kappa B binding to DNA. Furthermore, we clearly show that, using a nuclear Gal4 ''one-hybrid'' system, the MAPK inhibitors SB203580 and PD0980589 have a direct repressive effect on the transactivation pote ntial of the p65 kappa B subunit. Therefore, we conclude that, in addi tion to cytoplasmic activation and DNA binding of NF-kappa B, the p38 and extracellular signal-regulated kinase MAPK pathways act as necessa ry cooperative mechanisms to regulate TNF-induced IL-6 gene expression by modulating the transactivation machinery.