p38 and Extracellular Signal-regulated Kinase Mitogen-activated Protein Kinase Pathways Are Required for Nuclear Factor-κB p65 Transactivation Mediated by Tumor Necrosis Factor

Interleukin-6 (IL-6) is a pleiotropic cytokine, which is involved in inflammatory and immune responses, acute phase reactions, and hematopoiesis. In the mouse fibrosarcoma cell line L929, the nuclear factor (NF)- Kappa B plays a crucial role in IL-6 gene expression mediated by tumor necrosis factor (TNF). The levels of the activated factor do not, however, correlate with the variations of IL-6 gene transcription; therefore, 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 factors. Even the smallest deletion variant, containing almost exclusively a NF- Kappa B-responsive sequence preceding the IL-6 minimal promoter, as well as a recombinant construction containing multiple Kappa B-motifs, could still be stimulated with TNF. We observed that the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 was able to repress TNF-stimulated expression of the IL-6 gene, as well as of a Kappa B-dependent reporter gene construct, without affecting the levels of 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 potential of the p65 Kappa B subunit. Therefore, we conclude that, in addition to cytoplasmic activation and DNA binding of NF- Kappa B, the p38 and extracellular signal-regulated kinase MAPK pathways act as necessary cooperative mechanisms to regulate TNF-induced IL-6 gene expression by modulating the transactivation machinery.