LIPOPOLYSACCHARIDE SIGNALS ACTIVATION OF TUMOR-NECROSIS-FACTOR BIOSYNTHESIS THROUGH THE RAS RAF-1/MEK/MAPK PATHWAY/

Background: Lipopolysaccharide (LPS) is known to activate macrophages, causing the release of toxic cytokines that may provoke inflammation and shock. One of the most important and best studied of these cytokin es is tumor necrosis factor (TNF). Details of the signaling pathway le ading to TNF biosynthesis remain unclear. The pathway is branched in t he sense that TNF gene transcription and TNF mRNA translation are both strongly stimulated by LPS. Recent evidence has indicated that MAP ki nase homologs become phosphorylated in LPS-stimulated cells, suggestin g their possible involvement in signal transduction. We sought to test this hypothesis. Materials and Methods: Measurements of LPS-induced M EK and ERK2 activity were undertaken in LPS-sensitive and LPS-insensit ive cells. Transfection studies, in which dominant inhibitors of ras a nd raf-1 were used to block signaling to the level of MAP kinase, were carried out in order to judge whether the TNF gene transcription and TNF mRNA translation are modulated through this pathway. Results: In R AW 264.7 mouse macrophages, both ERK2 and MEK1 activity are induced by LPS treatment. In the same cell line, dominant negative inhibitors of ras and raf-1 block LPS-induced activation of the TNF promoter, as we ll as derepression of the translational blockade normally imposed by t he TNF 3'-untranslated region. A constitutively active form of raf-1 ( raf-BXB) was found to augment, but not replace, the LPS signal. In LPS -insensitive cells (RAW 264.7 X NIH 3T3 fusion hybrid cells and primar y macrophages derived from C3H/HeJ mice), ERK2 activity was found to b e refractory to induction by LPS. Conclusions: The ras/raf-1/MEK/MAPK pathway is chiefly responsible for transduction of the LPS signal to t he level of the TNF gene and mRNA. raf and raf-1 lie upstream from (or actually represent) the physical branchpoints of the transcriptional and translation activation signals generated by LPS. The lesions that prevent LPS signaling in macrophages from C3H/HeJ mice, or in RAW 264. 7 x NIH 3T3 fusion hybrid cells, occupy a proximal position in the sig naling pathway.