Lipopolysaccharide Signals Activation of Tumor Necrosis Factor Biosynthesis Through the Ras/Raf-1/MEK/MAPK Pathway

Abstract

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 cytokines is tumor necrosis factor (TNF). Details of the signaling pathway leading to TNF biosynthesis remain unclear. The pathway is branched in the sense that TNF gene transcription and TNF mRNA translation are both strongly stimulated by LPS. Recent evidence has indicated that MAP kinase homologs become phosphorylated in LPS-stimulated cells, suggesting their possible involvement in signal transduction. We sought to test this hypothesis. Materials and Methods: Measurements of LPS-induced MEK and ERK2 activity were undertaken in LPS-sensitive and LPS-insensitive cells. Transfection studies, in which dominant inhibitors of ras and raf-I 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 RAW 264.7 mouse macrophages, both ERK2 and MEKI 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 well as derepression of the translational blockade normally imposed by the 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 primary macrophages derived from C3H/HeJ mice), ERK2 activity was found to be refractory to induction by LPS. Conclusions: The ras/raf-1/MEK/MAPK pathway is chiefly responsible for transduction of the LPS signal to the 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 signaling pathway.

The Effect of Epigallocatechin Gallate on Lipopolysaccharide-Induced Acute Lung Injury in a Murine Model

“…A previous study reported that down-regulation of TLR 4 expression, using RNA interference techniques, decreased MAPKs activation, TNF-α and MIP-2 expression in RAW 264.7 cells stimulated with LPS [17]. However, there are conflicting reports concerning the physiological role of ERK1/2 in the TNF-α production [17,34,36,37]. For example, Means, et al reported that inhibition of ERK1/2 led to a decrease in the degree of TNF-α expression in murine alveolar macrophage cell line AMJ2C-8, not in the RAW264.7 cells [36].…”

“…JNK and ERK1/2 regulated TNF-α and MIP-2 expression and JNK (SP600125) or ERK1/2 inhibitor (PD98059) reduced these cytokines expression [14,15,[33][34][35]. A previous study reported that down-regulation of TLR 4 expression, using RNA interference techniques, decreased MAPKs activation, TNF-α and MIP-2 expression in RAW 264.7 cells stimulated with LPS [17].…”

Enhanced Phosphorylation of MAPKs by NE Promotes TNF-α Production by Macrophage Through α Adrenergic Receptor

“…In mammalian cells, MAPKs included three major groups, ERK, JNK, and p38. They could be activated by many proinflammatory stimuli and played important roles in the inflammatory process [10][11][12]. It was reported that MAPKs were involved in TNF-α biosynthesis in LPSstimulated macrophages [10][11][12].…”

TLR-independent induction of human monocyte IL-1 by phosphoglycolipids from thermophilic bacteria

“…LPS stimulates macrophages to release inflammatory cytokines, such as IL-1 and TNF-α. The molecular mechanism for IL-1 production includes the activation of ERK, JNK and p38, and the release of ROS, which play key roles in the LPS-mediated signal transductions between extracellular membrane stimulation, cytoplasmic response, and gene activation [44,62,63]. LPS-mediated PI3K/Rac/ p38 pathways play a dominant role in the regulation of proIL-1/IL-1; other pathways such as PKC/MEK/ERK and PI3K/Rac/JNK are less important [44].…”