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Regulation of TNF expression by multiple mitogen-activated protein kinase pathways

W Zhu1, J S Downey, J Gu

  • 1Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA.

Journal of Immunology (Baltimore, Md. : 1950)
|June 8, 2000
PubMed

Insights

Bacterial endotoxin (LPS) activates four mitogen-activated protein (MAP) kinase pathways in macrophages, crucial for TNF production. Simultaneous activation of these MAP kinase pathways cooperatively enhances TNF gene expression, requiring the C-terminal domain of RNA polymerase II.

Area of Science:

  • Immunology
  • Molecular Biology
  • Cell Signaling

Background:

  • Macrophages are key immune cells that produce tumor necrosis factor (TNF) upon stimulation with bacterial endotoxin (LPS).
  • TNF production is regulated by complex intracellular signaling pathways, including mitogen-activated protein (MAP) kinases.

Purpose of the Study:

  • To investigate the roles of four specific MAP kinase pathways (ERK, JNK/SAPK, p38, BMK/ERK5) in LPS-induced TNF production.
  • To elucidate the mechanism by which MAP kinases regulate TNF gene expression, focusing on promoter elements and transcriptional machinery.

Main Methods:

  • Stimulation of macrophages with LPS.
  • Analysis of MAP kinase pathway activation using specific inhibitors or activators.
  • Reporter assays to measure TNF promoter activity.
  • Use of RNA polymerase II mutants with C-terminal domain (CTD) deletions to assess its role in TNF production.

Main Results:

  • LPS activates ERK, JNK/SAPK, p38, and BMK/ERK5 pathways in macrophages.
  • Individual MAP kinase activation has a modest effect on TNF promoter activity, but simultaneous activation leads to dramatic induction.
  • A 40-bp sequence containing the TATA box, not previously recognized MAP kinase-responsive elements, is critical for MAP kinase-induced TNF gene upregulation.
  • Deletion of the CTD of RNA polymerase II (Delta31) significantly reduces MAP kinase-mediated TNF production (>90%).

Conclusions:

  • Multiple MAP kinase pathways cooperate to regulate TNF production in LPS-stimulated macrophages.
  • The TATA box-proximal region, rather than canonical MAP kinase-responsive elements, mediates this regulation.
  • The C-terminal domain of RNA polymerase II is essential for executing MAP kinase signaling in TNF gene expression.

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