Sensitivity of TLR4- and -7-induced NF kappa B1 p105-TPL2-ERK pathway to TNF-receptor-associated-factor-6 revealed by RNAi in mouse macrophages

Mol Immunol. 2007 Jul;44(15):3715-23. doi: 10.1016/j.molimm.2007.04.002. Epub 2007 May 15.


Tumor necrosis factor (TNF)-receptor-associated-factor-6 (TRAF6) is an adaptor protein involved in Toll-like receptor (TLR) signaling. Recent studies using macrophages from TRAF6 knockout mice have revealed that TRAF6 is required for TLR7 signaling. However, an essential role of TRAF6 in TLR4 signaling and cytokine production is slightly controversial. Using an RNAi approach to reduce the cellular levels of TRAF6, we tested the role of this adaptor protein on the sensitivity of the various components of the ERK pathway mediated by TLR4 and -7 in Raw264.7, a mouse macrophage cell line. ERK activation in macrophages by TLR4 and -7 is mediated via a MAP3K, called TPL2/COT, which under unstimulated conditions is associated with NF kappa B1 p105, a member of the I kappa B family of proteins. Upon stimulation with TLR ligands, p105 is phosphorylated by I kappa B kinase (IKK) complex and partially degraded, which releases TPL2. The free TPL2 is active and stimulates the ERK pathway via MEK1/2. The free TPL2, however, is also unstable and is targeted for degradation. We demonstrate here that reduced level of TRAF6 ( approximately 80% decrease) in macrophages does not significantly affect any of the components of the TLR4-stimulated ERK pathway, including p105 phosphorylation, TPL2 degradation and ERK1/2 phosphorylation. Surprisingly, however, TLR4-induced JNK1/2 phosphorylation is significantly blocked by TRAF6 knockdown, suggesting that ERK and JNK pathways are differentially sensitive to TRAF6 levels. Furthermore, although TLR4-mediated IKK-induced p105 phosphorylation is not sensitive to TRAF6 knockdown, I kappa B alpha phosphorylation (also, IKK-induced) is significantly blocked, suggesting that TLR4 activation results in a TRAF6-sensitive and -insensitive IKK activation in macrophages. In contrast to TLR4 signaling, TLR7 activation of ERK, JNK pathways and phosphorylation of p105 and I kappa B alpha are completely inhibited in TRAF6 knockdown cells. Compared to the signaling data, while TLR4-induced TNFalpha mRNA expression is not significantly inhibited by TRAF6 knockdown, TLR7-induced TNFalpha mRNA is significantly blocked. In contrast, both TLR4- and TLR7-induced IL6 mRNA are significantly blocked by TRAF6 knockdown. These results suggest that while TRAF6 is absolutely essential for TLR7 activation of ERK, JNK and NF kappa B pathways, TLR4-induced ERK, JNK pathways and IKK-mediated phosphorylation of I kappa B family members as well as cytokine expression are differentially sensitive to the cellular levels of TRAF6. These results have important implications in terms of therapeutic targeting of TRAF6 complexes in diseases where TLR4 and -7 are involved.

MeSH terms

  • Aminoquinolines / pharmacology
  • Animals
  • Cytokines / genetics
  • Cytokines / metabolism
  • Enzyme Activation / drug effects
  • Extracellular Signal-Regulated MAP Kinases / metabolism*
  • Gene Expression Regulation / drug effects
  • I-kappa B Proteins / metabolism
  • Imiquimod
  • JNK Mitogen-Activated Protein Kinases / metabolism
  • Lipopolysaccharides / pharmacology
  • MAP Kinase Kinase Kinases / metabolism*
  • Macrophages / drug effects
  • Macrophages / enzymology*
  • Membrane Glycoproteins / metabolism*
  • Mice
  • Models, Immunological
  • NF-KappaB Inhibitor alpha
  • NF-kappa B p50 Subunit / metabolism*
  • Phosphorylation / drug effects
  • Proto-Oncogene Proteins / metabolism*
  • RNA Interference
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • TNF Receptor-Associated Factor 6 / deficiency
  • TNF Receptor-Associated Factor 6 / metabolism*
  • Toll-Like Receptor 4 / metabolism*
  • Toll-Like Receptor 7 / metabolism*


  • Aminoquinolines
  • Cytokines
  • I-kappa B Proteins
  • Lipopolysaccharides
  • Membrane Glycoproteins
  • NF-kappa B p50 Subunit
  • Nfkbia protein, mouse
  • Proto-Oncogene Proteins
  • RNA, Messenger
  • TNF Receptor-Associated Factor 6
  • Tlr4 protein, mouse
  • Tlr7 protein, mouse
  • Toll-Like Receptor 4
  • Toll-Like Receptor 7
  • NF-KappaB Inhibitor alpha
  • Nfkb1 protein, mouse
  • Extracellular Signal-Regulated MAP Kinases
  • JNK Mitogen-Activated Protein Kinases
  • MAP Kinase Kinase Kinases
  • Map3k8 protein, mouse
  • Imiquimod