Coordinate Regulation of TPL-2 and NF-κB Signaling in Macrophages by NF-κB1 p105

Abstract

The role of IκB kinase (IKK)-induced proteolysis of NF-κB1 p105 in innate immune signaling was investigated using macrophages from Nfkb1^SSAA/SSAA mice, in which the IKK target serines on p105 are mutated to alanines. We found that the IKK/p105 signaling pathway was essential for TPL-2 kinase activation of extracellular signal-regulated kinase (ERK) mitogen-activate protein (MAP) kinase and modulated the activation of NF-κB. The Nfkb1^SSAA mutation prevented the agonist-induced release of TPL-2 from its inhibitor p105, which blocked activation of ERK by lipopolysaccharide (LPS), tumor necrosis factor (TNF), CpG, tripalmitoyl-Cys-Ser-Lys (Pam₃CSK), poly(I · C), flagellin, and R848. The Nfkb1^SSAA mutation also prevented LPS-induced processing of p105 to p50 and reduced p50 levels, in addition to decreasing the nuclear translocation of RelA and cRel. Reduced p50 in Nfkb1^SSAA/SSAA macrophages significantly decreased LPS induction of the IκBζ-regulated Il6 and Csf2 genes. LPS upregulation of Il12a and Il12b mRNAs was also impaired although specific blockade of TPL-2 signaling increased expression of these genes at late time points. Activation of TPL-2/ERK signaling by IKK-induced p105 proteolysis, therefore, induced a negative feedback loop to downregulate NF-κB-dependent expression of the proinflammatory cytokine interleukin-12 (IL-12). Unexpectedly, TPL-2 promoted soluble TNF production independently of IKK-induced p105 phosphorylation and its ability to activate ERK, which has important implications for the development of anti-inflammatory drugs targeting TPL-2.

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ACKNOWLEDGMENTS

We thank P. Tsichlis (Tufts University, Boston, MA) and Thomas Jefferson University, Philadelphia, PA, for the Map3k8^−/− mice; Philip Cohen for MEK inhibitor (University of Dundee, Scotland); Emilie Jacque (NIMR), George Kassiotis (NIMR), Ben Seddon (NIMR), Steve Smale (UCLA), and Victor Tybulewicz (NIMR) for advice and critical reading of the manuscript; Hamish Allen (Abbott Bioresearch Center, Worcester, MA) for encouragement at the start of the project; the NIMR Photographics department, NIMR Biological Services, NIMR flow cytometry service, and other members of the Ley laboratory for help during the course of this work.

This work was supported by the United Kingdom Medical Research Council.