GSE116220
The TPL-2 – ERK1/2 signalling pathway inhibits interferon beta expression in macrophages via TCF-dependent Fos transcription
Studies in mice have indicated that TPL2 kinase plays important roles in immune responses to pathogens and in various models of inflammatory disease. We set out to determine how TPL2 (Map3k8) regulates transcription using an in vitro system of LPS-stimulated primary macrophages. TPL2 regulated genes are involved in a number of different processes including inflammation as well as reproduction, cell cycle and signal transduction. Differentially expressed genes were used in a K means analysis, grouping based on expression profile, which identified 12 different expression profiles. There was a strong correlation between rapidly-induced genes regulated by TPL2 kinase activity and their expression in cells treated with the MKK1/2 inhibitor (PD032590)1 or from cells where phosphorylation of P105 by IKK2 is blocked. Cluster 1 genes were found to be enriched for TCF family members. TPL2 was shown to regulate ELK1 phosphorylation and known TCF targets genes such as Egr1. TPL2 is known to regulate transcription of multiple cytokines and chemokines including IFNb. Like Map3k8[D270A] macrophages, expression of Ifnb1 was increased in Elk1-/-Elk4-/- macrophages compared to WT macrophages following LPS stimulation. In fact, this increase was also seen in MKK1/2 inhibitor treated macrophages. Expression of Fos was decreased in both Map3k8[D270A] and Elk1-/-Elk4-/- macrophages. Macrophages generated from mice with myeloid-specific deletion of Fos also had elevated levels of Ifnb1 compared to controls. Similar results were obtained following activation of TLR1/2, TLR9 and TNF-R in macrophages. Furthermore, LPS or TNF stimulation of MEFs lacking Elk1, Elk3 and Elk4 had reduced FOS protein levels and increased Ifnb1 mRNA suggesting a general role for ERK1/2 > TCF pathway regulating Ifnb1 levels through Fos expression. Overall design: Bone marrow derived macrophages (BMDMs) from WT (C57BL/6), Map3k8[D270A], Nfkb1[SS/AA], WT+PD0325901, WT+RSKi47 and Elk1-/- Elk4-/- mice were generated and stimulated with LPS to determine the genes and processes controlled by TPL2 (Map3k8) signalling. BMDMs were stimulated with LPS (100ng/ml) for various times up to 2 hours or left unstimulated. PD0325901 (MKK1/2 inhibitor) and RSKi47 (RSK inhibitor) were added prior to stimulation for 10mins at 0.1 µM and 2 µM respectively