The inflammatory kinase IKKα phosphorylates and stabilizes c-Myc and enhances its activity.

BACKGROUND: The IκB kinase (IKK) complex, comprising the two enzymes IKKα and IKKß, is the main activator of the inflammatory transcription factor NF-κB, which is constitutively active in many cancers. While several connections between NF-κB signaling and the oncogene c-Myc have been shown, functional links between the signaling molecules are still poorly studied. METHODS: Molecular interactions were shown by co-immunoprecipitation and FRET microscopy. Phosphorylation of c-Myc was shown by kinases assays and its activity by improved reporter gene systems. CRISPR/Cas9-mediated gene knockout and chemical inhibition were used to block IKK activity. The turnover of c-Myc variants was determined by degradation in presence of cycloheximide and by optical pulse-chase experiments.. Immunofluorescence of mouse prostate tissue and bioinformatics of human datasets were applied to correlate IKKα- and c-Myc levels. Cell proliferation was assessed by EdU incorporation and apoptosis by flow cytometry. RESULTS: We show that IKKα and IKKß bind to c-Myc and phosphorylate it at serines 67/71 within a sequence that is highly conserved. Knockout of IKKα decreased c-Myc-activity and increased its T58-phosphorylation, the target site for GSK3ß, triggering polyubiquitination and degradation. c-Myc-mutants mimicking IKK-mediated S67/S71-phosphorylation exhibited slower turnover, higher cell proliferation and lower apoptosis, while the opposite was observed for non-phosphorylatable A67/A71-mutants. A significant positive correlation of c-Myc and IKKα levels was noticed in the prostate epithelium of mice and in a variety of human cancers. CONCLUSIONS: Our data imply that IKKα phosphorylates c-Myc on serines-67/71, thereby stabilizing it, leading to increased transcriptional activity, higher proliferation and decreased apoptosis.

Ikk2-mediated inflammatory activation of arterial endothelial cells promotes the development and progression of atherosclerosis.

BACKGROUND AND AIMS: Inflammatory activation of endothelial cells is considered to be the first step in the development of atherosclerosis. Here, we determined the consequences of chronic endothelial activation via the NF-κB activator Ikk2 (Inhibitor of nuclear factor kappa-B kinase 2, Ikk-beta) on the development and progression of atherosclerosis. METHODS: We established a conditional transgenic mouse model, expressing a tamoxifen-inducible, constitutively active form of Ikk2 exclusively in arterial endothelial cells (caIkk2EC mice) on an ApoE-deficient background. Mice were fed a Western-type diet and endothelial Ikk2 was activated either at early or late stages of atherosclerosis. RESULTS: En face preparations of isolated aortas revealed a significant increase in plaque area in caIkk2EC mice at 12 weeks of Western-type diet as compared to ApoE-deficient littermates. This was accompanied by increased infiltration of macrophages and T cells into the lesion. Several chemokine/cytokine and immune cell pathways were significantly upregulated in the aortic transcriptome of caIkk2EC mice. Of note, in mice with established atherosclerosis, activation of endothelial Ikk2 still further accelerated progression of atherosclerosis. This indicates that inflammatory endothelial activation is crucial during all stages of the disease. CONCLUSIONS: Our results show for the first time that chronic inflammatory activation of arterial endothelial cells accelerates the development and progression of atherosclerosis both at early and late stages of disease development. Thus, pharmacological targeting of endothelial inflammation emerges as a promising treatment approach.