In this study, we demonstrate that FOXO3 interferes with p65/RelA binding to the IFN-β promoter (Fig. 4D) and leads to reduction of its transcription (Fig. 4E). Together, our data and the results of others are in favor of the hypothesis that FOXO3 could sequester the proteins and interfere with their DNA binding to target gene. Further experiments will be needed

to dissect the molecular mechanisms of the FOXO3 suppressor action in detail, but it is likely to be a transcription factor- and gene-specific phenomenon, for example TLR-induced IRF7 mRNA expression, which is under the IRF3 control, is not affected by Palbociclib FOXO3 (data not shown). IKK-ε is an important mediator of the IFN type I response as it phosphorylates and activates IRF3 and IRF7 [[17, 18]] via phosphorylation of an extended sequence motif–SxSxxxS–common to IRF3 and IRF7 [[35]]. The C-terminus of FOXO3 contains three putative IKK-ε-phosphorylation sites (Ser349, Ser476, Ser584) in addition to the close-related phosphorylation site Ser644, previously shown to be important for IKK-β regulation [[16]]. Mutation

of this site was not sufficient to block IKK-ε-induced phosphorylation of FOXO3 (Supporting Information Fig. 2B), suggesting that FOXO3 contains a specific IKK-ε-targeted site. The presence of multiple serine and threonine phosphorylations also suggests that IKK-ε may target more than one of the phosphorylation sites and help to fine-tune the Volasertib ic50 Rutecarpine FOXO3 regulation during the immune response, by acting on different aspects of the protein activity and stability, but more work is needed to dissect their role in FOXO3 transactivation activity, protein localization, or protein–protein interaction. FOXO3 is a well-described tumor suppressor involved in triggering cell-cycle arrest and apoptosis and is inhibited in many cancers including prostate, ovarian, and breast cancer. IKK-ε was recently mapped

as a new oncogene and was found to be overexpressed in prostate, ovarian, and breast cancer [20, 21, 36]. Interestingly, IKK-ε can replace a PI3K activity to inhibit cell-cycle arrest and apoptosis [[20]] processes associated with FOXO3 activity [[16, 37]]. Thus, it is possible that IKK-ε-mediated inhibition of FOXO3 thwarts cell-cycle arrest and apoptosis in cancer cells. In addition, it would favor the production of normally FOXO3 negatively controlled proinflammatory cytokines IL-6 and IL-8 [10, 21, 29], facilitating tumorigenesis. In summary, we identify FOXO3 as a new IKK-ε-controlled check-point of IRF activation and regulation of IFN-β expression. FOXO3, which antagonizes NF-κB and IRF activities and hampers IFN-β and IFN-λ1 expression, is regulated by IKK-ε. Once the activating signal has been received, IKK-ε provides a positive regulatory signal to IRF3 and at the same time phosphorylates FOXO3, contributing to its inactivation.