3B). Adenoviral delivery had no significant effect on the resting cells []. The complementary experiment targeting endogenous FK228 chemical structure FOXO3a in MDDCs by
short interfering RNA (siRNA) duplexes resulted in upregulation of IFN-β mRNA expression (Supporting Information Fig. 5). Next, we examined if FOXO3-mediated inhibition of IFN transcription was due to its antagonizing effect on contributing regulatory factors. Both IFN-β and IFN-λ1 genes are regulated by NF-κB and IRF factors [[25, 28]]. Using NF-κB-luc gene-reporter construct, we found that, consistent with the published data [], FOXO3 inhibited LPS-induced activation of NF-κB (Fig. 4A). In addition, it also inhibited the activity of the ISRE-luc gene-reporter construct, driven by tandem IRF-binding elements (Fig. 4B), suggesting that FOXO3 may regulate more inflammatory pathways than initially described. A direct effect of FOXO3 on IRF signaling was confirmed by the ability of FOXO3 to inhibit IRF3/7-induced activation of a luciferase-reporter driven by the IFN-β promoter (Fig. 4C). The mechanism by which FOXO3 antagonizes NF-κB remains unclear. FOXO3 was implicated in regulation of NF-κB
inhibitors, IκBs [[11, 15]], with signaling pathway inhibition of FOXO3 resulting in attenuated expression of IL-8 in LPS-treated intestinal epithelia []. It has also been proposed that FOXO3 prevents NF-κB translocation to the nucleus []. However, we observed no difference in LPS-induced p65/RelA translocation in 293-TLR4 cells transduced with an adenovirus expressing FOXO3 protein (Supporting Information Fig. 7A). Moreover, FOXO3 had no effect on expression of RelA or IRF3 mRNA in MDDCs (data not shown). Another possibility is the sequestration of Amylase active NF-κB complexes, as described for FOXO4 []. Indeed, complex formation between HA-tagged FOXO3 and FLAG-tagged p65/RelA and IRF3 were detected in 293-TLR4 cells ectopically expressing
the aforementioned proteins (Supporting Information Fig. 7B), suggesting that FOXO3 may inhibit NF-κB and IRF-driven gene transcription via protein–protein interactions, acting as a co-repressor or blocking the sites needed for DNA binding or signal transmission. To further examine these possibilities, the recruitment of ectopically expressed p65/RELA to the endogenous IFN-β promoter was analyzed in 293-TLR4 cells by ChIP and demonstrated a noticeable reduction in the presence of ectopically expressed FOXO3 (Fig. 4D). Thus, the sequestration of p65/RelA by FOXO3 can thwart its recruitment to the target promoters. Moreover, the recruitment of polymerase II to the IFN-β promoter, which reflects on the rate of gene transcription, was blocked in the presence of FOXO3 (Fig. 4E). In summary, our data indicate that FOXO3-mediated inhibition of the p65/RelA-driven gene transcription is likely to be via interfering with p65/RELA DNA-binding to the target promoters.