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Abstract
Although the proximal cytoplasmic signaling events that control the activation of the NFκB transcription factor are understood in considerable detail, the subsequent intranuclear events that regulate the NFκB-mediated transcriptional response, especially the regulation of NFκB by nuclear IκBα remain poorly defined. Macrophages are one of the primary producers of pro-inflammatory cytokines such as TNF, IL-8, IL-1β and IL-6. The transcription of these pro-inflammatory cytokines is regulated by NFκB. In this study, we show that LMB-induced nuclear IκBα binds to p65 NFκB in the nucleus and inhibits its DNA binding activity in LPS-stimulated human macrophages. While mRNA expression and cellular release of TNFα, IL-1β and IL-6 are inhibited by the LMB-induced nuclear IκBα, IL-8 mRNA expression and cellular release are not significantly affected, indicating that the regulation of NFκB-dependent pro-inflammatory genes by nuclear IκBα is gene specific. Analysis of in vivo recruitment of p65 NFκB to NFκB-regulated promoters in U937 macrophages and human peripheral blood mononuclear cells (PBMCs) showed that while the p65 recruitment to TNFα, IL-1β and IL-6 promoters was inhibited by the nuclear IκBα, p65 recruitment to IL-8 promoter was not repressed. Chromatin immunoprecipitation (ChIP) analyses using IκBα and S536 phospho-specific p65 NFκB antibodies demonstrated that while the newly synthesized IκBα induced by post-induction repression was recruited to TNFα, IL-1β and IL-6 promoters but not to IL-8 promoter, S536 phosphorylated p65 was recruited to IL-8 promoter, but not to TNFα, IL-1β or IL-6 promoters. In addition, the p65 recruited to TNFα, IL-1β or IL-6 promoters in LPS-stimulated macrophages was acetylated at K310, whereas p65 recruited to the IL-8 promoter was not acetylated. ChIP analyses using non-canonical NFκB members, rel-B, c-rel and p52, demonstrated that in LPS-stimulated U937 macrophages, rel-B and c-rel were not recruited to any of the above promoters, while p52 was recruited to TNFα, IL-6 and IL-1β promoters, but not to the promoter of IL-8.
Together, these data indicate that the inhibition of NFκB-dependent transcription by nuclear IκBα in LPS stimulated macrophages is gene specific, and depends on the S536 phosphorylation status of the recruited p65 NFκB. S536 phosphorylation of p65 NFκB at IL-8 promoter seems to inhibit acetylation at K310 and also binding of other NFκB members. More importantly, the S536 phosphorylation of p65 interferes with nuclear NFκB-IκBα binding, thus indicating an IκBα–independent NFκB regulation.
The overall significance of this work is that, understanding mechanisms by which nuclear IκBα regulates NFκB–dependent transcription of pro-inflammatory genes would lead to the development of specific therapies targeted to diseases characterized by excessive cytokine release.
