The major finding of this study is that the nuclear IκBα has a differential effect on the in vivo expression of NF-κB–regulated pro-inflammatory genes. Whereas transcription of TNF-α, IL-1β, and IL-6 in LPS-stimulated human macrophages is inhibited by the nuclear IκBα, transcription of IL-8 is not. We have previously shown that the increased nuclear accumulation of IκBα inhibits in vitro NF-κB DNA binding and expression of NF-κB–dependent anti-apoptotic genes (29
). In this study, we show that LMB inhibits proinflammatory cytokine release from stimulated human macrophages () by inducing nuclear accumulation of IκBα (), which then associates with p65 NF-κB in the nucleus (), resulting in the inhibition of NF-κB DNA-binding activity () and inhibition of transcription (). However, quantitative ChIP analysis of the in vivo p65 NF-κB recruitment to NF-κB–dependent promoters revealed that the regulation of NF-κB–dependent transcription by LMB-induced nuclear IκBα is gene specific. Whereas the LMB-induced nuclear IκBα inhibits p65 NF-κB recruitment to TNF-α, IL-1β, and IL-6 promoters (), thus inhibiting transcription of these genes (), it does not remove p65 NF-κB from IL-8 promoter () and does not inhibit IL-8 transcription (). Importantly, our results indicate that an identical or a similar mechanism is operational in primary human leukocytes, because the LMB-induced nuclear IκBα inhibited p65 NF-κB recruitment to the TNF-α but not the IL-8 promoter in LPS-stimulated human PBMCs (). In addition, ChIP analysis using Abs specific against the S536-phosphorylated form of p65 NF-κB demonstrated that the S536 p65 is specifically recruited to IL-8 promoter, but not to TNF-α, IL-1β, or IL-6 promoters (), indicating that this phosphorylation may represent one of the mechanisms responsible for the gene-specific inhibition of NF-κB–dependent transcription by nuclear IκBα.
The regulation of NF-κB recruitment to target genes is extremely complex and depends on the cell stimulus and on the cell type that is mediating the inflammatory response (10
). For example, in LPS-stimulated murine macrophages, p65 NF-κB is recruited to the IL-6 promoter 2 h poststimulation, whereas in murine fibroblasts, it takes only 15 min (47
). Using quantitative ChIP analysis of the in vivo binding of NF-κB and IκBα proteins to TNF-α, IL-1β, IL-6, and IL-8 promoters, we demonstrate in this paper that both p65 and p50 NF-κB are recruited to IL-1β and IL-6 promoters in 6-h LPS-stimulated human macrophages, whereas TNF-α and IL-8 promoters contain predominantly p65 NF-κB (). These data indicate that the in vivo transcription of IL-1β and IL-6 genes in LPS-stimulated macrophages is regulated by p50/65 NF-κB heterodimers, whereas the synthesis of TNF-α and IL-8 is controlled by p65 homodimers. Interestingly, the TNF-α and IL-8 genes that recruited predominantly p65 NF-κB () are induced earlier () than the IL-1β and IL-6 genes () that recruited both p65 and p50 NF-κB (). In addition, the IL-1β and IL-6 genes are induced to a considerably higher level than the TNF-α and IL-8 genes (), indicating that the p50/65 heterodimer is associated with a higher level of transcription.
In macrophages stimulated 6 h with LPS, the newly synthesized IκBα induced by postinduction repression was recruited to TNF-α, IL-1β, and IL-6 promoters, but not to the IL-8 promoter (). These results suggest that the IL-8 promoter is not regulated by IκBα in vivo and that the regulation of NF-κB–dependent transcription by nuclear IκBα is promoter specific. This is further supported by the fact that the LMB-induced nuclear IκBα does not inhibit IL-8 transcription, whereas it inhibits transcription of TNF-α, IL-1β, and IL-6 genes (). Consistent with this finding, it has been previously shown in vitro that the IL-8 promoter binds p65 NF-κB homodimers, whereas it does not bind p50/65 heterodimers (49
). In addition, a recent study using ChIP analysis demonstrated a selective recruitment of p65 NF-κB to the IL-8 promoter in dendritic cells (47
). It is interesting to note, however, that in Jurkat cells, the portion of p65 that is specifically targeted to the IL-8 promoter is phosphorylated on S536, resulting in its inability to bind IκBα in vitro (40
Our results indicate that the S536 phosphorylation of p65 also regulates its interaction with IκBα in vivo in LPS-stimulated macrophages, and this phosphorylation may represent one of the mechanisms responsible for the gene-specific inhibition of NF-κB–dependent transcription by nuclear IκBα. The unphosphorylated p65 that associates with TNF-α, IL-1β, and IL-6 promoters is removed by binding to nuclear IκBα, whereas the S536-phosphorylated portion of p65 binds, as a homodimer, to the IL-8 promoter, independently of the nuclear IκBα. This S536 phosphorylation of p65 NF-κB has been reported to be mediated by the enzymes of IκB kinase complex and has been shown to regulate p65 acetylation and transcription activity (50
). It might occur before p65 binds to DNA, either in the cytoplasm or in the nucleus, or after, as a part of the preinitiation complex assembly.
In addition, the strength of the nuclear IκBα-p65 NF-κB interaction might be influenced by the DNA sequence of κB response elements in the regulatory regions of NF-κB–dependent genes. In this model, the IL-8 promoter sequence may allow a stronger binding of the S536-phosphorylated p65 NF-κB, which has a significantly lower affinity for the nuclear IκBα. This would be consistent with a recent study demonstrating that a single nucleotide can influence the recruitment of specific NF-κB dimers and the required cofactors for efficient gene transcription (44
). In this context, we have compared the NF-κB promoter sequences of IL-1β, IL-6, TNF-α, and IL-8 genes used in this study (). Interestingly, the TNF-α and IL-8 NF-κB binding sites that recruited predominantly p65 NF-κB had C and T at the 8th and 10th position, respectively; however the IL-8 site differs from the TNF-α promoter sequence in the third position, having A instead of G (). Thus, according to this model, A in the third position would increase affinity for the S536-phosphorylated p65 binding or recruitment of the corresponding kinase, which phosphorylates p65 on S536, thus decreasing its affinity for the nuclear IκBα. Future studies should determine whether p65 is phosphorylated on S536 before its binding to the IL-8 promoter or whether the corresponding kinase is preferentially recruited to IL-8 promoter, where it phosphorylates p65, thus inhibiting its interaction with IκBα.
LMB belongs to a group of CRM1 inhibitors that have been investigated for their ability to increase the nuclear accumulation of IκBα, p53, and the oncoprotein BCR-ABL in cells of myeloid origin, suggesting their anticancer and anti-inflammatory potential (54
). In stimulated human leukocytes, LMB induces apoptosis by inhibiting the NF-κB and caspase 3 activities (29
). One of the earliest events in the LMB-induced changes is probably the increased accumulation of IκBα in the nucleus and its gene-specific interaction with NF-κB–dependent promoters. Although most promoters will presumably behave similarly to TNF-α, IL-1β, or IL-6 promoters, and the NF-κB recruitment will be inhibited by the nuclear IκBα, IL-8 may not be the only gene that is not repressed by LMB and nuclear IκBα. It will be important in future to analyze the genes that preferentially associate with the S536-phosphorylated form of p65 similar to IL-8, because these genes will be expected to be resistant to postinduction repression and inhibition by the endogenous nuclear IκBα. Differences in the transcriptional regulation by nuclear IκBα might hold the key for development of more specific therapies for inflammatory disorders and cancers characterized by the excessive activation of NF-κB and cytokine release.