Gene-Specific Repression of Proinflammatory Cytokines in Stimulated Human Macrophages by Nuclear IBa

We have previously shown that increased nuclear accumulation of IBa inhibits NF-B activity and induces apoptosis in human leukocytes. In this study, we wanted to explore the possibility that the nucleocytoplasmic distribution of IBa can be used as a therapeutic target for the regulation of NF-B-dependent cytokine synthesis. Treatment of LPS-stimulated human U937 macrophages with an inhibitor of chromosome region maintenance 1-dependent nuclear export, leptomycin B, resulted in the increased nuclear accumulation of IBa and inhibition of NF-B DNA binding activity, caused by the nuclear IBa-p65 NF-B interaction. Surprisingly, however, whereas mRNA expression and cellular release of TNF-a, the b form of pro-IL-1 (IL-1b), and IL-6 were inhibited by the leptomycin B-induced nuclear IBa, IL-8 mRNA expression and cellular release were not significantly affected. Analysis of in vivo recruitment of p65 NF-B to NF-B-regulated promoters by chromatin immunoprecipitation in U937 cells and human PBMCs indicated that although the p65 recruitment to TNF-a, IL-1b, and IL-6 promoters was inhibited by the nuclear IBa, p65 recruitment to IL-8 promoter was not repressed. Chromatin immunoprecipitation analyses using IBa and S536 phosphospecific p65 NF-B Abs demonstrated that although the newly synthesized IBa induced by postinduction repression is recruited to TNF-a, IL-1b, and IL-6 promoters but not to the IL-8 promoter, S536-phosphorylated p65 is recruited to IL-8 promoter, but not to TNF-a, IL-1b, or IL-6 promoters. Together, these data indicate that the inhibition of NF-B-dependent transcription by nuclear IBa in LPS-stimulated macrophages is gene specific and depends on the S536 phosphorylation status of the recruited p65 NF-B.