title: Gene-specific repression of proinflammatory cytokines in stimulated human macrophages by nuclear IκBα.
abstract: We have previously shown that increased nuclear accumulation of IkappaBalpha inhibits NF-kappaB activity and induces apoptosis in human leukocytes. In this study, we wanted to explore the possibility that the nucleocytoplasmic distribution of IkappaBalpha can be used as a therapeutic target for the regulation of NF-kappaB-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 IkappaBalpha and inhibition of NF-kappaB DNA binding activity, caused by the nuclear IkappaBalpha-p65 NF-kappaB interaction. Surprisingly, however, whereas mRNA expression and cellular release of TNF-alpha, the beta form of pro-IL-1 (IL-1beta), and IL-6 were inhibited by the leptomycin B-induced nuclear IkappaBalpha, IL-8 mRNA expression and cellular release were not significantly affected. Analysis of in vivo recruitment of p65 NF-kappaB to NF-kappaB-regulated promoters by chromatin immunoprecipitation in U937 cells and human PBMCs indicated that although the p65 recruitment to TNF-alpha, IL-1beta, and IL-6 promoters was inhibited by the nuclear IkappaBalpha, p65 recruitment to IL-8 promoter was not repressed. Chromatin immunoprecipitation analyses using IkappaBalpha and S536 phosphospecific p65 NF-kappaB Abs demonstrated that although the newly synthesized IkappaBalpha induced by postinduction repression is recruited to TNF-alpha, IL-1beta, and IL-6 promoters but not to the IL-8 promoter, S536-phosphorylated p65 is recruited to IL-8 promoter, but not to TNF-alpha, IL-1beta, or IL-6 promoters. Together, these data indicate that the inhibition of NF-kappaB-dependent transcription by nuclear IkappaBalpha in LPS-stimulated macrophages is gene specific and depends on the S536 phosphorylation status of the recruited p65 NF-kappaB.
authors.affiliation: Department of Biological Sciences, St. John's University, New York, NY 11439, USA.
authors.forename: Chandra C
authors.initials: CC
authors.lastname: Ghosh
authors.forename: Sitharam
authors.initials: S
authors.lastname: Ramaswami
authors.forename: Ashish
authors.initials: A
authors.lastname: Juvekar
authors.forename: Hai-Yen
authors.initials: HY
authors.lastname: Vu
authors.forename: Luciano
authors.initials: L
authors.lastname: Galdieri
authors.forename: Dennis
authors.initials: D
authors.lastname: Davidson
authors.forename: Ivana
authors.initials: I
authors.lastname: Vancurova
mesh_terms: D000328:Adult; D016207:Cytokines / Q000037:antagonists & inhibitors* / Q000502:physiology; D015536:Down-Regulation / Q000276:immunology*; D005786:Gene Expression Regulation / Q000276:immunology*; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism / Q000502:physiology*; D018836:Inflammation Mediators / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000502:physiology; D053583:Interleukin-1beta / Q000235:genetics / Q000378:metabolism; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism; D008262:Macrophage Activation / Q000235:genetics / Q000276:immunology*; D000072000:NF-KappaB Inhibitor alpha; D009687:Nuclear Proteins / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000276:immunology; D051996:Transcription Factor RelA / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism; D020298:U937 Cells
chemical_list: D016207:Cytokines; D020672:I-kappa B Proteins; D018836:Inflammation Mediators; D053583:Interleukin-1beta; D015850:Interleukin-6; D016209:Interleukin-8; C000606204:NFKBIA protein, human; D009687:Nuclear Proteins; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha