title: The sequestosome 1/p62 attenuates cytokine gene expression in activated macrophages by inhibiting IFN regulatory factor 8 and TNF receptor-associated factor 6/NF-kappaB activity.
abstract: Sequestosome 1/p62 (p62) is a scaffold/adaptor protein with multiple functions implicated for neuronal and bone diseases. It carries a ubiquitin binding domain through which it mediates proteasome-dependent proteolysis. In addition, p62 is reported to regulate NF-kappaB activity in some cells. To date, however, the role of p62 in innate immunity has not been fully elucidated. In this study, we report that IFN-gamma plus TLR signaling stimulates late expression of p62 in murine macrophages. Overexpression of p62 inhibited expression of multiple cytokines, IL-12p40, TNF-alpha, IL-1beta, IL-6, and IFN-beta, whereas p62 underexpression by small hairpin RNA markedly elevated their expression, indicating that p62 is a broad negative regulator of cytokine expression in stimulated macrophages. We show that p62 interacts with IFN regulatory factor 8 and Ro52, the transcription factor and ubiquitin E3 ligase that are important for IL-12p40 expression. This interaction, detectable at a late stage in stimulated macrophages, led to increased polyubiquitination and destabilization of IFN regulatory factor 8. We also show that upon macrophage stimulation, p62 binds to TNFR-associated factor 6, another E3 ligase important for NF-kappaB activation, but later this interaction was replaced by the recruitment of the deubiquitinating enzyme, cylindromatosis, an inhibitor of NF-kappaB activity. Recruitment of cylindromatosis coincided with reduced TNFR-associated factor 6 autoubiquitination and lower NF-kappaB activation. Our results indicate that p62 orchestrates orderly regulation of ubiquitin modification processes in macrophages to ensure attenuation of cytokine transcription postactivation. Together, p62 may provide a mechanism by which to control excessive inflammatory responses after macrophage activation.
authors.affiliation: Laboratory of Molecular Growth Regulation, Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Ji Young
authors.initials: JY
authors.lastname: Kim
authors.forename: Keiko
authors.initials: K
authors.lastname: Ozato
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000276:immunology* / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D016207:Cytokines / Q000096:biosynthesis; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression / Q000276:immunology; D005786:Gene Expression Regulation / Q000276:immunology*; D006360:Heat-Shock Proteins / Q000276:immunology* / Q000378:metabolism; D015151:Immunoblotting; D047468:Immunoprecipitation; D050835:Interferon Regulatory Factors / Q000276:immunology* / Q000378:metabolism; D053762:Interleukin-12 Subunit p40 / Q000096:biosynthesis / Q000235:genetics; D008262:Macrophage Activation / Q000276:immunology; D008264:Macrophages / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000276:immunology* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000071456:Sequestosome-1 Protein; D048029:TNF Receptor-Associated Factor 6 / Q000276:immunology* / Q000378:metabolism; D014162:Transfection; D054875:Ubiquitination; D000099185:Interferon Regulatory Factor-8
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D016207:Cytokines; D006360:Heat-Shock Proteins; D050835:Interferon Regulatory Factors; D053762:Interleukin-12 Subunit p40; D016328:NF-kappa B; D000071456:Sequestosome-1 Protein; D048029:TNF Receptor-Associated Factor 6; D000099185:Interferon Regulatory Factor-8; C101343:Sqstm1 protein, mouse