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The Sequestosome 1/p62 Attenuates Cytokine Gene Expression in Activated Macrophages by Inhibiting IFN Regulatory Factor 8 and TNF Receptor-Associated Factor 6/NF-B Activity¹

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

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-B 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- plus TLR signaling stimulates late expression of p62 in murine macrophages. Overexpression of p62 inhibited expression of multiple cytokines, IL-12p40, TNF-, IL-1β, IL-6, and IFN-β, 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-B activation, but later this interaction was replaced by the recruitment of the deubiquitinating enzyme, cylindromatosis, an inhibitor of NF-B activity. Recruitment of cylindromatosis coincided with reduced TNFR-associated factor 6 autoubiquitination and lower NF-B 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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the intramural program of National Institute of Child Health and Human Development and in part by the Intramural AIDS Targeted Antiviral Program of the National Institutes of Health.

² Address correspondence and reprint requests to Dr. Keiko Ozato, Building 6, Room 2A01, Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development, National Institutes of Health, 6 Center Drive, Bethesda, MD 20892-2753. E-mail address: ozatok{at}nih.gov

³ Abbreviations used in this paper: IRF, IFN regulatory factor; Co-IP, coimmunoprecipitation; CYLD, cylindromatosis; HA, hemagglutinin; IKK, inhibitor of IB kinase; qRT-PCR, quantitative RT-PCR; shRNA, small hairpin RNA; TRAF6, TNFR-associated factor 6.

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