title: Intracellular IL-1 receptor antagonist type 1 inhibits IL-1-induced cytokine production in keratinocytes through binding to the third component of the COP9 signalosome.
abstract: The IL-1 receptor antagonist (IL-1Ra) exists in four isoforms, three of which lack signal peptides and are primarily intracellular proteins. The biologic roles of the intracellular isoforms of IL-1Ra have remained unknown. The objective of these studies was to determine whether the major intracellular isoform of IL-1Ra 18-kDa type 1 (icIL-1Ra1), mediated unique functions inside cells. A yeast two-hybrid screen with HeLa cell lysates revealed specific binding of icIL-1Ra1, and not of the other IL-1Ra isoforms, to the third component of the COP9 signalosome complex (CSN3). This binding was confirmed by Far Western blot analysis, sedimentation on a glycerol gradient, glutathione pull-down experiments, and coimmunoprecipitation. In addition to binding specifically to CSN3, icIL-1Ra1 inhibited phosphorylation of p53, c-Jun, and IkappaB by the crude CSN-associated kinase and of p53 by recombinant protein kinase CK2 and protein kinase D, both associated with CSN3. The biologic relevance of the interaction between icIL-1Ra1 and CSN3 was demonstrated in the keratinocyte cell lines KB and A431, both possessing abundant CSN3. A431 cells exhibited high levels of icIL-1Ra1 but lacked both detectable IL-1alpha-induced IL-6 and IL-8 production and phosphorylation of p38 MAPK. KB cells displayed the opposite pattern which was reversed after transfection with icIL-1Ra1 mRNA. Inhibition of CSN3 or of icIL-1Ra1 production through gene knockdown with specific small interfering RNA in A431 cells each led to an inhibition of IL-1alpha-induced IL-6 and IL-8 production. Thus, icIL-1Ra1 exhibits unique anti-inflammatory properties inside cells through binding to CSN3 with subsequent inhibition of the p38 MAPK signal transduction pathway.
authors.affiliation: Division of Rheumatology, Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 20262, USA.
authors.forename: Nirmal K
authors.initials: NK
authors.lastname: Banda
authors.forename: Carla
authors.initials: C
authors.lastname: Guthridge
authors.forename: Devon
authors.initials: D
authors.lastname: Sheppard
authors.forename: Kelly S
authors.initials: KS
authors.lastname: Cairns
authors.forename: Michele
authors.initials: M
authors.lastname: Muggli
authors.forename: Dawadschargal
authors.initials: D
authors.lastname: Bech-Otschir
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Dubiel
authors.forename: William P
authors.initials: WP
authors.lastname: Arend
mesh_terms: D001665:Binding Sites; D000075686:COP9 Signalosome Complex; D016207:Cytokines / Q000096:biosynthesis*; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1 / Q000494:pharmacology; D015850:Interleukin-6 / Q000096:biosynthesis; D016209:Interleukin-8 / Q000096:biosynthesis; D015603:Keratinocytes / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*; D009687:Nuclear Proteins; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012795:Sialoglycoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: C477661:COPS3 protein, human; D016207:Cytokines; C507763:IL1RN protein, human; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1; D015850:Interleukin-6; D016209:Interleukin-8; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D012795:Sialoglycoproteins; D011494:Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D000075686:COP9 Signalosome Complex