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

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 intracellul...

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Bibliographic Details
Published in:	The Journal of immunology (1950) Vol. 174; no. 6; pp. 3608 - 3616
Main Authors:	Banda, Nirmal K, Guthridge, Carla, Sheppard, Devon, Cairns, Kelly S, Muggli, Michele, Bech-Otschir, Dawadschargal, Dubiel, Wolfgang, Arend, William P
Format:	Journal Article
Language:	English
Published:	United States Am Assoc Immnol 15-03-2005
Subjects:	Binding Sites COP9 Signalosome Complex Cytokines - biosynthesis HeLa Cells Humans In Vitro Techniques Interleukin 1 Receptor Antagonist Protein Interleukin-1 - pharmacology Interleukin-6 - biosynthesis Interleukin-8 - biosynthesis Keratinocytes - drug effects Keratinocytes - immunology Keratinocytes - metabolism Nuclear Proteins p38 Mitogen-Activated Protein Kinases - metabolism Phosphorylation Protein Kinases - genetics Protein Kinases - immunology Protein Kinases - metabolism Proto-Oncogene Proteins - genetics Proto-Oncogene Proteins - immunology Proto-Oncogene Proteins - metabolism Recombinant Proteins - genetics Recombinant Proteins - immunology Recombinant Proteins - metabolism RNA, Small Interfering - genetics Sialoglycoproteins - genetics Sialoglycoproteins - immunology Sialoglycoproteins - metabolism Two-Hybrid System Techniques Yeasts
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Summary:	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.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2
ISSN:	0022-1767 1550-6606
DOI:	10.4049/jimmunol.174.6.3608