title: Cryopyrin-induced interleukin 1beta secretion in monocytic cells: enhanced activity of disease-associated mutants and requirement for ASC.
abstract: Several autoinflammatory disorders are associated with missense mutations within the nucleotide-binding oligomerization domain of cryopyrin. The mechanism by which cryopyrin mutations cause inflammatory disease remains elusive. To understand the molecular bases of these diseases, we generated constructs to express three common cryopyrin disease-associated mutations, R260W, D303N, and E637G, and compared their activity with that of the wild-type protein. All cryopyrin mutant proteins tested were found to induce potent NF-kappaB activity when compared with the wild-type protein. This activation was dependent on the expression of ASC, an adaptor protein previously suggested to mediate cryopyrin signaling. When the disease-associated mutants were expressed in monocytic THP-1 cells (which express endogenous ASC), each induced spontaneous IL-1beta secretion, whereas wild-type protein did not. In the absence of stimuli, wild-type cryopyrin was unable to bind to ASC, whereas the three mutants coimmunoprecipitated with ASC, suggesting a mechanism involved in the constitutive activation of mutant proteins. The induction of cryopyrin activity by enforced oligomerization in THP-1 cells resulted in ASC binding and the secretion of IL-1beta, an effect that was abolished by the inhibition of ASC expression with small interfering RNAs. Thus, cryopyrin-mediated IL-1beta secretion requires ASC in monocytic cells. Further, these results indicate that cryopyrin disease-associated mutants are constitutively active and able to induce NF-kappaB activation and IL-1beta secretion at least in part by an increased ability to interact with ASC.
authors.affiliation: Department of Pathology and Comprehensive Cancer Center, The University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
authors.forename: Theresa A
authors.initials: TA
authors.lastname: Dowds
authors.forename: Junya
authors.initials: J
authors.lastname: Masumoto
authors.forename: Li
authors.initials: L
authors.lastname: Zhu
authors.forename: Naohiro
authors.initials: N
authors.lastname: Inohara
authors.forename: Gabriel
authors.initials: G
authors.lastname: Núñez
mesh_terms: D017209:Apoptosis; D053468:CARD Signaling Adaptor Proteins; D002352:Carrier Proteins / Q000494:pharmacology*; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007375:Interleukin-1 / Q000235:genetics / Q000378:metabolism*; D009000:Monocytes / Q000378:metabolism*; D009154:Mutation; D020125:Mutation, Missense; D016328:NF-kappa B / Q000378:metabolism; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation
chemical_list: D053468:CARD Signaling Adaptor Proteins; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D007375:Interleukin-1; D016328:NF-kappa B; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C453881:NLRP3 protein, human; C402202:PYCARD protein, human; D034741:RNA, Small Interfering