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G Protein Subunit Β 1 Negatively Regulates NLRP3 Inflammasome Activation

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G Protein Subunit Β 1 Negatively Regulates NLRP3 Inflammasome Activation Cutting Edge: G Protein Subunit β 1 Negatively Regulates NLRP3 Inflammasome Activation This information is current as Tomohiko Murakami, Lerdluck Ruengsinpinya, Eriko of September 26, 2021. Nakamura, Yoshifumi Takahata, Kenji Hata, Hiroaki Okae, Shun'ichiro Taniguchi, Masafumi Takahashi and Riko Nishimura J Immunol 2019; 202:1942-1947; Prepublished online 18 February 2019; Downloaded from doi: 10.4049/jimmunol.1801388 http://www.jimmunol.org/content/202/7/1942 Supplementary http://www.jimmunol.org/content/suppl/2019/02/15/jimmunol.180138 http://www.jimmunol.org/ Material 8.DCSupplemental References This article cites 20 articles, 3 of which you can access for free at: http://www.jimmunol.org/content/202/7/1942.full#ref-list-1 Why The JI? Submit online. by guest on September 26, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Cutting Edge: G Protein Subunit b 1 Negatively Regulates NLRP3 Inflammasome Activation Tomohiko Murakami,* Lerdluck Ruengsinpinya,* Eriko Nakamura,* † ‡ Yoshifumi Takahata,*x Kenji Hata,* Hiroaki Okae, Shun’ichiro Taniguchi, Masafumi Takahashi, and Riko Nishimura* The NLRP3 inflammasome has important roles in inflammasome consists of a sensor molecule, NLRP3, an the pathogenesis of various inflammatory diseases. adaptor protein, ASC, and caspase-1. Activation of the NLRP3 However, the regulatory mechanisms of the NLRP3 inflammasome has important roles in the pathogenesis of var- inflammasome are not fully understood. In this study, ious inflammatory diseases including autoimmune disorders, we attempted to identify molecules that interact with gout, type 2 diabetes, obesity, and Alzheimer disease (2). NLRP3 upon its activation. We identified G protein The NLRP3 inflammasome senses various types of exoge- Downloaded from subunit b 1(GNB1),adownstreammoleculeof nous and endogenous danger signals, including environmental G protein–coupled receptors (GPCRs), which regulates irritants, endogenous damage-associated molecular patterns, the NLRP3 inflammasome activation. GNB1 was phys- and organelle damage such as lysosome rupture, mitochondria ically associated with NLRP3 via the pyrin domain of damage, and endoplasmic reticulum stress (3). The NLRP3 inflammasome is activated by a two-step process (1–3). Signal 1, NLRP3. Activation of the NLRP3 inflammasome was http://www.jimmunol.org/ also known as the priming signal for the NLRP3 inflamma- enhanced in GNB1-knockdown or GNB1-deficient some, is mediated by microbial ligands recognized by TLR. murine macrophages, although a lack of GNB1 did Signal 1 activates the NF-kB pathway, resulting in the in- not affect activation of the AIM2 inflammasome. ASC creased expression of NLRP3 and pro–IL-1b. Signal 2, also oligomerization induced by NLRP3 was enhanced by known as the activation signal for the NLRP3 inflammasome, GNB1 deficiency. Conversely, NLRP3-dependent ASC is mediated by pathogen-associated molecular patterns or oligomerization was inhibited by the overexpression damage-associated molecular patterns, and promotes the as- of GNB1. This study indicates that GNB1 negatively sembly of ASC and pro–caspase-1, leading to activation of regulates NLRP3 inflammasome activation by suppress- the NLRP3 inflammasome. Several cellular signals such as by guest on September 26, 2021 ing NLRP3-dependent ASC oligomerization, and K+ efflux, Ca2+ signaling, mitochondria dysfunction, and re- it provides a regulatory mechanism of the NLRP3 active oxygen species have been proposed as the proximal triggers inflammasome. The Journal of Immunology, 2019, for NLRP3 inflammasome activation (2–5). These proximal 202: 1942–1947. triggers have been reported to be mediated by at least P2X7R, pore forming toxins, Ca2+ channels, and G protein– coupled receptors (GPCRs) (3, 6, 7) in response to NLRP3 nflammasomes are multiprotein complexes that are re- inflammasome activators. Additionally, recent studies iden- sponsible for the activation of inflammatory responses tified NEK7 as a new critical regulator of the NLRP3 I and which promote maturation of the proinflammatory inflammasome (8). However, the regulatory mechanisms of cytokine IL-1b and cell death by activating caspase-1 (1). the NLRP3 inflammasome are still elusive. Several nucleotide-binding domain and leucine-rich repeat In this study, to further understand the regulatory mecha- (LRR)–containing receptor (NLR) family proteins form nisms of the NLRP3 inflammasome, we searched for molecules inflammasomes in response to their specific stimulators. The that interact with NLRP3 in response to NLRP3 inflammasome NLR family pyrin domain (PYD)–containing 3 (NLRP3) activators and identified G protein subunit b 1(GNB1),a *Department of Molecular and Cellular Biochemistry, Osaka University Graduate Address correspondence and reprint requests to Dr. Tomohiko Murakami, Department of School of Dentistry, Osaka 565-0871, Japan; †Department of Informative Genetics, Molecular and Cellular Biochemistry, Osaka University Graduate School of Dentistry, 1-8 Environment and Genome Research Center, Tohoku University Graduate School of Yamada-Oka, Suita, Osaka 565-0871, Japan. E-mail address: [email protected] Medicine, Sendai 980-8575, Japan; ‡Department of Comprehensive Cancer Therapy, x The online version of this article contains supplemental material. Shinshu University School of Medicine, Nagano 390-8621, Japan; and Division of Inflammation Research, Center for Molecular Medicine, Jichi Medical University, Abbreviations used in this article: BMDM, bone marrow–derived macrophage; DSS, Tochigi 329-0498, Japan disuccinimidyl suberate; GNB1, G protein subunit b 1; GNB1 shRNA, GNB1 short hairpin RNA; GPCR, G protein–coupled receptor; IP3, inositol trisphosphate; KO, ORCIDs: 0000-0003-4367-9343 (K.H.); 0000-0003-3245-6670 (S.T.); 0000-0003- knockout; LRR, leucine-rich repeat; MSU, monosodium urate; NLRP3, NLR family 2716-7532 (M.T.). pyrin domain–containing 3; NP-40, Nonidet P-40; PLA, proximity ligation assay; PLC, Received for publication October 17, 2018. Accepted for publication January 30, 2019. phospholipase C; PYD, pyrin domain; shRNA, short hairpin RNA; WT, wild-type. This work was supported by Japan Society for the Promotion of Science grants-in-aid for Ó scientific research, the Nakajima Foundation, the Naito Foundation, the Kanae Foun- Copyright 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 dation for the Promotion of Medical Science, the Senri Life Science Foundation, and the Astellas Foundation for Research on Metabolic Disorders. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801388 The Journal of Immunology 1943 downstream molecule of GPCRs (9). We showed that GNB1 Plasmids and transfection negatively regulated NLRP3 inflammasome activation by inhib- Expression vectors for mouse GNB1 were purchased from GenScript. Mouse iting ASC oligomerization, which is necessary for full activation NLRP3, ASC, and AIM2 expression vectors were used as described previously of the NLRP3 inflammasome. Our findings reveal a regulatory (12). Deletion mutants of NLRP3 were generated by PCR and standard ligation procedures and were subcloned into pcDNA4 Myc-His (Invitrogen). mechanism of the NLRP3 inflammasome by G protein subunit. Cells were transfected with the indicated plasmids using X-tremeGENE 9 (Sigma-Aldrich). Materials and Methods Lentivirus infection Mice 2 2 Lentiviral pLKO.1 plasmids targeting Gnb1 (GNB1 short hairpin RNA C57BL/6J (B6) mice were obtained from Japan SLC. ASC-deficient (Asc / , 2/2 [GNB1 shRNA]) (TRCN0000034431) were purchased from Sigma-Aldrich. B6 background) and GNB1-deficient (Gnb1 , B6 background) mice were GFP shRNA was used as a negative control. Lentiviral particle preparation generated and genotyped as described previously (10, 11). All animal exper- and infection were performed according to the manufacturer’s protocol iments were approved by the Osaka University Institute Animal Experiment (Sigma-Aldrich). BMDMs were infected with control or GNB1 shRNA and Committee and performed in accordance with the regulatory guidelines. selected by 4 mg/ml puromycin in culture. Chemicals, Abs, and other reagents Immunoblotting and ELISA LPS (L3024), ATP (A6419), and calf thymus DNA (D3664) were purchased Immunoblotting analyses were performed as previously described (4). Im- from Sigma-Aldrich. Nigericin (145-07263) was purchased from Wako Pure munoblotting with anti–b-actin Ab was performed to assess the amount of Chemical. Monosodium urate (MSU; AG-CR1-3950) was purchased from protein in each sample. The ASC oligomerization assay was performed as AdipoGen. FliC (ALX-522-058) was purchased from Enzo Life Sciences. described previously (13). Pellets from whole-cell lysates were cross-linked Downloaded from For immunoblotting, anti-NLRP3 (Cryo-2, 1:5000) and anti-ASC
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