After stimulation, cells were fixed, permeabilized, and then stained with anti-ASC ((N-15)-R) antibody, followed by Alexa Fluor 488-conjugated anti-rabbit IgG and Hoechst 33342. triggers various cellular responses, such as cell death, NLRP3 inflammasome activation, and autophagy. The NLRP3 inflammasome is a multiple-protein complex comprising NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase 1, and the activation of this complex, in turn, activates caspase 1, which cleaves pro-IL-1 or pro-IL-18, generating the mature forms of these inflammatory cytokines, IL-1 or IL-18 (1). The NLRP3 inflammasome regulates multiple aspects of inflammation, and the dysregulation of this complex leads to undesirable inflammatory states. Limited by lysosome rupture, NLRP3 inflammasome activation has been associated with various human inflammatory diseases such as infection, pneumonia, gout, and atherosclerosis. Although lysosome rupture-induced NLRP3 inflammasome activation is BIRC3 considered the primary cause of inflammation, the underlying SKF38393 HCl mechanism is not fully understood. Recent studies have demonstrated that some kinases contribute to inflammasome activation. For example, the double-stranded RNA-dependent protein kinase (PKR) is activated through inflammasome-activating stimuli and kinase activity-dependent interactions with NLRP3, NLRP1, AIM2, and NLRC4, leading to the complete activation of the inflammasome (2). In response to infection, PKC phosphorylates the Ser-533 residue of NLRC4 to activate this inflammasome (3). In addition, it has SKF38393 HCl been shown recently that Syk and JNK are required for the activation of the inflammasomes NLRP3 and AIM2 through the regulation of ASC phosphorylation and oligomerization (4). There are abundant kinase inhibitor compounds available, and some kinase-targeted drugs have been used as clinical cues. Therefore, elucidating the regulatory mechanism of inflammasome activation through kinases might lead to new therapeutic developments. The stress-responsive MAPK pathway is activated through various stresses, such as oxidative stress and infection SKF38393 HCl (5, 6). Here we confirmed that JNK, a stress-responsive MAPK, is activated after lysosome rupture and that JNK inhibition suppresses NLRP3 inflammasome activation. Although the involvement of JNK in NLRP3 inflammasome activation has been verified, the mechanism underlying how lysosome rupture induces JNK activation remains poorly understood. In this study, we identified the lysosome rupture-induced Ca2+-CaMKII-TAK1-JNK pathway, which regulates NLRP3 inflammasome activation, using an siRNA screen for mitogen-activated protein kinase kinase kinases (MAP3Ks) and a screen for inhibitors. The results suggest that these inhibitors and kinases might be potential drug candidates and targets for regulating NLRP3 inflammasome activation. EXPERIMENTAL PROCEDURES Reagents and Antibodies Oxozeaenol, SB202190, SP600125, Bay11-7082, KN-93 water-soluble, KN-92 (Merck Millipore, Billerica, MA), LPS (O55:B5), CA-074ME, E-64d, bafilomycin A1, ATP, poly(dA:dT), disuccinimidyl suberate, dantrolene (Sigma-Aldrich, St. Louis, MO), l-leucyl-l-leucine methyl ester (LLME) (Chem-Impex International, Wood Dale, IL), calyculin A (LC Laboratories, Boston, MA), bis(2-aminophenyl)ethyleneglycol-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), Hoechst 33342 (Dojindo, Kumamoto, Japan), phorbol 12-myristate 13-acetate (PMA), xestospongin C (Wako Pure Chemical Industries, Osaka, Japan), and Texas Red-Dextran (Invitrogen) were purchased. Antibodies for p-TAK1 (Thr-184/187) (Cell Signaling Technology, catalog no. 4508), p-JNK (Thr-183/Tyr-185) (Cell Signaling Technology, catalog no. 9251), p-p38 (Thr-180/Tyr-182) (Cell Signaling Technology, catalog no. 9211), cleaved IL-1 (Cell Signaling Technology, catalog no. SKF38393 HCl 2021), p38 (L53F8, Cell Signaling Technology, catalog no. 9228), cleaved caspase 1 (Asp-297, D57A2, (Cell Signaling Technology, catalog no. 4199), TAK1 (M-579, Santa Cruz Biotechnology), caspase 1 p10 (C-20, Santa Cruz Biotechnology), caspase 1 caspase recruitment domain (A-19, Santa Cruz Biotechnology), JNK (FL, Santa Cruz Biotechnology), p38 (C-20-G, Santa Cruz Biotechnology), IB (C-21, Santa Cruz Biotechnology), ASC ((N-15)-R, Santa Cruz Biotechnology), ASC (TMS-1, Medical and Biological Laboratories, Nagoya, Aichi, Japan), p62 CT (Progen Biotechnik GmbH, Heidelberg, Germany), LC3 (Cosmo Bio, Tokyo, Japan), FLAG (1E6, Wako), FLAG (M2, Sigma), Actin (AC-40, Sigma), CD16/32 (mouse BD Fc block, BD Pharmingen), and PE Ly-6G (1A8, BioLegend, San Diego, CA) were purchased. Cell Culture THP-1, HEK293A, and HEK293FT cells were obtained from RIKEN, the ATCC, and Invitrogen, respectively. THP-1 cells were maintained in RPMI 1640 medium supplemented with 10% FBS. The HEK293A and HEK293FT cells were maintained in DMEM (4500 mg/liter glucose) supplemented with 10% FBS. THP-1 cells were infected with lentivirus carrying ASC-FLAG and selected in the presence of 0.5 g/ml puromycin for at least 2 weeks to generate ASC-FLAG-stable THP-1 cells. THP-1 macrophages were differentiated using 10 ng/ml PMA for 3 days, and the cells were changed to fresh medium containing 10 ng/ml PMA 1 day after treatment. Plasmid Construction.