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J. Biol. Chem., Vol. 279, Issue 21, 21924-21928, May 21, 2004
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Secretion in Monocytic Cells
¶
From the
Department of Pathology and Comprehensive Cancer Center, The University of Michigan Medical School, Ann Arbor, Michigan 48109
Received for publication, February 3, 2004 , and in revised form, March 9, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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B 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-1
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-1, an effect that was abolished by the inhibition of ASC expression with small interfering RNAs. Thus, cryopyrin-mediated IL-1 secretion requires ASC in monocytic cells. Further, these results indicate that cryopyrin disease-associated mutants are constitutively active and able to induce NF-B activation and IL-1 secretion at least in part by an increased ability to interact with ASC. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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More than 20 of the identified missense cryopyrin disease-associated mutations described in patients with FCAS, MWS, and NOMID are found within the centrally located NOD (1, 7, 8). The NOD contains several distinct motifs including nucleotide-binding and Mg2+-binding sites, referred to as the Walker A and B motifs, respectively. Many of the disease-associated mutants are clustered near the predicted Mg2+-binding site (1, 7). Notably, similar missense mutations have been found in the NOD of Nod2 in patients with Blau syndrome, another autosomal-dominant autoinflammatory syndrome (8, 9). Interestingly, the R260W cryopyrin mutation (identified in both FCAS and MWS) and the R334W mutation (in Blau syndrome) involve amino acid residues at analogous sequence positions, suggesting a common molecular mechanism for the development of autoinflammatory disease (8, 9).
The signaling pathways activated through cryopyrin have just begun to be identified. Previous studies have shown that the PD of cryopyrin interacts with ASC, a PD/CARD-containing adaptor molecule that has been suggested to mediate cryopyrin signaling (5, 10). Oligomerization through the NOD has been suggested to be critical to interaction with downstream molecules and the function of several NOD family members including Apaf-1, Nod1, and Nod2 (3, 11). Similarly, oligomerization of cryopyrin PD in the presence of ASC has been shown to induce NF-B activation and apoptosis (10). Other studies have reported that overexpression of ASC promotes caspase-1 activation through a CARD-CARD interaction, an event that can result in the processing of pro-IL-1 to mature IL-1 (12, 13). In addition, cryopyrin and ASC have also been suggested to act as inhibitors of NF-B and IL-1 secretion, depending on the amount expressed in the cells (14–16). Thus, the role and requirement of ASC for cryopyrin-mediated IL-1 production remain unclear.
Several lines of evidence suggest that IL-1 is an important mediator of inflammation in patients with cryopyrin mutations (17–19). IL-1 was found to be up-regulated in unstimulated monocytes from patients with NOMID syndrome (17). More significantly, clinical studies involving nonrelated MWS patients harboring the cryopyrin disease-associated R260W variant had a rapid clinical and serologic response following injection with an IL-1 receptor antagonist (18, 19). In the current studies, we compared the functional activity of wild-type and disease-associated cryopyrin mutants to understand the molecular basis of FCAS, MWS, and NOMID diseases. We found that cryopyrin mutants act as constitutively active proteins, providing a mechanism whereby cryopyrin mutations lead to dominant autoinflammatory disease. In addition, we provide evidence that ASC is required for IL-1 secretion induced through cryopyrin activation in monocytic THP-1 cells.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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Cell Culture and Transfection—Human embryonic kidney (HEK)293T cells were maintained and transfected with expression plasmids as described by Koseki et al. (20). The total amount of transfected plasmid DNA was adjusted with pcDNA3 to be consistent within individual experiments. THP-1 cells were cultured in RPMI 1640 medium (Invitrogen), 10% heat-inactivated fetal bovine serum, penicillin, and streptomycin. The Amaxa NucleofectorTM (Amaxa, Cologne, Germany) was used to transfect 1 x 106 cells with 3 µg of plasmid DNA as described by the manufacturer's protocol. 24 h post-transfection, analysis of green fluorescent protein (GFP) and -galactosidase expression was used to gauge transfection efficiency. For the generation of vector or R260W stable cell lines, selection was carried out using 0.8 µg/ml G418 (Invitrogen) for 18 days.
The THP-1 cryopyrinPD-Fpk3 and Fpk3 vector stable cell lines were generated by retrovirus delivery. Briefly, 293T cells were transfected by calcium phosphate method with pMSCVpuro-cryopyrinPD-Fpk3-Myc or pMSCVpuro-Fpk3-Myc. The supernatants from these cells were used to infect 1 x 107 THP-1 cells. Post-infection, puromycin-resistant THP-1 cells were selected by 0.5 µg/ml puromycin for 14 days. In some experiments, 200 nM AP1510 (Ariad Pharmaceuticals) or 1 ng/ml lipopolysaccharide from Escherichia coli 055B5 (Sigma) was used to treat the THP-1 cells.
NF-B Assay—1 x 105 HEK293T cells were co-transfected with a construct of interest in the presence of 33 ng of pEF1BOS--gal and 2.2 ng of pBxVI-luc reporter. NF-B luciferase reporter activity was measured 24 h post-transfection, and the values were normalized to -galactosidase from triplicate cultures. The results are given as the mean ± S.D.
IL-1 ELISA—Assays were performed using matched Ab pairs (BD Biosciences) and the manufacturer's recommended protocol. ELISAs were developed for 5 min using tetramethylbenzidine substrate (Bio F/X, Owings Mills, MD) and then stopped with 2 N H2SO4. Within 30 min, the assay plates were read at 450 nm with a correction at 570 nm and analyzed with KC Jr. software (Bio-Tek Instruments, Winooski, VT).
Immunoprecipitation and Detection of Proteins—1 x 108 THP-1 stably expressing cryopyrinPD-Fpk3-Myc or Fpk3-Myc were incubated in the presence or absence of 200 nM AP1510 for 16 h. The cells were rinsed with phosphate-buffered saline and lysed in Nonidet P-40 lysis buffer for immunoprecipitations, which were performed as described previously (10). The antibodies used for immunoprecipitation and immunodetection were anti-FLAG monoclonal (Santa Cruz), anti-Myc polyclonal (Santa Cruz), anti-ASC polyclonal (
ProSci), anti-ASC monoclonal (21), and anti-tubulin monoclonal (Sigma).
Small Interfering RNA—Sense and antisense oligonucleotides corresponding to the following cDNA sequences were purchased from Dharmacon: 5'-AACTGGACCTGCAAGGACTTG-3' (nucleotides 508–528) for ASC and 5'-GGCTACGTCCAGGAGCGCACC-3' for GFP used as control. THP-1 stably expressing cryopyrinPD-Fpk3-Myc or Fpk3-Myc were transfected with Amaxa NucleofectorTM (Amaxa, Cologne, Germany) according to the manufacturer's protocol with siRNA. In these experiments, 10 h post-transfection with siRNA, the cells were treated with AP1510 (Ariad Pharmaceuticals) where indicated followed by IL-1 ELISA of supernatants 12 h post-treatment with ligand.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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B in the Presence of ASC—We engineered full-length cryopyrin expression plasmids containing R260W, D303N, or E627G missense mutations commonly found in patients with FCAS, MWS, and NOMID to compare their activity to that of the wild-type protein. The R260W and D303N mutations are located near the Walker A and B boxes, respectively, whereas the E627G mutation in the NOD is proximal to the LRRs (Fig. 1A). Because HEK293T cells lack endogenous ASC, we transiently co-expressed constructs producing cryopyrin wild-type or disease-associated mutant proteins with ASC and measured NF-B activity. To minimize the effect of overexpression, we transfected very low amounts of cryopyrin and ASC expression plasmids and found that all three of the disease-associated mutations induced potent activation of NF-B, whereas the wild-type cryopyrin did not induce any detectable NF-B activation (Fig. 1B). This finding could not be explained by the differential expression of mutant and wild-type cryopyrin as determined by immunoblotting analysis (Fig. 1C). To confirm the functionality of the wild-type construct, we transfected a higher concentration of plasmid reported previously to induce NF-B activation in the presence of ASC (10). This increased amount of wild-type cryopyrin induced a significant but low level of NF-B activation, at least 10-fold less than that observed with the disease-associated mutants.2 Thus, at all plasmid concentrations tested, the ability of the cryopyrin mutants to induce NF-B activation was greatly enhanced when compared with the wild-type protein. In the absence of ASC, neither the wild-type nor the disease-associated cryopyrin mutants (at any concentration tested) induced detectable NF-B activation (Fig. 1B).
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Expression of Cryopyrin Disease-associated Mutants Induces Secretion of IL-1 in THP-1 Cells—In addition to NF-B activation, cryopyrin has been suggested to regulate a signaling pathway leading to the production of IL-1 (5, 15). Therefore, we compared the ability of disease-associated mutants and wild-type cryopyrin to induce the secretion of IL-1 in human monocytic THP-1 cells. THP-1 cells express endogenous ASC; therefore we transfected only the disease-causing mutants R260W, D303N, and E627G or wild-type cryopyrin and measured IL-1 production in the culture supernatants 24 h posttransfection. All three disease-associated mutants, as well as a mutant cryopyrin lacking the LRR that exhibits enhanced activity (5, 10), induced secretion of IL-1, whereas wild-type cryopyrin did not (Fig. 2A). Consistent with these results, THP-1 cells stably transfected with the disease-associated R260W mutant spontaneously produced IL-1, whereas cells transfected with wild-type cryopyrin or vector alone did not (Fig. 2B). In control experiments, stimulation of THP-1 cells expressing wild-type cryopyrin or vector alone with lipopolysaccharide induced the production of IL-1, indicating that the cells had the ability to produce IL-1.2 These studies demonstrate that expression of the disease-associated mutants in monocytic cells induced IL-1 secretion in a constitutively active manner without a stimulating ligand. Consistent with this, monocytes from a NOMID patient harboring the D303N cryopyrin mutation expressed up-regulated amounts of IL-1 as well as tumor necrosis factor, IL-3, IL-5, and IL-6 in the absence of stimulation (17).
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The Cryopyrin Disease-associated Mutants Exhibit Increased Binding to ASC—It has been suggested that cryopyrin mediates its activity by binding to ASC, although the interaction of full-length cryopyrin with ASC has not been detected (5, 10). Consistent with this, when we tested the ability of wild-type cryopyrin and the disease-associated mutants to interact with ASC in co-immunoprecipitation assays, wild-type cryopyrin failed to bind to ASC (Fig. 3). In contrast, the cryopyrin mutants R260W, D303N, E627G, or 
LRR readily co-immunoprecipitated with ASC (Fig. 3). These results indicate that the disease-associated cryopyrin mutants exhibit an enhanced ability to interact with ASC in the absence of stimulating ligand. This increased association with ASC could explain, at least in part, the constitutive activity exerted by the mutant cryopyrin proteins. The amino acid residues mutated in these inflammatory syndromes could mediate the release of the inhibitory function, resulting in increased availability of the PD for interaction with ASC. Furthermore, a computer-generated model of the cryopyrin structure has suggested that the Arg-260 and Asp-303 residues are located along the nucleotide-binding cleft (7). Thus, the R260W and D303N missense mutations may cause alterations in nucleotide binding and/or hydrolysis, all of which could result in conformational changes leading to increased ASC binding and deregulated signaling. Notably, the R260W cryopyrin mutation corresponds to the identified R334W Nod2 mutation in Blau syndrome, another dominant autoinflammatory disease (8, 9). Thus, mutations in different NOD proteins may share a common mechanism to induce protein activation and inflammatory disease.
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Secretion—The findings with the disease-associated cryopyrin mutants suggested that the interaction of cryopyrin with ASC is important for signaling. However, the role of ASC in cryopyrin-mediated IL-1 secretion has not been determined in monocytic cells that express endogenous ASC. To examine this, we developed a THP-1 cell line that stably expressed a chimeric protein composed of the effector PD of cryopyrin fused to three tandem Fkbp domains (cryopyrinPD-Fpk3) that can be oligomerized by the cell-permeable ligand AP1510 (Fig. 4A) (10). Using this inducible system of cryopyrin activation, we found that cryopyrinPD-Fpk3 co-immunoprecipitated with endogenous ASC only in the presence of AP1510, suggesting that oligomerization of cryopyrin PD is required for endogenous ASC binding (Fig. 4B). Moreover, this interaction correlated with the ability of these stable THP-1 cells to secrete IL-1 after incubation with the dimerizer AP1510 (Fig. 4C). In contrast, THP-1 cells expressing Fpk3 alone did not bind to endogenous ASC or secrete IL-1 in the presence or absence of AP1510 (Fig. 4, B and C).
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Secretion—To establish a direct role for ASC in cryopyrin-mediated IL-1 secretion, we used siRNAs to inhibit the expression of endogenous ASC in cryopyrinPD-Fpk3 or Fpk3 THP-1 stable cells and measured the IL-1 secretion in the presence or absence of AP1510 (Fig. 5, A and B). We tested four siRNAs to target different ASC sequences, and one of them reduced ASC expression in a dose-dependent manner (Fig. 5A). At a dose of 60 pmol of ASC siRNA, there was no detectable amount of ASC protein in THP-1 cells, whereas the same amount of control siRNA did not lower the level of ASC protein either in the presence or absence of AP1510 (Fig. 5A). The ASC siRNA did not influence cell viability2 and was specific in that expression of tubulin was not affected (Fig. 5A). Importantly, inhibition of ASC expression by siRNA decreased the amount of IL-1 secretion in cryopyrinPD-Fpk3 THP-1 cells in a dose-dependent manner in the presence of AP1510. In contrast, the same cells retained their ability to secrete IL-1 when incubated with control siRNA (Fig. 5B). In addition, the three cryopyrin siRNAs, which were unable to alter ASC expression, did not reduce IL-1 secretion, providing further evidence for the specificity of the results.2 These findings indicate that ASC is required for cryopyrin-mediated secretion of IL-1 in THP-1 monocytic cells.
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B activation and IL-1 secretion. The observed phenotype is consistent with the dominant mode of genetic transmission of FCAS, MWS, and NOMID (1). The molecular mechanism that confers constitutive activation to the disease-associated mutations is unclear. NOD family members are thought to be kept in an inactive conformation by interactions with the LRRs, a state that is relieved by recognition of stimulating ligands via the LRRs. For example, exposure of HeLa cells to Shigella activates Nod1, resulting in the recruitment of RIP-like interacting CLARP kinase (RICK) and NF-B activation (23). In this regard, the disease-associated mutations are constitutively active and exhibit an increased binding to ASC, an adaptor molecule shown here to be essential for cryopyrin-mediated IL-1 secretion in THP-1 cells. Thus, the mutations in cryopyrin associated with disease may mimic activation induced by a microbial ligand, which remains to be identified. In addition to dysregulated IL-1 secretion, disease-associated cryopyrin mutants exhibited enhanced NF-B activation. An important role for IL-1 in cryopyrin-mediated inflammation is suggested by clinical improvement induced by treatment of MWS patients with IL-1 receptor antagonist (18, 19). The latter observation suggests that secretion of IL-1 is more relevant than NF-B activation in cryopyrin-mediated inflammatory disease. However, enhanced NF-B activity is likely to contribute to overproduction of IL-1 in MWS patients. For example, IL-1 expression is induced transcriptionally through NF-B sites located in the IL-1 promoter (24).
Our current studies have focused on NF-B and IL-1 secretion induced by cryopyrin. There is also evidence that cryopyrin and ASC promote apoptosis (10). Thus, it is also possible that abnormal apoptosis induced by disease-associated cryopyrin mutations could contribute to inflammatory disease. Further studies are needed to fully understand the role of NF-B, IL-1 secretion, and apoptosis in cryopyrin-mediated autoinflammatory syndromes.
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FOOTNOTES |
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¶ To whom correspondence should be addressed. Tel.: 734-764-8514; Fax: 734-647-9654; E-mail: bclx{at}umich.edu.
1 The abbreviations used are: NALP, NACHT, LRR, and PYD domains; CATERPILLER, CARD, transcription enhancer, R (purine)-binding, pyrin, lots of leucine repeats; PD, pyrin domain; CARD, caspase-recruitment domain; NOD, nucleotide-binding oligomerization domain; LRR, leucine-rich repeat; FCAS, familial cold autoinflammatory syndrome; MWS, Muckle-Wells syndrome; NOMID, neonatal-onset multisystem inflammatory disease; IL, interleukin; NF-B, nuclear factor-B; siRNA, small interfering RNA; Ab, antibody; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; ASC, apoptosis-associated speck-like protein containing a CARD.
2 T. A. Dowds, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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