NLRP2, an Inhibitor of the NF-κB Pathway, Is Transcriptionally Activated by NF- κB and Exhibits a Nonfunctional Allelic Variant

This information is current as Ana Fontalba, Olga Gutierrez and Jose L. Fernandez-Luna of September 27, 2021. J Immunol 2007; 179:8519-8524; ; doi: 10.4049/jimmunol.179.12.8519 http://www.jimmunol.org/content/179/12/8519 Downloaded from

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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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

NLRP2, an Inhibitor of the NF-␬B Pathway, Is Transcriptionally Activated by NF-␬B and Exhibits a Nonfunctional Allelic Variant1

Ana Fontalba, Olga Gutierrez, and Jose L. Fernandez-Luna2

NLRP2 has been shown to inhibit the NF-␬B signaling pathway, and thus may contribute to modulate the inflammatory response, where NF-␬B plays a major role. In this study, we report that expression of NLRP2 is induced upon differentiation of CD34؉ hemopoietic progenitors into granulocyte or monocyte/macrophages. We also found that NLRP2 was up-regulated following differentiation of mesenchymal stem cells toward adipocytes. Notably, stimulation of HEK293T cells with TNF-␣ or overexpres- sion of the p65 subunit of NF-␬B resulted in up-regulation of NLRP2 and the formation of NF-␬B-NLRP2 promoter complexes. Moreover, ectopic expression of p65 but not of other transcriptional regulators induced transactivation of the NLRP2 promoter. Downloaded from Thus, NLRP2 may control NF-␬B activation through a regulatory loop. changes within the NACHT domain of other NLRP have been associated with hereditary fever syndromes and chronic inflammatory diseases. We identified five single nucleotide polymorphisms present in the NACHT domain of NLRP2 by sequencing genomic DNA from 319 healthy controls. The frequencies of the rare alleles varied between 0.2 and 10%. Of note, one of these variants, I352S was unable to block the transcriptional activity of NF-␬B and the formation of NF-␬B-DNA-binding complexes following stimulation with TNF-␣. Overall, ␬

our findings provide molecular insight into the expression of NLRP2 by NF- B and suggest that a polymorphism within the http://www.jimmunol.org/ NACHT domain of NLRP2 may contribute to the amplification of inflammatory responses due to a reduction of inhibitory signals on the NF-␬B pathway. The Journal of Immunology, 2007, 179: 8519–8524.

here is increasing evidence that NF-␬B is a major, if not NLRP3, and NLRP4, regulate the activity of NF-␬B (4). This fam- the main transcription factor controlling inflammation. ily is comprised of proteins grouped in subfamilies based on the T NF-␬B also regulates the expression of involved in domain architecture and provide positive and negative signals for the immune and stress responses, cell adhesion, and cell survival the control of immune and inflammatory responses (5). (1, 2). This transcription factor is regulated by the inhibitor of Members of the NLRP (pyrin domain-containing NLR proteins)

NF-␬B(I␬B) kinase complex containing the kinases I␬B kinase subfamily, including NLRP2, NLRP3, NLRP4, and NLRP10 have by guest on September 27, 2021 (IKK)3 ␣ and IKK␤ and the regulatory subunit IKK␥ or NEMO been shown to inhibit NF-␬B signaling by mechanisms involving (NF-␬B essential modulator). This kinase complex phosphorylates the interaction with proteins that control the activity of I␬B (6–9). I␬B, which is subsequently ubiquitinated and degraded, thus lead- NLRP4 associates with IKK␣ and suppresses cytokine-mediated ing to the activation of NF-␬B. In addition, some members of the activation of this kinase, which plays a critical role in controlling nucleotide-binding domain and leucine-rich repeat-containing degradation of I␬B, thus releasing NF-␬B (8). NLRP3 exerts an (NLR) (3) family, as recently designated by the new Human Ge- inhibitory effect on TNF and TNFR-associated factor 6-induced nome Organization nomenclature committee (formerly NF-␬B activation (7), and more recently NLRP2 has been de- known as CATERPILLER family) (3), including NOD1, NOD2, scribed to associate with the IKK complex and to inhibit I␬B deg- radation induced by TNF-␣ (6). On the contrary, studies using genetically modified cells have revealed that NOD1 and NOD2 Unidad de Genetica Molecular, Hospital Universitario Marques de Valdecilla, San- activate NF-␬B by means of the serine/threonine kinase RICK/ tander; Spain Rip2 (10). Thus, the emerging view is of a complex balance be- Received for publication May 30, 2007. Accepted for publication October 2, 2007. tween inducers and inhibitors of the NF-␬B signaling pathway that The costs of publication of this article were defrayed in part by the payment of page in the proper context serve to amplify or suppress inflammatory charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. processes. However, the contribution of each single gene to the response to inflammatory signals and its association with inflam- 1 This work was supported by Grants PI050169 and Instituto de Salud Carlos III-Red Tematica de Investigacion eu Cancer RD06/0020 from the Spanish Fondo de Inves- matory disorders still awaits further investigation. It has been tigacion Sanitaria and API/06/02 from Fundacion Marques de Valdecilla. shown that genetic variants of NLRP3 are associated with the fa- 2 Address correspondence and reprint requests to Dr. Jose L. Fernandez-Luna, Unidad milial cold autoinflammatory syndrome (FCAS), Muckle-Wells de Genetica Molecular, Hospital Universitario Marques de Valdecilla, Edificio Es- cuela Universitaria de Enfermeria, Av. Valdecilla s/n, 39008 Santander, Spain. E-mail syndrome (MWS), and chronic infantile neurologic cutaneous and address: fl[email protected] articular syndrome (CINCA) (11). Consistently, macrophages 3 Abbreviations used in this paper: IKK, I␬B kinase; NLR, nucleotide-binding domain from MWS patients spontaneously secrete active IL-1␤ (12). Ad- and leucine-rich repeat-containing family; NLRP, pyrin domain-containing NLR pro- ditionally, a frameshift and two nucleotide polymor- tein; NACHT, nucleotide-binding domain; SNP, single nucleotide polymorphism; FCAS, familial cold autoinflammatory syndrome; MWS, Muckle-Wells syndrome; phisms in the coding region of NOD2 have been associated with CINCA, chronic infantile neurologic cutaneous and articular syndrome; BMSC, bone susceptibility to Crohn’s disease, a chronic inflammatory disorder marrow-derived mesenchymal stem cell; ADSC, adipose tissue-derived mesenchymal of the intestinal tract (13). Therefore, or polymorphisms stem cell. in genes that can act as regulators of NF-␬B activation are associated Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 with persistent autoinflammatory and fever diseases. Interestingly,

www.jimmunol.org 8520 NLRP2 IS TRANSACTIVATED BY NF-␬B

␬ ␤ NOD2 is transcriptionally induced by NF- B, suggesting that up-reg- where n is the threshold cycle value of 2-microglobulin minus the thresh- ulation of NOD2 may be part of a positive regulatory loop triggered old cycle value of NLRP2, and normalized by the value of the sample with by inflammatory stimuli (14). the lowest expression level of NLRP2. Specificity of the desired PCR prod- ucts was determined with melting curve analysis. Previous studies showed that NLRP2 is broadly expressed in tissues and tumor cell lines (6, 15). In this study, we report that Transfections and gene reporter assays NLRP2 is up-regulated during differentiation of hemopoietic and A genomic PCR fragment of 665 bp from the promoter region of NLRP2, mesenchymal progenitors. Significantly, NLRP2 levels are in- starting 28 bases downstream from the transcription start site and deletion creased in response to TNF-␣ stimulation or transfection with the fragments obtained by digestion with PvuII or BlpI, were cloned the pGL2- NF-␬B subunit p65. Furthermore, the NLRP2 promoter responds basic luciferase reporter vector (Promega). Site-directed mutagenesis of the ␬ to these NF-␬B activators. We also found an allelic variant of putative NF- B binding sites in the pGL2-NLRP2 promoter was conducted ␬ by using the QuickChange mutagenesis kit (Stratagene) with the following NLRP2 that generates a lacking the NF- B inhibition ca- primers for site 1 (5Ј-TATCTGTTACTCTAGAAAGA-3Ј and 5Ј-TCTT pacity which suggests that this polymorphism may be associated TCTAGAGTAACAGATA-3Ј) and site 2 (5Ј-GTCTGTTCAGTCACAGT with the individual response to inflammatory stimuli. GTA-3Ј and 5Ј-TACACTGTGACTGAACAGAC-3Ј). The DNA inserts were sequenced to verify the mutation. HEK293T cells were cotransfected Materials and Methods with 1 ␮g of pGL2-NLRP2 promoter constructs and 0.2 ␮g of pRSV-␤- galactosidase by lipofection using Superfect (Qiagen). When indicated, Cell lines and primary cells cells were also cotransfected with pcDNA3-p53, pcDNA3-Gfi (both pro- HEK293T cells were maintained in RPMI 1640 medium (Biochrom) sup- vided by G. Nunez, University of Michigan, Ann Arbor, MI), pRc/CMV- plemented with 10% FCS (Flow Laboratories). When indicated, cells were p65 (provided by I. Udalova, University of Oxford, U.K.), pcDNA3-Stat3C

treated with TNF-␣ (Sigma-Aldrich). Mesenchymal stem cells from bone (17), pcDNA3-E2F1 (18), or pcDNA3 empty vector. In another set of Downloaded from marrow (BMSCs) and adipose tissue (ADSCs) (provided by F. Prosper, experiments, HEK293T cells were cotransfected with NLRP2 cDNA ␮ University of Pamplona, Pamplona, Spain) were maintained in ␣MEM cloned into pHA-EAK vector (15), 0.25 g of the reporter plasmid pBVIx- ␬ with 10% FCS. Confluent cultures of primary ADSCs were induced to Luc, containing six NF- B recognition sites within the promoter sequence ␮ ␤ undergo adipogenesis in the presence of 50 ␮M indomethacin, 0.5 mM linked to the luciferase gene (19), and 0.2 g of pRSV- -galactosidase. isobutyl-1-methylxanthine, and 1 ␮M dexamethasone (Sigma-Aldrich). Polymorphic variants of NLRP2 were generated by site-directed mutagen- Cells were maintained in culture for up to 21 days, with the maintenance esis of the pHA-EAK-NLRP2 vector (15) with 18-bp primers containing medium replaced every 3 days. Cultures were fixed in formalin solution the nucleotide change in the middle of the sequence. All cDNA inserts and adipocyte differentiation was determined by staining of neutral lipids were sequenced to verify the mutation. http://www.jimmunol.org/ with Oil Red O (Sigma-Aldrich). Twenty-four hours posttransfection, cells were incubated with 10 ng/ml ␣ Peripheral blood progenitors were obtained from normal donors under- TNF- (Sigma-Aldrich) for 6 h. In all cases, cell extracts were prepared going mobilization for allogeneic progenitor cell transplantation. CD34ϩ and analyzed for the relative luciferase activity by a dual-light reporter cells were selected from the mononuclear cell population and induced to gene assay system (Applied Biosystems). Results were normalized for ␤ undergo granulocyte or monocyte/macrophage differentiation as previously transfection efficiency with values obtained with pRSV- -gal. described (16). Western blot analysis Genotyping Cell extracts (50 ␮g of protein) were separated on a 8% polyacrylamide gel We analyzed single nucleotide polymorphisms (SNPs) present in the and transferred to nitrocellulose as previously described (17). Blots were NACHT domain of the NLRP2 gene in 319 DNA samples obtained from blocked with 3% BSA and incubated with rabbit Abs against Stat3, p65, by guest on September 27, 2021 unrelated subjects with no known history of serious disease, including au- and p53 (Santa Cruz Biotechnology) or NLRP2 (Abcam and Santa Cruz), toimmune or chronic infectious disorders. DNA was extracted from whole mouse anti-E2F1, or anti-␣-tubulin or goat anti-Gfi1 Abs (all from Santa blood by using the QIAamp DNA blood kit (Qiagen) and amplified with Cruz Biotechnology), followed by incubation with goat anti-rabbit or anti- primers for human NLRP2 5Ј-TGGGTAACTGATTGCATCCTC-3Ј and mouse or rabbit anti-goat Abs conjugated to alkaline phosphatase (Sigma- 5Ј-GCGTTGCTCCTCATTAGCTC-3Ј. The amplified fragment (753 bp) Aldrich). Bound Ab was detected by a chemiluminescence system was sequenced in both directions with the same primers used for amplifi- (Applied Biosystems). cation and with two internal primers: 5Ј-CACATCCTAGCCCAAG CAC-3Ј and 5Ј-GTAACATCACCCTGTTCAGC-3Ј. In all cases, we ver- Results ified the authenticity of the polymorphism in an independent amplification. The expression of NLRP2 is up-regulated during differentiation The study was approved by the Hospital Universitario Marques de Valdecilla Research Ethics Committee and all subjects gave informed consent before of hemopoietic and mesenchymal progenitors participation in this study. Initial studies showed that NLRP2 is widely expressed in tissues EMSA and tumor cell lines (6, 15). However, the expression of NLRP2 in progenitor cells and its regulation during differentiation of these Cells were lysed and nuclear fractions were resuspended in 20 mM HEPES ϩ (pH 7.9), 420 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 20% glycerol. progenitors is not known. We purified CD34 progenitor cells Nuclear extracts (10 ␮g of total protein) were incubated with a 32P-labeled from peripheral blood, and the selected population was cultured dsDNA probe from the promoter region of the NLRP2 gene (5Ј- with either G-CSF or M-CSF to induce granulocyte or monocyte/ GGGCAGTCCC-3Ј) or a probe corresponding to the consensus NF-␬B site macrophage maturation. The granulocytic cell population Ј Ј (5 -GGGAATTTCC-3 ). Samples were run on a 5% nondenaturing poly- (CD34ϪCD15ϩ) increased to Ͼ85% after 20 days of culture and acrylamide gel in 200 mM Tris-borate and 2 mM EDTA. Gels were dried and visualized by autoradiography. Supershifts were performed using rab- showed morphologic features of mature granulocytes. When the bit polyclonal Abs specific for p50 and p65 (Santa Cruz Biotechnology). cells were cultured in the presence of M-CSF, a clear pattern of monocyte/macrophage maturation was observed and, by day 20, RT-PCR analysis the majority of cells were mature CD14ϩ cells (data not shown). Total RNA was prepared using TRIzol reagent (Invitrogen Life Technol- We then analyzed the levels of NLRP2 mRNA in these cell pop- ogies). To assess mRNA expression, a semiquantitative RT-PCR)method ulations and found that CD34ϩ progenitors expressed low levels of was used as previously described (14). The generated cDNA was amplified ␤ Ј NLRP2, which increased ϳ4-fold by day 20 of culture in the by using primers for human cIAP-1 (14), 2-microglobulin (5 -GAGA CATGTAAGCAGCATCA-3Ј and 5Ј-AGCAACCTGCTCAGATACAT- monocytic lineage. During the granulocytic differentiation, NLRP2 3Ј), and NLRP2 (5Ј-CCGACAATGAGCTTCTGGATG-3Ј and 5Ј-AGC reached the highest expression level at day 10 (ϳ6-fold) of culture, AAGGTCCTTGCAATTGG-3Ј). The expected PCR fragments were size and by day 20 the expression was similar to that of CD34ϩ cells, fractionated onto a 2% agarose gel and stained with ethidium bromide. Quantitative real-time PCR was performed in a 7000 Sequence Detec- as determined by semiquantitative RT-PCR and real-time RT-PCR tion System (Applied Biosystems). The ratio of the abundance of NLRP2 (Fig. 1A). To probe whether this expression pattern was specific ␤ n transcripts to that of 2-microglobulin transcripts was calculated as 2 , for hemopoietic progenitors, we analyzed the expression of The Journal of Immunology 8521

FIGURE 1. Analysis of NLRP2 mRNA along differentiation of primary progenitor cells. A, CD34ϩ cells were cultured in the presence or in the absence of M-CSF or G-CSF to undergo monocytic or granulocytic dif- ferentiation, respectively. B, ADSCs were induced to undergo adipogenesis for the indicated period of time. Total RNA was obtained from progenitor FIGURE 2. TNF-␣ and p65 up-regulate NLRP2 and promote binding of and differentiated cells and analyzed for NLRP2 mRNA levels by real-time Downloaded from ␬ (upper panels) and semiquantitative (lower panels) RT-PCR. Real-time NF- BtotheNLRP2 promoter. A, HEK293T cells were incubated with or ␣ data were normalized using the value of the sample with the lowest level without 10 ng/ml TNF- for 24 h or transfected with p65 and analyzed 48 h ␤ posttransfection. Following treatment, total RNA was extracted to deter- of NLRP2. 2-microglobulin was used as an amplification control. Histo- grams represent the means Ϯ SD of three independent experiments. mine the expression levels of NLRP2 by RT-PCR. The expression of ␬ ␤ cIAP1, a known target of NF- B, was also included for comparison. 2- Microglobulin was used as an amplification control. B, HEK293T cells ␣ were cultured in the presence of TNF- at different time intervals and then http://www.jimmunol.org/ NLRP2 in mesenchymal stem cells obtained from bone marrow NLRP2 protein was analyzed by Western blot. A control of protein ex- (BMSCs) and adipose tissue (ADSCs), and in adipocytic cells de- pression using the lysate from HEK293T cells transfected with NLRP2 ␣ rived from ADSCs (Fig. 1B). Immunophenotypic characterization were included (lane C). The levels of -tubulin were also determined to ␣ based on flow cytometry revealed that mesenchymal stem cells assure equal loading. C and D, ADSCs were incubated with TNF- for the indicated time points and the NLRP2 mRNA (C) and protein (D) levels were positive for stromal cell-associated markers (CD73, CD90, were analyzed as described above. E, Sequence of the two putative NF- CD13, CD44) and negative for typical hemopoietic markers ␬B-binding elements in the NLRP2 promoter aligned with the NF-␬B con- ϳ (CD34, CD45) (data not shown). The levels of NLRP2 were 2- sensus sequence. F, HEK293T cells were either treated with 10 ng/ml fold higher in ADSCs than in BMSCs. Then, ADSCs were cul- TNF-␣ for1hortransfected with p65. Formation of binding complexes tured in adipogenic medium and by 21 days cells were intensely was determined by EMSA using radiolabeled probes from the NLRP2 pro- by guest on September 27, 2021 stained with Oil Red O, which is retained by lipid vacuoles, and moter or a NF-␬B consensus probe (cons). Nuclear extracts from ADSCs showed increased levels (3.5-fold) of NLRP2 mRNA compared treated or not with TNF-␣ were also analyzed for the binding of NF-␬Bto with those in undifferentiated progenitors. Thus, both hemopoietic site 2 of the NLRP2 promoter. Supershift analyses were performed using ␬ and mesenchymal models of differentiation show an up-regulated the indicated Abs to determine the NF- B subunit composition of the expression of NLRP2 in the more mature cells. complexes.

NLRP2 is transcriptionally activated by NF-␬B-p65 and TNF It has been shown that some modulators of NF-␬B activity (I␬B␣, (Fig. 2B). Based on the up-regulation of NLRP2 induced by TNF-␣, NOD2) are in turn transcriptionally induced by NF-␬B NF-␬B activators, we searched for consensus sites within the (14, 20, 21). In view of this, we analyzed the expression of NLRP2 NLRP2 promoter region and found two putative NF-␬B recogni- mRNA in HEK293T cells after treatment with TNF-␣ and found a tion sequences 244 and 271 bases upstream from the transcription significant increase in the levels of NLRP2 (Fig. 2A). This expres- start site (Fig. 2E). Then we tested whether NF-␬B binds to these sion pattern was similar to that of the cIAP1 gene, a known target sites in response to TNF-␣ exposure or p65 overexpression by of NF-␬B. To further confirm the involvement of this transcription using an EMSA. As shown in Fig. 2F, both treatments resulted in factor in the expression of NLRP2, we transfected HEK293T cells the formation of a protein-DNA complex only when the proximal with the NF-␬B subunit p65 and, 48 h later, RT-PCR analyses sequence (site 2) was used as a probe. The shifted bands obtained revealed a high up-regulation of NLRP2 mRNA (Fig. 2A). Similar with anti-p65 and anti-p50 Abs demonstrated the presence of p65- data were obtained at the protein level following treatment of cells p65 and p50-p65 dimers in the complex. The binding of NF-␬Bto with TNF-␣ for 48 h (Fig. 2B). The expression pattern of NLRP2 site 2 of the NLRP2 promoter was also detected using nuclear mRNA and protein in response to TNF-␣ was reproduced in extracts from ADSCs treated with TNF-␣ (Fig. 2F), which ADSCs (Fig. 2, C and D) and in the monocytic cell line THP1 strengthens the physiological relevance of this interaction. To as- (data not shown). As shown in Fig. 2C, the mRNA levels of sess the transcriptional activity of the NLRP2 promoter, a 665-bp NLRP2 increased after6hoftreatment (ϳ3-fold as determined by fragment containing the NF-␬B consensus site of the NLRP2 pro- real-time RT-PCR) and reached the highest expression level (Ͼ5- moter and two deletion fragments (Fig. 3A) were cloned into a fold) following 24 h of incubation with TNF-␣. Although it has promoterless luciferase vector, and these constructs were tran- been previously shown that NLRP2 protein is induced at 4 h after siently transfected into HEK293T cells. Cotransfection with p65 stimulation with TNF-␣ (6), we failed to detect the endogenous increased the luciferase activity ϳ9-fold compared with cells protein at earlier times in all cell models (Fig. 2, B and D), most transfected with the empty expression vector (Fig. 3B). Deletion of likely because of a lower affinity of the polyclonal Abs used, since the fragment from Ϫ637 to Ϫ331 did not significantly affect lu- they readily revealed the overexpressed protein in transfected cells ciferase activity. However, further deletion to Ϫ55 abrogated the 8522 NLRP2 IS TRANSACTIVATED BY NF-␬B

FIGURE 4. Allelic polymorphisms in the NACHT domain of NLRP2. A, Allele frequencies of the polymorphic variants found in the NACHT domain of NLRP2. nd, Not described in the National Center for Biotech- nology Information SNP database. B, Alignment of the NACHT domain of human NLRP2 protein with the corresponding segments of two members of the NLR family. Alignment was performed using the ClustalW program. Downloaded from Polymorphic residues in NLRP2 and disease-associated variants in NLRP3 FIGURE 3. p65 transactivates the NLRP2 promoter. A, Schematic rep- and NOD2 are circled. The alignment columns with strictly conserved resentation of the NLRP2 promoter that illustrates the position of each residues are highlighted in gray boxes. CD, Crohn’s disease. deletion relative to the transcription start site (TSS). The NF-␬B sites are also included. B, HEK293T cell line was transfected with a luciferase re- porter vector containing sequential deletion fragments of the NLRP2 pro-

moter, either in the presence or in the absence of p65. Following 24 h of NOD2, are associated with hereditary fever syndromes and chronic http://www.jimmunol.org/ transfection, cell extracts were analyzed for the relative luciferase activity. inflammatory diseases (24). Of note, most of these mutations are C, Reporter vector containing mutant site 1 or site 2 was introduced into clustered in the highly conserved nucleotide-binding NACHT do- HEK293T cells and luciferase activity in response to p65 was determined main. To reveal the presence of single nucleotide changes in the (cont, wild-type promoter fragment). Units of luciferase activity were nor- NACHT domain of NLRP2 that may give rise to a functionally malized based on values of pRSV-␤-galactosidase activity to control for transfection efficiency. D, Overexpressed transcription factors (TF) were altered protein, we sequenced the entire NACHT domain in 319 detected by Western blot with the specific Abs. The first lane (Ϫ) repre- DNA samples from normal donors. We identified five non-synon- sents cells transfected with the pcDNA3 empty vector. E, Cells were co- ymous SNP variants that promoted amino acid changes, T221M, transfected with the indicated transcription factors and the NLRP2 pro- I352S, I354V, F359L, and R364K. The frequency of the rare al- moter, and 24 h later were analyzed for luciferase activity. Data are leles in the studied population varied between 0.2 and 10%, with by guest on September 27, 2021 presented as the mean of triplicate experiments Ϯ SD. two alleles having frequencies lower than 1% (Fig. 4A). Two of the nucleotide changes were annotated polymorphisms in the National Center for Biotechnology Information human SNP database. This promoter capacity of the cloned sequence (Fig. 3B). To test the database included a SNP, E302Q, that was not detected in the functionality of the NF-␬B binding sites, reporter constructs con- population studied here. The alignment of NACHT domain se- taining the mutated site 1 or site 2 were assayed for luciferase quences of the NLR family members NLRP2, NLRP3, and NOD2 activity in response to p65 (Fig. 3C). Consistent with the EMSA showed that the SNPs found in NLRP2 are clustered within a re- experiments, mutation of site 1 did not modify the luciferase ac- gion aligned with sequences that contain disease-associated amino tivity. By contrast, mutation of site 2 dramatically reduced the acid changes in NLRP3 and NOD2 (Fig. 4B). Comparison with the response of the NLRP2 promoter to p65. Of note, the constitutive structure-based model predicted for NLRP3 after alignment of activity of the promoter remained unchanged in the mutant con- multiple NACHT domain-containing proteins (25) indicated that structs, indicating the contribution of other transcriptional regula- the polymorphic variants of NLRP2 lie in a region that plays crit- tors. More than 20 putative transcription factor binding sites were ical roles in oligomerization and NTP hydrolysis. To study the identified within the promoter region spanning Ϫ331 to Ϫ55, functional impact of the identified amino acid changes, we intro- which retained the basal promoter activity of the entire 665-bp duced the rare nucleotide for each one of the six polymorphisms in fragment. Among them, at least two are consensus sites for pro- the NLRP2 cDNA by site-directed mutagenesis and analyzed the teins (Stat and Gfi1) that have been previously associated with capacity of the different variants to inhibit NF-␬B activation in inflammation or chronic inflammatory disorders (22, 23). Thus, we response to TNF-␣. HEK293T cells were cotransfected with the tested the capacity of Stat3 and Gfi1 as well as two other tran- NLRP2 variants and a luciferase reporter gene driven by a pro- scriptional inducers, p53 and E2F1, used here as a specificity con- moter sequence containing six NF-␬B-responsive elements. Im- trol to regulate the activity of the NLRP2 promoter. However, munoblot analysis of the lysates confirmed production of the overexpression of these proteins (Fig. 3D) did not modify the con- NLRP2 variants at comparable levels (Fig. 5A). The overexpressed stitutive promoter activity of NLRP2 as determined by luciferase wild-type and most of the variant proteins reduced significantly the assay (Fig. 3E). Thus, although this result does not clarify the activation of NF-␬B; however, the I352S change generated a pro- contribution of other transcription factors, it strengthens the role of tein lacking the NF-␬B suppressor capacity (Fig. 5B). To further NF-␬B to the transcriptional regulation of NLRP2. confirm this result, we studied the formation of DNA-protein bind- ing complexes after TNF-␣ exposure in the presence of the dif- ␬ An allelic variant of NLRP2 lacking NF- B inhibition activity ferent NLRP2 variants. Consistent with the gene reporter data, the It has been shown that mutations in the other two members of the protein containing the I352S change did not interfere with the NLR family with NF-␬B modulatory properties, NLRP3 and binding of NF-␬B to its recognition site (Fig. 5C). The specificity The Journal of Immunology 8523

the proximal site proved to be functional and accounted for the capacity of the NLRP2 promoter to respond to NF-␬B proteins. However, chromatin immunoprecipitation analyses showed no en- richment of fragments containing the NF-␬B site, suggesting that our chromatin immunoprecipitation procedure failed to effectively enrich the cross-linked DNA material; although we cannot rule out the existence of another NF-␬B-binding sequence not included in the promoter fragment studied here. In contrast, the constitutive promoter activity seems to rely on other transcription factors. Among the potential regulators of the NLRP2 gene, we showed that Stat3 and Gfi1 did not modify the promoter activity, but other factors including Egr-1, which promotes the expression of inflam- matory cytokines (29), or Sp1, a proinflammatory protein involved in the expression of mediators of inflammation (30), should be considered in future studies. Because NLRP2 inhibits NF-␬B, up- regulation of NLRP2 may be part of a negative regulatory loop induced via inflammatory stimuli. A similar NF-␬B-dependent mechanism has been described for NOD2 (14) and TLR2, which

activates the NF-␬B signaling pathway following recognition of Downloaded from microbial components (31), and for the NF-␬B inhibitor I␬B␣ after stimulation with TNF-␣ or transfection with the p65 subunit of NF-␬B (21), which has been proposed as an inducible autoregu- latory pathway. FIGURE 5. The I352S polymorphism abrogates the NF-␬B inhibition There is mounting evidence that NF-␬B plays a major role in

capacity of NLRP2. HEK293T cells were cotransfected with wild-type (wt) autoimmune and inflammatory disorders. Therefore, mis-regula- http://www.jimmunol.org/ NLRP2 or its polymorphic variants and a reporter vector driven by six tion of proteins that control the NF-␬B pathway is likely to con- ␬ NF- B binding sites. A, After 24 h of transfection, cell extracts were an- tribute to disease susceptibility or severity. In line with this, mu- alyzed for protein expression by Western blotting. B, Transfected cells tations in the NOD2 gene have been identified in up to 50% of were stimulated with 10 ng/ml TNF-␣ for 6 h and then cell extracts were Crohn’s disease patients, but the risk alleles are also present in the prepared and analyzed for luciferase activity. C, Empty expression vector. Histograms show the means Ϯ SD of three independent experiments. C, healthy population albeit at lower frequencies than in disease. The Nuclear extracts from cells transfected with the NLRP2 variants and stim- rare allele frequency of NOD2 may be up to 18% depending on ulated with TNF-␣ for 1 h were analyzed for the formation of protein- the ethnic group (32). We have identified five SNPs within the DNA-binding complexes by EMSA using a NF-␬B consensus probe. Ex- NACHT domain of NLRP2 in a population of 319 healthy indi- tracts from control (empty vector-transfected) cells treated with TNF-␣ viduals. One of these polymorphisms (allele frequency of 0.4%) by guest on September 27, 2021 were preincubated with anti-p65 and irrelevant anti-GATA1 Abs. gave rise to an amino acid change (I352S) that disrupted the ca- pacity of NLRP2 to inhibit the transcriptional activity of NF-␬B and the formation of NF-␬B-DNA-binding complexes. Of note, of the protein-DNA complex was confirmed by supershift analysis patients with hereditary fever syndromes and chronic inflamma- with anti-p65 and irrelevant anti-GATA1 Abs. tory diseases who carry mutations in the NACHT domain of NLRP3 have been recently described (11, 33). Additionally, poly- Discussion morphisms in both alleles of the NOD2 gene increase the risk for Deciphering the mechanisms that regulate the activity of NF-␬Bis developing Crohn’s disease, and those patients carrying the rare of major importance for understanding the response to inflamma- alleles of NOD2 were characterized by a younger age at onset and tory stimuli. In this study, we show that NLRP2, an inhibitor of the a less frequent colonic involvement than were seen in patients who NF-␬B pathway, is up-regulated during differentiation of hemo- had no mutation (34). Genetic variants in other NF-␬B regulators, poietic progenitors toward granulocyte or monocyte/macrophage including I␬B␣ and NEMO, that abolish or reduce the activation of lineages and after differentiation of mesenchymal stem cells into NF-␬B have been associated with a number of inherited genetic adipocytes. Interestingly, NOD2, another NLR family member that disorders, mainly immunodeficiency syndromes (35). Moreover, a activates NF-␬B signaling, is also up-regulated during the my- truncating and inactivating polymorphism in CARD8, which is an elomonocytic differentiation of CD34ϩ progenitor cells (14). inhibitor of the NF-␬B pathway structurally related to NLRP1, has Based on these data, both positive and negative regulators appear been associated with susceptibility to inflammatory bowel disease to be less dispensable in mature cells where they may establish a (36). Most of the disease-associated polymorphisms in NLRP3 and balance that controls the activity of NF-␬B. NF-␬B has been some in NOD2 were located within the NACHT domain. We shown to protect osteoclast progenitors from (26) and to aligned the amino acid sequences surrounding the polymorphisms be activated in bone marrow and cord blood progenitor cells (27). identified in the NACHT domain of NLRP2 with the correspond- Additionally, it has been described that NF-␬B activation can pre- ing sequences in NLRP3 and NOD2 proteins. Both NLRP3 and vent chondrogenesis and myogenesis from mesenchymal cells NOD2 are predicted to have similar secondary structures (25). The (28). Taken together, these data along with our findings highlight I352S polymorphic change that gives rise to a nonfunctional the interest of studying the NF-␬B regulators in progenitor and NLRP2 protein lies close to four polymorphisms in NLRP3 asso- stem cells and deciphering their role in the differentiation programs ciated with CINCA, MWS, or FCAS and is also located near res- toward different lineages. We also found that NLRP2 regulation idue R345, corresponding to an arginine finger as predicted involves a transcriptional activation mechanism through NF-␬B. by alignment of the NTPase domains of NLRP proteins and p97 Although two putative NF-␬B binding sites were identified within ATPase (25). This finger plays a critical role in cooperative NTP a region spanning ϳ1400 bp from the transcription start site, only hydrolysis (37). I352S is also close to the position aligned with 8524 NLRP2 IS TRANSACTIVATED BY NF-␬B

A374 in NLRP3, which has been associated with CINCA. Con- 14. Gutierrez, O., C. Pipaon, N. Inohara, A. Fontalba, Y. Ogura, F. Prosper, sistent with our functional data, I352S is the only polymorphism G. Nunez, and J. L. Fernandez-Luna. 2002. Induction of Nod2 in myelomono- cytic and intestinal epithelial cells via nuclear factor-␬B activation. J. Biol. Chem. among all identified in the NACHT domain of NLRP2 that local- 277: 41701–41705. izes in a position aligned with a disease-associated variation 15. Kinoshita, T., Y. Wang, M. Hasegawa, R. Imamura, and T. Suda. 2005. PYPAF3, a PYRIN-containing APAF-1-like protein, is a feedback regulator of caspase-1- (E441K in NOD2) in either NLRP3 or NOD2 proteins. It has been dependent interleukin-1␤ secretion. J. Biol. Chem. 280: 21720–21725. shown that the E441K change has an impact on the NOD2-medi- 16. Sanz, C., A. Benito, M. Silva, B. Albella, C. Richard, J. C. Segovia, A. Insunza, ated activation of NF-␬B in response to bacterial peptidoglycan J. A. Bueren, and J. L. Fernandez-Luna. 1997. The expression of Bcl-x is down- regulated during differentiation of human hematopoietic progenitor cells along and is considered a rare polymorphism associated with Crohn’s the granulocyte but not the monocyte/macrophage lineage. Blood 89: 3199–3204. disease (38). Taking into account the complex control on the ac- 17. Real, P. J., A. Sierra, A. De Juan, J. C. Segovia, J. M. Lopez-Vega, and tivation of NF-␬B and the increasing number of proteins involved, J. L. Fernandez-Luna. 2002. Resistance to chemotherapy via Stat3-dependent overexpression of Bcl-2 in metastatic breast cancer cells. Oncogene 21: analyses of deleterious or activating polymorphisms in candidate 7611–7618. genes will be needed to decipher their contribution to chronic in- 18. Real, P. J., C. Sanz, O. Gutierrez, C. Pipaon, A. M. Zubiaga, and J. L. Fernandez-Luna. 2006. Transcriptional activation of the proapoptotic bik flammatory disorders or autoimmune diseases. gene by E2F proteins in cancer cells. FEBS Lett. 580: 5905–5909. Overall, our observations shed light on the expression of NLRP2 19. Inohara, N., Y. Ogura, F. F. Chen, A. Muto, and G. Nunez. 2001. Human during differentiation of progenitor or stem cells and provide ev- confers responsiveness to bacterial lipopolysaccharides. J. Biol. Chem. 276: ␬ 2551–2554. idence on the regulation of NLRP2 through NF- B, which sug- 20. May, M., and S. Ghosh. 1998. Signal transduction through NF-␬B. Immunol. gests that this protein may be involved in a negative regulatory Today 19: 80–88. ␬ loop to limit the response of NF-␬B. Our data also identify an 21. Sun, S. C., P. A. Ganchi, D. W. Ballard, and W. C. Greene. 1993. NF- B controls expression of inhibitor I␬B␣: evidence for an inducible autoregulatory pathway. allelic variant of NLRP2 that lacks the capacity to inhibit the Science 259: 1912–1915. Downloaded from NF-␬B transcriptional pathway, providing the rationale for study- 22. Sampath, D., M. Castro, D. C. Look, and M. J. Holtzman. 1999. Constitutive activation of an epithelial signal transducer and activator of transcription (STAT) ing the association between this inactivating polymorphism and pathway in asthma. J. Clin. Invest. 103: 1353–1361. disease activity or an inherited predisposition to autoimmune or 23. Karsunky, H., H. Zeng, T. Schmidt, B. Zevnik, R. Kluge, K. W. Schmid, chronic inflammatory disorders. U. Duhrsen, and T. Moroy. 2002. Inflammatory reactions and severe neutropenia in mice lacking the transcriptional repressor Gfi1. Nat. Genet. 30: 295–300. 24. Tschopp, J., F. Martinon, and K. Burns. 2003. NALPs: a novel protein family

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