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The Journal of Immunology, 2007, 179, 8519 -8524
Copyright © 2007 by The American Association of Immunologists, Inc.
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NLRP2, an Inhibitor of the NF-{kappa}B Pathway, Is Transcriptionally Activated by NF-{kappa}B and Exhibits a Nonfunctional Allelic Variant1

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

Unidad de Genetica Molecular, Hospital Universitario Marques de Valdecilla, Santander; Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
NLRP2 has been shown to inhibit the NF-{kappa}B signaling pathway, and thus may contribute to modulate the inflammatory response, where NF-{kappa}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-{alpha} or overexpression of the p65 subunit of NF-{kappa}B resulted in up-regulation of NLRP2 and the formation of NF-{kappa}B-NLRP2 promoter complexes. Moreover, ectopic expression of p65 but not of other transcriptional regulators induced transactivation of the NLRP2 promoter. Thus, NLRP2 may control NF-{kappa}B activation through a regulatory loop. Nucleotide changes within the NACHT domain of other NLRP proteins 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-{kappa}B and the formation of NF-{kappa}B-DNA-binding complexes following stimulation with TNF-{alpha}. Overall, our findings provide molecular insight into the expression of NLRP2 by NF-{kappa}B and suggest that a polymorphism within the NACHT domain of NLRP2 may contribute to the amplification of inflammatory responses due to a reduction of inhibitory signals on the NF-{kappa}B pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
There is increasing evidence that NF-{kappa}B is a major, if not the main transcription factor controlling inflammation. NF-{kappa}B also regulates the expression of genes involved in the immune and stress responses, cell adhesion, and cell survival (1, 2). This transcription factor is regulated by the inhibitor of NF-{kappa}B (I{kappa}B) kinase complex containing the kinases I{kappa}B kinase (IKK)3 {alpha} and IKKβ and the regulatory subunit IKK{gamma} or NEMO (NF-{kappa}B essential modulator). This kinase complex phosphorylates I{kappa}B, which is subsequently ubiquitinated and degraded, thus leading to the activation of NF-{kappa}B. In addition, some members of the nucleotide-binding domain and leucine-rich repeat-containing (NLR) (3) family, as recently designated by the new Human Genome Organization gene nomenclature committee (formerly known as CATERPILLER family) (3), including NOD1, NOD2, NLRP3, and NLRP4, regulate the activity of NF-{kappa}B (4). This family is comprised of proteins grouped in subfamilies based on the domain architecture and provide positive and negative signals for the control of immune and inflammatory responses (5).

Members of the NLRP (pyrin domain-containing NLR proteins) subfamily, including NLRP2, NLRP3, NLRP4, and NLRP10 have been shown to inhibit NF-{kappa}B signaling by mechanisms involving the interaction with proteins that control the activity of I{kappa}B (6, 7, 8, 9). NLRP4 associates with IKK{alpha} and suppresses cytokine-mediated activation of this kinase, which plays a critical role in controlling degradation of I{kappa}B, thus releasing NF-{kappa}B (8). NLRP3 exerts an inhibitory effect on TNF and TNFR-associated factor 6-induced NF-{kappa}B activation (7), and more recently NLRP2 has been described to associate with the IKK complex and to inhibit I{kappa}B degradation induced by TNF-{alpha} (6). On the contrary, studies using genetically modified cells have revealed that NOD1 and NOD2 activate NF-{kappa}B by means of the serine/threonine kinase RICK/Rip2 (10). Thus, the emerging view is of a complex balance between inducers and inhibitors of the NF-{kappa}B signaling pathway that in the proper context serve to amplify or suppress inflammatory processes. However, the contribution of each single gene to the response to inflammatory signals and its association with inflammatory disorders still awaits further investigation. It has been shown that genetic variants of NLRP3 are associated with the familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and chronic infantile neurologic cutaneous and articular syndrome (CINCA) (11). Consistently, macrophages from MWS patients spontaneously secrete active IL-1β (12). Additionally, a frameshift mutation and two nucleotide polymorphisms in the coding region of NOD2 have been associated with susceptibility to Crohn’s disease, a chronic inflammatory disorder of the intestinal tract (13). Therefore, mutations or polymorphisms in genes that can act as regulators of NF-{kappa}B activation are associated with persistent autoinflammatory and fever diseases. Interestingly, NOD2 is transcriptionally induced by NF-{kappa}B, suggesting that up-regulation of NOD2 may be part of a positive regulatory loop triggered by inflammatory stimuli (14).

Previous studies showed that NLRP2 is broadly expressed in tissues and tumor cell lines (6, 15). In this study, we report that NLRP2 is up-regulated during differentiation of hemopoietic and mesenchymal progenitors. Significantly, NLRP2 levels are increased in response to TNF-{alpha} stimulation or transfection with the NF-{kappa}B subunit p65. Furthermore, the NLRP2 promoter responds to these NF-{kappa}B activators. We also found an allelic variant of NLRP2 that generates a protein lacking the NF-{kappa}B inhibition capacity which suggests that this polymorphism may be associated with the individual response to inflammatory stimuli.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines and primary cells

HEK293T cells were maintained in RPMI 1640 medium (Biochrom) supplemented with 10% FCS (Flow Laboratories). When indicated, cells were treated with TNF-{alpha} (Sigma-Aldrich). Mesenchymal stem cells from bone marrow (BMSCs) and adipose tissue (ADSCs) (provided by F. Prosper, University of Pamplona, Pamplona, Spain) were maintained in {alpha}MEM with 10% FCS. Confluent cultures of primary ADSCs were induced to undergo adipogenesis in the presence of 50 µM indomethacin, 0.5 mM isobutyl-1-methylxanthine, and 1 µM dexamethasone (Sigma-Aldrich). Cells were maintained in culture for up to 21 days, with the maintenance medium replaced every 3 days. Cultures were fixed in formalin solution and adipocyte differentiation was determined by staining of neutral lipids with Oil Red O (Sigma-Aldrich).

Peripheral blood progenitors were obtained from normal donors undergoing mobilization for allogeneic progenitor cell transplantation. CD34+ cells were selected from the mononuclear cell population and induced to undergo granulocyte or monocyte/macrophage differentiation as previously described (16).

Genotyping

We analyzed single nucleotide polymorphisms (SNPs) present in the NACHT domain of the NLRP2 gene in 319 DNA samples obtained from unrelated subjects with no known history of serious disease, including autoimmune or chronic infectious disorders. DNA was extracted from whole blood by using the QIAamp DNA blood kit (Qiagen) and amplified with primers for human NLRP2 5'-TGGGTAACTGATTGCATCCTC-3' and 5'-GCGTTGCTCCTCATTAGCTC-3'. The amplified fragment (753 bp) was sequenced in both directions with the same primers used for amplification and with two internal primers: 5'-CACATCCTAGCCCAAGCAC-3' and 5'-GTAACATCACCCTGTTCAGC-3'. In all cases, we verified the authenticity of the polymorphism in an independent amplification. The study was approved by the Hospital Universitario Marques de Valdecilla Research Ethics Committee and all subjects gave informed consent before participation in this study.

EMSA

Cells were lysed and nuclear fractions were resuspended in 20 mM HEPES (pH 7.9), 420 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 20% glycerol. Nuclear extracts (10 µg of total protein) were incubated with a 32P-labeled dsDNA probe from the promoter region of the NLRP2 gene (5'-GGGCAGTCCC-3') or a probe corresponding to the consensus NF-{kappa}B site (5'-GGGAATTTCC-3'). Samples were run on a 5% nondenaturing polyacrylamide gel in 200 mM Tris-borate and 2 mM EDTA. Gels were dried and visualized by autoradiography. Supershifts were performed using rabbit polyclonal Abs specific for p50 and p65 (Santa Cruz Biotechnology).

RT-PCR analysis

Total RNA was prepared using TRIzol reagent (Invitrogen Life Technologies). To assess mRNA expression, a semiquantitative RT-PCR)method was used as previously described (14). The generated cDNA was amplified by using primers for human cIAP-1 (14), β2-microglobulin (5'-GAGACATGTAAGCAGCATCA-3' and 5'-AGCAACCTGCTCAGATACAT-3'), and NLRP2 (5'-CCGACAATGAGCTTCTGGATG-3' and 5'-AGCAAGGTCCTTGCAATTGG-3'). The expected PCR fragments were size fractionated onto a 2% agarose gel and stained with ethidium bromide.

Quantitative real-time PCR was performed in a 7000 Sequence Detection System (Applied Biosystems). The ratio of the abundance of NLRP2 transcripts to that of β2-microglobulin transcripts was calculated as 2n, where n is the threshold cycle value of β2-microglobulin minus the threshold cycle value of NLRP2, and normalized by the value of the sample with the lowest expression level of NLRP2. Specificity of the desired PCR products was determined with melting curve analysis.

Transfections and gene reporter assays

A genomic PCR fragment of 665 bp from the promoter region of NLRP2, starting 28 bases downstream from the transcription start site and deletion fragments obtained by digestion with PvuII or BlpI, were cloned the pGL2-basic luciferase reporter vector (Promega). Site-directed mutagenesis of the putative NF-{kappa}B binding sites in the pGL2-NLRP2 promoter was conducted by using the QuickChange mutagenesis kit (Stratagene) with the following primers for site 1 (5'-TATCTGTTACTCTAGAAAGA-3' and 5'-TCTTTCTAGAGTAACAGATA-3') and site 2 (5'-GTCTGTTCAGTCACAGTGTA-3' and 5'-TACACTGTGACTGAACAGAC-3'). The DNA inserts were sequenced to verify the mutation. HEK293T cells were cotransfected with 1 µg of pGL2-NLRP2 promoter constructs and 0.2 µg of pRSV-β-galactosidase by lipofection using Superfect (Qiagen). When indicated, cells were also cotransfected with pcDNA3-p53, pcDNA3-Gfi (both provided by G. Nunez, University of Michigan, Ann Arbor, MI), pRc/CMV-p65 (provided by I. Udalova, University of Oxford, U.K.), pcDNA3-Stat3C (17), pcDNA3-E2F1 (18), or pcDNA3 empty vector. In another set of experiments, HEK293T cells were cotransfected with NLRP2 cDNA cloned into pHA-EAK vector (15), 0.25 µg of the reporter plasmid pBVIx-Luc, containing six NF-{kappa}B recognition sites within the promoter sequence linked to the luciferase gene (19), and 0.2 µg of pRSV-β-galactosidase. Polymorphic variants of NLRP2 were generated by site-directed mutagenesis of the pHA-EAK-NLRP2 vector (15) with 18-bp primers containing the nucleotide change in the middle of the sequence. All cDNA inserts were sequenced to verify the mutation.

Twenty-four hours posttransfection, cells were incubated with 10 ng/ml TNF-{alpha} (Sigma-Aldrich) for 6 h. In all cases, cell extracts were prepared and analyzed for the relative luciferase activity by a dual-light reporter gene assay system (Applied Biosystems). Results were normalized for transfection efficiency with values obtained with pRSV-β-gal.

Western blot analysis

Cell extracts (50 µg of protein) were separated on a 8% polyacrylamide gel and transferred to nitrocellulose as previously described (17). Blots were blocked with 3% BSA and incubated with rabbit Abs against Stat3, p65, and p53 (Santa Cruz Biotechnology) or NLRP2 (Abcam and Santa Cruz), mouse anti-E2F1, or anti-{alpha}-tubulin or goat anti-Gfi1 Abs (all from Santa Cruz Biotechnology), followed by incubation with goat anti-rabbit or anti-mouse or rabbit anti-goat Abs conjugated to alkaline phosphatase (Sigma-Aldrich). Bound Ab was detected by a chemiluminescence system (Applied Biosystems).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The expression of NLRP2 is up-regulated during differentiation of hemopoietic and mesenchymal progenitors

Initial studies showed that NLRP2 is widely expressed in tissues and tumor cell lines (6, 15). However, the expression of NLRP2 in progenitor cells and its regulation during differentiation of these progenitors is not known. We purified CD34+ progenitor cells from peripheral blood, and the selected population was cultured with either G-CSF or M-CSF to induce granulocyte or monocyte/macrophage maturation. The granulocytic cell population (CD34–CD15+) increased to >85% after 20 days of culture and showed morphologic features of mature granulocytes. When the cells were cultured in the presence of M-CSF, a clear pattern of monocyte/macrophage maturation was observed and, by day 20, the majority of cells were mature CD14+ cells (data not shown). We then analyzed the levels of NLRP2 mRNA in these cell populations and found that CD34+ progenitors expressed low levels of NLRP2, which increased ~4-fold by day 20 of culture in the monocytic lineage. During the granulocytic differentiation, NLRP2 reached the highest expression level at day 10 (~6-fold) of culture, and by day 20 the expression was similar to that of CD34+ cells, as determined by semiquantitative RT-PCR and real-time RT-PCR (Fig. 1A). To probe whether this expression pattern was specific for hemopoietic progenitors, we analyzed the expression of NLRP2 in mesenchymal stem cells obtained from bone marrow (BMSCs) and adipose tissue (ADSCs), and in adipocytic cells derived from ADSCs (Fig. 1B). Immunophenotypic characterization based on flow cytometry revealed that mesenchymal stem cells were positive for stromal cell-associated markers (CD73, CD90, CD13, CD44) and negative for typical hemopoietic markers (CD34, CD45) (data not shown). The levels of NLRP2 were ~2-fold higher in ADSCs than in BMSCs. Then, ADSCs were cultured in adipogenic medium and by 21 days cells were intensely stained with Oil Red O, which is retained by lipid vacuoles, and showed increased levels (3.5-fold) of NLRP2 mRNA compared with those in undifferentiated progenitors. Thus, both hemopoietic and mesenchymal models of differentiation show an up-regulated expression of NLRP2 in the more mature cells.


Figure 1
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  		FIGURE 1. Analysis of NLRP2 mRNA along differentiation of primary progenitor cells. A, CD34+ cells were cultured in the presence or in the absence of M-CSF or G-CSF to undergo monocytic or granulocytic differentiation, respectively. B, ADSCs were induced to undergo adipogenesis for the indicated period of time. Total RNA was obtained from progenitor and differentiated cells and analyzed for NLRP2 mRNA levels by real-time (upper panels) and semiquantitative (lower panels) RT-PCR. Real-time data were normalized using the value of the sample with the lowest level of NLRP2. β2-microglobulin was used as an amplification control. Histograms represent the means ± SD of three independent experiments.

 
NLRP2 is transcriptionally activated by NF-{kappa}B-p65 and TNF

It has been shown that some modulators of NF-{kappa}B activity (I{kappa}B{alpha}, TNF-{alpha}, NOD2) are in turn transcriptionally induced by NF-{kappa}B (14, 20, 21). In view of this, we analyzed the expression of NLRP2 mRNA in HEK293T cells after treatment with TNF-{alpha} and found a significant increase in the levels of NLRP2 (Fig. 2A). This expression pattern was similar to that of the cIAP1 gene, a known target of NF-{kappa}B. To further confirm the involvement of this transcription factor in the expression of NLRP2, we transfected HEK293T cells with the NF-{kappa}B subunit p65 and, 48 h later, RT-PCR analyses revealed a high up-regulation of NLRP2 mRNA (Fig. 2A). Similar data were obtained at the protein level following treatment of cells with TNF-{alpha} for 48 h (Fig. 2B). The expression pattern of NLRP2 mRNA and protein in response to TNF-{alpha} was reproduced in ADSCs (Fig. 2, C and D) and in the monocytic cell line THP1 (data not shown). As shown in Fig. 2C, the mRNA levels of NLRP2 increased after 6 h of treatment (~3-fold as determined by real-time RT-PCR) and reached the highest expression level (>5-fold) following 24 h of incubation with TNF-{alpha}. Although it has been previously shown that NLRP2 protein is induced at 4 h after stimulation with TNF-{alpha} (6), we failed to detect the endogenous protein at earlier times in all cell models (Fig. 2, B and D), most likely because of a lower affinity of the polyclonal Abs used, since they readily revealed the overexpressed protein in transfected cells (Fig. 2B). Based on the up-regulation of NLRP2 induced by NF-{kappa}B activators, we searched for consensus sites within the NLRP2 promoter region and found two putative NF-{kappa}B recognition sequences 244 and 271 bases upstream from the transcription start site (Fig. 2E). Then we tested whether NF-{kappa}B binds to these sites in response to TNF-{alpha} exposure or p65 overexpression by using an EMSA. As shown in Fig. 2F, both treatments resulted in the formation of a protein-DNA complex only when the proximal sequence (site 2) was used as a probe. The shifted bands obtained with anti-p65 and anti-p50 Abs demonstrated the presence of p65-p65 and p50-p65 dimers in the complex. The binding of NF-{kappa}B to site 2 of the NLRP2 promoter was also detected using nuclear extracts from ADSCs treated with TNF-{alpha} (Fig. 2F), which strengthens the physiological relevance of this interaction. To assess the transcriptional activity of the NLRP2 promoter, a 665-bp fragment containing the NF-{kappa}B consensus site of the NLRP2 promoter and two deletion fragments (Fig. 3A) were cloned into a promoterless luciferase vector, and these constructs were transiently transfected into HEK293T cells. Cotransfection with p65 increased the luciferase activity ~9-fold compared with cells transfected with the empty expression vector (Fig. 3B). Deletion of the fragment from –637 to –331 did not significantly affect luciferase activity. However, further deletion to –55 abrogated the promoter capacity of the cloned sequence (Fig. 3B). To test the functionality of the NF-{kappa}B binding sites, reporter constructs containing the mutated site 1 or site 2 were assayed for luciferase activity in response to p65 (Fig. 3C). Consistent with the EMSA experiments, mutation of site 1 did not modify the luciferase activity. By contrast, mutation of site 2 dramatically reduced the response of the NLRP2 promoter to p65. Of note, the constitutive activity of the promoter remained unchanged in the mutant constructs, indicating the contribution of other transcriptional regulators. More than 20 putative transcription factor binding sites were identified within the promoter region spanning –331 to –55, which retained the basal promoter activity of the entire 665-bp fragment. Among them, at least two are consensus sites for proteins (Stat and Gfi1) that have been previously associated with inflammation or chronic inflammatory disorders (22, 23). Thus, we tested the capacity of Stat3 and Gfi1 as well as two other transcriptional inducers, p53 and E2F1, used here as a specificity control to regulate the activity of the NLRP2 promoter. However, overexpression of these proteins (Fig. 3D) did not modify the constitutive promoter activity of NLRP2 as determined by luciferase assay (Fig. 3E). Thus, although this result does not clarify the contribution of other transcription factors, it strengthens the role of NF-{kappa}B to the transcriptional regulation of NLRP2.


Figure 2
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  		FIGURE 2. TNF-{alpha} and p65 up-regulate NLRP2 and promote binding of NF-{kappa}B to the NLRP2 promoter. A, HEK293T cells were incubated with or without 10 ng/ml TNF-{alpha} for 24 h or transfected with p65 and analyzed 48 h posttransfection. Following treatment, total RNA was extracted to determine the expression levels of NLRP2 by RT-PCR. The expression of cIAP1, a known target of NF-{kappa}B, was also included for comparison. β2-Microglobulin was used as an amplification control. B, HEK293T cells were cultured in the presence of TNF-{alpha} at different time intervals and then NLRP2 protein was analyzed by Western blot. A control of protein expression using the lysate from HEK293T cells transfected with NLRP2 were included (lane C). The levels of {alpha}-tubulin were also determined to assure equal loading. C and D, ADSCs were incubated with TNF-{alpha} for the indicated time points and the NLRP2 mRNA (C) and protein (D) levels were analyzed as described above. E, Sequence of the two putative NF-{kappa}B-binding elements in the NLRP2 promoter aligned with the NF-{kappa}B consensus sequence. F, HEK293T cells were either treated with 10 ng/ml TNF-{alpha} for 1 h or transfected with p65. Formation of binding complexes was determined by EMSA using radiolabeled probes from the NLRP2 promoter or a NF-{kappa}B consensus probe (cons). Nuclear extracts from ADSCs treated or not with TNF-{alpha} were also analyzed for the binding of NF-{kappa}B to site 2 of the NLRP2 promoter. Supershift analyses were performed using the indicated Abs to determine the NF-{kappa}B subunit composition of the complexes.

 

Figure 3
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  		FIGURE 3. p65 transactivates the NLRP2 promoter. A, Schematic representation of the NLRP2 promoter that illustrates the position of each deletion relative to the transcription start site (TSS). The NF-{kappa}B sites are also included. B, HEK293T cell line was transfected with a luciferase reporter vector containing sequential deletion fragments of the NLRP2 promoter, either in the presence or in the absence of p65. Following 24 h of transfection, cell extracts were analyzed for the relative luciferase activity. C, Reporter vector containing mutant site 1 or site 2 was introduced into HEK293T cells and luciferase activity in response to p65 was determined (cont, wild-type promoter fragment). Units of luciferase activity were normalized based on values of pRSV-β-galactosidase activity to control for transfection efficiency. D, Overexpressed transcription factors (TF) were detected by Western blot with the specific Abs. The first lane (–) represents cells transfected with the pcDNA3 empty vector. E, Cells were cotransfected with the indicated transcription factors and the NLRP2 promoter, and 24 h later were analyzed for luciferase activity. Data are presented as the mean of triplicate experiments ± SD.

 
An allelic variant of NLRP2 lacking NF-{kappa}B inhibition activity

It has been shown that mutations in the other two members of the NLR family with NF-{kappa}B modulatory properties, NLRP3 and NOD2, are associated with hereditary fever syndromes and chronic inflammatory diseases (24). Of note, most of these mutations are clustered in the highly conserved nucleotide-binding NACHT domain. To reveal the presence of single nucleotide changes in the NACHT domain of NLRP2 that may give rise to a functionally altered protein, we sequenced the entire NACHT domain in 319 DNA samples from normal donors. We identified five non-synonymous SNP variants that promoted amino acid changes, T221M, I352S, I354V, F359L, and R364K. The frequency of the rare alleles in the studied population varied between 0.2 and 10%, with 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 database included a SNP, E302Q, that was not detected in the population studied here. The alignment of NACHT domain sequences of the NLR family members NLRP2, NLRP3, and NOD2 showed that the SNPs found in NLRP2 are clustered within a region aligned with sequences that contain disease-associated amino acid changes in NLRP3 and NOD2 (Fig. 4B). Comparison with the structure-based model predicted for NLRP3 after alignment of multiple NACHT domain-containing proteins (25) indicated that the polymorphic variants of NLRP2 lie in a region that plays critical roles in oligomerization and NTP hydrolysis. To study the functional impact of the identified amino acid changes, we introduced the rare nucleotide for each one of the six polymorphisms in the NLRP2 cDNA by site-directed mutagenesis and analyzed the capacity of the different variants to inhibit NF-{kappa}B activation in response to TNF-{alpha}. HEK293T cells were cotransfected with the NLRP2 variants and a luciferase reporter gene driven by a promoter sequence containing six NF-{kappa}B-responsive elements. Immunoblot analysis of the lysates confirmed production of the NLRP2 variants at comparable levels (Fig. 5A). The overexpressed wild-type and most of the variant proteins reduced significantly the activation of NF-{kappa}B; however, the I352S change generated a protein lacking the NF-{kappa}B suppressor capacity (Fig. 5B). To further confirm this result, we studied the formation of DNA-protein binding complexes after TNF-{alpha} exposure in the presence of the different NLRP2 variants. Consistent with the gene reporter data, the protein containing the I352S change did not interfere with the binding of NF-{kappa}B to its recognition site (Fig. 5C). The specificity of the protein-DNA complex was confirmed by supershift analysis with anti-p65 and irrelevant anti-GATA1 Abs.


Figure 4
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  		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 Biotechnology 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. Polymorphic residues in NLRP2 and disease-associated variants in NLRP3 and NOD2 are circled. The alignment columns with strictly conserved residues are highlighted in gray boxes. CD, Crohn’s disease.

 

Figure 5
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  		FIGURE 5. The I352S polymorphism abrogates the NF-{kappa}B inhibition capacity of NLRP2. HEK293T cells were cotransfected with wild-type (wt) NLRP2 or its polymorphic variants and a reporter vector driven by six NF-{kappa}B binding sites. A, After 24 h of transfection, cell extracts were analyzed for protein expression by Western blotting. B, Transfected cells were stimulated with 10 ng/ml TNF-{alpha} for 6 h and then cell extracts were prepared and analyzed for luciferase activity. C, Empty expression vector. Histograms show the means ± SD of three independent experiments. C, Nuclear extracts from cells transfected with the NLRP2 variants and stimulated with TNF-{alpha} for 1 h were analyzed for the formation of protein-DNA-binding complexes by EMSA using a NF-{kappa}B consensus probe. Extracts from control (empty vector-transfected) cells treated with TNF-{alpha} were preincubated with anti-p65 and irrelevant anti-GATA1 Abs.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Deciphering the mechanisms that regulate the activity of NF-{kappa}B is of major importance for understanding the response to inflammatory stimuli. In this study, we show that NLRP2, an inhibitor of the NF-{kappa}B pathway, is up-regulated during differentiation of hemopoietic progenitors toward granulocyte or monocyte/macrophage lineages and after differentiation of mesenchymal stem cells into adipocytes. Interestingly, NOD2, another NLR family member that activates NF-{kappa}B signaling, is also up-regulated during the myelomonocytic differentiation of CD34+ progenitor cells (14). Based on these data, both positive and negative regulators appear to be less dispensable in mature cells where they may establish a balance that controls the activity of NF-{kappa}B. NF-{kappa}B has been shown to protect osteoclast progenitors from apoptosis (26) and to be activated in bone marrow and cord blood progenitor cells (27). Additionally, it has been described that NF-{kappa}B activation can prevent chondrogenesis and myogenesis from mesenchymal cells (28). Taken together, these data along with our findings highlight the interest of studying the NF-{kappa}B regulators in progenitor and stem cells and deciphering their role in the differentiation programs toward different lineages. We also found that NLRP2 regulation involves a transcriptional activation mechanism through NF-{kappa}B. Although two putative NF-{kappa}B binding sites were identified within a region spanning ~1400 bp from the transcription start site, only the proximal site proved to be functional and accounted for the capacity of the NLRP2 promoter to respond to NF-{kappa}B proteins. However, chromatin immunoprecipitation analyses showed no enrichment of fragments containing the NF-{kappa}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-{kappa}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 inflammatory 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-{kappa}B, up-regulation of NLRP2 may be part of a negative regulatory loop induced via inflammatory stimuli. A similar NF-{kappa}B-dependent mechanism has been described for NOD2 (14) and TLR2, which activates the NF-{kappa}B signaling pathway following recognition of microbial components (31), and for the NF-{kappa}B inhibitor I{kappa}B{alpha} after stimulation with TNF-{alpha} or transfection with the p65 subunit of NF-{kappa}B (21), which has been proposed as an inducible autoregulatory pathway.

There is mounting evidence that NF-{kappa}B plays a major role in autoimmune and inflammatory disorders. Therefore, mis-regulation of proteins that control the NF-{kappa}B pathway is likely to contribute to disease susceptibility or severity. In line with this, mutations in the NOD2 gene have been identified in up to 50% of Crohn’s disease patients, but the risk alleles are also present in the healthy population albeit at lower frequencies than in disease. The rare allele frequency of NOD2 may be up to 18% depending on the ethnic group (32). We have identified five SNPs within the NACHT domain of NLRP2 in a population of 319 healthy individuals. One of these polymorphisms (allele frequency of 0.4%) gave rise to an amino acid change (I352S) that disrupted the capacity of NLRP2 to inhibit the transcriptional activity of NF-{kappa}B and the formation of NF-{kappa}B-DNA-binding complexes. Of note, patients with hereditary fever syndromes and chronic inflammatory diseases who carry mutations in the NACHT domain of NLRP3 have been recently described (11, 33). Additionally, polymorphisms in both alleles of the NOD2 gene increase the risk for developing Crohn’s disease, and those patients carrying the rare alleles of NOD2 were characterized by a younger age at onset and a less frequent colonic involvement than were seen in patients who had no mutation (34). Genetic variants in other NF-{kappa}B regulators, including I{kappa}B{alpha} and NEMO, that abolish or reduce the activation of NF-{kappa}B have been associated with a number of inherited genetic disorders, mainly immunodeficiency syndromes (35). Moreover, a truncating and inactivating polymorphism in CARD8, which is an inhibitor of the NF-{kappa}B pathway structurally related to NLRP1, has been associated with susceptibility to inflammatory bowel disease (36). Most of the disease-associated polymorphisms in NLRP3 and some in NOD2 were located within the NACHT domain. We aligned the amino acid sequences surrounding the polymorphisms identified in the NACHT domain of NLRP2 with the corresponding sequences in NLRP3 and NOD2 proteins. Both NLRP3 and NOD2 are predicted to have similar secondary structures (25). The I352S polymorphic change that gives rise to a nonfunctional NLRP2 protein lies close to four polymorphisms in NLRP3 associated with CINCA, MWS, or FCAS and is also located near residue R345, corresponding to an arginine finger as predicted by alignment of the NTPase domains of NLRP proteins and p97 ATPase (25). This finger plays a critical role in cooperative NTP hydrolysis (37). I352S is also close to the position aligned with A374 in NLRP3, which has been associated with CINCA. Consistent with our functional data, I352S is the only polymorphism among all identified in the NACHT domain of NLRP2 that localizes in a position aligned with a disease-associated variation (E441K in NOD2) in either NLRP3 or NOD2 proteins. It has been shown that the E441K change has an impact on the NOD2-mediated activation of NF-{kappa}B in response to bacterial peptidoglycan and is considered a rare polymorphism associated with Crohn’s disease (38). Taking into account the complex control on the activation of NF-{kappa}B and the increasing number of proteins involved, analyses of deleterious or activating polymorphisms in candidate genes will be needed to decipher their contribution to chronic inflammatory disorders or autoimmune diseases.

Overall, our observations shed light on the expression of NLRP2 during differentiation of progenitor or stem cells and provide evidence on the regulation of NLRP2 through NF-{kappa}B, which suggests that this protein may be involved in a negative regulatory loop to limit the response of NF-{kappa}B. Our data also identify an allelic variant of NLRP2 that lacks the capacity to inhibit the NF-{kappa}B transcriptional pathway, providing the rationale for studying the association between this inactivating polymorphism and disease activity or an inherited predisposition to autoimmune or chronic inflammatory disorders.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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 Investigacion Sanitaria and API/06/02 from Fundacion Marques de Valdecilla. Back

2 Address correspondence and reprint requests to Dr. Jose L. Fernandez-Luna, Unidad de Genetica Molecular, Hospital Universitario Marques de Valdecilla, Edificio Escuela Universitaria de Enfermeria, Av. Valdecilla s/n, 39008 Santander, Spain. E-mail address: fluna{at}humv.es Back

3 Abbreviations used in this paper: IKK, I{kappa}B kinase; NLR, nucleotide-binding domain and leucine-rich repeat-containing family; NLRP, pyrin domain-containing NLR protein; NACHT, nucleotide-binding domain; SNP, single nucleotide polymorphism; FCAS, familial cold autoinflammatory syndrome; MWS, Muckle-Wells syndrome; CINCA, chronic infantile neurologic cutaneous and articular syndrome; BMSC, bone marrow-derived mesenchymal stem cell; ADSC, adipose tissue-derived mesenchymal stem cell. Back

Received for publication May 30, 2007. Accepted for publication October 2, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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