The Regulation of MEFV Expression and Its Role in Health and Familial Mediterranean Fever

REVIEW The regulation of MEFV expression and its role in health and familial Mediterranean fever

S Grandemange1, I Aksentijevich2, I Jeru3, A Gul4 and I Touitou1 1Unite´ me´dicale des maladies auto-inflammatoires, CHRU de Montpellier, INSERM U844, UM1, Hopital Arnaud de Villeneuve, Montpellier, France; 2National Human Genome Research Institute, Bethesda, MD, USA; 3INSERM U933, Universite´ Pierre et Marie Curie-Paris6 UMR S933, Paris, France and 4Istanbul Faculty of Medicine, Division of Rheumatology, Department of Internal Medicine, Istanbul University, Istanbul, Turkey

Familial Mediterranean fever (FMF) is a hereditary recurrent fever associated with mutations in the gene MEFV encoding pyrin. It is expressed mainly in neutrophils and macrophages, and modulates the production of the potent pro-inflammatory cytokine interleukin-1b through regulation of nuclear factor-kB and caspase-1. The MEFV gene expression depends on multiple levels of regulation. Sequence variants located in the promoter and at the 30-untranslated region of the gene modulate this expression. Two studies demonstrated decreased mRNA levels in FMF patients compared with healthy subjects, whereas two others found no significant differences. The diverse experimental settings may have resulted in variable quantification of the 15 splice variants that have been identified recently. Some of these isoforms are regulated by nonsense-mediated decay in both cell- and transcript-specific manner, and may be differentially translated in THP1 cells. In addition, pyrin may be cleaved by caspase 1. The full-length pyrin was less abundant than the cleaved fragment in mononuclear cells from FMF patients than in controls, whereas the opposite was observed in granulocytes. Altogether, the regulation of MEFV expression is more complex than anticipated in both physiological and pathological conditions. Its deregulation is likely to alter the inflammasome function and subsequently result in uncontrolled inflammation as seen in FMF. Genes and Immunity (2011) 12, 497–503; doi:10.1038/gene.2011.53; published online 21 July 2011

Keywords: expression; MEFV; FMF

Introduction The gene responsible for FMF, named MEFV, encodes pyrin2 (alternatively known as marenostrin,3) a protein of Familial Mediterranean fever (FMF) is a recessively 781 amino-acids (Figure 1). Initial computer analysis of inherited systemic auto-inflammatory disease. Auto- pyrin/marenostrin sequence revealed that this protein inflammatory diseases are an expanding group of innate contains two putative overlapping nuclear binding immunity diseases that have been recently revisited as a motifs and shows high homology with the B30/2 rfp continuum, which includes diseases with some features transcription factor family. This observation led to the of adaptive immune deregulation. They include heredi- hypothesis that pyrin was a nuclear transcriptional tary recurrent fevers, pyogenic disorders such as the factor. Later, sequence alignments and secondary struc- deficiency of interleukin (IL)-1 receptor and pyogenic ture prediction highlighted that the first 100 N-terminal sterile arthritis pyoderma gangrenosum and acne (PAPA), residues of pyrin constituted a novel protein domain granulomatous diseases such as Crohn’s disease and Blau named PYrin Domain (PYD),4–6 and revealed that PYD syndrome. FMF is considered the prototype among the was the fourth member of the death domain fold hereditary recurrent fevers as the most frequent and the superfamily. PYD orthologs were also identified in first whose gene has been identified. FMF patients suffer several species such as mouse, cow and zebrafish. from recurrent and seemingly unprovoked attacks of The PYD is an N-terminal adapter domain that is found fever, often associated with peritonitis, pleuritis, arthritis in more than 20 human proteins involved in inflamma- and localized erysipelas-like erythema. The attacks tion and apoptosis, such as NLRP3 (NLR family, PYD usually last 12–72 h.1 The inflammatory reaction is containing 3), similar to other death domain fold- characterized by a massive influx of polymorphonuclear containing proteins (for a review see Kohl et al.7) Recent leukocytes into the affected tissues. studies indicate that pyrin modulates the production of the potent pro-inflammatory cytokine IL-1b probably through two major pathways: (i) the N-terminal frag- Correspondence: Professor I Touitou, Unite´ me´dicale des maladies ment of pyrin may regulate nuclear factor (NF)-kB auto-inflammatoires, CHRU de Montpellier, INSERMU844, UM1, activation through interactions with an adapter protein Hopital Arnaud de Villeneuve, Montpellier, 371, Avenue du Doyen named apoptosis-associated speck-like protein with a Giraud 34295, Montpellier cedex 5, France. 8–10 E-mail: [email protected] caspase-recruitment domain (ASC) and/or with Received 20 May 2011; accepted 27 June 2011; published online 21 NFkB-p65,11 and (ii) pyrin regulates caspase-1/IL-1b July 2011 activation through its interaction with ASC.12 Addition- MEFV expression in FMF S Grandemange et al 498 MEFV DNA: 14600bp 5’flanking 3’ flanking 1 2 3 4 5 6 7 8 9 10

mRNA: 3499bp 1 2 3 4 5 67 8 9 10

Protein: 781aa PyD B-Box CC B30.2/SPRY

TRIM20 DDF TRIpartite Motif family Viral Proteins? Inflammasome Figure 1 Schematic representation of the FMF gene (MEFV) and its expression products at the RNA and protein levels. Exons are boxed, introns are depicted as lines. The protein domains are illustrated along with their putative functions. The death domain fold at the N-terminal domain is involved in regulating inflammation via homotypic interactions with inflammasomes. Interestingly, residues (680 and 694) important for the conservation of the pyrin structure are precisely those which, when mutated, are associated with a severe form of the disease.39 The SPRY domain is present in several proteins of the tripartite motif family. This domain may interact with viral components. A full colour version of this figure is available at the Genes and Immunity journal online.

ally, the C-terminal B30.2 domain of pyrin, also known as FMF mutations may also lead to dysregulation in the SPRY, directly interacts with caspase-1 to modulate IL-1b MEFV expression affecting pyrin level. production.13,14 This domain, together with the B-Box The past decade has witnessed the explosion of and coiled-coil domains, constitutes a tripartite motif, reviews on auto-inflammatory diseases written by which is contained in proteins involved in viral recogni- scientists and clinicians from a variety of disciplines. tion. Thus, pyrin is also known as TRIM20.15 Most of them attempted to correlate clinical features with The MEFV gene contains 10 exons. Nearly 200 genotypes in patients with auto-inflammatory diseases, sequence variants, almost all single-nucleotide substitu- or to dissect the underlying inflammatory pathways. tions, have been recorded in Infevers,16 a database However, there has been no review on the regulation of dedicated to auto-inflammatory mutations (http:// MEFV gene expression in physiological or pathological fmf.igh.cnrs.fr/ISSAID/infevers/). Only a small number conditions. In this manuscript, we review the complex of these variants are unambiguously pathogenic, such as regulation of MEFV expression at the RNA and protein M694V, a severe mutation with a founder effect, and no levels, summarize the data obtained from various cell more than half of the reported MEFV variations are lines along with expression data from FMF patients, and associated with the FMF phenotype. The vast majority of discuss impaired regulation of MEFV expression as a genuine FMF-associated mutations are found in the possible mechanism of the disease. SPRY domain of the protein. How mutations in the MEFV gene can result in so variable an inflammatory phenotype has been only partially elucidated.17 Most MEFV expression at the mRNA level molecular mechanisms that have been hypothesized suggest an impaired or dysregulated interaction of Cellular expression mutant pyrin protein with its partners, resulting in Initial expression studies using northern blot experi- activation of the NLRP3 inflammasomes.18 Inflamma- ments detected MEFV mRNA only in peripheral blood somes are cytoplasmic multiprotein complexes essential leukocytes (PBLs) and in a colorectal adenocarcinoma 2 for the maturation of proinflammatory cytokines IL-1b, cell line, SW480. Specific analysis of leukocyte fractions IL-18 and IL-33. Several inflammasomes have been from bone marrow and PBLs showed that MEFV was described on the basis of their constituent stress-sensing highly expressed in neutrophils, and to some extent in 20,21 components. In NLRP inflammasomes, ASC works as an eosinophils and monocytes, but not in lymphocytes. adapter molecule that connects the stress-sensing com- MEFV is also expressed in dendritic cells and in ponent and pro-caspase-1 through its N-terminal PyD fibroblasts from synovium, peritoneum and to a lesser 22 domain and C-terminal CARD domain, respectively (for extent from skin. This highly specific cellular expres- reviews see Chae et al.18 and Schroder and Tschopp19). sion pattern likely accounts for the predilection of FMF This brings two molecules of procaspase-1 into close patients to develop inflammation in serosal, synovial and proximity, leading to proteolytic activation and the skin tissues. subsequent release of the active catalytic domains, p20 and p10. Active caspase-1, in turn, cleaves the 31-kDa Transcriptional regulation precursor form of IL-1b into its biologically active 17-kDa Numerous mediators, such as lipopolysaccarides, pro- fragment. Given that ASC also interacts with pyrin inflammatory or anti-inflammatory cytokines, can mod- through PyD, it is possible that pyrin is somehow ulate the mRNA expression of the MEFV gene. However, involved in several inflammasomes as a modulator, or its expression profile is very much dependent on the pyrin itself is a component of an inflammasome. Thus, cellular sources and experimental conditions (Table 1). most studies related to pyrin’s function in the innate Most studies have used only semi-quantitative immune system have been focused on the regulation of approaches to measure the MEFV expression. Several the caspase-1 activation and subsequent IL-1b secretion. investigators have examined the role of regulatory A growing body of evidence suggests, however, that regions upstream and downstream of the MEFV coding

Genes and Immunity MEFV expression in FMF S Grandemange et al 499 Table 1 Effect of various soluble mediators on MEFV mRNA levels

Cells Effect Reference

Colchicine Neutrophils None Centolla et al.20 Colchicine Peritoneal fibroblasts Increase Abedat et al.37 Colchicine + IFN-a or + IFN-g Neutrophils Increase Centolla et al.20 PMA Cutaneous and peritoneal fibroblasts None Matzner et al.38 PBLs, PBMCs, monocytes, THP1 LPS Synovial fibroblasts Increase Centolla et al.,20 Papin et al.26 and Matzner et al.38 IFN-g Neutrophils, peritoneal fibroblasts None Centolla et al.20 and Abedat et al.37 IFN-g Monocytes, neutrophils, peritoneal Increase Centolla et al.20 and Papin et al.26 fibroblasts TGFb Monocytes Decrease Centolla et al.20 TNFa Monocytes, peritoneal fibroblasts Increase Centolla et al.20 and Abedat et al.37 TNFa Neutrophils None Abedat et al.37 IL1b Cutaneous and peritoneal fibroblasts Increase Centolla et al.20 and Abedat et al.37 IL1b Neutrophils None Abedat et al.37 IL4 Monocytes Decrease Centolla et al.20 IL10 Monocytes Decrease Centolla et al.20

Abbreviations: IFN, interferon; IL, interleukin; LPS, lipopolysaccarides; PBLs, peripheral blood leukocytes; PBMCs, peripheral blood mononuclear cells; PMA, phorbol myristate acetate; TGF, tumor growth factor; TNF, tumor necrosis factor.

NFB C/EBP IFN c-myb AML IFN PU1 -571 -414 -286 -41 enhancer minimal promoter c.-614G>C c.-382C>T ATG c.*836_837ins haplotypes 5’-region Coding region 3’-region Figure 2 Schematic representation of MEFV regulatory elements. The 1000-bp region upstream of the translation initiation site contains a putative enhancer and a minimal promoter. The broken arrow stands for the transcription initiation site. Sites common in myeloid-specific promoters are represented by green boxes (AML, acute myeloid leukemia; cEBP, CCAAT/enhancer binding protein) and those required for cytokine activation by purple boxes (IFN, interferon). The circle arrow shows the synergistic activation of C/EBP and NF-kB-p65 for responsiveness to tumor necrosis factor. The functional polymorphisms with possible role in FMF are depicted in red. A full colour version of this figure is available at the Genes and Immunity journal online. sequence. The promoter exhibits a number of consensus 36 Turkish FMF patients without MEFV coding region binding sites for known transcription factors, for mutations did not find any variants in the putative example, sites common in myeloid specific promoters MEFV promoter region.25 However, sequencing of the and sites required for cytokine activation (Figure 2). 30-untranslated region revealed several single-nucleotide Papin et al.23 reported that the MEFV 50-flanking region is polymorphisms within two Alu repeats, which were necessary for responsiveness to tumor necrosis factor, expressed in the MEFV mRNA. The respective single- and that it is dependent on a synergistic activation of nucleotide polymorphisms were clustered into two C/EBPbeta (factor required for cell responsiveness to haplotypes, and the frequency of heterozygote genotypes tumor necrosis factor) and NFkB-p65 (activating factor was significantly higher in the patients without coding acting in synergy with C/EBPbeta). We have character- region mutations compared with healthy controls.25 ized the minimal MEFV promoter region between These data suggest a role of the 50- and 30-regulatory nucleotide residues c-41 and c-286, and identified a sequences in the expression of MEFV and pathogenesis region with a putative enhancer extending from c-414 to of FMF. c-571.24 Further investigation of the 1000-bp sequence upstream of the initiation codon identified a couple of Post-transcriptional regulation nucleotide variants that may have a role in regulation of MEFV is transcribed into a major full-length transcript of the MEFV expression. The novel c-614C4G variant that 3.5 kb. The first MEFV splice isoform was described by was found in a heterozygous state in an FMF patient of Papin et al.26 This variant is generated by in-frame Arab descent resulted in a 70% decrease in promoter alternative splicing of exon 2 (2D) and expressed in PBLs. activity as measured by luciferase reporter experiments. Seven more transcript isoforms that introduce various The other promoter variant, c-382C4T found in a non- combinations of novel exons 2a and 4a, and a 30 Ashkenazi-Jewish asymptomatic individual induced a extension of exon 8 (8ext) were identified in synovial 100% increased activity. Using electrophoretic mobility fibroblasts by Diaz et al.22 Recently, our group27 and a shift assay experiments, we observed specific DNA– Lebanese group28 detected six more weakly expressed protein complexes at both –382 and –614 wild-type transcripts, with various deletions in exon 2, 3, 4, 7 and 8, regions, indicating that nuclear protein(s) were able to or a 50 extension of exon 9 (9ext) in PBLs, making the bind to these sequences and perhaps contribute to the total number of known MEFV transcript isoforms regulation of enhancer activity.24 A separate analysis of hitherto equal to 15. Most of them, if translated, would

Genes and Immunity MEFV expression in FMF S Grandemange et al 500 conduct to a premature stop codon (Figure 3). The and responsiveness to treatment.30 We hypothesize that physiological relevance of these alternatively spliced individual variations in NMD efficiency for alternatively isoforms is still unclear. spliced MEFV transcripts may have a role in FMF To determine which of the transcripts could potentially pathophysiology and, thereby, may account for part of be expressed at the protein level, we have examined the the phenotypic heterogeneity observed in FMF patients. effect of nonsense-mediated decay (NMD) inhibition on The MEFV gene expression in FMF patients and the expression of the MEFV transcripts in various cells.27 controls has been investigated using both semi-quanti- Alternative splicing and NMD pathways are generally tative and quantitative PCR analysis. MEFV mRNA coupled in the post-transcriptional regulation of gene levels were decreased in PBLs of symptom-free French expression (for a review see Neu-Yilik and Kulozik29). FMF patients of Mediterranean origin compared with Indeed, NMD is a mechanism that controls mRNA controls.31 This effect correlated with the number and the quality, prevents translation of unnecessary and aberrant type of mutations, the lowest mRNA expression being transcripts, for example, mutations generating premature observed in patients homozygous for the most severe terminal codon, and modulates protein level in a cell- FMF-associated mutation, M694V. The second study in a specific manner. The indirect inhibition of NMD by Turkish cohort showed significantly lower mRNA levels cycloheximide in THP1 cells and neutrophils had no in PBLs of asymptomatic FMF patients compared with effect on the full-length transcript, but dramatically non-FMF controls, and a further decrease in expression increased the expression of exon 4a transcripts. The was observed during an acute peritonitis attack. How- expression of exon 8ext and exon 4a transcripts was ever, the MEFV gene expression did not correlate with increased in monocytes, supporting the role for NMD in the patient’s genotypes. Interestingly, MEFV expression regulation of MEFV expression in these cells. Direct was significantly decreased in non-FMF control patients inhibition of NMD using small interfering RNA against with acute appendicitis during the peri-operative period Upf1, one of the components of the NMD machinery, compared with their asymptomatic states.32 In a recent showed that in THP1 cells, NMD has a role in the study, MEFV gene expression levels were marginally regulation of a subset of the MEFV isoforms, including lower in Greek FMF patients compared with healthy transcripts exon 4a and del234. All together, these results subjects.33 In contrast, a study performed in the USA demonstrated that the expression of MEFV is regulated cohort found a tendency for an increased level of the by NMD in both a cell- and a transcript-specific manner. MEFV expression in 19 asymptomatic FMF patients Previous reports suggested that variation in NMD compared with controls, but this result was not statisti- efficiency is associated with numerous genetic disorders cally significant.34 These apparent discrepancies are

MEFV gene 2a 4a 8ext 9ext 12 34 5 678910 93 96 98 23 33 33 317 633 350 231 115 118 175 Splicing variants 1667 kDa FL 86.4 4a 53.6 8ext 67.7

2 64.4 2-4a 31.7 2-8ext 45.7 2-9ext 47.4

2a 67.6 2a-4a 34.8 2a-8ext 49

del2.3.4 10.8

del2.3.4 del7 10.8

del2.3.4 del7.8 10.8

del2.3.4.5 11.8

del3.4 del7.8 33.2

coding region Figure 3 Schematic representation of the 15 currently known MEFV transcripts isoforms. Exons are boxed, introns are depicted as lines. The open-reading frames of each transcript are greyed and their corresponding untranslated regions are represented by white boxes. Only FL, 2D and 2a transcripts (underlined) retain the full open-reading frame; all other transcripts have a premature stop codon. The sequences gained are blacked. FL, full-length; del or D, deletion; ext, extension. The expected size of the corresponding protein is on the right in kilodaltons (kDa).

Genes and Immunity MEFV expression in FMF S Grandemange et al 501 likely partly accountable to the different experimental degradation of I-kappa-B-alpha (IKB-a).11,18 The C- conditions (sample size, cell type used for RNA extrac- terminal-cleaved fragment, where most FMF mutations tions, instrument, primer pairs used for quantitative lie, remains cytoplasmic, whereas the N-terminal- reverse transcriptase-PCR, methods used for data analy- cleaved fragment translocates along with p65 to the sis) that may inconsistently track the different MEFV nucleus. Moreover, the pyrin cleavage is increased in transcripts, especially those subjected to NMD regula- peripheral blood mononuclear cells from FMF patients, tion. Alternatively, the variable prevalence of putative whereas full-length pyrin seems to be less expressed in regulatory variants in different ethnic cohorts may patients. However, the pyrin cleavage appears to be cell- account for some variability in gene expression among dependent, and in granulocytes, the full-length pyrin these studies. was shown to be more abundant in FMF patients than in controls. MEFV expression at the protein level Cellular localization Conclusions Data at the protein level are scarce; however, pyrin protein expression is also highly variable and dependent This bibliographical work highlighted that MEFV on cell type, experimental conditions and protein iso- expression is dependent on cell type and inflammatory form. In Chinese hamster ovary and HeLa cells stably status. This is probably accounted for by the multiple transfected with green fluorescent protein-tagged pyrin, levels of regulation that the MEFV gene and its products the full-length protein is cytoplasmic, but the form undergo. At the transcriptional level, MEFV is regulated lacking exon 2 is mainly nuclear.26,35 In synovial by numerous factors such as interferon a and NF-kB. fibroblasts transiently transfected with myc-tagged pyr- At the post-transcriptional level, 15 splicing isoforms in, the full-length and 2a transcripts are cytoplasmic and have been described so far, some of them being subjected the 2D isoform showed a mixed pattern.22 The native to NMD regulation. At the post-translational level, pyrin is nuclear in synovial fibroblasts, neutrophils and pyrin can be phosphorylated and cleaved. Quantitative dendritic cells, but cytoplasmic in monocytes.22 Thus, the and qualitative differences between controls and FMF expression of endogenous pyrin is likely related to the patients have been observed at all levels, although existence of various protein isoforms. some of the available data need to be confirmed. Despite rapidly growing data about the regulation of MEFV, several questions remain to be addressed. Are Translational regulation there quantitative and qualitative variations of these Reasoning that differential protein expression could be alternative MEFV transcripts in physiological and FMF the result of differential mRNA regulation, we recently conditions? What is the effect of pro- and anti-inflam- conducted western blot analyses of endogenous proteins matory cytokines and colchicine, the mainstay treatment expressed in THP1 cells.27 Parallel overexpression of of FMF, on the expression of these transcripts? Could the various cDNAs in HEK293 cells provided us with the possible existence of several MEFV protein variants migration pattern of each isoform. We obtained bands explain the controversial results of sub-cellular localiza- compatible with the size of the full-length, 2D8ext tion studies? and 2a4a products demonstrating that, as expected, at Altogether, the regulation of MEFV expression is much least some of these alternatively spliced MEFV tran- more complex than initially thought, and it is an scripts are translated into protein variants. Moreover, in important issue, because its deregulation is likely to be agreement with the results obtained at the mRNA level, one of the mechanisms involved in FMF pathogenesis. the putative 2D8ext was insensitive to NMD, whereas Even if the exact role of pyrin in innate immunity is expression of the putative 2a4a was increased after controversial, it probably has a direct and/or indirect direct inhibition of NMD. Western blots did not crucial role in inflammasome regulation, and any demonstrate a significant difference in pyrin levels qualitative or quantitative modification of its expression between single and double variant patients. However, may have a critical consequence in the balance between FMF patients of both types showed higher protein pro- and anti-inflammatory signaling pathways, which expression, compared with controls and non-FMF may subsequently result in uncontrolled inflammation. patients with active inflammation.34

Post-translational regulation Pyrin expression was shown to be dependent to at least Conflict of interest two post-translational modification processes. Pyrin phosphorylation mediates the binding to 14.3.3 at three The authors declare no conflict of interest serine residues located in exon 2.36 14.3.3 is thought to have a role in apoptosis. As a result of this interaction, full-length pyrin is retained in the cytoplasm. The lack of Acknowledgements interaction with the D2-pyrin isoform results in protein translocation to the nucleus. These data reconcile some This work was supported by the CHRU of Montpellier observations made in vitro about the different subcellular (PHRC2005), the National Human Genome Research localization of various isoforms. Institute, INSERM and Istanbul Faculty of Medicine. We Pyrin may also be cleaved by caspase 1 at the residue thank the clinicians for providing patient samples, J Tazy Asp330, producing an N-terminal fragment that in- for helpful discussion and M Vittal for English editing of creases ASC-independent NF-kB activation through the manuscript.

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