Hematopoietic-specific expression of MEFV, the gene mutated in familial Mediterranean fever, and subcellular localization of its corresponding protein, pyrin

Cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). NB4 cells were generously provided by Dr M. Lanotte (St. Louis Hospital, Paris, France). ML-1 cells were a gift from Dr M. Kastan (Johns Hopkins University, Baltimore, MD). Kasumi1 and Kasumi3 cells were established by Dr Hiroya Asou (Hiroshima University, Hiroshima, Japan). Adherent and suspension cells were grown in DMEM containing 10% to 20% fetal calf serum (FCS) and in RPMI containing 10% FCS medium, respectively, in a humidified atmosphere with 5% carbon dioxide at 37°C. HL-60 cells were incubated in the presence of 1.25% (v/v) DMSO for up to 4 days to induce granulocytic differentiation.

Polymorphonuclear cells (neutrophils) were isolated from anticoagulated blood using PMN solution (Robbins Scientific Corp, Sunnyvale, CA) according to the 1-step density-gradient centrifugation method.9 10 Briefly, whole blood was layered over PMN solution and centrifuged for 25 minutes at 500g. Mononuclear cells and neutrophils were separated into 2 distinct bands, whereas erythrocytes pelleted to the bottom of the tube. Neutrophils were obtained from the lower band and washed twice with serum-free medium. Monocytes were separated from the mononuclear cell fraction by adherence to plastic cell-culture dishes. Viability of the cells was tested by trypan blue dye exclusion.

First, mononuclear cells were separated from heparinized blood by Ficoll-Paque (Pharmacia Biotech, Piscataway, NJ) density gradient centrifugation. Individual populations of peripheral blood leukocytes were further isolated by flow cytometry (FACStar, Becton Dickinson, Mountain View, CA) using monoclonal mouse antibodies against CD19 and CD3 conjugated to fluorescein isothiocyanate and PE, respectively (Dako, Carpinteria, CA).

Total RNA was isolated using TRIzol reagent (Gibco BRL, Gaithersburg, MD) according to the method described by Chomczynski and Sacchi11; 2 μg of total RNA were reverse transcribed using SuperscriptII (Gibco BRL) and random primers (Gibco BRL) according to the manufacturer's instructions. Polymerase chain reaction (PCR) was performed with Taq DNA polymerase (Gibco BRL) under standard conditions using MEFV-specific oligonucleotides (5′-ATCCAACTCCTCCACCAGAA-3′ and 5′-AGTGTTGGGC-ATTCAGTCAG-3′) that amplify a 690–base pair (bp) fragment. All PCR primer pairs span an exon/intron boundary to allow discrimination between complementary DNA (cDNA)- and DNA-generated fragments. Oligonucleotides specific for β-actin (5′-TACATGGCTGGGGTGTTGAA-3′ and 5′-AAGAGAGGCATCCTCACCCT-3′) amplify a 218-bp fragment, and oligonucleotides for lactoferrin (5′-GTTCAGTGGTGCGCCGTATC-3′ and 5′-CACCACAGCCACGGCATAAT-3′) generate a 279-bp fragment. Each cycle of PCR consisted of denaturation (1 minute at 95°C), annealing (1 minute at 60°C, 55°C, or 64°C forMEFV, lactoferrin, and β-actin, respectively), and extension (1 minute at 72°C) and was repeated 22 to 30 times, as indicated in “Results.” PCR-amplified DNA fragments were visualized by ethidium bromide staining after agarose gel electrophoresis.

Gel-separated PCR products were blotted onto a positively charged nylon membrane (Boehringer Mannheim, Indianapolis, IN, or Amersham Life Science, Arlington Heights, IL) by capillary transfer in 20xSCC. Prehybridization (30-45 minutes) and hybridization (1-2 hours) occurred at 42°C in DIG (digoxigenin) Easy-Hyb solution (Boehringer Mannheim). As a hybridization probe, internal oligonucleotides specific for MEFV5′-GACAGC-ATGGATCCTGGGAGCCTGCAAG-3′, lactoferrin 5′-CCCTTGATGGTGGTTTCA-TATACGA-3′, or β-actin 5′-ATCGAGCACGGCATCGTCAC-3′ were labeled with DIG using the DIG 3′ End Labeling Kit (Boehringer Mannheim). Washes were repeated twice in 2xSCC for 5 minutes at room temperature and in 0.5xSCC for 5 minutes at 42°C. The membrane was blocked (1% blocking reagent in maleic acid buffer) for 30 minutes prior to incubation with anti-DIG antibodies coupled to alkaline phosphatase (30 minutes). After washing twice in maleic acid buffer, detection was performed by incubating with the chemiluminescent substrate CDP-Star (Tropix, Bedford, MA). Autoradiographic film (X-OMAT/AR, Eastman Kodak, Rochester, NY) was exposed to the blot for several seconds up to 30 minutes.

In a 2-step cloning procedure, the cDNAs of EGFP and pyrin were cloned into the pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA). First, PCR-amplified EGFP cDNA (Clontech, Palo Alto, CA) was cloned into the vector using recognition sites for restriction endonucleasesHindIII and KpnI. Second, the full-length cDNA of pyrin was amplified by PCR using Pfu polymerase (Stratagene, La Jolla, CA) and the following primers that contain recognition sites forBglII and EcoRI, respectively: 5′-GCATATAGATCTATGGCTAAGACCCCTAG-3′ and 5′-GCATATGAATTCGGCAT-TCAGTCAGGCC-3′. PCR fragments were digested, gel-purified, and cloned in frame 3′ of EGFP cDNA into the EcoRI and BglII sites of a pCDNA3.1(+) expression vector. Transformants were tested for the correct insert by restriction enzyme digestion and DNA sequencing. Either EGFP or EGFP-pyrin plasmid (15 μg) were transfected into COS-1 cells using Superfect reagent (Qiagen, Valencia, CA). Transfected cells were plated on cover slips and analyzed 2 to 3 days after transfection by fluorescent microscopy using a 510-nm filter.

Transfected cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors on day 2 after transfection and separated by SDS-PAGE on a 4% to 15% acrylamide gradient gel. Proteins were transferred onto a nitrocellulose membrane and detected with a monoclonal anti-GFP antibody (Clontech). Incubation with a secondary goat anti-mouse antibody coupled to horseradish peroxidase and subsequent incubation with ECL1 and ECL2 substrate (Amersham Life Science) was followed by exposure to autoradiographic film (Biomax-MR, Eastman Kodak) for several seconds or minutes.

MEFV was recently identified as a disease-related gene in patients with FMF.3,4 So far, little is known about its function. To begin to address this issue, we studied the expression ofMEFV mRNA in different tissues and cell lines. Expression ofMEFV mRNA previously was described in peripheral blood leukocytes.3 4 In this report, we analyzed MEFVexpression in different populations of peripheral blood leukocytes. PCR was performed using specific oligonucleotides for MEFV, lactoferrin, and β-actin. Hybridization with gene-specific internal oligonucleotides confirmed the specificity of the PCR products.MEFV mRNA could be amplified in neutrophils, monocytes, and also weakly in CD19⁺ B cells and CD3⁺ T cells (Figure 1). Amplification of β-actin served as a control for cDNA quality (Figure 1). Lactoferrin is a specific granule marker and was chosen to exclude potential contamination of neutrophils to the other cell populations. Only neutrophils showed strong expression of lactoferrin mRNA, whereas CD19⁺ B lymphocytes, CD3⁺ T cells, and monocytes were negative for lactoferrin mRNA (Figure 1).

Next, we examined expression of MEFV in a panel of 22 leukemic cell lines. Interestingly, among the 11 myeloid leukemic cell lines, 8 cell lines showed MEFV expression after PCR amplification for 30 cycles followed by hybridization (Table1). Only 1 (Jurkat) of 11 lymphoid cell lines showed expression of MEFV mRNA.

The finding that MEFV is preferentially expressed in myeloid cells and cell lines led us to analyze its expression during hematopoietic differentiation. HL-60 cells were induced to differentiate toward granulocytes by incubation with DMSO for up to 96 hours. Induction of MEFV mRNA was analyzed by semiquantitative reverse transcription (RT)-PCR with amplification for 25 instead of 30 cycles and subsequent hybridization with an MEFV-specific oligonucleotide (Figure 2). Expression of MEFVin unstimulated HL-60 cells was detected only after prolonged exposure of the film to the blot (30 minutes). Regular exposure times (1 minute) revealed MEFV mRNA beginning at 12 hours of incubation with DMSO and peaking by 48 hours. In contrast, the level of MEFVexpression was not altered in ML-1 cells incubated in the presence of tetradecanoyl phorbol acetate to induce monocytic differentiation (data not shown).

To investigate nonhematopoietic tissue distribution of MEFV mRNA, expression was analyzed in normal human tissues. Expression of MEFV was detected in human muscle, lung, and spleen (Figure 3).

Initially, MEFV was reported to be expressed in the colon cancer cell line SW 480.6 We tested a panel of 11 colon cancer cell lines. Expression of MEFV in mRNA from SW 480 was confirmed. Additionally, MEFV mRNA was detected in the colon cancer cell line SW 837 and, at a very low level, it was detected in HuTu 80 and SW 1417 cells (Figure 4). Furthermore, we analyzed another 10 solid tumor–derived cell lines. Three prostate cancer cell lines (LNCaP, PC-3, and DU 145) expressed MEFV mRNA, whereas expression was not detected in 3 breast cancer cell lines (SK-BR2, MDA-MB-231, MCF7) tested (Table 1).

Determining the intracellular localization of a protein is important for understanding its function. Initial reports suggested that pyrin might act as a nuclear DNA binding factor based on analysis of its predicted amino acid sequence.3,4 12 To address the question of intracellular localization of pyrin, we fused MEFVcDNA to EGFP in a mammalian expression vector and analyzed expression of its corresponding protein, pyrin. COS-1 cells were transfected with an expression vector containing either EGFP or EGFP-pyrin. Western blot analysis of transfected COS-1 cells using a monoclonal anti-EGFP antibody showed that the cells expressed either the 27-kd EGFP protein or a 127-kd protein that corresponded to the calculated molecular mass of the EGFP-pyrin fusion protein (Figure5A). Fluorescent microscopy of transiently transfected cells demonstrated a diffuse expression in cells that were transfected with EGFP alone (Figure 5B). In contrast, COS-1 cells expressing the EGFP-pyrin fusion protein revealed a highly specific expression pattern consisting of distinct patches in the cytoplasm localized around the nucleus (Figure 5B). A similar expression pattern was found for EGFP-pyrin transiently expressed in U937 myeloid cells, which express low levels of endogenous pyrin (data not shown).

In patients suffering from FMF, several point mutations have been reported.3-5 We introduced either of 2 of the most common point mutations (Met694Val and Val726Ala) into theMEFV cDNA by site directed mutagenesis. Mutated MEFVwas fused to EGFP and expressed in COS-1 cells. The intracellular localization of EGFP-pyrin was not affected by the presence of the point mutations. The mutants maintained the distinct perinuclear expression in the cytoplasm like the wild-type pyrin protein.

FMF is an inherited disease that is characterized by recurrent attacks of fever. Positional cloning identified MEFV, the gene that is likely to cause FMF.3,4 So far, the function of the corresponding protein, named pyrin, remains unknown. Analysis of the predicted amino acid sequence of pyrin suggested that pyrin might be expressed as a nuclear factor that could regulate transcription.3,4,12 In contrast to this prediction, we demonstrate that the EGFP-pyrin fusion protein is expressed in the cytoplasm of COS-1 cells. To exclude that EGFP may interfere with nuclear localization, we expressed a known nuclear protein, cyclin A1, fused 3′ of EGFP, and found that it was clearly expressed in the nucleus. Our data suggest that pyrin is expressed in the cytoplasm and therefore may evoke functions other than a transcription factor. Alternatively, the intracellular localization of pyrin may change in response to appropriate stimuli, and pyrin might be translocated into the nucleus upon stimulation. For example, signal transducers and activators of transcription (STAT) proteins are expressed as monomeric cytoplasmic proteins and, in response to growth factors, they dimerize and translocate into the nucleus to act as transcription factors.13 Also, the transcription factor NFκB, when expressed as a GFP fusion protein in COS-7 cells, undergoes translocation from the cytoplasm to the nucleus upon stimulation with tumor necrosis factor.14 Although a variety of stimuli has been tested, none induced pyrin-EGFP to migrate into the nucleus of stably transfected U937 myeloid cells.

The distinct expression pattern of EGFP-pyrin in transfected COS cells might be correlated with the Golgi apparatus, as has been shown for another disease gene product.15 The Batten disease protein CLN3p is a Golgi integral membrane protein that displays a juxtanuclear asymmetric localization pattern when expressed as a GFP fusion protein in COS-7 cells.15 Our data suggest that pyrin might be expressed in association with the Golgi apparatus as well. This predicts a potential role for pyrin in Golgi-mediated protein assembly or transport.

Expression of MEFV mRNA was analyzed previously by Northern blot and detected in peripheral blood neutrophils and in 1 colon cancer cell line, SW 480.3 4 Hence, we tested both hematopoietic and nonhematopoietic normal human tissue and cell lines for expression of MEFV mRNA. Among the hematopoietic cell populations,MEFV mRNA was strongly expressed in neutrophils and was less pronounced in monocytes, CD19⁺ B cells, and CD3⁺ T cells. This low-level expression of MEFV may not be of physiologic relevance. So far, the expression levels of the corresponding protein, pyrin, have not been studied, and it remains an open question whether low levels of MEFV mRNA result in a significant amount of pyrin protein.

In nonhematopoietic tissues, MEFV mRNA was detected predominantly in spleen, lung, and muscle mRNA. This indicates that expression of MEFV is regulated in a tissue-specific manner with preferential expression in hematopoietic cells. Expression ofMEFV in lung and muscle might to some extent result from infiltrating macrophages or contaminating neutrophils.

To address a potential role for MEFV in tumorigenesis, we tested 43 human tumor cell lines for MEFV mRNA expression. Among the hematopoietic cell lines tested, we found expression ofMEFV predominantly in myeloid leukemic cell lines (8 of 11). Furthermore, we demonstrated prominent induction of MEFV mRNA during granulocytic differentiation of HL-60 cells. Expression ofMEFV in the colon cancer cell line SW 480 was confirmed, andMEFV expression was also found in 3 other colon cancer cell lines. In addition, MEFV transcripts were detectable in 3 prostate cancer cell lines. However, no dramatic differences inMEFV expression were observed between normal and matched cancerous colon or prostate samples from the same individuals (data not shown). At this time, we do not know if the relatively high expression of MEFV mRNA in several colon and prostate cancer cell lines has any clinical relevancy.

Missense mutations of the MEFV gene have been described in patients with FMF, but how these mutations affect the function of pyrin is unknown. Both activation and inactivation of pyrin as well as changes in either its localization or interaction with other proteins might result from the mutations. We expressed in COS-1 cells EGFP-pyrin bearing either of the 2 of the most frequent mutations in FMF patients. The perinuclear expression of EGFP-pyrin was not altered by the point muations. Interestingly, the mutations described so far primarily occur in the C-terminal B30.2 domain of pyrin.3-5 This domain is predicted to obtain a globular conformation when expressed as a protein and is present in different families of proteins (eg, nuclear factor Staf5016 or ret finger protein17) and might be involved in protein-protein interactions. The identification and characterization of interacting proteins might elucidate the functional role of pyrin.