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• W2034302810 abstract "MEFV is a gene expressed specifically in myeloid cells and whose mutations underlie an autosomal recessive auto-inflammatory disease, called familial Mediterranean fever (FMF), characterized by recurrent episodes of serosal inflammation. This gene, which encodes a protein with unclear physiological functions, has been shown to be up-regulated by the pro-inflammatory cytokine tumor necrosis factor α (TNFα). However, the mechanism of this regulation is unknown, and the MEFV promoter is still to be characterized. Here, we show that 243 bp of the 5′-flanking region of the human MEFV gene are sufficient to direct high level expression of MEFV in TNFα-treated cells. The TNFα-induced expression of MEFV is dependent on both NFκB p65 and C/EBPβ that bind to evolutionarily conserved sites located, in the human promoter, at positions –163 and –55, respectively. As shown by a series of transcription and gel shift assays performed with wild-type and mutated promoter sequences, these two transcription factors act differently on the TNFα-dependent transcription of MEFV: C/EBPβ is the key regulatory factor required to confer cell responsiveness to TNFα, whereas NFκB p65 increases this response by means of a synergistic interaction with C/EBPβ that is dependent on the integrity of the identified –55 C/EBP binding site. Given the phenotype of patients with FMF, this C/EBP-NFκB interaction may represent a key step in the control of an inflammatory response that is abnormally high in this disease. These data, which shed novel light on the pathophysiology of FMF, represent an unusual example of cross-talk between C/EBP and NFκB pathways in TNFα signaling. MEFV is a gene expressed specifically in myeloid cells and whose mutations underlie an autosomal recessive auto-inflammatory disease, called familial Mediterranean fever (FMF), characterized by recurrent episodes of serosal inflammation. This gene, which encodes a protein with unclear physiological functions, has been shown to be up-regulated by the pro-inflammatory cytokine tumor necrosis factor α (TNFα). However, the mechanism of this regulation is unknown, and the MEFV promoter is still to be characterized. Here, we show that 243 bp of the 5′-flanking region of the human MEFV gene are sufficient to direct high level expression of MEFV in TNFα-treated cells. The TNFα-induced expression of MEFV is dependent on both NFκB p65 and C/EBPβ that bind to evolutionarily conserved sites located, in the human promoter, at positions –163 and –55, respectively. As shown by a series of transcription and gel shift assays performed with wild-type and mutated promoter sequences, these two transcription factors act differently on the TNFα-dependent transcription of MEFV: C/EBPβ is the key regulatory factor required to confer cell responsiveness to TNFα, whereas NFκB p65 increases this response by means of a synergistic interaction with C/EBPβ that is dependent on the integrity of the identified –55 C/EBP binding site. Given the phenotype of patients with FMF, this C/EBP-NFκB interaction may represent a key step in the control of an inflammatory response that is abnormally high in this disease. These data, which shed novel light on the pathophysiology of FMF, represent an unusual example of cross-talk between C/EBP and NFκB pathways in TNFα signaling. In humans, mutations in the MEFV gene have been shown to be responsible for familial Mediterranean fever (FMF) 1The abbreviations used are: FMFfamilial Mediterranean feverTNFtumor necrosis factorLPSlipopolysaccharideDTTdithiothreitolEMSAelectrophoretic mobility shift assayntnucleotide(s)ILinterleukin.1The abbreviations used are: FMFfamilial Mediterranean feverTNFtumor necrosis factorLPSlipopolysaccharideDTTdithiothreitolEMSAelectrophoretic mobility shift assayntnucleotide(s)ILinterleukin. (1.The French FMF Consortium Nat. Genet. 1997; 17: 25-31Crossref PubMed Scopus (1262) Google Scholar, 2.The International FMF Consortium Cell. 1997; 90: 797-807Abstract Full Text Full Text PDF PubMed Scopus (1317) Google Scholar), an autosomal recessive disorder that affects essentially populations of Mediterranean extraction, and is characterized by recurrent episodes of fever and serosal inflammation manifested by sterile peritonitis, arthritis, and/or pleurisy, which may lead to unnecessary invasive investigations. Although the first mutations have been identified 6 years ago, the pathophysiology of this unusual disease remains unclear, even though several lines of indirect evidence strongly suggest the involvement of MEFV in inflammatory and apoptotic pathways both in humans and in mice (3.Richards N. Schaner P. Diaz A. Stuckey J. Shelden E. Wadhwa A. Gumucio D.L. J. Biol. Chem. 2001; 276: 39320-39329Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 4.Gumucio D.L. Diaz A. Schaner P. Richards N. Babcock C. Schaller M. Cesena T. Clin. Exp. Rheumatol. 2002; 20: S45-S53PubMed Google Scholar, 5.Bertin J. DiStefano P.S. Cell Death Differ. 2000; 7: 1273-1274Crossref PubMed Scopus (159) Google Scholar, 6.Chae J.J. Komarow H.D. Cheng J. Wood G. Raben N. Liu P.P. Kastner D.L. Mol. Cell. 2003; 11: 591-604Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). Indeed, the protein predicted by MEFV, called pyrin or marenostrin, was shown to belong to an expanding family of molecules containing a protein:protein interaction domain called pyrin domain (5.Bertin J. DiStefano P.S. Cell Death Differ. 2000; 7: 1273-1274Crossref PubMed Scopus (159) Google Scholar, 7.Pawlowski K. Pio F. Chu Z. Reed J.C. Godzik A. Trends Biochem. Sci. 2001; 26: 85-87Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 8.Fairbrother W.J. Gordon N.C. Humke E.W. O'Rourke K.M. Starovasnik M.A. Yin J.P. Dixit V.M. Protein Sci. 2001; 10: 1911-1918Crossref PubMed Scopus (125) Google Scholar, 9.Martinon F. Hofmann K. Tschopp J. Curr. Biol. 2001; 11: R118-R120Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar), which, in the case of the pyrin/marenostrin protein, is able to interact with ASC (apoptosis-associated Speck-like protein containing a CARD), a protein involved in apoptosis regulation (3.Richards N. Schaner P. Diaz A. Stuckey J. Shelden E. Wadhwa A. Gumucio D.L. J. Biol. Chem. 2001; 276: 39320-39329Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 10.Conway K.E. McConnell B.B. Bowring C.E. Donald C.D. Warren S.T. Vertino P.M. Cancer Res. 2000; 60: 6236-6242PubMed Google Scholar). In addition, FMF shares striking clinical similarities with a number of other inflammatory syndromes defining the group of hereditary recurrent fevers. Because several of these related diseases have been shown to result from mutations in the CIAS1 gene (i.e. Muckle-Wells syndrome (11.Hoffman H.M. Mueller J.L. Broide D.H. Wanderer A.A. Kolodner R.D. Nat. Genet. 2001; 29: 301-305Crossref PubMed Scopus (1266) Google Scholar), familial cold urticaria (11.Hoffman H.M. Mueller J.L. Broide D.H. Wanderer A.A. Kolodner R.D. Nat. Genet. 2001; 29: 301-305Crossref PubMed Scopus (1266) Google Scholar), chronic infantile neurological cutaneous and articular syndrome (12.Feldmann J. Prieur A.M. Quartier P. Berquin P. Cortis E. Teillac-Hamel D. Fischer A. Am. J. Hum. Genet. 2002; 71: 198-203Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar)) or the TNFR gene (TNF receptor 1A-associated periodic syndromes or TRAPS) (13.McDermott M. Aksentijevich I. Galon J. McDermott E. Ogunkolade B. Centola M. Mansfield E. Gadina M. Karenko L. Pettersson T. McCarthy J. Frucht D. Aringer M. Torosyan Y. Teppo A. Wilson M. Karaarslan H. Wan Y. Todd I. Wood G. Schlimgen R. Kumarajeewa T. Cooper S. Vella J. Kastner D. Cell. 1999; 97: 133-144Abstract Full Text Full Text PDF PubMed Scopus (1088) Google Scholar)), it is tempting to speculate the existence of pathophysiological mechanisms that may be common to those genetic disorders and involving the underlying genes. In this regard, it is noteworthy that cryopyrin, the protein encoded by CIAS1, which contains a pyrin domain, has been shown to be a potential regulator of the NFκB transcription factor (14.Manji G.A. Wang L. Geddes B.J. Brown M. Merriam S. Al-Garawi A. Mak S. Lora J.M. Briskin M. Jurman M. Cao J. DiStefano P.S. Bertin J. J. Biol. Chem. 2002; 277: 11570-11575Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar), whereas TNFα, the ligand of TNFR1A, is a well known cytokine involved in the acute-phase response to a wide range of inflammatory stimuli (15.Baud V. Karin M. Trends Cell Biol. 2001; 11: 372-377Abstract Full Text Full Text PDF PubMed Scopus (1356) Google Scholar). Furthermore, the MEFV gene displays a highly restricted pattern of expression: MEFV transcripts are found specifically in myeloid cells, essentially in monocytes and neutrophils, two cell types that participate in the inflammatory infiltrates found in patients with FMF (1.The French FMF Consortium Nat. Genet. 1997; 17: 25-31Crossref PubMed Scopus (1262) Google Scholar, 2.The International FMF Consortium Cell. 1997; 90: 797-807Abstract Full Text Full Text PDF PubMed Scopus (1317) Google Scholar). These transcripts, which are expressed during myelomonocytic differentiation (16.Tidow N. Chen X. Muller C. Kawano S. Gombart A.F. Fischel-Ghodsian N. Koeffler H.P. Blood. 2000; 95: 1451-1455Crossref PubMed Google Scholar, 17.Centola M. Wood G. Frucht D.M. Galon J. Aringer M. Farrell C. Kingma D.W. Horwitz M.E. Mansfield E. Holland S.M. O'Shea J.J. Rosenberg H.F. Malech H.L. Kastner D.L. Blood. 2000; 95: 3223-3231Crossref PubMed Google Scholar), are also present in a number of cell lines, including the early myeloblastic KG1 cells, as well as in HeLa cells (16.Tidow N. Chen X. Muller C. Kawano S. Gombart A.F. Fischel-Ghodsian N. Koeffler H.P. Blood. 2000; 95: 1451-1455Crossref PubMed Google Scholar). Most importantly, several inflammatory mediators are known to increase the expression of the human MEFV gene: TNFα and LPS in monocytes, and interferon-γ in both monocytic and granulocytic cells (17.Centola M. Wood G. Frucht D.M. Galon J. Aringer M. Farrell C. Kingma D.W. Horwitz M.E. Mansfield E. Holland S.M. O'Shea J.J. Rosenberg H.F. Malech H.L. Kastner D.L. Blood. 2000; 95: 3223-3231Crossref PubMed Google Scholar). However, the mechanism by which those cytokines regulate MEFV gene expression is entirely unknown, and, from a more general viewpoint, the MEFV promoter is still to be characterized.To gain insights into the pathophysiology of FMF, we have investigated the regulation of the human MEFV gene that, on the basis of in silico means, contains in its 5′-flanking region putative binding sites for a variety of transcription factors, including NFκB and CCAAT/enhancer binding proteins (C/EBP). The NFκB family includes the p50, p65, and c-Rel proteins, which exist as homo- or heterodimers. In unstimulated cells, the NFκB dimers are bound to the inhibitory protein IκB, which masks the nuclear localization signal of NFκB, thereby retaining the dimers in the cytoplasm. The binding of TNFα to TNFR1 leads to the activation of the IKK complex, which, in turn, phosphorylates IκB. The subsequent ubiquitination of IκB, followed by its degradation by the proteasome, leads to its dissociation from NFκB, which results in activation and nuclear translocation of the NFκB dimers that interact with the promoter region of a number of target genes (for review, see Refs. 18.Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Crossref PubMed Scopus (3250) Google Scholar and 19.Baldwin Jr., A.S. J. Clin. Invest. 2001; 107: 3-6Crossref PubMed Google Scholar). The C/EBP family of transcription factors contains six members (C/EBPα, β, γ, δ, ϵ, ζ) and is characterized by the presence of a COOH-terminal basic DNA binding domain and a leucine zipper domain that promotes homo- and heterodimerization of the different family members. These latter factors, which are induced by several cytokines, play key roles in numerous cellular responses, including immune and inflammatory processes, by regulating cytokine and acute phase response genes (for review, see Ref. 20.Ramji D.P. Foka P. Biochem. J. 2002; 365: 561-575Crossref PubMed Google Scholar). The presence of putative binding sites for both NFκB and C/EBP in the 5′-flanking region of the human MEFV gene prompted us to assess the role of those proteins in the regulation of MEFV transcription.EXPERIMENTAL PROCEDURESPlasmid Constructs—Four MEFV promoter constructs were generated by PCR amplification using human genomic DNA (Boehringer) as template. Standard PCR reactions, with cycling temperature of 94, 55, and 72 °C, were performed using one of the following forward primers (–1013) 5′-TATAAAGATCTTGAGTAGCTGGGAGTACAGGT-3′, (–483) 5′-TATAAAGATCTCTCACAGGACTGTATTCAGTG-3′, (–243) 5′-TATAAAGATCTGGTCTAGTGTGGAGGCGAGGT-3′, or (–133) 5′-TATAAAGATCTCACAGCCGGCATGGACACACC-3′ (the number in parentheses indicates the position of the 5′ base of the primer within the MEFV promoter sequence) and the reverse primer (+37) 5′-TATAAAGCTTCTGAGCAGGAGAGGCTCGAGC-3′. The forward primers contain a HindIII restriction enzyme site, whereas the reverse primer contains a BglII restriction site. The PCR products were digested by HindIII and BglII enzymes and cloned into the luciferase vector pGL3 linearized using the same enzymes. The four generated MEFV promoter constructs were designated p1013-MEFV-Luc, p483-MEFV-Luc, p243-MEFV-Luc, and p133-MEFV-Luc, according to the forward primer used in the PCR reaction (Fig. 1). The responsive elements identified in silico by means of the TFSEARCH (Yutaka Akiyama: TFSEARCH: Searching Transcription Factor Binding Sites, available at www.rwcp.or.jp/papia/) and the MATINSPECTOR (Genomatix, available at www.genomatix.de) software were subjected to point-directed mutagenesis, using the mutated oligonucleotides described in Fig. 1A and according to the manufacturer's instructions (Stratagene). The mutated derivative constructs were named p243-MEFVMut NFκB-Luc, p243-MEFVMut C/EBP-Luc, p243-MEFVMut NFκB/C/EBP-Luc, p243-MEFVMut C/EBP2-Luc, and p243-MEFVMut NFκB/C/EBP2-Luc (Fig. 1B), according to the oligonucleotides used. The C/EBPβ cDNA was obtained by reverse transcription-PCR performed with total RNA extracted from HeLa cells as template and the following primers (forward: 5′-TTCATGCAACGCCTGGTGGCCTGG-3′ and reverse: 5′-GCGCTAGCAGTGGCCGGAGGAGGC-3′). The reverse transcription-PCR products were subsequently cloned into the TOPO-V5 expression vector (Invitrogen). The NFκB p65 expression vector used in this study has been described previously (21.Thanos D. Maniatis T. Cell. 1992; 71: 777-789Abstract Full Text PDF PubMed Scopus (557) Google Scholar). All constructs were verified by sequencing.Cell Growth, Treatment, and Transfection—KG1 cells were maintained in minimal essential medium α (Invitrogen) supplemented with 10% fetal calf serum. HeLa and HEK293 cell lines were maintained in Dulbecco's modified Eagle medium (Invitrogen) supplemented with 10% fetal calf serum. 3 × 105 HeLa or 4.5 × 105 HEK293 cells were cultured 24 h in 6-wells culture plates and transfected using 350 ng of the MEFV promoter construct or the empty pGL3 vector, with or without C/EBPβ and/or NFκB p65 expression vectors (100 ng of each plasmid construct), according to the manufacturer's instructions (LipofectAMINE, Invitrogen). Total amount of transfected DNA was kept constant by adding empty vector. When indicated, 18 h after transfection, cells were treated with TNFα (50 ng/ml, Tebu). The cell extracts were prepared and assayed for luciferase activity using the Promega assay system. Luciferase activity was normalized to protein concentration, which was measured using the Coomassie Plus Protein Assay Reagent Kit (Pierce), rather than to a second reporter plasmid, because, as shown by others (22.Huszar T. Mucsi I. Terebessy T. Masszi A. Adamko S. Jeney C. Rosivall L. J. Biotechnol. 2001; 88: 251-258Crossref PubMed Scopus (21) Google Scholar), the use of such internal standards in transient experiments may lead to a systematic error. Each transfection experiment was performed independently at least three times. Results of one representative experiment done in triplicate are presented.Statistical Analysis—All data were expressed as mean values ± S.D. Differences among groups were analyzed by means of the analysis of variance test, and statistical significance was accepted when p < 0.05.Electrophoretic Mobility Shift Assays—KG1 and untransfected HeLa cells were maintained in minimal essential medium α or Dulbecco's modified Eagle medium (Invitrogen), respectively, in the absence or in the presence of 50 ng/ml TNFα. Nuclear extracts were prepared as follows: cells were resuspended and incubated for 15 min at 4 °C in 400 μl of buffer A (10 mm HEPES, pH 7.9, 10 mm KCl, 0.1 mm EDTA, pH 8, 0.1 mm EGTA, 1 mm DTT, and proteases inhibitors). 25 μl of buffer B (Nonidet P-40 10%) were added to HeLa extracts; all extracts were centrifuged at 12,000 rpm for 30 s. The pellets, which contain nuclear fractions, were then resuspended in 40 μl of buffer C (20 mm HEPES, pH 7.9, 400 mm NaCl, 1 mm EDTA, pH 8, 1 mm DTT, and protease inhibitors), vortexed for 15 min at 4 °C, and centrifuged at 12,000 rpm for 5 min; the resulting supernatants correspond to nuclear extracts. DNA binding reactions were performed at room temperature in a 20-μl reaction mixture containing 5–20 μg of nuclear extracts, 5 μl of 4× binding buffer (10% glycerol, 20 mm HEPES, 60 mm KCl, 1 mm MgCl2, 20 mm EDTA, 0.5 mm DTT), 1 μg of poly(dI-dC) (Roche Applied Science), 1 ng of probe end-labeled with [γ-32P]ATP (3,000 μCi/mmol, Amersham Biosciences), and T4 polynucleotide kinase (Invitrogen). For NFκB EMSAs in KG1 cells, protein extracts were prepared using a lysis buffer consisting of 20 mm HEPES, pH 7, 150 mm NaCl, 1 mm EDTA, Igepal 1%, and protease inhibitors. For competition assays, a 100-fold molar excess of unlabeled oligonucleotides was added prior to the binding reaction. For supershift experiments, protein extracts were preincubated with 2 μg of antibodies directed against either C/EBPα, C/EBPβ, C/EBPδ, NFκB p65, or NFκB p50 for 1 h at 4 °C prior to the binding reaction. Anti-C/EBPα and anti-C/EBPβ antibodies were kindly provided by R. Barouki (INSERM, Unit 490, Paris, France); antibodies against C/EBPδ, NFκB p65, and NFκB p50 were purchased from Santa Cruz Biotechnology. DNA·protein complexes were separated by electrophoresis on a 6% polyacrylamide gel in 0.5× TBE at 150 V, and analyzed by autoradiography. The oligonucleotides used for EMSAs are described in Fig. 1A.RESULTSThe 1-kb 5′-Flanking Sequence of MEFV Is Sufficient to Confer Responsiveness of HeLa Cells to TNFα—Because MEFV transcription is known to be up-regulated by TNFα and NFκB is the main transcription factor involved in TNFα signaling, we screened the 1-kb MEFV sequence upstream of the transcriptional start site (17.Centola M. Wood G. Frucht D.M. Galon J. Aringer M. Farrell C. Kingma D.W. Horwitz M.E. Mansfield E. Holland S.M. O'Shea J.J. Rosenberg H.F. Malech H.L. Kastner D.L. Blood. 2000; 95: 3223-3231Crossref PubMed Google Scholar) for the presence of potential NFκB binding sites. Three potential binding sites for NFκB (consensus sequence: 5′-GGGGAATCCC-3′) were identified, at positions –594 (5′-GGAAGAACCA-3′), –428 (5′-GGAAGGTCCC-3′), and –163 (5′-GGAAAATCCT-3′) (Fig. 1B). To assess the effect of TNFα on MEFV transcription, we therefore first cloned a 1013-bp MEFV fragment encompassing these three sites upstream of the transcriptional start site into the luciferase reporter vector pGL3. Promoter activity was determined by luciferase assays after transfection of the resulting plasmid designated p1013-MEFV-Luc into the human HeLa cell line that is known to express MEFV (16.Tidow N. Chen X. Muller C. Kawano S. Gombart A.F. Fischel-Ghodsian N. Koeffler H.P. Blood. 2000; 95: 1451-1455Crossref PubMed Google Scholar) and compared with the activity of transfected cells exposed to TNFα (Fig. 2). All luciferase values were normalized to cell protein concentration and compared with those for the promoterless vector pGL3. The luciferase activity of transfected cells exposed to TNFα was found to be 5- to 10-fold greater than that of untreated cells; in contrast the luciferase activity of cells transfected with pGL3 was extremely low (about 15-fold lower than that of cells transfected with p1013-MEFV-Luc) and remained unchanged after TNFα treatment. These results, therefore, show that the 1013-bp fragment of the 5′-flanking sequence of MEFV displays a basal promoter activity and that this sequence is sufficient to confer responsiveness of HeLa cells to TNFα.Fig. 2Delineation of the human MEFV promoter region involved in TNFα activation. HeLa cells were transfected either with one MEFV promoter construct (p1013-MEFV-Luc, p483-MEFV-Luc, p243-MEFV-Luc, or p133-MEFV-Luc), or with the empty luciferase pGL3-Luc vector. Cells were treated with 50 ng/ml TNFα for 6 h (TNFα) or left untreated (UT). Relative luciferase activity was determined as described under “Experimental Procedures.” Results are represented as mean values ± S.D. of a representative experiment done in triplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Functional Importance of nt –243 to +37 for the TNFα-dependent Activation of the MEFV Promoter—To map the functionally important sites for the responsiveness of the human MEFV promoter to TNFα, we generated a series of three deleted constructs of the 5′-flanking region of MEFV with respect to the three potential binding sites for NFκB located at positions –594, –428, and –163 (Fig. 1B). Transfection of these constructs into HeLa cells revealed the existence of a TNFα-inducible promoter activity in p1013-MEFV-Luc, p483-MEFV-Luc, and p243-MEFV-Luc (Fig. 2). In contrast, further deletion of the MEFV promoter between nt –243 and –133 resulted in a significant decrease (2- to 3-fold) of TNFα inducibility of the MEFV promoter, without affecting basal promoter activity (Fig. 2), whereas targeted disruption of the –594 or –428 NFκB putative site in the context of the intact 1-kb promoter did not affect significantly MEFV responsiveness to TNFα (data not shown).These results argue against the involvement of the –594 and –428 putative NFκB binding sequences in MEFV responsiveness to TNFα, while indicating the presence of a TNFα-responsive element located within the proximal region of the MEFV promoter (nt –243 to –133), which contains the third putative binding site for NFκB (at nt –163). However, although TNFα inducibility of the p133-MEFV-Luc promoter was substantially reduced when compared with that of constructs containing larger promoter regions, it was not completely abrogated, indicating the presence of additional site(s) involved in TNFα responsiveness and located within the first 133 bp of the promoter region (Fig. 2). Interestingly, this region contains a putative binding site for C/EBP at position –55.Both the –163 NFκB and –55 C/EBP Binding Sites Are Required for the TNFα-dependent Activation of the MEFV Promoter—To examine the possible contribution of the putative binding sites for NFκB (–163) and C/EBP (–55) to the responsiveness of the MEFV promoter to TNFα, we constructed two mutant reporter genes: p243-MEFVMut NFκB-Luc, in which the NFκB site was disrupted by two point mutations, and p243-MEFVMut C/EBP-Luc, in which the C/EBP site was disrupted by a single nucleotide substitution (Fig. 1). As shown in Fig. 3, mutation of the NFκB site decreased the ability of the MEFV promoter to respond to TNFα, in a range (2–3-fold) similar to that documented with the deletion construct (p133-MEFV-Luc) encompassing this sequence (Fig. 2). Most importantly, disruption of the C/EBP binding site resulted in a complete loss of TNFα inducibility of the MEFV promoter without affecting basal promoter activity (Fig. 3); similar results were obtained when the same point mutation was introduced in the –55 C/EBP site of all other MEFV promoter constructs (data not shown).Fig. 3Contribution of the putative binding sites for NFκB (at position –163) and C/EBPβ (at nt –55) to the TNFα-dependent transcriptional activation of the MEFV promoter. HeLa cells were transfected either with the wild-type p243-MEFV-Luc construct or the mutated constructs p243-MEFVMut NFκB-Luc or p243-MEFVMut C/EBP-Luc. Cells were treated with 50 ng/ml TNFα for 6 h (TNFα) or left untreated (UT). Relative luciferase activity was determined as described under “Experimental Procedures.” Results are represented as mean values ± S.D. of a representative experiment done in triplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Taken together, these results strongly suggest that member(s) of the C/EBP protein family would play a key role in mediating TNFα inducibility of MEFV expression. More precisely, the putative binding site for C/EBP located at position –55 appears to be necessary for conferring responsiveness of the MEFV promoter to TNFα, whereas the –163 NFκB site would enhance the C/EBP-mediated stimulating effect of TNFα on MEFV transcription.NFκB p65 and C/EBPβ Bind the –163 NFκB and –55 C/EBP Sites of the Human MEFV Promoter, Respectively— Electrophoretic mobility shift assays (EMSAs) were used to determine whether the promoter contained bona fide NFκB and C/EBP binding sites. Radiolabeled 20-mer oligonucleotides spanning the region from nt –59 to –39 and containing the putative binding site for C/EBP, designated MEFVC/EBP, were incubated with nuclear protein extracts prepared from untreated and TNFα-treated HeLa cells. As shown in Fig. 4A (lanes 1 and 2), this probe revealed a complex in both conditions, whereas no shift was observed when nuclear extracts were incubated with the MEFVMut C/EBP oligonucleotide (an oligonucleotide differing by a single nucleotide critical for C/EBP binding) as the probe (lanes 3 and 4). Binding specificity was further determined by competition assays (Fig. 4B): the formation of the complex could be inhibited by an excess of unlabeled wild-type oligonucleotides corresponding either to the MEFVC/EBP site or to a consensusC/EBP site (Fig. 4B, lanes 2 and 4), but not by the MEFVMut C/EBP oligonucleotide (lane 3).Fig. 4Binding of C/EBPβ and NFκB p65 factors to the MEFV promoter assessed by gel shift assays. EMSAs were performed with protein extracts obtained from HeLa or KG1 cells left untreated (–) or treated (+) with TNFα, and different probes corresponding to the wild-type oligonucleotide MEFVC/EBP, the mutated oligonucleotide MEFVMut C/EBP, or the wild-type oligonucleotide MEFVNFκB. A, nuclear extracts from HeLa cells treated with TNFα for 6 h (lanes 2 and 4) or left untreated (lanes 1 and 3) were incubated with a radiolabeled oligonucleotide containing the normal C/EBP binding site (MEFVC/EBP) (lanes 1 and 2) or a mutated C/EBP binding site (MEFVMut C/EBP) (lanes 3 and 4). B, nuclear extracts from HeLa cells treated with TNFα for 6 h were incubated with the MEFVC/EBP oligonucleotide as probe, and competition experiments were performed using a 100-fold excess of one of the following cold oligonucleotides: MEFVC/EBP (lane 2), MEFVMut C/EBP (lane 3), or ConsensusC/EBP (lane 4). C, nuclear extracts from HeLa and KG1 cells treated with TNFα for 24 h (lanes 2–5 and lanes 7–12, respectively) or left untreated (lanes 1 and 6, respectively) were preincubated with antibodies directed against C/EBPα (lanes 3 and 10), β (lanes 4 and 11), or δ (lanes 5 and 12), or with an excess of cold MEFVC/EBP (lane 8) or ConsensusC/EBP (lane 9) competitor oligonucleotides, and then incubated with the wild-type MEFVC/EBP oligonucleotide as probe. D, nuclear extracts from HeLa cells treated with TNFα for 30 min (lanes 2–5) or left untreated (lane 1) were incubated with the MEFVNFκB probe, and competition experiments were performed using an excess of cold MEFVNFκB, MEFVMut NFκB, or ConsensusNFκB oligonucleotides. E, nuclear extracts from HeLa cells treated with TNFα for 30 min (lanes 2–4) or left untreated (lane 1) were preincubated with antibodies directed against either NFκB p65 (lane 3) or NFκB p50 (lane 4) and then incubated with the MEFVNFκB oligonucleotide as probe. Similar experiments were performed with protein extracts obtained from KG1 cells (lanes 5–8). The identity of the shifted bands indicated by an arrowhead in panels D and E is discussed in the text.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To identify which C/EBP member binds to the proximal region of the MEFV promoter, we performed supershift experiments using antibodies directed against C/EBPα, C/EBPβ, and C/EBPδ (Fig. 4C). Nuclear extracts of HeLa cells were preincubated with each of these antibodies before the addition of the labeled MEFVC/EBP probe. The whole complex was retarded by a C/EBPβ antibody (lane 4), whereas anti-C/EBPα and C/EBPδ antibodies had no effect on the mobility of the complex (lanes 3 and 5). Altogether, these data indicate that C/EBPβ is the protein member of the C/EBP family whose specific binding to a site located at position –55 of the human MEFV promoter is required for the TNFα-dependent activation of MEFV gene expression.To determine whether C/EBPβ is also able to bind the –55 MEFV promoter site in hematopoietic cells, we performed similar EMSAs using nuclear extracts of KG1 cells, a myeloblastic cell line that e" @default.
• W2034302810 created "2016-06-24" @default.
• W2034302810 creator A5004921372 @default.
• W2034302810 creator A5037042892 @default.
• W2034302810 creator A5067108473 @default.
• W2034302810 creator A5083664722 @default.
• W2034302810 creator A5087211499 @default.
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• W2034302810 date "2003-12-01" @default.
• W2034302810 modified "2023-09-26" @default.
• W2034302810 title "The Tumor Necrosis Factor α-dependent Activation of the Human Mediterranean Fever (MEFV) Promoter Is Mediated by a Synergistic Interaction between C/EBPβ and NFκB p65" @default.
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