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Characterization of the Promoter for the Human Long Pentraxin PTX3

Characterization of the Promoter for the Human Long Pentraxin PTX3 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 13, Issue of March 28, pp. 8172–8178, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Characterization of the Promoter for the Human Long Pentraxin PTX3 ROLE OF NF-kB IN TUMOR NECROSIS FACTOR-a AND INTERLEUKIN-1b REGULATION* (Received for publication, November 19, 1996, and in revised form, January 23, 1997) Andrea Basile‡, Antonio Sica‡, Elisabetta d’Aniello‡, Ferruccio Breviario‡, Gabino Garrido‡, Marina Castellano‡, Alberto Mantovani‡§, and Martino Introna‡ From the ‡Istituto di Ricerche Farmacologiche “Mario Negri,” via Eritrea, 62, 20157 Milan, Italy and the §Section of General Pathology and Immunology, University of Brescia, 25123 Brescia, Italy the 59 half of the protein does not show significant homology The “long pentraxins” are an emerging family of genes that have conserved in their carboxy-terminal halves a with other known proteins. PTX3 is indeed the first isolated pentraxin domain homologous to the prototypical acute member of a new group of proteins, known as “long pentrax- phase protein pentraxins (C-reactive protein and serum ins,” which have different 59-termini upstream from their pen- amyloid P component) and acquired novel amino-termi- traxin domains (5–9). nal domains. In this report, a genomic fragment of 1371 While the classical pentraxins CRP and SAP are almost nucleotides from the human “long pentraxin” gene exclusively produced by the liver in response to IL-6 in combi- PTX3 is characterized as a promoter on tumor necrosis nation with IL-1 and TNF, PTX3 shows a more promiscuous factor-a (TNFa) and interleukin (IL)-1b exposure in response in that its expression in vitro can be induced in transfected 8387 human fibroblasts by chloramphenicol endothelial cells, hepatocytes, fibroblasts, and monocytes. In acetyltransferase and RNase protection assays. In the all cases, the gene is rapidly and directly induced by exposure same cells, the PTX3 promoter does not respond to IL-6 to IL-1b, TNFa, and lipopolysaccharide (LPS), but not by IL-6, stimulation. Furthermore, IL-1b and TNFa responsive- the mRNA peaking 4 – 6 h after the stimulation (1, 4, 10). This ness is not seen in the Hep 3B hepatoma cell line. The induction is paralleled by de novo transcription of the gene (10) minimal promoter contains one NF-kB element which is and is transient in that no more message is detectable after shown to be necessary for induction and able to bind p50 24 h (1, 10). homodimers and p65 heterodimers but not c-Rel. Mu- The mouse homologue, mPTX3, shows a similar exon/intron tants in this site lose the ability to bind NF-kB proteins organization and 82% identity at the amino acid level with and to respond to TNFa and IL-1b in functional assays. hPTX3 (4). When C57BL mice were injected i.v with LPS to Sp1- and AP-1 binding sites lying in proximity to the induce an acute phase response, mPTX3 expression was mark- NF-kB site do not seem to play a major role for cytokine edly induced in vivo after4hin several muscular organs, responsiveness. Finally, cotransfection experiments with expression vectors validate that the natural pro- including the heart and the thigh (4). In situ hybridization moter contains a functional NF-kB site. studies showed that endothelial cells within the muscular tis- sues were the major responder cell type. Interestingly, in strik- ing contrast with CRP and SAP, no mRNA for mPTX3 could be The human gene hPTX3 has been recently cloned from in- detected by Northern analysis in the liver (4). terleukin-1b (IL-1b) -stimulated endothelial cells (1) and from Similar promiscuous in vivo expression has also been ob- tumor necrosis factor-a (TNFa)-stimulated fibroblasts (2). served for the other “long pentraxins” in organs as diverse as PTX3 belongs to the family of pentraxins (so named because the brain and the testis (6 –9). they are assembled in pentamers) that include C-reactive pro- To begin to understand the molecular mechanisms underly- tein (CRP) and serum amyloid P component (SAP) from several ing the regulated expression of the first cloned long pentraxin different species (3) and which are markers of the acute phase. PTX3, we cloned and characterized the promoter of hPTX3. Moreover, while the 39 half of PTX3 can be aligned with the EXPERIMENTAL PROCEDURES full-length sequences of CRP and SAP (4) (pentraxin domain), Cell Culture—The human fibrosarcoma 8387 (11) and the human hepatoma Hep 3B (12) cell lines were maintained in Dulbecco’s modified * This work was supported in part by Piano Nazionale Farmaci II, Eagle’s medium (Seromed, Biochrom KG, Berlin, Germany), supple- tema 5; F. B. was supported by NATO Grant CRG 941228. The costs of mented with 10% fetal calf serum (Hyclone, Logan, UT), 50 mg/ml publication of this article were defrayed in part by the payment of page gentamycin (Life Technologies, Inc., Paisley, Renfewshire, United King- charges. This article must therefore be hereby marked “advertisement” dom), and 20 mML-glutamine (Seromed). in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Plasmid Construction—A 1.37-kilobase EcoRI-PvuII genomic frag- The nucleotide sequence(s) reported in this paper has been submitted ment from lP2 phage (1), which spans nucleotides 21317 to 154 TM to the GenBank /EBI Data Bank with accession number(s) X97748. relative to the transcription start site, was blunted and subcloned into ¶ To whom requests for reprints should be addressed: Laboratory of the XhoI-digested and blunted pBL CAT 3 vector (13) (giving 21317- Molecular Immunohematology, Department of Immunology and Cell CAT). Following PstI-XbaI digestion, the latter plasmid was used as a Biology, Istituto di Ricerche Farmacologiche “Mario Negri,” via Eritrea, substrate to generate a set of deletion clones with ExoIII (Stratagene, 62, 20157 Milan, Italy. Tel.: 0039/2/39014547; Fax: 0039/2/33200231. 1 La Jolla, CA) digestion, mung (Stratagene) blunting, and subsequent The abbreviations used are: IL, interleukin; TNFa, tumor necrosis religation. Deletion clones covering the whole 59-flanking region of the factor-a; CRP, C-reactive protein; SAP, serum amyloid P component; hPTX3 gene were sequenced by the Sanger dideoxy method (14). Three LPS, lipopolysaccharide; CAT, chloramphenicol acetyltransferase; of them, 2387-CAT, 2180-CAT, and 274-CAT, were subsequently used 2180 m NF-kB-CAT, plasmid carrying a mutation in the 296 NF-kB in functional assays. site; PCR, polymerase chain reaction; bp, base pair; PIPES, 1,4-pipera- The plasmid carrying a mutation in the 296 NF-kB site (2180 m zinediethanesulfonic acid; HIV-I, human immunodeficiency virus type I; LTR, long terminal repeat. NF-kB-CAT) was produced by polymerase chain reaction (PCR) tech- 8172 This paper is available on line at http://www-jbc.stanford.edu/jbc/ This is an Open Access article under the CC BY license. PTX3 Promoter 8173 nique. The plasmid 2180-CAT was used as a substrate for amplification of the CAT gene (up to the EcoRI site) were subcloned into the pGEM-4 with four synthetic oligonucleotides (Duotech srl, Milan, Italy): oligo- plasmid (Promega). The plasmid was linearized with HindIII, and a nucleotide I (59-CTATTACGCCAGCTGGCG-39) was designed on a P-labeled RNA probe was generated with SP6 RNA polymerase and pUC-derived sequence from the pBL CAT 3 vector in sense orientation; [ P]UTP according to the manufacturer’s instructions. oligonucleotide IV (59-CAACGGTGGTATATCCAGTG-39) was designed Six plates per plasmid were transfected with the same precipitation on the CAT sequence in antisense orientation. Oligonucleotide II (59- mixture, and then three were treated with TNFa (500 units/ml) for 4 h TGCTCTAGAATCTGAATTTGGTGGGGGAGG-39) was designed in an- and three were left untreated. At the end of the incubation time, the tisense orientation on hPTX3 promoter from nucleotide 2115 to nucle- cells were extracted in guanidinium isothiocyanate, and the RNA was otide 297 with an additional 11 nucleotides at the 59 end; 6 of them purified as described previously (18). (underlined) give rise to a XbaI restriction site. Oligonucleotide III 7.5 mg of total RNA or yeast tRNA control were hybridized with 2 3 (59-TGCTCTAGACCGTTACCGCAGTGCCACC-39) was designed in 10 cpm CAT riboprobe in 86% deionized formamide, 0.4 M NaCl, 1 mM sense orientation on the hPTX3 promoter from nucleotides 288 to 269; EDTA, 40 mM PIPES, pH 6.7. The hybridization was carried out for 18 h an additional tail of 9 nucleotides includes (underlined)a XbaI site. Two at 55 °C. The hybridization mix was then diluted 10 times in a buffer PCR reactions were performed with oligonucleotides I/II and III/IV, containing 10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.3 M NaCl, 0.5 respectively; the fragments obtained were separated on 5% native poly- unit/ml RNase A (Ambion, Austin, TX), and 100 units/ml RNase T1 acrylamide gel, eluted, and cut with HindIII/XbaI and XbaI/XhoI, re- (Ambion). Incubation was carried out for 45 min at 37 °C. In the same spectively. Finally, the digested fragments were inserted into a HindIII/ 5 hybridization mixture, 5 3 10 cpm of a b-actin riboprobe transcribed XhoI-digested pBL CAT 3 vector. The resulting construct (2180 m from the plasmid pTRIbACT-human (Ambion) by SP6 RNA polymerase NF-kB-CAT) carries a substitution of 8 bases at the NF-kB site (AT- were added. TCTAGA instead of GGGAACTC). After inactivation of the enzymes, the hybridization products were To obtain the mutant plasmid 2180 m Sp1-CAT, we used an oligo- extracted in phenol-chloroform, precipitated, and loaded onto an urea/ nucleotide carrying two mutations (underlined)inthe 2123 Sp1 site polyacrylamide 6% gel. (oligonucleotide V 59-CTCTCCCACCCATACCCCTCCCCCACCAAAT- Electrophoretic Mobility Shift Assay—Nuclear extracts were pre- 39, spanning from nucleotide 2131 to nucleotide 2101). This mutagenic pared from 8387 cells that were stimulated with TNFa (500 units/ml) primer, together with the flanking oligonucleotides I and IV and with for3hor left untreated as described (19). The oligonucleotides utilized the SmaI linearized wild-type 2180-CAT plasmid as a substrate were (Duotech) correspond to the NF-kB site at position 296 and its flanking used to produce a DNA fragment carrying the desired mutations ac- sequences both in a wild type (59-AATTCAGGGGAACTCCCGTTACC- cording to the PCR mutagenesis technique as described (15). Similarly, 39) and a mutated form (59-AATTCAGATTCTAGACCGTTACC-39). 10 to obtain the 2180 m AP-1-CAT mutant plasmid, we used a mutagenic mg of nuclear proteins were incubated with 50 pg of P-labeled oligo- primer (oligonucleotide VI 59-CCACCAGCATTACGTTTTCATC- nucleotides and 1 mg of poly(dIzdC) (Pharmacia Biotech, Uppsala, Swe- CCCATTC-39 spanning from nucleotide 274 to nucleotide 246) carry- den) in 15 ml of binding reaction buffer (40 mM Tris (pH 7.5), 120 mM ing three substitutions (underlined)inthe 265 AP-1 site. The double KCl, 8% Ficoll, 4 mM EDTA, 1 mM dithiothreitol, and 10% glycerol) for mutant 2180 m NF-kB/Sp1-CAT was obtained using oligonucleotides I, 20 min at room temperature. A 1000-fold molar excess of cold oligonu- IV, and V and the SmaI linearized 2180 m NF-kB-CAT plasmid as a cleotide was used for competition assays. Competition assays were substrate for PCR reaction. The mutated fragments obtained were performed using oligonucleotides carrying wild-type or mutant NF-kB purified by polyacrylamide gel electrophoresis, eluted, digested with sites and with an oligonucleotide carrying a functional NF-kB site from HindIII/XhoI, and cloned into a pBL CAT3 vector. All the PCR reactions human immunodeficiency virus type I (HIV-I) long terminal repeat above described were carried out using Pfu DNA polymerase (LTR) (59-GATCCAGAGGGGACTTTCCGAGAGGC-39) (20) which was (Stratagene). also used for a standard binding reaction as a positive control. The Transfection and CAT Assays—8387 human fibrosarcoma (11) and resulting complexes were separated from the free probe by electro- Hep 3B (12) human hepatoma cells were grown to confluence, collected phoresis in a 5% native polyacrylamide gel in 0.5% Tris-buffered EDTA. after trypsinization, and cultured at a density of 8 3 10 (8387 cells) or In supershift analysis we used serum 1141 raised against an amino- 1 3 10 (Hep 3B) in 100-mm dishes 24 h before the transfection. Cells terminal peptide of human p50 (21), serum 1226 raised against a were transfected by the calcium phosphate precipitation method (16) carboxy-terminal peptide of human p65 (21), and serum 1136 raised with 15 mg of CAT reporter plasmid together with 2 mg of pSV-b plasmid carrying the b-galactosidase gene under the control of the SV40 pro- against a carboxy-terminal peptide of human c-Rel (21); after 20 min of moter (Promega Corp., Madison, WI). Cells were left in contact with preincubation on ice, a standard binding assay was performed. DNA for 12 (8387 cells) or 16 (Hep 3B) h. The medium was then replaced with fresh medium. Cells were left to recover for 5 h and then RESULTS stimulated with human recombinant TNFa (500 U/ml, BASF/Knoll, Cloning and Sequencing of the 59-Flanking Region of Ludwigshafen am Rhein, Germany) or with human recombinant IL-1b hPTX3—To analyze the promoter region of the hPTX3 gene, (100 ng/ml, Dompe ´ , L’Aquila, Italy) for 24 h. Human recombinant IL-6 (Immunex Corp., Seattle, WA) was used at 50 units of Cess/ml. genomic DNA sequences upstream the cDNA-encoding se- The cotransfection assays were performed by transfecting 8387 cells quence were studied. An EcoRI-PvuII fragment, which spans with 15 mg of CAT containing constructs together with 0.5 mg of pRSPA nucleotides 21317 to 154 (and does not include the ATG) (Fig. expression vector containing the cDNAs coding for NF-kB p50 and p65 1A), was subcloned for further investigation in the pBL CAT 3 driven by Rous sarcoma virus promoter (17) (a kind gift of Dr. Gary J. expression vector (13) and identified as 21317-CAT (Fig. 1B). A Nabel), either alone or in combination in a 1:1 ratio. After transfection the cells were stimulated with TNFa (500 units/ml) or left untreated. series of deletion mutants obtained by ExoIII/mung directional Cells were harvested and lysed by three cycles of freezing and thaw- deletions was selected as shown in Fig. 1B. ing in 250 mM Tris buffer, pH 7.6. The transfection efficiencies were The sequence of the 59-flanking region of the hPTX3 gene measured by b-galactosidase activity determination in the same (Fig. 1A) revealed features of an eukaryotic promoter, such as amount of cell lysate (usually 40 mg of proteins), measured by an the presence of a number of potential binding sites for tran- enzymatic assay with chlorophenol red-b-D-galactopyranoside (Boeh- scription factors. We have identified one NF-IL 6, two NF-kB, ringer Mannheim GmbH, Mannheim, Germany) as a substrate. Protein concentrations were determined with the Bio-Rad Protein Assay (Bio- one AP-1, two Pu.1, three PEA 3, one Ets-1, and two Sp1 Rad, Richmond, CA). consensus sequences (Fig. 1A). No obvious TATA or CAAT CAT reactions were carried out at 37 °C for3hinan80-ml reaction consensus box was found. The previously identified transcrip- mixture containing 40 mg of cellular extracts, 0.5 mCi of [ C]chloram- tion start site (1), however, corresponds to a pyrimidine-rich phenicol (Amersham International, Little Chalfont, UK), and 5 mM 7-nucleotide consensus (22) sequence which has been reported acetyl-CoA (Boehringer Mannheim). The products were separated on a thin layer chromatography sheet, and the percentage of conversion to to act in TATA-less promoters and is underlined in Fig. 1A. the acetylated form of chloramphenicol was quantified by scintillation While this article was in preparation, the sequence of the counting. CAT activity values were normalized to the b-galactosidase mPTX3 became available (23), and the alignment shows an activity. overall 50.1% conservation, with the last 380 nucleotides show- RNase Protection Assay—A 382-base pair (bp) HindIII-EcoRI frag- ing a 66% conservation (Fig. 1A). Furthermore, two of the ment from the construct 274-CAT, comprising the nucleotides span- ning from 274 to 154 (PvuII site) of the hPTX3 promoter, and a portion reported potential binding sites, the NF-kB and the AP-1 sites 8174 PTX3 Promoter FIG.1. Alignment of the human and murine PTX3 promoters and scheme of the deletion mutants. A, nucleotide sequence of the hPTX3 promoter (h) aligned with the murine sequence (m). The transcription start sites are indicated as 11 and by arrows. Numbers on the right refer to the position in the 59 re- gions. Consensus sequences for binding of transcriptional factors and the pyrimi- dine-rich region are underlined. The translation initiation codon is indicated. The EcoRI and PvuII sites are shown. B, the different constructs shown were used for transfection. at positions 296 and 265, respectively, are maintained at with the 21317, 2387, and 2180 CAT constructs, but it is approximately the same positions in both sequences (Fig. 1A). completely inactive on the 274 construct. IL-1b exposure Functional Analysis of the Promoter—The deletion mutants (hatched bars) induced a quite similar effect in the conditions schematically shown in Fig. 1B were used for expression stud- tested. On the contrary, IL-6 assayed on 8387 cells transfected ies in 8387 human fibrosarcoma cells and in Hep 3B human with the 21317 construct was completely inactive and did not hepatoma cells. After calcium phosphate-mediated transfection modify the responsiveness to TNFa (data not shown). In the of the cells, cultures were left untreated or were treated with same experimental setting, an artificial CAT reporter construct TNFa or IL-1b for 24 h. Data from at least four separate containing four tandem NF-kB sites from IL-6 promoter (24) experiments are shown in Fig. 2. The 21317-CAT construct cloned into a pBL CAT 2 vector (13) gave a mean fold induction shows a 5.3-fold basal activity with respect to the empty vector, of 4.5 6 0.35 times on TNFa induction compared with the and a comparable level is observed also with the 2387 con- untreated cells (data not shown). struct, implying that the 1000 intervening nucleotides do not To validate these results with a different approach, we ana- contribute significantly to this basal activity. In contrast, the lyzed the 8387-transfected cells by RNase protection. As shown 2180 construct has a 2-fold higher basal level, while further in Fig. 3, lane 1, the undigested riboprobe corresponds to the deletion up to 274 abolishes almost completely the activity. predicted size of 383 nucleotides. In the transfected 8387 cells TNFa exposure (gray bars) results in a 2.5-fold induction (lanes 4 –13), the protected band is 309 bp in all cases, thus PTX3 Promoter 8175 FIG.2. CAT analysis of the transcriptional activity of the FIG.1—continued hPTX3 promoter. The different constructs were transfected in 8387 cells, subsequently treated or not with TNFa and IL-1b. Extracts were analyzed for CAT levels. Fold induction is referred to CAT levels with showing the use of the same transcriptional start site in the respect to empty vector (pBL CAT3)-transfected cells (which produces a artificial constructs as in the wild-type gene (1). mean value of 1.8% acetylation). Mean values and standard deviations While no precise measurement can be made for the baseline from four independent experiments are shown. values of the different constructs among them due to possible variation in transfection efficiency, for each experimental NF-kB proteins to the wild-type sequence (lane 4) and, on the group the untreated and treated cells can be compared. TNFa other hand, when used as a probe, did not show binding activity indeed increases the transcription of the three responsive con- in either untreated (lane 6) and TNFa-treated (lane 7) 8387 structs by 3– 6-fold (as determined by densitometric scanning) cells. (lanes 4 –9), whereas construct 274 shows no activity in both Supershifting with antibodies clearly indicated that the two unstimulated and stimulated cells (lanes 12 and 13). These bands correspond to the p50/p65 heterodimer and to the p50/ data have been reproduced in three separate experiments. p50 homodimer, respectively (Fig. 4B, lanes 3 and 4). Further- To study the cellular specificity of the TNFa/IL-1b respon- more, c-Rel is not present in this complex (lane 5) as demon- siveness, we transfected the same constructs in the human strated by the lack of supershifting, similar to what is observed hepatoma cell line Hep 3B; although PTX3 mRNA is inducible with an irrelevant antibody (lane 6). in these cells by TNFa and IL-1b exposure (1), we could not We further compared under the same experimental condi- observe, either in unstimulated or in TNFa-stimulated cells, a tions the binding activity of 8387 nuclear extracts on an oligo- significant CAT activity with respect to the empty vector-trans- nucleotide containing the PTX3 NF-kB site and on an oligonu- fected cells (data not shown) despite a good transfection effi- cleotide containing a canonical NF-kB-binding site from HIV-1 ciency. On the other hand, the artificial CAT reporter construct LTR, which has been previously reported to give rise to only containing four tandem NF-kB sites (see above) showed, under one retarded complex (20). As shown in Fig. 4C, while the the same experimental conditions, a full responsiveness to binding on the PTX3 oligonucleotide gave rise to two retarded TNFa (.15-fold; data not shown). All these data together imply complexes, corresponding to p50/p50 and p50/p65 homo- and that further genomic elements or posttranslational modifica- heterodimers, only the upper band was present in the binding tions may crucially contribute to the hepatic transcription of to the HIV-1 LTR NF-kB site. PTX3 detectable in vitro. The NF-kB Site Is Functionally Relevant in the hPTX3 Pro- NF-kB p50 and p65 Can Bind to the hPTX3 Promoter—The moter—To more directly assess the functional relevance of the sharp difference in the activity between 2180 and 274, the NF-kB site, we mutagenized this site in the 2180 construct, presence of an NF-kB element at position 296 (Fig. 1A), and (2180 m NF-kB-CAT). The mutated sequence is identical to the the known effect of NF-kBinTNFa- and IL-1b-mediated re- degenerated oligonucleotide that we had used in the gel retar- sponses prompted us to analyze the involvement of NF-kBin dation experiments (Fig. 4A, lanes 6 and 7). When the 2180 m PTX3 regulation by electrophoretic mobility shift assay. An NF-kB-CAT construct was analyzed by CAT analysis and oligonucleotide corresponding to the sequence from position RNase protection, it was evident that despite a detectable level 2103 to 281 of hPTX3 was utilized. A low level of binding of basal activity, cytokines exposure did not lead to any signif- activity was detectable in untreated 8387 cells as two separate icant induction of its transcription (Fig. 2, 2180 m NF-kB-CAT, bands (Fig. 4A, lane 1), and they were clearly increased after a and Fig. 3, lanes 10 and 11), thus implying an NF-kB mediated 3-h TNFa stimulation (lane 2). The specificity of the binding induction of the transcriptional activity of the hPTX3 promoter activity is documented by the complete competition with the by TNFa and IL-1b. cold specific oligonucleotides (lane 3) and with an oligonucleo- To validate the hypothesis that NF-kB is the key responsive tide containing a canonical NF-kB site from HIV-1 LTR (lane 5) element, we also mutagenized the Sp 1 and AP-1 sites which (20). are present in the minimal promoter (as shown in Fig. 1b). The We also generated a mutant hPTX3 NF-kB oligonucleotide 2180 m Sp 1-CAT was indeed still responsive (Fig. 2), although which did not contain a NF-kB-binding site. This mutated at a lower level compared with the wild type 2180-CAT con- oligonucleotide was unable to compete for the binding of the struct (1.7 mean fold induction over four separate experiments 8176 PTX3 Promoter FIG.3. RNase protection analysis of the transcriptional activity of hPTX3 promoter. Cells were transfected as in Fig. 2. The CAT mRNA levels in the ex- tracts were quantified by RNase protec- tion assay. That each sample pair con- tained similar levels of RNA was checked by hybridization with an antisense b-ac- tin (b-ACT) probe. FIG.4. NF-kB binding activity is induced by TNF exposure. A, nuclear proteins were extracted from 8387 cells treated with TNFa (lanes 2–5 and 7) or left untreated (lanes 1 and 6). The binding to wild-type (lanes 1–5) or mutant NF-kB oligonucleotide (lanes 6 and 7) was determined by electrophoretic mobility shift assay. The competition assays were performed with a 1000-fold molar excess of cold oligonucleotides containing the wild-type NF-kB site (lane 3), the mutant site (lane 4), or the HIV-I LTR-derived NF-kB site (lane 5). B, supershift assay. Nuclear proteins from TNFa-stimulated 8387 cells were incubated with antibodies against NF-kB p50 (lane 3), NF-kB p65 (lane 4), c-Rel (lane 5), or nonspecific antibody (lane 6) before a standard binding reaction with the oligonucleotide containing the 296 hPTX3 NF-kB site. Control binding of nuclear proteins extracted from either TNFa-stimulated (lane 2) or unstimulated cells (lane 1) is also shown. C, binding to hPTX3 NF-kB oligonucleotide compared with HIV-1 LTR NF-kB oligonucleotide. Lanes 1 and 2, binding reaction with hPTX3 NF-kB; lanes 3–5, binding reaction with HIV-1 LTR NF-kB; lane 5, competition assay performed with a 1000-fold molar excess of cold HIV-1 LTR NF-kB. Nuclear extracts were prepared from untreated (lanes 1 and 3)orTNFa-treated 8387 cells (lanes 2, 4, and 5). upon TNFa induction). As expected, also the double mutant alone or in combination. As shown in Fig. 5, top, cotransfection 2180 m NF-kB/Sp 1-CAT is not responsive to TNFa and IL-1b of p50 alone did not modify the basal CAT activity by the stimulation, although it retains a basal activity. On the other construct in the wild type configuration, as compared with cells hand, the AP-1 mutant 2180 m AP-1-CAT shows full cytokine transfected with the empty vector pRSPA, while p65 and the responsiveness (3.8 and 2.7 mean fold induction with TNFa combination of the two increased the basal activity by a factor and IL-1b, respectively), but a much lower basal level of CAT of 3.6 (p50/p65) to 3.9 (p65 alone). The addition of TNFa was expression (Fig. 2). effective in all the experimental conditions tested (3, 2.6, 1.7, To quantify the observed stimulations with respect to canon- and 1.7 fold induction over the unstimulated cells in pRSPA, ical NF-kB sites, we made use in the same experimental set- p50, p65, and p50/p65 transfected cells respectively). On the ting, of an artificial CAT reporter construct containing four other hand, overexpression of p65 alone, or in combination with tandem NF-kB sites derived from IL-6 promoter (24) and p50, as well as addition of TNFa had no effect on the 2180 m cloned in a pBL CAT 2 vector (13). This reporter gave a mean NF-kB-CAT construct carrying the mutation in the 296 NF-kB fold induction of 4.5 6 0.35 upon TNFa stimulation relatively site (Fig. 5, bottom). These data are consistent with the hypoth- to untreated cells (data not shown), therefore of comparable esis that the hPTX3 natural promoter contains a functional entity to those observed with the PTX3 constructs. NF-kB site. Cotransfection with Expression Vectors—To further substan- Under the same experimental conditions, the control re- tiate that the NF-kB site is indeed the main functional respon- porter plasmid containing four NF-kB binding sites from the sive element, we cotransfected 8387 cells with the 2180-CAT IL-6 promoter was induced by p50/p65 overexpression by 5.9- and with the 2180 m NF-kB-CAT constructs as reporters to- fold, while no induction was detectable against a pSV2 CAT gether with p50 and p65 NF-kB expression vectors (17) either reporter plasmid utilized as a negative control (not containing PTX3 Promoter 8177 PTX3 belongs structurally to the family of the classical acute phase protein pentraxins, which include CRP and SAP, in several animal species. Both genes are characteristically in- duced by IL-6 in combination with IL-1 and TNF mainly, if not exclusively, in hepatocytes (25–31). PTX3 was the first cloned member of the newly emerging group of “long pentraxins,” (5–9) because they show a long amino-terminal domain fused to the carboxy-terminal pentraxin domain (corresponding to most of the classical pentraxin sequence). The significance of this genetic acquisition is far from being understood, but all the long pentraxins do not show a liver-restricted expression pat- tern and seem to be expressed in a much wider spectrum of organs, such as the brain and the testis (6 –9). Furthermore, hPTX3 was shown to be transcribed after exposure to IL-1b, TNFa, and the bacterial product LPS (i.e. all prototypical proinflammatory signals) but not by IL-6, in several different cell types, including endothelial cells, fibroblasts, hepatocytes, and monocytes (1, 10). Furthermore, PTX3 expression induced by IL-1b is not modified in endothelial cells and hepatocytes by concomitant exposure to IL-6 (data not shown). The same gene was cloned in TNFa-stimulated fibroblasts, named TSG-14 (2), and demonstrated to be directly induced by TNFa (32, 33). Inducibility by IL-1b and TNFa, but not by IL-6, may corre- late well with the demonstrated role of NF-kB (for review, see Refs. 34 –36) and, furthermore, with the presence of only one NF-IL 6 binding site (37, 38) and with the absence of APRF elements (39 – 41) in the human promoter. Both elements, in FIG.5. Effect of NF-kB p50 and p65 overexpression on hPTX 3 fact, have been demonstrated to be necessary in multiple copies wild-type and mutant promoter. 8387 cells were cotransfected with for IL-6 inducibility (42). the 2180-CAT (top)orthe 2180 m NF-kB-CAT construct (bottom) and The murine gene (82% identical at the amino acid level) with expression vectors containing NF-kB p50 and p65 cDNAs driven by the Rous sarcoma virus promoter. Fold induction refers to the CAT shows a similar exon/intron organization and is localized on a activity of the different CAT constructs measured in presence of NF- syntenic chromosomal region (4). It is induced in vitro only in kB-containing plasmids relative to those measured with the empty peritoneal macrophages, in some fibroblasts, and in very few pRSPA vector only (to which a value of 1.0 was assigned). White and endothelial cell lines, but not in hepatocytes; on the other hand, gray bars, untreated and TNFa-treated cells, respectively. it was induced in vivo in several muscular tissues after LPS i.v. a NF-kB element) when cotransfected with p50 and p65 in injection (an acute phase experimental model), but not in the combination (data not shown). liver (4, 32). In addition, in situ hybridization studies have indicated that in the heart and in the thigh, the endothelial DISCUSSION cells were the most abundant producer cell type (4). In this report, we characterize the promoter of the human The alignment between the human and the murine promot- PTX3 gene (hPTX3). A genomic fragment of 1317 bp, located 59 ers shows a high overall degree of conservation, including few to the transcriptional start site, responds to TNFa and IL-1b hypothetical binding sites for transcription factors, in particu- stimulation in transiently transfected human 8387 fibroblasts lar the NF-kB site which is here demonstrated as functionally but not in human hepatoma Hep 3B cells, as measured by important for the hPTX3 gene. What are the structural reasons transfection and CAT assays (more than 2-fold induction) and for the differences in the expression of PTX3 between humans by RNase protection analysis (3– 6-fold induction). Deletion and mice is still unclear. mutants show that the 180 bp more proximal to the start site The reported lack of consensus sites for hepatic transcription are sufficient for TNFa- and IL-1b-inducible transcriptional factors in the murine promoter (and in the human promoter as activity. On the contrary, the last 74 bp are unresponsive. In well) may account in part for the absence of induction in the the intervening 106 bp we show that a classical NF-kB binding liver (another obvious difference with the classical CRP and site is present and furthermore that p50/p50 homodimers and SAP genes (3), but the positive elements required for its induc- p50/p65 heterodimers can bind to this element after incubation ibility in the endothelial cells of the muscular district have yet with nuclear extracts from 8387 fibroblasts. TNFa exposure to be elucidated. On the other hand, recent work with trans- increases this NF-kB activity, while the minimal construct genic animals has shown, in the case of CRP, that the precise carrying an inactivating mutation of this site loses the TNFa characterization of the functional elements required for the in inducibility in the same cells. Finally, we confirmed the hy- vivo “acute phase” inducibility may require a complex interac- pothesis that NF-kB proteins are functionally active on the tion between 59 and 39 elements (43), which was unexpected on hPTX3 promoter by cotransfection with p50 and p65 NF-kB the basis of previous in vitro studies (27–29, 31). expression vectors. On the contrary, Sp1 and AP-1 do not seem to play a major role for the cytokine inducibility of the gene. We have described the functional role of the NF-kB site in the promoter of the hPTX3 gene for TNFa inducibility in fibro- These data show for the first time that a classical NF-kB complex can functionally interact with the “long pentraxin” blasts. We can only speculate at the moment, on the basis of the large amount of published data, that this same site may be hPTX3 promoter in human fibroblasts after exposure to TNFa. The different methods utilized indicate a 2–5-fold transcrip- relevant also for LPS inducibility of the gene in fibroblasts as tional induction of the gene, which is in agreement with the well as in other cell types. NF-kB may interact with other observed increase in nuclear runoff experiments on isolated factors as suggested by others (44 – 48), particularly in view of monocytes (10). the presence of AP-1 and Sp1 sites in close proximity to the 8178 PTX3 Promoter Proc. Natl. Acad. Sci. U. S. 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Characterization of the Promoter for the Human Long Pentraxin PTX3

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 13, Issue of March 28, pp. 8172–8178, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Characterization of the Promoter for the Human Long Pentraxin PTX3 ROLE OF NF-kB IN TUMOR NECROSIS FACTOR-a AND INTERLEUKIN-1b REGULATION* (Received for publication, November 19, 1996, and in revised form, January 23, 1997) Andrea Basile‡, Antonio Sica‡, Elisabetta d’Aniello‡, Ferruccio Breviario‡, Gabino Garrido‡, Marina Castellano‡, Alberto Mantovani‡§, and Martino Introna‡ From the ‡Istituto di Ricerche Farmacologiche “Mario Negri,” via Eritrea, 62, 20157 Milan, Italy and the §Section of General Pathology and Immunology, University of Brescia, 25123 Brescia, Italy the 59 half of the protein does not show significant homology The “long pentraxins” are an emerging family of genes that have conserved in their carboxy-terminal halves a with other known proteins. PTX3 is indeed the first isolated pentraxin domain homologous to the prototypical acute member of a new group of proteins, known as “long pentrax- phase protein pentraxins (C-reactive protein and serum ins,” which have different 59-termini upstream from their pen- amyloid P component) and acquired novel amino-termi- traxin domains (5–9). nal domains. In this report, a genomic fragment of 1371 While the classical pentraxins CRP and SAP are almost nucleotides from the human “long pentraxin” gene exclusively produced by the liver in response to IL-6 in combi- PTX3 is characterized as a promoter on tumor necrosis nation with IL-1 and TNF, PTX3 shows a more promiscuous factor-a (TNFa) and interleukin (IL)-1b exposure in response in that its expression in vitro can be induced in transfected 8387 human fibroblasts by chloramphenicol endothelial cells, hepatocytes, fibroblasts, and monocytes. In acetyltransferase and RNase protection assays. In the all cases, the gene is rapidly and directly induced by exposure same cells, the PTX3 promoter does not respond to IL-6 to IL-1b, TNFa, and lipopolysaccharide (LPS), but not by IL-6, stimulation. Furthermore, IL-1b and TNFa responsive- the mRNA peaking 4 – 6 h after the stimulation (1, 4, 10). This ness is not seen in the Hep 3B hepatoma cell line. The induction is paralleled by de novo transcription of the gene (10) minimal promoter contains one NF-kB element which is and is transient in that no more message is detectable after shown to be necessary for induction and able to bind p50 24 h (1, 10). homodimers and p65 heterodimers but not c-Rel. Mu- The mouse homologue, mPTX3, shows a similar exon/intron tants in this site lose the ability to bind NF-kB proteins organization and 82% identity at the amino acid level with and to respond to TNFa and IL-1b in functional assays. hPTX3 (4). When C57BL mice were injected i.v with LPS to Sp1- and AP-1 binding sites lying in proximity to the induce an acute phase response, mPTX3 expression was mark- NF-kB site do not seem to play a major role for cytokine edly induced in vivo after4hin several muscular organs, responsiveness. Finally, cotransfection experiments with expression vectors validate that the natural pro- including the heart and the thigh (4). In situ hybridization moter contains a functional NF-kB site. studies showed that endothelial cells within the muscular tis- sues were the major responder cell type. Interestingly, in strik- ing contrast with CRP and SAP, no mRNA for mPTX3 could be The human gene hPTX3 has been recently cloned from in- detected by Northern analysis in the liver (4). terleukin-1b (IL-1b) -stimulated endothelial cells (1) and from Similar promiscuous in vivo expression has also been ob- tumor necrosis factor-a (TNFa)-stimulated fibroblasts (2). served for the other “long pentraxins” in organs as diverse as PTX3 belongs to the family of pentraxins (so named because the brain and the testis (6 –9). they are assembled in pentamers) that include C-reactive pro- To begin to understand the molecular mechanisms underly- tein (CRP) and serum amyloid P component (SAP) from several ing the regulated expression of the first cloned long pentraxin different species (3) and which are markers of the acute phase. PTX3, we cloned and characterized the promoter of hPTX3. Moreover, while the 39 half of PTX3 can be aligned with the EXPERIMENTAL PROCEDURES full-length sequences of CRP and SAP (4) (pentraxin domain), Cell Culture—The human fibrosarcoma 8387 (11) and the human hepatoma Hep 3B (12) cell lines were maintained in Dulbecco’s modified * This work was supported in part by Piano Nazionale Farmaci II, Eagle’s medium (Seromed, Biochrom KG, Berlin, Germany), supple- tema 5; F. B. was supported by NATO Grant CRG 941228. The costs of mented with 10% fetal calf serum (Hyclone, Logan, UT), 50 mg/ml publication of this article were defrayed in part by the payment of page gentamycin (Life Technologies, Inc., Paisley, Renfewshire, United King- charges. This article must therefore be hereby marked “advertisement” dom), and 20 mML-glutamine (Seromed). in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Plasmid Construction—A 1.37-kilobase EcoRI-PvuII genomic frag- The nucleotide sequence(s) reported in this paper has been submitted ment from lP2 phage (1), which spans nucleotides 21317 to 154 TM to the GenBank /EBI Data Bank with accession number(s) X97748. relative to the transcription start site, was blunted and subcloned into ¶ To whom requests for reprints should be addressed: Laboratory of the XhoI-digested and blunted pBL CAT 3 vector (13) (giving 21317- Molecular Immunohematology, Department of Immunology and Cell CAT). Following PstI-XbaI digestion, the latter plasmid was used as a Biology, Istituto di Ricerche Farmacologiche “Mario Negri,” via Eritrea, substrate to generate a set of deletion clones with ExoIII (Stratagene, 62, 20157 Milan, Italy. Tel.: 0039/2/39014547; Fax: 0039/2/33200231. 1 La Jolla, CA) digestion, mung (Stratagene) blunting, and subsequent The abbreviations used are: IL, interleukin; TNFa, tumor necrosis religation. Deletion clones covering the whole 59-flanking region of the factor-a; CRP, C-reactive protein; SAP, serum amyloid P component; hPTX3 gene were sequenced by the Sanger dideoxy method (14). Three LPS, lipopolysaccharide; CAT, chloramphenicol acetyltransferase; of them, 2387-CAT, 2180-CAT, and 274-CAT, were subsequently used 2180 m NF-kB-CAT, plasmid carrying a mutation in the 296 NF-kB in functional assays. site; PCR, polymerase chain reaction; bp, base pair; PIPES, 1,4-pipera- The plasmid carrying a mutation in the 296 NF-kB site (2180 m zinediethanesulfonic acid; HIV-I, human immunodeficiency virus type I; LTR, long terminal repeat. NF-kB-CAT) was produced by polymerase chain reaction (PCR) tech- 8172 This paper is available on line at http://www-jbc.stanford.edu/jbc/ This is an Open Access article under the CC BY license. PTX3 Promoter 8173 nique. The plasmid 2180-CAT was used as a substrate for amplification of the CAT gene (up to the EcoRI site) were subcloned into the pGEM-4 with four synthetic oligonucleotides (Duotech srl, Milan, Italy): oligo- plasmid (Promega). The plasmid was linearized with HindIII, and a nucleotide I (59-CTATTACGCCAGCTGGCG-39) was designed on a P-labeled RNA probe was generated with SP6 RNA polymerase and pUC-derived sequence from the pBL CAT 3 vector in sense orientation; [ P]UTP according to the manufacturer’s instructions. oligonucleotide IV (59-CAACGGTGGTATATCCAGTG-39) was designed Six plates per plasmid were transfected with the same precipitation on the CAT sequence in antisense orientation. Oligonucleotide II (59- mixture, and then three were treated with TNFa (500 units/ml) for 4 h TGCTCTAGAATCTGAATTTGGTGGGGGAGG-39) was designed in an- and three were left untreated. At the end of the incubation time, the tisense orientation on hPTX3 promoter from nucleotide 2115 to nucle- cells were extracted in guanidinium isothiocyanate, and the RNA was otide 297 with an additional 11 nucleotides at the 59 end; 6 of them purified as described previously (18). (underlined) give rise to a XbaI restriction site. Oligonucleotide III 7.5 mg of total RNA or yeast tRNA control were hybridized with 2 3 (59-TGCTCTAGACCGTTACCGCAGTGCCACC-39) was designed in 10 cpm CAT riboprobe in 86% deionized formamide, 0.4 M NaCl, 1 mM sense orientation on the hPTX3 promoter from nucleotides 288 to 269; EDTA, 40 mM PIPES, pH 6.7. The hybridization was carried out for 18 h an additional tail of 9 nucleotides includes (underlined)a XbaI site. Two at 55 °C. The hybridization mix was then diluted 10 times in a buffer PCR reactions were performed with oligonucleotides I/II and III/IV, containing 10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.3 M NaCl, 0.5 respectively; the fragments obtained were separated on 5% native poly- unit/ml RNase A (Ambion, Austin, TX), and 100 units/ml RNase T1 acrylamide gel, eluted, and cut with HindIII/XbaI and XbaI/XhoI, re- (Ambion). Incubation was carried out for 45 min at 37 °C. In the same spectively. Finally, the digested fragments were inserted into a HindIII/ 5 hybridization mixture, 5 3 10 cpm of a b-actin riboprobe transcribed XhoI-digested pBL CAT 3 vector. The resulting construct (2180 m from the plasmid pTRIbACT-human (Ambion) by SP6 RNA polymerase NF-kB-CAT) carries a substitution of 8 bases at the NF-kB site (AT- were added. TCTAGA instead of GGGAACTC). After inactivation of the enzymes, the hybridization products were To obtain the mutant plasmid 2180 m Sp1-CAT, we used an oligo- extracted in phenol-chloroform, precipitated, and loaded onto an urea/ nucleotide carrying two mutations (underlined)inthe 2123 Sp1 site polyacrylamide 6% gel. (oligonucleotide V 59-CTCTCCCACCCATACCCCTCCCCCACCAAAT- Electrophoretic Mobility Shift Assay—Nuclear extracts were pre- 39, spanning from nucleotide 2131 to nucleotide 2101). This mutagenic pared from 8387 cells that were stimulated with TNFa (500 units/ml) primer, together with the flanking oligonucleotides I and IV and with for3hor left untreated as described (19). The oligonucleotides utilized the SmaI linearized wild-type 2180-CAT plasmid as a substrate were (Duotech) correspond to the NF-kB site at position 296 and its flanking used to produce a DNA fragment carrying the desired mutations ac- sequences both in a wild type (59-AATTCAGGGGAACTCCCGTTACC- cording to the PCR mutagenesis technique as described (15). Similarly, 39) and a mutated form (59-AATTCAGATTCTAGACCGTTACC-39). 10 to obtain the 2180 m AP-1-CAT mutant plasmid, we used a mutagenic mg of nuclear proteins were incubated with 50 pg of P-labeled oligo- primer (oligonucleotide VI 59-CCACCAGCATTACGTTTTCATC- nucleotides and 1 mg of poly(dIzdC) (Pharmacia Biotech, Uppsala, Swe- CCCATTC-39 spanning from nucleotide 274 to nucleotide 246) carry- den) in 15 ml of binding reaction buffer (40 mM Tris (pH 7.5), 120 mM ing three substitutions (underlined)inthe 265 AP-1 site. The double KCl, 8% Ficoll, 4 mM EDTA, 1 mM dithiothreitol, and 10% glycerol) for mutant 2180 m NF-kB/Sp1-CAT was obtained using oligonucleotides I, 20 min at room temperature. A 1000-fold molar excess of cold oligonu- IV, and V and the SmaI linearized 2180 m NF-kB-CAT plasmid as a cleotide was used for competition assays. Competition assays were substrate for PCR reaction. The mutated fragments obtained were performed using oligonucleotides carrying wild-type or mutant NF-kB purified by polyacrylamide gel electrophoresis, eluted, digested with sites and with an oligonucleotide carrying a functional NF-kB site from HindIII/XhoI, and cloned into a pBL CAT3 vector. All the PCR reactions human immunodeficiency virus type I (HIV-I) long terminal repeat above described were carried out using Pfu DNA polymerase (LTR) (59-GATCCAGAGGGGACTTTCCGAGAGGC-39) (20) which was (Stratagene). also used for a standard binding reaction as a positive control. The Transfection and CAT Assays—8387 human fibrosarcoma (11) and resulting complexes were separated from the free probe by electro- Hep 3B (12) human hepatoma cells were grown to confluence, collected phoresis in a 5% native polyacrylamide gel in 0.5% Tris-buffered EDTA. after trypsinization, and cultured at a density of 8 3 10 (8387 cells) or In supershift analysis we used serum 1141 raised against an amino- 1 3 10 (Hep 3B) in 100-mm dishes 24 h before the transfection. Cells terminal peptide of human p50 (21), serum 1226 raised against a were transfected by the calcium phosphate precipitation method (16) carboxy-terminal peptide of human p65 (21), and serum 1136 raised with 15 mg of CAT reporter plasmid together with 2 mg of pSV-b plasmid carrying the b-galactosidase gene under the control of the SV40 pro- against a carboxy-terminal peptide of human c-Rel (21); after 20 min of moter (Promega Corp., Madison, WI). Cells were left in contact with preincubation on ice, a standard binding assay was performed. DNA for 12 (8387 cells) or 16 (Hep 3B) h. The medium was then replaced with fresh medium. Cells were left to recover for 5 h and then RESULTS stimulated with human recombinant TNFa (500 U/ml, BASF/Knoll, Cloning and Sequencing of the 59-Flanking Region of Ludwigshafen am Rhein, Germany) or with human recombinant IL-1b hPTX3—To analyze the promoter region of the hPTX3 gene, (100 ng/ml, Dompe ´ , L’Aquila, Italy) for 24 h. Human recombinant IL-6 (Immunex Corp., Seattle, WA) was used at 50 units of Cess/ml. genomic DNA sequences upstream the cDNA-encoding se- The cotransfection assays were performed by transfecting 8387 cells quence were studied. An EcoRI-PvuII fragment, which spans with 15 mg of CAT containing constructs together with 0.5 mg of pRSPA nucleotides 21317 to 154 (and does not include the ATG) (Fig. expression vector containing the cDNAs coding for NF-kB p50 and p65 1A), was subcloned for further investigation in the pBL CAT 3 driven by Rous sarcoma virus promoter (17) (a kind gift of Dr. Gary J. expression vector (13) and identified as 21317-CAT (Fig. 1B). A Nabel), either alone or in combination in a 1:1 ratio. After transfection the cells were stimulated with TNFa (500 units/ml) or left untreated. series of deletion mutants obtained by ExoIII/mung directional Cells were harvested and lysed by three cycles of freezing and thaw- deletions was selected as shown in Fig. 1B. ing in 250 mM Tris buffer, pH 7.6. The transfection efficiencies were The sequence of the 59-flanking region of the hPTX3 gene measured by b-galactosidase activity determination in the same (Fig. 1A) revealed features of an eukaryotic promoter, such as amount of cell lysate (usually 40 mg of proteins), measured by an the presence of a number of potential binding sites for tran- enzymatic assay with chlorophenol red-b-D-galactopyranoside (Boeh- scription factors. We have identified one NF-IL 6, two NF-kB, ringer Mannheim GmbH, Mannheim, Germany) as a substrate. Protein concentrations were determined with the Bio-Rad Protein Assay (Bio- one AP-1, two Pu.1, three PEA 3, one Ets-1, and two Sp1 Rad, Richmond, CA). consensus sequences (Fig. 1A). No obvious TATA or CAAT CAT reactions were carried out at 37 °C for3hinan80-ml reaction consensus box was found. The previously identified transcrip- mixture containing 40 mg of cellular extracts, 0.5 mCi of [ C]chloram- tion start site (1), however, corresponds to a pyrimidine-rich phenicol (Amersham International, Little Chalfont, UK), and 5 mM 7-nucleotide consensus (22) sequence which has been reported acetyl-CoA (Boehringer Mannheim). The products were separated on a thin layer chromatography sheet, and the percentage of conversion to to act in TATA-less promoters and is underlined in Fig. 1A. the acetylated form of chloramphenicol was quantified by scintillation While this article was in preparation, the sequence of the counting. CAT activity values were normalized to the b-galactosidase mPTX3 became available (23), and the alignment shows an activity. overall 50.1% conservation, with the last 380 nucleotides show- RNase Protection Assay—A 382-base pair (bp) HindIII-EcoRI frag- ing a 66% conservation (Fig. 1A). Furthermore, two of the ment from the construct 274-CAT, comprising the nucleotides span- ning from 274 to 154 (PvuII site) of the hPTX3 promoter, and a portion reported potential binding sites, the NF-kB and the AP-1 sites 8174 PTX3 Promoter FIG.1. Alignment of the human and murine PTX3 promoters and scheme of the deletion mutants. A, nucleotide sequence of the hPTX3 promoter (h) aligned with the murine sequence (m). The transcription start sites are indicated as 11 and by arrows. Numbers on the right refer to the position in the 59 re- gions. Consensus sequences for binding of transcriptional factors and the pyrimi- dine-rich region are underlined. The translation initiation codon is indicated. The EcoRI and PvuII sites are shown. B, the different constructs shown were used for transfection. at positions 296 and 265, respectively, are maintained at with the 21317, 2387, and 2180 CAT constructs, but it is approximately the same positions in both sequences (Fig. 1A). completely inactive on the 274 construct. IL-1b exposure Functional Analysis of the Promoter—The deletion mutants (hatched bars) induced a quite similar effect in the conditions schematically shown in Fig. 1B were used for expression stud- tested. On the contrary, IL-6 assayed on 8387 cells transfected ies in 8387 human fibrosarcoma cells and in Hep 3B human with the 21317 construct was completely inactive and did not hepatoma cells. After calcium phosphate-mediated transfection modify the responsiveness to TNFa (data not shown). In the of the cells, cultures were left untreated or were treated with same experimental setting, an artificial CAT reporter construct TNFa or IL-1b for 24 h. Data from at least four separate containing four tandem NF-kB sites from IL-6 promoter (24) experiments are shown in Fig. 2. The 21317-CAT construct cloned into a pBL CAT 2 vector (13) gave a mean fold induction shows a 5.3-fold basal activity with respect to the empty vector, of 4.5 6 0.35 times on TNFa induction compared with the and a comparable level is observed also with the 2387 con- untreated cells (data not shown). struct, implying that the 1000 intervening nucleotides do not To validate these results with a different approach, we ana- contribute significantly to this basal activity. In contrast, the lyzed the 8387-transfected cells by RNase protection. As shown 2180 construct has a 2-fold higher basal level, while further in Fig. 3, lane 1, the undigested riboprobe corresponds to the deletion up to 274 abolishes almost completely the activity. predicted size of 383 nucleotides. In the transfected 8387 cells TNFa exposure (gray bars) results in a 2.5-fold induction (lanes 4 –13), the protected band is 309 bp in all cases, thus PTX3 Promoter 8175 FIG.2. CAT analysis of the transcriptional activity of the FIG.1—continued hPTX3 promoter. The different constructs were transfected in 8387 cells, subsequently treated or not with TNFa and IL-1b. Extracts were analyzed for CAT levels. Fold induction is referred to CAT levels with showing the use of the same transcriptional start site in the respect to empty vector (pBL CAT3)-transfected cells (which produces a artificial constructs as in the wild-type gene (1). mean value of 1.8% acetylation). Mean values and standard deviations While no precise measurement can be made for the baseline from four independent experiments are shown. values of the different constructs among them due to possible variation in transfection efficiency, for each experimental NF-kB proteins to the wild-type sequence (lane 4) and, on the group the untreated and treated cells can be compared. TNFa other hand, when used as a probe, did not show binding activity indeed increases the transcription of the three responsive con- in either untreated (lane 6) and TNFa-treated (lane 7) 8387 structs by 3– 6-fold (as determined by densitometric scanning) cells. (lanes 4 –9), whereas construct 274 shows no activity in both Supershifting with antibodies clearly indicated that the two unstimulated and stimulated cells (lanes 12 and 13). These bands correspond to the p50/p65 heterodimer and to the p50/ data have been reproduced in three separate experiments. p50 homodimer, respectively (Fig. 4B, lanes 3 and 4). Further- To study the cellular specificity of the TNFa/IL-1b respon- more, c-Rel is not present in this complex (lane 5) as demon- siveness, we transfected the same constructs in the human strated by the lack of supershifting, similar to what is observed hepatoma cell line Hep 3B; although PTX3 mRNA is inducible with an irrelevant antibody (lane 6). in these cells by TNFa and IL-1b exposure (1), we could not We further compared under the same experimental condi- observe, either in unstimulated or in TNFa-stimulated cells, a tions the binding activity of 8387 nuclear extracts on an oligo- significant CAT activity with respect to the empty vector-trans- nucleotide containing the PTX3 NF-kB site and on an oligonu- fected cells (data not shown) despite a good transfection effi- cleotide containing a canonical NF-kB-binding site from HIV-1 ciency. On the other hand, the artificial CAT reporter construct LTR, which has been previously reported to give rise to only containing four tandem NF-kB sites (see above) showed, under one retarded complex (20). As shown in Fig. 4C, while the the same experimental conditions, a full responsiveness to binding on the PTX3 oligonucleotide gave rise to two retarded TNFa (.15-fold; data not shown). All these data together imply complexes, corresponding to p50/p50 and p50/p65 homo- and that further genomic elements or posttranslational modifica- heterodimers, only the upper band was present in the binding tions may crucially contribute to the hepatic transcription of to the HIV-1 LTR NF-kB site. PTX3 detectable in vitro. The NF-kB Site Is Functionally Relevant in the hPTX3 Pro- NF-kB p50 and p65 Can Bind to the hPTX3 Promoter—The moter—To more directly assess the functional relevance of the sharp difference in the activity between 2180 and 274, the NF-kB site, we mutagenized this site in the 2180 construct, presence of an NF-kB element at position 296 (Fig. 1A), and (2180 m NF-kB-CAT). The mutated sequence is identical to the the known effect of NF-kBinTNFa- and IL-1b-mediated re- degenerated oligonucleotide that we had used in the gel retar- sponses prompted us to analyze the involvement of NF-kBin dation experiments (Fig. 4A, lanes 6 and 7). When the 2180 m PTX3 regulation by electrophoretic mobility shift assay. An NF-kB-CAT construct was analyzed by CAT analysis and oligonucleotide corresponding to the sequence from position RNase protection, it was evident that despite a detectable level 2103 to 281 of hPTX3 was utilized. A low level of binding of basal activity, cytokines exposure did not lead to any signif- activity was detectable in untreated 8387 cells as two separate icant induction of its transcription (Fig. 2, 2180 m NF-kB-CAT, bands (Fig. 4A, lane 1), and they were clearly increased after a and Fig. 3, lanes 10 and 11), thus implying an NF-kB mediated 3-h TNFa stimulation (lane 2). The specificity of the binding induction of the transcriptional activity of the hPTX3 promoter activity is documented by the complete competition with the by TNFa and IL-1b. cold specific oligonucleotides (lane 3) and with an oligonucleo- To validate the hypothesis that NF-kB is the key responsive tide containing a canonical NF-kB site from HIV-1 LTR (lane 5) element, we also mutagenized the Sp 1 and AP-1 sites which (20). are present in the minimal promoter (as shown in Fig. 1b). The We also generated a mutant hPTX3 NF-kB oligonucleotide 2180 m Sp 1-CAT was indeed still responsive (Fig. 2), although which did not contain a NF-kB-binding site. This mutated at a lower level compared with the wild type 2180-CAT con- oligonucleotide was unable to compete for the binding of the struct (1.7 mean fold induction over four separate experiments 8176 PTX3 Promoter FIG.3. RNase protection analysis of the transcriptional activity of hPTX3 promoter. Cells were transfected as in Fig. 2. The CAT mRNA levels in the ex- tracts were quantified by RNase protec- tion assay. That each sample pair con- tained similar levels of RNA was checked by hybridization with an antisense b-ac- tin (b-ACT) probe. FIG.4. NF-kB binding activity is induced by TNF exposure. A, nuclear proteins were extracted from 8387 cells treated with TNFa (lanes 2–5 and 7) or left untreated (lanes 1 and 6). The binding to wild-type (lanes 1–5) or mutant NF-kB oligonucleotide (lanes 6 and 7) was determined by electrophoretic mobility shift assay. The competition assays were performed with a 1000-fold molar excess of cold oligonucleotides containing the wild-type NF-kB site (lane 3), the mutant site (lane 4), or the HIV-I LTR-derived NF-kB site (lane 5). B, supershift assay. Nuclear proteins from TNFa-stimulated 8387 cells were incubated with antibodies against NF-kB p50 (lane 3), NF-kB p65 (lane 4), c-Rel (lane 5), or nonspecific antibody (lane 6) before a standard binding reaction with the oligonucleotide containing the 296 hPTX3 NF-kB site. Control binding of nuclear proteins extracted from either TNFa-stimulated (lane 2) or unstimulated cells (lane 1) is also shown. C, binding to hPTX3 NF-kB oligonucleotide compared with HIV-1 LTR NF-kB oligonucleotide. Lanes 1 and 2, binding reaction with hPTX3 NF-kB; lanes 3–5, binding reaction with HIV-1 LTR NF-kB; lane 5, competition assay performed with a 1000-fold molar excess of cold HIV-1 LTR NF-kB. Nuclear extracts were prepared from untreated (lanes 1 and 3)orTNFa-treated 8387 cells (lanes 2, 4, and 5). upon TNFa induction). As expected, also the double mutant alone or in combination. As shown in Fig. 5, top, cotransfection 2180 m NF-kB/Sp 1-CAT is not responsive to TNFa and IL-1b of p50 alone did not modify the basal CAT activity by the stimulation, although it retains a basal activity. On the other construct in the wild type configuration, as compared with cells hand, the AP-1 mutant 2180 m AP-1-CAT shows full cytokine transfected with the empty vector pRSPA, while p65 and the responsiveness (3.8 and 2.7 mean fold induction with TNFa combination of the two increased the basal activity by a factor and IL-1b, respectively), but a much lower basal level of CAT of 3.6 (p50/p65) to 3.9 (p65 alone). The addition of TNFa was expression (Fig. 2). effective in all the experimental conditions tested (3, 2.6, 1.7, To quantify the observed stimulations with respect to canon- and 1.7 fold induction over the unstimulated cells in pRSPA, ical NF-kB sites, we made use in the same experimental set- p50, p65, and p50/p65 transfected cells respectively). On the ting, of an artificial CAT reporter construct containing four other hand, overexpression of p65 alone, or in combination with tandem NF-kB sites derived from IL-6 promoter (24) and p50, as well as addition of TNFa had no effect on the 2180 m cloned in a pBL CAT 2 vector (13). This reporter gave a mean NF-kB-CAT construct carrying the mutation in the 296 NF-kB fold induction of 4.5 6 0.35 upon TNFa stimulation relatively site (Fig. 5, bottom). These data are consistent with the hypoth- to untreated cells (data not shown), therefore of comparable esis that the hPTX3 natural promoter contains a functional entity to those observed with the PTX3 constructs. NF-kB site. Cotransfection with Expression Vectors—To further substan- Under the same experimental conditions, the control re- tiate that the NF-kB site is indeed the main functional respon- porter plasmid containing four NF-kB binding sites from the sive element, we cotransfected 8387 cells with the 2180-CAT IL-6 promoter was induced by p50/p65 overexpression by 5.9- and with the 2180 m NF-kB-CAT constructs as reporters to- fold, while no induction was detectable against a pSV2 CAT gether with p50 and p65 NF-kB expression vectors (17) either reporter plasmid utilized as a negative control (not containing PTX3 Promoter 8177 PTX3 belongs structurally to the family of the classical acute phase protein pentraxins, which include CRP and SAP, in several animal species. Both genes are characteristically in- duced by IL-6 in combination with IL-1 and TNF mainly, if not exclusively, in hepatocytes (25–31). PTX3 was the first cloned member of the newly emerging group of “long pentraxins,” (5–9) because they show a long amino-terminal domain fused to the carboxy-terminal pentraxin domain (corresponding to most of the classical pentraxin sequence). The significance of this genetic acquisition is far from being understood, but all the long pentraxins do not show a liver-restricted expression pat- tern and seem to be expressed in a much wider spectrum of organs, such as the brain and the testis (6 –9). Furthermore, hPTX3 was shown to be transcribed after exposure to IL-1b, TNFa, and the bacterial product LPS (i.e. all prototypical proinflammatory signals) but not by IL-6, in several different cell types, including endothelial cells, fibroblasts, hepatocytes, and monocytes (1, 10). Furthermore, PTX3 expression induced by IL-1b is not modified in endothelial cells and hepatocytes by concomitant exposure to IL-6 (data not shown). The same gene was cloned in TNFa-stimulated fibroblasts, named TSG-14 (2), and demonstrated to be directly induced by TNFa (32, 33). Inducibility by IL-1b and TNFa, but not by IL-6, may corre- late well with the demonstrated role of NF-kB (for review, see Refs. 34 –36) and, furthermore, with the presence of only one NF-IL 6 binding site (37, 38) and with the absence of APRF elements (39 – 41) in the human promoter. Both elements, in FIG.5. Effect of NF-kB p50 and p65 overexpression on hPTX 3 fact, have been demonstrated to be necessary in multiple copies wild-type and mutant promoter. 8387 cells were cotransfected with for IL-6 inducibility (42). the 2180-CAT (top)orthe 2180 m NF-kB-CAT construct (bottom) and The murine gene (82% identical at the amino acid level) with expression vectors containing NF-kB p50 and p65 cDNAs driven by the Rous sarcoma virus promoter. Fold induction refers to the CAT shows a similar exon/intron organization and is localized on a activity of the different CAT constructs measured in presence of NF- syntenic chromosomal region (4). It is induced in vitro only in kB-containing plasmids relative to those measured with the empty peritoneal macrophages, in some fibroblasts, and in very few pRSPA vector only (to which a value of 1.0 was assigned). White and endothelial cell lines, but not in hepatocytes; on the other hand, gray bars, untreated and TNFa-treated cells, respectively. it was induced in vivo in several muscular tissues after LPS i.v. a NF-kB element) when cotransfected with p50 and p65 in injection (an acute phase experimental model), but not in the combination (data not shown). liver (4, 32). In addition, in situ hybridization studies have indicated that in the heart and in the thigh, the endothelial DISCUSSION cells were the most abundant producer cell type (4). In this report, we characterize the promoter of the human The alignment between the human and the murine promot- PTX3 gene (hPTX3). A genomic fragment of 1317 bp, located 59 ers shows a high overall degree of conservation, including few to the transcriptional start site, responds to TNFa and IL-1b hypothetical binding sites for transcription factors, in particu- stimulation in transiently transfected human 8387 fibroblasts lar the NF-kB site which is here demonstrated as functionally but not in human hepatoma Hep 3B cells, as measured by important for the hPTX3 gene. What are the structural reasons transfection and CAT assays (more than 2-fold induction) and for the differences in the expression of PTX3 between humans by RNase protection analysis (3– 6-fold induction). Deletion and mice is still unclear. mutants show that the 180 bp more proximal to the start site The reported lack of consensus sites for hepatic transcription are sufficient for TNFa- and IL-1b-inducible transcriptional factors in the murine promoter (and in the human promoter as activity. On the contrary, the last 74 bp are unresponsive. In well) may account in part for the absence of induction in the the intervening 106 bp we show that a classical NF-kB binding liver (another obvious difference with the classical CRP and site is present and furthermore that p50/p50 homodimers and SAP genes (3), but the positive elements required for its induc- p50/p65 heterodimers can bind to this element after incubation ibility in the endothelial cells of the muscular district have yet with nuclear extracts from 8387 fibroblasts. TNFa exposure to be elucidated. On the other hand, recent work with trans- increases this NF-kB activity, while the minimal construct genic animals has shown, in the case of CRP, that the precise carrying an inactivating mutation of this site loses the TNFa characterization of the functional elements required for the in inducibility in the same cells. Finally, we confirmed the hy- vivo “acute phase” inducibility may require a complex interac- pothesis that NF-kB proteins are functionally active on the tion between 59 and 39 elements (43), which was unexpected on hPTX3 promoter by cotransfection with p50 and p65 NF-kB the basis of previous in vitro studies (27–29, 31). expression vectors. On the contrary, Sp1 and AP-1 do not seem to play a major role for the cytokine inducibility of the gene. We have described the functional role of the NF-kB site in the promoter of the hPTX3 gene for TNFa inducibility in fibro- These data show for the first time that a classical NF-kB complex can functionally interact with the “long pentraxin” blasts. We can only speculate at the moment, on the basis of the large amount of published data, that this same site may be hPTX3 promoter in human fibroblasts after exposure to TNFa. The different methods utilized indicate a 2–5-fold transcrip- relevant also for LPS inducibility of the gene in fibroblasts as tional induction of the gene, which is in agreement with the well as in other cell types. NF-kB may interact with other observed increase in nuclear runoff experiments on isolated factors as suggested by others (44 – 48), particularly in view of monocytes (10). the presence of AP-1 and Sp1 sites in close proximity to the 8178 PTX3 Promoter Proc. Natl. Acad. Sci. U. S. 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Published: Mar 1, 1997

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