Characterization of the long pentraxin PTX3 as a TNFα-induced secreted protein of adipose cells

• DOI: 10.1194/jlr.m200382-jlr200
• Publication Date: 2003-05-06T16:33:53Z
• Authors: Anissa Abderrahim...; Olivier Bezy; Chiara Chiellini; Margherita Maffei; Paul Grimaldi; Frédéric Bonino; Naïma Moustaid-Mo...; Fabio Pasqualini; Alberto Mantovani; Gérard Ailhaud; Ez-Zoubir Amri
• Keywords: PTX3

Exposure of preadipocytes to long-chain fatty acids induces the expression several markers adipocyte differentiation. In an attempt identify novel genes and proteins that are regulated by in preadipocytes, we performed a substractive hybridization screening identified PTX3, protein pentraxin family. PTX3 mRNA is transient during differentiation clonal cell lines absent fully differentiated cells. Stable overexpression has no effect on line with this, expressed stromal-vascular fraction adipose tissue, but not fraction; however, 3T3-F442A adipocytes, gene can be reinduced tumor necrosis factor α (TNFα) dose-dependent manner. This accompanied secretion from both adipocytes explants mouse tissue. levels found higher tissue genetically obese mice versus control mice, consistent their increased TNFα levels.In conclusion, appears as TNFα-induced provides new link between chronic low-level inflammatory state obesity. levels. 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Elevated overweight adults.JAMA. 282: 2131-2135Google 16Visser Low-grade inflammation children.Pediatrics. 107: E13-E18Google Clearly, confined previously reported leukemia inhibitory (LIF) transiently at time when cognate surface (LIF-R) also (17Aubert Dessolin Belmonte McKenzie F.R. Staccini Villageois Barhanin B. Vernallis Smith A.G. Ailhaud Dani Leukemia its promote mitogen-activated cascade.J. 274: 24965-24972Google LIF/LIF-R system then promotes extracellular signal-regulated kinases mediate CCAAT/enhancer binding β δ (18Belmonte Phillips B.W. Massiera Wdziekonski Saint-Marc Nichols Aubert Saeki K. Yuo Narumiya Activation CREB/ATF-1 beta delta preadipocytes.Mol. 15: 2037-2049Google Scholar), LIF ceases ongoing process. During course our investigations searching early-expressed, acid-responsive genes, encodes long form pentraxin, PTX3. Up now, known TSG14, member family extrahepatic sites, predominantly vascular cells heart skeletal muscle (19Breviario d'Aniello E.M. Golay Peri Bottazzi Bairoch Saccone Marzella Predazzi V. Rocchi Della Valle Dejana Mantovani Introna Interleukin-1-inducible Cloning related P component.J. 267: 22190-22197Google 20Introna Alles V.V. Castellano Picardi De Gioia Bottazzai Breviario Salmona Gregorio Dragani T.A. Srinivasan Blundell T.L. Hamilton sites.Blood. 1996; 87: 1862-1872Google 21Lee G.W. Lee T.H. Vilcek TSG-14, factor- IL-1-inducible protein, pentaxin proteins.J. Immunol. 150: 1804-1812Google We show herein that, despite fact becomes low, if any, induced dramatically exposure. models monogenic suggesting obesity.MATERIALS AND METHODSCell cultureCells were grown Dulbecco's modified Eagle's medium (DMEM) supplemented 8% fetal bovine serum, 200 U/ml penicillin, 50 μg/ml streptomycin (standard medium) confluence (hereafter referred Day 0). For differentiation, confluent shifted standard 5 nM 2 triiodothyronine (differentiation medium). experiments serum-free medium, first inoculated density 103 cells/cm2 washed 24 h later DMEM-Ham's F12 (50:50, v/v). Cells 4-F (22Gaillard Negrel Fetuin growth Ob17 preadipose hormone-supplemented medium.Biochim. Biophys. Acta. 1985; 846: 185-191Google Scholar) consisting (5 μg/ml), transferrin (10 submaxillary gland extract (SMGE) (2 fibroblast (25 ng/ml). At confluence, same absence SMGE, fetuin (100 μg/ml) hormone nM), (0.2 BRL 49653 (rosiglitazone, 10 ascorbate μM), selenite (20 nM). Confluent 3T3-L1 stimulated differentiate addition hormonal cocktail [0.5 mM 1-methyl-3-isobutylmethyl-xanthine, 0.5 μM dexamethasone, 170 (MDI)] days maintained thereafter only insulin. Typically, 6, more than 95% into adipocytes. Media changed every other day.Substractive hybridizationSubstractive RNA untreated acid-treated Ob1771 preadipocyte was use substraction kit (Stratagene, Netherlands) according manufacturer's instructions.Retrovirus transductionBOSC23 transfected viral DNA 50% FuGENE 6 reagent (Roche Biochemicals, France) described manufacturer. Forty-eight hours after transfection, supernatant harvested, centrifuged, filtered. Fifty percent transduced diluted one volume fresh presence Polybrene. On following day, split subjected neomycin (Sigma) selection (200 μg/ml). populations obtained 7–10 selection. After medium.Culture explantsAdipose epididymal fat pads dissected under sterile conditions, freed much possible blood capillaries, bathed DMEM containing antibiotics above. minced ∼100 mm3 pieces incubated 15 h.Stromal-vascular fractions already (23Cannon Nedergaard Cultures precursor brown brown-adipocyte-like lines.Methods Mol. 155: 213-224Google Scholar).Secretion mediaAdipose twice PBS (pH 7.4), once ITT (DMEM-Ham's insulin, triiodothyronine, transferring), h. Cytokines added 3 media collected. Secretion centrifuged min 2,000 rpm 4°C, concentrated either acetone precipitation centrifugation Amicon filters manufacturer (Millipore, France).Isolation analysis RNATotal extracted using Tri-Reagent™ (Euromedex, instructions analyzed Northern blot (24Amri E.Z. Bertrand Grimaldi Regulation Fatty inducers aP2 expression.J. Lipid Res. 1991; 32: 1449-1456Google Blots digital imaging (FujixBAS 1000).An amount 1 μg total RNA, digested DNaseI Biochemicals), RT-PCR analysis. RT reactions carried out pd(N)6 random hexamer units Moloney murine virus reverse transcriptase (Promega, supplied buffer, dNTPs (0.5 mM) RNase inhibitor (4 units) 37°C 25 μl reaction volume. Real-time PCR ABI Prism 7700 (Perkin-Elmer Applied Biosystem). each run, master mixture prepared ice 1× TaqMan Universal Master Mix Biosystem), 300 primer except (900 probe, (1:2, v/v) reverse-transcribed cDNA. conditions comprised 50°C, initial denaturation step 95° min, 40 cycles sec 95°C, 60°C. Gene quantified comparative-Ct method. Experiments triplicate data point. TATA-binding (TBP) used internal standard. oligonucleotides target interest, designed Primer Express software (Perkin-Elmer), (forward reverse): 5′-GACAACGAAATAGACAATGGACTTCA-3′, 5′-GCGAGTTCTCCAGCATGATGA-3′; TBP, 5′-ACCCTTCACCAATGACTCCTATG-3′, 5′-ATGATGACTGCAGCAAATCGC-3′. probes Fam/Tamra-labeled: 5′-CACCGAGGACCCCAC-3′ 5′-AGCTCTGGAATTGTACCGCAGCTTCAAAATA-3′.PlasmidsThe retroviral construct full-length cDNA derived pSG5-PTX3 cloned NotI site pAkvi-Bipe2 (gift Dr. Kristiansen, University Odense, Denmark).Oil red O stainingCulture dishes 7.4) fixed 3.7% formaldehyde h, followed staining Oil Red diluting stock solution g O) 100 ml isopropanol water (60:40, filtration. staining, plates photographed.Protein analysisWhole-cell extractsPlates Tris-HCl buffer 7.5) 150 NaCl. lysed 2.5% SDS, 10% glycerol, 6.8), dithiothreitol, β-glycerophosphate, NaF, 0.1 sodium orthovanadate, phenylmethylsulfonyl fluoride, complete protease (1:50 tablet per ml) Biochemicals). Lysis immediately boiling min. Lysates subsequently treated benzon nuclease (Merck, France). Whole-cell extracts stored −80°C. Protein concentrations determined Bradford method (Bio-Rad, France).Western blottingFifty hundred micrograms loaded lane. SDS-polyacrylamide gel electrophoresis, blotted onto polyvinylidene difluoride membranes (Amersham Biosciences, Bio-Rad semidry blotter. Equal loading/transfer confirmed Ponceau S membranes. Membranes blocked overnight TBS [10 Tris-Cl 7.5), NaCl] 0.1% Tween 20 (TBS-T) 5% nonfat dry milk. Incubation primary secondary antibodies TBS-T milk room temperature. incubation antibodies, TBS-T. Rat anti-human (25Bottazzi Vouret-Craviari Bastone Matteucci Spreafico Pausa D'Ettorre Gianazza Tagliabue Tedesco Multimer formation ligand recognition Similarities differences short pentraxins 1997; 272: 32817-32823Google cross-reacted Anti-TBP (Santa-Cruz) monitor equal loading. Secondary horseradish peroxidase-conjugated anti-rat (Jackson Laboratories) anti-rabbit (Promega). Enhanced chemiluminescence Biosciences) detection. Stripping done water.MaterialsCulture media, calf cytokines, geneticin Life Technologies, Inc. (France). Other chemicals purchased Sigma Aldrich Radioactive materials, priming kit, nylon Amersham Biosciences. kind gift (S. Michel U. Reicher, Galderma RandD, Sophia-Antipolis, Mice (C57BL/6J, ob/ob, db/db) Janvier (Le Genest Saint Isle, 6–8 weeks age. Animals killed cervical dislocation. All experimental protocols accordance recommendations French Accreditation Laboratory Animal Care.RESULTSIdentification deduced amino acid sequenceTo search early responsive acids, employed technique. Two libraries postconfluent (26Negrel Establishment pad ob/ob responds lipolytic hormones.Proc. Natl. Acad. Sci. USA. 1978; 75: 6054-6058Google exposed α-linolenate among various clones, clone contained fragment about 1.7 kb. Using this probe analysis, single transcript around kb observed very low (Fig. 1A). As was, contrast, treatment nearly sequenced, Blastn nucleic databases National Center Biotechnology Information look sequence homologies (27Altschul S.F. Madden Schaffer A.A. Zhang Z. Miller W. Lipman D.J. Gapped BLAST PSI-BLAST: generation database programs.Nucleic Acids 25: 3389-3402Google identical (GenBank™ accession number X83601). 42 kDa glycoprotein carboxy-terminal half shares high homology entire CRP which (28Pepys M.B. Baltz M.L. Acute special reference (pentaxins) protein.Adv. 1983; 34: 141-212Google 29Gewurz X.H. Lint T.F. Structure function pentraxins.Curr. 7: 54-64Google 30Goodman A.R. Cardozo Abagyan Altmeyer Wisniewski H.G. Long pentraxins: emerging group diverse functions.Cytokine Growth Factor Rev. 191-202Google whereas NH2-terminal part does any proteins.Expression differentiationWhite tissues express fraction, respectively. contrast peroxisome proliferator-activated γ (PPARγ) 1B, shown). led us examine (31Green Kehinde O. Spontaneous heritable changes leading conversion 3T3 cells.Cell. 1976; 105-113Google (32Green Meuth established pre-adipose culture.Cell. 1974; 3: 127-133Google Fig. 2A, growing became cells, decreased thereafter. Its kinetics appeared opposite those PPARγ, characterize terminal process (33Cook K.S. Hunt C.R. Developmentally mRNAs 3T3-adipocytes: transcriptional control.J. 100: 514-520Google 34Rosen E.D. adipogenesis.Annu. 16: 145-171Google Identical results 2A) (data differentiation-related event message induction 2A), weak continuous 3T3-C2 shown), nonadipocyte (35Green culture. II. Factors affecting conversion.Cell. 1975; 19-27Google Consistent interpretation, order accelerate process, disappearance faster pattern 2B). result excluded nonspecific effects components, including favored existence differentiation-specific event. Thus, finding family, prompted role, differentiation.Fig. 2PTX3 serum-containing medium. A: had serum-supplemented different intervals relative confluence. (+MDI) (−MDI) insulin) days. B: Total Twenty sample Materials Methods. representative two independent experiments.View Large Image Figure ViewerDownload (PPT)Differentiation stable overexpressing PTX3Overexpression accomplished infection 3control infected empty vector endogenous expected size coding signal 4 corresponding 3A, B). unchanged throughout period, similar 3A). 3B C, PTX3-overexpressing unimpaired compared assessed microscope examination markers, excluding However, owing involvement postulated defense against bacterial infections (36Muller Doni Torri Landmann Circulating correlate severity critically ill patients.Crit. Care Med. 29: 1404-1407Google 37Luchetti M.M. Piccinini Pomponio Fratini Fraticelli Sambo Di Loreto Gabrielli Expression rheumatoid arthritis (RA).Clin. Exp. 119: 196-202Google 38Peri Corradi Iacuitti Signorini Avanzini Pizzetti Maggioni A.P. Moccetti Metra Cas L.D. Ghezzi Sipe J.D. Re Olivetti Latini prototypical indicator myocardial infarction humans.Circulation. 102: 636-641Google studies response TNFα, expression, termed highly promoters (39Basile Sica Garrido Characterization promoter Role NF-kappaB interleukin-1beta regulation.J. 8172-8178Google 40Altmeyer Klampfer Goodman Promoter structure TSG-14 encoding factor/interleukin-1-inducible protein.J. 270: 25584-25590Google Scholar).Fig. 3Ectopic preadipocytes. 3T3-F442A-vector 3T3-F442A-PTX3 transfectants Days 0 13, Western blot. C: stained photographed. (PPT)Modulation expressionUpon exposure present nanomolar range concentrations, occurred within Peak reached somewhat 4). Interestingly, inactive up nM, lipopolysaccharide (LPS) active ng/ml. able respond 0.01 Likewise, enhanced treatment. clearly detectable 5). Collectively, emphasize secreted examined animals.Fig. 4Effect 7 (A) periods (TNFα), (B) (IL-6) indicated, ng/ml (LPS). three (PPT)Fig. 5PTX3 secr