Chandrasekar IL-17 Stimulates Mmp-1 Expression

 doi:10.1152/ajpheart.00928.2007 293:3356-3365, 2007. First published Oct 5, 2007; Am J Physiol Heart Circ Physiol Bjorn Steffensen, Matthew Vincenti, Jeffrey L. Barnes and Bysani Chandrasekar Dolores M. Cortez, Marc D. Feldman, Srinivas Mummidi, Anthony J. Valente, You might find this additional information useful... 34 articles, 13 of which you can access free at: This article cites 3 other HighWire hosted articles: Th
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   doi:10.1152/ajpheart.00928.2007 293:3356-3365, 2007. First published Oct 5, 2007;  Am J Physiol Heart Circ Physiol Bjorn Steffensen, Matthew Vincenti, Jeffrey L. Barnes and Bysani Chandrasekar Dolores M. Cortez, Marc D. Feldman, Srinivas Mummidi, Anthony J. Valente,   You might find this additional information useful... 34 articles, 13 of which you can access free at: This article cites other HighWire hosted articles: This article has been cited by [PDF] [Full Text] [Abstract] , May 1, 2008; 294 (5): H2078-H2087.  Am J Physiol Heart Circ Physiol K. Venkatachalam, S. Mummidi, D. M. Cortez, S. D. Prabhu, A. J. Valente and B. Chandrasekar  expression in primary mouse cardiac fibroblastsResveratrol inhibits high glucose-induced PI3K/Akt/ERK-dependent interleukin-17  [PDF] [Full Text] [Abstract] , January 1, 2009; 40 (1): 4-12.  Am. J. Res pir. Cell Mol. Biol. B. A. Mercer, A. M. Wallace, C. E. Brinckerhoff and J. M. D'Armiento Identification of a Cigarette Smoke-Responsive Region in the Distal MMP-1 Promoter  [PDF] [Full Text] [Abstract] , February 24, 2009; 2 (59): ra8-ra8. Sci. Signal. and S. L. Gaffen F. Shen, N. Li, P. Gade, D. V. Kalvakolanu, T. Weibley, B. Doble, J. R. Woodgett, T. D. Wood  Regulatory 2 DomainIL-17 Receptor Signaling Inhibits C/EBP{beta} by Sequential Phosphorylation of the including high-resolution figures, can be found at: Updated information and services can be found at:  AJP - Heart and Circulatory Physiology about Additional material and information information is current as of March 11, 2009 . 0363-6135, ESSN: 1522-1539. Visit our website at Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2005 by the American Physiological Society. intact animal to the cellular, subcellular, and molecular levels. It is published 12 times a year (monthly) by the Americanlymphatics, including experimental and theoretical studies of cardiovascular function at all levels of organization ranging from the publishes srcinal investigations on the physiology of the heart, blood vessels, and  AJP - Heart and Circulatory Physiology   onM ar  c h 1 1  ,2  0  0  9  a j   ph  e ar  t  . ph  y  s i   ol   o g y . or  gD  ownl   o a d  e d f  r  om   IL-17 stimulates MMP-1 expression in primary human cardiac fibroblasts viap38 MAPK- and ERK1/2-dependent C/EBP-  , NF-  B, and AP-1 activation Dolores M. Cortez, 1 Marc D. Feldman, 1,2 Srinivas Mummidi, 1,2 Anthony J. Valente, 1 Bjorn Steffensen, 3 Matthew Vincenti, 4 Jeffrey L. Barnes, 1,2 and Bysani Chandrasekar 1,2 1  Department of Veterans Affairs South Texas Veterans Health Care System, and Departments of   2  Medicine and   3 Periodonticsand Biochemistry, University of Texas Health Science Center, San Antonio, Texas; and   4  Department of Medicine, Dartmouth Medical School, Lebanon, New Hampshire Submitted 10 August 2007; accepted in final form 4 October 2007 Cortez DM, Feldman MD, Mummidi S, Valente AJ, SteffensenB, Vincenti M, Barnes JL, Chandrasekar B.  IL-17 stimulatesMMP-1 expression in primary human cardiac fibroblasts via p38MAPK- and ERK1/2-dependent C/EBP-  , NF-  B, and AP-1 activa-tion.  Am J Physiol Heart Circ Physiol  293: H3356–H3365, 2007. Firstpublished October 5, 2007; doi:10.1152/ajpheart.00928.2007.—Matrix metalloproteinases (MMPs) degrade collagen and mediatetissue remodeling. The novel cytokine IL-17 is expressed duringvarious inflammatory conditions and modulates MMP expression. Weinvestigated the effect of IL-17 on MMP-1 expression in primaryhuman cardiac fibroblasts (HCF) and delineated the signaling path-ways involved. HCF were treated with recombinant human IL-17.MMP-1 expression was analyzed by Northern blotting, RT-quantitative PCR, Western blotting, and ELISA; transcriptional in-duction and transcription factor binding by EMSA, ELISA, andreporter assay; and p38 MAPK and ERK1/2 activation by proteinkinase assays and Western blotting. Signal transduction pathwayswere investigated using pharmacological inhibitors, small interferingRNA (siRNA), and adenoviral dominant-negative expression vectors.IL-17 stimulated MMP-1 gene transcription, net mRNA levels, pro-tein, and promoter-reporter activity in HCF. This response wasblocked by IL-17 receptor-Fc chimera and IL-17 receptor antibodies,but not by IL-6, TNF-  , or IL-1  antibodies. IL-17-stimulated type Icollagenase activity was inhibited by the MMP inhibitor GM-6001and by siRNA-mediated MMP-1 knockdown. IL-17 stimulated acti-vator protein-1 [AP-1 (c-Fos, c-Jun, and Fra-1)], NF-  B (p50 andp65), and CCAAT enhancer-binding protein (C/EBP)-  DNA bindingand reporter gene activities, effects attenuated by antisense oligonu-cleotides, siRNA-mediated knockdown, or expression of dominant-negative signaling proteins. Inhibition of AP-1, NF-  B, or C/EBPactivation attenuated IL-17-stimulated MMP-1 expression. IL-17 in-duced p38 MAPK and ERK1/2 activation, and inhibition by SB-203580 and PD-98059 blunted IL-17-mediated transcription factoractivation and MMP-1 expression. Our data indicate that IL-17induces MMP-1 in human cardiac fibroblasts directly via p38 MAPK-and ERK-dependent AP-1, NF-  B, and C/EBP-   activation andsuggest that IL-17 may play a critical role in myocardial remodeling.cytokines; interleukins; matrix metalloproteinases; fibrosis EXTRACELLULAR MATRIX  (ECM) turnover in the normal heart is atightly regulated process. The alteration in the delicate balancebetween matrix metalloproteinases (MMPs) and their tissueinhibitors (TIMPs) during myocardial injury and inflammationresults in enhanced ECM degradation and remodeling (29, 30).MMPs belong to a family of related, but structurally distinct,zinc-dependent proteases that degrade various ECM proteins,including collagens, gelatins, fibronectin, and laminins (14).MMP-1 (EC, or collagenase type I, is the firstidentified metalloproteinase that degrades interstitial collagens(collagens I, II, III, and VII) in the myocardium (14).Sustained production of inflammatory cytokines plays acentral role in the initiation and progression of left ventricularhypertrophy to failure (11). Various cytokines have beenshown to regulate MMP-1 expression at transcriptional andposttranscriptional levels (5, 23). IL-17, a recently discoveredfamily of proinflammatory cytokines secreted mainly by asubset of T (Th17) cells, consists of six ligands (IL-17A, B, C,D, E, and F) that signal through five receptors (IL-17RA, B, C,D, and E) (4). IL-17 family members show little to no homol-ogy with other ILs and, therefore, constitute a family of theirown (4). Enhanced expression of IL-17 has been reported invarious models of inflammation, including rheumatoid arthri-tis, periodontitis, asthma, and organ rejection (4), and a causalrole for IL-17 has been demonstrated in experimental autoim-mune myocarditis (21, 28). However, a role for IL-17 inmyocardial ischemic injury, hypertrophy, and remodeling hasnot been described. Since remodeling is characterized byhypertrophy and fibrosis and since fibroblasts play a criticalrole in fibrosis through expression of MMPs, we investigatedwhether IL-17 regulates MMP-1 expression in primary humancardiac fibroblasts (HCF). MATERIALS AND METHODS  Materials.  Recombinant human IL-6 (catalog no. 206-IL-010) andIL-17 (catalog no. 317-IL-050), neutralizing antibodies against IL-6,IL-1  , and TNF-  , and normal goat IgG (Ab 108-C) were purchasedfrom R & D Systems (Minneapolis, MN). We previously reported theeffectiveness of the anti-cytokine neutralizing antibodies (6, 19).Anti-p38, phosphorylated p38 [PhosphoPlus p38 MAP kinase(Thr 180  /Tyr 182 ) antibody kit], ERK1/2 (catalog no. 9102), phosphor-ylated ERK1/2 (catalog no. 9101S), and anti-phosphorylated CCAATenhancer-binding protein (C/EBP)-   (catalog no. 3084S) antibodieswere obtained from Cell Signaling Technology (Beverly, MA).Cycloheximide [InSolution cycloheximide (CHX)], a protein synthe-sis inhibitor (10  g/ml in DMSO); SB-203580, a p38 MAPK inhibitor(1  M in DMSO for 30 min); PD-98059, an ERK1/2 inhibitor (10  Min DMSO for 1 h); and DMSO were purchased from EMD Bio-sciences (San Diego, CA). GM-6001, a nonspecific hydroxamic acid-based MMP inhibitor with potent inhibitory activity against collage-nase, gelatinases, and stromelysin (15) (10  M in DMSO for 15 min),was purchased from Upstate/Chemicon (Temecula, CA). Actinomy- Address for reprint requests and other correspondence: B. Chandrasekar,Medicine/Cardiology, The Univ. of Texas Health Science Center, 7703 FloydCurl Dr., San Antonio, TX 78229-3900 (e-mail: costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “ advertisement  ”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.  Am J Physiol Heart Circ Physiol  293: H3356–H3365, 2007.First published October 5, 2007; doi:10.1152/ajpheart.00928.2007.http://www.ajpheart.orgH3356   onM ar  c h 1 1  ,2  0  0  9  a j   ph  e ar  t  . ph  y  s i   ol   o g y . or  gD  ownl   o a d  e d f  r  om   cin D (ActD), an RNA synthesis inhibitor (2.5   g/ml in DMSO);  -tubulin polyclonal antibodies; and all other chemicals were pur-chased from Sigma-Aldrich (St. Louis, MO). Cell culture.  HCF (catalog no. 6300, ScienCell Research Labora-tories, San Diego, CA) were characterized by an immunofluorescentmethod using antibody to fibronectin (manufacturer’s technical datasheet). HCF were grown in fibroblast medium (FM) supplied by themanufacturer and supplemented with 2% FBS, fibroblast growthsupplement, and antibiotics (complete medium). At 70% confluency,the complete medium was replaced with FM containing 0.5% BSA.After overnight incubation (quiescent cells), IL-17 was added, and thecells were cultured for the indicated time periods. At the end of theexperimental period, culture supernatants were collected and snapfrozen. Cells were harvested, snap frozen, and stored at  80°C.Since IL-17 is a proinflammatory cytokine and induces the expres-sion of other cytokines (27) that are known to stimulate MMP-1expression (5, 23), we determined whether IL-17-mediated MMP-1expression is dependent on IL-1  , IL-6, or TNF-  . HCF were incu-bated with IL-1  , IL-6, or TNF-   neutralizing antibodies (10   g/mlfor 1 h; R & D Systems) before addition of IL-17. Normal goat/mouseIgG served as a control.  Adenoviral vectors and RNA interference.  Recombinant, replica-tion-deficient adenoviral vectors encoding green fluorescent protein(Ad-CMV-GFP), dominant-negative (dn) IKK-  , dnp65, anddnI  B-   (S32A/S36A) have been previously described (18). Ad-CMV-dnc-Jun was purchased from Vector Biolabs. Cells were in-fected at ambient temperature with adenoviruses in PBS at a multi-plicity of infection (MOI) of 100. After 1 h, the PBS solutioncontaining adenovirus was replaced with FM containing 0.5% BSA.Assays were carried out 48 h later, and knockdown of proteins wasconfirmed by Western blotting. C/EBP-   small interfering RNA(siRNA; identification no. 114496, catalog no. 16708) was purchasedfrom Ambion (Austin, TX). Cells at 70% confluency were transfectedwith siRNA (100 nM) using the Nucleofection kit (catalog no.VPI-1002) provided by Amaxa (Gaithersburg, MD). Among the fiverecommended programs, the T-16 program gave optimal transfectionefficiency (38%) with only 9% cell death. After overnight culture inmedium containing 0.5% BSA, dead cells were removed. Controlnegative siRNA (sense, 5  -CUC GGC GUU UCA UCU GUGGdTdT) served as a control.  MMP-1 promoter-reporter assays.  A 4,386-bp fragment (  4334/   52) of the 5  -flanking region of the  MMP1  gene was amplified fromhuman genomic DNA (catalog no. G3041, Promega) using the fol-lowing primers: 5  -acg cgt AGA TGT AAG AGC TGG AAA GGACGG-3   (sense) and 5  -ctc gag TCA GTG CAA GGT AAG TGATGG CTT C-3   (antisense). The sense primer contained an  Mlu  Irestriction site at the 5  end, and the antisense primer contained an  Xho I restriction site (lower case). The PCR product was cloned intopCR2.1-TOPO and subcloned into the pGL3-basic reporter vector inthe same restriction sites. The identity of the PCR product wasconfirmed by sequencing on both strands. To determine the roleof C/EBP, NF-  B, and AP-1, we generated deletion constructs lackingC/EBP, NF-  B, or AP-1 (  2685/   52, 5  -acg cgt AGA TGC TCCCAG AGG AAA C-3  for C/EBP;  1524/   52, 5  -acg cgt CAG GAATCC ATA AGG GGA GG  3  for C/EBP and NF-  B; and  62/   52,acg cgt ACC TCT GGC TTT CTG GAA GG-3  for C/EBP, NF-  B,and AP-1) and the antisense primer described above. mRNA expression: Northern blotting and real-time quantitativePCR.  DNA-free total RNA was extracted using the RNAqueous-4PCR kit (Ambion). RNA quality was assessed by capillary elec-trophoresis using the Agilent 2100 Bioanalyzer (Agilent Technolo-gies, Palo Alto, CA). All RNA samples used for quantitative PCR hadRNA integrity numbers   9.1 (on a scale of 1–10), as assigned bydefault parameters of the Expert 2100 Bioanalyzer software package(version 2.02). IL-17 receptor (IL-17R) type A (GenBank accessionno. NM_014339) expression was analyzed by RT-PCR using two setsof primers [5  -GAT GAC AGC TGG ATT CAC C-3   (sense) and5  -CTC ATA TTC CTG GTC AGG G-3   (antisense) for  set 1  and5  -GTC TGG TTA TCG TCT ATC C-3   (sense) and 5  -CAA ACTCCT GAC CTC AGA G-3  (antisense) for  set 2 ], which resulted in a300-bp amplification product.   -Actin [GenBank accession no.NM_001101; 668-bp amplification product, 5  -CGT GCG TGA CATTAA GGA GA-3   (sense) and 5  -CAC CTT CAC CGT TCC AGTTT-3  (antisense)] served as an internal control. Northern blot analysiswas carried out as previously described (6). MMP-1 cDNA (GenBank accession no. NM_002421.2) was amplified from reverse-transcribedHCF RNA by RT-PCR using the sense primer 5  -ATT CTA CTGATA TCG GGG CTT TGA-3  and the antisense primer 5  -ATG TCCTTG GGG TAT CCG TGT AG-3  . Expression of 28S rRNA was usedas an internal control.MMP-1 mRNA expression was also analyzed by real-time quanti-tative PCR [5  -CAT TGA TGG CAT CCA AGC C-3   (sense) and5  -GGC TGG ACA GGA TTT TGG G-3  (antisense)] using Quanti-Tect SYBR-Green Probe RT-PCR kit (Qiagen). Each sample wasassayed in triplicate. For relative quantification, the cycle threshold(C t ) method {ratio    2    [C t (MMP-1)    C t (GAPDH)]} was used,with GAPDH as a control. For copy number determination, a calibra-tion curve was obtained using serial dilutions of a linearized GAPDHcDNA with the GAPDH primer pair [5  -GAA GGT GAA GGT CGGAGT C-3  (forward) and 5  -GAA GAT GGT GAT GGG ATT TC-3  (reverse)].  MMP-1 levels.  MMP-1 levels in culture supernatants were ana-lyzed using an ELISA kit according to the manufacturer’s instructions(Amersham Biosciences). Western blot analysis.  ECM proteins (MMP-1, -2, -3, -8, -9, -10,and -13 and TIMP-1, -2, and -4) in the culture supernatants weredetermined by an antibody array (RayBio MMP antibody array 1,catalog no. AAH-MMP-1, RayBiotech, Norcross, GA) following themanufacturer’s protocol and quantified by densitometry (6). Proteinconcentrations were determined using the bicinchoninic acid method(Pierce, Rockford, IL).MMP-1 levels were confirmed by Western blotting (6, 18, 19).Proteins were separated by 10% PAGE and electroblotted onto aHybond-P polyvinylidene difluoride membrane (Amersham Bio-sciences). The membrane was incubated with rabbit anti-humanMMP-1 (1:2,000 dilution; Chemicon International, Temecula, CA)and subsequently with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (New England Biolabs, Beverly, MA). Theimmunoreactive bands were detected by chemiluminescence (ECLPlus, GE Healthcare). The blots were stripped and reprobed with  -tubulin antibodies to confirm equal protein loading.  EMSA, ELISA, and reporter assays.  NF-  B and AP-1 protein-DNAcomplex formation was assessed by EMSA (6, 18, 19) using HCFnuclear extracts and double-stranded consensus DNA for C/EBP(5  -TGC AGA TTG CGC AAT CTG CA-3  ), NF-  B (5  -AGT TGAGGG GAC TTT CCC AGG C-3  ), AP-1 (5  -CGC TTG ATG ACTCAG CCG GAA-3  ), mutant C/EBP (5  -TGC AGA GAC TAG TCTCTG CA-3  ), mutant NF-  B (5  -AGT TGA GGC GAC TTT CCCAGG C-3), and mutant AP-1 (5  -CGC TTG ATG ACT TGG CCGGAA-3  ; Santa Cruz Biotechnology). Activation of transcription fac-tors (TF) was confirmed by ELISA (20, 21, 24) [TransAM TF ELISAkits 43296 (NF-  B), 44196 (C/EBP), and 44296 (AP-1), ActiveMotif, Carlsbad, CA].TF activation was also analyzed by reporter assays using adenovi-ral transduction of NF-  B (Ad-NF-  B-Luc, 50 MOI; kindly providedby Dr. John F. Engelhardt) and has been previously described (19,25). Similarly, HCF were infected with Ad.AP-1-Luc (catalog no.1670, Vector Biolabs, Philadelphia, PA) reporter vector. Ad-MCS-Luc (Vector Biolabs) served as a control. Ad-  -Gal (50 MOI; VectorBiolabs) served as an internal control.   -Gal activity in cell extractswas determined using luminescent   -Gal detection kit II (BD Bio-sciences), and the results are expressed in relative light units as a ratioof firefly luciferase to  -Gal activity. C/EBP activation was confirmedusing a C/EBP reporter vector (2  C/EBP-Luc) that contains two H3357 IL-17 STIMULATES CARDIAC FIBROBLAST MIGRATION  AJP-Heart Circ Physiol  ã  VOL 293  ã  DECEMBER 2007  ã   onM ar  c h 1 1  ,2  0  0  9  a j   ph  e ar  t  . ph  y  s i   ol   o g y . or  gD  ownl   o a d  e d f  r  om   canonical C/EBP binding sites (kindly provided by Peter Johnson,National Cancer Institute, Frederick, MD). HCF were transfected withC/EBP-Luc (3   g) using the Nucleofection kit. Cells were cotrans-fected with Ad-  -Gal to normalize for variations in transfectionefficiency. Cells were then treated with IL-17 for 12 h. Fireflyluciferase and   -Gal activities were analyzed as described above.Neither pharmacological inhibitors, dominant-negative expressionvectors, nor siRNA affected cell viability for the indicated studyperiod.  Immune complex protein kinase assays.  p38 MAPK and ERKactivities were determined as described previously (6, 18, 19). Briefly,a commercially available colorimetric assay kit [p38 MAP kinaseassay kit (nonradioactive), Cell Signaling Technology] was used todetermine p38 MAPK activity in whole cell homogenates. The assayis based on phosphorylation of activating TF (ATF)-2 by the immu-noprecipitated phosphorylated p38 MAPK. ERK activity was deter-mined in whole cell homogenates using a commercially availablecolorimetric assay kit (ERK, p44/42 MAP kinase assay kit, CellSignaling Technology). Cell migration.  HCF migration was quantified as previously de-scribed (10) using Transwell chambers with 3-  m polycarbonatemembrane (Corning) precoated with 100   g/ml type I collagen orBSA on both sides of the membrane. HCF were trypsinized andsuspended in FM medium containing 0.5% BSA, and 1 ml containing2.0  10 5 cells/ml was layered on the coated insert filters. Cells werestimulated with IL-17 (10 ng/ml). The lower chamber contained IL-17at the same concentration. Plates were incubated at 37°C for 24 h.Membranes were washed with PBS, and noninvading cells on theupper surface were removed using cotton swabs. Cells migrating tothe lower surface of the membrane were determined at 540-nmabsorbance using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide assay.To determine the role of MMP-1 in IL-17-mediated cell migration,we treated HCF with GM-6001 (10   M in DMSO for 15 min) orMMP-1 siRNA (target sequence corresponding to nucleotides 153–171 downstream from starting codon; 100 nM for 48 h) beforeaddition of IL-17. siRNA against this target sequence has been shownto knock down MMP-1 mRNA and protein expression by  90% (34).siRNA, which will not target any genes in the human genome(5  -UUC UCC GAA CGU GUC ACG UdTdT-3  ; catalog no.1022076, Qiagen), and green fluorescent protein (GFP) siRNA (sense,5  -pGGCUACGUCCAGGAGCGCACC-3  ) served as controls.MMP-1 knockdown was confirmed by Western blotting. Cell death assays.  Quiescent HCF were treated with IL-17 (  100ng/ml) for  48 h. Cell death was analyzed by quantitation of mono-and oligonucleosomes in the cytoplasmic fraction of cell lysates by anELISA (Cell Death Detection ELISA PLUS kit, Roche Diagnostics) (6). Statistical analysis.  Values are means  SE. For statistical analy-sis, we used ANOVA followed by an appropriate post hoc multiplecomparison test (Tukey’s method). Data were considered statisticallysignificant at  P  0.05. RESULTS  IL-17 stimulates MMP-1 mRNA expression in human car-diac fibroblasts.  Since the proinflammatory cytokine IL-17signals via IL-17RA, we first used RT-PCR to determinewhether HCF express IL-17RA. Our results demonstrated thatHCF express IL-17RA under basal conditions (data notshown). We next investigated whether IL-17 can induceMMP-1 mRNA expression in HCF. HCF were treated withIL-17 (0–100   g/ml) for 12 h and analyzed by Northernblotting and densitometry. HCF express MMP-1 mRNA at lowlevels under basal conditions (Fig. 1  A ), and treatment withIL-17 for 12 h significantly increased MMP-1 expression, withpeak levels obtained with 10 ng/ml. Therefore, in all subse-quent experiments, IL-17 was used at 10 ng/ml. IL-17 inducedMMP-1 expression in a time-dependent manner, with peak levels of mRNA observed at 12 h (Fig. 1  B ). MMP-1 levelsremained at these high levels throughout the 48-h study period.IL-17-induced MMP-1 expression was also investigated byRT-quantitative PCR (qPCR). The specificity of the responseto IL-17 was verified by incubation of HCF with IL-17 orIL-17R neutralizing antibodies (10   g/ml) for 1 h beforeaddition of IL-17. Our results indicate that the potent inductionof MMP-1 mRNA expression by IL-17 can be blocked byIL-17 or IL-17R neutralizing antibodies (Fig. 1 C  ). These re-sults demonstrate that  1 ) HCF express IL-17RA,  2 ) IL-17 is apotent inducer of MMP-1 expression, and  3 ) IL-17 inducesMMP-1 expression in a time- and dose-dependent manner(Fig. 1).  IL-17 stimulates MMP-1 protein expression.  We next inves-tigated whether IL-17 also stimulates MMP-1 protein expres-sion. Western blot analysis of whole cell homogenates usingantibodies that recognize latent and active forms of MMP-1revealed that, under basal conditions, HCF expressed latent andactive forms of MMP-1 (Fig. 2  A ), whereas treatment withIL-17 resulted in a modest increase in the latent form but a Fig. 1. IL-17 stimulates matrix metalloproteinase (MMP)-1 expression.  A : Northern blot of MMP-1 mRNA expression in DNA-free total RNA isolatedfrom quiescent human cardiac fibroblasts (HCF) treated with IL-17 for 12 h.28S rRNA served as loading control.  B : kinetics of IL-17-mediated MMP-1expression shown in Northern blot of MMP-1 mRNA expression in quiescentHCF treated with IL-17 (10 ng/ml) for  48 h. C, control.  C  : RT-quantitativePCR (qPCR) analysis of MMP-1 mRNA expression in quiescent HCF treatedwith IL-17 or IL-17 receptor (IL-17R) neutralizing antibodies for 1 h before12 h of treatment with IL-17 (10 ng/ml). Normal goat/mouse IgG and GAPDHserved as internal controls. IL-17-mediated MMP-1 expression is blocked byIL-17 or IL-17 receptor (IL-17R) neutralizing antibodies. * P    0.01 vs.untreated (ANOVA). † P  0.01 vs. IL-17 (ANOVA). H3358  IL-17 STIMULATES CARDIAC FIBROBLAST MIGRATION  AJP-Heart Circ Physiol  ã  VOL 293  ã  DECEMBER 2007  ã   onM ar  c h 1 1  ,2  0  0  9  a j   ph  e ar  t  . ph  y  s i   ol   o g y . or  gD  ownl   o a d  e d f  r  om 
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