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Am J Physiol Heart Circ Physiol 293: H3356-H3365, 2007. First published October 5, 2007; doi:10.1152/ajpheart.00928.2007
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IL-17 stimulates MMP-1 expression in primary human cardiac fibroblasts via p38 MAPK- and ERK1/2-dependent C/EBP-β, NF-{kappa}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 Chandrasekar1,2

1Department of Veterans Affairs South Texas Veterans Health Care System, and Departments of 2Medicine and 3Periodontics and Biochemistry, University of Texas Health Science Center, San Antonio, Texas; and 4Department of Medicine, Dartmouth Medical School, Lebanon, New Hampshire

Submitted 10 August 2007 ; accepted in final form 4 October 2007


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Matrix metalloproteinases (MMPs) degrade collagen and mediate tissue remodeling. The novel cytokine IL-17 is expressed during various inflammatory conditions and modulates MMP expression. We investigated the effect of IL-17 on MMP-1 expression in primary human cardiac fibroblasts (HCF) and delineated the signaling pathways 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 induction and transcription factor binding by EMSA, ELISA, and reporter assay; and p38 MAPK and ERK1/2 activation by protein kinase assays and Western blotting. Signal transduction pathways were investigated using pharmacological inhibitors, small interfering RNA (siRNA), and adenoviral dominant-negative expression vectors. IL-17 stimulated MMP-1 gene transcription, net mRNA levels, protein, and promoter-reporter activity in HCF. This response was blocked by IL-17 receptor-Fc chimera and IL-17 receptor antibodies, but not by IL-6, TNF-{alpha}, or IL-1β antibodies. IL-17-stimulated type I collagenase activity was inhibited by the MMP inhibitor GM-6001 and by siRNA-mediated MMP-1 knockdown. IL-17 stimulated activator protein-1 [AP-1 (c-Fos, c-Jun, and Fra-1)], NF-{kappa}B (p50 and p65), and CCAAT enhancer-binding protein (C/EBP)-β DNA binding and reporter gene activities, effects attenuated by antisense oligonucleotides, siRNA-mediated knockdown, or expression of dominant-negative signaling proteins. Inhibition of AP-1, NF-{kappa}B, or C/EBP activation attenuated IL-17-stimulated MMP-1 expression. IL-17 induced p38 MAPK and ERK1/2 activation, and inhibition by SB-203580 and PD-98059 blunted IL-17-mediated transcription factor activation and MMP-1 expression. Our data indicate that IL-17 induces MMP-1 in human cardiac fibroblasts directly via p38 MAPK- and ERK-dependent AP-1, NF-{kappa}B, and C/EBP-β activation and suggest 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 a tightly regulated process. The alteration in the delicate balance between matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) during myocardial injury and inflammation results 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 3.4.24.7 [EC] ), or collagenase type I, is the first identified metalloproteinase that degrades interstitial collagens (collagens I, II, III, and VII) in the myocardium (14).

Sustained production of inflammatory cytokines plays a central role in the initiation and progression of left ventricular hypertrophy to failure (11). Various cytokines have been shown to regulate MMP-1 expression at transcriptional and posttranscriptional levels (5, 23). IL-17, a recently discovered family of proinflammatory cytokines secreted mainly by a subset 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 homology with other ILs and, therefore, constitute a family of their own (4). Enhanced expression of IL-17 has been reported in various models of inflammation, including rheumatoid arthritis, periodontitis, asthma, and organ rejection (4), and a causal role for IL-17 has been demonstrated in experimental autoimmune myocarditis (21, 28). However, a role for IL-17 in myocardial ischemic injury, hypertrophy, and remodeling has not been described. Since remodeling is characterized by hypertrophy and fibrosis and since fibroblasts play a critical role in fibrosis through expression of MMPs, we investigated whether IL-17 regulates MMP-1 expression in primary human cardiac fibroblasts (HCF).


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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 REFERENCES
 
Materials. Recombinant human IL-6 (catalog no. 206-IL-010) and IL-17 (catalog no. 317-IL-050), neutralizing antibodies against IL-6, IL-1β, and TNF-{alpha}, and normal goat IgG (Ab 108-C) were purchased from R & D Systems (Minneapolis, MN). We previously reported the effectiveness of the anti-cytokine neutralizing antibodies (6, 19). Anti-p38, phosphorylated p38 [PhosphoPlus p38 MAP kinase (Thr180/Tyr182) antibody kit], ERK1/2 (catalog no. 9102), phosphorylated ERK1/2 (catalog no. 9101S), and anti-phosphorylated CCAAT enhancer-binding protein (C/EBP)-β (catalog no. 3084S) antibodies were obtained from Cell Signaling Technology (Beverly, MA). Cycloheximide [InSolution cycloheximide (CHX)], a protein synthesis inhibitor (10 µg/ml in DMSO); SB-203580, a p38 MAPK inhibitor (1 µM in DMSO for 30 min); PD-98059, an ERK1/2 inhibitor (10 µM in DMSO for 1 h); and DMSO were purchased from EMD Biosciences (San Diego, CA). GM-6001, a nonspecific hydroxamic acid-based MMP inhibitor with potent inhibitory activity against collagenase, gelatinases, and stromelysin (15) (10 µM in DMSO for 15 min), was purchased from Upstate/Chemicon (Temecula, CA). Actinomycin D (ActD), an RNA synthesis inhibitor (2.5 µg/ml in DMSO); {alpha}-tubulin polyclonal antibodies; and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Cell culture. HCF (catalog no. 6300, ScienCell Research Laboratories, San Diego, CA) were characterized by an immunofluorescent method using antibody to fibronectin (manufacturer's technical data sheet). HCF were grown in fibroblast medium (FM) supplied by the manufacturer and supplemented with 2% FBS, fibroblast growth supplement, 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 the cells were cultured for the indicated time periods. At the end of the experimental period, culture supernatants were collected and snap frozen. Cells were harvested, snap frozen, and stored at –80°C.

Since IL-17 is a proinflammatory cytokine and induces the expression of other cytokines (27) that are known to stimulate MMP-1 expression (5, 23), we determined whether IL-17-mediated MMP-1 expression is dependent on IL-1β, IL-6, or TNF-{alpha}. HCF were incubated with IL-1β, IL-6, or TNF-{alpha} neutralizing antibodies (10 µg/ml for 1 h; R & D Systems) before addition of IL-17. Normal goat/mouse IgG served as a control.

Adenoviral vectors and RNA interference. Recombinant, replication-deficient adenoviral vectors encoding green fluorescent protein (Ad-CMV-GFP), dominant-negative (dn) IKK-β, dnp65, and dnI{kappa}B-{alpha} (S32A/S36A) have been previously described (18). Ad-CMV-dnc-Jun was purchased from Vector Biolabs. Cells were infected at ambient temperature with adenoviruses in PBS at a multiplicity of infection (MOI) of 100. After 1 h, the PBS solution containing adenovirus was replaced with FM containing 0.5% BSA. Assays were carried out 48 h later, and knockdown of proteins was confirmed by Western blotting. C/EBP-β small interfering RNA (siRNA; identification no. 114496, catalog no. 16708) was purchased from Ambion (Austin, TX). Cells at 70% confluency were transfected with siRNA (100 nM) using the Nucleofection kit (catalog no. VPI-1002) provided by Amaxa (Gaithersburg, MD). Among the five recommended programs, the T-16 program gave optimal transfection efficiency (38%) with only 9% cell death. After overnight culture in medium containing 0.5% BSA, dead cells were removed. Control negative siRNA (sense, 5'-CUC GGC GUU UCA UCU GUG GdTdT) 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 from human genomic DNA (catalog no. G3041, Promega) using the following primers: 5'-acg cgt AGA TGT AAG AGC TGG AAA GGA CGG-3' (sense) and 5'-ctc gag TCA GTG CAA GGT AAG TGA TGG CTT C-3' (antisense). The sense primer contained an Mlu I restriction site at the 5' end, and the antisense primer contained an Xho I restriction site (lower case). The PCR product was cloned into pCR2.1-TOPO and subcloned into the pGL3-basic reporter vector in the same restriction sites. The identity of the PCR product was confirmed by sequencing on both strands. To determine the role of C/EBP, NF-{kappa}B, and AP-1, we generated deletion constructs lacking C/EBP, NF-{kappa}B, or AP-1 (–2685/+52, 5'-acg cgt AGA TGC TCC CAG AGG AAA C-3' for C/EBP; –1524/+52, 5'-acg cgt CAG GAA TCC ATA AGG GGA GG'3' for C/EBP and NF-{kappa}B; and –62/+52, acg cgt ACC TCT GGC TTT CTG GAA GG-3' for C/EBP, NF-{kappa}B, and AP-1) and the antisense primer described above.

mRNA expression: Northern blotting and real-time quantitative PCR. DNA-free total RNA was extracted using the RNAqueous-4 PCR kit (Ambion). RNA quality was assessed by capillary electrophoresis using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). All RNA samples used for quantitative PCR had RNA integrity numbers >9.1 (on a scale of 1–10), as assigned by default parameters of the Expert 2100 Bioanalyzer software package (version 2.02). IL-17 receptor (IL-17R) type A (GenBank accession no. NM_014339) expression was analyzed by RT-PCR using two sets of primers [5'-GAT GAC AGC TGG ATT CAC C-3' (sense) and 5'-CTC ATA TTC CTG GTC AGG G-3' (antisense) for set 1 and 5'-GTC TGG TTA TCG TCT ATC C-3' (sense) and 5'-CAA ACT CCT GAC CTC AGA G-3' (antisense) for set 2], which resulted in a 300-bp amplification product. β-Actin [GenBank accession no. NM_001101; 668-bp amplification product, 5'-CGT GCG TGA CAT TAA GGA GA-3' (sense) and 5'-CAC CTT CAC CGT TCC AGT TT-3' (antisense)] served as an internal control. Northern blot analysis was carried out as previously described (6). MMP-1 cDNA (GenBank accession no. NM_002421.2) was amplified from reverse-transcribed HCF RNA by RT-PCR using the sense primer 5'-ATT CTA CTG ATA TCG GGG CTT TGA-3' and the antisense primer 5'-ATG TCC TTG GGG TAT CCG TGT AG-3'. Expression of 28S rRNA was used as an internal control.

MMP-1 mRNA expression was also analyzed by real-time quantitative PCR [5'-CAT TGA TGG CAT CCA AGC C-3' (sense) and 5'-GGC TGG ACA GGA TTT TGG G-3' (antisense)] using QuantiTect SYBR-Green Probe RT-PCR kit (Qiagen). Each sample was assayed in triplicate. For relative quantification, the cycle threshold (Ct) method {ratio = 2 – [Ct(MMP-1) – Ct(GAPDH)]} was used, with GAPDH as a control. For copy number determination, a calibration curve was obtained using serial dilutions of a linearized GAPDH cDNA with the GAPDH primer pair [5'-GAA GGT GAA GGT CGG AGT C-3' (forward) and 5'-GAA GAT GGT GAT GGG ATT TC-3' (reverse)].

MMP-1 levels. MMP-1 levels in culture supernatants were analyzed 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 were determined by an antibody array (RayBio MMP antibody array 1, catalog no. AAH-MMP-1, RayBiotech, Norcross, GA) following the manufacturer's protocol and quantified by densitometry (6). Protein concentrations 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 a Hybond-P polyvinylidene difluoride membrane (Amersham Biosciences). The membrane was incubated with rabbit anti-human MMP-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). The immunoreactive bands were detected by chemiluminescence (ECL Plus, GE Healthcare). The blots were stripped and reprobed with {alpha}-tubulin antibodies to confirm equal protein loading.

EMSA, ELISA, and reporter assays. NF-{kappa}B and AP-1 protein-DNA complex formation was assessed by EMSA (6, 18, 19) using HCF nuclear extracts and double-stranded consensus DNA for C/EBP (5'-TGC AGA TTG CGC AAT CTG CA-3'), NF-{kappa}B (5'-AGT TGA GGG GAC TTT CCC AGG C-3'), AP-1 (5'-CGC TTG ATG ACT CAG CCG GAA-3'), mutant C/EBP (5'-TGC AGA GAC TAG TCT CTG CA-3'), mutant NF-{kappa}B (5'-AGT TGA GGC GAC TTT CCC AGG C-3), and mutant AP-1 (5'-CGC TTG ATG ACT TGG CCG GAA-3'; Santa Cruz Biotechnology). Activation of transcription factors (TF) was confirmed by ELISA (20, 21, 24) [TransAM TF ELISA kits 43296 (NF-{kappa}B), 44196 (C/EBP), and 44296 (AP-1), Active Motif, Carlsbad, CA].

TF activation was also analyzed by reporter assays using adenoviral transduction of NF-{kappa}B (Ad-NF-{kappa}B-Luc, 50 MOI; kindly provided by 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-β-Gal (50 MOI; Vector Biolabs) served as an internal control. β-Gal activity in cell extracts was determined using luminescent β-Gal detection kit II (BD Biosciences), and the results are expressed in relative light units as a ratio of firefly luciferase to β-Gal activity. C/EBP activation was confirmed using a C/EBP reporter vector (2xC/EBP-Luc) that contains two canonical C/EBP binding sites (kindly provided by Peter Johnson, National Cancer Institute, Frederick, MD). HCF were transfected with C/EBP-Luc (3 µg) using the Nucleofection kit. Cells were cotransfected with Ad-β-Gal to normalize for variations in transfection efficiency. Cells were then treated with IL-17 for 12 h. Firefly luciferase and β-Gal activities were analyzed as described above. Neither pharmacological inhibitors, dominant-negative expression vectors, nor siRNA affected cell viability for the indicated study period.

Immune complex protein kinase assays. p38 MAPK and ERK activities were determined as described previously (6, 18, 19). Briefly, a commercially available colorimetric assay kit [p38 MAP kinase assay kit (nonradioactive), Cell Signaling Technology] was used to determine p38 MAPK activity in whole cell homogenates. The assay is based on phosphorylation of activating TF (ATF)-2 by the immunoprecipitated phosphorylated p38 MAPK. ERK activity was determined in whole cell homogenates using a commercially available colorimetric assay kit (ERK, p44/42 MAP kinase assay kit, Cell Signaling Technology).

Cell migration. HCF migration was quantified as previously described (10) using Transwell chambers with 3-µm polycarbonate membrane (Corning) precoated with 100 µg/ml type I collagen or BSA on both sides of the membrane. HCF were trypsinized and suspended in FM medium containing 0.5% BSA, and 1 ml containing 2.0 x 105 cells/ml was layered on the coated insert filters. Cells were stimulated with IL-17 (10 ng/ml). The lower chamber contained IL-17 at the same concentration. Plates were incubated at 37°C for 24 h. Membranes were washed with PBS, and noninvading cells on the upper surface were removed using cotton swabs. Cells migrating to the lower surface of the membrane were determined at 540-nm absorbance using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.

To determine the role of MMP-1 in IL-17-mediated cell migration, we treated HCF with GM-6001 (10 µM in DMSO for 15 min) or MMP-1 siRNA (target sequence corresponding to nucleotides 153–171 downstream from starting codon; 100 nM for 48 h) before addition of IL-17. siRNA against this target sequence has been shown to 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 (≤100 ng/ml) for ≤48 h. Cell death was analyzed by quantitation of mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates by an ELISA (Cell Death Detection ELISAPLUS kit, Roche Diagnostics) (6).

Statistical analysis. Values are means ± SE. For statistical analysis, we used ANOVA followed by an appropriate post hoc multiple comparison test (Tukey's method). Data were considered statistically significant at P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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IL-17 stimulates MMP-1 mRNA expression in human cardiac fibroblasts. Since the proinflammatory cytokine IL-17 signals via IL-17RA, we first used RT-PCR to determine whether HCF express IL-17RA. Our results demonstrated that HCF express IL-17RA under basal conditions (data not shown). We next investigated whether IL-17 can induce MMP-1 mRNA expression in HCF. HCF were treated with IL-17 (0–100 µg/ml) for 12 h and analyzed by Northern blotting and densitometry. HCF express MMP-1 mRNA at low levels under basal conditions (Fig. 1A), and treatment with IL-17 for 12 h significantly increased MMP-1 expression, with peak levels obtained with 10 ng/ml. Therefore, in all subsequent experiments, IL-17 was used at 10 ng/ml. IL-17 induced MMP-1 expression in a time-dependent manner, with peak levels of mRNA observed at 12 h (Fig. 1B). MMP-1 levels remained at these high levels throughout the 48-h study period. IL-17-induced MMP-1 expression was also investigated by RT-quantitative PCR (qPCR). The specificity of the response to IL-17 was verified by incubation of HCF with IL-17 or IL-17R neutralizing antibodies (10 µg/ml) for 1 h before addition of IL-17. Our results indicate that the potent induction of MMP-1 mRNA expression by IL-17 can be blocked by IL-17 or IL-17R neutralizing antibodies (Fig. 1C). These results demonstrate that 1) HCF express IL-17RA, 2) IL-17 is a potent inducer of MMP-1 expression, and 3) IL-17 induces MMP-1 expression in a time- and dose-dependent manner (Fig. 1).


Figure 1
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  		Fig. 1. IL-17 stimulates matrix metalloproteinase (MMP)-1 expression. A: Northern blot of MMP-1 mRNA expression in DNA-free total RNA isolated from 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-1 expression shown in Northern blot of MMP-1 mRNA expression in quiescent HCF treated with IL-17 (10 ng/ml) for ≤48 h. C, control. C: RT-quantitative PCR (qPCR) analysis of MMP-1 mRNA expression in quiescent HCF treated with IL-17 or IL-17 receptor (IL-17R) neutralizing antibodies for 1 h before 12 h of treatment with IL-17 (10 ng/ml). Normal goat/mouse IgG and GAPDH served as internal controls. IL-17-mediated MMP-1 expression is blocked by IL-17 or IL-17 receptor (IL-17R) neutralizing antibodies. *P < 0.01 vs. untreated (ANOVA). {dagger}P < 0.01 vs. IL-17 (ANOVA).

 
IL-17 stimulates MMP-1 protein expression. We next investigated whether IL-17 also stimulates MMP-1 protein expression. Western blot analysis of whole cell homogenates using antibodies that recognize latent and active forms of MMP-1 revealed that, under basal conditions, HCF expressed latent and active forms of MMP-1 (Fig. 2A), whereas treatment with IL-17 resulted in a modest increase in the latent form but a significant increase in the active form (Fig. 2A). Furthermore, IL-17 increased MMP-1 secretion from the HCF (Fig. 2B). These results demonstrate that IL-17 1) stimulates MMP-1 protein expression, with a modest increase in the latent form and a significant increase in the active form, and 2) stimulates MMP-1 secretion (Fig. 2).


Figure 2
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  		Fig. 2. IL-17 induces MMP-1 protein expression. A: Western blot analysis of MMP-1 protein levels in whole cell homogenates from quiescent HCF treated with IL-17 (10 ng/ml) for 24 h. {alpha}-Tubulin served as internal control. B: ELISA quantification of MMP-1 levels in culture supernatants from quiescent HCF treated as in described A. IL-17 stimulates MMP-1 secretion. *P < 0.001 vs. untreated (ANOVA).

 
IL-17 regulates MMP-1 expression at the transcriptional level. We next determined whether IL-17-mediated MMP-1 expression is regulated at the transcriptional and/or translational level. HCF were treated with CHX or ActD for 1 h, IL-17 (10 ng/ml for 12 h) was added, and RNA was isolated and analyzed for MMP-1 expression by RT-qPCR. IL-17-mediated MMP-1 expression was significantly inhibited by the RNA synthesis inhibitor ActD, but not by the protein synthesis inhibitor CHX (Fig. 3A). Further studies indicated that the rate of degradation of MMP-1 mRNA was similar in ActD-treated and untreated controls (data not shown), indicating that mRNA stabilization did not contribute significantly to the induction of MMP-1 by IL-17. We next determined whether IL-17 induces MMP-1 promoter-reporter gene activity. Consistent with our earlier observations, IL-17 stimulated the MMP-1 promoter-reporter (pGL3-4334) activity, and incubation with IL-17 or IL-17R neutralizing antibodies blocked this effect (Fig. 3B). It has been reported previously that several IL-17 responsive genes contain NF-{kappa}B, AP-1, and C/EBP binding elements in their cis-regulatory regions (27). Since these elements also play a role in cytokine- and growth factor-induced MMP-1 expression, we determined whether these elements mediate IL-17-stimulated MMP-1 transcription. Transfection of the deletion constructs lacking C/EBP (pGL3-2685), C/EBP and NF-{kappa}B (pGL3-1524), or all three binding sites (pGL3-62) and treatment with IL-17 showed that IL-17-induced MMP-1 promoter-reporter activity was significantly attenuated by the deletion of C/EBP or NF-{kappa}B, but this inhibition was significantly more pronounced when all three sites were deleted (Fig. 3C). These results demonstrate that 1) IL-17 does not affect MMP-1 mRNA stability, 2) IL-17 regulates MMP-1 expression at the transcriptional level, and 3) IL-17-mediated MMP-1 transcription is dependent on C/EBP, NF-{kappa}B, and AP-1 (Fig. 3).


Figure 3
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  		Fig. 3. IL-17 stimulates MMP-1 expression at transcriptional and posttranscriptional levels. A: quantification by RT-qPCR of MMP-1 expression in quiescent HCF treated with IL-17 and/or cyclohexamide (CHX, 10 µg/ml in DMSO) or actinomycin D (ActD, 10 µg/ml in DMSO) for 12 h. IL-17-mediated MMP-1 expression is regulated at transcriptional and posttranscriptional levels. B: IL-17 stimulation of MMP-1 promoter-reporter activity in quiescent HCF transfected with the full-length MMP-1 promoter-reporter vector (pGL3-4334; 3 µg) and 24 h later treated with IL-17 (10 ng/ml) for 12 h. Specificity of IL-17 was verified by 1 h of preincubation of cells with IL-17 or IL-17R neutralizing antibodies (10 µg/ml). pGL3-basic vector (3 µg) served as control. Cells were infected with adenovirus expressing β-galactosidase [Ad-β-Gal, 50 multiplicity of infection (MOI)]. Firefly luciferase and β-Gal activities were analyzed, and results are presented as fold increase relative to untreated and represent ratio of firefly luciferase to β-Gal activity. *P < 0.001 vs. untreated. {dagger}P < 0.01 vs. IL-17. C: deletion of CCAAT enhancer-binding protein (C/EBP), NF-{kappa}B, or activator protein (AP)-1 binding sites blunts IL-17-mediated MMP-1 promoter-reporter activity in quiescent HCF transfected with the full-length vector (pGL3-4334) or deletion constructs lacking C/EBP (pGL3-2685), NF-{kappa}B (pMMP1–1524), or all 3 binding sites (pGL3-62). Ad-β-Gal served as control. *P < 0.01 vs. untreated. {dagger}P < 0.05 vs. pGL3-4334. §P < 0.01 vs. pGL3-4334.

 
IL-17 activates NF-{kappa}B, AP-1, and c/EBP. Since we demonstrated that IL-17-mediated MMP-1 transcription is dependent on NF-{kappa}B, AP-1, and C/EBP (Fig. 3), we next investigated whether IL-17 induces activation of these TFs. TF activations were analyzed by EMSA and reporter assays, and their subunit composition was determined by ELISA. IL-17 induced NF-{kappa}B DNA binding activity, which was blunted by pretreatment with IL-17 or IL-17R neutralizing antibodies (Fig. 4A). Complementing these EMSA results, IL-17 also induced NF-{kappa}B-dependent reporter gene activity (Fig. 4B). ELISA of nuclear protein extracts revealed that p65 and p50 contribute to IL-17-mediated NF-{kappa}B activation (Fig. 4C). Similarly, IL-17 induced AP-1 DNA binding (Fig. 4D) and reporter gene activities (Fig. 4E), and c-Fos, FosB, c-Jun, Fra-1, and JunD contributed to its activation (Fig. 4F). IL-17 induced C/EBP DNA binding (Fig. 4G) and reporter gene activities (Fig. 4H), and C/EBP-β, but not C/EBP-{alpha}, contributed to its activation (Fig. 4I). These results demonstrate that IL-17 potently induces NF-{kappa}B (p65 and p50), AP-1 (c-Fos, FosB, c-Jun, Fra-1, and JunD), and C/EBP-β activation in HCF (Fig. 4).


Figure 4
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  		Fig. 4. IL-17 stimulates AP-1, NF-{kappa}B, and C/EBP activation. A: IL-17 stimulation of NF-{kappa}B DNA binding activity in quiescent HCF treated with IL-17 (10 ng/ml) for 2 h. Nuclear protein was extracted and analyzed by EMSA using labeled double-stranded consensus NF-{kappa}B oligodeoxynucleotides (ODNs). Specificity of IL-17 was verified by preincubation of HCF with IL-17 or IL-17R antibodies. B: IL-17 stimulation of NF-{kappa}B-dependent reporter gene activity in quiescent HCF transduced with Ad-NF-{kappa}B-Luc (50 MOI). Ad-MCS-Luc (50 MOI) served as control; Ad-β-Gal (50 MOI) served as internal control. After 24 h, cells were treated with IL-17 (10 ng/ml), and firefly luciferase and β-Gal activities were determined. C: contribution of p50 and p65 to IL-17-mediated NF-{kappa}B activation. Nuclear extracts from quiescent HCF were treated with IL-17 as described in A and analyzed by ELISA for p50 and p65. OD, optical density. D–I: IL-17-mediated AP-1 and C/EBP activation determined by EMSA [AP-1 (D) and C/EBP (G)], reporter [AP-1 (E) and C/EBP (H)] assays, and ELISA [AP-1 (F) and C/EBP (I)]. Arrows in A, D, and G indicate transcription factor-specific DNA-protein complexes. *P < 0.001 vs. respective untreated (B, E, and H); P < 0.01 vs. untreated (C, F, and I).

 
Targeting TF activation blunts IL-17-mediated MMP-1 expression. We have demonstrated that IL-17 induces NF-{kappa}B, AP-1, and C/EBP activation (Fig. 4). We have also shown that deletion of the NF-{kappa}B, AP-1, or C/EBP binding site blunts IL-17-mediated MMP-1 transcription (Fig. 3). We next investigated whether targeting NF-{kappa}B, AP-1, or C/EBP-β activation will inhibit IL-17-mediated MMP-1 expression. Activation of NF-{kappa}B, AP-1, and C/EBP-β was targeted by adenoviral transduction with dominant-negative expression vectors, phosphorothioated antisense oligonucleotides (ODN), or RNA interference. IL-17-induced MMP-1 promoter-dependent reporter gene activity was significantly attenuated by adenoviral transduction with dnIKK-β, dnp65, or dnI{kappa}B-{alpha} (S32A/S36A) (Fig. 5A). Similarly, c-Fos, c-Jun antisense ODN, or Ad.dnc-Jun significantly attenuated IL-17-dependent MMP-1 promoter reporter activity (Fig. 5B), as did siRNA-mediated C/EBP-β knockdown (Fig. 5C). Furthermore, targeting NF-{kappa}B, AP-1, or C/EBP-β attenuated IL-17-mediated MMP-1 mRNA expression (Fig. 5D). These results demonstrate that activation of NF-{kappa}B, AP-1, or C/EBP-β is a significant mechanism in IL-17-mediated MMP-1 transcription and mRNA expression (Fig. 5).


Figure 5
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  		Fig. 5. IL-17 stimulates MMP-1 promoter-reporter activity in an NF-{kappa}B-, AP-1-, and C/EBP-β-dependent manner. A: attenuation of IL-17-mediated MMP-1 promoter-reporter activity by dominant-negative (dn) IKK-β, dnp65, and dnI{kappa}B-{alpha} in quiescent HCF transfected with pGL3-4334 and transduced with adenoviral vector expressing dnIKK-β, dnp65, or dnI{kappa}B-{alpha} for 24 h and then treated with IL-17 for 12 h. Ad-β-Gal served as control. B: c-Fos and c-Jun antisense (AS) ODNs or adenoviral transduction of dnc-Jun block IL-17-mediated MMP-1 promoter-reporter activity in quiescent HCF transfected with pGL3-4334 and treated with phosphorothioated c-Fos or c-Jun AS ODN or transduced with Ad.dnc-Jun. Scrambled ODN or Ad-β-Gal served as control. C: attenuation of IL-17-mediated MMP-1 promoter-reporter activity by knockdown of C/EBP-β in quiescent HCF transfected with pGL3-4334 and treated with C/EBP-β small interfering RNA (siRNA; 100 nM) for 48 h and then with IL-17 (10 ng/ml). Scrambled siRNA served as control. Firefly luciferase and β-Gal activities were analyzed after 12 h. D: attenuation of IL-17-mediated MMP-1 mRNA expression by inhibition of AP-1, NF-{kappa}B, or C/EBP-β in quiescent HCF treated as described in A–C with AS ODN, dominant-negative expression vectors, or siRNA before 24 h of IL-17 (10 ng/ml) treatment. MMP-1 expression was quantified by RT-qPCR. GAPDH served as internal control. *P < 0.001 vs. untreated. {dagger}P < 0.01 (A); P < 0.05 (B, C, D) vs. IL-17. {dagger}{dagger}P < 0.01 vs. IL-17.

 
IL-17 induces p38 MAPK and ERK1/2 activation. MAPKs are important mediators of a variety of physiological and pathological cellular processes, including cell death, cell survival, proliferation, and migration (20). Since IL-17 induced MMP-1 transcription and mRNA expression via NF-{kappa}B, AP-1, and C/EBP-β activation (Fig. 5) and since these TFs serve as nuclear effectors of MAPKs, we investigated whether IL-17 induces MAPK activation in HCF. Quiescent HCF were treated with IL-17 for 30 min, and cleared cell lysates were then analyzed for MAPK activation by Western blotting using activation-specific antibodies. Kinase activity was determined by immune complex kinase assays. IL-17 induced p38 MAPK phosphorylation (Fig. 6A) and activity (Fig. 6B), which were blocked by the inhibitor SB-203580. Similarly, IL-17 induced ERK1/2 phosphorylation (Fig. 6C) and activity (Fig. 6D), which were blunted by PD-98059. These results demonstrate that IL-17 potently induces p38 MAPK and ERK1/2 activation in HCF (Fig. 6).


Figure 6
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  		Fig. 6. IL-17 induces p38 MAPK and ERK1/2 activation. A: inhibition of IL-17-mediated p38 MAPK activation by SB-203580 (SB). Quiescent HCF were treated with SB-203580 (1 µM in DMSO) for 30 min and then with IL-17 (10 ng/ml) for 30 min, and total and phosphorylated p38 (P-p38) MAPK levels were analyzed by Western blotting. B: IL-17 induction of p38 MAPK activity in quiescent HCF treated with SB-203580 for 30 min and then with IL-17. p38 MAPK activity was determined by immune complex kinase assay, with activating transcription factor (ATF)-2 used as a substrate. {alpha}-Tubulin served as internal control. C: inhibition of IL-17-induced ERK1/2 activation by PD-98059 (PD) in quiescent HCF treated with PD-98059 (10 µM in DMSO) for 1 h and then with IL-17. Total and phosphorylated ERK1/2 (pERK1/2) levels were analyzed by Western blotting. D: IL-17 induction of ERK1/2 activity in quiescent HCF treated with PD-98059 and then with IL-17. ERK1/2 activity was determined by immune complex kinase assay, with Elk used as substrate. {alpha}-Tubulin served as internal control. Experiments were performed ≥3 times.

 
IL-17 induces TF activation and MMP-1 mRNA expression via p38 MAPK and ERK1/2. Our results show that IL-17 potently induces NF-{kappa}B, AP-1, and C/EBP-β activation in HCF (Fig. 4). IL-17 also induced p38 MAPK and ERK1/2 activation (Fig. 6). Therefore, we next investigated whether IL-17 induces TF activation via p38 MAPK and ERK1/2. Quiescent HCF were treated with SB-203580 or PD-98059 and then with IL-17. TF activation was analyzed after 2 h by ELISA of nuclear protein extracts. MMP-1 mRNA expression was analyzed after 12 h by RT-qPCR using total RNA. IL-17 stimulated nuclear translocation of NF-{kappa}B p65, an effect significantly attenuated by SB-203580 and PD-98059 (Fig. 7A). Similarly, SB-203580 and PD-98059 attenuated IL-17-mediated AP-1 (c-Fos; Fig. 7B) and C/EBP (C/EBP-β; Fig. 7C) activation. Furthermore, SB-203580 and PD-98059 attenuated IL-17-mediated MMP-1 mRNA expression (Fig. 7D). These results demonstrate that IL-17 induces TF activation and MMP-1 mRNA expression via p38 MAPK and ERK1/2 activation (Fig. 7).


Figure 7
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  		Fig. 7. IL-17-mediated NF-{kappa}B, AP-1, and C/EBP-β activation and MMP-1 expression are dependent on p38 MAPK and ERK1/2 activation. A: attenuation of IL-17-mediated NF-{kappa}B activation by inhibition of p38 MAPK and ERK1/2 in quiescent HCF treated with SB-203580 (1 µM in DMSO for 30 min), PD-98059 (10 µM in DMSO for 1 h), or DMSO and then with IL-17 (10 ng/ml for 2 h). Nuclear p65 levels were quantified by ELISA. B: attenuation of IL-17-mediated AP-1 activation by inhibition of p38 MAPK and ERK1/2. Nuclear protein extracts isolated as in described A were analyzed for c-Fos levels by ELISA. C: attenuation of IL-17-mediated C/EBP-β activation by inhibition of p38 MAPK and ERK1/2. Nuclear protein extracts isolated as described in A were analyzed for C/EBP-β levels by ELISA. D: attenuation of IL-17-mediated MMP-1 mRNA expression by inhibition of p38 MAPK and ERK1/2 in quiescent HCF treated with SB-203580 or PD-98059 and then with IL-17. After 24 h, total RNA was isolated and MMP-1 expression was analyzed by RT-qPCR. *P < 0.001 (A, C, D); P < 0.01 (B) vs. untreated. {dagger}P < 0.01 (A, C); P < 0.05 (B); P < 0.001 (D) vs. IL-17.

 
Inhibition of MMP-1 expression blocks IL-17-mediated HCF migration. Fibroblast migration and proliferation are critical processes in wound healing and scar formation following ischemia, infarction, and inflammation (12). Since MMPs degrade ECM and facilitate cell migration, we investigated whether IL-17 induces HCF migration in an MMP-1-dependent manner. IL-17 does indeed stimulate HCF migration, and pretreatment with the broad-spectrum MMP inhibitor GM-6001 or siRNA-mediated MMP-1 knockdown attenuated IL-17-dependent HCF migration (Fig. 8A); knockdown of MMP-1 was confirmed by Western blotting (Fig. 8B). The effect of GM-6001 appeared to be more pronounced than was MMP-1 knockdown. However, neither control siRNA (Fig. 8A) nor GFP siRNA (data not shown) modulated IL-17-mediated HCF migration. These results demonstrate that IL-17 induces HCF migration, at least in part, in an MMP-1-dependent manner (Fig. 8).


Figure 8
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  		Fig. 8. IL-17 stimulates HCF migration in an MMP-1-dependent manner. A: quiescent HCF were layered on collagen type I-coated polycarbonate membrane and treated with the MMP-1 inhibitor GM-6001 (10 µM in DMSO or DMSO alone for 15 min) and then with IL-17. MMP-1 expression was also targeted by siRNA: quiescent HCF were treated with MMP-1 or control siRNA (100 nM for 48 h), layered on collagen-coated filters, and treated with IL-17. Cell migration was determined after 24 h. B: knockdown of MMP-1 protein was confirmed by Western blotting. {alpha}-Tubulin served as internal control. *P < 0.01 vs. untreated. {dagger}P < 0.05 vs. IL-17.

 
IL-17 induces MMP-2, -3, -9, and -13 and TIMP-1 expression in HCF. In addition to MMP-1, MMP-2, -9, and -13 also play critical roles in ECM degradation and myocardial remodeling. These MMPs also contain similar TF binding sites in their promoters and, therefore, may be transcriptionally regulated by IL-17. The proteolytic activity of MMPs is tightly regulated by various physiological inhibitors termed TIMPs. Since TIMPs such as TIMP-1 contain similar cis elements, including AP-1 and NF-{kappa}B, in their promoter regions, we hypothesized that IL-17 may regulate their expression as well. Therefore, we used an antibody array to investigate the expression of various MMPs and TIMPs (Fig. 9A). Confirming our earlier results obtained using ELISA (Fig. 2B), results in Fig. 9B show that IL-17 potently induces MMP-1 expression. In addition, IL-17 also induced the expression of MMP-2, -3, -9, and -13 (Fig. 9B). However, IL-17 failed to significantly affect MMP-8 and -10 expression. Similarly, IL-17 induced TIMP-1, but failed to modulate TIMP-2 and -4, expression (Fig. 9B). These experiments were performed three times, and the results are summarized in Fig. 9C. Together, these results indicate that IL-17 induces the expression of various MMPs and TIMP-1, which play a role in myocardial remodeling (Fig. 9).


Figure 9
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  		Fig. 9. IL-18 stimulates secretion of MMPs and tissue inhibitors of MMPs (TIMPs). A: antibody array that detects various MMPs and TIMPs simultaneously was used to assess whether IL-17 induces other MMPs and TIMPs. POS, positive control; NEG, negative control. B: IL-17 stimulates secretion of MMPs and TIMPs. Quiescent HCF were stimulated with saline or IL-17 (10 ng/ml) for 24 h, and culture supernatants were collected and analyzed for extracellular matrix proteins. C: quantitation of signals in B by image analysis. Intensity of signals was normalized to saline-treated control samples, and results are expressed as fold increases. *P < 0.05; **P < 0.001 vs. respective saline (by ANOVA).

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
The results from this study show that the novel cytokine IL-17 is a potent inducer of MMP-1 expression in primary HCF and stimulates MMP-1 expression independently of IL-1β, IL-6, and TNF-{alpha}. IL-17 regulates MMP-1 expression at the transcriptional level and is dependent on AP-1, NF-{kappa}B, and C/EBP-β activation. More importantly, IL-17 induces HCF migration in an MMP-1-dependent manner. Since MMPs degrade ECM and facilitate migration, our results suggest that IL-17 may be potentially important in myocardial injury, remodeling, and failure.

IL-17s constitute a newly discovered and unique family of cytokines that show no structural homology to other ILs (4). IL-17 is expressed mainly by a subset of CD4+ T cells, i.e., Th17 cells. Many cell types express IL-17Rs and are, therefore, targets of IL-17. Increased IL-17 levels have been detected in various models of inflammation, including rheumatoid arthritis and periodontitis (1, 4). However, few studies have reported the role of IL-17 in myocardial inflammation and injury. Sonderegger et al. (28) demonstrated that administration of IL-23 neutralizing antibodies attenuated experimental autoimmune myocarditis (EAM). Since IL-23 stimulates a pathogenic IL-17-producing T cell population, these authors hypothesized that targeting IL-17 would reduce EAM. They targeted IL-17 expression by an active vaccination approach that breaks B cell tolerance and found that neutralization of IL-17 effectively reduced heart autoantibody responses and myocardial inflammation (28). Recently, Rangachari et al. (21) showed that mice lacking T-bet, a T-box TF essential for Th1 lineage differentiation, develop severe EAM. Using T-bet–/– IL-12Rβ1–/– and T-bet–/– IL-12p35–/– mice and antibody depletion experiments, these authors reported that IL-23 and IL-17 are critical for EAM pathogenesis.

EAM serves as an animal model for postinfectious myocarditis and cardiomyopathy. Dilated cardiomyopathy is characterized by increased MMP-1 expression (13). It is therefore plausible that IL-17 may play a causal role in dilated cardiomyopathy via enhanced expression of various MMPs, including MMP-1. Although the above-mentioned studies demonstrate a role for IL-17 in myocarditis and cardiomyopathy, Li et al. (16) showed that local expression of soluble IL-17R-immunoglobulin chimera (sIL-17R-Ig) prolongs graft survival in rat cardiac allografts by suppressing cytokine responses and leukocyte infiltration. Together, these studies suggest that IL-17 may be a therapeutic target to reduce cardiac inflammation and injury.

Although the above-mentioned studies show that neutralization of IL-17 blunts myocardial inflammation mainly by regulating Th1 cell responses and attenuating inflammatory and immune cell infiltration (16, 21, 28), it is not known whether IL-17 affects myocardial biology directly. It is also not known whether myocardial constituent cells express IL-17 and whether IL-17 affects myocardial cells differentially. Using multiplexed immunoassays, we observed for the first time that human cardiac fibroblasts secrete various proinflammatory cytokines, including low levels (4.1 pg/ml) of IL-17 at basal conditions (unpublished observations). Since no cells of a non-HCF phenotype contaminate these cultures, our results suggest that cells other than Th17 lineage may secrete IL-17. Thus fibroblast-secreted IL-17 may affect fibroblasts and other myocardial cells via autocrine and paracrine mechanisms.

In the present study, we show that IL-17 induces MMP-1 expression in cardiac fibroblasts, and these stimulatory effects are independent of other proinflammatory cytokines. IL-17 induced MMP-1 expression via enhanced transcription, rather than MMP-1 mRNA stability. Furthermore, IL-17 stimulated MMP-1 transcription via NF-{kappa}B, AP-1, and C/EBP-β activation. It has been previously demonstrated that cytokines induce MMP-1 expression via c-Jun induction (22). Although we have not investigated whether IL-17 induces c-Jun expression, our studies show that IL-17-mediated AP-1 DNA binding involves various subunits including c-Jun, and treatment with c-Jun antisense ODN and adenoviral transduction of dnc-Jun attenuate IL-17-mediated MMP-1 transcription. Our results also show that IL-17-mediated AP-1 activation includes Fra-1. Since Fra-1 confers invasiveness and motility in various cancer cell lines (2), it is plausible that Fra-1 may function in a similar fashion in mediating IL-17-dependent fibroblast migration.

In addition to AP-1, our results indicate that IL-17-mediated MMP-1 expression is dependent on NF-{kappa}B and C/EBP-β activation. In support of our studies, Raymond et al. (23) recently demonstrated that NF-{kappa}B and C/EBP-β cooperatively induce IL-1β-mediated MMP-1 transcription in chondrocytes. They further demonstrated that IL-1β induces phosphorylation of C/EBP-β on threonine 235, enhancing C/EBP-β transactivation potential. In that study, IL-1β induced C/EBP-β phosphorylation via ERK activation (23). Our results show that IL-17 induces MMP-1 expression via p38 MAPK and ERK activation. Together, these studies demonstrate that p38 MAPK- and ERK-dependent coordinated activation of NF-{kappa}B, AP-1, and C/EBP-β plays a role in IL-17-mediated MMP-1 induction in cardiac fibroblasts. Since MMPs such as MMP-3 and -9 are also responsive to AP-1 and NF-{kappa}B activation (6, 26, 33), IL-17 may induce their expression and, thus, play a critical role ECM regulation and myocardial remodeling.

In fact, our results demonstrate that, in addition to MMP-1, IL-17 induces the expression of other members of the MMP family, such as MMP-2, -3, -9, and -13, which play critical roles in myocardial remodeling. Similar to MMP-1, these MMPs are also regulated predominantly at the transcriptional level. On the basis of the composition of cis-regulatory regions in their promoters, MMPs are arbitrarily grouped into three categories (32). Group 1 consists of MMP-1, -3, -7, -9, -10, -12, and -13, which contain a TATA box and an AP-1 binding site proximal to the transcriptional start site. MMP-9 also contains an NF-{kappa}B site at the distal region, and its expression is regulated at the transcriptional levels via AP-1 and NF-{kappa}B. Group 2 consists of MMP-8 and -11, which contain a TATA box but lack a proximal AP-1 site. Group 3 consists of MMP-2 and -14, which lack the TATA box and the proximal AP-1 site. Because of these variations, it is possible that these MMPs are regulated differently. Our results show that IL-17 potently induces the expression of various group 1 MMPs. However, lack of significant induction of MMP-10 expression suggests that its induction is not solely dependent on AP-1 activation. The MMP-8 promoter, which contains a TATA box but lacks a proximal AP-1 site, failed to respond to IL-17. In contrast to its effects on MMP-1 induction, IL-17 failed to induce MMP-8 expression, despite three potential C/EBP binding sites at –70, –112, and –164 in its cis-regulatory region. IL-17 also induces the expression of MMP-2 in HCF. MMP-2 contains neither a TATA box nor the proximal AP-1 site. However, MMP-2 contains two AP-2 binding sites at –61 and –1649. It is possible that IL-17 may induce MMP-2 via AP-2 activation. However, Bergman et al. (3) demonstrated that the MMP-2 promoter contains an AP-1 binding site at –1670 that binds FosB and JunB, suggesting that further critical analysis of TF binding sites is necessary.

Transcription is a complex process and is regulated at multiple stages. It is possible that IL-17 may regulate MMP expression at transcriptional and posttranscriptional levels. Despite few CpG islands in the promoter regions of MMPs, epigenetic mechanisms have recently been shown to significantly affect MMP expression (31). For example, increased promoter methylation was shown to suppress MMP-9 transcription (9). Therefore, it is possible that IL-17 may regulate expression of MMPs via genetic and epigenetic mechanisms. It is also possible that IL-17-induced MMP induction is mediated by an intermediary, inasmuch as IL-17 is a potent inducer of various proinflammatory cytokines that are known to induce MMP expression. However, in the present study, we have shown that neutralization of IL-β, IL-6, and TNF-{alpha} fails to inhibit IL-17-mediated MMP-1 induction.

MMPs are synthesized as proenzymes and, upon secretion, bind to various ECM components (31, 32). These stored proforms are immediately available and become activated during inflammation and injury. In addition to ECM degradation, MMPs also release ECM-bound growth factors and other biological molecules. For example, TGF-β, a growth factor readily expressed after myocardial ischemic injury (7), is secreted as a latent and inactive form due to an intact prodomain, the latency-associated peptide (17). MMP-1 degrades latency-associated peptide and releases mature TGF-β, and mature TGF-β downregulates MMP-1 expression. This dynamic coregulation and downregulation of MMP-1 expression may result in reduced tissue injury. In fact, active MMP-1 has been shown to induce cardiomyocyte death (8), and these cytotoxic effects are blunted by exogenous addition of mature TGF-β. However, studies are in progress to determine whether IL-17 coregulates MMP-1 and TGF-β expression.

Our studies have important clinical implications. 1) We have established for the first time that IL-17, a novel proinflammatory cytokine, induces primary cardiac fibroblast migration in an MMP-1-dependent manner. Since fibroblast migration and proliferation are two critical steps in cardiac fibrosis, our results indicate that IL-17 may play a role in myocardial remodeling. 2) IL-17 induced NF-{kappa}B, AP-1, and C/EBP-β activation. Therefore, IL-17 may upregulate NF-{kappa}B-, AP-1-, and C/EBP-β-responsive proinflammatory cytokines, chemokines, adhesion molecules, and MMPs in fibroblasts and other myocardial constituent cells. IL-17 may synergize with these mediators and induce myocardial inflammation and injury. 3) Neutrophils, at least initially, play a role in myocardial ischemic injury. IL-17, a potent inducer of neutrophil chemoattractants (24), may amplify the inflammatory cascade during ischemic injury via recruitment of neutrophils to the site of injury/inflammation. Therefore, targeting IL-17 expression may reduce fibrosis and remodeling following myocardial inflammation and injury.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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REFERENCES
 
This work was supported in part by the Research Service of the Department of Veterans Affairs (B. Chandrasekar) and National Institutes of Health Grants DE-017139 and K02 DE-016312 (B. Steffensen).


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. Chandrasekar, Medicine/Cardiology, The Univ. of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900 (e-mail: chandraseka{at}uthscsa.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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E. Kobayashi, M. Kobayashi, K. Tsuneyama, T. Fukami, M. Nakajima, and T. Yokoi
Halothane-Induced Liver Injury is Mediated by Interleukin-17 in Mice
Toxicol. Sci., October 1, 2009; 111(2): 302 - 310.
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 Sci SignalHome page
F. Shen, N. Li, P. Gade, D. V. Kalvakolanu, T. Weibley, B. Doble, J. R. Woodgett, T. D. Wood, and S. L. Gaffen
IL-17 Receptor Signaling Inhibits C/EBP{beta} by Sequential Phosphorylation of the Regulatory 2 Domain
Sci. Signal., February 24, 2009; 2(59): ra8 - ra8.
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 Am. J. Respir. Cell Mol. Bio.Home page
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
Am. J. Respir. Cell Mol. Biol., January 1, 2009; 40(1): 4 - 12.
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 Am. J. Physiol. Heart Circ. Physiol.Home page
K. Venkatachalam, S. Mummidi, D. M. Cortez, S. D. Prabhu, A. J. Valente, and B. Chandrasekar
Resveratrol inhibits high glucose-induced PI3K/Akt/ERK-dependent interleukin-17 expression in primary mouse cardiac fibroblasts
Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H2078 - H2087.
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