From the
Molecular Biology Program, University of
Montreal, and the Departments of ¶Medicine and
Anatomy and Cell Biology, McGill University,
Montreal, Quebec H3A 1A1, Canada
Received for publication, December 16, 2002 , and in revised form, May 12, 2003.
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ABSTRACT |
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INTRODUCTION |
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It has become increasing clear that interleukin-17 occupies an important
place in the hierarchy of proinflammatory cytokines associated with
inflammatory, immune, malignant, and arthritic diseases (reviewed in Refs.
7 and
8). Indeed, there is now wide
agreement, based on in vitro and in vivo studies, that the
T-cell-derived cytokine may play a fundamental role in the pathophysiology of
rheumatoid arthritis (RA) and possibly osteoarthritis (OA)
(710).
The cytokine is found in high levels in the synovial fluid of RA and OA
patients, and synovial tissues express and produce abundant IL-17
(1113),
likely from infiltrating T-cell populations. The gamut of target genes and
cells include the IL-17-dependent stimulation of the pro-inflammatory
cytokines TNF- and IL-1
by infiltrating macrophages, IL-6, IL-8,
granulocyte-macrophage colony-stimulating factor, GRO-
, and ICAM-1 from
mononuclear phagocytes, endothelial cells and fibroblasts, matrix-destructive
metalloproteases (e.g. collagenases and aggrecanases) from activated
synovial fibroblasts and cartilage chondrocytes, NO from endothelial cells and
chondrocytes, and inflammatory modulators such as eicosanoids (e.g.
PGE2) from may sources
(1422).
Thus, IL-17 stimulates tissue damage and cartilage degradation (joint failure)
directly or indirectly by recruiting activated inflammatory cells
(e.g. neutrophils via adhesion molecules) and inducing the synthesis
of proinflammatory cytokines in the inflamed tissue.
The effector cascades mediating the proinflammatory actions of IL-17 are
currently under study, and although the IL-17 receptor (IL-17R) is a type I
transmembrane protein (130 kDa) with no intrinsic kinase activity
(23), it can transduce a
signal through the activation of protein kinase A (PKA), JNK, ERK1/2,
JAK/STAT, and the NF-B cascades
(20,
2427).
Interleukin-17-treated bovine chondrocytes express increased levels of
inducible nitric-oxide synthase mRNA, inducible nitric-oxide synthase protein,
and NO release, a process associated with PKA, ERK1/2, and, to a lesser
extent, JNK activation (20).
Cyclooxgenase-2 mRNA expression and PGE2 release is concomitant
with the stimulation of JNK1 and JNK2 by IL-17 in bovine chondrocytes
(20). Interleukin-17 activated
release of MMP-9 by human monocyte/macrophages followed a time course
coincident with the phosphorylation of ERK1/2 and the transcription factors
STAT1 and STAT3 (12). Using
the same cell and culture conditions, IL-17 stimulated macrophagic release of
IL-1
, TNF-
, and IL-6 was related to rapid calcium flux and a more
delayed increase in NF-
B DNA binding
(14). Studies using TRAF-2 or
TRAF-6-deficient mouse embryonic fibroblasts suggest strongly that TRAF6 is a
critical mediator of IL-17 signaling, implying the involvement of NF-
B
and/or JNK cascades (26).
In the present study, we examined the IL-17-dependent signaling events using as a paradigm the IL-17 induction of COX-2 gene in human synovial fibroblasts, chondrocytes, and, where indicated, macrophages. The COX-2 protein represents the rate-limiting step in the activated biosynthesis of prostanoids, the latter playing a cardinal role as pleiotropic immune and inflammatory modulators (2830). The COX-2 gene is an inducible immediate early gene regulated at both transcriptional (promoter based) and post-transcriptional levels (3133) and, once induced, can be largely controlled by a positive feedback loop involving PGE2 (34). We report that the magnitude and duration of the induction of COX-2 mRNA, COX-2 protein, and PGE2 release by rhIL-17 is primarily the result of IL-17-dependent stabilization of COX-2 mRNA, although transcriptional mechanisms are also involved in the initial phase of induction. Essentially, rhIL-17 mitigates COX-2 mRNA decay normally mediated by the 3'-UTR of COX-2 mRNA. Finally, we provide evidence that the transcriptional and stabilization processes involve a restricted MAPK profile, the MKK3/6/SAPK2/p38 cascade.
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EXPERIMENTAL PROCEDURES |
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Specimen Selection and Cell CultureSynovial lining cells (human synovial fibroblasts (HSF)) were isolated from synovial membranes (synovia) and chondrocytes from articular cartilage. Both were obtained at necropsy from donors with no history of arthritic disease (mean age, 30 ± 27). Additional experiments were conducted with specimens obtained from OA and RA patients undergoing arthroplasty who were diagnosed based on the criteria developed by the American College of Rheumatology Diagnostic Subcommittee for OA/RA (mean age, 67 ± 19) (35). Human synovial fibroblasts and chondrocytes were released by sequential enzymatic digestion with 1 mg/ml Pronase (Roche Applied Science) for 1 h, followed by 6 h with 2 mg/ml collagenase (type IA; Sigma) at 37 °C in DMEM supplemented with 10% heat-inactivated FCS, 100 units/ml penicillin, and 100 µg/ml streptomycin (3638). Released HSF were incubated for 1 h at 37 °C in tissue culture flasks (Primaria 3824, Falcon, Lincoln Park, NJ), allowing the adherence of nonfibroblastic cells possibly present in the synovial preparation, particularly from OA and RA synovia. In addition, flow cytometric analysis (Epic II, Coulter, Miami, FL), using the anti-CD14 (fluorescein isothiocyanate) antibody, was conducted to confirm that no monocytes/macrophages were present in the synoviocyte preparation (3638). The cells were seeded in tissue culture flasks and cultured until confluence in DMEM supplemented with 10% FCS and antibiotics at 37 °C in a humidified atmosphere of 5% CO2, 95% air. The cells were incubated in fresh medium containing 0.51% fetal bovine serum for 24 h before the experiments, and only second or third passaged HSF were used. Human monocyte/macrophage cultures were prepared from the freshly drawn blood of healthy volunteers as previously described (14).
Preparation of Cell Extracts and Western
Blotting50100 µg of cellular protein extracted in RIPA
buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2
mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
each of aprotinin, leupeptin, and pepstatin, 1% Nonidet P-40, 1 mM
sodium orthovanadate, and 1 mM NaF) or hot SDS-PAGE loading buffer,
from control and treated cells, were subjected to SDS-PAGE through 10% gels
(final concentration of acrylamide, 16 x 20 cm) under reducing
conditions and transferred onto nitrocellulose membranes (Amersham
Biosciences). Following blocking with 5% BLOTTO for 2 h at room temperature
and washing, the membranes were incubated overnight at 4 °C with
polyclonal anti-human COX-2 (Cayman Chemical Co., Ann Arbor, MI; 1:7500
dilution) in TTBS containing 0.25% BLOTTO. The second anti-rabbit
antibody-horseradish peroxidase conjugate (1:10,000 dilution) was subsequently
incubated with membranes for 1 h at room temperature, washed extensively for
3040 min with TTBS, and then rinsed with Tris-buffered saline at room
temperature. Following incubation with an ECL chemiluminescence reagent
(Amersham Biosciences), the membranes were prepared for autoradiography,
exposed to Kodak (Rochester, NY) X-Omat film, and subjected to a digital
imaging system (Alpha G-Imager 2000; Canberra Packard Canada, Mississauga,
Canada) for semi-quantitative measurements. In addition to the anti-COX-2 and
COX-1 (Cayman) antisera, the following polyclonal antibodies were used (New
England Biolabs Ltd., Mississauga, ON, Canada): total (independent of
phosphorylation state) and anti-phospho-p38 MAPK
(Thr180/Tyr182), anti-phospho-MKK3/6
(Ser189/207), total and anti-phospho-IB-
(Ser32), anti-phospho-ATF-2 (Thr69/71),
anti-phospho-CREB-1 (Ser133), anti-phospho-c-Jun
(Ser63), anti-phospho-JNK/SAPK
(Thr183/Tyr185), anti-phospho-Mnk1
(Thr197/201), total and anti-phospho-eIF4E (Ser209),
total and anti-phospho-p44/42 (Thr202/Tyr204), and total
and anti-phospho-STAT3 (Ser727).
Northern Blot Analysis of mRNATotal cellular RNA was isolated (1 x 106 cells = 1020 µg of RNA) using the Trizol (Invitrogen) reagent. Generally, 5 µg of total RNA were resolved on 1.2% agarose-formaldehyde gel and transferred electrophoretically (30 V overnight at 4 °C) to Hybond-NTM nylon membranes (Amersham Biosciences) in 0.5x Tris/acetate/EDTA buffer, pH 7. After prehybridization for 24 h, the hybridizations were carried out at 5055 °C for 2436 h, followed by high stringency washing at 68 °C in 0.1x SSC, 0.1% SDS. The following probes, labeled with digoxigenin-dUTP by random priming, were used for hybridization: human COX-2 cDNA (1.8 kb; Cayman Chemical Co.) initially cloned into the EcoRV site of pcDNA 1 (Invitrogen) that was released by PstI and XhoI digestion resulting in a 1.2-kb cDNA fragment and a 780-bp PstI/XbaI fragment from GAPDH cDNA (1.2 kb; American Type Culture Collection, Rockville, MD) that was initially cloned into a PstI of pBR322 vector. This latter probe served as a control of RNA loading because GAPDH is constitutively expressed in cells used in these experiments. All of the blots were subjected to a digital imaging system (Alpha G-Imager 2000; Canberra Packard Canada) for semi-quantitative measurements, and changes in COX-2 expression were always considered as a ratio, COX-2/GAPDH mRNA.
Transfection ExperimentsTransient transfection experiments
were conducted in 4-, 6-, or 12-well cluster plates as previously described
(34,
38). Transfections were
conducted using the FuGENE 6TM (Roche Applied Science) or LipofectAMINE
2000TM reagents (Invitrogen) method for 6 h according to the
manufacturers' protocol with cells at around 3040% confluence. The
cells were re-exposed to a culture medium with 1% FCS for 2 h prior to the
addition of the biological effectors. Transfection efficiencies were
controlled in all experiments by co-transfection with 0.5 µg of
pCMV--gal, a
-galactosidase reporter vector under the control of
CMV promoter (Stratagene, La Jolla, CA). A COX-2 promoter (2390 to +
34)-LUC plasmid was kindly provided by Dr. Stephen Prescott (University of
Utah) (39), and site-directed
mutagenesis was performed with the Bsu36I COX-2 promoter fragment
(415 to + 34) using the QuikChangeTM (Stratagene) kit and involved
modifying the ATF-CRE site at bp 53/54 (CA
TC) and the
NF-
B site at bp215/216 (CC
GG). Chimeric
luciferase reporter plasmids fused with the human COX-2 mRNA 3'-UTR
(1451 bp), AU-rich elements (429 bp of which the first 116 bp contain an
AU-cluster), the 3'-UTR minus the AU-rich element cluster, or a
construct completely devoid of the COX-2 3'-UTR but containing the SV40
poly(A) signal (40). The
plasmids are designated LUC-3'-UTR, LUC-+ARE, LUC-
ARE,
LUC-
3'-UTR, respectively, and were a kind gift of Dr. D. Dixon
(University of Utah).
In our signal transduction pathway reporting systems (Stratagene), a
reporter plasmid containing the 17-bp (5x) GAL4 DNA-binding element
(UAS) fused to a TATA box upstream from the luciferase gene (pFR-LUC) was
co-transfected with a construct containing the transactivation domains of
transcription factors (e.g. ATF-2 and c-Jun) fused to GAL4
DNA-binding domain and driven by a CMV promoter (e.g. pFA-ATF-2). In
addition, plasmids (Stratagene) harboring NF-B or interferon-stimulated
response element enhancer elements (5x) fused to a TATA box upstream
from the luciferase gene were used to assess transactivation processes
activation by IL-17 in the cell phenotypes tested. Finally, wild-type and
activated MKK3/6 expression plasmids (Stratagene) were overexpressed to
determine the role on COX-2 expression. The luciferase values, expressed as
enhanced relative light units, were measured in a Lumat LB 9507 luminometer
(EG&G, Stuttgart, Germany) and normalized to the level of
-galactosidase activity (optical density at 450 nm after 24 h of
incubation) and cellular protein (bicinchoninic acid procedure; Pierce).
RT-PCR for Luciferase and GAPDHThe oligonucleotide primers for PCR were prepared with the aid of a DNA synthesizer (Cyclone Model, Biosearch Inc., Montreal, Canada) and used at a final concentration of 200 nmol/liter. The sequences for the Luciferase primers were as follows: 5'-ACGGATTACCAGGGATTTCAGTC-3' and 5'-AGGCTCCTCAGAAACAGCTCTTC-3' (antisense) for the luciferase fragment of 367 bp (40). The sequences for the GAPDH (which served as a standard of quantitation) primers were 5'-CAGAACATCATCCCTGCCTCT-3', which corresponds to position 604624 bp of the published sequence, and 5'-GCTTGACAAAGTGGTCGTTGAG-3', from positions 901922 bp, for an amplified product of 318 bp (36). Two µg of total RNA, extracted with the Trizol reagent, was reverse transcribed and then subjected to PCR as previously described (34). RT and PCR assays were carried out with the enzymes and reagents of the GeneAmP RNA PCR kit manufactured by PerkinElmer Life Sciences. Both the RT and PCR reactions were done in a Gene ATAQ Controller (Amersham Biosciences).
The amplification process was conducted over 1030 cycles to define the linear range of product amplification. The first cycle consisted of a denaturation step at 95 °C for 1 min, followed by annealing and elongation at 60 °C for 30 s, and 72 °C for 1.5 min, respectively. All subsequent cycles were executed under the same conditions, with the exception of the last cycle, where the elongation step was extended to 7 min. We found a linear range (log luciferase/GAPDH versus log cycle number) between 10 and 17; as such we chose 1113 cycles depending on the type of experiment.
The PCR products were analyzed and verified by electrophoresis on 1.15% agarose gels in a Tris-borate-EDTA buffer system as previously described (34). All gel photos were subjected to a digital imaging system (see above) for semi-quantitative measurements, and the results were expressed as a ratio of luciferase/GAPDH PCR fragments.
Extraction of Nuclear Proteins and EMSA ExperimentsConfluent control and treated cells in 4-well cluster plates (35 x 106 cells/well) were carefully scraped into 1.5 ml of ice-cold phosphate-buffered saline and pelleted by brief centrifugation. The nuclear extracts were prepared as previously described (36).
Double-stranded oligonucleotides containing wild-type and mutant sequences
were from Invitrogen, annealed in 100 nM Tris-HCl, pH 7.5, 1
M NaCl, 10 mM EDTA buffer at 65 °C for 10 min,
cooled for 12 h at room temperature, and finally end-labeled with
[-32P]ATP using T4 polynucleotide kinase (Promega, Madison,
WI). The sense sequences of the oligonucleotides tested were as follows:
NF-
B (COX-2), 5'-CAG GAG AGT GGG GAC TAC CCC CTC TGC
TC-3'; NF-
B mut, 5'-CAG GAG AGT GGC GAC TAG GCC CTC
TGC TC-3'; ATF/CRE (COX-2), 5'-GGC GGA AAG AAA CAG TCA TTT CGT
CAC ATG GGC TTG G-3'; ATF/CRE mut, 5'-GGC GGA AAG AAA CAG
TCA TTT CGT TCC ATG GGC TTG-3'; NF-IL6, 5'-CTA GGG CTT GCG
CAA TCT ATA TTC G-3'; and NF-IL6 mut, 5'-CTA GGG CTT GCT ACC CCT
ATA TTC G-3'. Binding buffer consisted of 10 mM Tris-HCl, pH
7.5, 50 mM NaCl, 0.5 mM dithiothreitol, 0.5
mM EDTA, 1 mM MgCl2, 4% glycerol, and 2.5
µg of poly(dI-dC). The binding reactions were conducted with 15 µg of
nuclear extract (± 1 µg c/EBP
/
/
/
antibodies in supershift analysis) and 100,000 cpm of 32P-labeled
oligonucleotide probe at 22 °C for 20 min in a final volume of 10 µl.
The binding complexes were resolved by nondenaturing polyacrylamide gel
electrophoresis through 6% gels in a Tris-borate buffer system, after which
the gels were fixed, dried, and prepared for autoradiography.
Statistical AnalysisAll of the results were expressed as the means ± S.D. or the means and the coefficient of variation of three to five separate experiments as indicated. The transfection experiments were performed in triplicate. Statistical treatment of the data was performed parametric (Student's t test) or by nonparametric (Mann-Whitney) analysis if Gaussian distribution of the data could not be confirmed. Significance was acknowledged when the probability that the Null Hypothesis was satisfied at <5%.
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RESULTS |
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SAPK2/p38 MAPK Activity and rhIL-17-dependent Regulation of COX-2 Gene
ExpressionPrevious studies in our laboratory and others
demonstrate that IL-17 may signal through activation of MAPK (e.g.
p44/42 MAPK) and/or NF-B cascades, although it is presently unclear how
the COX-2 gene responds (12,
14,
20). To delineate
post-receptor signaling pathways activated by IL-17, we chose, as a first
approach, to use cell-permeable chemical inhibitors. As shown in
Fig. 2A, SB202190 (SB,
SAPK2
/
/p38
/
MAPK inhibitor) suppressed
rhIL-17-induced COX-2 mRNA and protein synthesis by
90% (87 ± 9%,
mean ± S.D., n = 4); PGE2 release was suppressed by
greater than 95% (data not shown). The MEK1/2 inhibitor PD98059, and the
I
B kinase inhibitor, Bay 11-7802 were without significant effect,
whereas the PKA inhibitor KT-5720 had a modest albeit inconsistent inhibitory
activity. The clinically useful anti-inflammatory steroid, dexamethasone
completely suppressed rhIL-17-induced COX-2 mRNA and protein synthesis,
whereas pyrrolidinedithiocarbamate and
L-N6-(1-iminoethyl)lysine, 2HCl, inhibitors of
reactive oxygen radicals and nitric oxide production respectively, were
seemingly without effect (Fig.
2A). We previously reported that IL-1 induction of the
COX-2 gene was mediated by a PGE2-dependent feed-forward mechanism
and as such could be abrogated by co-incubating IL-1 with a preferential COX-2
inhibitor like NS-398 (34).
The present data show that NS-398 does not block to any significant degree the
rhIL-17 induction of the COX-2 gene (Fig.
2A). Identical results were obtained with HSF in culture
(data not shown).
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Given the apparent role of SAPK2/p38 in the induction of COX-2 by rhIL-17, we endeavored to determine the degree of sensitivity of the inductive process to SB202190 by carefully controlled dose-response studies. As shown in Fig. 2B, the inhibitor blocked rhIL-17 stimulation of steady-state levels of COX-2 mRNA in a dose-dependent fashion, and the IC50, calculated by plotting the log optical density COX-2/GAPDH mRNA versus the concentration of SB202190, was 36 ± 4 nM (n = 3).
Cell Signaling by rhIL-17 Is Restricted to the SAPK2/p38 MAPK
CascadeTo pursue further the results obtained with regard to
rhIL-17 and SAPK2/p38 activation, we performed additional investigative
experimentation. As shown in Fig.
3A, rhIL-17 triggered a bi-phasic pattern of MKK3/6
(Ser189/207) and SAPK2/p38 phosphorylation with the initial phase
reaching a zenith at 20 min and then falling to essentially control levels
within the next 40 min (i.e. 1 h post-stimulation). The second phase
was initiated within the next 60 min (i.e. 2 h post-stimulation) and
attained a maximum at 8 h. There were no changes in the cellular protein level
of SAPK2/p38 (Fig.
3A). In addition, ATF-2, a transcription factor whose
transcriptional activity is modulated by phosphorylation at Thr69
and Thr71 by SAPK2/p38
(41), was phosphorylated at
the latter sites in a time course essentially identical to that of
SAPK2/p38/MKK3/6 (Fig.
3A). A similar pattern was observed with CREB-1/ATF-1
(Ser133) phosphorylation following IL-17 stimulation, although the
second phase was less evident. Fragmentation of both SAPK2/p38 and ATF-2 was
observed in the initial phosphorylation phase. The downstream kinases
MAPK-APK2 and MSK1 were not phosphorylated by rhIL-17 treatment, although the
pro-inflammatory cytokine rhIL-1 phosphorylated both (data not shown).
The MAPK signal-integrating kinase-1 (Mnk1) and its putative substrate, the
eukaryotic initiation factor 4E (eIF-4E), were avidly phosphorylated by
rhIL-17 at Thr197/202 and Ser209, respectively
(Fig. 3B). The latter
rhIL-17-induced post-translational modifications were substantially blocked in
the presence of SB202190 (Fig.
3C). Finally, overexpression of a constitutively
activated MKK3 construct (p
MKK3) stimulated COX-2 protein synthesis, an
effect completely abrogated by SB202190
(Fig. 3D).
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To confirm whether rhIL-17 could increase the transactivational capacity of
ATF-2, we conducted experiments in which a luciferase reporter construct
harboring 5'-flanking GAL4 DNA-binding elements (5x) was
co-transfected with a chimeric plasmid containing the N-terminal (196)
ATF-2 transactivation domain fused to GAL4 DNA-binding domain and driven by a
CMV promoter. When the cells were then incubated with rhIL-17 for 6 h, a 5.3
± 0.7 increase (n = 3, mean ± S.D.) in luciferase
activity was observed (Fig.
4A), an effect completely blocked by co-incubations with
SB202190 (control, 2808 ± 312 RLU; SB202190, 2689 ± 675 RLU).
This response was quite specific because when co-transfections were performed
with chimeric constructs containing the transactivation domain of c-Jun
(1223), c-Fos (206313), or Elk-1 (307428), no increases
in luciferase activity were observed following rhIL-17 stimulation. In
contrast, phorbol 12-myristate 13-acetate potently stimulated transactivation
by c-Fos and c-Jun, whereas rhIL-1 activated the transactivational
capacity of Elk1 (Fig.
4B). Interestingly, rhIL-17 stimulated reporter
transactivation by CREB by a modest but statistically significant 2.1 ±
0.2, whereas the combination of forskolin (adenylate cyclase activator) and
rolipram (cAMP-dependent phosphodiesterase type IV inhibitor) provoked a 6.2
± 0.45 (n = 3, mean ± S.D.) increase
(Fig. 4A). The absence
of induction by rhIL-17 of c-Jun, c-Fos, or Elk-1 transactivational activity
was supported by the observations that the cytokine had little or no effect
(in contrast to rhIL-1
) on the phosphorylation (activity) of the
signaling intermediates ERK1/2 and JNK, which are known to phosphorylate and
increase the transactivational capacity of the latter transcription factors
(42,
43)
(Fig. 4C).
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Many studies have suggested that the IL-17 signal is transduced by the
JAK/STAT and NF-B cascades in a variety of cell types
(12,
20,
25). To address this issue in
the context of our cell culture models and the control of COX-2 gene
expression, we measured the phosphorylation (activation) state of critical
intermediates and performed transactivational analysis with reporter
constructs. As shown in Fig.
5A, rhIL-17 weakly stimulated the phosphorylation of
I
B-
, which reached a maximum after 10 min with no measurable
change in total cellular I
B-
. In contrast, TNF-
, a
prototypical pro-inflammatory cytokine and potent activator of the NF-
B
cascade potently induced I
B-
phosphorylation in less than 2 min
with a concomitant reduction in total I
B-
of more than 50%.
Interleukin-17 stimulated a time-dependent phosphorylation of the
transcription factor STAT3 (but not STAT1) with detectable Ser727
phosphorylation observed between 12 h, a zenith at 4 h, and gradual
decay thereafter. The prototypic STAT inducer IL-10 caused a similar magnitude
of phosphorylation but much sooner (after 20 min)
(Fig. 5B).
Furthermore, although TNF-
stimulated a 710-fold increase in
luciferase activity in cells transfected with a reporter construct harboring
five tandem NF-
B consensus sequences, rhIL-17 had no statistically
significant effect (Fig.
5C). However, when identical protocols were repeated in
transiently transfected HSF, rhIL-17 increased reporter activity by 1.78
± 0.31-fold (n = 4 determinations). 6 h post-stimulation,
rhIL-10, interferon-
and rhIL-17 all increased luciferase activity to a
significant degree in cells transfected with a reporter construct harboring
five interferon-stimulated response element tandem sequences
(Fig. 5C).
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COX-2 Promoter StudiesTo examine for elements of transcriptional control of the COX-2 gene via rhIL-17-stimulated SAPK2/p38 signaling, we conducted transient transfection analyses with a 416-bp (Bsu36I site) human COX-2 promoter construct harboring enhancer elements (44, 45) for critical transcription factors including a ATF/CRE site (58 to 53). We observed that rhIL-17 (10 ng/ml) stimulated a human COX-2 promoter-luciferase reporter construct by 1.76 ± 0.11 (mean ± S.D., n = 35)-fold (Fig. 6A). SB202190 abolished the induction completely (control, 11,739 ± 1,452 versus rhIL-17, 20,660 ± 1,291; rhIL-17 + SB202190; 7,635 ± 987 RLU). Companion experiments with a ATF/CRE mutant plasmid (see "Transfection Experiments" under "Experimental Procedures") revealed that the reporter activity was considerably lower than wild-type promoter plasmid (mutant, 6,722 ± 911 versus wild type (control), 11,739 ± 1,452 RLU, mean ± S.D., n = 3) and was refractory to rhIL-17 (compare 6,722 ± 911 versus rhIL-17, 6,945 ± 753 RLU, mean ± S.D., n = 3). To assess the role of ATF-2 in transcriptional activation of the COX-2 promoter, we used a previously described decoy strategy (46) in which the chimeric plasmid containing the N-terminal (196) transactivation domain of ATF-2 fused to the GAL4 DNA-binding domain was overexpressed prior to rhIL-17 stimulation. As can be seen in Fig. 6A, rhIL-17 induction of luciferase activity was completely suppressed, and indeed luciferase activity was observed to be below control values. Forced expression of other transcription factor-GAL4 chimers were without effect with the exception of pFA-CREB, where 30.3 ± 3.15%of rhIL-17-stimulated luciferase activity was blocked (Fig. 6A).
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Mutating the more proximal NF-B site (223/214) in the
human COX-2 promoter construct was without effect in terms of basal and
IL-17-stimulated luciferase activity (control, 10,639 ± 1,975
versus rhIL-17, 18,332 ± 2,070 RLU; 1.72 ± 0.13-fold
increase, n = 3; compare with wild type; see above) when
transfections were performed with human chondrocytes. However, when HSF were
used with the same protocol, basal reporter activity was reduced but not the
level of rhIL-17 induction (control, 8,117 ± 1,424 versus
rhIL-17, 14,998 ± 2,114 RLU; 1.85 ± 0.14-fold increase).
EMSA experiments using 32P-labeled ATF/CRE (COX-2)
oligonucleotides indicated a high level of endogenous nuclear protein binding
that was increased with rhIL-17 treatment
(Fig. 6B, upper
panel). Basal and induced binding was displaced by adding 10-fold excess
cold oligonucleotide but not by the mutant oligonucleotide (see above ATF/CRE
mutant COX-2 promoter studies). Furthermore, the addition of SB202190
abrogated induced and, to a large extent, basal oligonucleotide binding.
Confirming promoter studies, rhIL-17 stimulation of human chondrocytes
produced no increases in 32P-labeled NF-B (COX-2)
oligonucleotide binding in contrast to TNF-
(Fig. 6B, middle
panel). The human COX-2 promoter harbors a proximal NF-IL-6 site that
binds c/EBP transcription factors as either homodimers or heterodimers with
other transcription factors (e.g. NF-
B) and can increase
transcriptional activation
(44,
45). Time course studies
revealed that rhIL-17 increased NF-IL-6 binding after 30 min, reached a zenith
after 60 min, and thereafter declined so that at 90 min binding to the
oligonucleotide was similar to controls
(Fig. 6B, lower
panel). Supershifts with antibodies to the different isoforms of c/EBP
(see "Experimental Procedures") confirmed the presence of
c/EBP
only (data not shown).
Interleukin-17 Stabilizes COX-2 mRNAJudging by the
accumulated data (see above), it is unlikely that IL-17 modifies steady-state
COX-2 mRNA expression in our cell culture models exclusively at the
transcriptional level. As such we examined post-transcriptional mechanisms
involving strictly message stabilization and protein synthesis. As a first
approach, we employed classical techniques involving measuring COX-2 mRNA in
transcriptionally arrested cells (actinomycin D) in the absence or presence of
rhIL-17. We had previously reported
(34) that when HSF are
activated with rhIL-1 for 34 h (steady state) followed by
wash-out and a fresh change of medium, the elevated levels of COX-2 mRNA
declined rapidly such that within 2 h the levels were similar to control
non-stimulated cells (t
= 0.85 h). However, if
PGE2 was added to fresh medium (in the presence of actinomycin D),
COX-2 mRNA levels remained elevated (t
= 13 h)
(34). Similarly, as
represented in Fig. 7, rhIL-17
stabilized COX-2 mRNA and increased its half-life to
5.8 h, based on
multiple linear regressions of optical density of COX-2 mRNA versus
time (n = 3, y = 1.10.095x). The stabilizing
effect was abrogated by co-incubations with SB202190. We included
PGE2 in these experiments as a positive control and for purposes of
comparison.
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As mentioned earlier, the COX-2 mRNA has multiple copies of the Shaw-Kamen
AU-rich sequences that are believed to influence message stability. Recent
studies (40,
47) have provided evidence
that the AU-rich elements (6)
in the first 116 bp of the 3'-UTR may mediate COX-2 mRNA instability,
whereas we recently reported that proximal but also distal sequences mediate
PGE2-dependent stabilization
(34). To determine whether
rhIL-17-dependent COX-2 mRNA stabilization was manifested through the
3'-UTR sequences, we transfected HSF with CMV-driven chimeric expression
constructs containing luciferase cDNA (reporter) fused 3' to the human
COX-23'-UTR (Luc3'-UTR), AU-rich region (Luc+ARE),
AU-deleted region (LucARE-3'-UTR), or complete removal of the
3'-UTR region (Luc
3'-UTR). As shown in
Fig. 8A, cells
transfected with Luc
3'-UTR were refractive to any kind of
modulation. However, rhIL-17 increased luciferase activity 34-fold
(n = 5) in cells transfected with Luc3'-UTR and
Luc
ARE-3'-UTR constructs but not with Luc+ARE. If the cells were
washed out, and fresh medium was added, luciferase activity decreased
dramatically after 4 h. The latter decrease could be mitigated with the
addition of rhIL-17, a response that was apparently SAPK2/p38-mediated as the
induction was abrogated by co-incubations with SB202190. We obtained similar
results with PGE2, included as a positive control, with the sole
difference being that the ARE-rich region of the 3'-UTR is also
sensitive to eicosanoid stimulation (Fig.
8A).
|
Although COX-2 mRNA stabilization and protein synthesis are closely coupled
in our cell culture models, we verified whether our reporter system
(i.e. luciferase protein (activity) and mRNA) exhibited similar
coordination. As shown in Fig.
8B, in cells transfected with Luc3'-UTR and
stimulated with rhIL-17, luciferase mRNA decayed rapidly under wash-out
conditions but was stabilized with the addition of rhIL-17. Densitometric
scanning analysis (n = 4) revealed a t
for luciferase mRNA of under wash-out conditions of about 0.78 h (y =
2.151.38x) and t
in the presence
of rhIL-17 of
6.2 h (y = 2.40.195x). As shown in
Fig. 8B, the addition
of SB202190 blocked the stabilizing effect of rhIL-17.
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DISCUSSION |
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Normally not expressed in quiescent connective tissue cells or monocytes, the elevated levels of COX-2 mRNA, COX-2 protein, and PGE2 release observed in arthritis-affected synovial membranes have also been associated etiologically with the disease process (29, 30, 51). It has been suggested that the synovium, whether in a disease or normal state, is a PGE2-dependent tissue and that COX-2 behaves as a "master control gene" (34, 52). Thus, understanding the mechanisms responsible for dysregulated COX-2 expression is of considerable clinical concern, and the role of IL-17 in this regard takes on added significance from a therapeutic perspective.
To our knowledge, this is the first report associating IL-17 action with
post-transcriptional/translational control of any target gene, although the
SAPK/p38 pathway is connected with both levels of control as was shown
previously (33,
34,
47,
53). The bi-phasic pattern of
MKK3/6/SAPK/p38/ATF-2 phosphorylation induced by rhIL-17 was
observed previously with rhIL-1, and the second phase was attributed to
ambient accumulation of PGE2
(34). A bifurcation of the
IL-17-induced signaling pathway to Mnk1 occurs in a slower time frame (maximum
1 h) than MKK3/6/SAPK/p38/ATF-2 but is coincident with the Ser209
phosphorylation of the CAP (N7-methylguanosine)-binding
protein, eIF-4E. The latter post-translational modifications of both Mnk1 and
eIF-4E were apparently p38 MAPK-dependent and suggested a link between IL-17
action and translational control. However, because the role of eIF-4E
phosphorylation in translational control mechanisms is unclear
(5456),
and more targeted experimentation is required, such a suggestion would be
premature. Perhaps paradoxically, phosphorylation of eIF4E is sometimes
associated with a global inhibition of protein synthesis observed during cell
stress or serum deprivation
(5759).
This would provide a reasonable explanation for the conspicuous
phosphorylation of eIF4E after 16 h in our quiescent cultures (1% serum) both
in control and rhIL-17-treated wells (Fig.
3B).
Previous studies, using transformed cell lines for the most part, emphasized the role of proximal, AU-rich containing sequences in mediating COX-2 mRNA instability (6063). Pro-inflammatory cytokines, through SAPK/p38 activation, increase steady-state levels of COX-2 mRNA by stimulating the production of cognate RNA-binding proteins (e.g. HuR) that mitigate, through sequence-specific binding, mRNA degradation (Ref. 60; reviewed in Ref. 64). The precise mechanisms, however, remain ill-defined. We find that distal sequences were exclusively reactive to the stabilizing effects of IL-17 both in terms of mRNA (stabilization) and protein synthesis in our primary cell culture models. Indeed, the absence of the AU-rich element (123 nucleotides) had no effect on IL-17-dependent stabilization. Thus, the IL-17-modulated RNA-binding protein repertoire is likely to be different from that of other cytokines. In transient transfections of HSF with expression constructs of known RNA-binding proteins (AUF1, HuR, and tristetraprolin), only tristetraprolin destabilized distal COX-23'-UTR reporter constructs (65).2 Interestingly, tristetraprolin is targeted by SAPK/p38 for phosphorylation (60), which may alter its RNA binding properties and function.
There are a number of cis-elements found in the promoter region of
the COX-2 gene that may exert transcriptional control of which ATF/CRE, c/EBP
(132/124), and both NF-B sites (223/214 and
445/427) are the best studied
(44,
45,
66). In our cell culture
models, mutating the ATF/CRE site alone, is apparently sufficient to abrogate
IL-17 induction of COX-2 promoter activity and also to reduce basal promoter
activity compared with wild type. In many cell types, the ATF/CRE site is
activated by homodimers and heterodimers of c-Jun, c-Fos, and ATF/CREB family
members subsequent to serum, 12-O-tetradecanoylphorbol-13-acetate, or
growth factor stimulation (66,
67). However, rhIL-17 does not
stimulate the transactivating capacities of Fos/Jun proteins but clearly
favors those of ATF-2 and CREB-1. Decoy ATF-2 overexpression reduced all of
the induced (partial reduction with CREB-1) and some of the basal COX-2
promoter activity, although this does not necessarily mean that ATF-2
homodimers exclusively transactivate the COX-2 promoter at the ATF/CRE; such
results could be obtained if ATF-2-containing heterodimers were binding.
Judging by our EMSA studies, there is considerable basal binding activity at
the COX-2 ATF/CRE site, which is not surprising given that ATF-2 is expressed
constitutively at significant levels both in its native and, to a lesser
extent, phosphorylated forms in our cell cultures (see also Ref.
41). Induced binding by IL-17
was modest (although significant), and this may be a reflection of the fact
that IL-17 activation of the COX-2 promoter was less than 2-fold. More focused
experiments would be required to identify IL-17 induced ATF/CRE-binding
transactivating proteins.
As indicated above, several recent studies imply that the IL-17 signal is
transduced by the NF-B signaling cascade in a number of different cell
types (reviewed in Ref. 7). In
the present context, NF-
B may mediate transcriptional induction of the
COX-2 gene by IL-17 in bovine chondrocytes
(20). In contrast, we showed
here that in human chondrocyes/synovial fibroblasts, IL-17-dependent
activation of NF-
B is delayed and modest (compared with TNF-
)
and is not related temporally to COX-2 transcription, mRNA stabilization, or
protein synthesis. Basal COX-2 promoter activity was not affected when the
proximal NF-
B site was mutated in transfections using human
chondrocytes, nor did the mutation abrogate IL-17 induced COX-2 promoter
activity. The situation differed somewhat in HSF, because IL-17 can mildly
stimulate NF-
B transactivation activity, although the mutation in the
COX-2 proximal site did not compromise IL-17 induction of the COX-2 promoter.
Furthermore, using stably transfected HSF overexpressing dominant-negative
mutants of TRAF-2, TRAF6, or I
B-
, IL-17 activation of a
NF-
B reporter was abrogated to varying degrees but not the expression
of COX-2 mRNA.3 Taken
together, our data support the notion that the NF-
B signaling pathway
does not play an important role in mediating a response in chondrocytes to the
IL-17 signal. In support, IL-17 induction of the COX-2 gene in human
macrophages is indirect (delayed) and is dependent on IL-17-induced
TNF-
release, which in a feedback reaction (autocrine) activates
NF-
B (14). Similar
results were reported in osteoblasts where IL-17 induction of inducible
nitric-oxide synthase was mediated by NF-
B but only in combination with
TNF-
and not with IL-17 alone
(68).
Despite studies demonstrating that IL-17 can activate ERK and JNK pathways in chondrocytes (20), we could not, under carefully controlled conditions, reproduce these data in human chondrocytes or synovial fibroblasts. Our experiments with transactivation reporter systems show that downstream transcription factors such as Elk1, c-Jun, or c-Fos were not activated in our connective tissue cell culture models. Admittedly most of the previously reported studies were performed with nonhuman cell types, and thus species differences may be implicated, although it is possible that cell phenotype may also be important. For example, we showed that the cytokine could activate ERK1/2 in human monocyte/macrophage cultures, although this occurred only after 12 h of stimulation and did not coincide with COX-2 induction (12). This of course does not exclude the regulation of other target genes, and in this regard IL-17 is known to promote monocyte differentiation (7, 17). Furthermore, IL-17 is a potent stimulator of T-cell proliferation and differentiation, suggesting that in cells of lymphoid and myeloid origin, the mitogenic activity of the cytokine may be manifested through the MAPK pathway (7, 17, 69).
In summary, IL-17 is a widely acknowledged regulator of the immune and inflammatory response regulating directly key target genes like COX-2. The mechanisms controlling the COX-2 gene delineated in the present study involve highly coordinated regulation of both DNA-binding (transcriptional) and RNA-binding (post-transcriptional) proteins and may provide a molecular paradigm for the control of other IL-17 target genes. Interleukin-17 could prove to be an excellent therapeutic target in the clinical management of inflammatory diseases like RA using, for example, soluble IL-17 receptor preparations.
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FOOTNOTES |
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|| To whom correspondence should be addressed: Div. of Rheumatology and Clinical Immunology, Royal Victoria Hospital, McGill University Health Centre, 687 Pine Ave., W., Rm. M.11.22, Montréal, PQ H3A 1A1, Canada.
1 The abbreviations used are: IL, interleukin; MAPK, mitogen-activated
protein kinase; COX-2, cyclooxygenase-2; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; PGE2, prostaglandin E2; DMEM, Dulbecco's
modified Eagles medium; FCS, fetal calf serum; rh, recombinant human; SAPK,
stress-activated protein kinase; JNK, c-Jun N-terminal kinase; ATF-2,
activating transcription factor-2; PKA, cAMP-dependent protein kinase; CREB-1,
cAMP-response element binding protein; MEK3 or MKK3/6, p38 MAPK kinase; UTR,
untranslated region; ARE, AU-rich element; MnK1, MAPK interacting kinase;
eIF-4E, eukaryotic initiation factor 4E; IB-
, inhibitor of
NF-
B; JAK, Janus family tyrosine kinase; STAT, signal transducer and
activator of transcription; ERK1/2 (p42/44), extracellular signal-regulated
kinase; HC, human chondrocyte(s); HSF, human synovial fibroblast(s); CMV,
cytomegalovirus; RA, rheumatoid arthritis; OA, osteoarthritis; TNF-
,
tumor necrosis factor-
; RT, reverse transcription;
-gal,
-galactosidase; EMSA, electrophoretic mobility shift assay; c/EBP,
CCAAT-enhancer-binding protein; RLU, relative light unit.
2 W. H. Faour, A. Mancini, Q. W. He, and J. A. Di Battista, unpublished
observations.
3 W. H. Faour, A. Mancini, Q. W. He, and J. A. Di Battista, manuscript in
preparation.
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REFERENCES |
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