 |
INTRODUCTION |
Interleukin-18 (IL-18)1
is a proinflammatory cytokine associated with various pathological
conditions including rheumatoid arthritis (RA). IL-18 induces the
release of Th1 cytokines by T cells and macrophages and also stimulates
the production of inflammatory mediators, such as chemokines by
synovial fibroblasts or nitric oxide by macrophages and chondrocytes
(1, 2). Additionally, we have shown that IL-18 acts upon endothelial
cells to induce angiogenesis and cell adhesion (3, 4). IL-18 is mainly
produced by activated macrophages, whereas the IL-18 receptor (IL-18R)
is expressed on T lymphocytes, natural killer cells, macrophages,
neutrophils, and chondrocytes (1, 5, 6). The IL-18R complex is composed
of two protein chains 
and 
. The IL-18R is the extracellular
binding domain of the IL-18R complex, whereas the IL-18R is the
signal transducing chain. When IL-18 binds to the IL-18R, it induces
the formation of an IL-1R-associated kinase (IRAK)/TNF
receptor-associated factor 6 (TRAF-6) complex that subsequently
activates nuclear factor 
B (NFB) in Th1 cells (7) and in EL4/6.1
thymoma cells (8). IL-18 also operates through IRAK-independent
pathways that remain to be elucidated. For instance, IL-18 mediates
interferon-
(INF-) production by the mitogen-activated protein
kinases p38 and p42/44, also known as ERK1/2 (9).
Phosphatidylinositol 3-kinase (PI3-kinase) is a lipid kinase that
consists of catalytic (p110) and regulatory (p85) subunits. PI3-kinase
catalyzes the phosphorylation of the inositol phospholipids at position
3 to generate phosphatidylinositol 3-phosphates, phosphatidylinositol 3,4-biphosphates, and phosphatidylinositol 3-5-triphosphates. These
phosphorylated lipid products act as second messengers, activating
protein kinases such as Akt (also known as protein kinase B).
PI3-kinase is activated by a large spectrum of cytokines, growth
factors, and hormones (10). This activation of PI3-kinase is generally
regulated by receptor tyrosine kinase and non-receptor tyrosine kinase
(NRTK). PI3-kinase can also be activated by G-protein-coupled receptors
or by the small GTPase Ras (11, 12). PI3-kinase has been implicated as
a key signaling molecule for transcription factor activation, protein
synthesis, angiogenesis, and cell adhesion (13, 14). We recently showed
that the PI3-kinase inhibitor LY294002 inhibited RA synovial fibroblast
IL-18-induced VCAM-1 expression by 50% when used alone and by 85%
when used with the NFB inhibitor pyrrolidine dithiocarbamate (2).
These results suggested the existence of at least two independent
pathways involved in IL-18-induced adhesion molecule expression.
Further investigation of IL-18-induced signaling mechanisms revealed
involvement of Src, an NRTK that functions in ligand-induced cellular
responses, such as leukocyte survival, adhesion, migration, and
proliferation. Src has also been implicated in various cancers and in
bone resorption (15). Activation of Src requires phosphorylation at its
activation site Tyr-418 concomitantly with decreased phosphorylation at
its negative regulatory site Tyr-529; regulation also involves phosphorylation on other residues such as Tyr-215. c-Src activation by
tyrosine kinase receptors leads to translocation from the plasma membrane to the cytoskeleton, where Src interacts with a host of
proteins that orchestrate cell-matrix adhesion and cell migration (16).
Here we found that IL-18 directly activates Src with rapid kinetics,
and Src activation appears to be an early event common to the
PI3-kinase/Akt and ERK1/2 pathways.
There is precedence for the involvement of transcription factor
activation in IL-18-mediated immune and inflammatory functions. For
instance, IL-18 activates AP-1 and NFB in the Jurkat T cell leading
to IL-2 expression (17). We therefore examined involvement of the
PI3-kinase and ERK1/2 signaling pathways in AP-1 activation. Because
many cytokine-mediated functions, including IL-18-induced IFN-
expression, are regulated via NFB activation (18, 19), its
significance in IL-18-induced VCAM-1 expression was examined. One
approach for determining the role of the NFB pathway is by way of
IRAK involvement, because IRAK is known to be associated with NFB
upon IL-18 stimulation (20, 21). Furthermore, IRAK was initially cloned
and characterized as a kinase associated with the IL-1 receptor (22),
suggesting its importance to IL-18 signaling as IL-18 is a member of
the IL-1 family.
Because antisense oligodeoxynucleotides (ODNs) offer a potential gene
therapy strategy to block transcription or translation of specific
genes, antisense ODNs to relevant signaling molecules were employed to
examine RA synovial fibroblast signaling events following IL-18
stimulation. Ultimately, the treatment effect of antisense ODN on
IL-18-induced VCAM-1 expression implicated specific signaling molecules
involved in RA synovial fibroblast VCAM-1 expression. The ability of
these antisense ODNs to inhibit IL-18-induced activation of the
transcription factor NF-B and downstream VCAM-1 expression was also assessed.
We examined the signal transduction mechanisms by which IL-18 induces
VCAM-1 expression in RA synovial fibroblasts. Our findings demonstrate
that IL-18 induced VCAM-1 expression through Src kinase, PI3-kinase/Akt, and ERK1/2 pathways. The important role of the IRAK/NFB pathway in VCAM-1 expression was also elucidated. Finally, we describe a new signaling cascade involving Src/Ras/Raf/ERK/AP-1 in
IL-18-stimulated RA synovial fibroblasts.
| |
EXPERIMENTAL PROCEDURES |
Reagents--
Recombinant human IL-18 (specific activity
4.1 × 104 units/mg) was purchased from Peprotech
(Rocky Hill, NJ). Orthovanadate, para-nitrophenyl phosphate,
leupeptin, aprotinin, phenylmethylsulfonyl fluoride, dimethyl sulfoxide
(Me2SO), IGEPAL CA-630, protein A- and G-agarose,
pertussis toxin, and phosphatidylinositol were bought from Sigma.
Protease inhibitor mixture tablets were obtained from Roche Molecular
Biochemicals. Modified radioimmunoprecipitation (RIPA) lysis buffer was
prepared according to Upstate Biotechnology, Inc., protocol, with final
concentrations being Tris-HCl (50 mM, pH 7.4), Nonidet P-40
(1%), NaCl (150 nM), EDTA (1 mM),
phenylmethylsulfonyl fluoride (1 mM),
aprotinin/leupeptin/pepstatin (1 µg/ml each), NaF (1 mM).
The anti-Src-agarose beads, clone GD11, a mouse monoclonal IgG1, was
purchased form Upstate Biotechnology, Inc. (Lake Placid, NY), as was
Src kinase and the Src kinase assay kit, including Src kinase reaction
buffer (SrcRB), Src substrate peptide, Src manganese/ATP mixture, and
P81 phosphocellulose paper. The radioisotope [-32P]ATP
(3000 Ci/mmol) was obtained from PerkinElmer Life Sciences. Phosphoric
acid (0.75%) diluted from 85% stock solution and trichloroacetic acid
was from Sigma. LY294002, PP2, SB203580, and PD98059 were purchased
from Calbiochem. Monoclonal mouse anti-human VCAM-1, clone 4B9 that
recognizes domain 1 of VCAM-1, was a generous gift from Dr. Roy Lobb
(Biogen, Cambridge, MA); mouse IgG1 antibody (negative control) was
purchased from Coulter Clone (Hialeah, FL); goat anti-mouse PE (Jackson
ImmunoResearch) was used as secondary antibody for flow experiments.
Mouse monoclonal anti-phosphotyrosine antibody (clone 4G10), mouse
monoclonal anti-human phospho-ERK1/2 antibody, and the Ras activation
detection kit were purchased from Upstate Biotechnology, Inc. (Lake
Placid, NY). Polyclonal rabbit anti-human phospho-Src antibody
(Tyr(P)418) and polyclonal rabbit anti-human
phospho-Raf-1 (Tyr(P)340 and Tyr(P)341)
antibody were obtained from BIOSOURCE
International (Camarillo, CA). Mouse monoclonal anti-human phospho-Akt
(Ser(P)473) antibody was purchased from Cell Signaling
Technology (Beverly, MA) and BIOSOURCE
International. Mouse polyclonal anti-human IRAK antibody was obtained
from BD PharMingen, and rabbit polyclonal anti-human NFB p65
antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA). Mouse monoclonal anti-tubulin antibody was obtained from Oncogene
Research Products (Boston, MA). Goat anti-rabbit IgG horseradish
peroxidase-conjugated antibody was purchased from Sigma. Protein
estimation reagents (BCA kit) were from Pierce. Enhanced
chemiluminescence Western blotting detection reagents and sheep
anti-mouse IgG horseradish peroxidase conjugated antibody were obtained
from Amersham Biosciences. LipofectAMINE and LipofectAMINE PlusTM
Reagents were obtained from Invitrogen.
Cell Culture--
Fibroblasts were isolated from synovium
obtained from RA patients meeting the American College of Rheumatology
criteria for RA who had undergone total joint replacement surgery or
synovectomy (23). Fresh synovial tissues were minced and digested in
solution of dispase, collagenase, and DNase. Cells were used at passage 5 or older, at which time they were a homogeneous population of fibroblasts. Synovial fibroblasts were grown in 175-mm tissue culture
flasks (Falcon, Franklin Lakes, NJ) at 37 °C, in a humidified atmosphere with 5% CO2. Upon confluence, cells were
passaged by brief trypsinization as described previously (24).
Cell Lysis and Immunoblotting--
RA synovial fibroblasts
were plated onto 6- or 10-cm Petri dishes (Falcon) at 1 × 105 cells/ml and allowed to adhere for 24 h at
37 °C in 5% CO2 atmosphere. Alternatively, for ODN
experiments, fibroblasts were plated at 4 × 106
cells/well on 6-well plates. Fibroblasts were serum-starved for at
least 14 h before stimulation with IL-18 (10 nM) for
0, 5, 10, and 20 min. At the end of each period, supernatants were
gently aspirated, and fibroblasts were lysed in extraction buffer
containing 100 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM NaP2O4, 2 mM
Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mM phenylmethylsulfonyl
fluoride, and protease inhibitors (1 tablet/10 ml). For experiments
with signaling inhibitors, RA synovial fibroblasts were preincubated
with the respective inhibitor for 60-120 min before activation with
IL-18. For the ODN experiments the cells were treated as detailed and lysed similarly. Nuclei were pelleted (1250 × g at
4 °C for 5 min), and supernatants of different samples were
collected for determination of protein content using a BCA protein
assay kit. Cell lysates were mixed 1:1 with Laemmli's sample buffer,
boiled for 5 min, and then centrifuged at 10,000 × g
for 10 min. Equal amounts (or 15 µg) of each sample was subjected to
10% SDS-PAGE. Separated proteins were electrophoretically transferred
from the gel onto nitrocellulose membranes using a semi-dry
transblotting apparatus (Bio-Rad). To block nonspecific binding,
membranes were incubated with 5% milk in Tris-buffered saline
containing 0.1% Tween 20 (TBST) for 1 h at room temperature.
Blots were incubated in respective primary antibody in TBST + 5% milk
at 4 °C overnight. After washing with TBST, blots were incubated
with horseradish peroxidase-conjugated sheep anti-mouse IgG (1:10,000)
or with goat anti-rabbit IgG (1:10,000) for 1 h at room
temperature. An ECL detection system (Amersham Biosciences) was used to
detect specific protein bands. Different bands were then scanned and quantitated using the software UN-SCAN-IT version 5.1 (Silk Scientific, Orem, UT). Blots were subsequently stripped and restained with antibody
to tubulin to determine relative band densities.
PI3-Kinase Assay--
RA synovial fibroblasts (8 × 105 cells) were plated in 10 cm-dishes in RPMI 1640 containing 10% FBS, 1% penicillin/streptomycin. Once cells were
80-90% confluent, they were further incubated in serum-free RPMI 1640 for at least 14 h. RA synovial fibroblasts were then stimulated
with IL-18 (10 nM) for 0, 10, and 20 min at 37 °C. At
the end of the incubation, cell lysate was prepared. Protein content of
each sample was quantitated using a BCA protein assay kit and
normalized according to the protein concentration. 500 µg of each
sample in 500 µl of lysis buffer was incubated overnight at 4 °C
with 5 µl of rabbit anti-PI3-kinase antibody directed against the
85-kDa regulatory subunit. 60 µl of protein A-agarose conjugate (50%
slurry in PBS) was added to each sample and further incubated for
1 h at 4 °C. The immunoprecipitates were collected by
centrifugation at 14,000 × g for 10 s. The
immunoprecipitates were then washed 3 times with buffer A (137 mM NaCl, 20 mM Tris-HCl, pH 7.4, 1 mM CaCl2, 1 mM MgCl2,
and 0.1 mM sodium orthovanadate) containing 1% nonionic
detergent IGEPAL CA-630, followed by 3 washes with buffer B (0.1 M Tris-HCl, pH 7.4, 5 mM lithium chloride, and
0.1 mM sodium orthovanadate) and 3 washes with TNE (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 5 mM EDTA) containing 0.1 mM sodium
orthovanadate. To each sample, the following reagents were added
sequentially: 50 µl of TNE, 10 µl (20 µg) of phosphatidylinositol (PI, in 10 mM Tris-HCl, pH 7.4 containing 1 mM
EGTA), and 10 µl of 100 mM MgCl2. The PI
reaction was initiated with addition of 5 µl of
[-32P]ATP. The reaction mixture was incubated at
37 °C for 15 min with continuous agitation. The reaction was stopped
by addition of 20 µl of 6 N HCl. Radiolabeled lipid was
extracted from the reaction sample by adding 160 µl of
CH3Cl/MeOH (1:1), vortexing, and separating the organic and
aqueous phases by centrifugation for 10 min at 14,000 × g. 50 µl of radiolabeled lipid containing the lower
organic phase were spotted onto oxalate-treated TLC plates (Fisher) and
developed in CHCl3/MeOH/H2O/NH4OH
(60:47:11.3:2). TLC plates were dried and autoradiographed.
Ras Activation Assay--
Ras activation was studied using a Ras
activation kit (Upstate Biotechnology, Inc.). RA synovial fibroblasts
were stimulated with IL-18 (10 nM) for different times. At
each time point, synovial fibroblast extracts were prepared with cell
lysis buffer, and protein content in each sample was quantitated. The
Ras activation assay involved two steps. In the first step, the
activated form of Ras was immunoprecipitated from 800 µg of each
synovial fibroblast lysate sample with an immobilized Raf-1-Ras binding
domain (Raf-1-RBD) and subsequently run on 10% SDS-PAGE as
described above. The presence of activated Ras in samples was then
detected by probing with a specific mouse monoclonal anti-Ras antibody
(1 µg/ml). Different bands were then scanned and quantitated using an
imaging densitometer.
Src Kinase Activation Assay--
Src kinase was first
immunoprecipitated with agarose beads conjugated with anti-Src antibody
(mouse monoclonal IgG1). RA synovial fibroblasts were plated at 1 × 106 cells on 10-cm culture dishes, adhered overnight in
complete media, serum-starved overnight, and stimulated with IL-18 (10 nM) for 10 min. Cells were washed in cold PBS and lysed
with 0.5 ml of RIPA buffer. The total cell lysate was diluted to 1 µg/µl with PBS, and 1 mg was mixed with 4 µg (8 µl) of anti-Src
antibody-conjugated agarose beads and gently rocked at 4 °C for
2 h. Beads were collected by microcentrifuging (5 s at 14,000 × g), the supernatant was drained, and beads were then
washed (3 times) with ice-cold PBS. Samples containing agarose beads,
Src antibody, and Src protein in complex were then used to determine
Src kinase activation.
Direct activation of Src was examined with n Src kinase assay kit
(Upstate Biotechnology, Inc.) to measure the ability of activated Src
to act on known substrate. Stock solutions necessary for the assay were
prepared as follows: Src substrate peptide (600 µM,
diluted in SrcRB), purified Src kinase (p60 c-Src, 20units/10 µl/test), [-32P]ATP (PerkinElmer Life Sciences, 1 mCi/100 µl, 3000 Ci/mmol, further diluted to 1 µCi/µl with Src
manganese/ATP mixture), and 0.075% phosphoric acid (diluted from 85%
with PBS). Substrate peptide (10 µl, 150 µM final
concentration) was added to 10 µl of SrcRB, to which was added 10 µl of Src (p60, c-Src, 20 units) or immunoprecipitated sample (200 µg minimum) and 10 µl of diluted [-32P]ATP (10 µCi) in a microcentrifuge tube. The reagent mixture was
incubated for 10 min at 30 °C with agitation. To precipitate peptides, 20 µl of 40% trichloroacetic acid was added to each mixture and incubated at room temperature for 5 min. Onto the center of
P81 phosphocellulose paper squares, 25 µl of each sample was spotted.
Squares were washed with 0.75% phosphoric acid (5 times for 5 min) and
acetone (once for 3 min). Samples were read in a scintillation counter,
and counts/min of immunoprecipitated enzyme samples were compared with
counts/min of the background control samples (no enzyme). This assay
was performed with fibroblasts from 4 different RA donors.
Preparation of Oligonucleotides and Lipofection of RA Synovial
Fibroblasts--
Sequences of the ODNs employed in this study are
listed in Table I. Antisense ODNs were
selected for sequence target to c-Src (25), IRAK-1 (26), PI3-kinase
(27, 28), and ERK1/2 (29). The corresponding sense ODN was used as
control for each antisense ODN. The ODNs were synthesized and purified
by the Northwestern University Biotechnology Laboratory and modified
with phosphorothioate. Lipofection-encapsulated ODNs were prepared
using LipofectAMINE and LipofectAMINE Plus Reagent from Invitrogen.
Each ODN was reconstituted to 0.4 µg/µl concentration in double
distilled water and then diluted in Opti-MEM media as detailed. For
transient transfection of cells, ODN/Plus reagent-LipofectAMINE complex
was prepared with 40 µg of ODN/100 µl of serum-free Opti-MEM with
2.5 µl of Plus reagent and 4 µg of LipofectAMINE Reagent/100 µl
of serum-free Opti-MEM and incubation at room temperature for 30 min,
followed by dilution with another 800 µl of Opti-MEM serum-free
medium for an additional 15-min incubation. RA synovial fibroblasts
were plated at 90% confluency at 4 × 105 cells per
well on 6-well plates or per 6-cm dish and allowed to adhere in RPMI,
10% FBS, 1% penicillin and streptomycin. After attachment, cells were
then treated with 5 µM of antisense or sense ODN by
incubation with 1 ml of ODN/Plus reagent/LipofectAMINE complex for
5 h, followed by media change to complete RPMI, 10% FBS, 1%
penicillin and streptomycin overnight. The media were also changed to
serum-free RPMI, 1% penicillin and streptomycin for 8-10 h prior to
stimulation with IL-18 (10 nM, 10 min, for the Western blot
and Src kinase experiments; 5 nM, 8 h, for VCAM-1 expression by flow cytometry experiments).
Flow Cytometry--
For the chemical inhibition experiments, RA
synovial fibroblasts were plated onto 6-cm Petri dishes (Falcon) at
1 × 105 cells/ml and allowed to adhere overnight, and
cells were pretreated with pertussis toxin (100 ng/ml) for 12 h
before stimulation with IL-18 (10 nM) for 8 h. For the
ODN experiments, RA synovial fibroblasts were plated and treated as
detailed above prior to IL-18 (5 nM) stimulation for 8 h. Cells were harvested with a cell scraper and transferred to
fluorescence-activated cell sorting (FACS) tubes (BD PharMingen). Cells
were then treated with mouse anti-VCAM-1 or isotype-matched control (5 µg/ml) as primary antibody followed by incubation for 30 min with
PE-conjugated goat anti-mouse antibody (1.5 µg/ml). Samples were
washed twice with PBS, 1% FBS, and then fixed with 1%
paraformaldehyde. Samples were assayed using an Epics XL-MCL flow
cytometer (Beckman Coulter). Prior to data acquisition, the PE channel
was standardized using fluorescent beads (Rainbow Beads, Spherotech,
Libertyville, IL). Isotype-matched control values were subtracted from
the test results.
Cellular ELISA--
RA ST fibroblasts were plated on 96-well
tissue culture plates in RPMI 1640, 10% fetal calf serum and adhered
for 14 h. Cells were preincubated with specific inhibitors or
Me2SO vehicle control in RPMI 1640, 2% fetal calf serum
before stimulation with IL-18 (5 nM) for 8 h.
Inhibitors (LY294002, PP2, SB203580, and PD98059) or vehicle
Me2SO were applied to cells for 60 min, and then cells were
stimulated with IL-18 (5 nM) for 12 h. Cell viability
was judged by trypan blue exclusion and was >90%. RA synovial
fibroblasts were successively fixed in 3.7% formalin in PBS and
blocked in PBS, 1% bovine serum albumin, 5% goat serum for 15 min.
After successive incubations in mouse anti-human VCAM-1 or
isotype-matched control for 2 h, goat anti-mouse IgG
peroxidase-conjugated antibody was added for 1 h, and the ELISA
was developed with tetramethylbenzidine substrate. The reaction was
stopped with 1 N H2SO4 before
reading at 450 nm with a Bio-Rad model 550 microplate reader.
Electrophoretic Mobility Shift Assay--
RA synovial
fibroblasts (1 × 106) were grown on 10-cm dishes
(Falcon) and starved for more than 12 h in serum-free RPMI 1640, 1% penicillin/streptomycin, before stimulation with 10 nM
of IL-18 for 0, 1, 2, and 4 h. For experiments using specific
inhibitors, cells were pretreated for 2 h before stimulation with
IL-18 (10 nM). Cytoplasmic and nuclear extracts were
prepared as described previously (4), and 10 µg of nuclear extract
from each sample were incubated with 1.5 µg of poly(dI-dC) and 47 fmol of 32P-end-labeled ODN probe containing the
DNA-binding site for AP-1 (Promega, Madison, WI). Cold ODN competition
control was performed using labeled and unlabeled ODNs at a ratio of
1:2 and 1:4. Protein-DNA complexes were resolved on 4% polyacrylamide
gels and visualized by autoradiography.
Statistical Analysis--
Data were analyzed using the Wilcoxon
rank order test, and p values less than 0.05 were considered
statistically significant.
| |
RESULTS |
IL-18 Promotes PI3-kinase Activation--
We have shown previously
(4) that the PI3-kinase inhibitor LY294002 (10 µM)
inhibits RA synovial fibroblast VCAM-1 expression. These results
suggested PI3-kinase might be involved in IL-18-induced VCAM-1
expression. To confirm activation of PI3-kinase by IL-18, we employed
an in vitro kinase assay using phosphatidylinositol (PI) as
a substrate (Fig. 1). IL-18 (10 nM) stimulation resulted in a 5 ± 1.3-fold increase
of PI3-kinase activity, which peaked at 10 min. This IL-18-induced
increase in PI3-kinase activity declined rapidly at 20 min.
View larger version (42K):
[in this window]
[in a new window]
|
Fig. 1.
IL-18 activates PI3-kinase in RA synovial
fibroblasts. Serum-starved cells were stimulated for the indicated
times with IL-18 (10 nM). Extracts were subjected to
immunoprecipitation with a rabbit antibody against the p85 subunit of
PI3-kinase. PI3-kinase activity was determined using an in
vitro kinase assay as described under "Experimental
Procedures." Products were separated by thin layer chromatography.
Activity of PI3-kinase is presented as production of
phosphatidylinositol phosphate (PIP), indicated by
arrows. Experiments were repeated two times with essentially
identical results.
|
|
IL-18 Activates Phosphorylation of Akt through PI3-Kinase--
The
lipid products of PI3-kinase are known to activate serine-threonine
protein kinase Akt through phosphorylation of serine 473 and threonine
308 (30). We therefore examined the ability of IL-18 to induce Akt
phosphorylation by probing Western blots of proteins from
IL-18-stimulated RA synovial fibroblasts using antibodies to
phosphorylated Akt (serine 473). IL-18 induced Akt phosphorylation in a
time-dependent manner (Fig.
2, A and B), and
after 5 min there was a 4-fold increase in Akt activation, with maximum
activation (11-fold) observed at 20 min (p < 0.05, n = 3). This increase in Akt phosphorylation could be
blocked when RA synovial fibroblasts were preincubated with the
PI3-kinase inhibitor LY294002 (10 µM) (Fig. 2,
C and D). Pretreatment resulted in ~80 ± 17% (mean ± S.E.) decrease in Akt phosphorylation
(p < 0.05, n = 3). The role of
PI3-kinase in Akt activation was more specifically demonstrated through
inhibition with PI3-kinase AS ODN.
View larger version (28K):
[in this window]
[in a new window]
|
Fig. 2.
IL-18-induced RA synovial fibroblast Akt
phosphorylation and inhibition by the PI3-kinase inhibitor LY294002 or
PI3-kinase antisense ODN. A, serum-starved cells
were stimulated for the indicated times with IL-18 (10 nM).
Cell lysates were blotted for phospho-Akt (*p-Akt). The same
blot was stripped and reprobed with a mouse anti-human tubulin
antibody. B, blots were scanned and analyzed for
quantification with the UN-SCAN-IT software. Band intensities for
phospho-Akt were normalized to the corresponding band intensities for
tubulin. Data from three RA donors were averaged and are represented as
mean ± S.E., expressed as fold increase with respect to
nonstimulated (NS) cells; *, p < 0.05 versus time 0; n = number of donors.
C, RA synovial fibroblasts were serum-starved for
16 h before treatment with LY294002 (10 µM) or
Me2SO control. After 2 h of pretreatment with
inhibitors, IL-18 was added to the culture media for the indicated
times. Cell lysates were prepared and subjected to Western blot
analysis for activation of Akt. D, blots were scanned
and quantitated. Values for phospho-Akt were normalized to the
corresponding values for tubulin. Data from three RA donors were
averaged and are represented as the mean ± S.E., expressed as the
percentage of inhibition of Akt phosphorylation for cells pretreated
with Me2SO for 10 and 20 min; *, p < 0.05 versus time 10 or 20 min; n = number of
donors. E, antisense PI3-kinase
(PI3K) ODN inhibited RA synovial fibroblast Akt expression.
RA synovial fibroblasts were treated with antisense (AS)
PI3-kinase ODN or the corresponding control sense (S) ODN
and incubated with or without IL-18 (10 nM) for 20 min.
Total cell lysates were prepared for equal protein loading and
separation by Western blot. Phospho-Akt was detected with mouse
monoclonal anti-human phospho-Akt antibody. F, data
from four RA donors were scanned and analyzed for quantification with
the UN-SCAN-IT software, and band intensities for Akt were normalized
to tubulin to quantitate relative Akt protein expression and presented
as the mean ± S.E.; *, p < 0.05;
n = number of donors; S, sense ODN;
AS, antisense ODN.
|
|
Antisense PI3-Kinase Inhibits RA Synovial Fibroblast Akt
Expression--
To examine involvement of the Akt pathway in IL-18
signaling through a more specific approach, antisense ODN targeting
PI3-kinase and corresponding control sense ODN were used to treat RA
fibroblasts with subsequent IL-18 (10 nM) stimulation.
Levels of Akt expression were determined by Western blot of total cell
lysates. IL-18 (10 nM) stimulated Akt protein expression
from nearly undetectable basal levels. To confirm the above data and,
more importantly, to demonstrate specificity of inhibition by antisense
ODN, treatment with antisense PI3-kinase ODN reduced the level of Akt
protein expression almost completely versus treatment with
corresponding sense ODN, 100 ± 17.92 to 1.03 ± 0.11 (p < 0.05, n = 4), which is consistent
with the expectations for Akt as a target of PI3-kinase-activated signals (Fig. 2, E and F).
IL-18 Activates the Nonreceptor Protein Tyrosine Kinase
c-Src--
We next investigated signaling pathways involved in
PI3-kinase/Akt activation. Because PI3-kinase is generally activated by protein-tyrosine kinase (PTK), we examined PTKs activated by IL-18 using a mouse monoclonal antibody (4G10), which specifically reacts with tyrosine-phosphorylated proteins. IL-18 (10 nM)-induced protein tyrosine phosphorylation in RA synovial
fibroblasts in a time-dependent manner, showing maximum
activation at 10 and 20 min by Western blot. Two major bands were
detected at 50-75- and 35-50-kDa ranges (Fig.
3A). Proteins phosphorylated
at tyrosine residues corresponding to molecular mass ranges of
60-65 and 42-44 kDa are Src kinases and MAPK, respectively. Like
PI3-kinase, Src kinases are located near the cell membrane, and their
activation occurs relatively early in the signaling sequence (31).
Moreover, it has been demonstrated that c-Src recruits and activates
PI3-kinase in NIH3T3 fibroblasts (32). Thus, we examined the effect of
IL-18 stimulation on Src activation by Western blot and found that
IL-18 induces the phosphorylation of Src in a
time-dependent manner (p < 0.05, n = 3). As compared with nonstimulated RA synovial
fibroblasts, a 4-fold increase in phosphorylated Src was observed at 10 and 20 min (Fig. 3, B and C).
View larger version (30K):
[in this window]
[in a new window]
|
Fig. 3.
Protein tyrosine phosphorylation pattern and
c-Src phosphorylation in IL-18-stimulated RA synovial fibroblasts.
A, serum-starved RA synovial fibroblasts were
stimulated with IL-18 for various time points as indicated. Cells were
lysed with lysis buffer, and the protein content of each sample was
quantitated. Each sample (15 µg) was resolved by 10% SDS-PAGE and
probed with mouse monoclonal antibody 4G10 (750 ng/ml) to detect the
phosphorylated proteins. Blots were stripped and reprobed with mouse
anti-human tubulin antibody. Experiments were repeated two times with
essentially identical results. IL-18 induced tyrosine phosphorylation
of a number of proteins. An increase in tyrosine phosphorylation was
observed for 10 and 20 min in the molecular weight range of the Src
kinase family (50-60 kDa) (*p-Src) and the MAPK family
(35-45 kDa) (*p-MAPK). B, serum-starved
cells were stimulated for the indicated time with IL-18 (10 nM). Cell lysates were blotted for phospho-c-Src
(*p-c-Src). The same blot was stripped and reprobed with a
mouse anti-human tubulin antibody. C, blots were
scanned and analyzed for quantification with the UN-SCAN-IT software.
Band intensities for phospho-c-Src were normalized to the corresponding
band intensities for tubulin. Data from three RA donors were averaged
and are represented as the mean ± S.E., expressed as fold
increase with respect to nonstimulated (NS) cells; *,
p < 0.05 versus time 0; n = number of donors.
|
|
IL-18 Activates Src Kinase in RA Synovial Fibroblasts--
A
direct role for IL-18 activation of Src was examined by an enzymatic
assay. Confluent RA synovial fibroblasts were stimulated with IL-18 (10 nM) or control (media alone) for 20 min; their total
lysates were collected and immunoprecipitated to isolate Src; and the
Src kinase assay was performed with Src substrate peptide and
[-32P]ATP. Results represent fold increase in
counts/min versus non-stimulated control cells. IL-18 (10 nM) significantly induced Src activation greater than a
4-fold over basal activation (4.35 ± 1.92) (mean ± S.E.) in
the four different RA patient samples examined (p < 0.05) (Fig. 4A).
View larger version (19K):
[in this window]
[in a new window]
|
Fig. 4.
IL-18 directly activates Src kinase in RA
synovial fibroblasts, and antisense c-Src ODN treatment inhibits
IL-18-induced Akt phosphorylation. A,
serum-starved RA synovial fibroblasts were stimulated for 10 min with
IL-18 (5 nM). Cell lysates were prepared with RIPA buffer,
immunoprecipitated with anti-Src antibody-conjugated agarose beads, and
then assayed for Src kinase activity in the presence of peptide
substrate, [-32P]ATP and manganese/ATP mixture. Data
from five RA donors were averaged and are represented as mean ± S.E., expressed as fold increase relative to nonstimulated control; *,
p < 0.05; n = number of donors.
B, antisense c-Src ODN inhibits Akt expression. RA
synovial fibroblasts were treated with antisense (AS) c-Src
ODN or the corresponding control sense (S) ODN and incubated
with or without IL-18 (10 nM) for 20 min. Total cell
lysates were prepared for equal protein loading and separation by
Western blot. Akt was detected with mouse monoclonal anti-human
phospho-Akt antibody (*p-Akt). C, data from
four RA donors were scanned and analyzed for quantification with the
UN-SCAN-IT software, and band intensities for Akt were normalized to
tubulin to quantitate relative Akt protein expression and presented as
the mean ± S.E.; *, p < 0.05; n = number of donors; S, sense ODN; AS,
antisense ODN.
|
|
Antisense c-Src ODN Inhibits RA Synovial Fibroblast Phospho-Akt
Expression--
To examine the involvement of the Akt pathway in IL-18
signaling, antisense ODN targeting of the gene for c-Src and
corresponding control sense ODN were used to treat cells with
subsequent IL-18 (10 nM) stimulation. The level of
phospho-Akt expression was determined by Western blot of total cell
lysates. Again, IL-18 (10 nM) stimulated phospho-Akt
protein expression from nearly undetectable basal (or non-stimulated)
levels, similarly to the data in Fig. 2E. Antisense c-Src
ODN caused a reduction in the level of phospho-Akt expression,
apparently through specific reduction in c-Src (Fig. 4, B
and C). Antisense c-Src ODN inhibited phospho-Akt expression by 75%, from 100 ± 15.13 to 25.7 ± 10.96 (mean ± S.E.) (p < 0.05, n = 4). This finding
suggests that not only does IL-18-induced phospho-Akt expression occur
through the activation of c-Src but that this phospho-Akt expression
can be inhibited by c-Src blockade. With the knowledge that Akt is a
downstream substrate of PI3-kinase activity, which was confirmed above
to validate the use of antisense PI3-kinase ODN, these results showing
Akt down-regulation via c-Src ODN treatment support the critical
position of Src upstream of PI3-kinase/Akt.
IL-18-induced ERK1/2 Activation Is PI3-Kinase-independent but
Src-dependent--
Other studies have shown a role for
PI3-kinase in signaling ERK activation in a number of cell systems (33,
34). Hence, we looked at the kinetics of ERK activation in
IL-18-stimulated RA synovial fibroblasts by Western blot and found a
time-dependent response, which was maximal at 20 min
(p < 0.05, n = 3) (Fig. 5, A and B). To
determine whether ERK1/2 activation was dependent on Src kinases, we
studied the effect of the Src kinase inhibitor PP2 on IL-18-induced
ERK1/2 activation. PP2 (25 µM) strongly inhibited ERK1/2
activation (Fig. 5C). To assess the role of PI3-kinase in
ERK activation by IL-18, cells were preincubated with LY294002, a
potent inhibitor of PI3-kinase. LY294002 did not change IL-18-mediated ERK stimulation suggesting PI3-kinase is not involved in the signaling route leading to ERK activation (Fig. 5D).
View larger version (26K):
[in this window]
[in a new window]
|
Fig. 5.
IL-18-induced ERK1/2 phosphorylation in RA
synovial fibroblasts, and inhibition by the Src inhibitor PP2 or the
antisense c-Src ODN. A, serum-starved RA synovial
fibroblasts were stimulated with IL-18 for various time points as
indicated. Cell lysates were blotted for phospho-ERK1/2 with mouse
monoclonal anti-human phospho-ERK1/2 antibody (*p-ERK1/2).
The same blot was stripped and reprobed with a mouse anti-human tubulin
antibody. B, blots were scanned and analyzed for
quantification with the UN-SCAN-IT software. Band intensities for
phospho-ERK1/2 were normalized to the corresponding band intensities
for tubulin. Data from three RA donors were averaged and are
represented as the mean ± S.E., expressed as fold increase with
respect to nonstimulated (NS) cells. *, p < 0.05 versus time 0; n = number of donors.
C and D, synovial fibroblasts from four RA
donors (indicated RA number above the blot) were
serum-starved for 16 h before treatment with specific kinase
inhibitors (25 µM) or vehicle control Me2SO.
After 2 h of pretreatment with inhibitors, IL-18 was added to the
culture media for 20 min. Cells lysates were prepared and subjected to
Western blot analysis for the activation of ERK1/2
(*p-ERK1/2). The same blots were stripped and reprobed with
a mouse anti-human tubulin antibody. Blots were scanned and analyzed
for quantification. Shown in C are the blots for the cells
pretreated with PP2, and shown in D is the blot for cells
pretreated with LY294002. E, RA synovial fibroblasts
were treated with antisense (AS) ERK1/2 ODN or antisense
c-Src ODN or the corresponding control sense (S) ODNs and
incubated with or without IL-18 (10 nM) for 20 min. Total
cell lysates were prepared for equal protein loading and separation by
Western blot. ERK1/2 was detected with mouse monoclonal anti-human
phospho-ERK1/2 antibody (*p-ERK1/2). F, data
from four RA donors were scanned and analyzed for quantification with
the UN-SCAN-IT software, and band intensities for ERK1/2 were
normalized to tubulin to quantitate relative ERK1/2 protein expression
and presented as the mean ± S.E.; *, p < 0.05;
n = number of donors.
|
|
Antisense c-Src ODN Treatment Inhibits RA Synovial Fibroblast
IL-18-induced ERK1/2 Expression--
IL-18 (10 nM) greatly
enhanced ERK1/2 protein expression by about 25-35-fold from basal
levels (2.83 ± 1.51 to 3.72 ± 0.1) in RA synovial
fibroblasts. To determine whether treatment with antisense ERK1/2 ODN
specifically inhibits target gene expression, ERK1/2 protein in
antisense or sense ERK1/2 ODN-treated cells stimulated with IL-18 (10 nM) was detected by Western blot analysis. Antisense ERK1/2
ODN inhibited IL-18-induced ERK1/2 protein expression (Fig.
5E). The results showed a mean of 85% inhibition of ERK1/2 protein expression by antisense ERK1/2 ODN, whereas sense ERK1/2 ODN
was unable to block this expression in IL-18-stimulated cells, 15.13 ± 7.78 versus 100 ± 9.95 (mean ± S.E., p < 0.05, n = 4) (Fig.
5F). To examine the effect of c-Src inhibition on
IL-18-induced ERK1/2 expression in RA synovial fibroblasts, cells were
treated with antisense or sense c-Src ODN prior to IL-18 (10 nM) stimulation. Antisense c-Src ODN treatment inhibited
ERK1/2 protein expression by 32%, whereas sense c-Src ODN was unable
to block ERK1/2 protein expression in IL-18-stimulated cells,
68.43 ± 18.18 versus 100 ± 15.29 (mean ± S.E.) (p < 0.05, n = 4) (Fig. 5,
E and F).
IL-18 Activates the Ras/Raf Pathway--
A number of studies have
shown Raf kinase is a predominant MEK kinase which, in turn, is
activated by binding to Ras-GTP (35). Src family kinases have also been
shown to be linked to MAPK through Ras/Raf kinases (36, 37). To study
the activation of Ras/Raf kinases, we first immunoprecipitated
activated Ras with a Raf-1-Ras binding domain (RBD) conjugated to
agarose beads and subsequently probed with mouse monoclonal anti-Ras
antibody. IL-18 stimulation of RA synovial fibroblasts showed a
time-dependent activation of the Ras/Raf pathway with
maximal activation at 10 and 20 min (p < 0.05, n = 3) (Fig.
6A). We confirmed activation
of Raf by IL-18 using an antibody specific for phosphorylated Raf-1
(p < 0.05, n = 3) (Fig.
6B).
View larger version (21K):
[in this window]
[in a new window]
|
Fig. 6.
Time-dependent Ras and c-Raf
activation in RA synovial fibroblasts stimulated with IL-18.
Serum-starved RA synovial fibroblasts were stimulated with IL-18 for
various time points as indicated. Cells were extracted with cell lysis
buffer, and the protein content in each sample was estimated. Each
sample (100 µg) was immunoprecipitated with a Raf-1-RBD-agarose
conjugate and subsequently subjected to 10% SDS-PAGE.
A, the presence of activated Ras in the samples was
then detected by probing with a specific mouse monoclonal anti-Ras
antibody (1 µg/ml). C, phospho-c-Raf
(*p-raf-1) was detected by direct Western blot analysis.
Blots were stripped and reprobed with mouse anti-human tubulin.
B and D, in each case, blots were scanned and
analyzed for quantification, and data from three RA donors were
averaged and are represented as the mean ± S.E., expressed as
fold increase with respect to nonstimulated (NS) cells. *,
p < 0.05 versus time 0; n = number of donors.
|
|
IL-18 Activates AP-1 through ERK but Not PI3-Kinase--
Because
in our previous study inhibitors of the transcription factor NFB
were only able to reduce IL-18-induced VCAM-1 expression by 50%, we
explored whether other signaling pathways and transcription factors
might be involved in induction of VCAM-1 transcription. AP-1 was an
appropriate candidate because AP-1 is activated by PI3-kinase and MAPK
and has also been involved in the induction of adhesion molecule gene
transcription (38, 39). By using gel shift assays, we show that DNA
binding of AP-1 was potently induced by IL-18 at 1 h (Fig.
7A). The specificity of the
AP-1-DNA binding complexes was confirmed by the fact that their
formation could be eliminated by the addition of a 4 M
excess of unlabeled ODN (Fig. 7B). This AP-1 binding to DNA
was completely inhibited in RA synovial fibroblasts pretreated with
PD98059, but had no effect in cells pretreated with LY294002 or PDTC,
and had a partial effect with PP2 pretreatment (Fig. 7C).
PDTC served as a control due to its known absence of effect on AP-1
(40).
View larger version (47K):
[in this window]
[in a new window]
|
Fig. 7.
Time-dependent AP-1 DNA binding
activity in IL-18-stimulated RA synovial fibroblasts and inhibition of
AP-1 nuclear activation by PD98059. A, RA synovial
fibroblasts were stimulated with IL-18 (10 nM) for various
time points as indicated. Nuclear extracts were prepared and analyzed
for AP-1 binding activity by electrophoretic mobility shift assay. AP-1
denotes the specific AP-1-DNA complexes. B, competition
experiments used 2- and 4-fold molar excess of unlabeled ODN in nuclear
extracts of RA synovial fibroblasts stimulated for 1 h with 10 nM IL-18. C, serum-starved RA synovial
fibroblasts were pretreated with specific inhibitors LY294002, PD98059,
PP2 (25 µM), or Me2SO vehicle control for
2 h before stimulation with IL-18 (10 nM) for 1 h. For PDTC, cells were pretreated for 8 h at 300 µM. Nuclear extracts were analyzed by EMSA. Experiments
were repeated two times with essentially identical results.
LY corresponds to LY294002, PD to PD98059,
PDTC to pyrrolidine derivative of dithiocarbamate;
M = molar.
|
|
Antisense IRAK-1 ODN Inhibits IRAK-1 Gene Expression--
Initial
studies involved specificity determination to validate that the
antisense ODN could inhibit expression of its corresponding protein. To
verify that antisense IRAK-1 ODN inhibits target gene expression,
IRAK-1 protein in ODN-treated cells stimulated with IL-18 (10 nM) was detected by Western blot analysis. The expression of tubulin was examined as the housekeeping protein, and optical density measurement of the band representing the protein of interest was normalized to that of tubulin to quantitate relative protein expression (Fig. 8). Antisense IRAK-1 ODN
inhibited IL-18-induced IRAK-1 protein expression. Western blot
analysis showed 76% inhibition of IRAK-1 protein expression by
antisense IRAK-1 ODN, whereas sense IRAK-1 ODN was unable to block
IRAK-1 protein expression in IL-18-stimulated cells, 24.11 ± 1.23 versus 100 ± 18.1 (p < 0.05, n = 4). To demonstrate further the relative specificity of the antisense IRAK-1 ODN, as a control, cells were also treated with
antisense or sense c-Src ODN. c-Src ODNs were also unable to block
IRAK-1 protein expression, which was comparable with control (no ODN).
As shown, IL-18 clearly up-regulated IRAK-1 expression, whereas basal
levels by nonstimulated control cells were not readily detectable in
these Western blots.
View larger version (28K):
[in this window]
[in a new window]
|
Fig. 8.
Antisense IRAK-1 ODN inhibits
IL-18-induced IRAK-1 protein expression. A, RA
synovial fibroblasts were treated with antisense (AS) IRAK-1
ODN or the control sense (S) ODN and incubated with or
without IL-18 (10 nM) for 20 min. Lysates were prepared
with lysis buffer, and equal amounts of total protein were separated by
10% SDS-PAGE and transferred to nitrocellulose. IRAK-1 was detected
with mouse polyclonal anti-human IRAK antibody. B, data
from four RA donors were scanned and analyzed for quantification with
the UN-SCAN-IT software, and band intensities for IRAK-1 were
normalized to tubulin and presented as the mean ± S.E.; *,
p < 0.05; n = number of donors.
Antisense and sense c-Src ODN treatment effect on IRAK-1 was analyzed
similarly as shown.
|
|
Antisense IRAK-1 ODN Inhibits RA Synovial Fibroblast IL-18-induced
NF-B Activation--
IL-18 (10 nM) significantly
stimulated NF-B activation from basal levels of 25.7 ± 3.4 to
100 ± 7.5, a 4-fold increase. This IL-18-induced NF-B
activation was inhibited by antisense IRAK-1 ODN treatment for 5 h. The expression of NF-B was decreased by 32.9% from control
expression of 100 ± 7.5 to 67.12 ± 6.6 (p < 0.05, n = 4) (Fig. 9).
There was no difference between antisense and sense ODN nonstimulated
control cells. Again, as a control, cells were also treated with
antisense or sense c-Src ODN, neither of which was able to block
IL-18-induced NF-B activation, with similar values of 100 ± 10.8 and 101.75 ± 3.2, although both were increased from basal
levels of 32.37 ± 4.4 and 25.44 ± 2.4, respectively.
View larger version (34K):
[in this window]
[in a new window]
|
Fig. 9.
Antisense IRAK-1 ODN inhibits RA synovial
fibroblast IL-18-induced NF-B activation.
A, RA synovial fibroblasts were treated with antisense
IRAK-1 ODN or antisense c-Src ODN or the corresponding control sense
ODNs and incubated with or without IL-18 (10 nM) for 20 min. Lysates were prepared and analyzed as described in previous
figures. NF-B was detected with rabbit polyclonal anti-human NFB
p65 antibody. Shown is a representative Western blot, which was
stripped and reprobed with mouse anti-human tubulin antibody.
B, data from four RA donors were scanned and analyzed
for quantification with the UN-SCAN-IT software, and band intensities
for NF-B were normalized to tubulin and presented as the mean ± S.E.; *, p < 0.05; n = number of
donors.
|
|
Signaling Mechanisms Involved in RA Synovial Fibroblast VCAM-1
Expression--
To examine signaling pathways involved in RA synovial
fibroblast VCAM-1 production, we first tested the effect of different concentrations of inhibitors of PI3-kinase, Src kinase, and ERK on
IL-18-induced VCAM-1 expression by cell ELISA. The advantage of cell
ELISA to examine the effect of inhibitors is the requirement of only a
small number of cells. Thus, we were able to compare the effect of
several concentrations of different inhibitors concurrently on
IL-18-induced VCAM-1 expression. In the literature, effective concentrations used for the specific inhibitors tested are between 10 and 100 µM. The PI3-kinase inhibitor (LY294002) and the
Src inhibitor (PP2) at 25 µM significantly decreased
IL-18-induced VCAM-1 expression by 32 and 35% (0.329 ± 0.06 OD
for Me2SO versus 0.223 ± 0.04 OD for
LY294002, and 0.212 ± 0.03 OD for PP2, each value representing
the mean ± S.E., p < 0.05). The inhibition was
greater with 50 µM of these inhibitors, 43 and 46%,
respectively (data not shown). Interestingly, an inhibitor of ERK
(PD98059) at these concentrations did not have an effect on
IL-18-induced VCAM-1 expression measured by cell ELISA, which appeared
to suggest the lack of ERK involvement, but results of inhibition with
antisense ODN described below showed otherwise. Because studies have
suggested that G proteins can activate PI3-kinase and Src (41, 42), we
also examined the potential role of G proteins in IL-18-induced VCAM-1
expression. RA synovial fibroblasts were pretreated with the
Go/i protein inhibitor, pertussis toxin, before stimulation with IL-18. The expression of VCAM-1 on RA synovial fibroblasts was
measured by FACS analysis and was not different for cells in the
presence or absence of pertussis toxin (data not shown).
We further examined the effect of inhibiting these signaling pathways
by transfecting the RA synovial fibroblasts with antisense or sense ODN
to block gene translation/transcription directly through targeting
c-Src, PI3-kinase, IRAK, and ERK1/2. The ODN-treated cells were
stimulated with IL-18 (5 nM) for 8 h and analyzed by flow cytometry to examine the treatment effect on VCAM-1 surface expression. Results represent expression of VCAM-1 after antisense ODN
treatment relative to control sense ODN treatment (Fig.
10). Antisense c-Src ODN treatment
resulted in a 57% decrease in VCAM-1 expression (43.23 ± 9.61%
versus 100% for control sense ODN, p < 0.05, n = 4). Antisense IRAK ODN treatment reduced
VCAM-1 expression by 43% (57.13 ± 10.66% versus
100% for control sense ODN, p < 0.05, n = 5). Likewise, treatment with antisense ERK1/2 ODN
and antisense PI3-kinase ODN resulted in a 41 and 32% decrease in VCAM-1 expression compared with control sense ODN (59.41 ± 14.48 and 68.14 ± 16.07% versus 100% control,
respectively, p < 0.05, n = 5 for
each).
View larger version (18K):
[in this window]
[in a new window]
|
Fig. 10.
Inhibition of RA synovial fibroblast VCAM-1
expression with antisense ODN treatment. RA synovial fibroblasts
were treated with antisense (AS) or sense (S) ODN
to c-Src, PI3-kinase, IRAK, and ERK1/2 and then incubated with or
without IL-18 (10 nM) for 20 min. Cells were collected and
analyzed by flow cytometry using mouse anti-human VCAM-1 antibody or
isotype-matched control followed by PE-conjugated goat anti-mouse
antibody. Results represent the expression of VCAM-1 after antisense
ODN treatment relative to control sense ODN treatment (100%), and data
from the indicated number of experiments were averaged and shown as
mean ± S.E.; *, p < 0.05; n = number of donors; PI3K = PI3-kinase.
|
|
| |
DISCUSSION |
Because the PI3-kinase inhibitor partially blocked IL-18-induced
VCAM-1 expression, we hypothesized IL-18 could stimulate PI3-kinase
activity. Indeed, IL-18 potently stimulated PI3-kinase activity with
rapid kinetics. The serine-threonine protein kinase Akt is one of the
major targets of PI3-kinase-generated signals (43). In our experiments,
we showed that Akt becomes phosphorylated after IL-18 stimulation and
that the PI3-kinase-specific inhibitor LY294002 could inhibit such
stimulation. This was confirmed by nearly complete abrogation of
IL-18-induced Akt activation through antisense PI3-kinase ODN
treatment. These results demonstrate IL-18 is a novel activator of the
PI3-kinase/Akt signaling cascade. PI3-kinase activation was first
described in signaling of hormones and growth factors (11, 44). Most
recently, Reddy et al. (45, 46) reported that PI3-kinase was
also involved in signaling of inflammatory cytokines such as IL-1 and
TNF-. In comparison to these cytokines, the magnitude and kinetics
of induction of PI3-kinase by IL-18 are more comparable with those
observed for TNF- than IL-1.
Receptor and non-receptor tyrosine kinases and G protein-coupled
receptors are known to be main activators of PI3-kinase (11). There are
a number of reports demonstrating the role of NRTK Src kinase in
PI3-kinase activation (47, 48). We showed IL-18 induced a
time-dependent increase in Src kinase phosphorylation. In
addition, IL-18 directly activated Src kinase as shown by enzymatic assay. Members of the Src family NRPKs are critically involved in
triggering a number of intracellular signaling pathways. Affected pathways are involved in cell migration, adhesion, cell cycle, apoptosis, and phagocytosis (49, 50). Src has been implicated in a
variety of diseases, such as breast, liver and colon cancer, especially
those with liver metastasis (51-53). Some Src PTKs have tissue-restricted expression, which would provide some advantage as
therapeutic targets in inflammatory, autoimmune, allergic, and
malignant disease. Of note, our data showed a trend toward a greater
degree of inhibition of surface VCAM-1 expression by blocking
IL-18-stimulated RA synovial fibroblasts with c-Src antisense ODN
relative to blockade of other signaling molecules. This difference may
suggest a more prominent role for Src in IL-18 signaling and VCAM-1
expression, although the ability of the various ODNs to inhibit the
target gene of interest may be having more influence than would allow
for comparisons of relative roles. Inhibition of Src and
Src-dependent pathways may present potentially useful approaches for treatment of RA and diseases characterized by adhesion molecule up-regulation.
Interestingly, IL-18-induced Src phosphorylation was temporally
correlated with PI3-kinase and Akt activation. Experiments using
antisense c-Src ODN further confirmed the upstream position of Src in
this pathway. Our data indicate that the initial event in the IL-18
response is Src activation, which leads to activation of PI3-kinase and
ERK1/2. Our observations appear to support this hypothesis because the
Src kinase inhibitor PP2 strongly inhibits IL-18-induced ERK
activation, and inhibition with c-Src antisense ODN significantly
reduced ERK1/2 expression. Moreover, the Src inhibitor PP2 and the
PI3-kinase inhibitor LY294002 have very similar effects on
IL-18-induced VCAM-1 expression, suggesting that Src and PI3-kinases
belong to the same signaling cascade. However, ERK1/2 and
PI3-kinase/Akt are probably independent of each other, based on the
failure of the PI3-kinase inhibitor to alter IL-18-mediated ERK1/2 activation.
Our results are consistent with several lines of evidence suggesting
that the ERK1/2 and PI3-kinase signaling pathways do not cross-talk
(54, 55). Kalina et al. (9) reported previously that IL-18
activates ERK1/2 in the natural killer cell line 92, but the underlying
mechanism was unclear. Here we demonstrate IL-18 stimulates ERK1/2
through an Src/Ras/Raf pathway, as evidenced by decreased ERK1/2
expression by c-Src inhibition with antisense ODN. Src and PI3-kinases
may not be activated by G proteins, based on the fact that the
Go/i protein inhibitor pertussis toxin has no effect on
IL-18-induced VCAM-1 expression. These data suggest Src and G protein
are independent of each other. It is more likely that Src may recruit
and activate PI3-kinase via tyrosine phosphorylation of IL-18R. Indeed,
Src has already been shown to phosphorylate receptors such as epidermal
growth factor receptor (56), and it is known that phosphorylated
receptors like IL-1 are able to recruit PI3-kinase (57). Further
investigations are needed to clarify possible interactions between
activation of Src, PI3-kinase, and the IL-18R following IL-18
stimulation. Furthermore, the IL-18R chain has been shown to be
essential for IL-12-independent proinflammatory activity of
IL-18-induced IL-8 in COS-1 fibroblasts (58), suggesting the IL-18
receptor itself is potentially an important regulatory site in
signaling. The IL-18R components, IL-18R (previously IL-1R-related
protein, IL-1Rrpl) and IL-18R (or IL-1R accessory protein-like,
IL-1RacPL), confer a highly specific and unique IL-18 responsiveness
(59). Both have been cloned and shown to be critical for IL-18
signaling (60, 61), and their potential interactions with relevant
signaling molecules are of interest.
We next investigated downstream events of PI3-kinase/Akt and ERK1/2
activation. Our previous work (4) suggested that transcription factors other than NFB existed that may be involved in IL-18-induced VCAM-1 expression. AP-1 was of particular interest for three reasons. First, AP-1 is activated by PI3-kinase and ERK (45, 62, 63). Second,
AP-1 regulates the transcription of adhesion molecules (38, 39). Third,
AP-1 is an important transcription factor in RA synovial fibroblasts
(64). The data showing that IL-18 induces AP-1 binding activity made
this hypothesis plausible and is in accord with previous studies
reporting AP-1 activation by IL-18 in different cells types (17, 65,
66). Although the EMSAs only demonstrate AP-1 binding and cannot
provide information on its phosphorylation status, our data using
specific inhibitors suggest that, unlike PI3-kinase, ERK1/2 regulates
AP-1 activation in IL-18-stimulated synovial fibroblasts. For
PI3-kinase, the signaling event downstream of Akt remains unanswered.
STAT3 might be an interesting candidate because IL-18, c-Src, and
PI3-kinase have all been shown to stimulate STAT3 (9, 67, 68).
Interestingly, the nuclear translocation of STAT4 is strictly dependent
on the presence of IL-18 for IL-12/IL-18-induced IFN--mediated
nitric oxide production by mouse peritoneal macrophages (69).
Finally, we showed that PI3-kinase is involved in IL-18-induced VCAM-1
expression. In the literature, there are conflicting reports regarding
the effect of PI3-kinase on adhesion molecule expression. Radisavljevic
et al. (70) showed that vascular endothelial growth factor
up-regulates ICAM-1 expression via a PI3-kinase/Akt pathway in
endothelial cells. In contrast, using the same stimulus and a similar
cell type, Kim et al. (71) reported an up-regulation of ICAM-1, VCAM-1, and E-selectin expression by PI3-kinase inhibition. The experimental conditions including the different concentrations and
types of PI3-kinase inhibitors used by these and other authors may
explain this discrepancy. In our hands, blocking PI3-kinase with
LY294002 had a repressive effect on IL-18-induced RA synovial fibroblast VCAM-1 expression using two different techniques (FACS and
cell ELISA). More specific inhibition with PI3-kinase antisense ODN
treatment was confirmatory. These results are consistent with the known
properties of PI3-kinase and VCAM-1. Indeed, we and others (13, 14, 72,
73) have implicated these two molecules in cell adhesion and
angiogenesis. Moreover, we showed in recent reports (4) that IL-18
mediates leukocyte-fibroblast adhesiveness and stimulates endothelial
cell tube formation in vitro and in vivo (3).
Hence, it is possible that PI3-kinase may be a key signaling molecule
responsible for IL-18-induced VCAM-1 synthesis, and subsequently cell
adhesion and angiogenesis.
As with PI3-kinase, selective ERK1/2 inhibition with antisense ODN
treatment decreased IL-18-induced VCAM-1 expression. However, ERK
inhibitor PD98059 did not show significant inhibition, which perhaps
was due to the lack of ERK inhibition at the concentrations used
without affecting viability. Thus, the use of antisense ODN to block
signaling molecules specifically may be more appropriate for
investigating these pathways and for developing potential therapeutic
strategies, at least in the case of ERK1/2. In addition, antisense ODNs
may be an alternative to avoid the toxicity that prevents these
chemical inhibitors from becoming optimal therapeutics. Taken together,
our observations support the perspective that specifically targeting
PI3-kinase (71), or perhaps ERK1/2, may represent a valid approach to
blocking adhesion molecule expression, cell adhesion, and angiogenesis.
Furthermore, that c-Src has a signaling role in IL-18-induced VCAM-1
expression is consistent with the findings that c-Src activation by
IL-18 lies upstream to the induction of PI3-kinase and ERK1/2, both of
which we showed to affect VCAM-1 expression.
Additionally, the IRAK/NFB pathway, distinct from the
Src-dependent pathways, appears to play a significant role
in RA synovial fibroblast VCAM-1 expression. Indeed, antisense IRAK ODN
treatment inhibited IL-18-induced VCAM-1 expression. This finding is in accordance with the fact that the IRAK/NFB signaling pathway has
been implicated in various IL-18 functions. IL-18 binding to its
receptor induces IRAK recruitment, and IRAK activation leads to nuclear
translocation of NFB and subsequent expression of immune response
genes, such as IFN- in Th1 cells (20). This NFB activation is
diminished in fibroblasts from IRAK-deficient mice, although additional
IL-1 and IL-18 cytokine responses are independent of the IRAK pathway
(21), which could explain Src-mediated pathways. IRAK is reportedly
necessary for NFB and JNK activation in response to IL-18, as in the
mutant cell line I1A generated to lack IRAK protein; specifically, the
N-proximal region of IRAK is essential for NFB activation but not
for JNK activation, suggesting IRAK may serve as a branch point (74).
Guo and Wu (75) reported inhibition of IL-18-induced NFB activation
by blocking PI3-kinase with antisense ODN. Whether IRAK has a
regulatory role in uniting these pathways is one question to be
clarified. Impaired NFB and JNK activation in IRAK-deficient mice is
further evidence of an important role for IRAK in IL-18 signaling (76).
Upon IL-18 stimulation, in addition to IRAK recruitment and
phosphorylation, TRAF-6 associates with IRAK in mouse EL-4 cells (77).
Thus, the interplay of other key molecules with IRAK may also be
critical for regulation of IL-18 functions.
There is additional precedence for the involvement of transcription
factor activation in IL-18-mediated functions. For instance, IL-18
activates AP-1 and NFB in Jurkat T cells leading to IL-2 expression
(17). The significance of the NFB pathway in the regulation of
IL-18-induced IFN- production was shown by Kojima et al.
(18) in the human myelomonocytic cell line KG-1 cells. The essential
role of NFB activation in IL-18-induced IFN- production was also
shown in murine Th1 cells (19). Again, the possibility<
§,
TOP
ABSTRACT
INTRODUCTION
