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Platelet-Derived Growth Factor Receptor
Activation Promotes the Prodestructive
Invadosome-Forming Phenotype of
Synoviocytes from Patients with Rheumatoid
Arthritis
Martine Charbonneau, Roxane R. Lavoie, Annie Lauzier,
Kelly Harper, Patrick P. McDonald and Claire M. Dubois
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol published online 14 March 2016
http://www.jimmunol.org/content/early/2016/03/11/jimmun
ol.1500502
Published March 14, 2016, doi:10.4049/jimmunol.1500502
The Journal of Immunology
Platelet-Derived Growth Factor Receptor Activation Promotes
the Prodestructive Invadosome-Forming Phenotype of
Synoviocytes from Patients with Rheumatoid Arthritis
Martine Charbonneau,*,1 Roxane R. Lavoie,*,1 Annie Lauzier,* Kelly Harper,*
Patrick P. McDonald,† and Claire M. Dubois*
R
heumatoid arthritis (RA) is a systemic autoimmune disease that mainly affects the joints, leading to joint inflammation and erosive structural damages. Although
important progress has been made in managing the pain and inflammation associated with the disease, strategies to directly interfere with the process of erosion are lacking. The onset of RA
causes important morphological changes in joint lining, including
formation of an aggressive tumor-like synovial tissue that invades
and erodes cartilage and bone (1). A large body of evidence from
patients and experimental animal models indicated that fibroblastlike synoviocytes (FLS) are the main cell type that actively drives
*Immunology Division, Department of Pediatrics, Faculty of Medicine, University of
Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada; and †Pneumology Division,
Department of Medicine, Faculty of Medicine, University of Sherbrooke, Sherbrooke
Quebec J1H 5N4, Canada
1
M.C. and R.R.L. are cofirst authors.
ORCIDs: 0000-0001-6253-8090 (R.R.L.); 0000-0002-0751-2821 (C.M.D.).
Received for publication March 4, 2015. Accepted for publication February 15, 2016.
This work was supported by Canadian Institutes for Health Research (CIHR) Grants
MOP-86634 and MOP-286621 (to C.M.D.). C.M.D. is a member of the Fonds de la
Recherche en Santé du Québec–funded Centre de Recherche du Centre Hospitalier
Universitaire de Sherbrooke. K.H. is recipient of a scholarship from CIHR.
Address correspondence and reprint requests to Dr. Claire M. Dubois, Immunology
Division, Department of Pediatrics, Faculty of Medicine, Université de Sherbrooke,
3001 12th Avenue North, Sherbrooke, QC J1H 5N4, Canada. E-mail address: Claire.
[email protected]
Abbreviations used in this article: ACR, American College of Rheumatology;
ECM, extracellular matrix; FLS, fibroblast-like synoviocyte; LPA, lysophosphatidic
acid; NA, nonarthritis; OA, osteoarthritis; PDGF, platelet-derived growth factor;
PDGFR, PDGF receptor; PLC, phospholipase C; pY, phosphotyrosine; RA, rheumatoid
arthritis; RTK, receptor tyrosine kinase.
Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500502
inflammation and joint destruction (2–4). Arthritic FLS resemble
transformed mesenchymal cells that are highly invasive in vitro
and in vivo. This property correlates with elevated production of
inflammatory cytokines and proteolytic enzymes that sustain inflammation and joint matrix degradation. We have reported that
the ability of arthritic FLS to degrade the extracellular matrix
depends on the formation of plasma membrane structures that
resembled invadopodia in tumor cells (5, 6). These structures were
detected in fibroblast-like cells strategically located at the cartilage–synovial membrane interface. They were shown to contain
actin components, signaling molecules, such as Src, and high
levels of proteolytic enzymes known to be particularly efficient at
inducing cartilage damage. Importantly, interference with the
formation of invadosomes in arthritic FLS strongly inhibited
matrix degradation in vitro and ex vivo as well as cartilage degradation in a rat model of arthritis (5, 6). These observations
suggested that invadosomes were directly involved in joint degradation, leading to the conclusion that an in-depth understanding
of the mechanism of invadosome formation is of importance for
development of joint protection strategies for the clinical management of RA.
The mechanisms involved in invadosome formation in synovial
cells are not fully known. Invadosome formation and in vivo
cartilage degradation capability of synovial cells of collageninduced arthritis rats were shown to depend on an autocrine activation loop that involved TGF-b (6). Analysis of the protein and
mRNA in RA synovial tissues revealed that TGF-b was highly
expressed in RA patients (7–9). However, few studies have
addressed the role of TGF-b in the functions of synovial fibroblasts derived from these patients. TGF-b was shown to increase
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Fibroblast-like synoviocytes (FLS) play a major role in invasive joint destruction in rheumatoid arthritis (RA). This prodestructive
phenotype has been shown to involve autocrine TGF-b that triggers formation of matrix-degrading invadosomes through molecular mechanisms that are not fully elucidated. The platelet-derived growth factor (PDGF) receptor (PDGFR) family of receptor
tyrosine kinases (RTK) has been shown to cooperate with TGF-b in various pathological conditions. We therefore sought to
determine whether RTK activity played a role in invadosome biogenesis. We demonstrated that, among the common RTKs,
PDGFR-ab was specifically phosphorylated in FLS from RA patients. Phosphorylation of PDGFR-ab was also elevated in RA
synovial tissues. Interference with PDGFR activation or PDGF neutralization inhibited invadosome formation in RA synoviocytes,
indicating the presence of an autocrine PDGFR activation loop that involved endogenous PDGF. Among the PDGF-A–D isoforms,
only PDGF-B was found both significantly elevated in FLS lines from RA patients, and related to high-invadosome forming cells.
Addition of TGF-b upregulated invadosome formation, PDGF-B mRNA expression, and phosphorylation of PDGFR. All of these
functions were efficiently suppressed by TGF-b neutralization or interference with the Smad/TbR1or PI3K/Akt pathway. Among
the class 1 PI3K family proteins known to be expressed in RA synoviocytes, PI3Ka was selectively involved in PDGF-B expression,
whereas both PI3Ka and PI3Kd participated in invadosome formation. Our findings demonstrate that PDGFR is a critical RTK
required for the prodestructive phenotype of RA synovial cells. They also provide evidence for an association between autocrine
TGF-b and PDGFR-mediated invadosome formation in RA synoviocytes that involves the production of PDGF-B induced by
TGF-b. The Journal of Immunology, 2016, 196: 000–000.
2
PDGFR ACTIVATION PROMOTES INVADOSOME FORMATION IN RA-FLS
Materials and Methods
FLS cell lines
Human synovial cells derived from joint tissues (mostly knee joints) of
patients diagnosed with osteoarthritis (OA) and RA, or from control
nonarthritis (NA) individuals were purchased from Asterand (Detroit, MI).
The 1987 American College of Rheumatology (ACR) or 2010 ACR/
European League Against Rheumatism classification criteria were used for
diagnosing RA (32, 33), whereas the OA patients were classified using the
1986 ACR clinical criteria for OA (34). The NA cell lines were derived
from synovial tissues of cadavers who died of cardiovascular diseases. All
synovial tissues were obtained under approval of the local Institutional
Review Board and appropriate signed informed consent. Human synovial
tissue samples used for immunohistochemistry were obtained from OA or
RA patients undergoing total knee joint replacement surgery. The 1987
ACR or 2010 ACR/European League Against Rheumatism classification
criteria were used for diagnosing RA (32, 33), and OA patients were di-
agnosed using the 1986 ACR clinical criteria (34). The protocol was approved by the Centre Hospitalier Universitaire de Sherbrooke Ethics
Committee, and written consent was obtained from all participants.
Synoviocytes were isolated using standard procedures (35), and the
culture was maintained in DMEM-F12 medium supplemented with 10%
FBS and 40 mg/ml gentamicin. Cells were used between passages 3 and 8.
Synoviocyte cultures exhibited a classic spindle-shape fibroblastic morphology that formed parallel clusters at confluency. The cell surface
phenotypic marker analysis showed that they were consistently positive for
the stromal mesenchymal marker fibronectin (.99%) and negative (,1%)
for the macrophage marker CD68.
Plasmids and transfections
pLKO.1-puro short hairpin RNA targeting PI3Ka, PI3Kb, PI3Kd, and
control (scrambled) short hairpin RNA plasmids were from Sigma-Aldrich
(Oakville, ON, Canada). Viral particles were generated by transient transfection of 293T cells with the ViraPower lentiviral expression system
(Invitrogen Thermo Fisher Scientific, Burlington, ON, Canada). Experiments were conducted 48 h following lentiviral infection with Polybrene
(5 mg/ml; EMD Millipore). Transfected cells were selected by treatment
with puromycin for 72 h.
Immunofluorescence and confocal microscopy
Synoviocytes cultured on coverslips for 4 h were fixed in 2% paraformaldehyde, permeabilized with 0.05% saponin, and blocked with 2%
BSA. The following Abs or reagents were used to stain for actin
(phalloidin conjugated to Alexa647), cortactin (EMD Millipore), phosphotyrosine (pY) (EMD Millipore), and p-Src Tyr418 (Novus Biologicals,
Littleton, CO). Negative control slides were treated with isotypematched primary Abs, followed by secondary Abs. Confocal images
were acquired using a Fluoview 1000 scanning confocal microscope
(Olympus, Richmond Hill, ON, Canada) in line with an inverted
Olympus microscope equipped with a 340 oil immersion objective. Color
channels were scanned sequentially to avoid overlapping signals. A set of
z-stack images was collected at 0.25-mm intervals and reconstructed using
the FluoView software (Olympus).
Invadosome assays
Coverslips were prepared using Oregon Green488–conjugated gelatin (Life
Technologies, Burlington, ON, Canada) at a final concentration of 1%, as
described (36). Thirty thousand cells were seeded on each coverslip, cultured for 40–48 h, and fixed with 1% paraformaldehyde. Nuclei were
stained with DAPI, and F-actin was stained with Alexa647-conjugated
phalloidin (Life Technologies; dilution 1:50). To determine the percentage
of cells forming invadosomes, stained cells were visualized using a Zeiss
Axioskop fluorescence microscope and invadosomes were identified by
F-actin–enriched areas of matrix degradation. Three fields of 100 cells
(original magnification 340) were counted per coverslip. In selected experiments, we also measured the capacity of the cells to form actin/
cortactin-rich invadosomal structures. For this, cells were also stained
with anti–p-cortactin Abs, and clusters of p-cortactin/actin were calculated
for 25 cells per slide. The capacity of invadosomes to degrade gelatin was
also quantitated by measuring the areas of degradation per cell. Pictures of
fluorescent matrix were analyzed using the ImagePro software, and degradation areas associated with invadosome-positive cells were calculated in
pixels. A minimum of 25 cells was counted per coverslip. When specified
under the figure legends, RA synovial cells or control synoviocytes were
stimulated for 48 h with PDGF-BB or TGF-b1 (Peprotech, Montreal,
QC, Canada), in the presence or absence of imatinib mesylate (Cayman
Chemical, Cedarlane Laboratories, Burlington, ON, Canada), PDGFR tyrosine kinase inhibitor V (EMD Millipore, Billerica, MA), PDGF or
TGFb1-3 neutralizing Abs (R&D Systems, Minneapolis, MN), PI3K inhibitor LY294002 (Cayman), AKT inhibitor XI (EMD Millipore), phospholipase C (PLC) inhibitor U73122, or the control compound U73343
(inactive analog of U73122).
Western blotting
Whole-cell extracts were prepared by lysis of overnight serum-starved cells
in radioimmunoprecipitation assay buffer. When specified in the figure
legends, cells were incubated with imatinib mesylate (3 mM), PDGF-BB
(10 ng/ml), or TGF-b (5 ng/ml). Proteins were immunoblotted, as described (37), using anti-pPDGFRa (Tyr 849 )/pPDGFRb (Tyr 857 ) (Cell
Signaling Technology; dilution 1:2000), anti-PDGFRb (Cell Signaling
Technology; dilution 1:1000), anti–p-AKT (Ser473) (Cell Signaling Technology; dilution 1:2000), anti-AKT (Cell Signaling Technology; dilution
1:1000), or anti–a-tubulin (Sigma-Aldrich, Oakville, ON, Canada; dilution
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the expression of proinflammatory cytokines and metalloproteinases
in RA synoviocytes (9), an effect that was found to be dramatically potentiated by receptor tyrosine kinase (RTK)–dependent
signaling (10). These studies suggested a potential association
between TGF-b and RTK signaling to promote proarthritic
functions of synovial fibroblasts.
RTKs comprise a large family of cell surface receptors that are
essential components of signal transduction pathways that mediate cell survival, proliferation, differentiation, and motility, and
modulate cell metabolism (11). These transmembrane proteins
bind polypeptide ligands, mainly growth factors. Among the 58
RTK family members, epidermal growth factor receptor, plateletderived growth factor (PDGF) receptor (PDGFR), fibroblast
growth factor receptor, vascular endothelial growth factor receptor, hepatocyte growth factor receptor (c-Met), and stem cell
growth factor receptor (c-kit) have been shown to be expressed in
rheumatoid synovial tissues (11–19). On the basis of their roles as
growth factor receptors, several RTKs are the driving force for
onset or progression of various diseases such as malignancy and
arthritis and may represent key targets in therapeutic treatments.
For instance, in the case of arthritis management, immatinib
mesylate, a tyrosine kinase inhibitor initially used for treatment of
chronic myeloid leukemia, has been shown to reduce activation of
RA synoviocytes by interfering with PDGFR signaling (10, 20). In
experimental arthritis, imatinib has also been reported to markedly
reduce joint erosion when administered before onset of the disease
or during its progression (20–22). In RA patients, several reports
have confirmed the efficacy of imatinib, and this has lead, in some
cases, to complete clinical remission (23–26). Despite these encouraging results, the toxicity of imatinib was found to be high
(27), possibly due to its pan-effect on multiple RTKs. The bulk of
these observations suggested that identification of RTKs specifically involved in joint erosion would be invaluable to design
specific strategies for development of targeted therapies to minimize or prevent joint damage in RA.
The goal of the current study was to investigate whether RTKs
played a role in invadosome formation by synovial cells and to
identify the nature of these RTKs. RTKs, such as PDGFR, hepatocyte growth factor receptor, and epidermal growth factor
receptor, have been shown to direct matrix degradation and cell
invasion, a situation linked to invadosome formation in cancer
cells (28–31). In this study, we show that activation (phosphorylation) of PDGFR is specifically upregulated in RA synoviocytes and synovial tissues. We also show that PDGFR
activation involves TGF-b–induced PDGF-B upregulation mediated by TbR1/Smad and PI3K/AKT pathways. These findings
suggested the involvement of an overreactive TGF-b/PDGF-B/
PDGFR pathway in synoviocyte-driven extracellular matrix degradation in RA.
The Journal of Immunology
1:1000) Abs. Band intensities were analyzed using the Quantity One
software (Bio-Rad Laboratories, Mississauga, ON, Canada).
RTK arrays
Synovial cells were incubated in the presence or absence of imatinib
(3 mM) for 16 h in serum-free medium. A mixture of phosphatase
inhibitors (10 mM sodium fluoride, 1 mM sodium ortho-vanadate,
20 mM b-glycerophosphate) was added 20 min prior to cell lysis in the
cell lysis buffer. Extracted proteins (0.3 mg/ml) were analyzed with the
human PathScan RTK Signaling Antibody Array kit (Cell Signaling
Technology), according to the manufacturer’s instructions. A fluorescent image of the slide was captured using an Odyssey Infrared Imaging System, and fluorescence intensities were measured using the
ImageJ software.
3
Results
Invadosome generation and function are increased in
synoviocytes from RA patients and require RTK activity
We have recently shown that synovial cells from collagen-induced
arthritic rats spontaneously formed f-actin–enriched degradative
complexes that colocalized with zones enriched for the invadosome markers cortactin, an actin assembly protein; Src, a tyrosine
kinase involved in phosphorylation of actin-regulatory proteins;
and pY (5). In this study, we investigated whether similar observations could be made in the case of human synovial cells of
rheumatoid arthritic patients. Cells were plated on a cross-linked
fluorochrome-labeled gelatin matrix and left for 40 h at 37˚C.
Real-time PCR
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Synoviocytes were incubated in serum-free condition in the presence or
absence of TGF-b and in the presence or absence of PI3K inhibitor
LY294002 (Cayman Chemical), TbR1 inhibitors LY364947 (Tocris Bioscience), or SB431542 (Sigma-Aldrich). Total RNA was isolated using the
TRI-Reagent (Life Technologies) protocol, as previously described (38),
and quantitative real-time PCR was performed on a Rotor-Gene 3000
(Corbett Research). Primer sequences were as follows: PDGF-A forward
(59-CGTAGGGAGTGAGGATTCTTTG-39), PDGF-A reverse (59-GCTTCCTCGATGCTTCTCTT-39); PDGF-B forward (59-CCATTCCCGAGGAGCTTTATC-39), PDGF-B reverse (59-GGTCATGTTCAGGTCCAACTC-39);
PDGF-C forward (59-GTCAATGTGTCCCAAGCAAAG-39), PDGF-C
reverse (59-CCACGTCGGTGAGTGATTT-39); PDGF-D forward
(59-GAAATTGTGGCTGTGGAACTG-39), PDGF-D reverse (59-GGCCAGGCTCAAACTGTAATA-39); PAI-1 forward (59-AATCAGACGGCAGCACTGTCT-39), PAI-1 reverse (59-GGCAGTTCCAGGATGTCGTAGT-39);
PI3K-a forward (59-GTATCCCGAGAAGCAGGATTTAG-39), PI3K-a
reverse (59- CAGAGAGAGGATCTCGTGTAGAA-39); PI3K-b forward
(59-GCACTTGGTAATCGGAGGATAG-39), PI3K-b reverse (59-TTGTACTGAGACAGCAGGAATG-39); PI3K-d forward (59-CCCACAGGTGATCCTAACATATC-39), PI3K-d reverse (59-ACTTCTGGCTCTGTTGAGTTT39); RPLPO forward (59-GATTACACCTTCCCACTTGC-39), RPLO
reverse (59-CCAAATCCCATATCCTCGTCCG-39). Each reaction was
run in duplicate, and values were normalized against the RPLPO
housekeeping gene.
Immunohistochemistry
Paraffin-embedded synovial tissue sections were freed of paraffin and
rehydrated, and immunohistochemical staining was performed according
to the standard avidin–biotin immunoperoxidase complex technique by
using the following Abs: anti-pPDGFRa(Tyr849)/b(Tyr857) (Cell Signaling
Technology; dilution 1:75), anti-PDGFRb (Cell Signaling Technology;
dilution 1:100), or rabbit isotype IgG. Diaminobenzidine was used for the
detection of the labeled proteins, and the sections were counterstained with
Harris hematoxylin. Slides were scanned with a Hamamatsu Nanozoomer
2.0-RS scanner. For each patient, six random fields (at original magnification 320) were captured with the NPD viewing software, and intensity
of labeling in the synovial membrane was analyzed using the immunohistochemistry quantification technique described by Pham et al. (39).
Images were converted to CMYK with the FIJI software. Next, gray image
of the yellow (Y) channel was extracted, and chromogen intensities were
analyzed using the Image Pro software (Media Cybernetics, Bethesda,
MD). Results are expressed as the sum of labeling intensity (density)
relative to total area.
Measurement of intracellular calcium
Synovial cells were incubated with 3 mM Fura-2/AM (Molecular Probes)
for 30 min at room temperature in 1 mL HBSS containing 0.35 g/L
NaHCO3 and 10 mM HEPES (pH 7.0). The dye-loaded cells were then
washed and resuspended in HBSS containing 1.5 mM CaCl2, and baseline
fluorescence was measured at 0 s. Fluorescence released after lysophosphatidic acid (LPA) stimulation (10 mM) was recorded every 2 s for 4 min.
Where indicated, cells were pretreated with the PLC inhibitor U73122
(0.1 and 1 mM) before stimulation with LPA. Analysis was performed by
spectrophotometry using the Hitachi F2500 fluorescence spectrophotometer and fluorescence solutions software.
Statistical analysis
Comparison between two groups was analyzed using unpaired Student t
test. One-way ANOVA was used for comparison among three or more
groups. The p values ,0.05 were considered significant.
FIGURE 1. RA-FLS generate ECM-degrading invadosomes. RA-FLS
were cultured on Oregon Green488–conjugated gelatin for 4 h and stained
for invadopodia markers. Confocal microscopy images of the basal surface
of the cells showing colocalization of (A) F-actin (red) and cortactin (blue),
(B) F-actin and tyrosine-phosphorylated proteins (pY) (blue), and (C)
F-actin and p-Src (blue) to areas of Oregon Green488–conjugated gelatin
(green) degradation. Nuclei were stained with DAPI (blue). Boxed areas
outlining invadosomes are enlarged in the corresponding right panels. The
enlarged merged images also show fluorescence intensity profiles for
F-actin, Oregon Green488–conjugated gelatin, and the specified invadosome
marker at the cell–matrix interface. Representative images of three independent experiments generated with two independent RA-FLS lines are
shown. Scale bars, 10 mM. Original magnification 360 (insets).
4
PDGFR ACTIVATION PROMOTES INVADOSOME FORMATION IN RA-FLS
FIGURE 2. RA-FLS display increased invadosome-forming ability. (A and B) Synoviocytes of
three NA, four OA, and four RA individuals were
plated on Oregon Green 488 –conjugated gelatincoated coverslips and incubated for 48 h. (A) Percentage of invadosome-forming cells was calculated
for 300 cells in four independent experiments. Each
column represents the results of individual cell lines.
(B) The mean area of degradation was calculated
for 20 cells in three independent experiments. (C)
Synoviocytes were incubated on Oregon Green488
gelatin-coated coverslips for 6 h and stained for
phospho-cortactin and actin. The number of phospho-cortactin/actin clusters was counted for 25 cells
in three independent experiments. (D) Synovial cells
were plated on Oregon Green488 gelatin-coated
coverslips and incubated in the presence or absence
of imatinib mesylate, used at the indicated concentrations, for 48 h (n = 4 independent experiments).
*p , 0.05, **p , 0.01, ***p , 0.001.
Increased phosphorylation of PDGFR is a distinctive feature of
RA synovial cells and tissues
We next investigated the nature of RTKs responsible for the aggressive invadosome-forming phenotype. A phospho-specific RTK
Ab array was used to screen synovial cells for 28 common RTKs
that included EGFR, FGFR, INSR, NGFR, HGFR, PDGFR (c-kit,
PDGFR, FLT3, CSF-1R), EphR, and Axl family members and Tie
and VEGFR. High levels of phosphorylated PDGFR were observed
in RA synovial cells compared with cells of OA and NA individuals (Fig. 3A). In contrast, there were no significant changes in
their levels of phosphorylation in the case of other RTKs. Western
blot analysis of synovial cell lysates using a phospho-specific
mAb that recognized active (phosphorylated) PDGFR-ab confirmed the RTK array results with a 2.6-fold increase in PDGFR
phosphorylation in RA synoviocytes compared with control NA
cells, whereas levels of PDGFR did not change (Fig. 3B–D).
PDGFR phosphorylation in RA cells was significantly inhibited by
imatinib mesylate both in the phospho-RTK array and Western
blotting (Fig. 3A–D), suggesting that the observed inhibitory effect of imatinib mesylate on invadosome formation by RA synovial cells was most likely due to inhibition of PDGFR kinase
activity. We also examined pPDGFR expression levels in synovial
tissues. Results showed that pPDGFR is strongly expressed in the
rheumatoid synovium with prominent staining in the synovial
intimal lining (Fig. 3E). Quantification of staining intensity indicated that pPDGFR expression is significantly elevated (p , 0.05)
in RA compared with OA (Fig. 3F). Together, these results indicate that PDGFR could be part of a dominant RTK signaling
pathway involved in ECM degradation by RA synoviocytes.
Signaling through PDGFR is required for the enhanced
invadosome biogenesis observed in RA synovial cells
To investigate whether endogenous PDGF and PDGFR activity
were required for invadosome formation in RA synovial cells, we
first tested whether PDGF promotes invadosome formation in control nonarthritic cells. Results showed that addition of PDGF-B,
which binds PDGFR a and b receptors, induced a concentrationdependent increase of dot-like actin-rich invasive structures
(Fig. 4A, associated micrograph panel) similar to those observed
in RA synoviocytes (Fig. 1A–C). In contrast, PDGF-B had no effect on the capacity of OA cells to form invadosomes and did not
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Confocal analysis of the cells indicated that they extended dot-like
actin-rich degradative structures from the ventral side of the plasma
membrane in contact with the matrix (Fig. 1A). To confirm that the
observed structures were indeed invadosomes, we stained the cells
for invadosome markers cortactin, pY, and p-src (40, 41). Stacking
along the z-axis of the cell–matrix interface clearly showed that all
these components were located within the matrix in a configuration
similar to that of actin, suggesting that they were located within
the cell membrane protrusions (Fig. 1). Because invadosomes are
transient dynamic structures, some areas of gelatin degradation
were observed that were no longer associated with invadosome
markers at the time of fixation. Together, these results indicate that
synovial cells of arthritis patients produce typical invadosome
structures similar to those produced by cancer cells or synovial cells
from collagen-induced arthritis rats (5, 42, 43).
We next investigated the invadosome formation potential of
synovial cell lines from two types of arthritic patients, namely OA
and RA, and compared the results with cells from NA. Results
showed that synoviocytes of RA patients had a 3.4- and 2.8-fold
increase in the percentage of cell-forming invadosomes as compared with cells from NA and OA patients, respectively (Fig. 2A).
Quantification of the extent of extracellular matrix (ECM) degradation (a measure of invadosome function), as well as the
number of invadosomal structures formed per cell, identified by
the colocalization of actin and phospho-cortactin, two known
markers of invadosomes (44), showed a significant increase in
both invadosome formation and function in RA synoviocytes
compared with NA or OA cells (Fig. 2B, 2C). Of note, there were
no statistically significant differences between the OA and NA
groups of synovial cells, indicating that, in contrast to RA cells,
OA synovial cells displayed a nonaggressive invadosome-forming
phenotype.
The involvement of RTK in invadosome formation of RA synoviocytes was investigated by culturing cells in the presence or absence of imatinib mesylate, a RTK inhibitor that possesses strong
activity against PDGFR and c-Kit and weaker activity against
FLT3 and CSF-1R. Results showed that inhibition of RTK activity resulted in a concentration-dependent decrease in invadosome
formation by RA synoviocytes, reaching basal levels of OA and NA
control cells (Fig. 2D). These results led to the conclusion that RTK
were involved in invadosome formation by RA synovial cells.
The Journal of Immunology
5
increase further the number of invadosomes in RA cells (Fig. 4A).
We then blocked either PDGFR activity using PDGFR tyrosine
kinase inhibitor V, which inhibits ligand-induced PDGFR phosphorylation and kinase activity (45), or endogenous PDGFR ligands using PDGF-B and pan-PDGF neutralizing Abs. Results
showed that invadosome formation induced by PDGF-B in control
synovial cells or already elevated in RA cells was abolished by the
addition of PDGF neutralizing Abs or PDGFR tyrosine kinase
inhibitor V (Fig. 4B, 4C). In contrast, an isotype-matched negative
control Ab has no effect. We therefore concluded that activation of
PDGFR in RA synoviocytes triggers signals that result in increased invadopodia formation.
PI3K/AKT pathway is an effector of PDGF-driven invadosome
formation in RA synovial cells
PDGFR activation triggers the recruitment of signaling proteins
that contain Src homology 2, Src homology 3, and pTyr-binding
domains and that lead to the activation of several pathways with
the PI3K-AKT pathway being a major downstream effector
of both PDGFR signaling and invadosome formation (43, 46).
Consistent with previous studies (47, 48), the action of PDGF-B
on nonarthritic synovial cells results in a rapid phosphorylation
of AKT(S476), further suggesting that this pathway is directly
triggered by PDGFR activation in these cells (Fig. 5A). In
addition, treatment of RA synovial cells or PDGF-stimulated
nonarthritic cells with inhibitors of PI3K (LY294002) or AKT
(AKT inhibitor XI) resulted in a concentration-dependent inhibition of invadosome formation. In contrast, inhibition of
PLCg (U73122), a downstream mediator of PDGFR signaling
(49), had no effect (Fig. 5B). The efficiency of the PLC inhibitor in synoviocytes was confirmed by its ability to block
calcium mobilization induced by LPA (Fig. 5C). Furthermore,
knockdown of the class I PI3K family catalytic subunits known
to be expressed in human synoviocytes (47) indicated that
PI3Ka and PI3Kd were selectively involved in invadosome
formation in RA cells (Fig. 5D, 5E). These results suggest that
activation of the PI3K-AKT pathway is required for PDGFRinduced invadosome biogenesis in RA synoviocytes and further support the role of this pathway in synoviocyte invasive
capabilities (48).
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FIGURE 3. Phosphorylation of PDGFR is increased in RA synovial cells and tissue sections. Synovial cells of five controls (Ctl) (three NA and two OA)
and five RA individuals were incubated in serum-free medium for 16 h. Four RA-FLS lines were incubated in the presence or absence of imatinib mesylate
(3 mM). (A) Representative images of cell lysates hybridized to a human phospho-RTK array. Hybridization signals at the corners and within the middle of
the strip served as internal controls. Dots within squared boxes represent phospho-PDGFR. The associated graph shows mean phospho-PDGFR fluorescence intensities calculated for each cell lysate. (B) Cell lysates were analyzed by immunoblotting with the indicated Abs. The associated graphs show (C)
pPDGFRab/PDGFRb ratio or (D) PDGFRb/a tubulin ratio of four independent experiments. (E) Representative images (original magnification 340) of
PDGFRb, pPDGFRab immunostaining, and control rabbit IgG of synovial tissues of OA and RA patients. (F) The associated graph shows relative labeling
intensities in arbitrary units (au) for 11 OA and 5 RA patients. *p , 0.05, **p , 0.01.
6
PDGFR ACTIVATION PROMOTES INVADOSOME FORMATION IN RA-FLS
Relationship between TGF-b and PDGF in invadosome
formation in RA synovial cells
We have recently reported that autocrine TGF-b production was
part of the mechanism that explained the aggressive invadosomeforming phenotype of rat synoviocytes derived from collageninduced arthritis joints (6). TGF-b has also been shown to promote
proliferation of smooth muscle cells and to induce epithelial–
mesenchymal transition in mammary epithelial cells through a
PDGF-dependent mechanism (50, 51). These observations led
us to investigate the relationship between TGF-b and PDGFRmediated invadosome formation in RA cells. As observed in
the case of rat synovial cells (6), addition of TGF-b1 to human
synoviocytes increased invadosome formation in a dosedependent fashion (Fig. 6A). Addition of a neutralizing anti–
TGF-b1–3 Ab significantly decreased the high incidence of
invadosome formation in RA cells (Fig. 6A), indicating that
autocrine TGF-b production also drives invadosome formation in
these cells. To examine the contribution of PDGF to TGF-b–
induced invadosome formation, TGF-b1–stimulated control synoviocytes were incubated in the presence or absence of anti–PDGF-BB
or pan-PDGF neutralizing Abs. Invadosome formation was similarly diminished upon PDGF neutralization by either of the two
Abs, supporting a role for autocrine PDGF-B in invadosome formation triggered by TGF-b (Fig. 6).
Data described above suggested that TGF-b was part of an
activation loop in RA synoviocytes that contributed to invadosome
formation through regulation of expression of PDGFR ligands.
This possibility was first tested by analysis of mRNA expression
of PDGF-A–D in RA cells compared with nonarthritic cells. Results showed that only mRNA levels of PDGF-B and C were
significantly increased in RA synoviocytes compared with nonarthritic cells (Fig. 6C). Of interest, PDGF-B mRNA expression
was significantly increased in high-invadosome–producing RA
cell lines, but not in RA cell lines, with low production of invadosomes. In contrast, there was an absence of discriminatory
effect on PDGF-C mRNA expression (Fig. 6D). Furthermore,
incubation of RA synoviocytes with TGF-b1–3 neutralizing Abs
resulted in a strong decrease of PDGF-B mRNA expression,
whereas mRNA expression of PDGF-A, -C, and -D was not affected (Fig. 6E). These data demonstrate that TGF-b contributes
to PDGFR-induced invadosome formation in RA synoviocytes,
through upregulation of PDGFR ligand, PDGF-B.
Previous reports have shown that TGF-b can modulate mRNA
expression of PDGF ligands in macrophages and endothelial and
glioma cells (52–54). To obtain further evidence that TGF-b induced PDGF-B ligand expression in synovial cells, we analyzed
mRNA expression of PDGF-B in nonarthritic cells stimulated with
TGF-b. PDGF-A, -C, and -D mRNA expression were assessed for
comparison. Addition of TGF-b led to a strong and significant
upregulation of PDGF-B, whereas only moderate changes in
PDGF-A, PDGF-C, and PDGF-D were observed (Fig. 7A). This
was associated with the ability of TGF-b to increase the phosphorylation of the PDGFR (Fig. 7B), suggesting production of
functional PDGF ligands. Previous reports have shown that Smads
bind and transactivate the PDGF-B promoter in macrophages,
endothelial cells, and gliomas (52–54), and PI3K/Akt pathways
were shown to participate in invadosome production by synovial
cells (Fig. 5A, 5B) (6). To determine whether the Smad and/or
PI3K/Akt pathways were involved in the expression of PDGF-B in
synoviocytes, we treated the cells with the TbR1/ALK5 inhibitors,
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FIGURE 4. PDGF activity regulates invadosome formation by RA synoviocytes. (A–C) Synovial cells of three NA, two OA, and four RA individuals
were plated on Oregon Green488–conjugated gelatin-coated coverslips and incubated for 48 h in the presence or absence of (A) PDGF-BB used at the
indicated concentrations (n = 3 independent experiments). Associated micrographs show representative invadosome formation (stained for F-actin [red] and
DAPI [blue]) of control and PDGF-BB–treated cells. (B) PDGF-pan–specific neutralizing Ab, PDGF-BB–specific neutralizing Ab, or an isotype-matched
IgG used at saturating concentrations (20 mg/ml); NA synoviocytes were also incubated in the presence or absence of PDGF-BB (n = 4 independent
experiments). Scale bars, 10 mM. (C) PDGFR tyrosine kinase inhibitor V used at the indicated concentrations (n = 4 independent experiments). Graphs
represent mean + SEM of the percentage of invadosome-forming cells. **p , 0.01, ***p , 0.001.
The Journal of Immunology
7
LY364947 (55), or SB-431542, a TbR1 inhibitor that was also
shown to block the activation and nuclear translocation of the
Smads (56–58) or the PI3K inhibitor LY294002 and assessed
the induction of PDGF-B mRNA by TGFb. PAI-1, a Smad2/3regulated gene, was used as an internal control. Results indicated that pharmacological suppression of the TbR1/Smad or the
PI3K pathway impeded the induction of PDGF-B mRNA expression by TGF-b, whereas, as expected, PAI-1 induction was
selectively blocked by the TbR1/Smad inhibitors (Fig. 7C, 7D). In
addition, knockdown of p110a, p110b, or p110d indicates that
P13Ka isoform was selectively involved in PDGF-B production
by RA synoviocytes (Fig. 7E). These results suggest that PDGF-B
induction in synoviocytes was dependent on both TbR1/Smad and
PI3Ka activities.
Discussion
Among the cells involved in RA pathogenesis, FLS are recognized
as the primary effectors of cartilage degradation. These cells possess strong invasive properties and produce extensive quantities of
ECM-degrading enzymes. We have previously shown that this
aggressive behavior is largely dependent on their ability to form
the actin-rich structures invadosomes, which concentrate matrix
metalloproteinases to focal sites of matrix degradation (5, 6). The
tyrosine kinase c-Src and the autocrine transglutaminase-2/TGF-b
loop were previously identified as part of the mechanism that
triggered invadosome formation in arthritic FLS. In this work, we
show that, among the main RTKs known to be expressed in RA,
the RTK PDGFR, through activation of the PI3K/AKT pathway,
also participates in promoting invadosome formation and matrix
degradation in synovial cells from RA patients. Our data reveal an
association between activation of the RTK PDGFR and TGF-b for
invadosome formation in RA-FLS that involves induction of
PDGF-B by TGF-b, an event under the control of both TbR1/
Smad and PI3K pathways. These findings identify PDGFR as an
important RTK required for the aggressive invadosome-forming
phenotype of RA synovial cells and suggest the participation of an
overactive TGF-b/PDGFR pathway during cartilage degradation
processes.
One central finding of the current studies is the potent role of
PDGFR in invadosome formation in RA synoviocytes. PDGFRa
and PDGFRb, and their ligands PDGFs A–D, have previously
been implicated in RA-FLS aggressiveness. Increased levels of
PDGFRa and PDGFR ligands were detected in FLS cultures of
RA patients (59, 60), and PDGFRab was found to be expressed in
stromal cells in the synovial lining, and in smooth muscle cells
and capillary cells in RA synovium (14). Furthermore, PDGFs
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FIGURE 5. PI3K/AKT as effectors of invadosome formation in RA synovial cells. (A) NA synovial cells were serum starved for 16 h and incubated with
PDGF-BB (10 ng/ml) for 0–30 min. Total proteins were extracted and analyzed by Western blot analysis for pPDGFRab (a[Tyr849]/b[Tyr857]), PDGFRb,
pAkt(S476), and AKT. A representative blot of three independent experiments using three independent NA cell lines is shown. (B) Nonarthritic synovial cells
stimulated with PDGF-BB (25 ng/ml) and RA synovial cells were plated on Oregon Green488–conjugated gelatin-coated coverslips and incubated for 48 h
in the presence or absence of PI3K inhibitor LY294002, the AKT inhibitor XI, the PLC inhibitor U73122, or the inactive U73343 analog used at the
indicated concentrations (n = 3–4 independent experiments using 7 independent NA cell lines and 5 independent RA cell lines). Graphs represent mean +
SEM of the percentage of invadosome-forming cell. (C) Graph showing the ability of U73122 to block LPA-induced calcium mobilization in NA synoviocytes. LPA was used at 10 mM. (D and E) RA synoviocytes were transduced with lentivirus-expressing short hairpin RNAs targeting individual PI3Ka
(shp110a), PI3Kb (shp110b), and PI3Kd (shp110d), and (D) expression of the PI3K isoforms was determined by RT-PCR or (E) cells were submitted to the
invadosome assay (n = 4 independent experiments with 4 independent RA cell lines). *p , 0.05, **p , 0.01, ***p , 0.001.
8
PDGFR ACTIVATION PROMOTES INVADOSOME FORMATION IN RA-FLS
have been found to regulate synoviocyte proliferation, invasion,
anchorage-independent growth, and collagenase transcription (61–
63). Our finding that PDGFR activation/phosphorylation was a
main trigger of signaling events that led to invadosome formation
in RA-FLS adds an original facet to the role of PDGF/PDGFR in
synovial aggressiveness that is directly related to the key effector
functions of these cells in cartilage degradation.
Results described in this work showed that PDGFRab phosphorylation was specifically increased in FLS of RA patients and
strongly elevated in the lining layer of RA synovium. It can be
argued that mechanisms responsible for PDGFR phosphorylation
can be direct, that is PDGF driven or indirect, which would not
involve PDGFs. Direct activation involves PDGF ligand-induced
PDGFR dimerization, leading to a robust activation of RTK activity, and appears to be a main mechanism responsible for
PDGFR phosphorylation. In contrast, reactive oxygen species,
which could be induced by growth factors outside of the PDGF
family, can activate Src family kinases that have been reported to
act as intracellular mediators of PDGFR phosphorylation (49). In
arthritic synoviocytes, evidence suggests that at least two pathways may be involved in PDGFR phosphorylation and activation.
First, RA or growth factor–activated synoviocytes were shown to
secrete PDGF ligands and to express the intracellular (e.g., furin)
or extracellular (e.g., tPA, uPA) proteases required for their activation (59, 60, 64–66). In this study, the findings that RA FLS
overexpressed PDGF-B and that the invadosome-forming phenotype of RA cells was inhibited using PDGF-BB–specific or PANPDGF– neutralizing Abs suggest direct phosphorylation of
PDGFR as a result of ligation by autocrine PDGF-B. Such
autoregulatory loop most likely involves TGF-b expression and
activity because PDGF-B production was blocked by TGF-b
neutralization in RA cells. Furthermore, addition of TGF-b to
synovial cells increased PDGF-B expression as well as PDGFR
phosphorylation/activation and thereby recapitulated the RA
phenotype. Second, we have reported that the active form of Src
was elevated in synovial tissues and FLS of collagen-induced
arthritis rats and that c-Src activity mediated invadosome formation in arthritic cells (5). Data indicating that Src inhibition efficiently blocked invadosome formation in arthritic FLS (5), along
with the known association of PDGFR and Src family kinases and
its site-specific phosphorylation by c-Src (67, 68), raised the
possibility that activation of Src kinases in arthritic FLS could also
contribute to PDGFR phosphorylation.
TGF-b and PDGFs are prime cytokines involved in the induction of an aggressive behavior by synovial cells (10). Based on
a TGF-b neutralization approach, autocrine TGF-b production
contributed to PDGFR-induced invadosome formation in RA patients through PDGF-B production. In accordance with previous
reports that TGF-b induced PDGF ligands in various cell types
(69–71), including synovial cells (72), PDGF-B and PDGF-C
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FIGURE 6. Involvement of autocrine TGF-b
in PDGFR-induced invadosome formation in
RA synovial cells. (A and B) Synovial cells of
NA and RA individuals were plated on Oregon
Green488–conjugated gelatin-coated coverslips
and incubated for 48 h in the presence or absence of the following reagents: NA synoviocytes were incubated in the presence or
absence of TGF-b used at the indicated concentrations, TGF-b1–3–neutralizing Ab,
PDGF pan-specific neutralizing Ab, PDGFBB–specific neutralizing Ab, or an isotypematched IgG. RA synoviocytes were incubated
in the presence or absence of TGF-b1–3–
neutralizing Ab or an isotype-matched IgG (n =
4 independent experiments using 4 NA and
4 RA cell lines). Graph represents mean +
SEM of the percentage of invadosome-forming
cells. (C–E) RT-PCR quantification of PDGF
mRNA expression in synovial cells of four
NA and/or six RA individuals. (C) PDGF-A–
D mRNA expression levels. (D) PDGF-B and
PDGF-C expression in RA synoviocytes
expressing low (,2-fold increase compared
with control synoviocytes, n = 3 independent
experiments) and high (.2-fold increase
compared with control synoviocytes, n = 4
independent experiments) invadosome-forming
capability. (E) PDGF-A–D expression in three
independent RA FLS lines previously exposed
to TGF-b1–3–neutralizing Ab or matching IgG
used at 20 mg/ml for 16 h (n = 3 independent
experiments). Graphs show the mean + SEM.
*p , 0.05, **p , 0.01, ***p , 0.001.
The Journal of Immunology
9
mRNA were found overexpressed in RA synoviocytes, whereas
PDGF-B was strongly induced by TGF-b in nonarthritic cells. Our
results are consistent with a recent report that PDGF-B and TGF-b
synergized to strongly potentiate the response of FLS to cytokines,
leading to the development of an aggressive proinflammatory
phenotype (10).
It is of note that OA synoviocytes were not able to form invadosomes upon PDGF stimulation, whereas an efficient response
was observed in synoviocytes from nonarthritic patients (Fig. 4A).
The exact reason for the lack of PDGF response in OA cells is
unknown but may involve, among others, alterations in the TGFb/
PDGF pathway in OA. Among the candidate genes that have been
reported as harboring risk alleles for OA are GDF5, a growth factor
that is part of the TGF-b superfamily, and SMAD3, which also
operates in the TGF-b pathway (73, 74). Both GDF5 and SMAD3
are involved in synovial joint development, maintenance, and repair,
and may thereby be involved in invadosome formation (75).
Formation of invadopodia in cancer cells or podosomes in Srctransformed fibroblasts requires activation of Smads and PI3K
(76–79). In this work, we showed that pharmacological inhibition
of PI3K blocked invadosome-mediated ECM degradation in RA
synoviocytes or in cells stimulated with PDGF. We also showed
that PI3K and Smad activation was required for PDGF-B mRNA
expression. PI3Ks comprise a family of signaling molecules that
have been involved in a wide range of cellular functions, including
cell growth, apoptosis, survival, and migration (80). Among them,
class I PI3K isoforms (PI3Ka, PI3Kb, PI3Kϒ, and PI3Kd) are the
best studied and are emerging drug targets in the treatment of
arthritis (23297340). Mice lacking PI3Kd were shown to be resistant to the development of experimental arthritis (47, 81), and
PI3Kϒ blockade by both genetic and pharmacological approaches
suppressed joint inflammation and cartilage damage in various
mice models of RA (47, 81, 82). It was therefore of interest to
know which isoforms contributed to invadosome formation.
Knockdown of class I PI3Ka, PI3Kb, and PI3Kd isoforms, which
are known to be expressed in human synoviocytes (47), indicated
that both PI3Ka and PI3Kd were selectively involved in invadosome formation in RA cells. In contrast, only PI3Ka participated
in autocrine PDGF-B production. These observations are consistent with previous findings indicating a role for PI3Kd in RA
synoviocyte invasiveness and joint destruction (47). They also
describe a new function for PI3Ka in growth factor gene expression. PI3Ka is an oncogene frequently mutated in human
cancer and has been shown to play a role in tumor progression
(83). New findings that the PI3Ka isoform is also involved in
PDGF-mediated prodestructive functions of RA synovial cells
suggest that dual inhibition PI3Ka and PI3Kd may protect from
cartilage damage by modulating the overactive prodestructive
functions of synoviocytes.
Another interesting finding of our investigation was the fact that
both PI3K and Smad activity were required in the regulation of
PDGF-B expression by TGF-b. Although TGF-b is known to
activate PI3K in a number of cell types, including synovial cells
(Fig. 7B), the PI3K/AKT pathway was shown to antagonize
Smad-mediated effects in most TGF-b–induced responses. This
effect was due to the ability of AKT to interfere with phosphorylation and nuclear localization of Smad3 (84, 85) or to counteract
the activity of the Smad3/FoxO nuclear complexes (86, 87). In this
work, findings that inhibition of PI3K or TbR1/Smad pathway in
synoviocytes blocked PDGF-B mRNA expression induced by
TGF-b suggest positive cooperation between both signaling
pathways. Cross-talk between Smad and PI3K pathways remains
largely unexplored. In the late phase of tumor progression, enhancement of the PI3K/AKT pathway has been shown to switch
TGF-b into a tumor-promoting signal through inactivation of
glycogen synthase kinase GSK3b that promotes Smad3 stabili-
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FIGURE 7. Implication of the
TbR1/Smad pathway in PDGF-B expression induced by TGF-b. (A and B)
Four NA synoviocyte lines were serum
starved and incubated in the presence or
absence of TGF-b (5 ng/ml) in serumfree condition for 6 or 24 h. (A) RT-PCR
quantification of PDGF-A–D mRNA expression (n = 3). (B) Western blot analysis of pPDGFRab (a[Tyr849]/b[Tyr857]),
PDGFRb, pAkt (S476), and AKT.
Representative blot of three independent experiments. (C and D) RT-PCR
quantification of (C) PDGF-B and (D)
PAI-1 mRNA expression in NA cells
incubated for 6 h with TGF-b (5 mg/ml)
in the presence or absence of PI3K inhibitor LY294002 (10 mM), TbR1 inhibitors LY364947 (1 mM), or SB431542
(1 mM) (n = 3 independent experiments).
(E) RA synoviocyte lines from four patients were transduced with lentivirusexpressing short hairpin RNAs targeting
individual PI3Ka (shp110a), PI3Kb
(shp110b), and PI3Kd (shp110d), and
expression of PDGF-B was determined
by RT-PCR (n = 3 independent experiments). Graphs represent the mean
+ SEM. *p , 0.05, **p , 0.01, ***p ,
0.001.
10
PDGFR ACTIVATION PROMOTES INVADOSOME FORMATION IN RA-FLS
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
Acknowledgments
22.
We thank Dr. Gilles Dupuis for critical reading of the manuscript and
helpful comments. We also thank Dr. Léonid Volkov for expert assistance
with confocal microscopy.
23.
Disclosures
The authors have no financial conflicts of interest.
24.
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zation (88, 89). Overexpressed Smad3 can in turn transactivate the
PDGF-B promoter (53), suggesting that one possible way PI3K
and Smads positively cooperate for PDGF-B expression in synoviocytes is through an increase in Smad3 protein stability. Interestingly, Smad3, but not Smad2, seems to be the primary target of
regulation by PI3K/Akt. This observation was consistent with our
previous findings of the selective role of Smad3 in invadopodia
formation mediated by TGF-b (79). Although further studies are
needed to delineate the exact downstream targets of PI3K and
TbR1/Smad activities and how these pathways interact with each
other, our results clearly suggest that both pathways play an essential role in the development of the prodestructive function of
RA synoviocytes.
Results of clinical trials have shown that current therapies for the
treatment of RA have only a moderate effect on joint destruction
(90–93). Although encouraging results have been reported using a
combination of the classical drug, methotrexate, with new biological treatments, such as TNF inhibitors, these new therapies are
only effective in a subset of patients, indicating a need for alternative treatments (94). Interestingly, TNF resistance has recently
been associated with a distinct synovial fibroid phenotype
enriched for gene sets associated with TGF-b/SMAD signaling, as
well as cell projection processes (95). In this work, we show that
blockade of the TbR1/PDGFR axis or downstream PI3K-AKT
pathway impaired invadosome formation, a process previously
linked to cartilage damage (5, 6). Various small-molecule inhibitors of TGF-b are being evaluated in preclinical and clinical trials
(96). Furthermore, several inhibitors that target PI3Ks are currently being tested in clinical trials for treatment of human cancers
(97, 98) and are under consideration for the treatment of RA (99).
Although further investigation of the interplay between the TGF-b
and PDGF pathways and downstream PI3K signaling pathway is
needed to define the most promising target(s), the potential action
of these inhibitors on both TGF-b pathways and PDGF-B induction highlights their therapeutic potential for treatment of joint
destruction in RA.
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