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Up-regulated transforming growth factor inducible gene h3 in rheumatoid arthritis mediates adhesion and migration of synoviocytes through ╨Ю┬▒v 3 integrinRegulation by cytokines.

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ARTHRITIS & RHEUMATISM
Vol. 54, No. 9, September 2006, pp 2734–2744
DOI 10.1002/art.22076
© 2006, American College of Rheumatology
Up-Regulated Transforming Growth Factor ␤–Inducible Gene
h3 in Rheumatoid Arthritis Mediates Adhesion and Migration
of Synoviocytes Through ␣v␤3 Integrin
Regulation by Cytokines
Eon Jeong Nam,1 Keum Hee Sa,1 Dong Wan You,1 Jang Hee Cho,1 Jae Seok Seo,1
Seung Woo Han,1 Jae Yong Park,1 Sung Il Kim,2 Hee Soo Kyung,1
In San Kim,1 and Young Mo Kang1
Objective. To delineate the expression of transforming growth factor ␤–inducible gene h3 (␤IG-H3) in
rheumatoid synovitis and to determine the regulatory
role of ␤IG-H3 in the adhesion and migration of
fibroblast-like synoviocytes (FLS).
Methods. Synovial tissue was obtained from patients with rheumatoid arthritis (RA) during joint replacement surgery, and FLS were isolated using enzymatic treatment. Immunohistochemical staining was
performed to show the expression of ␤IG-H3 within
rheumatoid synovium. Synthesis of ␤IG-H3 from FLS
was determined by semiquantitative reverse
transcription–polymerase chain reaction, Western blot
analysis, and enzyme-linked immunosorbent assay. Cell
adhesion and migration assays were performed using
the YH18 peptide in the fourth fas-1 domain of ␤IG-H3
and function-blocking antibodies to integrins.
Results. Expression of ␤IG-H3 was up-regulated
in RA synovial tissue compared with synovial tissue
from patients with osteoarthritis. FLS isolated from RA
synovial tissue constitutively produced ␤IG-H3, which
was up-regulated by transforming growth factor ␤1,
interleukin-1␤, and tumor necrosis factor ␣. Although
FLS expressed a variety of integrins, ␤IG-H3 mediated
adhesion and migration of FLS through interaction with
␣v␤3 integrin. Cytokines failed to affect the ␤IG-H3–
mediated adhesion. However, migration of FLS guided
by ␤IG-H3 was enhanced by interferon-␥ and plateletderived growth factor type BB. The YH18 peptide in the
fourth fas-1 domain of ␤IG-H3 inhibited adhesion and
migration in a dose-dependent manner.
Conclusion. The results suggest that ␤IG-H3,
which is abundantly expressed in RA synovial tissue,
plays a regulatory role in chronic destructive inflammation through the modulation of the adhesion and migration of FLS. This finding indicates the relevance of
␤IG-H3 and ␣v␤3 integrin–interacting motifs as potential therapeutic targets in this disease.
Supported by a grant from the Korea Research Foundation
(KRF-2004-041-E00169) funded by the Korean Government
(MOERD).
1
Eon Jeong Nam, MD, Keum Hee Sa, MS, Dong Wan You,
MD, Jang Hee Cho, MD, Jae Seok Seo, MD, Seung Woo Han, MD,
Jae Yong Park, MD, Hee Soo Kyung, MD, PhD, In San Kim, MD,
PhD, Young Mo Kang, MD, PhD: Kyungpook National University
School of Medicine, Daegu, Republic of Korea; 2Sung Il Kim, MD:
Pusan National University School of Medicine, Pusan, Republic of
Korea.
Dr. Nam and Ms. Sa contributed equally to this work.
Address correspondence and reprint requests to Young Mo
Kang, MD, PhD, Division of Rheumatology, Department of Internal
Medicine, Kyungpook National University Hospital, Samduk 2-Ga,
Junggu, Daegu 700-721, Republic of Korea. E-mail: ymkang@
knu.ac.kr.
Submitted for publication January 11, 2006; accepted in
revised form June 9, 2006.
Rheumatoid arthritis (RA) is a chronic inflammatory joint disease which is characterized by synovial
hyperplasia and progressive destruction of cartilage and
bone. Fibroblast-like synoviocytes (FLS) may mediate
joint destruction as final effector cells in a cytokine and
cell cascade in rheumatoid synovial tissue (1–3). Unlike
the synovial tissue of healthy joints, RA synovium is
characterized by hyperplasia of synoviocytes, which is
the result of enhanced survival, proliferation, and guided
migration (4).
The extracellular matrix (ECM) provides the
2734
␤IG-H3 IN RHEUMATOID ARTHRITIS
mechanical scaffolding within which tissue is assembled.
During inflammation, it is likely that a number of
integrins expressed on the surface of activated FLS
regulate critical adhesive interactions with a variety of
ECM proteins. These adhesive interactions anchor cells
and transduce signals that are critical for distinct biologic events such as adhesion, migration, proliferation,
and differentiation (5). Inhibition of integrin–ECM interactions suppresses cellular growth or induces apoptotic cell death (6,7).
Transforming growth factor ␤–inducible gene h3
(␤IG-H3) is a recently identified ECM protein that can
be induced by transforming growth factor ␤ (TGF␤) in
several cell types, including human melanoma cells,
epithelial cells, keratinocytes, and lung fibroblasts (8,9).
␤IG-H3 consists of 683 amino acids containing 4 homologous internal repeat domains, denoted fas-1 domains.
␤IG-H3 has been suggested to function mainly in cellular growth (9), differentiation (10), adhesion, migration
(8), and angiogenesis (11), which are mediated through
interaction with integrins ␣3␤1, ␣v␤3, and ␣v␤5. The
interactions with integrins ␣v␤3 and ␣v␤5 require at
least 18 amino acids, including highly conserved tyrosine
and leucine residues, which are denoted YH18 peptide
(11,12). Previous reports have suggested that ␤IG-H3 is
associated with diabetic nephropathy (13,14), cyclosporine-induced nephropathy (15,16), and astrocyte response to brain injury (17). To date, however, expression
of ␤IG-H3 protein has not been demonstrated in RA
synovial tissue, and it is unclear whether ␤IG-H3 is
produced by FLS and whether it mediates the functions
of RA FLS.
The aims of the present study were to delineate
the expression of ␤IG-H3 within RA synovial tissue and
to determine the regulatory role of ␤IG-H3 in the
functions of an important stromal cell, the FLS. We
found that ␤IG-H3 protein was expressed abundantly
within RA synovial tissue, and the synthesis of ␤IG-H3
from FLS was enhanced by TGF␤1, interleukin-1␤
(IL-1␤), and tumor necrosis factor ␣ (TNF␣). ␤IG-H3
mediated the adhesion and migration of FLS through
interactions with ␣v␤3 integrin, which were effectively
blocked by the YH18 peptide. Together, these results
indicate that ␤IG-H3, up-regulated within RA synovial
tissue, may play a role in the pathogenesis of RA through
the regulation of the functional activities of FLS.
PATIENTS AND METHODS
Patients. Synovial tissue was obtained during joint
replacement surgery from patients with RA, defined according
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to the 1987 revised criteria of the American College of
Rheumatology (formerly, the American Rheumatism Association) (18). Synovial tissue was also obtained from patients
with osteoarthritis (OA) who were undergoing knee joint
replacement surgery. The RA patients ranged in age from 32
years to 59 years (mean ⫾ SD 46.4 ⫾ 11.6 years). The OA
patients ranged in age from 65 years to 71 years (mean ⫾ SD
69.2 ⫾ 2.8 years). All RA patients were taking nonsteroidal
antiinflammatory drugs; 6 patients were taking methotrexate
and 4 were taking low-dose steroids at the time of the
operation. The RA patients had a mean ⫾ SD disease duration
of 13.8 ⫾ 7.5 years.
Cell isolation and culture. FLS were isolated by enzymatic dispersion of synovial tissue as previously described (19).
Cells were cultivated in Dulbecco’s modified Eagle’s medium
containing 4.5 gm/liter glucose (BioWhittaker, Walkersville,
MD) supplemented with 100 units/ml penicillin, 100 ␮g/ml
streptomycin, 2 mM ␥-glutamine, and 10% fetal calf serum,
and were maintained in a humidified atmosphere of 5% CO2
at 37°C. Upon reaching confluence, cells were detached with
trypsin/EDTA (Gibco BRL, Carlsbad, CA) and split. FLS
between passages 3 and 8 were used for the experiments.
Normal dermal fibroblasts were established from foreskin
specimens (kindly provided by Dr. Ho Yun Chung, Kyungpook
National University).
Antibodies and recombinant products. The bacterial
expression vector for wild-type ␤IG-H3 has been described
previously. Recombinant human ␤IG-H3 protein was induced
and purified as described previously (11). Monoclonal antibody to human ␤IG-H3 (clone 7A6) was prepared using
recombinant human ␤IG-H3. Polyclonal antibody was generated by immunizing rabbits with recombinant human ␤IG-H3.
YH18, YH18 control, RGD, and RGE peptides were synthesized at Peptron (Daejeon, Korea). YH18 peptide in the fourth
fas-1 domain of wild-type ␤IG-H3 was selected. Monoclonal
antibodies against CD68 (EBMII) and biotinylated rabbit
anti-mouse Ig were purchased from Dako (Glostrup, Denmark), polyclonal antibody against fibronectin was purchased
from Sigma (St. Louis, MO), and phycoerythrin-labeled antibodies against CD3 (SK7), CD14 (M␸P9), and CD20 (L27)
were purchased from BD Biosciences (San Jose, CA). Neutralizing polyclonal anti-human TGF␤1 and recombinant human cytokines including interferon-␥ (IFN␥), TNF␣, IL-1␤,
TGF␤1, IL-4, IL-10, basic fibroblast growth factor (bFGF),
platelet-derived growth factor type BB (PDGF-BB), insulinlike growth factor 1, and epidermal growth factor were purchased from R&D Systems (Minneapolis, MN). Monoclonal
function-blocking anti-␣2␤1 (BHA2.1), anti-␣3␤1 (M-KID2),
anti-␣5␤1 (JBS5), anti-␣v␤3 (LM609), anti-␣v␤5 (P1F6), and
anti-␤4 (ASC-3) antibodies were obtained from Chemicon
International (Temecula, CA).
Immunohistochemical staining. Sections of synovial
tissue were prepared from frozen tissue embedded in OCT
compound and fixed in acetone for 10 minutes. Tissue sections
were incubated with primary antibodies after blocking endogenous peroxidase with 0.03% H2O2/NaN3 buffer and nonspecific protein binding with 5% normal goat serum. Biotinylated
secondary antibodies, VECTASTAIN statin Elite ABC reagents (Vector Laboratories, Burlingame, CA), and 3,3⬘diaminobenzidine tetrahydrochloride (Dako) were used for
2736
development. Sections were mounted with VectaMount (Vector Laboratories) and analyzed under light microscopy.
Measurement of ␤IG-H3 in the culture supernatant.
The level of ␤IG-H3 was measured using the enzyme-linked
immunosorbent assay (ELISA) method as described previously (13). Briefly, a 96-well flat-bottom microtiter plate was
coated with wild-type ␤IG-H3 protein overnight at 4°C. Lyophilized culture media and standard ␤IG-H3 protein were
preincubated with anti–␤IG-H3 antibody and transferred to
the precoated plate and incubated for 30 minutes at room
temperature. After reaction with horseradish peroxidase
(HRP)–conjugated secondary antibody, substrate solution was
added for the color reaction. The reaction was stopped with 8N
H2SO4, and the absorbance was determined at 490 nm using a
model 550 microplate reader (Bio-Rad, Hercules, CA).
Flow cytometry analysis. FLS or dermal fibroblasts
were incubated under adherent conditions in medium alone or
in the presence of recombinant human TGF␤1 at 37°C, when
indicated. Cells were detached and incubated with the primary
antibodies at 4°C for 30 minutes. After washing in phosphate
buffered saline (PBS) with 0.5% bovine serum albumin (BSA),
cells were labeled with fluorescein isothiocyanate (FITC)–
conjugated goat anti-mouse Ig (BD Biosciences) at 4°C for 30
minutes and analyzed with a FACSCalibur (BD Biosciences).
RNA extraction and semiquantitative reverse
transcription–polymerase chain reaction. Total RNA was
extracted from FLS using TRIzol reagent (Gibco BRL) according to the manufacturer’s protocol. Complementary DNA
was obtained from 5 ␮g of total RNA using oligo(dT) and
AMV reverse transcriptase (Roche, Basel, Switzerland) and
amplified with primers (Bioneer, Daejeon, Korea) and LightCycler DNA master SYBR Green I (Roche). The amplification was conducted for 45 cycles at a denaturation temperature
of 95°C for 1 second, an annealing temperature of 55°C for 3
seconds, and an extension temperature of 72°C for 8 seconds,
in a LightCycler (Roche). After the final cycle, the numbers of
transcripts were determined using melting curve analysis and
an external standard curve. The following primers were used:
for ␤IG-H3, 5⬘-AGATCGAGGACACCTTTGAG-3⬘ (sense)
and 5⬘-TTGTTCAGCAGGTCTCTCAG-3⬘ (antisense); for
GAPDH, 5⬘-GGAGTCAACGGATTTGGTCG-3⬘ (sense) and
5⬘-GACGGTGCCATGGAATTTGC-3⬘ (antisense).
Immunofluorescence. FLS were mounted on a cover
glass coated with poly-L-lysine (Sigma) and cultured in media
alone or in the presence of TGF␤1. At the end of the
incubation period, cells were fixed with 4% paraformaldehyde
and permeabilized in PBS with 0.2% Triton X-100. After
blocking nonspecific protein binding, cells were incubated
serially with monoclonal anti-human ␤IG-H3 (7A6) and FITCconjugated goat anti-mouse Ig (Jackson ImmunoResearch,
West Grove, PA), and nuclei were identified by 4⬘,6diamidino-2-phenylindole. Nonimmunized IgG was used as a
negative control. The pattern of fluorescence was evaluated
under a fluorescence microscope (Zeiss, Jena, Germany).
Western blot analysis. Culture supernatants were concentrated by precipitation with trichloroacetic acid. Protein (10
␮g) from each sample was solubilized at 100°C for 5 minutes in
sample buffer and separated by electrophoresis in 10% polyacrylamide gels, then transferred to a nitrocellulose membrane. The membranes were blocked with 10% nonfat dried
milk in PBS containing 0.1% Tween 20 (PBST) and then
incubated with the primary antibody at room temperature for
NAM ET AL
Figure 1. Immunohistochemical staining of transforming growth factor ␤–inducible gene h3 (␤IG-H3) in synovial tissue from patients with
rheumatoid arthritis (RA) and osteoarthritis (OA). Expression of
␤IG-H3, fibronectin, and CD68 was examined in serial sections of RA
synovial tissue (A, C, and E, respectively) and OA synovial tissue (B, D,
and F, respectively). As a negative control, nonimmunized isotypematched antibody was used for staining (G and H). (Hematoxylin
counterstained; original magnification ⫻ 200.)
1 hour. Membranes were washed 3 times with PBST and
probed with HRP-conjugated goat anti-rabbit Ig in 5% nonfat
milk in PBST. The signals were developed using a Supersignal
Immunodetection System (Pierce, Rockford, IL).
Cell adhesion assay. The cell adhesion assay was
performed as described previously (12). Briefly, flat-bottom
96-well ELISA plates (Costar, Cambridge, MA) were coated
with recombinant human ␤IG-H3 proteins or purified human
plasma fibronectin and then blocked for 1 hour at room
temperature with PBS containing 2% BSA. Cells were suspended in culture media at a density of 3 ⫻ 105/ml, and 0.1 ml
of the cell suspension was added to each well of the plate. For
the inhibition assay, monoclonal antibodies to integrins and
YH18, YH18 control, RGD, and RGE peptides were preincu-
␤IG-H3 IN RHEUMATOID ARTHRITIS
2737
Figure 2. Induction of transforming growth factor ␤–inducible gene h3 (␤IG-H3) synthesis in fibroblast-like synoviocytes (FLS) by transforming
growth factor ␤1 (TGF␤1). A, Western blot, showing that ␤IG-H3 (68 kd) was secreted constitutively from FLS of 3 patients with rheumatoid
arthritis (RA). B, Intracellular synthesis of ␤IG-H3 in RA FLS. FLS were cultured on poly-L-lysine–coated cover glass and stimulated with TGF␤1
(0.5 ng/ml) for 24 hours. After fixation of cells with paraformaldehyde and subsequent permeabilization with 0.2% Triton X-100, FLS were stained
with anti–␤IG-H3 antibody and fluorescein isothiocyanate–conjugated goat anti-mouse IgG. Cells were examined under fluorescence microscopy.
Results are representative of 3 independent experiments (original magnification ⫻ 200). C, The expression of ␤IG-H3 mRNA was quantified by
reverse transcription–polymerase chain reaction. The number of ␤IG-H3 transcripts was divided by the number of GAPDH transcripts to normalize
values between experiments. FLS were treated with TGF␤1 (0.5 ng/ml) for 2, 4, 12, 24, and 48 hours. Results are representative of 3 independent
experiments. D, Concentrations of secreted ␤IG-H3 from FLS were measured by enzyme-linked immunosorbent assay with (solid bars) or without
(open bars) TGF␤1 stimulation (0.5 ng/ml) for 12, 24, 48, and 96 hours. E, FLS were stimulated with different concentrations of TGF␤1 for 48 hours.
Values are the mean ⫾ SEM of triplicate experiments.
bated at 37°C for 30 minutes with FLS before FLS were added
to the coated plates. After incubation for 1 hour at 37°C,
unattached cells were removed by rinsing twice with PBS.
Citrate buffer containing 3.75 mM p-nitrophenyl-N-acetyl ␤-Dglucosaminide (hexosaminidase substrate) and 0.25% Triton
X-100 was added to each well and incubated for 1 hour at 37°C.
Absorbance was measured at 405 nm in a model 550 microplate reader after blocking enzyme activity with 50 mM glycine
buffer (pH 10.4) containing 5 mmoles/liter EDTA.
Migration assay. Cell migration assays were performed
as described previously (11). Briefly, the undersurface of the
membrane in a transwell plate of 8-␮m pore size (Costar) was
coated overnight at 4°C with recombinant ␤IG-H3 proteins
and then blocked for 1 hour at room temperature with PBS
containing 2% BSA. FLS were suspended in medium at a
density of 5 ⫻ 105/ml, and 100 ␮l of the cell suspension was
added to the upper compartment of the filter. In some
experiments, FLS were preincubated for 30 minutes at 37°C
with function-blocking monoclonal antibodies or YH18, YH18
control, RGD, or RGE peptides. After allowing cells to
migrate for 6 hours at 37°C, migration was terminated by
removing cells from the upper chamber. FLS on the lower
surface of the membrane were fixed and stained with crystal
violet. The number of cells was determined in 9 randomly
selected high-power fields by using light microscopy.
Statistical analysis. Differences between groups were
examined for statistical significance using the Mann-Whitney
U test. P values less than 0.05 were considered significant.
2738
NAM ET AL
Figure 3. Regulation of ␤IG-H3 production in FLS by cytokines. FLS were stimulated with cytokines in the presence or absence of TGF␤1 (0.5
ng/ml) for 48 hours. Cell-free supernatants were collected, and the concentrations of ␤IG-H3 were determined using enzyme-linked immunosorbent
assay. A, Effect of neutralizing antibody (Ab) against TGF␤1 on the production of ␤IG-H3 induced by 0.5 ng/ml TGF␤1. ⴱ ⫽ P ⬍ 0.05 versus
TGF␤1-stimulated FLS in the absence of neutralizing antibody. B and C, RA FLS were stimulated with interferon-␥ (IFN␥) (B) or with
interleukin-1␤ (IL-1␤) or tumor necrosis factor ␣ (TNF␣) (C). D, FLS were treated with dexamethasone (Dexam; 1.0 and 100 ␮M) or cyclosporin
A (CSA; 10 and 1,000 ng/ml) for 48 hours in the presence of 0.5 ng/ml TGF␤1. ⴱ ⫽ P ⬍ 0.05 versus TGF␤1-stimulated FLS. Values are the mean
and SEM. Results are representative of 3 independent experiments. See Figure 2 for other definitions.
RESULTS
Up-regulated expression of ␤IG-H3 in RA synovial tissue compared with that in OA synovial tissue.
Synovial tissue sections obtained from 7 patients with
RA and 5 patients with OA were stained with monoclonal anti–␤IG-H3 antibody or with isotype-matched nonimmunized IgG antibodies as a negative control.
␤IG-H3 was highly up-regulated in all RA synovial
tissue (Figure 1A). While fibronectin showed a diffuse
distribution within both synovial lining and sublining
layers (Figures 1C and D), ␤IG-H3 showed a more
intense staining within the lining layer. Endothelial cells
within RA synovial tissue also expressed ␤IG-H3. In
contrast, the expression of ␤IG-H3 was markedly lower
in OA synovial tissue, in which the lining layer was the
main source of ␤IG-H3 (Figure 1B). The distribution of
␤IG-H3 was similar to that of CD68-positive cells within
both RA and OA synovial tissue (Figures 1E and F).
Tonsil tissue was stained with anti–␤IG-H3 antibody as
a positive control and showed well-demarcated interfollicular distribution (results not shown).
Regulation of ␤IG-H3 production in FLS by
cytokines. In an attempt to identify the cellular source of
␤IG-H3, which was abundantly expressed within RA
synovial tissue, we selected FLS as the primary candidate because of their capacity to produce other ECM
proteins such as fibronectin, type IV and VI collagens,
and laminin (20). As the first step, we used Western blot
analysis to examine the presence of ␤IG-H3 protein in
cultured unstimulated FLS. FLS from RA patients con-
␤IG-H3 IN RHEUMATOID ARTHRITIS
2739
Figure 4. Role of ␣v␤3 integrin as a functional receptor for ␤IG-H3–mediated adhesion of FLS. A and B, Adhesion of FLS on surfaces coated with
bovine serum albumin (BSA) and different concentrations of ␤IG-H3. A, Different concentrations of ␤IG-H3 were used for coating plates. The cells
were fixed with 8% paraformaldehyde, stained with crystal violet, and observed under the inverted microscope (original magnification ⫻ 200). B,
Ninety-six–well enzyme-linked immunosorbent assay plates coated with BSA or ␤IG-H3 were incubated with FLS at 37°C for 2 hours. Cells attached
to the surfaces were quantified by hexosaminidase assay. Values are the mean and SD of triplicate experiments. C, Flow cytometric analysis of the
surface expression of ␤4, ␣2␤1, ␣3␤1, ␣5␤1, ␣v␤3, and ␣v␤5 integrins by unstimulated FLS. The data are expressed as cell number plotted as a
function of fluorescence intensity and are representative of 3 independent experiments. Dotted histograms represent the negative control, and
shaded histograms represent the surface expression of each integrin. D, FLS were preincubated with function-blocking antibodies (Ab) to integrins
at 10 ␮g/ml for 30 minutes at 37°C and then added to the precoated wells with 10 ␮g/ml ␤IG-H3. Values are the mean and SEM of at least 3
independent experiments. ⴱ ⫽ P ⬍ 0.05 versus ␤IG-H3 alone. See Figure 2 for other definitions.
stitutively expressed ␤IG-H3 protein (Figure 2A). With
stimulation by TGF␤1 (0.5 ng/ml), FLS showed more
intense intracellular ␤IG-H3 staining with concomitant
changes in size and shape (Figure 2B). Stimulation of
FLS with TGF␤1 (0.5 ng/ml) up-regulated the expression of ␤IG-H3 transcript and protein in a timedependent manner (Figure 2C). ␤IG-H3 protein was
secreted constitutively when cultured in serum-free media. Synthesis of ␤IG-H3 protein was increased in a
dose-dependent manner after stimulation with TGF␤1 and
peaked at 0.5 ng/ml of TGF␤1 (Figure 2E).
Rheumatoid synovium is a reservoir of abundant
cytokines, both proinflammatory and antiinflammatory.
To test whether these cytokines regulate ␤IG-H3 production, FLS were stimulated with a single cytokine or
with a combination of cytokines. Among proinflammatory cytokines, IFN␥ at a lower concentration (10 ng/ml)
up-regulated the production of ␤IG-H3 induced by
TGF␤1, while IFN␥ at a higher concentration (100
ng/ml) down-regulated this production. Stimulation with
IFN␥ alone did not change the production of ␤IG-H3
(Figure 3B). TNF␣ (10 ng/ml) and IL-1␤ (10 ng/ml),
which are known to be the most influential in the
generation and maintenance of RA, up-regulated
2740
NAM ET AL
Figure 5. Regulation of ␤IG-H3–mediated migration by cytokines. Migration of FLS was assayed using 2-compartment transwells in which the
undersurface of the filter was coated with ␤IG-H3. FLS (5 ⫻ 104) in 0.1 ml medium were added to the upper chamber of transwells. A, The
undersurfaces of the transwell membrane were coated with different concentrations of ␤IG-H3. B, FLS were suspended in medium containing
interferon-␥ (IFN␥) or platelet-derived growth factor type BB (PDGF-BB) and added into the upper compartments of the transwell plate. BSA ⫽
bovine serum albumin. C and D, FLS were preincubated with function-blocking antibodies (Ab) to integrins at the concentration indicated in Figure
4 and added to the upper chambers of transwells. Cell migration was quantified by counting migrated cells in 9 microscope fields. Values are the
mean and SEM of at least 3 independent experiments. ⴱ ⫽ P ⬍ 0.01 versus ␤IG-H3 alone. See Figure 2 for other definitions. (Original
magnification ⫻ 200 in A and C.)
␤IG-H3 synthesis (Figure 3C). Treatment with neutralizing antibody against TGF␤1 at a concentration that
could block the concentration of TGF␤1 effective for
inducing ␤IG-H3 production (Figure 3A) did not inhibit
the effect of TNF␣ (10 ng/ml) and IL-1␤ (10 ng/ml),
showing that these cytokines stimulate the production of
␤IG-H3 in a TGF␤1-independent pathway (data not
shown). ␤IG-H3 synthesis induced by TGF␤1 was inhibited efficiently with a high dose of dexamethasone (100
␮M), but not with cyclosporin A (Figure 3D).
Mediation of FLS adhesion by ␤IG-H3 through
interaction with ␣v␤3 integrin. ␤IG-H3 has been reported to mediate cellular adhesion through interaction
with integrins. Interestingly, the integrins responsible for
␤IG-H3–mediated cellular adhesion differ with different
cell types. The ␣3␤1 integrin is a functional receptor in
epithelial cells, ␣v␤5 integrin in fibroblasts, and ␣v␤3
integrin in endothelial cells (8,11,12).
In the present study, we found that ␤IG-H3
mediated adhesion of FLS in a dose-dependent manner
(Figures 4A and B). Adhesion of FLS on coated ␤IG-H3
at a concentration of 10 ␮g/ml was similar to that on
coated fibronectin at a concentration of 5 ␮g/ml (data
not shown). To identify the integrin responsible for the
␤IG-H3–mediated adhesion, we tested several functionblocking antibodies against integrins. On flow cytometric
␤IG-H3 IN RHEUMATOID ARTHRITIS
Figure 6. YH18 peptide inhibits adhesion and migration of FLS mediated by ␤IG-H3. A, YH18 peptide (spanning amino acids 563–580 in the
fourth fas-1 domain [D-IV] of ␤IG-H3) (see ref. 12), YH18 control
peptide (YH18-con), and RGD (GRGDSP) and RGE (GRGESP)
peptides were prepared as described in Patients and Methods. B,
Inhibition of adhesion by YH18 peptide. FLS were preincubated with
RGE, RGD, YH18 control, or YH18 peptides at 500 ␮M for 30
minutes at 37°C and added to the ␤IG-H3–coated wells. Values are the
mean and SEM of at least 3 independent experiments. ⴱ ⫽ P ⬍ 0.01
versus YH18 control peptide; ⴱⴱ ⫽ P ⬍ 0.01 versus RGE peptide. C,
Effect of the YH18 and RGD peptides on ␤IG-H3–mediated migration of FLS. Cells were preincubated with the RGE, RGD, YH18, or
YH18 control peptides for 30 minutes before being added into the
upper compartments of the transwell plate. Cells that migrated
through the filter were stained and quantified by counting migrated
cells. Values are the mean and SEM; unstimulated migration has been
normalized to 100. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01, YH18 peptide versus
YH18 control peptide and RGD peptide versus RGE peptide. See Figure
2 for other definitions.
analysis, unstimulated FLS expressed ␣2␤1, ␣3␤1, ␣5␤1,
␣v␤3, and ␣v␤5, but not ␤4 (Figure 4C). The adhesion
of FLS on ␤IG-H3 was specifically blocked by function-
2741
blocking antibody against ␣v␤3, but not by functionblocking antibodies against ␣3␤1 and ␣v␤5 (Figure 4D).
␤IG-H3–mediated adhesion was not blocked by other
types of function-blocking antibodies against integrins
such as ␣2␤1, ␣5␤1, and ␤4 (data not shown). In
contrast, adhesion of normal dermal fibroblasts, which
also express ␣3␤1, ␣v␤3, and ␣v␤5 integrins, was efficiently blocked by antibody to ␣3␤1 integrin instead of
antibody to ␣v␤3 integrin (data not shown). To determine whether cytokines modulate ␤IG-H3–mediated
adhesion, FLS were incubated with the cytokines TNF␣,
IL-1␤, TGF␤1, bFGF, and PDGF-BB. None of these
mediators affected ␤IG-H3–mediated adhesion of FLS
(data not shown).
Mediation of FLS migration by ␤IG-H3 through
the ␣v␤3 integrin. Integrins, the major receptors connecting cells to the surrounding ECM, not only support
cell attachment but also act to regulate survival, differentiation, proliferation, and migration (21,22). To test
whether ␤IG-H3 mediates directed migration in FLS,
we performed a transmigration assay using a
2-compartment transwell system.
␤IG-H3 mediated migration of FLS in a dosedependent manner (Figure 5A). The number of migrating FLS was sharply increased up to a ␤IG-H3 concentration of 10 ␮g/ml, which was used in subsequent
experiments. To determine whether inflammatory mediators, which are known to be involved in the pathogenesis of RA, regulate ␤IG-H3–mediated migrations of
FLS, we studied the role of cytokines and growth factors.
As shown in Figure 5B, IFN␥ and PDGF-BB substantially enhanced the migratory response of FLS, which
was increased 1.5–3-fold compared with basal transmigration on a ␤IG-H3–coated filter. In contrast, IL-1␤
(10 ng/ml) slightly inhibited ␤IG-H3–mediated migration of FLS (data not shown). Other cytokines such as
TNF␣, TGF␤1, and bFGF did not exert a significant
effect (data not shown). Enhanced migration mediated
by ␤IG-H3 was effectively blocked by the functionblocking antibody to the ␣v␤3 integrin, but not by
antibodies to the ␣3␤1 and ␣v␤5 integrins (Figures 5C
and D). These results demonstrate that ␤IG-H3 mediates FLS migration through the ␣v␤3 integrin, and that
this migration is synergistically enhanced by IFN␥ and
PDGF-BB.
Inhibition of ␤IG-H3–mediated FLS adhesion
and migration by YH18 peptide. Previous reports
(11,12) noted that the 18–amino-acid peptide YH18,
which includes tyrosine, histidine, and flanking leucine/
isoleucine residues within a fragment corresponding to
2742
NAM ET AL
amino acids 548–614 of the fourth fas-1 domain, effectively inhibited cell adhesion to the ␤IG-H3 protein. The
YH18 peptide inhibited ␤IG-H3–mediated adhesion of
both MRC-5 fibroblasts (12) and endothelial cells (11),
although the integrins involved in the cell adhesion are
␣v␤5 and ␣v␤3, respectively. To examine whether the
YH18 peptide blocks ␤IG-H3–mediated FLS adhesion,
we used YH18 synthetic peptides of the fourth fas-1
domain in a cell adhesion assay (Figure 6A). This
peptide inhibited FLS adhesion to ␤IG-H3 at a concentration of 500 ␮M. Cell adhesion to ␤IG-H3 was also
inhibited by RGD peptide in a dose-dependent manner,
but not by RGE peptide (Figure 6B). These results
indicate that the YH18 peptide contains a sequence
essential for FLS adhesion to ␤IG-H3. Using a transwell
system, we then tested whether the RGD and YH18
peptides are required for ␤IG-H3–mediated migrations
of FLS. Both the RGD and YH18 peptides inhibited
FLS migration toward ␤IG-H3 in a dose-dependent
manner (Figure 6C).
DISCUSSION
In the present study, we demonstrated that
␤IG-H3 is abundantly expressed within RA synovial
tissue and produced by RA FLS. We also found that
␤IG-H3 mediates adhesion and migration of RA FLS
through ␣v␤3 integrin, which can be efficiently blocked
by YH18 peptide. These findings indicate that ␤IG-H3 is
involved in the pathogenesis of RA by mediating cell
trafficking, and that it may be a potential therapeutic
target in RA.
In RA synovial tissue, there is hyperplasia of
resident stromal cells such as FLS, which are the main
effector cells of joint-destroying pannus (23). FLS are
the major resident cells producing an abundance of
ECM proteins such as collagen, fibronectin, hyaluronic
acid, and laminin. In previous studies that have observed
ECM expression in RA synovium, fibronectin and vitronectin have been reported to be present at increased
levels (24–26). We showed that the expression of
␤IG-H3 is substantially increased in the synovial lining
and sublining layers in RA synovial tissue compared with
OA synovial tissue. In our study, FLS isolated from RA
patients produced ␤IG-H3 constitutively. The production of ␤IG-H3 from FLS was most efficiently induced by
TGF␤1 and was also induced by other cytokines such as
TNF␣, IL-1␤, and IFN␥. The source of abundant ␤IG-H3
within RA synovial tissue may be at least partially ex-
plained by the proliferated FLS and the up-regulation of
␤IG-H3 production by cytokines from these cells.
In RA, TGF␤1 was found predominantly in the
thickened synovial lining layer and was also detected at
the cartilage–pannus junction (27,28). Although systemically administered TGF␤1 suppresses development of
experimental arthritis (29), localized injections of
TGF␤1 into the articular space induced apparent arthritis which was characterized by pronounced synovial
hyperplasia (30,31). Antagonism of TGF␤1 with a neutralizing antibody blocked pathologic development in
the tissue in an experimental model of chronic erosive
arthritis (32). The role of TGF␤1 in inflammation is only
partially known and involves recruitment and activation
of immature leukocyte populations (33). Considering
that TGF␤1 is one of the main stimuli for ECM production, accumulation of ECM may be involved in regulating inflammation through interactions with stromal resident cells as well as with infiltrating inflammatory
leukocytes. As a TGF␤1-inducible ECM protein,
␤IG-H3 may also mediate inflammation by directly or
indirectly participating in the regulation of cellular adhesion, migration, and activation of FLS. In addition,
ECM–FLS interactions are important because the synovial lining cells and the pannus are susceptible to shear
stress (34). We demonstrate that ␤IG-H3 mediates the
adhesion of FLS as effectively as fibronectin. The ECM–
cell interactions, coupled to cytoskeletal elements, provide a potent anoikis resistance factor (35). The interaction between FLS and ␤IG-H3 may provide an
additional signal against apoptosis, and needs further
study.
The present study revealed that ␤IG-H3 mediates the adhesion of RA FLS via interaction with ␣v␤3
integrin. ␤IG-H3 has multiple cell adhesion motifs
within the fas-1 domains that can mediate interactions
with several cell types through different integrins, including ␣3␤1 (8,36), ␣v␤3 (11), and ␣v␤5 (12). Although
FLS expressed all these integrins, only ␣v␤3 was used for
adhesion on ␤IG-H3. In contrast, adhesion of normal
dermal fibroblasts, which also expressed all 3 integrins,
was blocked mainly by antibody to ␣3␤1 integrin. The
choice of the integrin may depend on the activation state
of the integrins in each cell type (12). Intracellular
molecular events may modulate the affinity and avidity
of integrins for their ECM ligand (37,38). In FLS, ␣v␤3,
but not ␣3␤1 or ␣v␤5, may have an appropriate conformation to engage the binding motif on ␤IG-H3. The
ability of extracellular domains of integrin to bind
ligands can be activated on a time scale of less than 1
second by signals within the cell, which is particularly
␤IG-H3 IN RHEUMATOID ARTHRITIS
evident with integrins on leukocytes (39). In the present
study, pretreatment of FLS with cytokines and growth
factors did not influence the ␤IG-H3–mediated adhesion.
The conserved consensus sequence of ␤IG-H3
that binds to both ␣v␤3 and ␣v␤5 integrins contains
highly conserved tyrosine and histidine residues that are
designated YH motif (11,12). At least 18 amino acids
including these conserved residues are required to mediate fibroblast and endothelial cell adhesion. We demonstrated that adhesion of FLS to ␤IG-H3 was effectively blocked by both RGD peptide and YH18 peptide.
Because the ␣v␤3 and ␣v␤5 integrins are known to share
ligands (40), and because their interactions with the
YH18 peptide have been shown to be RGD dependent
(11,12), the YH18 and RGD peptides may interact with
the ␣v␤3 and ␣v␤5 integrins in a similar manner.
We have shown that migration of FLS induced by
␤IG-H3 is markedly enhanced by IFN␥ and PDGF-BB
in a dose-dependent manner. In addition to supporting
cell adhesion to the ECM, integrins act in concert with
receptors for soluble factors to regulate cell migration,
survival, proliferation, and gene transcription (21,41,42).
This cooperation occurs at many levels, ranging from
membrane-proximal interactions, in which the different
types of receptors influence each other’s activity, to
multiple inputs into common pathways (43). In contrast,
TGF␤1 and bFGF, which can drive FLS proliferation
(31,44,45), had no effect on ␤IG-H3–mediated migration of FLS. These results indicate that ␤IG-H3 synthesis is not directly linked to migratory stimulation of FLS.
Within RA synovial tissue, a large pool of ECM
supports the architecture and also regulates the functions of a variety of cell types. ␤IG-H3 has been isolated
from articular cartilage and has been suggested to play a
role in osteogenesis, since ␤IG-H3 transcripts were
observed in proliferating chondrocytes and areas of
endochondral ossification (46). There is a possibility that
␤IG-H3 expressed in the bone may interact with ␣v␤3expressing osteoclasts; if this is true, such an interaction
could be blocked by YH18 peptides. Although the
overall relevance of ␤IG-H3 in the pathogenesis of RA
is still not clear, it might be important in the hierarchy of
synovial inflammation considering its abundance within
rheumatoid synovial tissue and its regulatory effects on
FLS. Therefore, further investigation is required to
understand the functions of ␤IG-H3 in rheumatoid
synovial inflammation and to define the therapeutic
potential of ␤IG-H3–derived peptides.
2743
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