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Lectin-like oxidized low-density lipoprotein receptor 1 mediates matrix metalloproteinase 3 synthesis enhanced by oxidized low-density lipoprotein in rheumatoid arthritis cartilage.

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ARTHRITIS & RHEUMATISM
Vol. 50, No. 11, November 2004, pp 3495–3503
DOI 10.1002/art.20581
© 2004, American College of Rheumatology
Lectin-Like Oxidized Low-Density Lipoprotein Receptor 1
Mediates Matrix Metalloproteinase 3 Synthesis
Enhanced by Oxidized Low-Density Lipoprotein in
Rheumatoid Arthritis Cartilage
Takumi Kakinuma,1 Tadashi Yasuda,2 Takefumi Nakagawa,3 Teruko Hiramitsu,3
Miki Akiyoshi,3 Masao Akagi,4 Tatsuya Sawamura,5 and Takashi Nakamura3
bation with neutralizing anti–LOX-1 antibody. MMP-3
synthesis by chondrocytes in explant cartilage was evaluated by immunofluorescence, and protein secretion
into conditioned medium was monitored by immunoblotting and enzyme-linked immunosorbent assay.
Results. The majority of the RA chondrocytes
stained positively with both anti–LOX-1 and anti–oxLDL antibodies; however, no positive cells were found in
OA and normal cartilage specimens. Anti–LOX-1 antibody suppressed the binding of DiI-labeled ox-LDL to
chondrocytes in explant culture, suggesting that the
interaction was mediated by LOX-1. In contrast to
native LDL, ox-LDL induced MMP-3 synthesis by articular chondrocytes in association with the induction of
LOX-1, which resulted in enhanced secretion of MMP-3
into the culture medium. Anti–LOX-1 antibody reversed
ox-LDL–stimulated MMP-3 synthesis to control levels.
Conclusion. Ox-LDL, principally mediated by
LOX-1, enhanced MMP-3 production in articular chondrocytes. Increased accumulation of ox-LDL with elevated expression of LOX-1 in RA cartilage indicates a
specific role of the receptor–ligand interaction in cartilage pathology in RA.
Objective. To investigate for the presence of oxidized low-density lipoprotein (ox-LDL) and lectin-like
oxidized LDL receptor 1 (LOX-1) in cartilage specimens
from rheumatoid arthritis (RA) joints and to determine
whether the interaction of ox-LDL with LOX-1 can
induce matrix metalloproteinase 3 (MMP-3) in articular cartilage explant culture.
Methods. Human articular cartilage specimens
obtained from patients with RA, osteoarthritis (OA),
and femoral neck fractures were examined for LOX-1
and ox-LDL by confocal fluorescence microscopy. The
association between ox-LDL and LOX-1 was evaluated
by immunofluorescence analysis. Articular cartilage
specimens from patients with femoral neck fractures
were incubated with ox-LDL, with or without preincuDr. Yasuda’s work was supported by a grant-in-aid for
scientific research from the Japan Society for the Promotion of
Science. Dr. Sawamura’s work was supported by grants from the
Ministry of Education, Culture, Sports, Science, and Technology of
Japan; the Ministry of Health, Labor, and Welfare of Japan; the
Organization for Pharmaceutical Safety and Research; Senri Life
Science Foundation; Mitsubishi Pharma Foundation; and Suzuki
Memorial Foundation.
1
Takumi Kakinuma, MD: Kyoto University Graduate School
of Medicine, Kyoto, Japan, and National Cardiovascular Center
Research Institute, Osaka, Japan; 2Tadashi Yasuda, MD, PhD: Kyoto
University Graduate School of Medicine, Kyoto, Japan, and Faculty of
Health, Budo, and Sports Studies, Tenri University, Tenri, Japan;
3
Takefumi Nakagawa, MD, PhD, Teruko Hiramitsu, MD, Miki Akiyoshi, MD, Takashi Nakamura, MD, PhD: Kyoto University Graduate
School of Medicine, Kyoto, Japan; 4Masao Akagi, MD, PhD: Kinki
University School of Medicine, Osaka, Japan; 5Tatsuya Sawamura,
MD, PhD: National Cardiovascular Center Research Institute, Osaka,
Japan.
Address correspondence and reprint requests to Tadashi
Yasuda, MD, PhD, Department of Orthopaedic Surgery, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin,
Sakyo-ku, Kyoto 606-8507, Japan. E-mail: tyasuda-jsb@umin.ac.jp.
Submitted for publication October 20, 2003; accepted in
revised form July 13, 2004.
Rheumatoid arthritis (RA) is a chronic inflammatory joint disease characterized by synovial proliferation and destruction of cartilage and bone around the
inflamed joint. Lipid metabolism in RA patients has
been reported to differ from that in healthy subjects (1),
and it might be one of the causes of a high incidence of
cardiovascular disease in such patients. The lipids and
lipoproteins in synovial fluid from inflamed joints of RA
patients are oxidized (2–4), as is fatty acid in oxidized
serum (5). It has also been shown that oxidized lowdensity lipoprotein (ox-LDL) accumulates in inflamed
3495
3496
joints as foam cells (6) similar to those found in vascular
atherosclerotic lesions. In the formation of atherosclerotic plaques, inflammatory cytokines, including
interleukin-1 (IL-1), control immune-mediated inflammatory processes, which result in the accumulation of
ox-LDL at the lesions (7). Lipid peroxidation and oxLDL affect gene expression in endothelial cells and
atherogenic changes in vivo. Recent studies have implicated the involvement of lipid peroxides in cartilage
degradation (8) because lipid peroxides cause structural
destabilization of cartilage matrix (9). In addition, reactive oxygen species (ROS) mediate collagenase expression by IL-1␤ in chondrocytes (10). Thus, the relationship between RA and atherosclerosis has been
recognized with regard to immunologic aspects as well as
lipid metabolism and redox status (8). Although ox-LDL
apparently plays a central role in the pathogenesis of
atherosclerosis (11,12), there are no clear data on the
pathologic roles of ox-LDL in RA.
Lectin-like ox-LDL receptor 1 (LOX-1), originally cloned from vascular endothelial cells, is one of the
ox-LDL receptors. This 273–amino acid molecule has
been named OLR1 (oxidized LDL) by the Human
Genome Organisation Gene Nomenclature Committee
at the University College London. It is a singletransmembrane protein with the carboxyl-terminal end
containing an extracytoplasmic C-type lectin domain
(13). LOX-1 is expressed in various cells, including
endothelial cells, macrophages, vascular smooth muscle
cells, and dendritic cells, and its expression is enhanced
by oxidative stress, stimulation by mediators of inflammation such as IL-1␤ and tumor necrosis factor ␣
(TNF␣) (13–16), and fluid shear stress (17). LOX-1 has
been implicated in the initiation and development of
atherosclerosis, the promotion of phagocytosis of aged
or apoptotic cells by endothelium, and antigen processing in dendritic cells. We have recently demonstrated
that ox-LDL up-regulates LOX-1 messenger RNA
(mRNA) in rat articular chondrocytes in vitro (18).
Using a rat zymosan-induced arthritis (ZIA) model, we
have also shown that LOX-1 promotes joint inflammation and cartilage destruction with the accumulation of
ox-LDL in articular chondrocytes (19). Currently, however, the presence of LOX-1 in human RA cartilage
remains unclear.
Matrix metalloproteinases (MMPs) are the critical enzymes involved in the destruction of articular
cartilage in various arthritic diseases, including RA and
osteoarthritis (OA). Among the MMPs, MMP-3 is active
against cartilage matrix components such as proteoglycan and fibronectin, and can activate proMMPs (20).
KAKINUMA ET AL
Higher levels of MMP-3 (stromelysin 1) are found in the
synovial fluid of RA patients than in that of OA patients
(21). Serum levels of MMP-3 have been shown to
correlate with the severity of RA (22,23).
The present study was designed to identify
LOX-1 expression in chondrocytes in RA cartilage and
elucidate the pathogenic role of ox-LDL binding to
LOX-1 in MMP-3 production by chondrocytes. We
showed that the association between ox-LDL and
LOX-1, both of which were elevated in RA cartilage,
enhanced MMP-3 production in articular cartilage explant culture.
MATERIALS AND METHODS
Reagents and antibodies. Phosphate buffered saline
(PBS), 10% neutralized formalin solution, and ionophore
monensin were purchased from Sigma-Aldrich (Tokyo, Japan).
Biotin-blocking reagent for immunofluorescence study was
obtained from Dako (Kyoto, Japan). Block Ace for immunostaining and immunoblotting was obtained from Dainippon
(Osaka, Japan). Rabbit anti-mouse IgG antibody conjugated
with Alexa Fluor 488, streptavidin–Alexa Fluor 488, and
DiIC18(3) were purchased from Molecular Probes (Eugene,
OR). Mouse monoclonal anti–ox-LDL antibody (clone OXL
41.1) was obtained from NeoMarkers (Fremont, CA). Control
mouse IgG, rabbit anti–MMP-3 antibody, and goat anti-rabbit
IgG antibody conjugated with alkaline phosphatase were purchased from Dako. Human recombinant IL-1␤ was obtained
from R&D Systems (Minneapolis, MN).
Preparation of LDL and ox-LDL. Human LDL (density 1.019–1.063 gm/ml) was isolated from fresh donated
plasma by sequential ultracentrifugation, as previously described (13). Briefly, the fresh plasma was centrifuged twice in
a KBr gradient solution for 16 hours at 4°C to obtain native
LDL. After dialysis in PBS overnight at 4°C, the native LDL
solution was reacted with 7.5 ␮M CuSO4 for 20 hours at 37°C
for oxidation. The oxidative state was confirmed by measuring
the amount of thiobarbituric acid–resistant substances (⬃10
nmoles malondialdehyde equivalent per mg protein in oxLDL). Agarose gel electrophoresis showed increased electrophoretic mobility and minimal aggregation of ox-LDL particles. DiI was conjugated with ox-LDL according to the
manufacturer’s instructions (Molecular Probes). Our previous
study showed that the DiI-labeled ox-LDL prepared with
CuSO4 is actively taken up by Chinese hamster ovary cells that
express LOX-1 (13).
Articular cartilage explant culture. Human articular
cartilage with no significant arthritic changes was obtained
from non–weight-bearing regions of the femoral head from
patients undergoing replacement surgery for femoral neck
fracture. OA cartilage specimens were obtained from non–
weight-bearing areas of the distal femur and the proximal tibia
from patients undergoing total knee replacement surgery who
were diagnosed as having OA based on the American College
of Rheumatology (ACR; formerly, the American Rheumatism
Association) criteria (24). RA cartilage specimens were obtained from non–weight-bearing regions of the posterior con-
ROLE OF LOX-1 IN MMP-3 PRODUCTION IN RA
dyles of the distal femur during total knee replacement surgery
in patients who fulfilled the ACR 1987 revised criteria (25).
The cartilage was transferred to 24-well plates (⬃100 mg/well;
Corning, Corning, NY) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 mM HEPES
buffer, 100 units/ml penicillin, 100 units/ml streptomycin (all
from Gibco BRL, Grand Island, NY), and 3.7 gm/liter
NaHCO3. The cartilage was precultured in serum-free DMEM
for 2 days at 37°C in a humidified atmosphere of 5% CO2/95%
air, with or without pretreatment of cartilage explant with 15
␮g/ml neutralizing anti–LOX-1 antibody (JTX-92) or with 15
␮g/ml normal mouse IgG for 24 hours. After preculture, the
cartilage pieces were incubated for 2 days in serum-free
DMEM supplemented with DiI-labeled ox-LDL (10 ␮g/ml).
To measure MMP-3 in culture medium, articular cartilage was
preincubated in the presence or absence of 15 ␮g/ml neutralizing anti–LOX-1 antibody or control mouse IgG for 24 hours,
followed by coincubation with ox-LDL (10, 40, or 100 ␮g/ml)
or native LDL (150 ␮g/ml) for 5 days. The conditioned
medium was collected and stored at ⫺20°C.
Measurement of the activity of lactate dehydrogenase
(LDH). The effects of ox-LDL on cell viability were determined by measuring LDH activity released into the culture
medium (LDH C II test kit; Wako, Osaka, Japan). The assay
was performed in accordance with the manufacturer’s protocol
using cartilage explant cultures in the presence or absence of
ox-LDL for 5 days.
Immunofluorescence study. Articular cartilage specimens were obtained as described above. The cartilage samples
were fixed immediately after harvest in 10% neutralized
formalin and frozen in Tissue-Tek OCT compound (Sakura,
Westhaven, CA). Frozen sections of 8 ␮m thickness were
prepared with a cryostat (CM1850; Leica Microsystems, Wetzlar, Germany) on silane-coated glass plates (Matsunami Glass,
Osaka, Japan). The specimens were air-dried and nonspecific
reactivity was blocked by treatment with Block Ace. The
sections were reacted with mouse monoclonal anti–ox-LDL
antibody (1:100 in PBS–10% Block Ace) at room temperature
for 1 hour, and thereafter with secondary anti-mouse IgG
antibody conjugated with Alexa Fluor 488 (1:1,000 in PBS) at
room temperature for 1 hour. To visualize LOX-1 in chondrocytes, the specimens were treated with a biotin-blocking reagent before reacting with biotinylated anti-human LOX-1
monoclonal antibody or control mouse IgG (15 ␮g/ml in
PBS–10% Block Ace), followed by streptavidin–Alexa Fluor
488 (1:1,000 in PBS).
To detect LOX-1 and MMP-3 by immunofluorescence
analysis, the cartilage was incubated with ox-LDL or native
LDL for 48 hours in a 24-well plate (⬃100 mg/well) in DMEM
after preculture for 2 days. The ionophore monensin was
added to the cultures at 5 ␮M for the last 12 hours to prevent
the secretion of newly synthesized proteins. Cryostat sections
were permeabilized for 10 minutes at room temperature with
0.15% Triton X-100 in PBS, and then incubated with biotinylated anti-human LOX-1 monoclonal antibody (15 ␮g/ml in
PBS–10% Block Ace) followed by streptavidin–Alexa Fluor
488 (1:1,000 in PBS), or alternatively with rabbit anti–MMP-3
antibody (1:200 in PBS–10% Block Ace) followed by secondary anti-rabbit antibody conjugated with Alexa Fluor 488
(1:1,000 in PBS). The specimens were washed with PBS–0.05%
3497
Tween 20 for 20 minutes between each procedure. Images
were obtained with a FluoView confocal microscope (Olympus, Tokyo, Japan) and prepared with Photoshop software
(Adobe Systems, San Jose, CA).
Evaluation of MMP-3. The collected medium, normalized by the wet weight of the cartilage piece in each well for
culture, was separated by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis with 4–20% gradient gel
(Daiichi Pharmaceutical, Tokyo, Japan). The proteins were
electrotransferred to polyvinylidene difluoride membranes
(Immobilon-P; Millipore, Bedford, MA), which were immersed in methanol and air-dried to block nonspecific reactivity, followed by reacting with rabbit anti–MMP-3 antibody (1
␮g/ml) at 4°C overnight. After an extensive wash with PBS–
Tween 20, the membranes were incubated with goat anti-rabbit
antibody conjugated with alkaline phosphatase (0.5 ␮g/ml),
followed by the addition of nitroblue tetrazolium/BCIP to
visualize MMP-3. The membranes containing MMP-3 were
scanned and the intensity of the protein band was quantified
using Photoshop and Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). The experiments were repeated 4
times. In each experiment, the signal intensity of the samples
was divided by that of the normal control to yield a normalized
value for the relative signal intensity.
MMP-3 levels in the conditioned medium were also
evaluated using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (The Binding Site, Birmingham, UK) for proMMP-3 according to the manufacturer’s
instructions.
Statistical analysis. Comparisons of the relative protein band intensities of immunoblotting for MMP-3 and the
determinations of MMP-3 by ELISA were analyzed using
Student’s t-tests with StatView software (SAS Institute, Cary,
NC). Data are expressed as the mean ⫾ SD. P values less than
0.05 were considered significant.
RESULTS
Expression of LOX-1 in chondrocytes in RA
cartilage. Initially, the level of LOX-1 expression was
determined in chondrocytes in RA cartilage. Articular
cartilage specimens that were obtained from 3 RA patients
were incubated with anti–LOX-1 antibody and
streptavidin–Alexa Fluor 488. Immunofluorescence microscopic analyses demonstrated intense immunolocalization
of LOX-1 in association with chondrocytes in the 2 of 3 RA
cartilage specimens (Figure 1). LOX-1 was localized in
chondrocytes throughout the RA cartilage, while the signal
intensity of LOX-1 appeared to be strong near the articular
surface. The remaining specimen showed weak but clear
localization of LOX-1 in some chondrocytes, whereas
when OA and normal cartilage specimens were examined,
no immunoreactive staining was found with anti–LOX-1
antibody. Control nonspecific mouse IgG yielded no positive staining in chondrocytes.
3498
Figure 1. Identification of lectin-like oxidized low-density lipoprotein
receptor 1 (LOX-1) on chondrocytes in rheumatoid arthritis (RA)
cartilage. Cryostat sections of RA, osteoarthritis (OA), and normal
cartilage were stained with biotinylated mouse monoclonal anti–
LOX-1 antibody and streptavidin–Alexa Fluor 488. Articular surfaces
are at the top. Shown are RA and OA cartilage specimens obtained
from 3 patients and a representative of 3 normal specimens. Nonspecific mouse IgG was used as a negative control. Note that the
fluorescent signals around the chondrocyte lacunae in the deep layer
were artifactual. Bar ⫽ 200 ␮m.
Association of ox-LDL with chondrocytes in RA
cartilage. The cartilage specimens used for LOX-1 staining were also examined for ox-LDL by immunofluorescence microscopic studies with mouse monoclonal anti–
ox-LDL antibody and secondary anti-mouse IgG
conjugated with Alexa Fluor 488. In all 3 RA specimens,
strong staining of ox-LDL was observed in chondrocytes
(Figure 2). In contrast, no detectable ox-LDL was found
in OA or normal cartilage specimens. Control mouse
IgG showed no positive staining (results not shown).
Effects of neutralizing anti–LOX-1 antibody on
ox-LDL binding to chondrocytes. Further experiments
were performed to clarify the involvement of LOX-1 in
binding of ox-LDL to chondrocytes. When normal cartilage explants were incubated with 10 ␮g/ml DiI-labeled
ox-LDL for 48 hours following preculture in serum-free
DMEM, confocal microscopic images showed that the
DiI-labeled ox-LDL penetrated the cartilage matrix and
associated with chondrocytes in a punctated pattern
(Figure 3A). The association of ox-LDL was more
apparent in chondrocytes near the surface of the cartilage explants. Preincubation of articular cartilage with
KAKINUMA ET AL
Figure 2. Identification of oxidized low-density lipoprotein (ox-LDL)
in rheumatoid arthritis (RA) cartilage. Cryostat sections of RA,
osteoarthritis (OA), and normal cartilage were stained with mouse
monoclonal anti–ox-LDL antibody and Alexa Fluor 488–conjugated
anti-mouse secondary antibody. Shown are RA and OA cartilage
specimens obtained from 3 patients and a representative of 3 normal
specimens. Bar ⫽ 100 ␮m.
neutralizing anti–LOX-1 antibody at 15 ␮g/ml for 24
hours before addition of DiI-labeled ox-LDL completely
blocked the accumulation of DiI-labeled ox-LDL in
chondrocytes (Figure 3B). In contrast, control IgG failed
to inhibit ox-LDL accumulation (Figure 3C). These
findings suggest that LOX-1 principally mediates the
binding of ox-LDL to articular chondrocytes, which is
consistent with our previous observation using rat articular chondrocytes (18).
Figure 3. Association of oxidized low-density lipoprotein (ox-LDL)
with chondrocytes via lectin-like oxidized low-density lipoprotein
receptor 1 (LOX-1). Normal cartilage was incubated with DiI-labeled
ox-LDL at 10 ␮g/ml under serum-free conditions for 48 hours, fixed,
and examined by confocal microscopy (A). Following preincubation
with neutralizing anti–LOX-1 antibody at 15 ␮g/ml (B) or control
mouse IgG at 15 ␮g/ml (C) for 24 hours, cartilage was incubated for 48
hours with DiI-labeled ox-LDL at 10 ␮g/ml in the presence of the
antibody at the same concentration. Bars ⫽ 20 ␮m.
ROLE OF LOX-1 IN MMP-3 PRODUCTION IN RA
Figure 4. Induction of LOX-1 in ox-LDL–stimulated chondrocytes in
cartilage explant culture. Normal cartilage specimens were incubated
for 2 days with no additives (A), with native LDL at 150 ␮g/ml (B), or
with ox-LDL at 40 ␮g/ml (C). The ionophore monensin was added for
the final 12 hours. Cryostat sections were stained with anti–LOX-1
monoclonal antibody and streptavidin–Alexa Fluor 488. Bar ⫽ 50 ␮m.
See Figure 3 for definitions.
Induction of LOX-1 in ox-LDL–stimulated chondrocytes. To examine the effects of ox-LDL on LOX-1 in
explant culture, normal cartilage was incubated with or
without ox-LDL at 40 ␮g/ml or with native LDL at 40 or
150 ␮g/ml for 2 days. The ionophore monensin was used
to prevent the secretion of newly synthesized proteins.
Immunofluorescence analysis with anti–LOX-1 monoclonal antibody revealed that LOX-1 was rarely found in
untreated chondrocytes (Figure 4A) or in those treated
with native LDL (Figure 4B). In contrast, ox-LDL
enhanced LOX-1 accumulation in chondrocytes in normal cartilage explant culture (Figure 4C).
Enhanced production of MMP-3 in ox-LDL–
stimulated chondrocytes. Finally, the functional role of
the interaction between ox-LDL and LOX-1 was investigated. Normal articular cartilage was incubated with
ox-LDL at 40 ␮g/ml or with native LDL at 40 or 150
␮g/ml, and the ionophore monensin was added 12 hours
before fixation. IL-1␤ (2 ng/ml) was used as a positive
control to detect MMP-3. Since monensin caused intracellular accumulation of newly synthesized MMP-3 by
blocking the secretion of proteins, immunofluorescence
microscopic analyses with anti–MMP-3 antibody demonstrated that IL-1␤ treatment resulted in a marked
increase in MMP-3 in chondrocytes (Figure 5A). Control cultures with no additives showed no positive staining of MMP-3 (Figure 5E). Ox-LDL at 40 ␮g/ml also
stimulated the intracellular accumulation of MMP-3
(Figure 5C). In contrast, native LDL at 40 or 150 ␮g/ml
failed to induce MMP-3 (Figure 5D). When articular
cartilage was pretreated with neutralizing anti–LOX-1
antibody at 15 ␮g/ml, we found no enhanced accumulation of MMP-3 induced by ox-LDL at 40 ␮g/ml (Figure
5B). Control mouse IgG failed to alter the effect of
ox-LDL on enhanced synthesis of MMP-3 (results not
shown).
Levels of secreted MMP-3 after treatment with
3499
ox-LDL were also evaluated by immunoblotting with
anti–MMP-3 antibody. When normal cartilage was incubated with ox-LDL at 10, 40, or 100 ␮g/ml, we found that
40 ␮g/ml of ox-LDL was sufficient to induce increased
secretion of MMP-3 into conditioned medium (Figure
6A). Levels of secreted MMP-3 induced by ox-LDL at 40
and 100 ␮g/ml (mean ⫾ SD 2.07 ⫾ 1.17 and 2.22 ⫾ 1.18,
respectively) (Figure 6B, lanes 4 and 5) were significantly (P ⬍ 0.05) higher than those induced by native
LDL (0.91 ⫾ 0.07) (Figure 6B, lane 2). There was a
tendency for ox-LDL at 10 ␮g/ml to increase MMP-3
production (1.44 ⫾ 1.41) (Figure 6B, lane 3), although it
was not statistically significant compared with native
LDL. Preincubation with neutralizing anti–LOX-1 antibody at 15 ␮g/ml almost completely blocked enhanced
MMP-3 secretion by ox-LDL at 40 ␮g/ml (0.92 ⫾ 0.41;
P ⬍ 0.05 versus ox-LDL at 40 ␮g/ml) (Figure 6B, lane 6).
Control IgG had no effect on increased MMP-3 secretion induced by ox-LDL at 40 ␮g/ml (mean ⫾ SD 2.13 ⫾
1.85; data not shown). IL-1␤ at 2 ng/ml caused a stronger
secretion of MMP-3 (5.62 ⫾ 1.92) (Figure 6B, lane 7)
compared with the levels induced by ox-LDL treatment
and controls (P ⬍ 0.01).
MMP-3 levels in the conditioned medium determined by ELISA (Figure 6C) were consistent with the
results of immunoblot analysis (Figure 6B). Compared
with the level in LDL-treated cultures (5.12 ⫾ 1.16
ng/ml/mg of cartilage), treatment with ox-LDL at 40
Figure 5. Identification of enhanced matrix metalloproteinase 3
(MMP-3) production mediated by LOX-1 in ox-LDL–stimulated cartilage explant culture. Normal cartilage specimens were incubated with
interleukin-1␤ (IL-1␤) at 2 ng/ml (A), with 40 ␮g/ml ox-LDL with (B)
or without (C) anti–LOX-1 antibody at 15 ␮g/ml, with 150 ␮g/ml native
LDL (D), or with no additives (control) (E) for 2 days. The ionophore
monensin was added for the final 12 hours. Cryostat sections were
stained with anti–MMP-3 antibody. Bar ⫽ 100 ␮m. See Figure 3 for
other definitions.
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KAKINUMA ET AL
Figure 6. Evaluation of enhanced matrix metalloproteinase 3 (MMP-3) production in ox-LDL–stimulated cartilage explant
culture. A and B, Normal cartilage was incubated for 5 days with no additives (control, lane 1), with 150 ␮g/ml native LDL
(lane 2), with 10, 40, or 100 ␮g/ml ox-LDL (lanes 3, 4, and 5, respectively), with 40 ␮g/ml ox-LDL with 15 ␮g/ml anti–LOX-1
antibody (lane 6), or with 2 ng/ml interleukin-1␤ (IL-1␤) (lane 7). Conditioned medium was subjected to immunoblotting
with anti–MMP-3 antibody. The amount of sample applied for sodium dodecyl sulfate–polyacrylamide gel electrophoresis
was normalized with the wet weight of the cartilage specimen in each well. Four separate experiments were performed, with
similar results. A representative result is shown in A. The relative signal intensity of the protein band for MMP-3 was
determined against a control. Values in B are the mean ⫾ SD of 4 determinations. ⴱ ⫽ P ⬍ 0.05 versus lanes 1, 2, and 6;
# ⫽ P ⬍ 0.05 versus lane 4; ⴱⴱ ⫽ P ⬍ 0.01 versus all the other lanes. C, Normal cartilage was incubated for 5 days with no
additives (control), with 150 ␮g/ml native LDL, 40 ␮g/ml ox-LDL, 40 ␮g/ml ox-LDL with 15 ␮g/ml anti–LOX-1 antibody,
or 2 ng/ml IL-1␤. MMP-3 protein levels in the conditioned medium were determined by enzyme-liked immunosorbent assay.
Values are the mean ⫾ SD of 4 determinations. ⴱ ⫽ P ⬍ 0.05 versus native LDL–treated cultures; ⴱⴱ ⫽ P ⬍ 0.05 versus
ox-LDL–treated cultures, by t-test. See Figure 3 for other definitions.
␮g/ml resulted in a significant increase in MMP-3
(13.6 ⫾ 3.90 ng/ml/mg cartilage; P ⬍ 0.05), which was
significantly reversed with preincubation with anti–
LOX-1 antibody at 15 ␮g/ml (8.12 ⫾ 2.56 ng/ml/mg
cartilage; P ⬍ 0.05).
When LDH levels in the conditioned medium
were assayed after treatment with or without ox-LDL at
40 or 100 ␮g/ml for 5 days, there was no significant
difference between the cultures with and without oxLDL treatment (data not shown).
DISCUSSION
It has been suggested that lipid peroxidation is
involved in the pathogenesis of arthritis. In contrast to
synovial fluid from healthy subjects, RA synovial fluid
contains large amounts of lipoproteins (26). The concentration of lipoproteins in synovial fluid from an RA
patient increases by ⬃50% over that in the serum from
the same patient, in contrast to ⬍10% in healthy synovial fluid (27,28), possibly because lipoproteins can easily
permeate the synovial membrane in RA joints (28). In
addition, cholesterol crystals in synovial fluid are occasionally found in RA patients (29,30). LDL can be
oxidatively modified under inflammatory conditions in
vivo (31). Accordingly, the lipids and lipoproteins in RA
synovial fluid can be oxidized. Indeed, ox-LDL has been
identified in RA synovial fluid (2–4), although ox-LDL
concentration has not yet been determined because no
standard quantitative assay has been developed for
ox-LDL generated in vivo.
ROLE OF LOX-1 IN MMP-3 PRODUCTION IN RA
There are no reports in the current literature
showing the presence of ox-LDL in RA cartilage. Consistent with our previous findings in articular cartilage of
rats with ZIA (19), this is the first study to demonstrate
that ox-LDL associates with articular chondrocytes in
human RA cartilage. Because even such a large molecule as hyaluronan (HA) can penetrate cartilage matrix
after IL-1 treatment (32), ox-LDL penetration could
occur in degraded RA cartilage. We have also shown
that fluorescent dye–labeled ox-LDL permeates the
cartilage matrix and binds to chondrocytes. Similarly,
HA penetrates normal cartilage explants and binds to
the cells (33), although there is no clear explanation as
to why HA can penetrate articular cartilage. Thus, it is
likely that LDL diffusing from serum into joint fluid
could be modified oxidatively in the inflamed joint cavity
and the resultant ox-LDL could penetrate cartilage
matrix and associate with chondrocytes.
The ox-LDL receptor, LOX-1, is mainly expressed in vivo in vascular endothelial cells and vascularrich organs such as the placenta and lungs (13). Although we have already shown that ox-LDL induces
LOX-1 expression in rat chondrocytes in monolayer
culture (18), the presence of LOX-1 in cartilage, which is
an avascular tissue, has never been examined. The
current study is the first to identify LOX-1 protein in
chondrocytes in RA cartilage.
Various factors are known to regulate the expression of LOX-1 in endothelial cells (34). Shear stress can
up-regulate LOX-1 protein and mRNA in the cells (17).
In addition, mechanical strain such as cyclic tensile
stretch enhances ROS generation in chondrocytes (35),
which may lead to cartilage degradation. Thus, it is
possible that mechanical stress may induce LOX-1 in
chondrocytes in the weight-bearing regions of cartilage.
In contrast to normal and OA cartilage from non–
weight-bearing regions, we found that chondrocytes
expressed LOX-1 in non–weight-bearing areas in RA
cartilage (Figure 1). This indicates that LOX-1 could be
up-regulated in RA cartilage through other mechanism(s). The present study demonstrated that LOX-1
can be induced by its ligand, ox-LDL, in chondrocytes
(Figure 4), consistent with previous findings using rat
primary chondrocytes (18) and human coronary artery
endothelial cells (36). Therefore, increased expression of
LOX-1 in RA cartilage may involve such an induction
mechanism.
There is evidence that elevated MMP-3 levels in
the serum and synovium from RA patients are an
indicator of inflammation and are associated with joint
damage. In RA synovial fluid, MMP-3 levels are signif-
3501
icantly higher than those in OA (21). Furthermore, the
serum levels of MMP-3 are related to the activity of RA
and predict the outcome of early RA (22,23,37,38). In
human articular chondrocytes, MMP-3 can be induced
by IL-1␤, TNF␣, phorbol myristate acetate, and histamine (39,40). Recent studies have demonstrated that
ox-LDL enhances the expression of MMP-1 and MMP-3
via LOX-1 in human coronary artery endothelial cells in
vitro (41).
This study is the first to show that ox-LDL can
up-regulate MMP-3 production in normal articular
chondrocytes (Figure 5). While LOX-1 in chondrocytes
was undetectable with immunofluorescence staining in
normal cartilage immediately after harvest (Figure 1) or
in explant culture (Figure 4), LOX-1 can be induced by
ox-LDL stimulation in normal cartilage explant culture
(Figure 4).
Our previous study also showed LOX-1 upregulation by ox-LDL in monolayer chondrocytes that
constitutively express the receptor without ox-LDL stimulation (18). Pretreatment with anti–LOX-1 antibody
resulted in significant suppression of ox-LDL–
stimulated MMP-3 production (Figures 5 and 6). Taken
together, these findings suggest that LOX-1 could mediate ox-LDL action on MMP-3, even in normal cartilage. Thus, intense localization of LOX-1 and ox-LDL in
RA cartilage (Figures 1 and 2) indicates that ox-LDL
may contribute to MMP-3 induction through LOX-1 in
RA chondrocytes. In addition to MMP-3, MMP-13 is
thought to play a major role in cartilage destruction
through type II collagen cleavage in diseased cartilage
(42,43). Immunoblot analysis using the same samples as
with MMP-3 revealed that MMP-13 from ox-LDL–
stimulated cartilage explants was below detectable levels
(data not shown). Further studies are required to elucidate the effects of ox-LDL on MMP-13 as well as other
MMPs in cartilage.
Information regarding the pathophysiologic roles
of LOX-1 is accumulating. Physiologically, LOX-1 may
work as a scavenger or remove cellular debris and other
related materials (44,45). Under pathologic conditions,
the binding of LOX-1 to ox-LDL and cellular ligands
may result in the activation of endothelial cells, transformation of smooth muscle cells, and accumulation of
lipids in macrophages, which are all involved in the
promotion of atherosclerosis (45–47). In addition, the
receptor–ligand interaction may cause cell death. OxLDL at 10–100 ␮g/ml, the concentrations that stimulate
MMP-1 and MMP-3 expression in human coronary
artery endothelial cells (41), induces apoptosis of cells
(36) in association with NF-␬B activation (48). Ox-LDL
3502
KAKINUMA ET AL
at 40–100 ␮g/ml, the concentrations used in the present
study, which induce MMP-3 (Figure 5), causes nonapoptotic cell death through the Akt pathway in rat
articular chondrocytes in monolayer culture (18).
In contrast, the present finding that ox-LDL
failed to alter LDH activity suggests that ox-LDL ligation with LOX-1 caused little cytotoxic effect on human
chondrocytes in cartilage explant culture. While ox-LDL
can penetrate cartilage matrix (Figure 3), ox-LDL added
in the culture medium could not reach chondrocytes
surrounded by cartilage matrix in explants as readily as
in monolayer culture. In RA and OA, loss of chondrocytes is found initially at the articular surface and later
throughout the cartilage (49). Degraded cartilage matrix
in RA and OA could provide easier access of ox-LDL to
LOX-1. Thus, reduced cellularity in diseased cartilage
may involve the interaction between ox-LDL and
LOX-1. This is currently being investigated.
In rat ZIA, intravenous administration of anti–
LOX-1 antibody suppressed cartilage destruction, possibly by blocking LOX-1 on the endothelium of synovial
vessels, resulting in a decrease in leukocyte infiltration
into the arthritic joints (19). Foam cells containing
ox-LDL are found in the synovium in RA (6), and
macrophage-like type A synoviocytes from RA patients
could take up acetylated LDL (50). Lipoproteins that
are increased in RA synovial fluid are assumed to have
some role in the development of synovitis (51). However, LOX-1 expression in RA synovium has not yet
been investigated. Further study of the levels of LOX-1
in RA synovium is needed for increased understanding
of the pathologic roles of ox-LDL and LOX-1 in RA
joints.
ACKNOWLEDGMENTS
The authors thank Naoko Honda for technical assistance, and Lisbeth Stewart for critical reading. We thank the
Japanese Red Cross Society for providing us with fresh plasma
for the experiments.
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