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T cells regulate the expression of matrix metalloproteinase in human osteoblasts via a dual mitogen-activated protein kinase mechanism.

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ARTHRITIS & RHEUMATISM
Vol. 48, No. 4, April 2003, pp 993–1001
DOI 10.1002/art.10872
© 2003, American College of Rheumatology
T Cells Regulate the Expression of Matrix Metalloproteinase
in Human Osteoblasts via a Dual Mitogen-Activated
Protein Kinase Mechanism
Leonard Rifas1 and Sophia Arackal2
Objective. To investigate the role of T cell induction of matrix metalloproteinase 13 (MMP-13) production by human osteoblasts in order to better understand
the process of bone loss in rheumatoid arthritis (RA).
Methods. Activated T cell–conditioned medium
(ACTTCM) was used to mimic the physiologic conditions of inflammation. MMP-13 production by human
osteoblasts was assessed using a specific enzyme-linked
immunosorbent assay. Specific inhibitors of the p38
mitogen-activated protein (MAP) kinase and the extracellular signal–regulated kinase 1/2 (ERK-1/2) MAP
kinase signaling pathways were used to assess their
roles in T cell–mediated MMP-13 production. Finally,
recombinant cytokines representative of the major components in ACTTCM were assessed for their ability to
induce MMP-13.
Results. ACTTCM powerfully induced MMP-13
in human osteoblasts. Inhibition of p38 activity abolished, while inhibition of ERK-1/2 activity enhanced,
MMP-13 production. We next investigated physiologic
levels of the T cell cytokines tumor necrosis factor ␣
(TNF ␣ ), transforming growth factor ␤ (TGF ␤ ),
interferon-␥ (IFN␥), and interleukin-17 (IL-17) for
their roles in MMP-13 induction. Although individual
cytokines had no significant effect, the combination of
TNF␣, TGF␤, IFN␥, and IL-17 resulted in a dramatic
p38-dependent induction of MMP-13 identical to that
produced by ACTTCM.
Conclusion. These studies demonstrate for the
first time that human osteoblasts produce MMP-13.
The results also show that under conditions of chronic
inflammation, multiple T cell cytokines synergize to
induce high levels of MMP-13 via a mechanism that is
dependent on activated p38 MAP kinase and is suppressed by activated ERK-1/2. Selective inhibition of
p38 activity may offer a target for pharmacologic inhibition of bone loss in RA.
Osteoporosis occurs frequently in patients with
rheumatoid arthritis (RA) and is observed in 2 characteristic patterns: juxtaarticular bone loss, occurring
around inflamed joints, and generalized bone loss with
reduced bone mass (for review, see refs. 1–3). It has
been proposed that T cells are continuously involved in
the pathogenesis of RA, from its initiation phase
through to the chronic stage (4).
Recent studies have defined a role for T cells in
the severe joint inflammation and bone and cartilage
destruction that are the hallmarks of RA (5,6). Elevated
T cell populations have been identified in the synovial
cavity of patients with RA but not in those with osteoarthritis (7–9), and elevated levels of cytokines such as
tumor necrosis factor ␣ (TNF␣) (10), transforming
growth factor ␤ (TGF␤) (11), interferon-␥ (IFN␥) (10),
and interleukin-17 (IL-17) (12) have been described as
well. Furthermore, the combination of TNF␣ blockade
with IL-1 and IL-17 blockade is more effective than is
TNF␣ blockade alone for controlling synovial inflammation and cartilage degradation in RA (6), suggesting that
disease progression is regulated by the synergistic action
of multiple cytokines. However, the process by which T
cells induce bone matrix degradation has yet to be
identified.
Bone resorption is complex, requiring not only
the recruitment of osteoclasts to the site of resorption,
but also the removal of collagen from the bone surface,
through the action of matrix metalloproteinase 13
Supported by NIH grants AR-32087 and AR-46370.
1
Leonard Rifas, MS: Washington University School of Medicine, St. Louis, Missouri; 2Sophia Arackal, BS: Barnes-Jewish Hospital, St. Louis, Missouri.
Address correspondence and reprint requests to Leonard
Rifas, MS, Washington University School of Medicine, Barnes-Jewish
Hospital North, 216 South Kings Highway, St. Louis, MO 63110.
E-mail: lrifas@imgate.wustl.edu.
Submitted for publication June 18, 2002; accepted in revised
form December 19, 2002.
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(MMP-13; collagenase 3), to allow for osteoclast attachment (13,14). MMP-13 is secreted by cytokinestimulated cartilage cells (15) and cleaves fibrillar collagens (with a preference for type II collagen over type I
collagen and type III collagen), and displays ⬎40-fold
stronger gelatinase activity compared with MMP-1 (interstitial collagenase) and MMP-8 (neutrophil collagenase) (16). Human MMP-13 is present in the pannus of
rheumatoid synovium, suggesting a role in both bone
remodeling and inflammation-mediated bone erosion
(17). MMP-13 expression is under the control of
mitogen-activated protein (MAP) kinases (18), serinethreonine kinases that are activated by phosphorylation
of a Thr-Xxx-Tyr motif in the activation loop of the
molecule by dual-specificity MAP kinase kinases
(MEKs). MEKs are themselves activated by MAP kinase
kinase kinases (MEKKs) (19,20).
At present, the 3 major MAP kinase cascades
that are known are as follows: the extracellular signal–
related kinase 1/2 (ERK-1/2; also known as p44/42 MAP
kinase), the c-Jun N-terminal kinase (JNK) or stressactivated protein kinases (SAPKs) 1 and 2, and the p38
group of protein kinases. The ERK-1/2 pathway (Raf13 MEK-1/23 ERK-1/2) is strongly activated by growth
factor receptor occupancy but is indirectly activated by
cytokines and environmental stress through secondary
protein kinases (e.g., Src family tyrosine kinases). In
contrast, the main stimuli for the JNK (MEKK-1–
33 MEK-4/SAPK/ERK kinase 1, MEK-73 JNK/SAPK)
and p38 (MEKK3 MEK-3/63p38) pathways are cytokines and environmental stress, whereas mitogenic
growth factors have no or little effect (21).
In order to examine the role of T cell cytokines in
the process of inflammation-induced bone degradation
(such as occurs in inflammatory diseases such as RA),
we used activated T cell–conditioned medium
(ACTTCM) to examine, in vitro, the interactions and
intracellular signaling pathways of the cytokine repertoire produced by activated T cells that leads to MMP-13
production. Furthermore, because MAP kinase pathways have been shown to regulate the transcription
factors necessary for MMP-13 gene regulation, we also
studied these signaling events to determine their contribution to the production of MMP-13 in normal human
osteoblasts. We show that during chronic inflammation,
activated T cells produce 4 major cytokines, TNF␣,
TGF␤, IFN␥, and IL-17, which alone do not affect
MMP-13, but in combination synergistically and potently
stimulate production of MMP-13 in normal human
osteoblasts. We also demonstrate that MMP-13 expression is regulated by the p38 MAP kinase pathway.
RIFAS AND ARACKAL
MATERIALS AND METHODS
Antibodies. Mouse anti-human CD3 monoclonal antibody (clone HIT3a) and mouse anti-human CD28 monoclonal
antibody (clone 28.2) were obtained from PharMingen (San
Diego, CA), as sterile, azide-free, and low-endotoxin preparations.
Recombinant cytokines. Recombinant human TNF␣,
TGF␤1, IFN␥, and IL-17 were obtained from R&D Systems
(Minneapolis, MN).
MAP kinase inhibitors. The specific MEK-1/2 inhibitor, PD98059, and the specific p38 MAP kinase inhibitor,
SB203580, were purchased from Calbiochem (San Diego, CA)
and were prepared as stock solutions in DMSO (SigmaAldrich, St. Louis, MO) at 40 mM and 20 mM, respectively.
Isolation of T cells. Peripheral blood mononuclear
cells (PBMCs) were obtained in the form of buffy coats from
the American Red Cross (St. Louis, MO) and were further
purified by separation on Histopaque-1077 (1.077 gm/ml)
lymphocyte separation medium (Sigma-Aldrich), as previously
described, with some modification (22). Briefly, the buffy coat
preparations were diluted 1:1 in phosphate buffered saline
(PBS). Twenty milliliters of diluted buffy coat were overlayered onto 15 ml of Histopaque and centrifuged at 400g for 30
minutes at room temperature. Mononuclear cells were recovered from the interface, washed twice with PBS (Ca⫹2-,
Mg⫹2-free; Sigma-Aldrich) by centrifugation at 300g for 5
minutes, and then T cells were isolated by positive selection
from the PBMCs using CD4⫹ Dynabeads (Dynal, Lake Success, NY ) according to the manufacturer’s instructions. This
process results in a population of T cells enriched ⬎95%.
T cell cultures. T cells were cultured at 1 ⫻ 106 cells/ml
in AIM-V serum-free medium (Gibco, Grand Island, NY). T
cells were activated by the addition of mouse anti-human CD3
monoclonal antibody (1 ␮g/ml) and mouse anti-human CD28
monoclonal antibody (5 ␮g/ml). After a 72-hour incubation
period, the ACTTCM was harvested and frozen at ⫺80°C until
use in the experimental protocols.
Preparation of human osteoblast cultures. Human rib
specimens from 10 different donors were obtained from the
Missouri Transplantation Services (St. Louis, MO) as donor
tissue. Use of these tissues (and buffy coats) was approved by
the Human Studies Committee, Washington University Medical Center. Human osteoblast cultures were prepared from rib
specimens, as previously described (22). The cells have the
characteristics of osteoblasts, as previously described (22–24).
Induction of MMP-13 in human osteoblasts. Cells
(2 ⫻ 104 cells/well) were seeded into 48-well tissue culture
plates in ␣-minimum essential medium (␣-MEM; Mediatech,
Herndon, VA) containing 10% heat-inactivated fetal bovine
serum (HIFBS), and were incubated for 4 days. The confluent
cells were washed with PBS and then were incubated for 24
hours in either ␣-MEM containing 0.2% HIFBS or AIM-V
medium. Cytokines or ACTTCM was added, and the conditioned media were collected after the appropriate incubation
times, as noted in the figure legends. To examine the regulation of MMP-13 by MAP kinases, DMSO (vehicle control),
PD98059, or SB203580 was added to the cultures, at the
specified concentrations, 1 hour before addition of test agents.
The DMSO concentration was 0.1% in all cases. Media were
collected after the indicated times and were frozen at ⫺80°C
until assayed.
T CELLS AND HUMAN OSTEOBLAST MMP-13 PRODUCTION
Protein assays. After collection of the conditioned
media, the cell layers were washed 3 times with Tris buffered
saline (50 mM Tris HCl, pH 7.4, 150 mM NaCl), then
solubilized in 0.1% sodium dodecyl sulfate (SDS). Protein
assays were performed using a DC Protein Assay Kit (Bio-Rad
Laboratories, Hercules, CA).
Enzyme-linked immunosorbent assays (ELISAs). Human IL-1␤, TNF␣, and MMP-13 were assayed using specific
ELISA kits obtained from Amersham Biosciences (Piscataway,
NJ). TGF␤ was assayed using an ELISA kit from Promega
(Madison, WI). IFN␥ was assayed using an ELISA kit obtained
from R&D Systems.
Whole cell lysates and Western blotting. Human osteoblasts (1 ⫻ 106 cells) were subcultured into 100-mm tissue
culture plates in ␣-MEM containing 10% HIFBS and incubated for 4 days. The cell layers were then rinsed with PBS and
incubated for 24 hours in ␣-MEM containing 0.2% HIFBS.
After a further 24 hours of incubation, the medium was
changed, and the cells were preincubated with either DMSO or
SB203580 (20 ␮M) for 1 hour before adding either medium
alone or 25% ACTTCM.
After 24 hours of incubation, the cell layers were
rapidly rinsed with PBS, extracted with boiling lysis buffer (1%
SDS, 1.0 mM sodium orthovanadate, 10 mM Tris HCl, pH 7.4),
and then the cell layers were scraped. The lysed cells were then
passed several times through a 22-gauge syringe needle, boiled
for 5 minutes, followed by centrifugation at 15,000g in order to
remove insoluble material. Aliquots of whole cell extracts
containing 30 ␮g of protein were diluted 3:1 (volume/volume)
with 4⫻ SDS–polyacrylamide gel electrophoresis (SDSPAGE) sample buffer, boiled again for 5 minutes, then
separated by 10% SDS-PAGE and blotted onto polyvinylidene
difluoride membranes (Immunoblot; Bio-Rad), using a semidry transfer method.
After transfer, the membranes were dried overnight,
rewetted in methanol, washed 2 times in Tris buffered saline–
Tween (TBST; 50 mM Tris HCl, 150 mM NaCl, 0.1% Tween
20, pH 7.4) then probed with the specified primary antibodies
at 1:1,000 dilution in Superblock (Pierce, Rockford, IL) containing 0.1% Tween 20 (SBT). After 3 washes with TBST, the
membranes were incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (1:50,000) in SBT for 1
hour. Proteins were visualized using SuperSignal chemiluminescence substrate (Pierce) and exposure to Hyperfilm MP
(Amersham Biosciences). The blots were stripped with Restore Western blotting stripper (Pierce), according to the
manufacturer’s protocol, and were successively reprobed using
different antibodies.
Whole cell lysate preparation for kinase assays. Human osteoblasts (4 ⫻ 105 cells) were subcultured into 60-mm
tissue culture plates in ␣-MEM containing 10% HIFBS and
incubated for 4 days. The medium was changed to ␣-MEM
containing 0.2% HIFBS and incubated for 24 hours. The
medium was again changed, and the cells were preincubated
with either vehicle control or SB203580 (20 ␮M) for 1 hour
before adding either medium alone or 25% ACTTCM. After
24 hours of incubation, the cell layers were rapidly rinsed with
PBS, then the cells were lysed in a cold lysis buffer (10 mM Tris
HCl, pH 7.4, 1.0% Triton X-100, 0.5% Nonidet P40, 150 mM
NaCl, 20 mM sodium fluoride, 0.2 mM sodium orthovanadate,
1.0 mM EDTA, 1.0 mM EGTA, 0.2 mM phenylmethylsulfonyl
fluoride, 4 mM 4-(2-aminoethyl)benzenesulfonylfluoride, 3.2
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␮M aprotinin, 84 ␮M leupeptin, 0.14 mM bestatin, 60 ␮M
pepstatin, 56 ␮M E-64) in the culture dish for 30 minutes at
4°C. The lysed cells were then scraped off the dish and the
lysate passed several times through a 26-gauge needle to
disperse any large aggregates. The lysate was centrifuged at
16,000g for 30 minutes at 4°C. The supernatant (total cell
lysate) was collected for kinase assays. Aliquots were assayed
for protein as described above.
Immunoprecipitation for kinase assays. Cell lysates
(200 ␮l containing 200 ␮g total protein) were added to 20 ␮l
immobilized phospho–p38 MAP kinase (Thr180/Tyr182)
monoclonal antibody. Each mixture was incubated with gentle
rocking overnight at 4°C. The samples were then microcentrifuged for 30 seconds, and the pellet was washed twice with 500
␮l of ice cold 1⫻ lysis buffer (20 mM Tris, pH 7.5, 150 mM
NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM
sodium pyrophosphate, 1 mM ␤-glycerolphosphate, 1 mM
sodium orthovanadate, 2.1 ␮M leupeptin) and kept on ice.
Finally, the pellet was washed twice with 500 ␮l of ice cold 1⫻
kinase buffer (25 mM Tris, pH 7.5, 5 mM ␤-glycerolphosphate,
2 mM dithiothreitol, 0.1 mM sodium orthovanadate, 10 mM
MgCl2) at 4°C.
Kinase assays. For the p38 MAP kinase assay, the
pellet was suspended in 50 ␮l 1⫻ kinase buffer supplemented
with 200 ␮M adenosine triphosphate and 2 ␮g activating
transcription factor 2 (ATF-2) fusion protein and incubated for
30 minutes at 30°C. The reaction was terminated with 25 ␮l of
3⫻ SDS-PAGE sample buffer, then samples were boiled for 5
minutes, vortexed, and microcentrifuged for 2 minutes. Thirty
microliters of each sample was subjected to SDS-PAGE and
analyzed by Western blotting using phospho–ATF-2 antibody,
according to the manufacturer’s instructions.
Statistical analysis. Group mean values were compared by analysis of variance (ANOVA). Subsequent multiple
comparisons were performed using Fisher’s protected least
significance difference test. The 5% significance level was
used.
RESULTS
Human osteoblasts produce MMP-13 in response to T cell–derived inflammatory cytokines. T
cell–derived cytokines are known to participate in the
destruction of articular cartilage by inducing MMP-13 in
chondrocytes (15,25,26). We observed that although
MMP-13 is not produced constitutively in human osteoblasts, upon their exposure to ACTTCM for a 72-hour
period, abundant MMP-13 was produced in a dosedependent manner (Figure 1). Because MMP-13 induction by 25% ACTTCM was not significantly different
from that by 50% ACTTCM, 25% ACTTCM was chosen for the experiments described below. A time course
analysis of MMP-13 production after exposure to ACTTCM (Figure 2) revealed that MMP-13 was not expressed in significant quantities until 24 hours (mean ⫾
SEM 16.83 ⫾ 0.81 ng/mg protein), but there was a
dramatic, 44-fold linear increase in MMP-13 production
at 48 hours (mean ⫾ SEM 131.24 ⫾ 6.23 ng/mg protein),
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Figure 1. Dose-dependent induction of matrix metalloproteinase 13
(MMP-13) in human osteoblasts by activated T cell–conditioned
medium (ACTTCM). Human osteoblasts were cultured in ␣-minimum
essential medium (␣-MEM) containing 10% heat-inactivated fetal
bovine serum (HIFBS) until confluent (5 days). The medium was
changed to ␣-MEM containing 0.2% HIFBS and incubated for 24
hours. The medium was changed again, the ACTTCM was added at
the indicated concentrations, and cells were incubated for 72 hours.
Medium was collected and assayed for MMP-13 by a specific enzymelinked immunosorbent assay. Proteins were assayed using the Bio-Rad
DC protein assay kit. Values are the mean and SEM of 3 independent
cultures. P ⬍ 0.01 for all comparisons, by analysis of variance.
and a 71-fold increase at 72 hours (mean ⫾ SEM
212.97 ⫾ 21.65 ng/mg protein), compared with the
8-hour time point.
MMP-13 expression in human osteoblasts is
regulated via p38 MAP kinase and ERK-1/2 MAP
kinase. To assess the role of MAP kinases in the
regulation of MMP-13, human osteoblasts were exposed
to ACTTCM in the presence or absence of increasing
doses of the specific ERK-1/2 pathway inhibitor
PD98059 (0–40 ␮M) and the specific p38 MAP kinase
inhibitor SB203580 (0–20 ␮M). Increasing concentrations of PD98059 resulted in production of an increased
amount of MMP-13 (Figure 3A), demonstrating that the
ERK pathway inhibited expression of the enzyme. In
contrast, SB203580 (Figure 3B) inhibited MMP-13 secretion ⬎50% at 0.1 ␮M, the lowest dose tested, and
almost completely inhibited MMP-13 secretion at 20
␮M, demonstrating that activated p38 MAP kinase is a
potent stimulator of MMP-13 production in human
osteoblasts.
To confirm that p38 MAP kinase is critical in
MMP-13 production, human osteoblasts were treated
with ACTTCM in the presence or absence of 20 ␮M
SB203580 over a time course of 0–96 hours, and
RIFAS AND ARACKAL
MMP-13 was assayed by a specific ELISA. During the
0–96-hour time course, ACTTCM increased MMP-13
protein production 51-fold, while only a 2.6-fold increase
was observed in the presence of SB203580 (ACTTCM
versus ACTTCM plus SB203580: at 8 hours, mean ⫾
SEM 9.76 ⫾ 0.52 versus 9.39 ⫾ 0.33 ng/mg protein; at 96
hours, mean ⫾ SEM 493.96 ⫾ 75.66 versus 24.63 ⫾ 1.00
ng/mg protein; P ⬍ 0.001 by ANOVA [n ⫽ 4 cultures]).
These results demonstrate that p38 MAP kinase is
essential in the process of MMP-13 expression.
Activation of p38 depends on phosphorylation.
Therefore, we investigated whether ACTTCM increases
phosphorylation of p38, and whether SB203580 inhibits
its kinase activity. To do so, we examined the phosphorylation state of p38 MAP kinase in the cells using
Western blot analysis, and the kinase activity of activated p38 directly using ATF-2 as substrate. Our results
(Figure 4) show that after 24 hours, ACTTCM increased
p38 phosphorylation as determined by Western blot
analysis. Surprisingly, SB203580, in the presence of
ACTTCM, also enhanced p38 phosphorylation. To determine whether SB203580 inhibited the activity of
ACTTCM-induced phospho-p38, we analyzed the kinase activity of p38 directly using ATF-2 as substrate.
The results (Figure 4) show that ACTTCM induced p38
Figure 2. Effects of ACTTCM on MMP-13 production in human
osteoblasts over time. Human osteoblasts were cultured as described in
Figure 1. The medium was changed again, and the cells were stimulated with 25% ACTTCM. Medium was collected at 8, 16, 24, 48, and
72 hours and assayed for MMP-13 by a specific enzyme-linked
immunosorbent assay. Proteins in the cell layers were assayed as
described in Figure 1. Values are the mean ⫾ SEM of 3 independent
cultures. ⴱ ⫽ P ⬍ 0.001 versus 8 hours, by analysis of variance. See
Figure 1 for definitions.
T CELLS AND HUMAN OSTEOBLAST MMP-13 PRODUCTION
997
kinase activity, as demonstrated by its ability to phosphorylate its substrate, ATF-2. However, the presence of
SB203580 completely inhibited p38 kinase activity (Figure 4).
Figure 4. Western blot analysis of the effect of SB203580 on the
activation (phosphorylation) and kinase activity of p38 mitogenactivated protein (MAP) kinase in human osteoblasts treated with
ACTTCM. Human osteoblasts were grown as described in Figure 1.
Human primary osteoblasts were incubated for 1 hour with the vehicle
(DMSO) or SB203580 (20 ␮M) before stimulation with ACTTCM for
24 hours. p38 or its phosphorylated (activated) isoform (phospho-p38)
was identified by immunoblotting, using specific polyclonal antibodies.
To determine the activity of p38, cellular extracts were analyzed for
their ability to phosphorylate the p38-specific substrate, activating
transcription factor 2 (ATF-2). Phosphorylated ATF-2 (p-ATF-2) was
identified by immunoblotting using specific polyclonal antibodies as
described in Materials and Methods. See Figure 1 for other definitions.
Figure 3. Regulation of MMP-13 expression in human osteoblasts by
PD98059 and SB203580. Human osteoblasts were cultured until
confluent (5 days) in ␣-MEM containing 10% HIFBS. The medium
was changed to ␣-MEM containing 0.2% HIFBS and incubated for 24
hours. After the 24-hour incubation period, cells were rinsed with
phosphate buffered saline, then ␣-MEM containing 0.2% HIFBS was
added to control wells or medium containing PD98059 (0–40 ␮M) or
SB203580 (0–40 ␮M). Cells were incubated for 1 hour with the
inhibitors, then stimulated with 25% ACTTCM for 48 hours. Media
from osteoblasts treated with either PD98059 (A) or SB203580 (B)
were assayed for MMP-13 by enzyme-linked immunosorbent assay.
Protein concentrations were determined as described in Figure 1.
PD98059 up-regulated and SD203580 down-regulated expression of
MMP-13. Values are the mean ⫾ SEM of 3 independent cultures. ⴱ ⫽
P ⬍ 0.01 as determined by analysis of variance. See Figure 1 for
definitions.
T cell–derived cytokines synergistically induce
MMP-13 in human osteoblasts. Activated T cells produce numerous inflammatory cytokines, including
TNF␣ (22), TGF␤ (27), IFN␥ (27,28), and IL-17 (28).
The mean ⫾ SEM amount of each cytokine in 25%
ACTTCM (as used in our experiments) was as follows:
for TNF␣ 116.61 ⫾ 2.1 pg/ml, for TGF␤ 285.5 ⫾ 3.0
pg/ml, and for IFN␥ 5.88 ⫾ 0.6 ng/ml. Fossiez et al
reported that under the same activating conditions as
those used in our studies, IL-17 was secreted at levels of
⬃8 ng/ml (29). Therefore, 25% ACTTCM would contain ⬃2 ng/ml. Thus, we examined these factors individually and in combinations.
Figure 5 shows that when the cytokines were
tested either individually or in any combination of two,
no significant induction of MMP-13 secretion was observed. However, when TNF␣, TGF␤, and IFN␥ were
added in combination, a significant synergistic (⬃6-fold)
induction of MMP-13 was observed. Furthermore, addition of IL-17 increased the synergistic activity to ⬃100fold over control levels, and the level of MMP-13
induction by this combination of 4 cytokines was identical to that achieved with induction by ACTTCM. When
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Figure 5. Effect of T cell cytokines and SB203580 on matrix metalloproteinase 13 (MMP-13) production in human osteoblasts. Human
osteoblasts were cultured as described in Figure 3, then incubated with
25% activated T cell–conditioned medium (ACTTCM) or recombinant human tumor necrosis factor ␣ (TNF␣) (120 pg/ml), transforming
growth factor ␤ (TGF␤) (300 pg/ml), interleukin-17 (IL-17) (2 ng/ml),
or interferon-␥ (IFN␥) (10 ng/ml), alone or in combinations as shown,
for 48 hours at 37°C. After the incubation period, the conditioned
media were collected and assayed for MMP-13 by enzyme-linked
immunosorbent assay. Values are the mean and SEM of 3 independent
cultures. a ⴝ P ⬍ 0.05 versus control; b ⫽ P ⬍ 0.05 versus cytokines
tested in combinations of two; c ⫽ P ⬍ 0.05 versus cytokines tested in
combinations of three; d ⫽ not significantly different; e ⫽ P ⬍ 0.05
versus all 4 cytokines combined, as determined by analysis of variance.
the combination of all 4 cytokines was added to cells
pretreated with SB203580, MMP-13 production was
almost completely inhibited (Figure 5), demonstrating
that these 4 cytokines are responsible for the MMP-13–
inducing activity present in ACTTCM.
DISCUSSION
Under normal physiologic conditions, bone homeostasis is governed by the balance between the process of resorption of bone by osteoclasts and the formation of bone by osteoblasts. These 2 processes are
coupled in space and time such that there is no net loss
of bone. However, under conditions of chronic inflammation, such as occurs in RA, there is an unbridled
release of cytokines from T cells, which activates osteoclastogenesis (30) and induces osteoblastic cytokine
production (22,24). The result is an uncoupling of
resorption and formation, a process that favors bone
resorption and ultimately net bone loss.
The major cytokines that have been identified as
inducing increased osteoclast formation and activity are
RIFAS AND ARACKAL
IL-1, TNF␣, and receptor activator of nuclear factor ␬B
ligand (30). However, in order for osteoclasts to degrade
bone, collagen covering the surface must first be degraded so that osteoclasts can attach. The primary
enzyme that is responsible for cleaving collagen is
collagenase. MMP-13 can degrade type I, type II, and
type III collagen (with a preference for type II collagen)
and also acts as a gelatinase to further degrade the initial
cleavage products of collagenolysis to small peptides
(17,31) that can interact with osteoclasts, resulting in
their activation (13).
Recently, MMP-13 has been detected in human
osteoblasts, but only during fetal development (32).
However, we did not detect MMP-13 in adult human
osteoblasts in the absence of cytokine stimulation. Now,
we report that in the adult osteoblast, inflammatory
cytokines are major inducers of MMP-13. These results
point to a role for the T cell in the osteopenia associated
with RA, and perhaps other inflammatory autoimmune
diseases as well, by inducing osteoblast MMP-13 locally.
The result of this high secretion of MMP-13 would be
the release of collagen peptides and denuding of the
bone surface to allow activated osteoclasts to attach and
resorb the calcified matrix.
The activated T cell has only recently been identified as playing a major role in the pathophysiology of
RA (4,33,34), with destruction of articular cartilage
representing the major thrust of investigation (35). We
now report that T cells produce a repertoire of cytokines
that potently stimulate MMP-13 in osteoblasts as well.
Our data demonstrate for the first time that activated T
cells in the local milieu may play a major role in bone
destruction in RA due to the release of multiple cytokines that, by themselves, have little effect, but as a
result of synergistic activity have a profound effect. We
demonstrate that 4 major cytokines present in
ACTTCM—TNF␣, TGF␤, IFN␥, and IL-17—are responsible for modulating MMP-13 production in human
osteoblasts. Reported measurements of these 4 cytokines in synovial fluid from RA patients include the
following: for TNF␣ 157 pg/ml and for IFN␥ 17 pg/ml
(10), for TGF␤ 2–20 ng/ml (11), and for IL-17 12–5,000
pg/ml (12,36).
These data support our observation that only
small amounts of each of these cytokines need be
present to have a powerful effect. Individually, many of
these cytokines have been examined for their role in the
regulation of MMP-13 in osteoblasts or osteoarthritic or
rheumatoid cartilage (37–40). Of interest is that in our
studies, IFN␥ played a major role in the synergistic
induction of MMP-13. This finding was surprising, because IFN␥ has been reported to be a potent inhibitor of
T CELLS AND HUMAN OSTEOBLAST MMP-13 PRODUCTION
TGF␤-induced MMP-13 (41). IL-17 has been shown to
participate in the bone erosion associated with RA
(42–44), primarily as a function of cytokine induction via
interaction with TNF␣ (43,45). However, we now show
for the first time that IL-17 may play yet a further role in
bone resorption in RA by enhancing T cell cytokine
induction of MMP-13 production in osteoblasts. This
finding may open new avenues for the treatment of bone
loss in inflammatory diseases such as RA.
Our results show that stimulation of human osteoblasts with ACTTCM or inflammatory cytokines
results in the coordinate activation of at least 2 separate
MAP kinase pathways: the ERK-1/2 pathway and the
p38 pathway. Of interest is that stimulation of MMP-13
by ACTTCM was potently up-regulated by the ERKspecific inhibitor, PD98059, while the p38-specific inhibitor, SB203580, blocked production of MMP-13 over a
96-hour period. We used inhibitor concentrations that
are specific, as described by other investigators who have
analyzed the roles of both the p38 MAP kinase pathway
(46–48) and the ERK MAP kinase pathway (49–51) in
osteoblasts and other cell types. However, SB203580 has
been reported to inhibit certain isoforms of JNK at
levels higher than the reported specificity of this inhibitor for p38 (⬃1–3 ␮M) (52). We did observe that
concentrations of SB203580 as low as 0.1–1.0 ␮M resulted in a 50–70% inhibition of MMP-13 production,
which is well within the specificity of the inhibitor for
p38. Furthermore, ACTTCM did not induce phosphoJNK over a time course of 0–48 hours (data not shown).
Of interest was the fact that SB203580 inhibited
production of MMP-13 by ACTTCM despite the fact
that the phosphorylated form of p38 was enhanced.
These observations are in agreement with those of other
investigators who have shown that SB203580 induces
enhanced phosphorylation of p38 in the presence of
specific stimulators (53,54). Our data confirm these
observations, because we observed phospho-p38 to be
increased, rather than decreased, in the presence of
ACTTCM and SB203580, demonstrating that this compound can inhibit the activity, but not the phosphorylation, of p38. We have substantiated this by the finding
that ACTTCM induces p38 activity, as demonstrated by
phosphorylation of its substrate ATF-2, while SB203580
abolished this activity.
The importance of the MAP kinase pathway in
regulating cytokine-induced MMP-13 has been demonstrated in multiple cell types, and the particular pathway
that regulates MMP-13 expression is cell specific. For
example, TNF␣ induction of MMP-13 production in
transformed keratinocytes requires p38 MAP kinase but
is not inhibited by ERK-1/2 (55). In human dermal
999
Figure 6. Signaling pathways mediating regulation of matrix metalloproteinase 13 (MMP-13) expression in human osteoblasts by activated
T cell cytokines. Binding of cytokines to their respective receptors
results in the synergistic activation of the extracellular signal–regulated
kinase 1/2 (ERK-1/2) and p38 mitogen-activated protein (MAP)
kinase pathways. Because cytokines are weak inducers of the ERK
pathway, although strong activators of the p38 pathway, an imbalance
of p38 over ERK-1/2 favors the production of MMP-13 gene activation, most likely through activation of activator protein 1 (AP-1) and
Cbfa-1. Inhibition of ERK-1/2 by PD98059 further favors the production of MMP-13, while inhibition of p38 by SB203580 leads to loss of
MMP-13 production. SB203580 is a specific inhibitor of p38 MAP
kinase; PD98059 is a specific inhibitor of MAP kinase kinase 1
(MEK-1) and MEK-2. TGF␤ ⫽ transforming growth factor ␤;
TNF␣ ⫽ tumor necrosis factor ␣; MEKK ⫽ MAP kinase kinase
kinase.
fibroblasts, induction of MMP-13 by collagen, through
the integrins ␣1␤1 and ␣1␤2 is induced by p38 and
inhibited by ERK-1/2, demonstrating that integrin signaling regulates MMP-13 via a dual MAP kinase mechanism (49).
However, recent evidence points to diverging
roles of MAP kinases with respect to specific MMP
activation. In articular chondrocytes, IL-1 induction of
MMP-13 requires p38 activity, JNK activity, and nuclear
factor ␬B (NF-␬B) translocation, while in chondrosarcoma cells MMP-1 induction by IL-1 is dependent on
p38 and MEK (ERK pathway) but does not require JNK
(56). Although the requirement for NF-␬B in MMP-13
expression is essential in rabbit chondrocytes or human
1000
RIFAS AND ARACKAL
chondrosarcoma cells (56,57), its role in human osteoblast MMP-13 expression is yet to be defined. However,
our data clearly show that in human osteoblasts, p38
MAP kinase is both necessary and sufficient to regulate
MMP-13. However, further studies must be undertaken
in order to determine whether other signaling pathways
may play a regulatory role.
Based on our collective data, we propose a model
(Figure 6) in which the release of cytokines from activated T cells results in binding to their respective
receptors, leading to activation of the ERK-1/2 and p38
MAP kinase pathways. Because cytokines are weak
inducers of the ERK pathway while being strong activators of the p38 pathway, an imbalance of p38 over
ERK-1/2 favors the production of MMP-13 gene activation, most likely through activation of activator protein 1
and Runx2/Cbfa-1 (58,59).
In conclusion, our data support the role of T cells
in the process of bone resorption observed in patients
with RA. The net MMP-13 production from stimulated
osteoblasts appears to be the result of the balance
between the two MAP kinase pathways, suggesting that
selective inhibition of active p38 may prove to be a
pivotal point for the pharmacologic inhibition of bone
loss in inflammatory disease such as RA.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
ACKNOWLEDGMENTS
We wish to thank Ms Aurora Fausto and Ms Linda
Halstead for preparation of the human osteoblast. The authors
also wish to thank Drs. M. Neale Weitzmann and Yousef
Abu-Amer for their critical reading of this manuscript.
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