From the
Departments of Biochemistry and
Internal Medicine, Section of Rheumatology, Rush
Medical College, Rush-Presbyterian-St. Luke's Medical Center, Chicago,
Illinois 60612, the ¶Department of Cell Biology,
University of Massachusetts Medical School, Worcester, Massachusetts 01655,
and the ||Department of Medicine, University of
Miami School of Medicine, Miami, Florida 33101
Received for publication, February 26, 2003 , and in revised form, May 2, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Accumulating evidence demonstrates that the bone morphogenetic proteins
(BMPs), a subfamily of the transforming growth factor- superfamily, are
inhibitors of MMP-13 expression in human fetal chondrocytes
(7) or rat osteoblast-enriched
cells (8,
9). Upon ligand binding, the
specific serine/threonine kinase activity of BMP receptors (type I or II)
transduce signals (10) by
allowing the association of Smad1 and Smad4 in the cytoplasm followed by the
translocation of the Smad1/4 complexes into the nucleus
(11). Smads up- or
down-regulate the transcription of a subset of target genes that includes
Runx2 (12). BMP-7, also known
as the osteogenic protein-1 (OP-1), has been found to induce new cartilage and
bone formation in vitro and in vivo
(1315)
and regulate cell proliferation and differentiation
(16,
17). OP-1 blocks cartilage
damage caused by Fn-f and may promote repair by enhancing proteoglycan and
collagen synthesis (18). More
recently, it has been demonstrated that OP-1 is expressed in human articular
chondrocytes and in osteoarthritis cartilage
(19). Taken together, these
findings imply potential involvement of endogenous OP-1 in cartilage repair
mechanisms, either by enhancing matrix synthesis
(18) or by repressing the
pro-inflammatory expression of cytokines, such as IL-6 and IL-1
, as well
as the chemokines IL-8 and monocyte chemotatic protein-1
(20). It has been proposed
that the effects of OP-1 in chondrocytes are mediated through type I
serine/threonine kinase receptors (activin receptor-like kinase), ALK/BMPR-IB
(21).
Synergistic stimulation of cell differentiation and proliferation by insulin-like growth factor (IGF-1) and OP-1 has been demonstrated in rat osteoblastic cells (22) and in human primary chondrocytes.2 Yeh and colleagues (17, 23) have presented data indicating that IGF-1 gene expression may be up-regulated by OP-1 at both transcriptional and post-transcriptional level in osteoblastic cells. IGF-1 is a polypeptide that exerts anabolic/anticatabolic effects on cartilage. The overexpression of human IGF-1 promoted new tissue formation in an ex vivo model of articular chondrocyte transplantation (24). The anabolic effects of IGF-1 appeared to be mediated by stimulating proteoglycan synthesis in articular chondrocytes (24, 25) or by down-regulating the transcriptional induction of MMP enzymes (e.g. MMP-1, MMP-3, MMP-8, and MMP-13) and inflammatory cytokines (26, 27).
In the present study, we assessed the inhibitory effects of IGF-1 and OP-1
on the basal as well as Fn-f- and IL-1-induced expression of MMP-13. We
demonstrated that the combination of IGF-1 and OP-1 was effective at
inhibiting both the Fn-f- and IL-1
-stimulated MMP-13 gene expression and
defined the molecular mechanisms underlying the observed inhibitory
effects.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmid Construction and TransfectionA series of human
MMP-13 promoter deletional DNA fragments, 1600MMP-13, 736MMP-13,
370MMP-13, and 186MMP-13, were subcloned into a firefly
luciferase promoterless vector, pGL2-Enhancer (Promega) at the sites of
SacI and BglII. The human MMP-1 promoter region
(562/+1) was subcloned into the same vector at the sites of
XhoI and HindIII. For transfection, the cells were plated 24
h prior to transfection at a density of 1 x 106 cells/well in
6-well plates and transiently transfected with 2 µg of promoter-reporter
constructs or 500 ng of cDNA expression vectors using FuGENE 6 transfection
reagent (Roche Applied Science) following the manufacturer's instructions. The
promoterless vector, pGL2-Enhancer was used as a control, and a
Renilla luciferase construct was included as an internal control for
transfection efficiency. After 24 h incubation, the cells were rinsed in
phosphate-buffered saline and changed to serum-free condition for 20 24
h followed by treatment with the 110-kDa Fn-f (1 µM) (Chemicon,
Temecula, CA), IL-1 (25 ng/ml) (R&D System, Minneapolis, MN), IGF-1
(100 ng/ml) (a gift from Chiron Cooperation, Emeryville, CA), or OP-1 (100
ng/ml) (provided by Stryker Biotech, Hopkinton, MA). Inhibition of IL-1
was performed using 100 ng/ml IL-1Ra (Anakinra; a gift from Amgen, Thousand
Oakes, CA). A neutralizing antibody to human IGF-1 (100 µg/ml) (R&D
Systems, Minneapolis, MN) or IGF-1 receptor monoclonal antibody (100 µg/ml)
(Oncogene, San Diego, CA) and IgG (100 µg/ml) for negative control (Sigma)
were added directly to the medium. The cells were harvested after 24 h, and
luciferase activity was assayed using a dual luciferase reporter assay system
(Promega). All of the transfection experiments were repeated at least three
times in duplicate.
An IGF-1 receptor cDNA construct, CVNIGF-1R, and its dominant negative,
hIGF-1RDN, were provided by Dr. R. Baserga (Thomas Jefferson University,
Philadelphia, PA). The human IGF-1 promoter reporter construct, IGF-1Luc, was
provided by Dr. Peter S. Rotwein (Oregon Health Sciences University, Portland,
OR). The constitutive active AP-1 factors, c-Jun and c-Fos cDNA constructs,
LNCXc-Fos, and LNCXc-Jun were provided by Dr. Stephen Murphy (Mayo Clinic,
Rochester, MN). NF-B dominant negative, pCMV-IkB
M, and
cis-reporting systems of pNF-
B-Luc, pAP-1-Luc, were purchased
from Clontech (Palo Alto, CA) and Stratagene (La Jolla, CA).
RNA and Genomic DNA ExtractionTotal cellular RNA and genomic DNA were isolated using the RNeasy® Mini kit and DNeasy® Tissue kit (Qiagen), respectively, according to the manufacturer's instructions. All of the samples were stored at 80 °C until analyzed.
Reverse Transcription and Semi-quantitative Polymerase Chain
ReactionReverse transcription (RT) was carried out with 2 µg of
total cellular RNA using the ThermoScriptTM RT-PCR System (Invitrogen)
for first strand cDNA synthesis in 50 µl of reaction volume. The sequences
for the primers and the conditions for their use are summarized in
Table I. For all experiments,
the conditions were determined to be in the linear range for the PCR
amplification as described previously
(29). Briefly, all of the
samples were subjected to RT at the same time, and subsequently, all of the
samples of cDNA were amplified by PCR at the same time to avoid any potential
experiment to experiment variation in efficiency. Each RT sample was assessed
for GAPDH cDNA. The levels of GAPDH mRNA did not vary with time after the
addition of Fn-f, cytokine, or growth factors following 24 PCR cycles. Genomic
DNA was included for the PCR to ensure that there was no genomic DNA
contamination in the total RNA samples. The cDNA was amplified by PCR using 24
cycles of 95 °C for 30 s, 5869 °C for 30 s, and 72 °C for 30
s in the presence of Taq polymerase (Invitrogen), 50 pmol of sense
and antisense primers. PCR primers and annealing temperature used is shown in
Table I. PCR products were
resolved on 1.5% agarose gels and visualized by staining with ethidium bromide
and UV transillumination. Integrated density values for the genes in question
were normalized to the GAPDH values to yield a semi-quantitative
assessment.
|
MMP-13 Analysis by Western BlotConditioned media from control and treated chondrocytes were concentrated (10:1) using Centricon YM 10 filters (Millipore, Bedford, MA) and centrifugation. The soluble protein concentration was determined with BCA reagent (Pierce), and the samples containing equal amounts of total protein were separated by SDS-PAGE followed by transferring to nitrocellulose for immunoblotting. The detection of MMP-13 was performed by using antibody to MMP-13 and ECL system (Amersham Biosciences). MMP-13 antibody L2916 was generously provided by Dr. Gillian Murphy (Norwich, UK).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
IGF-1 and OP-1 Down-regulate Chondrocyte-associated Basal MMP-13 Gene
ExpressionTranscriptional down-regulation of MMP-13 expression by
IGF-1 has been previously reported in rat bone cell cultures
(26) and in bovine nasal
cartilage (27). We
investigated whether the treatment of IGF-1, OP-1, or the combination of IGF-1
and OP-1 is capable of down-regulating the basal level of the MMP-13
expression in human articular chondrocytes. As shown in
Fig. 2A, the
full-length of MMP-13 promoter construct, 1600MMP-13, demonstrated the
strongest response to IGF-1 or OP-1 with an 30% reduction in the promoter
activity. Further deletions (736, 370, and 186MMP-13)
resulted in the gradual decrease of the inhibitory effect of IGF-1 and OP-1 by
down-regulating the promoter activities approximately from 20% to 15%
(Fig. 2A). These
results suggest that not a single motif but multiple regulatory motifs within
the MMP-13 promoter are responsible for the maximum inhibitory effect of IGF-1
and/or OP-1. The combination of IGF-1 and OP-1 further reduced the basal
promoter activity of MMP-13 (60% reduction) compared with control. The
inhibitory effects of IGF-1, OP-1, and especially the combination of the two
growth factors on MMP-13 transcription were further confirmed by Western blot
analysis using primary human chondrocytes
(Fig. 2, B and
C).
|
The inhibitory effect of IGF-1 and OP-1 on MMP-13 promoter activity was concentration-dependent. The maximum inhibitory activity of IGF-1 or OP-1 was observed at the concentration of 100 ng/ml, whereas the treatment with more than 200 ng/ml of either IGF-1 or OP-1 alone showed no further dose-dependent inhibitory effect on MMP-13 expression (Fig. 2D). Individual treatment of IGF-1 or OP-1 at the concentration of 10 ng/ml showed no effect on the MMP-13 promoter activity. However, it was noted that when a fixed amount of IGF-1 or OP-1 (each 100 ng/ml) was preincubated, the concentration of 10 ng/ml of the other growth factor was able to further down-regulate MMP-13 promoter activity. These results suggest that one growth factor enhances the cellular sensitivity for the other growth factor.
IGF-1 and OP-1 Inhibit IL-1- and Fn-f-elicited MMP-13
ExpressionUp-regulation of MMP-13 expression in response to Fn-f
or IL-1
has been observed in chondrocytes. Therefore, we examined
whether IGF-1 and OP-1 had suppressive effects on Fn-f- or
IL-1
-stimulated MMP-13 expression by transient transfection assays.
IGF-1 and OP-1 alone and in combination showed a stronger inhibitory effect on
Fn-f-induced MMP-13 promoter activity (Fig.
3A) than the activity induced by IL-1
(Fig. 3B). The
combination of IGF-1 and OP-1 almost completely inhibited Fn-f-stimulated
activity and partially inhibited IL-1
-stimulated activity of MMP-13
promoter expression. Similar results were obtained in cells transfected with
the 562MMP-1 promoter construct. Taken together, we conclude that IGF-1
and OP-1 exert a combinatory effect in the suppression of basal as well as
Fn-f- or IL-1
-stimulated MMP-13 gene expression in human
chondrocytes.
|
The Combined Effect of IGF-1 and OP-1 on MMP-13 Expression Is Due to
Enhancement of the IGF-1 Autocrine System by OP-1Individual
treatment with the increased amounts of either IGF-1 (2 µg) or OP-1 (2
µg) did not show any further inhibitory effect on the promoter activity of
MMP-13 (Fig. 2D). In
addition, the preincubated fixed amount (100 ng/ml) of IGF-1 or OP-1 allowed
the cells to respond as to low a concentration as 10 ng/ml of IGF-1 or OP-1 by
increasing the cellular sensitivity. Therefore, studies were initiated to
understand the molecular mechanisms by which the combination of two growth
factors, IGF-1 and OP-1, exerts the most prominent effect on the expression of
MMP-13 gene in chondrocytes. The elevated mRNA levels of rat IGF-1 and
IGF-1-binding proteins but not IGF-1 receptors by human OP-1 have been
reported in rat osteoblast cells
(23). Thus, we postulated that
IGF-1 and OP-1 may influence each other, stimulating the cellular autocrine
system for either IGF-1 or OP-1. To study the influence of OP-1 on the
expression of the IGF-1 gene, a human IGF-1 promoter construct containing a
luciferase reporter gene was utilized. Transient transfection of the IGF-1
promoter-luciferase construct followed by the treatment with OP-1 increased
the IGF-1 promoter activity 100% in immortalized chondrocytes and 60% in
primary human chondrocytes above controls
(Fig. 4A). The
induction of IGF-1 expression by OP-1 was further supported by
semi-quantitative RT-PCR assay. The addition of OP-1 increased the mRNA levels
of not only IGF-1 but also the IGF-1 receptor in immortalized chondrocytes
(Fig. 4B). Treatment
with IGF-1 for 36 h, on the other hand, showed no significant effect on the
mRNA or protein levels of OP-1 or OP-1 receptors including ActR-I, BMPR-1A,
BMPR-1B, and BMPRII (data not shown). Taken together, these results suggest
that OP-1 may stimulate the IGF-1 autocrine system at the level of
transcription in human chondrocytes.
|
To determine whether the stimulated autocrine system of IGF-1 caused the suppression of the MMP-13 gene, we tested the effect of a blocking monoclonal antibody to the IGF-1 receptor and an IGF-1 neutralizing antibody on the inhibitory activity of IGF-1. Treatment with nonspecific IgG (100 µg/ml) was included as a negative control. Indeed, the treatment with either the antibody to IGF-1 receptor or the IGF-1 neutralizing antibody completely abolished the IGF-1 effect on MMP-13 promoter activity (Fig. 5A). The treatment with control IgG did not alter the MMP-13 promoter activity. Next, we examined the effect of the same antibodies on the inhibitory activity of OP-1. Importantly, we found that the same antibodies also blocked the inhibitory activity of OP-1 on MMP-13 gene expression. Similar results were obtained by transient transfection studies performed by using human IGF-1 receptor dominant negative (hIGF-1RDN), which blocks IGF-1-induced cellular signaling pathway. The forced expression of the hIGF-1RDN almost completely abolished cellular responses to not only IGF-1 but also OP-1 (Fig. 5B). The empty vector that was used for negative control did not show any significant effect on the promoter activity. These results suggest that OP-1 was acting through stimulation of the IGF-1 system to down-regulate MMP-13 gene expression. However, it was worth noting that the antibodies to IGF-1, IGF-1 receptor, or the co-expression of hIGF-1RDN only partially abolished the combinatory effects of IGF-1 and OP-1 on MMP-13 expression (Fig. 5).
|
The fact that increasing the amount of IGF-1 above 100 ng/ml had no further inhibitory effect on MMP-13 expression, along with the fact that OP-1 increases the IGF-1 receptor expression, suggested that the IGF-1 receptor could be a limiting factor in this system. If the cellular level of IGF-1 receptor is the limiting factor in the response to IGF-1, we may observe dose-dependent down-regulation of MMP-13 gene expression by forced expression of the IGF-1 receptor. Transient transfection experiments were performed to express human IGF-1 receptor (pCVNIGF-1R) followed by the treatment of increasing IGF-1 up to 2 µg/ml in chondrocytes. We observed an enhanced cellular response to IGF-1 (100 ng/ml) by the forced expression of IGF-1 receptor (Fig. 6A). Higher concentration of IGF-1 (2 µg/ml) with the forced expression of IGF-1 receptor did not show a further enhanced cellular response. No significant enhancement of the IGF-1 effect on MMP-13 promoter activity was observed when the OP-1 receptor (BMPR-1B) was expressed followed by the addition of IGF-1. On the other hand, the forced expression of either IGF-1 receptor or OP-1 receptor enhanced the inhibitory effect of OP-1 on MMP-13 promoter activity (Fig. 6B). Taken together, we concluded that OP-1-mediated up-regulation of IGF-1 and the IGF-1 receptor, thereby stimulating the IGF-1 autocrine system, was responsible in part for the combinatory effect of IGF-1 and OP-1 on MMP-13 expression in human chondrocytes.
|
IGF-1 and OP-1 Mediate Repression of Inflammatory Cytokines in Human
ChondrocytesIt has been reported that IGF-1 and OP-1 suppress
inflammatory cytokine expression in primary human proximal tubule epithelial
cells or in skin (20,
30). The pro-inflammatory
cytokines are also strong inducers for MMPs
(3133).
Therefore, we postulated that the potent inhibitory effects of IGF-1 and OP-1
on MMP-13 expression may in part result from the suppression of inflammatory
cytokine expression and/or interference with the inflammatory
cytokine-associated intermediate gene product signaling pathways in human
chondrocytes. IL-1 or Fn-f stimulation significantly increased the mRNA
level of MMP-13 as well as inflammatory cytokines in immortalized chondrocytes
(Fig. 7A). These
included IL-1
, IL-6, and IL-8. Upon the addition of the combination of
IGF-1 and OP-1, we observed decreased expression of endogenous inflammatory
cytokines that were induced by Fn-f or IL-1
(Fig. 7, B and
C). Several studies have demonstrated that the inhibition
of inflammatory cytokinestimulated AP-1 and NF-
B transcription factors
down-regulates MMP-13 gene expression in chondrosarcoma cells or human
chondrocytes (32,
33), showing the
direct/indirect effects of these transcriptional factors in modulating MMP-13
expression. Thus, we tested whether the blocking of NF-
B or
overexpression of AP-1 factors, transcription factors required for the
induction of MMP-13 by IL-1
(34), can modulate the IGF-1
and OP-1 effect on MMP-13 expression. Co-transfection of mutant I
B cDNA
construct, pCMV-IkB
M, which can block NF-
B signaling,
dramatically diminished the basal level as well as the IL-1
- or
Fn-f-induced MMP-13 promoter activity (Fig.
7D). The inhibition of NF-
B signaling pathway
could cause apoptosis in chondrocytes
(35). Therefore, we performed
DNA fragmentation analysis to make sure that the reduced promoter activity of
MMP-13 was not due to cell death. We observed no sign of apoptosis from the
cells transfected with pCMV-IkB
M (data not shown). In addition, the
co-expression of AP-1 factors, such as c-Fos and c-Jun, abrogated the IGF-1-
and OP-1-mediated suppression of MMP-13 promoter activity
(Fig. 7D).
|
In parallel, transient transfection with the NF-B-responsive control
plasmid, pNF-
B-Luc or AP-1 control plasmid, pAP1-Luc, which contains
three tandem repeats of NF-
B or AP-1 consensus sequences, respectively,
was performed as a control for the responsiveness to IL-1
, mutant
I
B (pCMV-I
B
M), or the AP-1 expression vectors. Without
stimulation, the basal expression pattern of these control plasmids was not
significantly modulated in response to IGF-1, OP-1, or the combination of the
two growth factors. However, when chondrocytes were stimulated with Fn-f or
IL-1
, the combination of IGF-1 and OP-1 significantly reduced
IL-1
- or Fn-f-elicited activity of pNF-
B-Luc and pAP-1-Luc
(Fig. 8). The co-expression of
pCMV-IkB
M, which blocks the NF-
B signaling pathway, completely
abolished the IL-1
-stimulated and, to a lesser extent, Fn-f-stimulated
promoter activity of pNF-
B-Luc, whereas the co-expression of AP-1
factors (c-Fos/c-Jun) dramatically increased the promoter activity of pAP1-Luc
(16-fold). In addition, the co-expression of AP-1 factors abrogated the
suppressive effect of IGF-1 and OP-1 on the promoter activity of pAP1-Luc.
Taken together, these results suggest that NF-
B and AP-1 factors are
downstream regulators involved in the stimulation of MMP-13 expression by Fn-f
or IL-1
in human chondrocytes. Therefore, the down-regulation of
inflammatory cytokine expression and/or their intermediate signaling molecules
may be part of the mechanism by which IGF-1 and OP-1 exert their suppressive
effects on MMP-13 expression in human chondrocytes.
|
The Effects of the IL-1Ra on Fn-f- or IL-1-stimulated
MMP-13 Expression in Human ChondrocytesThere have been
controversial reports on whether the Fn-f-induced MMP-13 expression is
IL-1-dependent. Several studies have shown that Fn-f increase MMP expression
via an IL-1 autocrine loop
(36,
37). On the other hand,
stimulated expression of MMPs mediated by an IL-1-independent mechanism has
been reported (38).
Previously, we reported that the IL-1 signal transduction pathway may at least
in part control Fn-f-induced MMP-13 expression
(6), suggesting the cross-talk
between Fn-f and IL-1 signaling molecules leading to the stimulation of MMP-13
expression. Therefore, we determined the effect of IL-1Ra on Fn-f-induced
MMP-13 gene expression compared with that of IL-1
in the presence or
absence of IGF-1 and OP-1. IL-1Ra almost completely abolished
IL-1
-induced MMP-13 promoter activity, whereas it decreased Fn-f-induced
MMP-13 expression
25% in immortalized human chondrocytes
(Fig. 9A). When
combined with IGF-1 and OP-1, IL-1Ra further reduced Fn-f- or
IL-1
-induced MMP-13 promoter activity to the basal levels. In parallel
experiments, similar results were obtained in cells transfected with the MMP-1
promoter construct (data not shown). Our transient transfection results were
further confirmed by semi-quantitative RT-PCR
(Fig. 9B). Consistent
with the transient transfection results, the addition of IL-1Ra significantly
reduced IL-1
-induced MMP-13 mRNA, and to a lesser extent, Fn-f-induced
MMP-13 mRNA levels. IL-1Ra combined with IGF-1 and OP-1 further down-regulated
MMP-13 mRNA levels, completely abolishing the induction of MMP-13 expression
medicated by Fn-f or IL-1
. Taken together with our previous findings in
which IL-1Ra significantly reduced IL-1
-induced MMP-13 protein level
and, to a lesser extent, Fn-f-induced MMP-13 protein expression by human
primary chondrocytes (6), the
results suggest that the Fn-f-induction of MMP-13 occurs through two
mechanisms, via IL-1-dependent and IL-1-independent pathways.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our data indicate that the combined effect of IGF-1 and OP-1 on MMP-13 expression is mediated, at least in part by (a) the OP-1-elicited stimulation of the IGF-1 cellular autocrine system in human chondrocytes and (b) the ability of IGF-1 and OP-1 to reduce the expression of inflammatory cytokines and the activity of the inflammatory cytokine-associated intermediate gene products. This concept is based on several findings. First, recombinant human OP-1 increases the transcription of IGF-1 and the IGF-1 receptor, whereas no significant induction of OP-1 or OP-1 receptor mRNA synthesis was observed by IGF-1. Second, an excess amount of antibodies to IGF-1 and the IGF-1 receptor or forced expression of the human IGF-1 receptor dominant negative completely abolishes the inhibitory effect of IGF-1 as well as OP-1 on MMP-13 promoter activity. Third, forced expression of the human IGF-1 receptor enhances the inhibitory effect of IGF-1 and OP-1; yet forced expression of an OP-1 receptor (BMPR-1B) shows no significant enhancement of the IGF-1 effect on MMP-13 promoter activity. Further investigation would be necessary for other OP-1 receptor components such as BMPR-1A or BMPR-II. The enhanced OP-1 effect upon elevating IGF-1 receptor protein levels by forced expression may be due to the stimulated IGF-1 autocrine system by OP-1, which reflects cellular conditions similar to adding the combination of IGF-1 and OP-1. Finally, down-regulated expression of inflammatory cytokines or the reduced activation of the inflammatory cytokine-associated gene products by the combination of IGF-1 and OP-1 may directly/indirectly reinforce the combined effect of IGF-1 and OP-1.
Our observations also suggest that a complicated process through cross-talk between the signaling molecules of IGF-1 and OP-1 in chondrocytes may be involved in the combined effects of IGF-1 and OP-1. For example, an excess amount of antibodies to IGF-1 and the IGF-1 receptor completely abrogates the individual inhibitory effect of IGF-1 and OP-1 but only partially blocks the combined effect of IGF-1 and OP-1. Furthermore, the preincubation of cells with IGF-1 (100 ng/ml) followed by a gradual increase in the amount of OP-1 or vice versa sensitizes cells to OP-1 or IGF-1. Preincubated chondrocytes by either IGF-1 or OP-1 were able to respond to concentrations of as low as 10 ng/ml of IGF-1 or OP-1. Furthermore, preliminary data revealed that IGF-1 treatment significantly down-regulates Smad7 gene expression, causing interference with the OP-1 signaling pathway.3 This observation suggests that IGF-1 may also in turn stimulate the OP-1 autocrine system through indirect mechanisms in human chondrocytes. Defining the molecular basis for the cross-talk between the IGF-1 and OP-1 signaling pathways should be undertaken as future studies for better understanding of the inhibitory mechanism of IGF-1 and OP-1 on MMP-13 gene expression in chondrocytes.
Transient transfection of a series of deletion promoter constructs of
MMP-13 into immortalized or human primary chondrocytes showed the highest
luciferase activity driven by the full-length MMP-13 promoter,
1600MMP-13. The 1600MMP-13 promoter encodes multiple putative
recognition motifs for transcription factors, including the core binding sites
for AP-1 and Ets, as well as two copies of Runx2 (Cbfa I), HRE, NF-B,
and several Sox family members. With further deletion of the 5'-flanking
region, we observed decreased MMP-13 promoter activity. This observation
suggested that although the proximal core binding sites are critical for basal
promoter activity (39), the
multiple putative recognition sites in the distal 5'-flanking region of
MMP-13 gene may be necessary to mediate maximal MMP-13 promoter activity in
human chondrocytes.
Our data also revealed that the full-length of MMP-13 promoter construct,
1600MMP-13, was most responsive to the treatment of IGF-1 and OP-1. The
response was gradually decreased as the promoter deletion progressed. The
shortest promoter construct, 186MMP-13, showed the minimal response to
the growth factors. These results suggested that the inhibitory effects of the
growth factors may not be mediated by a single response element but are rather
mediated through multiple cis-acting elements present in the distal
5'-flanking promoter region of the MMP-13 gene. Although the DNA-protein
interaction of AP-1 factors in the regulation of MMP-13 gene is well known
(3941),
the molecular mechanism of NF-B, including the cis-acting
element on MMP-13 promoter region, is still not clear. Our gene analysis
identified the putative NF-
B motif in the proximal 5'-flanking
MMP-13 promoter region between 736 and 1600, and we propose that
this region would be a potential candidate to study for the
NF-
B-mediated MMP-13 gene expression.
Mengshol et al.
(31) reported that transiently
transfected MMP-13 promoters were not IL-1-inducible. In our system, however,
using either primary or immortalized human chondrocytes, promoter activity of
MMP-13 was responsive to treatment with IL-1. The IL-1
-stimulated
MMP-13 promoter activity was abolished upon the addition of IL-1Ra, which is
known to antagonize activity of the IL-1 receptor. These results suggest that
the MMP-13 promoter construct is responsive to treatment with IL-1
. The
difference in our results from the previous report may be due to either the
cell system used for the experiments or from the way in which the
promoter-reporter constructs were generated. We utilized a firefly luciferase
reporter vector, pGL2-Enhancer, which is promoterless but contains an enhancer
region reflecting much higher sensitivity than the pGL3-Basic that was used by
the other group.
In summary, the present study demonstrates the inhibitory effects of IGF-1
and OP-1 on basal as well as on the Fn-f- and IL-1-induced expression of
the MMP-13 gene in immortalized human chondrocytes in a
concentration-dependent manner. The combination of these two growth factors
shows prominent inhibition of MMP-13 expression at the transcriptional level
with corresponding decreases in MMP-13 protein. The molecular mechanisms by
which OP-1 and IGF-1 exert their combined effects on MMP-13 appeared to be in
part through OP-1-mediated stimulation of the IGF-1 autocrine system and the
suppression of pro-inflammatory cytokines by IGF-1 and OP-1 in human
chondrocytes. Our findings suggest that IGF-1 and OP-1 could be important
physiological regulators of MMP-13 expression, and the combination of IGF-1
and OP-1 may be useful in controlling the catabolic activity in arthritis.
![]() |
FOOTNOTES |
---|
** To whom correspondence should be addressed: Rheumatology, Rush-Presbyterian-St. Luke's Medical Center, 1725 W. Harrison, Suite 1017, Chicago, IL 60612. Tel.: 312-942-8994; Fax: 312-942-3053; E-mail: rloeser{at}rush.edu.
1 The abbreviations used are: MMP, matrix metalloproteinase; IL, interleukin;
Fn-f, fibronectin fragment; IGF-1, insulin-like growth factor-1; OP-1,
osteogenic protein-1; BMP, bone morphogenetic protein; IL-1Ra, IL-1 receptor
antagonist; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse
transcription.
2 Loeser, R. F., Pacione, C. A., and Chubinskaya, S. (2003) Arthritis
Rheum., in press.
3 H.-J. Im, C. Pacione, S. Chubinskaya, and R. F. Loeser, unpublished
observation.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|