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J. Biol. Chem., Vol. 277, Issue 10, 7865-7874, March 8, 2002
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From the
Received for publication, June 28, 2001, and in revised form, December 17, 2001
Neutral matrix metalloproteinases (MMPs) play an
important role in bone matrix degradation accompanied by bone
remodeling. We herein show for the first time that macrophage migration
inhibitory factor (MIF) up-regulates MMP-13 (collagenase-3) mRNA of
rat calvaria-derived osteoblasts. The mRNA
up-regulation was seen at 3 h in response to MIF (10 µg/ml), reached the maximum level at 6-12 h, and returned to the
basal level at 36 h. MMP-13 mRNA up-regulation was preceded by
up-regulation of c-jun and c-fos mRNA.
Tissue inhibitor of metalloproteinase (TIMP)-1 and MMP-9 (92-kDa type
IV collagenase) were also up-regulated, but to a lesser extent. The
MMP-13 mRNA up-regulation was significantly suppressed by
genistein, herbimycin A and
4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. Similarly, a selective mitogen-activated protein kinase (MAPK) kinase
(MEK)1/2 inhibitor (PD98059) and c-jun/activator protein (AP)-1 inhibitor (curcumin) suppressed MMP-13 mRNA
up-regulation induced by MIF. The mRNA levels of c-jun
and c-fos in response to MIF were also inhibited by
PD98059. Consistent with these results, MIF stimulated phosphorylation
of tyrosine, autophosphorylation of Src, activation of Ras, activation
of extracellular signal-regulated kinases (ERK) 1/2, a MAPK, but not
c-Jun N-terminal kinase or p38, and phosphorylation of c-Jun.
Osteoblasts obtained from calvariae of newborn JunAA mice, defective in
phosphorylation of c-Jun, or newborn c-Fos knockout (Fos Matrix metalloproteinases
(MMPs)1 are a family of
proteolytic enzymes, including collagenases, gelatinases, and
stromelysins (1-3). Collagenases cleave fibrillar collagens at neutral
pH and play an important role in matrix remodeling. Collagenases are
largely categorized into three classes: collagenases 1, 2, and 3, which
are mainly secreted from fibroblasts and osteoblasts, neutrophils, and
breast carcinoma cells, respectively (1, 3). In rodents, rat
osteoblasts and rat osteosarcoma cells have the potential to express
collagenase 3 such as MMP-13, but not MMP-1 (4-6). With regard to
collagenase production, several hormones and cytokines stimulate MMP-1
synthesis in human and MMP-13 in rat osteoblasts (4, 7, 8). From the
data available to date, it is considered that most molecules
potentially inducing bone resorption often stimulate MMP-1 production
in humans and MMP-13 production in rodents. In this context, it is
conceivable that MMP-13 may play a pivotal role in bone remodeling in rats.
Macrophage migration inhibitory factor (MIF) was originally identified
as a soluble factor in culture medium of activated-T cells (9, 10);
however, its precise biological function was largely unknown for nearly
30 years. Following the cloning of human MIF cDNA (11), an array of
novel biological functions of this protein has been reported (12, 13).
MIF is released as a hormone by the anterior pituitary gland in
endotoxin shock (14) and a glucocorticoid-induced immunomodulator
released from macrophages in response to a variety of inflammatory
stimuli (15). Regarding the potential role of MIF in induction of MMPs,
we revealed that MIF could stimulate MMP-1 and MMP-3 mRNA
expression in synovial fibroblasts obtained from rheumatoid arthritis
patients (16), suggesting its pathological role for direct destruction
of joint tissues in autoimmune disease.
In a previous study, we demonstrated that mouse osteoblasts expressed
high amounts of MIF (17); however, its pathophysiological role in the
bone tissues remains to be elucidated. We herein report for the first
time that MIF enhances the MMP-13 mRNA level in osteoblasts
obtained from newborn rat calvariae. Following this discovery, we
further investigated the signal transduction pathway of MIF in the
event of MMP-13 mRNA up-regulation to elucidate its intracellular
mechanism of action.
Materials--
The following materials were obtained from
commercial sources. Collagenase, staurosporine, genistein, and
herbimycin A were purchased from Wako (Osaka, Japan); H-7 and H-8 from
Seikagaku Kogyo (Tokyo, Japan);
4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) from Calbiochem (La Jolla, CA); cycloheximide, indomethacin, and
tyrphostin A25 from Sigma; [
Recombinant rat MIF was expressed in Escherichia coli
BL21/DE3 (Novagen, Madison, WI) and purified as described (18). It contained less than 1 pg of endotoxin/µg of protein, as determined by
the chromogenic Limulus amoebocyte assay (BioWhittaker,
Walkersville, MD).
Cells--
Rat calvarial osteoblasts were prepared by serial
collagenase digestion as described previously (19). In brief, newborn rat calvariae (day 1) were removed from soft tissues and digested with
0.1% collagenase and 0.2% dispase in DMEM containing 100 µM NEAA for 10 min at 37 °C five times (fractions
1-5), of which fractions 3-5 were used. After centrifugation and
washing with the medium, cells were resuspended in DMEM supplemented
with 10% FCS and NEAA in 100 mm culture dishes in a humidified 5%
CO2 atmosphere at 37 °C. After 48 h, nonadherent
cells were removed, and adherent cells were harvested after treatment
with 0.25% trypsin/EDTA and were successively passaged.
Rat synovial fibroblasts were prepared by 0.2% collagenase digestion
of rat knee synovial tissues. The murine osteoblastic cell line
MC3T3-E1 was purchased from RIKEN Cell Bank (Tsukuba, Japan) and
cultured in
Rat articular chondrocytes were obtained by collagenase digestion of
minced articular cartilage tissues from the femoral chondyle, femoral
head, and humeral head of 8-week-old rats. Rat skin fibroblasts were
obtained by harvesting outgrowing cells from explant cultures of minced
skin tissues from newborn rats. All of these cells were cultured in
DMEM supplemented with 10% FCS and NEAA.
To examine the effect of MIF on the expression of MMP mRNA,
production of MIF protein, phosphorylation of tyrosine, Src kinase activity, MAPK activity, and Ras activation, cultured rat osteoblasts of the third passages were used throughout the experiment. After reaching confluence (10-14 days after initial plating), the
osteoblasts were rinsed with phosphate-buffered saline (PBS),
serum-starved for 24 h, and challenged with 100 ng/ml, 1 µg/ml,
and 10 µg/ml MIF for 6 h in 10 ml of serum-free DMEM containing
NEAA. Rat PTH (10
For the time-course study, parallel cultures of rat osteoblasts were
treated simultaneously with or without 10 µg/ml MIF, and harvested at
indicated intervals after stimulation. Following this, the cells were
subjected to Northern blot analysis to evaluate mRNA levels of
MMP-13, TIMP-1, and type I (
To evaluate involvement of c-Jun phosphorylation in the signaling
pathway of MIF, calvarial osteoblasts retrieved from newborn JunAA mice
were also examined. In JunAA mice, serine residues 63 and 73 of the
endogenous Jun allele were substituted with alanine residues (21).
JunAA mice were a kind gift from Dr. E. F. Wagner (Research
Institute of Molecular Pathology, Vienna, Austria). Calvarial
osteoblasts derived from native strain C57BL/6 mice were used as
wild-type osteoblasts for positive controls. We also assessed the
involvement of c-Fos in the signaling pathways of MIF using calvarial
osteoblasts retrieved from newborn mice that were homozygous for the
Fos mutation (Fos Inhibition of MMP-13 mRNA Up-regulation by Reagents--
To
investigate the signal transduction pathways regarding up-regulation of
MMP-13 mRNA in response to MIF, effects of various reagents on rat
osteoblasts were tested. After reaching confluence following serum
starvation for 24 h, the cells were challenged with MIF (10 µg/ml) 30 min after the addition of cycloheximide (3.6 µM), indomethacin (10 µM), genistein (10 and 100 µM), herbimycin A (1 and 10 µM),
PP2 (1, 10, and 50 µM), tyrphostin A25 (10 and 100 µM), staurosporine (10 and 100 nM), H-7 (1 and 10 µM), H-8 (1.5 and 15 µM), PD98059 (5 and 25 µM), SB203580 (1, 10, and 20 µM), or
curcumin (1 and 10 µM) in serum-free medium. After 6 h, the cells were harvested and subjected to Northern blot analysis for
MMP-13 mRNA.
Northern Blot Analysis--
Rat c-jun cDNA and
rat col(I)
Total RNA was isolated from rat osteoblasts using an Isogen RNA
extraction kit according to the manufacturer's protocols. RNA was
quantitated using a spectrophotometer, and equal amounts of RNA (20 µg) from control and test samples were loaded on a formaldehyde-agarose gel. The gel was stained with ethidium bromide to
visualize RNA standards, and the RNA was transferred onto a nylon
membrane. Fragments obtained by restriction enzyme treatments for
MMP-13, MMP-2, MMP-9, TIMP-1, col(I) Immunoblot Analysis--
Cells (1 × 106 cells)
were disrupted with a Polytron homogenizer (Kinematica, Lucerne,
Switzerland). The protein concentrations of the cell homogenates were
quantitated using a Micro BCA protein assay reagent kit. Equal amounts
of homogenates were dissolved in 20 µl of Tris-HCl, 50 mM
(pH 6.8), containing 2-mercaptoethanol (1%), sodium dodecyl sulfate
(SDS) (2%), glycerol (20%), and bromphenol blue (0.04%), and heated
at 100 °C for 5 min. The samples were subjected to
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as described (22) and
transferred electrophoretically onto a nitrocellulose membrane. The
membranes were blocked with 1% nonfat dry milk in PBS, probed with 1 µg/ml anti-phosphotyrosine mAb or anti-Src mAb, and reacted with the
goat anti-mouse IgG Ab coupled with horseradish peroxidase. The
resultant complexes were processed for detection by enhanced
chemiluminescence using ECL Western blotting detection system according
to the manufacturer's protocol.
Moreover, we performed immunoblot analysis not only for the MMP-13
protein level in response to MIF, but also for three distinct MAPKs to
further elucidate their involvement in the signal transduction of MIF
for expression of MMP-13. Immunoblot analysis was carried out as
described above except for the use of antibodies against rat MMP-13 and
MAPKs, including ERK1/2, JNK, and p38.
Immune Complex Kinase Assay--
The immune complex kinase assay
was performed as described previously (23). In brief, cells were lysed,
precleared by incubating with nonimmune mouse IgG plus Pansorbin cells
and Protein A-Sepharose 4B overnight. The supernatant was incubated
with the anti-mouse Src mAb (10 µg) and then with 20 µl of Protein
A-Sepharose 4B for 2 h. These procedures were performed at
4 °C. The beads were incubated in a kinase buffer containing 370 kBq
of [ Transfection--
Primary osteoblasts were transfected with a
recombinant adenovirus carrying a kinase-defective rat C-terminal Src
family kinase (Csk). The recombinant virus contains the cytomegalovirus
immediate early enhancer, chicken [32P]Orthophosphate Labeling of Cells--
Rat
osteoblasts were labeled as described previously (25). Briefly,
2.0 × 106 cells were washed twice with 50 mM HEPES containing 0.1% bovine serum albumin. The cells
were propagated for 15 min in phosphate-free medium (Irvine Scientific,
Santa Ana, CA), labeled with 0.5 mCi of
[32P]orthophosphate for 3 h at 37 °C, and then
treated with MIF (10 µg/ml) at the indicated intervals. After
labeling, the cells were washed twice with cold PBS containing 20 mM sodium pyrophosphate, 100 mM sodium
fluoride, 4 mM EDTA, and then solubilized for 10 min on ice
in RIPA lysis buffer containing 20 mM sodium pyrophosphate, 100 mM sodium fluoride, 4 mM EDTA, and 2 mM phenylmethylsulfonyl fluoride. The cell lysates were
cleared by ultracentrifugation at 100,000 × g for 45 min. Then the supernatants were subjected to immunoprecipitation with
an anti-c-Jun antibody and Protein G-Sepharose, and to SDS-PAGE. The
gel was dried, exposed to an imaging plate for 30 min, and analyzed
with a BAS2000 imaging system.
Ras Activation Assay--
The activation of Ras was evaluated
using a Ras activation assay kit according to the manufacturer's
protocol. Briefly, rat primary osteoblasts (2.0 × 106
cells) were serum-starved for 24 h, treated with MIF (10 µg/ml) at the indicated intervals, washed twice with cold PBS, and lysed with
1 ml of Mg2+ Lysis/Wash Buffer (MLB). Then the lysate was
precleared by glutathione-agarose, 10 µg of Raf-1 Ras-binding domain
(RBD)-conjugated agarose was added, and it was incubated at 4 °C for
30 min. Raf-1 RBD-conjugated agarose is a glutathione
S-transferase fusion protein, corresponding to the human Ras
binding domain (residues 1-149) of Raf-1, provided bound to
glutathione-agarose; it specifically binds to and precipitates Ras-GTP
from cell lysates. After washing the beads three times with MLB, they
were suspended in 2× Laemmli sample buffer, subjected to SDS-PAGE and
immunoblot analysis using 1 µg/ml anti-Ras mAb as a primary antibody,
and visualized using the ECL Western blotting detection system.
Dose-response Study of MIF Effects on MMP-13, MMP-2, MMP-9, TIMP-1,
and col(I)
To compare expression levels of MMP-13, TIMP-1, and col(I) Time-course Study of MMP-13, TIMP-1, col(I) Effects of Various Reagents on Induction of MMP-13 mRNA by
MIF--
To examine whether the effect of MIF on rat MMP-13 mRNA
levels depended on de novo protein synthesis or was the
result of changes in prostaglandin synthesis, rat osteoblasts were
treated with MIF in the presence or absence of cycloheximide or
indomethacin. Cycloheximide or indomethacin minimally affected the
expression of MMP-13 without MIF stimulation (Fig.
4a). When the cells were stimulated with MIF, cycloheximide (3.6 µM) significantly
suppressed up-regulation of MMP-13 mRNA (45.8% of the
MIF-stimulated level), suggesting that the induction of MMP-13 mRNA
required de novo MIF protein synthesis. On the other hand,
indomethacin (10 µM) did not suppress the up-regulation
of MMP-13 mRNA. This result suggested that MMP-13 induction by MIF
was irrelevant to prostaglandin synthesis.
Next, we examined whether protein kinases were involved in
up-regulation of MMP-13 mRNA using protein kinase C inhibitors (staurosporine, H-7), a cyclic AMP-dependent kinase
inhibitor (H-8), and tyrosine kinase inhibitors (genistein, herbimycin
A, PP2, tyrphostin A25). When these inhibitors were added to rat osteoblasts 30 min prior to MIF stimulation (10 µg/ml), staurosporine (10 nM, 100 nM), H-7 (1 µM, 10 µM), and H-8 (1.5 µM, 15 µM)
showed no inhibitory effect on up-regulation of the mRNA of MMP-13
(Fig. 4b). On the other hand, genistein (100 µM) and herbimycin A (1 µM, 10 µM) significantly suppressed the MIF-induced MMP-13
mRNA expression (Fig. 4c). Similarly, PP2, a selective
inhibitor for Src-related tyrosine kinase (26, 27), markedly suppressed the up-regulation at the dose of 50 µM, whereas
tyrphostin A25, an inhibitor for epidermal growth factor receptor-type
tyrosine kinase, did not inhibit the induction at doses ranging from 10 to 100 µM (Fig. 4d). These results indicated
that MMP-13 mRNA induction by MIF might depend on Src-related
tyrosine kinase.
Effect of MIF on the Phosphorylation of Tyrosine--
We analyzed
levels of tyrosine phosphorylation of intracellular proteins in
response to MIF by immunoblot analysis using an anti-phosphotyrosine
mAb. MIF treatment resulted in phosphorylation of multiple cellular
proteins observed at 60, 34, and 30 kDa in comparison with controls (0 min) (Fig. 5). The enhancement of tyrosine phosphorylation occurred at 1-5 min after stimulation by MIF,
and the phosphorylated 60-kDa protein was considered to be an
autophosphorylated member of the Src-protein trosine kinase family.
Effect of MIF on the Autophosphorylation of Src--
To determine
whether MIF could enhance kinase activity of Src, we performed an
immune complex kinase assay using cell lysates of serum-starved rat
osteoblasts after precipitation with the anti-Src mAb.
Autophosphorylation of Src was enhanced at 3 min after stimulation with
MIF (10 µg/ml). The phosphorylation level was sustained at least up
to 1 h (Fig. 6a). On the
other hand, the amount of Src protein obtained in parallel from rat
osteoblasts in the presence of MIF was not changed as determined by
immunoblot analysis (Fig. 6b). These results suggested that
the enhanced autophosphorylation of Src was a result of up-regulation
of Src kinase activity, but not of increased Src protein level.
Effect of Dominant Negative Csk Overexpression on the MIF-induced
MMP-13 Expression--
Src family kinases are known to be negatively
regulated by Csk, and overexpression of kinase-defective Csk was
reported to cause activation of Src family kinases (24). To investigate the potential involvement of Src family kinases in the induction of
MMP-13 expression by MIF stimulation, we assessed the effect of
kinase-defective Csk overexpression on MIF-induced up-regulation of
MMP-13. Primary osteoblasts were transfected with adenovirus vector
pAxCATcsk carrying kinase-defective Csk or with a control vector,
pAx1w1. Overexpression of the mutant Csk was confirmed by immunoblot
analysis (data not shown). The basal and MIF-induced MMP-13 mRNA
levels were similar in the wild-type and pAx1w1-transfected rat
osteoblasts, whereas there was significant enhancement of the MMP-13
mRNA level in the kinase-defective Csk-transfected cells (9.3-fold
increase compared with the level of pAx1w1) (Fig. 6c). The
data indicated that overexpression of kinase-defective Csk, leading to
activation of Src family kinases, could enhance the effect of MIF on
MMP-13 expression. This fact also suggested the possibility that MAPK
was involved in the signaling pathway of MIF-induced MMP-13
up-regulation.
Effect of MIF on the Activation of MAP Kinases--
We performed
immunoblot analysis for three distinct MAP kinases to further assess
their involvement in the signal transduction of MIF for induction of
MMP-13. We found that ERK1/2 was significantly activated in response to
MIF, whereas p38 and JNK were not (Fig. 7a). The level of the
phosphorylated form of ERK1/2 was significantly enhanced at 15 min by
MIF (1.7-fold increase compared with the value of time 0), and returned
to the basal level at 1 h, whereas the levels of
phosphorylated-form JNK and p38 were minimally changed. The enhanced
ERK1/2 activity was inhibited by the Src family-specific tyrosine
kinase inhibitors PP2 (50 µM) and herbimycin A (1 µM) (Fig. 7b). The results suggested that
activation of ERK1/2 by MIF could be regulated downstream of Src family
kinases.
Effect of PD98059 and SB203580 on MIF-induced MMP-13
Expression--
We further examined whether activation of ERK1/2 was
involved in the up-regulation of MMP-13 using a MAPK/ERK kinase (MEK) 1/2-specific inhibitor (PD98059) and p38-specific inhibitor (SB203580). When these inhibitors were added to rat osteoblasts 30 min prior to MIF
treatment for 6 h, PD98059 (5 and 25 µM)
significantly suppressed MMP-13 up-regulation, whereas SB203580 (1, 10, and 20 µM) showed no inhibitory effect (Fig.
7c). Thus, it is conceivable that MMP-13 mRNA induction
by MIF depends on the activation of ERK1/2.
Effect of Curcumin on MIF-induced MMP-13 mRNA
Expression--
Next, we tested the effects of curcumin, an inhibitor
of c-jun/activator protein (AP)-1, to examine whether
c-jun/AP-1 was involved in the up-regulation of MMP-13
mRNA expression in response to MIF. It is known that curcumin
inhibits gene expression of c-jun induced by TPA, but not
that of the c-fos gene (28). This inhibitor suppresses the
TPA-induced TRE binding activity of AP-1 protein. We found that
curcumin suppressed the amounts of MIF-induced MMP-13 mRNAs at the
dose of 1 µM (Fig. 8). This
result suggested that up-regulation of MMP-13 by MIF depended on
activation of c-jun/AP-1.
Effects of PD98059 and SB203580 on MIF-induced c-jun and c-fos
Expression--
We examined whether the activation of ERK1/2 was
involved in the transcription of c-jun and c-fos
using PD98059 and SB203580. When these inhibitors were added to rat
osteoblasts and incubated with MIF for 30 min or 1 h, PD98059
significantly suppressed the up-regulation of c-jun mRNA
at a dose of 25 µM, and c-fos mRNA at
doses of 5 and 25 µM, whereas SB203580 (1 and 10 µM) had no inhibitory effect (Fig.
9, a and b). The
data suggested that c-jun and c-fos mRNA
induction by MIF depended on the activation of ERK1/2.
MMP-13 Induction by MIF in Calvarial Osteoblasts Obtained from
JunAA Mice and Fos Knockout Mice--
We assessed whether inhibition
of phosphorylation of Jun might result in the abolishment of MMP-13
induced by MIF using phosphorylation-defective JunAA mice.
Osteoblasts obtained from wild-type C57BL/6 mice showed marked
up-regulation of MMP-13 in response to MIF similar to rat osteoblasts.
On the other hand, the induction was significantly reduced in
osteoblasts obtained from JunAA mice (34.4% of the wild-type level)
(Fig. 10a). The data
suggested that up-regulation of MMP-13 by MIF required, at least in
part, phosphorylation of c-jun. We also assessed the role of
c-Fos in the MMP-13 induction by MIF using Fos knockout mice
(Fos Effect of MIF on the Phosphorylation of c-Jun--
We analyzed
levels of c-Jun phosphorylation in response to MIF using
[32P]orthophosphate labeling of the cells,
immunoprecipitation, and SDS-PAGE. MIF treatment resulted in the
phosphorylation of c-Jun (Fig. 11).
c-Jun phosphorylation was mostly enhanced at 15 min after stimulation
by MIF (3.8-fold increase compared with the level at time 0), and
decreased at 1 h.
Effect of MIF on the Activation of Ras--
To determine whether
MIF could enhance activity of Ras, we performed a Ras activation assay
using cell lysates of serum-starved rat osteoblasts. The active form of
Ras (Ras-GTP) was increased in response to MIF (10 µg/ml), and the
maximal increase was identified at 5 min (5.2-fold increase compared
with the level at time 0). The activation returned to the basal level
at 1 h after stimulation (Fig.
12).
The expression of MMP-13 is regulated at the transcriptional level
by various cytokines and other mediators in a positive or negative
manner in physiological conditions. In this study, we showed for the
first time that MMP-13 mRNA in rat primary osteoblasts was
significantly up-regulated by MIF. It has been reported that MMP-13
secreted by osteoblasts plays an important role in bone resorption. In
brief, collagenase degrades the osteoid layer covering the surface of
bone that results in exposure of calcified bone matrix so that
osteoclasts can steadily adhere to the bone surface (29, 30). In this
context, the digestion and resorption of osteoid initiates bone
resorption. Supporting this notion, bone resorption induced by PTH is
strikingly hampered in collagenase-resistant mutant mice (31).
Moreover, type I collagen, accounting for 90% of bone matrix produced
by osteoblasts, is essential for differentiation of osteoblasts and
mineralization of bone matrix (32). Accordingly, an increase of
collagenolytic activity accompanied by enhanced degradation of matrix
proteins leads not only to enhanced bone resorption, but also to
suppression of bone formation.
Enzyme activities of MMPs are also post-transcriptionally
regulated by activation of the latent proenzymes as well as interaction with their specific inhibitors called TIMPs (33). Biosynthesis of
MMP-13 is up-regulated by hormones/cytokines such as PTH, IL-1, IL-6,
retinoic acid, basic fibroblast growth factor, cortisol, leukemia
inhibitory factor, oncostatin M, and platelet-derived growth factor-BB
in rat osteoblasts or mouse calvariae (6, 8, 34-38). On the other
hand, MMP-13 is down-regulated by transforming growth factor- It should be noted that MIF is constitutively expressed in various
cells and is readily released in response to various stimuli (13).
Circulatory concentrations of MIF in humans and mice range from 5 to 20 ng/ml and increase to more than 100 ng/ml, which is extraordinarily
high in comparison with other cytokines (20). Thus, it is speculated
that a higher concentration of MIF may be required to elicit its
biological function. In fact, we needed a large amount of MIF (10 µg/ml) to activate osteoblasts in this study. To activate
synoviocytes obtained from patients with rheumatoid arthritis to
stimulate MMP-1 production, 1 µg/ml MIF was required (16). Cellular
responsiveness to MIF may change depending on the pathological state,
but this needs further evaluation. Although the receptor of MIF has not
yet been isolated, it is hypothesized that the requirement for a high
dose of MIF may be the result of a small number of MIF receptors, or
its low affinity to the MIF ligand. To confirm the specific action of
MIF regarding induction of MMP-13, we demonstrated that MIF-induced
MMP-13 mRNA up-regulation was suppressed by the anti-MIF monoclonal antibody.
Regarding the signaling pathways of MIF for induction of MMP-13, we
found that Src-related tyrosine kinase was profoundly involved in this
inductive process. Both herbimycin A and PP2, selective inhibitors for
Src-related tyrosine kinase, suppressed up-regulation of the MMP-13
mRNA level, whereas an epidermal growth factor receptor-type
tyrosine kinase inhibitor, tyrphostin A25, failed to inhibit the
induction. These data indicate that Src-related tyrosine kinase, but
not receptor-type tyrosine kinase, is utilized in intracellular
signaling in response to MIF. To date, it has been elucidated that Src
family kinases consist of nine members, including Blk, c-Fgr, Fyn, Hck,
Lck, Lyn, c-Src, c-Yes, and Yrk, and each molecule encoding a
cytoplasmic protein-tyrosine kinase is involved in the transmembrane
signal transduction. Among the Src family members, c-Src or v-Src is
reported to regulate members of MMP families in various types of cells
(45-47). In addition, c-Src is known to contribute to the Ras-MAPK
signaling pathway (48), in which tyrosine phosphorylation of Shc, one
of the Src substrates binding Grb2 in v-Src-transformed cells, is a
potential cause of Ras activation (49). Activation of the Ras-MAPK
signaling pathway induces various transcription factors, including
c-jun and c-fos (50). Recently, another MAPK
pathway involving Src kinases has been reported (51). These findings
suggest that c-Src may transactivate transcription factor AP-1 in
several pathways. Our current data showed enhanced autophosphorylation
of Src at 3 min in response to MIF, whereas the levels of Src protein
were essentially unchanged. In addition, overexpression of the
kinase-defective Csk gene up-regulated both the basal and MIF-induced
levels of MMP-13 mRNA perhaps via increases of activity of Src
family kinase activities. Furthermore, we observed the activation of
Ras in response to MIF with a slight delay from Src activation. These facts strongly indicated that MIF might up-regulate MMP-13 mRNA through activation of Src or Src family kinases.
Next, we investigated the involvement of MAP kinases in the induction
of MMP-13 in response to MIF. It has been reported that MIF activates
ERK1/2 in NIH3T3 fibroblasts (52). Consistent with this report, MIF
increased phosphorylated-form ERK1/2; however, two other MAPK members,
JNK and p38, were not phosphorylated. Moreover, phosphorylation of
ERK1/2 was inhibited by addition of herbimycin A and PP2. Thus, it is
conceivable that the activation of ERK1/2 may be located downstream of
Src activation in the signaling pathway of MIF. Furthermore, induction
of MMP-13 was also inhibited by PD98059, a selective inhibitor of
MEK1/2, whereas SB203580, a specific inhibitor of p38, failed to
suppress the MMP-13 up-regulation. Based on these data, it is
considered that activation of ERK1/2, but not JNK or p38, could be
essential for the up-regulation of MMP-13 in response to MIF.
The rat MMP-13 promoter region contains several consensus transcription
factor recognition sequences such as C/EBP, Cbfa1, p53, PEA-3, AP-2,
and AP-1 (53). On the other hand, the TIMP-1 promoter contains binding
sites for AP-1, PEA-3, and Sp-1 (54). As for the MMP-9 promoter region,
it has binding sites for AP-1, PEA-3, NF Concerning transcription factors, up-regulation of c-jun and
c-fos mRNA was seen prior to MMP-13 induction by MIF in
the time-course study. As for c-fos induction,
phosphorylation of Elk-1 was reported to facilitate formation of the
ternary complex composed of a serum-responsive factor, SRE and itself
(60), to activate transcription. Because the phosphorylation of Elk-1
is caused by ERK1/2, this mechanism may account of MIF-induced
c-fos transcription. This notion concerning the signaling
pathway is supported by the facts that MIF phosphorylates Elk-1 in
NIH3T3 fibroblasts (52), and that c-fos mRNA induction was suppressed by PD98059, a MEK1/2 inhibitor, but not by SB203580, a
p38 MAPK inhibitor, as shown in this study.
On the other hand, the mechanism of c-jun induction by MIF
seems more complex. Phosphorylation of c-Jun and ATF2, which is essential for the transcription of c-jun, is generally
mediated by JNK of MAPKs; however, our current data indicated that JNK might not be activated by MIF. Moreover, induction of c-jun
mRNA by MIF was inhibited by PD98059, not by SB203580. It is likely that induction of c-jun mRNA by MIF is mediated by
ERK1/2, but not by JNK or p38. Recently, Leppä et al.
(61) showed that ERK1/2 can induce phosphorylation of c-Jun via several
pathways including serine 63 and serine 73, using PC12 cells, and
activation of ERK1/2 resulted in the efficient induction of c-Jun. In
this study, we demonstrated an increase of phosphorylation of c-Jun by
MIF stimulation. Consistent with these findings, we observed a
significant decrease in MMP-13 up-regulation by MIF in osteoblasts retrieved from the JunAA mouse in comparison with wild-type mouse osteoblasts, demonstrating the importance of c-Jun phosphorylation in
this pathway. Thus, ERK1/2, but not JNK or p38, may phosphorylate c-Jun
and Elk-1, leading to c-jun and c-fos expression,
respectively, followed by an increase of AP-1 activity in rat
osteoblasts in response to MIF.
As for the Src/AP-1 signaling pathway with regard to MMP-13 induction,
v-Src-mediated signals to AP-1 has been reported to regulate gene
expression of MMP-9 (45). On the other hand, Vincenti et al.
(62) reported v-Src activation of the MMP-1 promoter through PEA3, but
not AP-1. Despite these controversial findings, activation of the
c-Src/AP-1 pathway seems important for the induction of MMP-13 in
response to MIF in rat osteoblasts. Accordingly, it is considered that
signals from MIF activate Src family kinases, followed by Ras/ERK1/2
activation and increased AP-1 activity, which leads to the enhancement
of MMP-13 mRNA.
In conclusion, we demonstrated that MIF up-regulated MMP-13 mRNA in
rat osteoblasts via Src-related tyrosine kinase, ERK1/2, and
AP-1-dependent signaling pathways. This intracellular
signaling pathway may play an important role in the degradation of the
collagen matrix and in bone remodeling. The current results should give further insights into the biological aspects of MIF in bone, and MIF
could be a target molecule for therapeutic use in destructive bone
diseases such as osteoporosis or rheumatoid arthritis.
We are grateful to Asagi Matsumura for
excellent technical assistance.
*
This work was supported in part by grants-in aid from the
Hokkaido Foundation for the Promotion of Scientific and Industrial Technology and from the Nakatomi Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Research fellow of the Japan Society for the Promotion of Science.
Published, JBC Papers in Press, December 20, 2001, DOI 10.1074/jbc.M106020200
2
S. Onodera, J. Nishihira, K. Iwabuchi, Y. Koyama, K. Yoshida, S. Tanaka, and A. Minami, unpublished observation.
The abbreviations used are:
MMP, matrix
metalloproteinase;
AP-1, activator protein 1;
DMEM, Dulbecco's
modified Eagle's medium;
ERK, extracellular signal-regulated kinase;
FCS, fetal calf serum;
GAPDH, glyceroaldehyde-3-phosphate
dehydrogenase;
IL, interleukin;
JNK, c-Jun N-terminal kinase;
mAb, monoclonal antibody;
MAP, mitogen-activated protein;
MAPK, mitogen-activated protein kinase;
MEK, mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase;
MEM, minimal
essential medium;
MIF, macrophage migration inhibitory factor;
NEAA, nonessential amino acid(s);
PBS, phosphate-buffered saline;
PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine;
PTH, parathyroid hormone;
RBD, Ras-binding domain;
TIMP, tissue
inhibitor of matrix metalloproteinases;
TPA, tetradecanoyl phorbol
acetate;
TRE, TPA-responsive element.
Macrophage Migration Inhibitory Factor Up-regulates Matrix
Metalloproteinase-9 and -13 in Rat Osteoblasts
RELEVANCE TO INTRACELLULAR SIGNALING PATHWAYS*
§,
,,
Department of Orthopaedics, ¶ Central
Research Institute, Department of
Biochemistry, and §§ Department of
Ophthalmology, Hokkaido University Graduate School of Medicine, Sapporo
060-8638, the ** Division of Immunobiology, Institute for
Genetic Medicine, Hokkaido University, Sapporo 060-0815, and the
¶¶ Department of Orthopaedic Surgery, Faculty of Medicine,
University of Tokyo, Tokyo 113-0033, Japan
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/) mice,
showed much less induction of MMP-13 with the addition of MIF than
osteoblasts obtained from wild-type or littermate control mice. Taken
together, these results suggest that MIF increases the MMP-13 mRNA
level of rat osteoblasts via the Src-related tyrosine kinase-,
Ras-, ERK1/2-, and AP-1-dependent pathway.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP and
[32P]orthophosphate (8 mCi/ml, carrier-free) from
PerkinElmer Life Sciences (Wilmington, DE); Dulbecco's modified
Eagle's medium (DMEM) and parathyroid hormone (PTH) from ICN
Biomedicals (Aurora, OH); fetal calf serum (FCS) from HyClone (Logan,
UT); 
-MEM and nonessential amino acids (NEAA) from
Invitrogen; Isogen RNA extraction kit and GenePure from Nippon
Gene (Toyama, Japan); Hybond N nylon membrane and ECL Western blotting
detection system from Amersham Biosciences, Inc.; horseradish
peroxidase-conjugated antibody from Bio-Rad; nonimmune mouse IgG plus
Pansorbin cells from Calbiochem-Novabiochem (La Jolla, CA);
Ex-Taq DNA polymerase and DNA random primer labeling kit
from Takara (Kyoto, Japan); curcumin from Nakarai Tesque (Kyoto, Japan); pT7 vector from CLONTECH (Palo Alto, CA);
anti-mouse Src monoclonal antibody (mAb) (GD11; mouse IgG1) and Ras
activation assay kit from Upstate Biotechnology (Lake Placid, NY);
antibodies for phosphorylated-form mitogen-activated protein kinases
(MAPK), including extracellular signal-regulated kinase (ERK), c-Jun
N-terminal kinase (JNK) and p38, from New England Biolabs (Beverly,
MA), and anti-phosphotyrosine mAb (PY20; mouse IgG1) from ICN
ImmunoBiologicals (Costa Mesa, CA); mouse anti-rat MMP-13
(collagenase-3) monoclonal antibody from Chemicon (Temecula, CA);
anti-c-Jun (H-79) from Santa Cruz Biotechnology (Santa Cruz, CA), and
Micro BCA protein assay reagent kit from Pierce. All other chemicals
were of analytical grade.
-MEM supplemented with 10% FCS. Rat osteosarcoma cell
line UMR106 was purchased from the American Type Culture Collection
(ATCC) (Rockville, MD), and cultured in Eagle's MEM supplemented with
10% FCS and NEAA.
9 M and 108
M) and tetradecanoyl phorbol acetate (TPA)
(106 M) were used as positive controls. Two
sets of the third-passage rat synovial fibroblasts, MC3T3-E1 cells,
UMR106 cells, second-passage rat articular chondrocytes, and
second-passage rat skin fibroblasts were used as control cells.
) procollagen (col(I) (1)), and also
to immunoblot analysis and in vitro kinase assay. To exclude
possible endotoxin contamination in recombinant MIF, the effect of
heat-denatured recombinant MIF treated at 65 °C for 1 h was
also assessed. Then neutralizing effect of an anti-MIF monoclonal
antibody (IgG1) on MIF-induced MMP up-regulation was also evaluated.
The anti-MIF monoclonal antibody was prepared by immunizing mice with
recombinant rat MIF as described elsewhere (20).
/) and littermate wild type (Fos+/+) mice as
controls. Mice that were heterozygous for Fos mutation (Fos+/) (B6,
129S-Fostm1Pa female and male mice) were purchased from the
Jackson Laboratory (Bar Harbor, ME). After mating, the genotypes of the
newborn mice obtained were determined by reverse
transcription-polymerase chain reaction (PCR) analysis of the lysed
tail from each of them. These murine calvarial osteoblasts were
retrieved in a manner similar to rat osteoblasts, and were cultured in
-MEM supplemented with 10% FCS.
(1) cDNA probes were kindly provided by Dr. M. Sakai
(Department of Biochemistry, Hokkaido University Graduate School of
Medicine) and Dr. M. Shibanuma (Department of Microbiology, Showa
University School of Pharmaceutical Sciences), respectively. The
templates for Northern blot analyses of rat MMP-13, MMP-2, MMP-9,
TIMP-1, c-fos, and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) cDNA were obtained by reverse transcription-PCR from a
cDNA library of rat synovial fibroblasts. Preparation of each
template was as follows: MMP-13 (424 bp), sense primer
5'-GCGGGAATCCTGAAGAAGTCTAC-3' (143-165) and antisense primer
5'-TTGGTCCAGGAGGAAAAGCG-3' (547-566) (GenBankTM M60616); MMP-2 (217 bp), sense primer 5'-GCTGATACTGACACTGGTACTG-3' (1642-1663) and
antisense primer 5'-CAATCTTTTCCGGGAGCTC-3' (1840-1858) (GenBankTM
U65656); MMP-9 (280 bp), sense primer 5'-AAGGATGGTCTACTGGCAC-3' (553-571) and antisense primer 5'-AGAGATTCTCACTGGGGC-3' (815-832) (GenBankTM U24441); TIMP-1 (413 bp), sense primer
5'-CAGATATCCGGTTCGCCTACACC-3' (149-171) and antisense primer
5'-CAGGCAAGGTGACGGGACTGGAAGC-3' (539-561) (GenBankTM L29512);
c-fos (897 bp), sense primer 5'-CTGAAAGAGAAGGAAAAACTGGA-3' (693-715) and antisense primer 5'-TGGCTCACATGCTACTAACTACC-3'
(1567-1589) (GenBankTM X06769); GAPDH (983 bp), sense primer
5'-TGAAGGTCGGTGTCAACGGATTTGGC-3' (35-60) and antisense primer
5'-CATGTAGGCCATGAGGTCCACCAC-3' (994-1017) (GenBankTM M17701). Each
PCR product was separated by 1% agarose gel, purified by GenePure, and
subcloned into a pT7 plasmid vector by TA cloning. The subcloned
plasmids were transformed into DH5-competent cells. After
amplification, each insert was prepared by restriction enzyme
digestion, checked by a sequencing analyzer (ABI 377A), and used as a
probe for Northern blot analysis.
(1), c-jun,
c-fos, and GAPDH were labeled with
[-32P]deoxycytidine triphosphate using a DNA random
primer labeling kit. Hybridization was carried out at 42 °C for
24-48 h. Post-hybridization washing was performed in 0.1% SDS, 0.2×
standard saline citrate (SSC) (1× SSC: 0.15 M NaCl, 0.015 M sodium citrate) at 65 °C for 15 min. The radioactive
bands were visualized by autoradiography on Kodak X-AR5 film and
quantitatively analyzed using the NIH Image system. The results were
normalized by GAPDH mRNA levels.
-32P]ATP at 30 °C for 10 min. Thereafter, the
beads were washed, boiled for 5 min, and subjected to SDS-PAGE. The gel
was dried, exposed to an imaging plate for 30 min, and analyzed with a
BAS2000 imaging system (Fuji Film, Tokyo, Japan).
-actin promotor, and rabbit
-globin poly(A) signal and AxCATcsk (K222R), in which the lysine 222 is replaced by an arginine residue, as described previously (24). The
control virus Ax1w1 contains no foreign genes. The third-passage rat
primary osteoblasts were infected with the recombinant adenovirus as
follows. The cells were cultured with small amounts of DMEM containing
recombinant adenovirus for 1 h at 37 °C at an indicated multiplicity of infection. Then, more than 10 volumes of medium with
10% FCS was added to the cells. After 48 h of the infection, the
cells were washed, serum-starved for 24 h, and treated with MIF
(10 µg/ml) for 6 h. Then they were collected and subjected to
Northern blot analysis.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
(1) mRNA Expression of Rat Osteoblasts--
In rat
calvaria-derived osteoblasts, up-regulation of MMP-13 mRNA at
6 h after the challenge with MIF was slight at the dose of 1 µg/ml, but it became apparent at the dose of 10 µg/ml (12.8-fold increase compared with the level of nonstimulated control) (Fig. 1a). The induction of MMP-13
by MIF was much higher than that by PTH (109 to
108 M) (2.1-fold increase at the dose of
108 M), a representative physiological
inducer of MMP-13. TPA (106 M), a positive
control, most strongly enhanced MMP-13 mRNA (25.2-fold increase at
the dose of 106 M). The TIMP-1 mRNA level
was also up-regulated, but to a much lesser extent, at doses ranging
from 0.1 µg/ml to 10 µg/ml. The type I procollagen (col(I) (1))
mRNA level was higher at the basal level and was essentially
unchanged with addition of MIF. Moreover, we assessed the
responsiveness of MMP-2 and MMP-9 mRNA to MIF. MMP-9 mRNA was
significantly up-regulated, similar to MMP-13 but to a lesser extent
(4.5-fold increase compared with the level of the nonstimulated control
at 10 µg/ml MIF), whereas MMP-2 mRNA was slightly increased.
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Fig. 1.
Effects of MIF on mRNA expression of
MMP-13, MMP-2, MMP-9, TIMP-1, and
col(I) (1). Total RNAs of rat osteoblasts
treated with various concentrations of MIF in serum-free medium for
6 h were subjected to Northern blot analysis. The blots were
hybridized with 32P-labeled rat cDNA probes of MMP-13,
MMP-2, MMP-9, TIMP-1, col(I)(1), and GAPDH, and then visualized by
autoradiography. a, effects of MIF (0.1-10 µg/ml) on rat
primary osteoblasts. PTH (109 to 108
M) and TPA (106 M) were used as
positive controls. Cont, nonstimulated control.
b, comparison of MMP-13 mRNA levels between rat
osteoblasts, rat synovial fibroblasts, murine MC3T3-E1 cells, and rat
UMR-106 cells in response to MIF (10 µg/ml). c, comparison
of MMP-13 mRNA levels between rat osteoblasts, rat articular
chondrocytes, and rat skin fibroblasts in response to MIF (10 µg/ml).
d, effect of anti-MIF monoclonal antibody (50 µg/ml) on
MMP-13 up-regulation induced by MIF (10 µg/ml). Nonimmune mouse IgG
(50 µg/ml) was used as a control.
(1) among
different cell types in response to MIF, rat primary osteoblasts, rat
primary synovial fibroblasts, murine MC3T3-E1 cells, and rat UMR-106
cells were examined. MMP-13 mRNA was up-regulated by MIF (10 µg/ml) in rat primary osteoblasts and in rat synovial fibroblasts
(Fig. 1b). It was minimally changed in MC3T3-E1 cells and
UMR-106 cells, in which the basal expression level of MMP-13 in UMR-106
was very high. On the other hand, TIMP-1 mRNA was slightly up-regulated by MIF in rat primary osteoblasts, but not changed in
synovial fibroblasts, MC3T3-E1 cells, or UMR-106 cells. The mRNA
level of col(I) (1) was essentially not affected by MIF stimuli in
all these cells. Furthermore, we examined mRNA expression levels in
rat articular chondrocytes and rat skin fibroblasts. MMP-13 mRNA
was up-regulated in the articular chondrocytes in response to MIF, but
not in skin fibroblasts (Fig. 1c). A slight increase of
TIMP-1 mRNA in articular chondrocytes was also observed. To assess
the structure-specific action of MIF protein, we treated MIF at
65 °C for 1 h. The potential of MIF for induction of MMP-13 mRNA was significantly reduced by the heat treatment (data not shown). We also assessed the neutralizing effect of an anti-MIF monoclonal antibody on MMP-13 mRNA expression. Addition of 50 µg/ml antibody 30 min prior to addition of MIF significantly
suppressed the induction of MMP-13 and TIMP-1 mRNAs, though the
neutralizing effect was incomplete (Fig. 1d). Nonimmune
mouse IgG (IgG1) minimally affected the expression of MMP-13 mRNA.
We performed immunoblot analysis of MMP-13 at the protein level to
confirm the results regarding the mRNA level. We found that the
MMP-13 protein level was increased in response to MIF (10 µg/ml) by
24 h treatment as seen for the PTH (108
M) and TPA (106 M) treatment used
as positive controls (Fig. 2).
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Fig. 2.
Effect of MIF on the MMP-13 protein levels in
rat osteoblasts. Cell lysates of rat primary osteoblasts (1 × 106 cells) treated with MIF (1 and 10 µg/ml) for
24 h were subjected to immunoblot analysis using an anti-rat
MMP-13 monoclonal antibody as the primary antibody. PTH
(10 8 M) and TPA (106
M) were used as positive controls. Cont,
control.
(1) c-jun, and c-fos
mRNA Expression in Rat Primary Osteoblasts--
MMP-13 mRNA
expression of rat primary osteoblasts increased at 3 h after
stimulation in response to MIF (10 µg/ml), reached the maximal level
at 6-12 h (13.4-fold increase compared with the level of time 0), and
returned to the unstimulated basal level at 36 h (Fig.
3). The mRNA level of TIMP-1 was
slightly up-regulated in response to MIF at 3 h after stimulation
and then decreased. The mRNA level of col(I) (1) was essentially
unchanged throughout the time course. Prior to the up-regulation of the
MMP-13 mRNA level in response to MIF, transient increases of
c-jun and c-fos mRNA levels were observed.
The c-jun mRNA was transiently up-regulated at 1 h
after stimulation, and then decreased to the basal level at 3 h.
The c-fos mRNA was markedly up-regulated at 30 min after stimulation, and immediately returned to the basal level at 1 h.
No significant changes of MMP-13 and TIMP-1 mRNA levels were observed in the absence of MIF (data not shown).
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Fig. 3.
Time-course study for the effect of MIF on
mRNA levels of MMP-13, TIMP-1, col(I) (1),
c-jun, and c-fos in
rat primary osteoblasts. Total RNA of rat primary osteoblasts in
serum-free medium treated with MIF (10 µg/ml) at various time
intervals was subjected to Northern blot analysis. The transferred RNA
was hybridized with 32P-labeled rat cDNA probes of
MMP-13, TIMP-1, col(I)(1), c-jun, c-fos, and
GAPDH, and visualized by autoradiography.
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Fig. 4.
Effects of reagents on MIF-induced MMP-13
mRNA expression. Rat primary osteoblasts were preincubated for
30 min in the presence or absence of inhibitors prior to challenge with
MIF. Then the cells were incubated for 6 h in the presence or
absence of inhibitors and MIF (10 µg/ml), and subjected to Northern
blot analysis. The blots were hybridized with 32P-labeled
rat cDNA probes of MMP-13 and GAPDH, and visualized by
autoradiography. a, cycloheximide (3.6 µM) or
indomethacin (10 µM); b, two protein kinase C
inhibitors, staurosporine and H-7, and a cyclic
AMP-dependent protein kinase inhibitor, H-8; c,
two tyrosine kinase inhibitors, genistein and herbimycin A;
d, two tyrosine kinase inhibitors, PP2 and tyrphostin
A25.
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Fig. 5.
Effect of MIF on tyrosine phosphorylation of
cellular proteins. Cell lysates of rat primary osteoblasts treated
with MIF (10 µg/ml) for 0, 1, 5, 15, and 60 min were subjected to
immunoblot analysis using anti-phosphotyrosine mAb (PY-20) as a primary
antibody. Arrowheads indicate three proteins that are
significantly phosphorylated at tyrosine residues compared with those
of controls collected at time 0.
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Fig. 6.
Analysis of the involvement of Src kinase in
response to MIF. a, effect of MIF on the Src kinase
activity examined by immune complex kinase assay. Cell lysates of rat
primary osteoblasts treated with MIF (10 µg/ml) for 0, 1, 3, 5, 10, 15, 30, and 60 min were subjected to immune complex kinase assay.
b, immunoblot analysis of Src protein in response to MIF (10 µg/ml). The arrow indicates a 60-kDa band corresponding to
Src. c, effect of dominant-negative Csk overexpression on
the MIF-induced expression of MMP-13. Northern blot analysis was
carried out on the cells transfected with recombinant viruses carrying
a kinase-defective Csk expression vector (pAxCATcsk) (K222R) or control
vector (pAx1wl) before MIF (10 µg/ml) treatment for 6 h.
WT, nontransfected wild-type.
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Fig. 7.
Analysis for involvement of MAP kinases in
response to MIF. a, effects of MIF on activation of MAP
kinases. Cell lysates of rat primary osteoblasts treated with MIF (10 µg/ml) for 0 min, 15 min, and 1 h were subjected to immunoblot
analysis using anti-phospho-ERK1/2, anti-phospho-JNK, and
anti-phospho-p38 as primary antibodies. b, effects of
specific inhibitors of Src family kinases on the activation of ERK1/2
induced by MIF (10 µg/ml). Rat primary osteoblasts were preincubated
for 30 min in the presence or absence of PP2 (50 µM) or
herbimycin A (1 µM). They were coincubated for 15 min
with or without MIF (10 µg/ml) and subjected to immunoblot analysis
using anti-phospho-ERK1/2 as a primary antibody. c, effects
of specific inhibitors of MEK1/2 or p38 on the MIF-induced MMP-13
mRNA expression. Rat primary osteoblasts were preincubated for 30 min in the presence or absence of PD98059 or SB203580. Then the
osteoblasts were coincubated for 6 h with or without MIF (10 µg/ml) and subjected to Northern blot analysis. The blots were
hybridized with 32P-labeled rat cDNA probes of MMP-13
and GAPDH, and visualized by autoradiography.
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Fig. 8.
Effect of an inhibitor of
c-jun/AP-1 on MIF-induced MMP-13 mRNA
expression. Rat primary osteoblasts were preincubated for 30 min
in the presence or absence of an inhibitor of c-jun/AP-1,
curcumin. Then, the osteoblasts were coincubated for 6 h with or
without MIF (10 µg/ml) and subjected to Northern blot analysis. The
blots were hybridized with 32P-labeled rat cDNA probes
of MMP-13 and GAPDH, and visualized by autoradiography.
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Fig. 9.
Effects of specific inhibitors of MEK1/2 and
p38 on MIF-induced c-jun and c-fos
mRNA expression. Rat primary osteoblasts were
preincubated for 30 min in the presence or absence of PD98059 or
SB203580. Then the osteoblasts were coincubated for 30 min or 1 h
with or without MIF (10 µg/ml) and subjected to Northern blot
analysis. The blots were hybridized with 32P-labeled rat
cDNA probes of c-jun and GAPDH (a) and of
c-fos and GAPDH (b), and visualized by
autoradiography.
/). The induction of MMP-13 was reduced in osteoblasts obtained
from newborn Fos/ mice (1.4-fold increase compared with the level
of the nonstimulated control) compared with cells from the littermate
Fos+/+ mice (3.1-fold increase compared with the level of the
nonstimulated control) (Fig. 10b), suggesting that c-Fos is
involved, at least in part, in up-regulation of MMP-13 by MIF.
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Fig. 10.
Effects of defective c-Jun phosphorylation
and defective c-Fos gene on MMP-13 mRNA levels in response to
MIF. a, mouse primary osteoblasts obtained from
wild-type (WT) or JunAA mice harboring a wild-type allele or
mutant Jun allele with Ser-63 
Ala and Ser-73 Ala, respectively,
were incubated for 6 h with or without MIF (10 µg/ml) and
subjected to Northern blot analysis. The blots were hybridized with
32P-labeled rat cDNA probes of MMP-13 and GAPDH, and
visualized by autoradiography. WT, C57BL/6 wild-type mouse.
b, mouse primary osteoblasts obtained from Fos knockout mice
(Fos/) and littermate control mice (Fos+/+) were incubated for
6 h with or without MIF (10 µg/ml) and subjected to Northern
blot analysis. The blots were hybridized with 32P-labeled
rat cDNA probes of MMP-13 and GAPDH and visualized by
autoradiography.
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Fig. 11.
Effect of MIF on the phosphorylation of
c-Jun. Rat osteoblasts (2.0 × 106 cells) were
washed twice with 50 mM HEPES containing 0.1% bovine serum
albumin. The cells were propagated for 15 min in phosphate-free medium
and then labeled with 0.5 mCi of [32P]orthophosphate for
3 h at 37 °C, and then treated with MIF (10 µg/ml) at the
indicated intervals. After labeling, the cells were washed twice with
cold PBS containing 20 mM sodium pyrophosphate, 100 mM sodium fluoride, 4 mM EDTA, and then
solubilized for 10 min on ice in RIPA lysis buffer containing 20 mM sodium pyrophosphate, 100 mM sodium
fluoride, 4 mM EDTA, and 2 mM
phenylmethylsulfonyl fluoride. After retrieval, the cell lysates were
immunoprecipitated with an anti-c-Jun antibody, subjected to SDS-PAGE,
and then visualized by autoradiography.
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Fig. 12.
Effect of MIF on Ras activation. Rat
primary osteoblasts (2.0 × 106 cells) were
serum-starved for 24 h, treated with MIF (10 µg/ml) at the
indicated intervals, washed twice with cold PBS, and lysed with 1 ml of
Mg2+ Lysis/Wash Buffer. Then, the lysates were precleared
by glutathione-agarose, 10 µg of Raf-1 RBD-conjugated agarose was
added, and they were incubated at 4 °C for 30 min. Raf-1
RBD-conjugated agarose is a glutathione S-transferase fusion
protein, corresponding to the human Ras binding domain of Raf-1.
Following SDS-PAGE and immunoblotting, Ras-GTP was visualized by the
ECL Western blotting detection system.
DISCUSSION
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DISCUSSION
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,
insulin-like growth factors, and bone morphogenetic protein (39-42).
Regarding these bioactive molecules, bone-resorbing agents tend to
enhance MMP-13 expression, whereas mineralization-stimulating agents
inhibit the expression in osteoblasts. The role of MIF in the process
of bone formation and resorption has been poorly understood. Our
current results showed that MIF more potently up-regulated MMP-13
mRNA than PTH, but minimally up-regulated mRNA expression of
col(I) (1), which is a putative marker for bone formation and early
differentiation of osteoblasts. We also assessed the expression of
MMP-2 and MMP-9 mRNA in response to MIF, because MMP-2 and MMP-9
are involved in the development and osteoclastic resorption of bone
(37). We found significant up-regulation of MMP-9 mRNA, and a
slight increase of MMP-2 mRNA, consistent with a previous finding
(43). Considering these facts, including our previous report revealing
high expression of MIF in murine osteoblasts (17), it is likely that
MIF may act on osteoblasts as an autocrine factor promoting bone
resorption rather than bone formation. Indeed, MIF-overexpressing
transgenic mouse (16-week-old) shows signs of
osteoporosis.2 Similar
effects of MIF were observed in rat synovial fibroblasts, and also in
rat articular chondrocytes as demonstrated in this study. These facts
suggest that MIF potentially causes cartilage destruction in
inflammatory diseases such as rheumatoid arthritis, because expression
of endogenous MMPs from chondrocytes was profoundly related to
cartilage destruction in this disease (44). On the other hand, this
effect was not seen in rodent osteoblastic cell lines MC3T3-E1 and
UMR-106, despite their possessing the capacity to produce MMP-13 upon
stimulation with PTH or IL-6 (4, 36). These cells may have lost
responsiveness to MIF in the process of immortalization or transformation.
B, and Sp-1 (55, 56). These
findings indicate that MMP-13, TIMP-1, and MMP-9 promoter regions
contain two common regulatory elements, i.e. an AP-1 binding
site (TRE) and PEA-3. In this context, it is expected that MMP-13,
TIMP-1, and MMP-9 could be up-regulated in accordance with activation
of TRE or PEA-3. Indeed, AP-1, alone or in concert with Cbfa-1,
regulates the transcription of MMP-13 in response to platelet-derived
growth factor (57), basic fibroblast growth factor (58), and PTH (59).
Consistent with these findings, we observed that MIF-induced up-regulation of MMP-13 mRNA was inhibited by curcumin, an
inhibitor of c-jun/AP-1. Furthermore, we found a significant
decrease of the MMP-13 mRNA level in osteoblasts obtained from
Fos/ mice compared with osteoblasts from littermate Fos+/+ control
mice. Because AP-1 is known to be a homodimer or heterodimer of c-Jun and c-Fos, these results strongly indicated an essential role of AP-1
for MMP-13 induction by MIF.
ACKNOWLEDGEMENT
FOOTNOTES
To whom correspondence should be addressed: Central
Research Inst., Hokkaido University School of Medicine, Sapporo
060-8638, Japan. Tel.: 81-11-706-6081; Fax: 81-11-706-7864;
E-mail: j_nisihi@med.hokudai.ac.jp.
ABBREVIATIONS
REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Mauviel, A.
(1993)
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