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Departments of Biochemistry (Y.K.-O.,T.K., M.N., Y.Ka.), Removable Prosthodontics (H.S., Y.A.), and Endodontology and Periodontology (S.N.), Hiroshima University School of Dentistry, Hiroshima 734; the Department of Orthopedic Surgery, Faculty of Medicine, the University of Tokyo (Y.Ku.), Tokyo 113; the Department of Biochemistry, Aichi-Gakuin University School of Dentistry (T.H.), Nagoya 464; and the Department of Molecular Immunology and Pathology, Cancer Research Institute, Kanazawa University (Y.O.), Kanazawa 920, Japan; and the Department of Biochemistry, Institute of Endemic Disease, Norman Bethune University of Medical Sciences (W.Y.), Changchun 1300–21, China
Address all correspondence and requests for reprints to: Dr. Yukio Kato, Department of Biochemistry, Hiroshima University School of Dentistry, 1–2-3 Kasumi, Minami-ku, Hiroshima 734, Japan. E-mail: ykato{at}ipc.hiroshima-u.ac.jp
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many hormones and growth factors have been shown to modulate the proliferation, matrix synthesis, and expression of the hypertrophy-related phenotypes (alkaline phosphatase, 1,25-dihydroxyvitamin D3 receptor, and type X collagen synthesis) (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). The hormones and growth factors that regulate cartilage matrix degradation before ossification, in contrast, are unknown.
Matrix metalloproteinase (MMP) is thought to be crucial for cartilage matrix degradation, because MMP inhibitors prevent cartilage resorption and proteoglycan loss in arthritic joints in vivo (13, 14). MMP-1 (collagenase) degrades native collagen; MMP-2 (gelatinase A) and -9 (gelatinase B) degrade gelatin, elastin, and fibronectin; and MMP-3 (stromelysin-1) degrades proteoglycan, link protein, laminin, and fibronectin at neutral pH (15).
We are interested in the actions of PTH and PTH-related peptide (PTH-rp) on MMP, because PTH/PTH-rp plays an important role in endochondral bone formation. PTH/PTH-rp enhances DNA and aggrecan syntheses in growth plate chondrocytes (3, 16, 17) and suppresses their hypertrophy and apoptosis in vitro and in vivo (9, 11, 17, 18, 19, 20, 21, 22, 23). PTH-rp is synthesized in various tissues, including cartilage and perichondrium (17, 20), and binds to the PTH/PTH-rp receptor (24). The PTH/PTH-rp receptor level is much higher in the growth plate than in permanent cartilage (9, 25). Furthermore, a null mutation of the PTH-rp gene causes dwarfism and growth plate dysplasia without appreciable histological changes in articular cartilage and nonskeletal tissues (11, 17). The crucial role of PTH/PTH-rp in endochondral bone formation is also indicated by a link between a constitutively active mutation of the PTH/PTH-rp receptor gene and Jansen-type metaphyseal chondrodysplasia (26).
We report here that PTH/PTH-rp induces MMP-2, -3, and -9 in growth plate chondrocyte cultures, whereas it has little effect on MMP levels in articular chondrocyte cultures. The enhancement of these major MMPs by PTH/PTH-rp may be involved in the programed cartilage matrix degradation during endochondral bone formation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-modification
(MEM), and FBS were obtained from Sanko Jyunyaku (Tokyo, Japan) and
Mitsubishi Kagaku (Tokyo, Japan), respectively.
Immunohistochemistry studies
Paraffin sections (4 µm thick) of rib cartilage obtained from
4-week-old male Japanese White rabbits were incubated at room
tempera-ture with 1 U/ml chondroitinase avidin-biotin-peroxidase
complex (Seikagaku Kogyo, Tokyo, Japan) in 50 mM Tris-HCl,
pH 7.4, containing 0.15 M NaCl for 3 h. These sections
were incubated with a mouse monoclonal antibody (mAb; IgG) to human
MMP-2 (42-5D11), MMP-9 (56-2A4), or type II collagen (II-4C11; 10
µg/ml; Fuji Chemical Industries, Takaoka, Japan) or with control
nonimmune mouse IgG (10 µg/ml) for 1 h after blocking of
endogenous peroxidase with 0.3% H2O2 and 0.1%
NaN3 in methanol and blocking of nonspecific IgG binding
with 10% normal horse serum. The sections were washed with PBS,
incubated with biotinylated horse IgG to mouse IgG (diluted 1:200;
Vector Laboratories, Burlingame, CA) for 30 min, then stained with an
avidin-biotin-peroxidase complex (Vector Laboratories). Color was
developed with 0.03% 3,3'-diaminobenzidine tetrahydrochloride in 50
mM Tris-HCl, pH 7.6, containing 0.006%
H2O2, and the sections were counterstained with
hematoxylin.
Chondrocyte cultures
Chondrocytes were isolated from the rib growth plate and the
surface (0.2 mm) of articular cartilage of the femur at knee joints of
4-week-old male Japanese White rabbits, as previously described (27, 28). The cells were seeded at 2 x 105, 3 x
104, or 104 cells/35-, 16-, or 6-mm plastic
tissue culture dish, respectively, and maintained in 2, 0.5, or 0.1 ml
MEM supplemented with 10% FBS, 50 µg/ml ascorbic acid, 32 U/ml
penicillin, and 40 µg/ml streptomycin (medium A) at 37 C under 5%
CO2 in air. The cultures were fed fresh medium A 3 days
after seeding, and thereafter the medium was changed every other
day.
Determination of alkaline phosphatase activity
Chondrocytes grown in 16-mm wells were homogenized in a glass
homogenizer in 1 ml 0.9% NaCl-0.2% Triton X-100 at 4 C and
centrifuged for 15 min at 12,000 x g. Alkaline
phosphatase activity in the supernatant was measured by a modification
of the method of Bessey et al., using
p-nitrophenyl phosphate as the substrate, as described
previously (2, 29). One unit was defined as the activity catalyzing
hydrolysis of 1 µmol p-nitrophenyl phosphate/µg DNA·30
min.
Zymography
Chondrocytes in 16-mm wells were exposed to 0.5 ml serum-free
MEM supplemented with various hormones, growth factors, or cytokines
for 48 h. The media (1 or 4 µg protein/lane) conditioned by
chondrocytes were mixed with concentrated Laemmli buffer (30) without
reducing agent. Protein in the samples was resolved in 10%
polyacrylamide gels containing 0.5 mg/ml gelatin (Wako Pure Chemical
Industry, Osaka, Japan) or 0.5 mg/ml casein (Wako Pure Chemical
Industry) by SDS-PAGE. After the gels were washed in 2.5% Triton X-100
for 30 min to remove SDS and in 50 mM Tris-HCl (pH 8.0) for
10 min, they were incubated for 8–24 h in 50 mM Tris-HCl
(pH 8.0) containing 5 mM CaCl2, 0.2
M NaCl, and 0.02% NaN3 at 37 C. The gels were
then stained with Coomassie brilliant blue. The enzymatic activity was
seen as negatively stained bands.
Sequential slices (the width of a slice, 
0.3 mm; slice 1, the
hypertrophic zone; slice 2, the matrix-forming zone; slice 3, the
proliferating zone) of the rib growth plate were obtained from three
4-week-old male Japanese White rabbits, as described previously (9, 28). Resting cartilage of the rib and articular cartilage of the femur
at knee joints was also obtained from three 4-week-old male Japanese
White rabbits (9, 28). The tissue was minced and homogenized in 10 vol
Laemmli buffer (30). Protein in the samples (2 µg protein/lane) was
resolved in the gels containing gelatin by SDS-PAGE, as described
above.
Immunoblotting
Chondrocytes in 6-mm wells were exposed to 0.1 ml serum-free
MEM supplemented with various hormones, growth factors, or cytokines
for 48 h. The media conditioned by chondrocytes were mixed with
concentrated Laemmli buffer (30) without reducing agent.
The sequential growth plate slices and resting cartilage of the rib and articular cartilage of the femur at knee joints obtained from three 4-week-old male Japanese White rabbits were minced and homogenized in 10 vol sodium acetate buffer, pH 5.8, containing 4 M guanidine HCl, 1 mM phenylmethylsulfonylfluoride, 10 µM amidinophenylmethylsulfonylfluoride, 10 mM N-ethylmaleimide, 1 mM EDTA, and 0.1 mM pepstatin A in a Polytron homogenizer (Kinematica, Littau, Switzerland) at 4 C. The homogenate was incubated at 4 C for 18 h, then centrifuged at 5000 x g for 10 min at 4 C. The supernatant was dialyzed against water containing protease inhibitors (10 µM amidinophenylmethylsulfonylfluoride, 10 mM N-ethylmaleimide, 10 µM pepstatin A, and 1 mM EDTA) at 4 C, freeze-dried, and then mixed with Laemmli buffer (30) without reducing agent.
Proteins (2 µg/lane) in the samples (the conditioned media or cartilage extracts) were resolved in a 4–20% polyacrylamide gradient gel by SDS-PAGE under nonreducing conditions, then electrophoretically transferred to a nitrocellulose membrane (31). Nonspecific binding was blocked in a milk solution (5% nonfat dry milk in PBS) at room temperature for 1 h. The blots were exposed to a primary mouse mAb against human MMP-2 (42-5D11, Fuji Chemical Industries) at 4 C for 12 h (32) and then exposed to 125I-labeled sheep antimouse IgG-F(ab')2 fragment (Amersham, Aylesbury, UK) in PBS for 3 h at room temperature. (Antibodies that cross-react with rabbit MMP-3 or -9 in immunoblots are not available.)
Northern analysis
Total RNA was extracted by the guanidine HCl method (33).
Samples of total RNA (5–15 µg) were denatured in the presence of 2.2
M formaldehyde, electrophoresed on a 1% agarose gel
containing formaldehyde, and transferred to a Nytran membrane filter
(Schleicher and Schuell, Keene, NH) (34). The RNA bound to the Nytran
filter was prehybridized at 68 C with buffer containing 6 x SSC,
0.5% SDS, 5 x Denhardt’s solution, 0.1 mg/ml sonicated salmon
DNA, and 10 mM EDTA for 1 h. The hybridization was
carried out at 68 C for 15 h in the same buffer with a
32P-labeled 1.5-kilobase (kb) human MMP-2 complementary DNA
(cDNA) probe (35) or a 32P-labeled 0.6-kb rabbit
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe, which was
labeled with an oligolabeling kit (Pharmacia Japan, Tokyo). The
membrane was washed with 0.2 x SSC containing 0.1% SDS and
exposed to x-ray film.
RT-PCR/Southern blot analysis
MMP-3 cDNA (336 bp) and GAPDH cDNA (613 bp) were obtained by
RT-PCR from total RNA of rabbit cartilage using pairs of
oligonucleotides: 5'-CTGGAGGTTTGATGAGAAGA-3' and
5'-CAGTTCATGCTCGAGATTCC-3' for MMP-3, and 5'-GTCAAGGCTGAGAACGGGAA-3'
and 5'-GCTTCACCACCTTCTTGATG-3' for GAPDH. These were synthesized based
on the nucleotide sequences of rabbit MMP-3 (36) and rabbit and human
GAPDH cDNAs (37), respectively. The PCR products were subcloned into
pCRII vector (Stratagene, La Jolla, CA) and identified by sequencing by
means of dideoxy chain termination (38).
The MMP-3 and GAPDH messenger RNA (mRNA) levels were estimated by RT-PCR/Southern blotting using the pairs of primers. The PCR products (25 and 28 cycles for MMP-3 and GAPDH, respectively) were separated on a 1% agarose gel, then transferred to Nytran. The MMP-3 and GAPDH cDNAs were labeled with an oligolabeling kit (Pharmacia Japan, Tokyo, Japan). Hybridization proceeded under the same conditions as those described above.
Gelatinolytic and stromelysin activities in conditioned
media
Chondrocytes in 16-mm wells were incubated with 0.5 ml MEM
supplemented with various concentrations of hormones or cytokines for
48 h. The assay for gelatinase was carried out using conditioned
media in the presence of 1 mM 4-aminophenylmercuric acetate
(an activator of latent MMPs) against heat-denatured
14C-acetylated collagen (type I) as a substrate, as
previously described (39). For the determination of stromelysin
activity, the media were preincubated for 20 h with 1.5
mM 4-aminophenylmercuric acetate at 37 C. The stromelysin
activity was then measured using reduced and carboxymethylated
[3H]transferrin as a substrate, as previously described
(40). MMP-3, but not the other MMPs tested, degraded transferrin (40).
These assays were performed in the presence of 2 mM
phenylmethylsulfonylfluoride and 5 mM
N-ethylmaleimide to inhibit serine and cystein
proteinases. One unit of these enzymes degrades 1 µg substrate/min at
37 C.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, a and b). The growth plate and resting
cartilage equally reacted with antitype II collagen mAb (data not
shown). No staining was evident in the cartilage samples incubated with
control IgG (Fig. 1c).
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).
The protein in the hypertrophic zone (slice 1), the matrix-forming zone
(slice 2), and the proliferating zone (slice 3) of the growth plate as
well as the resting zone and articular cartilage was extracted with 4
M guanidine or Laemmli’s buffer. Immunoblotting analyses
of the guanidine extracts showed that although the level of pro-MMP-2
(66 kDa) was almost constant among the growth plate slices, it was much
higher in the growth plate than in the resting zone or articular
cartilage (Fig. 2a). The zymographical analyses of the SDS extracts
also showed that the level of pro-MMP-2 was much higher in the growth
plate than in the resting zone or articular cartilage (Fig. 2b). In
addition, Fig. 2b shows that pro-MMP-9 (92 kDa) was expressed in the
hypertrophic zone.
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).
Effects of PTH on MMP-2 expression by chondrocytes
The zymographical and immunoblotting analyses showed that in
triplicate cultures of rabbit growth plate chondrocytes, PTH increased
the secreted levels of 66-kDa pro-MMP-2 and 62-kDa MMP-2 (an active
form that is produced by partial proteolysis of pro-MMP-2) in the
absence of serum (Fig. 3, a and b). The
zymographical analyses did not provide quantitative data, but detected
both 66- and 62-kDa MMP-2. On the other hand, the immunoblotting
analyses did not detect 62-kDa MMP-2 at low levels. The lack of the
62-kDa band in some samples seems to be due to a low sensitivity of the
antibody (Fig. 3, b and c).
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Figure 3c shows that the effect of PTH on MMP-2 levels could be
observed at 10-9 M and reached a maximum at
10-8 M.
Figure 4a shows that PTH increased the
level of MMP-2 transcripts in the growth plate chondrocytes at 24 and
48 h.
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). (Bu)2cAMP also increased the protein
level of pro-MMP-2 (Fig. 5). However,
TGFß, BMP-2, bFGF, EGF, T3, 1,25-dihydroxyvitamin
D3, IL-1, IGF-I, and insulin did not increase the pro-MMP-2
level (Fig. 5).
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and 2b), we examined the effect of PTH on MMP-9 expression by
cultured chondrocytes on days 14 (the matrix-forming and early
hypertrophic stage) and 28 (the hypertrophic stage). We used
chondrocytes derived from the entire rib growth plate. Thus, the
initial cell population is heterogeneous. Nonetheless, these cells
reinitiate proliferation and are capable of recapitulating all stages
of differentiation in vitro (43). Previous studies have
shown that rabbit chondrocytes from the entire rib growth plate undergo
proliferation, matrix formation, and hypertrophy in vitro,
and that the cell changes proceed in the same order as those in the
growth plate in vivo (44). In these cultures, alkaline
phosphatase activity increases from day 12 and reaches a maximum on day
28 (44). In the present study, the cultures became confluent on day 7,
and the alkaline phosphatase activity on day 28 (2.6 U) was 3-fold
higher than that (0.9 U) on day 14. PTH induced 92-kDa MMP-9 in the
chondrocyte cultures on day 28 in all independent studies (Fig. 6; data not shown), but this effect of
PTH on MMP-9 was not always observed on day 14 (Fig. 3a; data not
shown). On the other hand, PTH induced MMP-2 expression on days 14 and
28 in all independent studies (Figs. 3a and 6; data not shown). In
another series of studies, PTH induced MMP-2, but not MMP-9, on day 10
before the induction of alkaline phosphatase (data not shown). These
findings suggest that PTH enhances MMP-2 synthesis throughout the
matrix-forming and hypertrophic stages and stimulates MMP-9 synthesis
after chondrocytes becoming hypertrophic.
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). In this experiment (Fig. 6), MMPs were
incubated in the gelatin-containing gels for a short time to stop the
reaction before saturation; thus, MMP-2 activity was hardly detectable
in the samples from the PTH-free cultures (lanes 1 and 3).
Effects of PTH-rp on MMP-2 and -9 levels
The zymographical analyses showed that PTH-rp-(1–84) and
PTH-rp-(1–141) as well as PTH-(1–84) increased the level of 92-kDa
MMP-9 in growth plate chondrocyte cultures (Fig. 7a). Our immunoblotting analyses showed
that these peptides increased the level of pro-MMP-2 (Fig. 7b).
However, PTH-rp-(15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) had little effect on the level of MMP-9 or
pro-MMP-2 (Fig. 7).
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).
In this assay, PTH also increased the MMP-2 level dose dependently
(Fig. 8a). Furthermore, PTH induced the expression of MMP-3 mRNA in
chondrocytes within 24 h (Fig. 8b).
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, open bars) or presence of
PTH (closed bars) or IL-1ß (hatched bars). The
gelatinase activity (upper panel) and stromelysin activity
(lower panel) released into the media were determined, using
14C-acetylated gelatin or reduced and carboxymethylated
[3H]transferrin as a substrate, respectively (39, 40).
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, upper panel). In the absence of PTH and IL-1,
stromelysin activity was undetectable on days 7, 14, and 28
(lower panel). The addition of PTH markedly enhanced the
production of gelatinase activity on days 14 and 28 (upper
panel). PTH also enhanced the production of stromelysin activity
on day 28 (lower panel). The effect of PTH on gelatinase
production was similar to that of IL-1ß (upper panel),
whereas PTH was less effective on stromelysin production than was
IL-1ß (lower panel).
In cultures of articular chondrocytes from the same rabbits, PTH had
little effect on the production of gelatinase (Fig. 9a, upper
panel) or stromelysin (Fig. 9a, lower panel), although
IL-1ß induced the production of both gelatinase and stromelysin by
the cells. The gelatin zymograms showed that PTH had little effect on
the level of MMP-2 or -9 in articular chondrocyte cultures, whereas
IL-1ß induced MMP-9 in the cultures (Fig. 9b). These findings
differentiated the action of PTH on MMPs from that of IL-1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Unlike other MMPs, MMP-2 is constitutively expressed in several tissues and is not usually induced by inflammatory stimuli (41, 48). In almost all evaluated cells, many growth factors and cytokines do not enhance MMP-2 synthesis even when they induce other MMPs in vitro (48, 49, 50, 51). Thus, MMP-2 may be involved in normal turnover of the extracellular matrix in several tissues. However, in a few cell types, TGFß and IL-1 enhance MMP-2 synthesis. TGFß stimulates MMP-2 synthesis in gingival fibroblasts and some tumor cells (50, 51), and IL-1 stimulates MMP-2 synthesis in glomerular mesangial cells (49). PTH induced MMP-2 in growth plate chondrocytes. These findings suggest that MMP-2 is involved in the remodeling of several tissues in some situations.
The action of PTH/PTH-rp on MMP may be critical, particularly for the control of cartilage matrix degradation during endochondral bone formation, because PTH/PTH-rp had no effect on the level of MMP-2, -3, or -9 in cultures of articular chondrocytes that did not undergo endochondral bone formation. The selective action of PTH/PTH-rp on growth plate chondrocytes is partly explained by the 10-fold increases in PTH/PTH-rp receptor (41,000 receptors/cell) (9) and its mRNA (25) levels in the growth plate relative to those in permanent cartilage.
If the PTH-rp stimulation of MMP synthesis observed in vitro
is relevant to matrix degradation in vivo, then
PTH-rp-depleted mice should show an abnormal accumulation of the matrix
in the growth plate. This is indeed what occurs. In the PTH-rp-depleted
growth plate, type II collagen is abnormally accumulated in the
hypertrophic zone. A fraction (40%) of the terminal chondrocytes
(alkaline phosphatase-producing cells) are unable to increase their
cell size, perhaps because of impaired degradation of the pericellular
matrix. The small terminal cells, but not the large ones, are
surrounded by an intact type II collagen matrix (17). In addition, the
chondrocyte lacunae in the PTH-rp-depleted growth plate become more
resistant to vascular invasion (17). These findings taken together with
our present observations suggest that PTH-rp promotes
chondrocyte-mediated matrix degradation during endochondral bone
formation in vivo.
IL-1 induces MMP-1, 3, and -9 in various chondrocytes (45, 52, 53), and this action of IL-1 is thought to be crucial for cartilage matrix degradation in arthritic cartilage. However, in contrast to PTH, IL-1 inhibits the proliferation (54) and differentiation (19, 53) of chondrocytes at all stages and has little effect on MMP-2 synthesis by growth plate chondrocytes. Thus, IL-1 may not be crucial for the cartilage resorption before ossification.
It is noteworthy that unlike PTH/PTH-rp, bFGF and 1,25-dihydroxyvitamin D3 had little effect on the MMP-2 level in growth plate chondrocyte cultures, although these compounds are as potent as PTH/PTH-rp in inhibiting chondrocyte hypertrophy in vitro and in vivo (6, 12, 28, 55). These findings suggest that PTH/PTH-rp and the other inhibitors of hypertrophy have different roles in the remodeling of the extracellular matrix during endochondral bone formation.
In growth plates, PTH/PTH-rp enhances DNA synthesis at the proliferative stage (16, 17), stimulates the syntheses of aggrecan (3) and type II collagen at the matrix-forming stage (56), and induces MMP-2, -3, and -9 at the hypertrophic stage. PTH-rp stimulation of the synthesis and degradation (turnover) of the cartilage matrix in the growth plate may facilitate bone elongation.
In conclusion, the findings in the present study showed that PTH/PTH-rp markedly enhances the expressions of MMP-2, -3, and -9 in maturing chondrocytes. This action of PTH/PTH-rp may be critical in the control of programed cartilage resorption during skeletal development and repair.
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Acknowledgments |
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Footnotes |
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2 These authors contributed equally to this work.
3 Present address: Structural Biology Center, AIST-National Institute
of Bioscience and Human Technology, Tsukuba).
Received October 27, 1997.
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