EPILOGUE
Triiodothyronine induces collagenase-3 and gelatinase B
expression in murine osteoblasts
Renata C.
Pereira1,3,
Vanda
Jorgetti3, and
Ernesto
Canalis1,2
1 Departments of Research and
Medicine, Saint Francis Hospital and Medical Center, Hartford 06105;
2 University of Connecticut School
of Medicine, Farmington, Connecticut 06030; and
3 Laboratorio de Fisiopatologia
Renal, Universidade de Sao Paulo, Sao Paulo, Brazil
01246-903
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ABSTRACT |
Triiodothyronine
(T3) increases bone resorption,
but its effects on matrix metalloprotease (MMP) expression in bone are
unknown. We tested the effects of
T3 on collagenase-3 and gelatinase
A and B expression in MC3T3 osteoblastic cells.
T3 at 1 nM to 1 µM for
24-72 h increased collagenase-3 and gelatinase B mRNA levels, but
it did not increase gelatinase A transcripts. In addition, T3 increased immunoreactive
collagenase and gelatinase activity. Cycloheximide prevented the
stimulatory effect of T3 on
collagenase-3 but not on gelatinase B transcripts. Indomethacin did not
prevent the effect of T3 on either
MMP. T3 did not alter the decay of collagenase-3 or gelatinase B mRNA in transcriptionally arrested MC3T3
cells, and it increased the rate of collagenase-3 and gelatinase B gene
transcription. Although T3
enhanced the expression of the tissue inhibitor of metalloproteinase-1
in MC3T3 cells, it increased collagen degradation in cultured intact
rat calvariae. In conclusion, T3 increases collagenase-3 and
gelatinase B synthesis in osteoblasts by transcriptional mechanisms.
This effect may contribute to the actions of
T3 on bone matrix remodeling.
bone remodeling; thyroid hormone; metalloprotease; collagen
degradation
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INTRODUCTION |
THYROID HORMONES have important effects on bone
metabolism and enhance bone remodeling. Thyroid hormones increase bone
resorption, although their major target cell in bone probably is a cell
of the osteoblastic lineage (6, 45, 59, 65). In fact, the presence of
osteoblasts is required to detect an increase in bone resorption (6).
Thyroid hormones also increase the replication of cells of the
osteoblastic lineage and suppress the differentiation of
osteoprogenitor cells to osteoblasts (13, 29, 47). Selected effects of
thyroid hormones on bone metabolism may be mediated by the production
of local factors, and triiodothyronine
(T3) enhances the synthesis of
prostaglandin E2 and of
insulin-like growth factor (IGF) I by skeletal cells (31, 55).
The administration of exogenous T3
to humans results in increased bone remodeling, and postmenopausal
females exposed to thyroid hormone excess display decreased bone
mineral density (BMD) of the spine and hip as well as of cortical bone
(1, 25, 37, 61). Some studies have failed to demonstrate marked changes in BMD, and there appears to be a relationship between the dose of
thyroid hormone administered and changes in BMD (4, 22, 23, 39, 61). In
addition to changes in BMD, patients with hyperthyroidism have
increased urinary excretion of pyridinoline cross-links, confirming the
stimulatory actions of thyroid hormone on bone remodeling and
suggesting an effect on collagen matrix breakdown (21, 24). This may
cause a decrease in a collagen matrix available for mineralization, and
in conjunction with an increase in bone resorption, it might explain
the decrease in BMD observed in these patients. Although it is tempting
to believe that the major actions of thyroid hormone are on bone
resorption, its direct effects on bone matrix degradation have not been
explored. Bone resorption is a process mediated by the osteoclast,
whereas bone matrix degradation is a process mediated by the secretion of proteases by the osteoblast. However, the two processes are closely
regulated, and agents that modify bone resorption frequently alter bone
collagen degradation by changing the expression of matrix
metalloproteinases (MMP) and their inhibitors (9, 48). Furthermore,
collagenase, an MMP, appears to play an indirect role in bone
resorption (26, 27). Consequently,
T3 might alter bone collagen
degradation and the expression of collagenase and related metalloproteases.
MMPs are a family of related proteolytic enzymes including
collagenases, gelatinases, and stromelysins (19, 41, 42). Collagenases
cleave fibrillar collagen at neutral pH and are considered important in
matrix remodeling. Three collagenases have been described: collagenase-1, secreted by stimulated human fibroblasts and osteoblasts and by human chondrocytes from osteoarthritic cartilage; collagenase-2, secreted by neutrophils; and collagenase-3, secreted by human breast
carcinoma cells, human chondrocytes, and rodent osteoblasts (19, 43,
38, 50, 52). Unstimulated normal human osteoblasts do not secrete
detectable levels of collagenase, although human osteosarcoma cell
lines synthesize collagenase, and normal human osteoblasts exposed to
parathyroid hormone and selected cytokines express the protease (52).
Normal rat osteoblasts and rat osteosarcoma cells express
collagenase-3, but rodent cells do not express collagenase-1 (48, 50).
Type II collagen is preferentially hydrolyzed by collagenase-3, and
collagenases-1, -2, and -3 degrade fibrillar type I collagen with
similar efficiency (32). Two gelatinases have been described:
gelatinase A, a 72-kDa MMP secreted by multiple cell types; and
gelatinase B, a 92-kDa MMP secreted by connective tissue cells,
monocytes, and various tumor cells (42). Gelatinase A and B are also
expressed by skeletal cells and are known to degrade elastin, collagens
IV, V, VII, X, and XI, and additional minor components of the
extracellular matrix (38, 42). Agents known to enhance bone resorption
increase the synthesis of gelatinases by the osteoblast (33, 38). These
observations suggest a role for collagenase-3 and gelatinases in
connective tissue remodeling in the skeleton, and we postulated that
T3 might regulate their expression
in osteoblasts.
In the present study, we examined the actions of
T3 on the expression of
collagenase-3, gelatinase A and B, and on tissue inhibitors of MMPs
(TIMP)-1, -2, and -3 in cultures of osteoblastic MC3T3 cells and
determined possible consequences in cultures of intact rat calvariae.
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MATERIALS AND METHODS |
Culture models. MC3T3 cells, clonal
mouse osteoblastic cells derived from newborn mouse calvariae, were
plated at a density of 8,000-12,000
cells/cm2 and cultured in a
humidified 5% CO2 incubator at
37°C until reaching confluence (~50,000
cells/cm2; Ref. 57). Cells were
cultured in
-modified Eagle's medium (Life Technologies, Grand
Island, NY) supplemented with 10% fetal bovine serum (Summit
Biotechnology, Fort Collins, CO). MC3T3 cells were grown to confluence,
transferred to serum-free medium for 20 to 24 h, and exposed to test or
control medium in the absence of serum for 2-72 h as indicated in
the text and Figs. 1-11;
3,3',5'-triiodo-L-thyronine sodium salt (Sigma, St. Louis, MO) was added directly to the culture medium. Cycloheximide, 5,6-dichlorobenzimidazole riboside (DRB), and
indomethacin (Sigma) were dissolved in ethanol and diluted 1:200 and
1:1,000, respectively, in
-modified Eagle's medium. An equal amount
of solvent was added to control cultures. In experiments lasting longer
than 24 h, the culture medium was replaced with fresh solutions every
24 h. At the end of the incubation, the medium was harvested in the
presence of 0.1% polyoxyethylenesorbitan monolaurate (Tween,
Pierce, Rockford, IL) for Western blot analyses and in its
absence for gelatin zymograms and then was stored at
80°C
before analysis. The cell layer was extracted for RNA analysis and
stored at
80°C, and nuclei were obtained by Dounce
homogenization for nuclear run-on assays.
Cultures of intact rat parietal bones were used to determine changes on
collagen degradation, because of the greater accumulation of
collagenous protein in the matrix of intact bone than in that of
monolayer cultures (54). Parietal bones were obtained from 22-day-old
fetal rats immediately after the mothers were killed by blunt trauma to
the nuchal area. The project was approved by the
Institutional Animal Care and Use Committee of Saint Francis Hospital
and Medical Center. Half-calvariae were cultured in flasks containing
Biggers, Gwatkin, and Judak medium in the absence of serum. The flasks
were gassed with 5% CO2, sealed
and placed in a shaking water bath at 37°C for a 24-h period, and
labeled with 5 µCi/ml of
[2,3-3H]proline
(specific activity of 40 Ci/mmol; Du Pont, Wilmington, DE). Bones were transferred to control or test medium in
10 mM proline for a 24- to 72-h "chase period." Calvariae and
corresponding culture medium samples were obtained for
[3H]hydroxyproline
analysis (54).
Northern blot analysis. Total cellular
RNA was isolated by RNeasy kit per instructions of the manufacturer
(Qiagen, Chatsworth, CA). The RNA recovered was quantitated by
spectrophotometry, and equal amounts of RNA from control or test
samples were loaded on a formaldehyde-agarose gel after denaturation.
The gel was stained with ethidium bromide to visualize RNA standards
and ribosomal RNA, before and after transfer, documenting equal RNA
loading of the samples. The RNA was blotted onto GeneScreen Plus
charged nylon (Du Pont). Restriction fragments containing a 2.6-kb rat interstitial collagenase-3 cDNA (kindly provided by Cheryl Quinn, St.
Louis, MO), a 1.1-kb human gelatinase A cDNA (American Type Culture
Collection, Rockville, MD), a 1.4-kb murine gelatinase B cDNA (kindly
provided by Ghislain Opdenakker, Leuven, Belgium), an 825-bp murine
TIMP-1 cDNA, a 700-bp murine TIMP-2 cDNA, a 750-bp murine TIMP-3 cDNA
(all TIMP cDNAs kindly provided by Dylan Edwards, Calgary, Alberta,
Canada), and an 800-bp rat glyceraldehyde-3-phosphate dehydrogenase
(GAPD) cDNA (kindly provided by Ray Wu, Ithaca, NY) were labeled with
[
-32P]dCTP and
[
-32P]dATP
(specific activity of 3,000 Ci/mmol; Du Pont) with the random
hexanucleotide primed second strand synthesis method (12, 14, 35, 36,
40, 50, 60). Hybridizations were carried out at 42°C for 16-72
h. Posthybridization washes were performed in 1× saline sodium
citrate (SSC) at 65°C for collagenase-3 and TIMP-1, -2, and -3 cDNAs in 0.2× SSC at 65°C for gelatinase A and B and in
0.5× SSC at 65°C for GAPD cDNA. The bound radioactive material was visualized by autoradiography on Kodak X-AR5 or Biomax film (Eastman Kodak, Rochester, NY) or Du Pont reflection film employing intensifying screens. Relative hybridization levels were
determined by densitometry. Northern analyses shown are representative of three or more cultures.
Nuclear run-on assay. To examine
changes in the rate of transcription, nuclei were isolated by Dounce
homogenization in a Tris buffer containing 0.5% Nonidet P-40. Nascent
transcripts were labeled by incubation of nuclei in a reaction buffer
containing 500 µM each of ATP, CTP, and guanidine triphosphate, 150 U
of RNasin (Promega), and 250 µCi of
[
-32P]uridine
triphosphate (specific activity of 3,000 Ci/mmol; Du Pont; Ref. 3). RNA
was isolated by treatment with DNase I and proteinase K, followed by
phenol-chloroform extraction and ethanol precipitation. Linearized
plasmid DNA containing ~1 µg of cDNA was immobilized onto
GeneScreen Plus by slot blotting according to the directions of the
manufacturer (Du Pont). The plasmid vector pUC18 (Life Technologies)
was used as a control for nonspecific hybridization, and GAPD cDNA was
used to estimate uniformity of radioactive counts applied to the
membrane. Equal counts per minute of
32P-labeled RNA from
each sample were hybridized to cDNAs with the same conditions as for
Northern blot analysis and were visualized by autoradiography.
Western immunoblot analysis. Medium
samples were fractionated by polyacrylamide gel electrophoresis with
denaturing and nonreducing conditions and were transferred onto
Immobilon P membranes (Millipore, Bedford, MA). After being blocked
with 2% BSA, the membranes were exposed to a 1:1,000 dilution of
rabbit antiserum raised against rat collagenase-3 (kindly provided by
John Jeffrey, Albany, NY), previously characterized for specificity and
immunoreactivity, followed by the addition of goat anti-rabbit IgG
conjugated to horseradish peroxidase (28). The blots were washed and
developed with a horseradish peroxidase chemiluminescence detection
reagent (Du Pont), visualized by autoradiography on Du Pont reflection film employing reflection intensifying screens, and analyzed by densitometry. Data shown are representative of three cultures.
Gelatin zymogram. To assess gelatinase
activity, aliquots of conditioned medium were mixed with sample buffer
containing 2% sodium dodecyl sulfate. Samples were loaded on a 7.5%
polyacrylamide gel containing 1 mg/ml of gelatin and fractionated by
electrophoresis as described (33). The gels were incubated 1 h in 50 mM
Tris · HCl, 5 mM
CaCl2, and 1 µM
ZnCl2 buffer (pH 7.5) containing
2.5% Triton X-100 (Sigma). Gels were incubated in the same buffer
without Triton X-100 at 37°C overnight. Proteolytic activity was
visualized by staining the gels with 0.1% Coomassie blue in 50%
methanol and 20% acetic acid and destaining with 30% methanol and 1%
formic acid. Data shown are representative of three cultures.
Collagen degradation assay. Calvariae
were homogenized in 0.5 M acetic acid, and aliquots of the homogenate
and the respective culture medium were hydrolyzed in vacuo in 6 N HCl
at 107°C for 24 h. The samples were derivatized with
phenylisothiocyanate, and
[3H]proline and
[3H]hydroxyproline
were separated by reverse-phase HPLC with a
C18 Nova-Pak column and an
acetonitrile solvent system, as previously described (54). Although the
method used to hydrolyze collagen from calvariae and medium samples may
result in some destruction of hydroxyproline, this effect is minimal
because it achieves nearly 100% recovery of hydroxyproline (54).
[3H]hydroxyproline was
measured in calvaria and medium samples by liquid scintillation counting.
Statistical methods. Data on
collagenase-3 and gelatinase B mRNA and protease levels in MC3T3 cells
and collagen degradation levels in calvaria are expressed as means ± SE. Statistical differences were calculated by ANOVA, and post
hoc examination was performed by the Ryan-Einot-Gabriel-Welch
F test (64). Data on collagenase-3 and
gelatinase B mRNA decay were analyzed by linear regression, and the
slopes of the regression lines obtained for control and treated cells
were compared for significant differences with the method of Sokal and
Rohlf (56).
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RESULTS |
Northern blot analysis of total RNA from MC3T3 cells revealed a
collagenase-3 transcript of 2.9 kb (Fig.
1). Continuous treatment of MC3T3 cells
with T3 at 10 nM caused a
time-dependent increase in collagenase steady-state transcripts. No
stimulation was observed after 2 or 6 h (not shown) of treatment with
T3, whereas at 24 h collagenase-3
mRNA levels were increased by (means ± SE;
n = 6) 2.8 ± 0.3-fold. The effects
were maximal after 48 h and sustained for 72 h, when
T3 increased collagenase
transcripts by 9.8 ± 2.7- and 11.3 ± 0.8-fold,
respectively (Fig. 1). The effect of
T3 was dose dependent, and
continued exposure of MC3T3 cells to
T3 at 1-1,000 nM for 72 h
increased collagenase transcripts by 3.7- to 11.3-fold (Fig.
2). T3
at 10 nM for 24, 48, or 72 h increased the levels of immunoreactive
interstitial collagenase-3 in the culture medium of MC3T3 cells by
(means ± SE; n = 3) 1.6 ± 0.3, 6.1 ± 1.1, and 4.5 ± 1.2, respectively (Fig.
3). Collagenase-3 was identified by
comigration with a purified rat procollagenase-3 standard.

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Fig. 1.
Effect of triiodothyronine (T3)
at 10 nM on collagenase-3 [matrix metalloprotease (MMP)-13]
mRNA levels in cultures of MC3T3 cells treated for 24, 48, or 72 h.
Total RNA from control (-) or
T3-treated (+) cultures was
subjected to Northern blot analysis and hybridized with an
-32P-labeled collagenase-3
cDNA. Blot was stripped and rehybridized with an
-32P-labeled
glyceraldehyde-3-phosphate dehydrogenase (GAPD) cDNA. Collagenase-3
transcripts of 2.9 kb (top) and GAPD
mRNA (bottom) were visualized by
autoradiography.
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Fig. 2.
Effect of T3 at 1-1,000 nM on
collagenase-3 (MMP-13) mRNA levels in cultures of MC3T3 cells treated
for 72 h. Total RNA from control or
T3-treated cultures was subjected
to Northern blot analysis and hybridized with an
-32P-labeled collagenase-3
cDNA. Blot was stripped and rehybridized with an
-32P-labeled GAPD cDNA.
Inset, autoradiographic visualization
of collagenase-3 (top) and GAPD
transcripts (bottom). Graph shows
densitometric analysis of collagenase-3 mRNA levels. Values are
treated-to-control ratios and are means ± SE for 3-6
observations.
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Fig. 3.
Effect of T3 at 10 nM on
procollagenase-3 secretion in MC3T3 cell cultures treated for 24, 48, or 72 h. Western blot analysis was performed with equal amounts of
culture medium from control (-) or
T3-treated (+) cultures.
Procollagenase (MMP) was detected with a rabbit anti-rat collagenase-3
antibody and a horseradish peroxidase chemiluminescence detection
system, and its migration is indicated by arrow on
right. Molecular mass markers
(left) are in
kDa.
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To determine whether the effect of
T3 was specific for collagenase-3,
we examined its actions on other MMPs in MC3T3 cell cultures.
T3 at 10-1,000 nM for 24, 48, or 72 h did not cause a detectable change in gelatinase A mRNA levels
in MC3T3 cells (not shown). In contrast,
T3 caused a time- and
dose-dependent increase on gelatinase B mRNA levels. MC3T3 cells
expressed 2.5- and 3.2-kb gelatinase B transcripts and
T3 at 10 nM increased gelatinase B
mRNA levels by (means ± SE; n = 6) 2.4 ± 0.2-, 3.5 ± 0.2-, and 4.3 ± 0.3-fold after 24, 48, and 72 h,
respectively (Fig. 4). There was no
apparent increase after 2 or 6 h (not shown). The stimulatory effect on
gelatinase B was dose dependent and was observed at
T3 concentrations of 1-1,000
nM, which increased gelatinase B mRNA by 2- to 5-fold after 72 h (Fig.
5). Conditioned medium from control and
T3-treated MC3T3 cell cultures
contained gelatinase activity migrating with approximate molecular
masses of 68 and 88 kDa (Fig. 6).
Incubation of conditioned medium with 4 p-aminophenylmercuric
acetate for 48 h resulted in the disappearance of the
88-kDa form and an increase in the 68-kDa form of the enzyme (not
shown). This indicates that the predominant gelatinolytic activity
corresponded to gelatinase B, which generates a 68-kDa form after
activation, and not to gelatinase A, which generates a 60-kDa active
form and is fully degraded by
p-aminophenylmercuric acetate after 48 h (44). T3 at 10 nM increased
gelatinase B activity after 48 and 72 h (Fig. 6).

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Fig. 4.
Effect of T3 at 10 nM on
gelatinase B (MMP-9) mRNA levels in cultures of MC3T3 cells treated for
24, 48, and 72 h. Total RNA from control (-) or
T3-treated (+) cultures was
subjected to Northern blot analysis and hybridized with an
-32P-labeled gelatinase B cDNA.
Blot was stripped and rehybridized with an
-32P-labeled GAPD cDNA.
Gelatinase B transcripts of 2.5 and 3.2 kb
(top) and GAPD mRNA
(bottom) were visualized by
autoradiography.
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Fig. 5.
Effect of T3 at 1-1,000 nM on
gelatinase B (MMP-9) mRNA levels in cultures of MC3T3 cells treated for
72 h. Total RNA from control or
T3-treated cultures was subjected
to Northern blot analysis and hybridized with an
-32P-labeled gelatinase B cDNA.
Blot was stripped and rehybridized with an
-32P-labeled GAPD cDNA.
Inset, autoradiographic visualization
of gelatinase B transcripts of 2.5 and 3.2 kb
(top) and GAPD mRNA
(bottom). Graph shows densitometric
analysis of gelatinase B mRNA levels. Values are treated-to-control
ratios and are means ± SE for 3 observations.
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Fig. 6.
Effect of T3 10 nM on gelatinase
activity secreted by MC3T3 cell cultures treated for 24, 48, or 72 h.
Gelatin zymogram was performed with equal amounts of culture medium
from control (-) or T3-treated (+)
cultures. Migration of protease activity in kDa is indicated on
left.
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To determine whether the effect of
T3 on collagenase-3 and gelatinase
B mRNA levels was dependent on protein synthesis, confluent cultures of
MC3T3 cells were treated with T3
in the presence or absence of cycloheximide at doses known to inhibit
protein synthesis (8). Cycloheximide at 7.2 µM decreased the basal
expression and the stimulatory effect of
T3 on collagenase-3 (Fig.
7) but not on gelatinase B mRNA (not
shown). To determine whether the effects of
T3 were due to changes in
prostaglandin synthesis, T3 was
tested in the presence and absence of indomethacin at 10 µM.
Indomethacin did not modify the basal expression of collagenase-3 or
gelatinase B transcripts in MC3T3 cell cultures and did not prevent the
stimulatory effects of T3,
indicating that they were independent of
T3 action on prostaglandin
synthesis (not shown).

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Fig. 7.
Effect of T3 at 10 nM in the
presence or absence of cycloheximide (Cx) at 7.2 µM on collagenase-3
(MMP-13) mRNA levels in cultures of MC3T3 cells treated for 24 h. Total
RNA from control (-) or treated (+) cultures was subjected to Northern
blot analysis and hybridized with an
-32P-labeled collagenase-3
cDNA. Blot was stripped and rehybridized with an
-32P-labeled GAPD cDNA.
Collagenase-3 transcripts of 2.9 kb
(top) and GAPD mRNA
(bottom) were visualized by
autoradiography.
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To determine whether the effects of
T3 on collagenase-3 and gelatinase
B mRNA levels were due to changes in transcript stability, MC3T3 cells
were exposed to control or
T3-containing medium for 24 h and
then treated with the RNA polymerase II inhibitor DRB at 75 µM in the
presence or absence of T3 for 24 h
(66). The half-lives of collagenase-3 and gelatinase B mRNAs in
transcriptionally arrested MC3T3 cells were 16 and 20 h, respectively,
in control cultures (Fig. 8). Slope
analysis indicated that T3 did not
change the stability of collagenase-3 or gelatinase B mRNA. To confirm that T3 modified collagenase-3 and
gelatinase B mRNA by transcriptional mechanisms, the rate of gene
transcription was measured by nuclear run-on assay.
T3 at 10 nM increased the
transcriptional rate of the collagenase-3 gene by 13-fold and the rate
of the gelatinase B gene by 2.5-fold after 48 h (Fig.
9).


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Fig. 8.
Effect of T3 at 10 nM on
collagenase-3 (MMP-13; A) and
gelatinase B (MMP-9; B) mRNA decay
in MC3T3 cells. Confluent cultures of MC3T3 cells were serum deprived
and exposed to control or
T3-containing medium for 24 h
before the addition of 5,6-dichlorobenzimidazole riboside (DRB) at 75 µM. Total RNA, obtained 0-24 h after DRB or
T3 and DRB addition, was subjected
to Northern blot analysis and hybridized with an
-32P-labeled collagenase-3 or
gelatinase B cDNA. Collagenase-3 and gelatinase B mRNA was visualized
by autoradiography and quantitated by densitometry. Data from control
( ) and T3-treated ( ) cells
are means ± SE for 6 cultures and are a percentage of mRNA levels
present before addition of DRB.
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Fig. 9.
Effect of T3 at 10 nM on
collagenase-3 (MMP-13) and gelatinase B (MMP-9) transcription rates in
cultures of MC3T3 cells treated for 48 h. Nascent transcripts from
control (-) or T3-treated (+)
cultures were labeled in vitro with
[ -32P]uridine
triphosphate, and the labeled RNA was hybridized to immobilized cDNA
for collagenase-3 and gelatinase B. GAPD cDNA was used to demonstrate
uniformity of radioactive sample applied, and pUC18 vector DNA was used
as a control for nonspecific hybridization. Transcripts were visualized
by autoradiography.
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MC3T3, like rat Ob, cells expressed a TIMP-1 transcript of 0.9 kb, two
TIMP-2 transcripts of 1.0 and 3.5 kb, respectively, and two TIMP-3
transcripts of 2.5 and 4.5 kb, respectively (7). T3 at 10 nM increased the
expression of TIMP-1 by (means ± SE; n = 3) 2.5 ± 0.3 to 3.0 ± 0.2 after 24-72 h (Fig. 10). This effect was of smaller magnitude than the effect of
T3 on collagenase mRNA.
Furthermore, T3 at 10 nM for
24-72 h did not modify the expression of TIMP-2 or TIMP-3 (not
shown).

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Fig. 10.
Effect of T3 at 10 nM on tissue
inhibitor of metalloproteinase (TIMP-1) mRNA expression in cultures
of MC3T3 cells treated for 24, 48, or 72 h. Total RNA from control
(-) or T3-treated (+) cultures was
subjected to Northern blot analysis and hybridized with an
-32P-labeled murine TIMP-1
cDNA. Blot was stripped and rehybridized with an
-32P-labeled GAPD cDNA. TIMP-1
transcripts of 0.9 kb (top) and GAPD
mRNA (bottom) were visualized by
autoradiography.
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Collagen degradation rates, examined during a "pulse chase"
experiment, revealed that T3 at 10 nM for 72 h increased the amount of
[3H]hydroxyproline
released by intact calvariae to the culture medium by 1.6-fold (Fig.
11). Nearly 100% of the
[3H]hydroxyproline
detected in the culture medium is acid soluble, suggesting that it
represents degraded collagen (54). Over a 72-h period, the amount of
newly synthesized collagen released from bone to culture medium in
control cultures was ~29%, and it was increased by 1.5-fold by
T3 at 10 nM (Fig. 11).

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Fig. 11.
Effect of T3 on collagen
degradation. Intact rat calvariae were cultured for 24 h in the absence
of unlabeled proline and in the presence of
[3H]proline (5 µCi/ml), rinsed and transferred to medium containing 10 mM proline,
and "chased" in absence ( ) or presence ( ) of
T3 at 10 nM for indicated periods
of time. Symbols and vertical lines are means ± SE for 5-6
half-calvariae. Data are picomoles of total
[3H]hydroxyproline
released to the culture medium (A)
or as a percentage of newly synthesized
[3H]hydroxyproline
released to culture medium (B).
* Significantly different from control,
P < 0.05.
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DISCUSSION |
The present investigation was undertaken to determine whether
T3 regulates the expression of
selected MMPs and TIMP-1, -2, and -3 in cultures of osteoblastic MC3T3
cells. T3 caused a time- and
dose-dependent stimulation of collagenase-3 mRNA and protease levels.
T3 also increased the expression
of gelatinase B transcripts and activity without modifying gelatinase A
mRNA levels. Although T3 increased
TIMP-1 mRNA levels, the effect was modest in relation to the effect on
MMPs, and T3 did not stimulate the
expression of TIMP-2 or 3. Because TIMPs bind MMPs in a 1:1
stoichiometric fashion, our results suggest that the actions of the
increased collagenase-3 and gelatinase B will remain unopposed by the
limited expression of TIMPs in the bone microenvironment (32). This is
further supported by the demonstration of an induction of active gelatinase B by T3, as detected by
zymography. The stimulatory effect of
T3 on collagenase and gelatinase B
may be relevant to its stimulatory actions on bone resorption and
probably explains the increase in bone collagen degradation observed in
intact calvariae (27). However, it is important to note that this is a
different model, and intact calvaraie contain not only osteoblasts but
a mixed cell population and a structured matrix. Therefore, other factors in addition to the induction of collagenase-3 and gelatinase B
may play a role in the collagen degradation induced by
T3 in this model. The effect of
T3 on collagenase transcripts was
dependent on de novo protein synthesis, whereas that on gelatinase B
was not, indicating that different mechanisms regulate the expression of the two MMPs by T3. Although
T3 is known to induce
prostaglandin E2 in osteoblasts
and prostaglandins are known to enhance collagenase synthesis, we found
that the stimulation of collagenase-3 and gelatinase B by
T3 in MC3T3 cells was not
dependent on prostaglandin synthesis (10, 31). This is in agreement
with the actions of T3 on bone
resorption, which can be independent of prostaglandin synthesis (31).
T3 did not alter collagenase-3 or
gelatinase B mRNA stability in transcriptionally arrested Ob
cells but did increase the rate of transcription of the
collagenase-3 and gelatinase B genes. These results indicate that
T3 stimulates rat collagenase-3 and gelatinase B expression by transcriptional mechanisms. The effect
of T3 on gelatinase B expression
is not surprising because other agents known to induce collagenase-3
synthesis, such as interleukin-1 and -6, also increase gelatinase A or
B production by skeletal cells (18, 38).
T3 induces the transcription of
the collagenase-3 and gelatinase B gene in osteoblasts, but the gene
sequences responsible for the effects have not been determined. In
nonskeletal cells, T3 activation
of other genes involves a T3
response element (TRE; Refs. 5, 30). The traditional half-site contains
the sequence 5'-AGGTCA-3', although optimal binding of
T3 receptors may require additional sequences and the formation of a heterodimeric and not of a
homodimeric complex. Furthermore, retinoic acid receptor-binding motifs
may play a role in T3-receptor
binding (46). Examination of the human collagenase-3 gene revealed four
TRE consensus sequences in the region of bp
1,550 to
590
(58). The sequence of the rat collagenase-3 gene is known from bp
456 to +62 and that of the murine gene is not known (51). It is
possible that rodent collagenase-3 genes contain TREs in a location
similar to that described for the human gene that are responsible for
the transcriptional effects observed. However, the exact elements
responsible for the T3 effects are
not known. T3 receptors may also
act by regulating activator protein-1 or -2 sequences, present in the
collagenase-3 gene, as it has been reported for the activation and
suppression of other genes (20, 49). Examination of the gelatinase B
gene promoter reveals the presence of a TRE and activator protein-1 and
-2 binding sequences, which might be responsible for the effect of
T3 (53). Additional studies will
be required to define the exact elements responsible for the induction
of collagenase-3 and gelatinase B by
T3.
The synthesis of collagenase-1 and -3 by human and rat osteoblasts is
regulated by systemic hormones and by cytokines present in the bone
microenvironment. Consequently, the apparent constitutive level of
collagenase expression by the osteoblast is in fine balance and depends
on the exposure of the cell to factors that stimulate and factors that
inhibit collagenase synthesis (7, 10, 11, 17, 48, 58, 62, 63). It is
possible that T3 interacts with
other cytokines, such as interleukin-1 or -6, to enhance collagenase-3
expression as it has been reported for the effects of
T3 on bone resorption (59).
Because the effect of T3 on MMP expression is slow in onset, we tested whether or not
T3 induced the expression of local
factors known to stimulate collagenase expression in osteoblasts.
Treatment of MC3T3 cells with T3
at 10 nM for 2-72 h did not increase the expression of
platelet-derived growth factor BB, fibroblast growth factor 2, IL-1, or
IL-6, all known stimulators of collagenase-3 expression (Pereira and
Canalis, unpublished observations; Refs. 10, 17, 33, 62, 63). The
stimulatory effect of T3 on bone
resorption requires the presence of osteoblasts and depends on
receptors expressed by osteoblastic cells (6, 65). Consequently, it is
not surprising that T3 regulates
MMP expression in osteoblasts. T3
was tested at concentrations of 1 nM and higher because previous
studies from other investigators revealed that
T3 was not effective in cultures
of intact calvariae, long bones, and cells of the osteoblastic lineage
at lower doses (31, 45, 47, 49). It is possible that under the
conditions used in these experiments, concentrations lower than 1 nM
could have been effective, although they were not tested. The effects of T3 on MMP expression reported
here were observed at 1 nM, and this concentration is ~200-fold
higher than the concentrations of free
T3 found in the serum of normal
humans and 50- to 100-fold higher than the concentrations found in
patients with hyperthyroidism (34). The effect of
T3 on collagenase and gelatinase
expression reported here probably explains the increase in bone
collagen degradation that we observed in rat calvariae and may be
relevant to its stimulatory actions on bone resorption reported
previously (6, 45, 59). It may also explain the increased bone
remodeling and increased urinary excretion of collagen cross-links in
vivo after thyroid hormone administration and be important in the
pathogenesis of the osteopenia after thyroid hormone excess (4, 21,
24). The actions of T3 on MMP
expression could have additional effects in bone metabolism because
collagenase was recently implied in the fragmentation of IGF-binding
protein-5, a binding protein with known stimulatory effects on bone
formation (2, 14, 15, 16). Although the activity of these fragments
remains to be defined, their appearance in conjunction with an increase in IGF-I might be relevant to selected anabolic actions of
T3 in the skeleton (29, 55).
In conclusion, the present studies demonstrate that
T3 increases collagenase-3 and
gelatinase B expression in osteoblasts by transcriptional mechanisms.
It is probable that these effects play a role in the degradation of the
collagen matrix and in the osteopenia observed in conditions of thyroid
hormone excess.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Cheryl Quinn for the rat collagenase cDNA, Dr.
Ghislain Opdenakker for the murine gelatinase B cDNA, Dr. Dylan Edwards
for the murine TIMP-1, -2 and -3 cDNAs, Dr. Ray Wu for GAPD cDNA, and
Dr. John Jeffrey for the rat interstitial collagenase antibody. We also
thank Cathy Boucher, Deena Durant, and Sheila Rydziel for technical
assistance and Charlene Gobeli and Margaret Nagle for secretarial help.
 |
FOOTNOTES |
This work was supported by Grant AR-21707 from the National Institute
of Arthritis and Musculoskeletal and Skin Diseases. Renata C. Pereira
is a recipient of a Fundaç
o Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior Student
Fellowship Grant from the Universidade de Sao Paulo.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: E. Canalis,
Dept. of Research, Saint Francis Hospital and Medical Center, 114 Woodland St., Hartford, CT 06105-1299.
Received 12 February 1999; accepted in final form 29 April 1999.
 |
REFERENCES |
1.
Adlin, E. V.,
A. H. Maurer,
A. D. Marks,
and
B. J. Channick.
Bone mineral density in postmenopausal women treated with L-thyroxine.
Am. J. Med.
90:
360-366,
1991[Medline].
2.
Andress, D. L,
and
R. S. Birnbaum.
Human osteoblast-derived insulin-like growth factor (IGF) binding protein-5 stimulates osteoblast mitogenesis and potentiates IGF action.
J. Biol. Chem.
267:
22467-22472,
1992[Abstract/Free Full Text].
3.
Ausubel, F. M.,
R. Brent,
R. E. Kingston,
D. D. Moore,
J. G. Siedman,
J. A. Smith,
and
K. Struhl.
Current Protocols in Molecular Biology. New York: John Wiley & Sons, 1995, 4.10.1-4.10.10.
4.
Baran, D. T.
Detrimental skeletal effects of thyrotropin suppressive doses of thyroxine: fact or fantasy?
J. Clin. Endocrinol. Metab.
78:
816-817,
1994[Medline].
5.
Brent, G. A.
The molecular basis of thyroid hormone action.
N. Engl. J. Med.
331:
847-853,
1994[Free Full Text].
6.
Britto, J. M.,
A. J. Fenton,
W. R. Holloway,
and
G. C. Nicholson.
Osteoblasts mediate thyroid hormone stimulation of osteoclastic bone resorption.
Endocrinology
134:
169-176,
1994[Abstract].
7.
Canalis, E.,
S. Rydziel,
A. Delany,
S. Varghese,
and
J. Jeffrey.
Insulin-like growth factors inhibit interstitial collagenase synthesis in bone cell cultures.
Endocrinology
136:
1348-1354,
1995[Abstract].
8.
Centrella, M.,
T. L. McCarthy,
and
E. Canalis.
Glucocorticoid regulation of transforming growth factor
, (TGF
) activity and binding in osteoblast-enriched cultures from fetal rat bone.
Mol. Cell. Biol.
11:
4490-4496,
1991[Medline].
9.
Civitelli, R.,
K. A. Hruska,
J. J. Jeffrey,
A. J. Kahn,
L. V. Avioli,
and
N. C. Partridge.
Second messenger signaling in the regulation of collagenase production by osteogenic sarcoma cells.
Endocrinology
124:
2928-2934,
1989[Abstract].
10.
Clohisy, J. C.,
T. J. Connolly,
K. D. Bergman,
C. O. Quinn,
and
N. C. Partridge.
Prostanoid-induced expression of matrix metalloproteinase-1 messenger ribonucleic acid in rat osteosarcoma cells.
Endocrinology
135:
1447-1454,
1994[Abstract].
11.
Delany, A. M.,
S. Rydziel,
and
E. Canalis.
Autocrine down-regulation of collagenase-3 in rat bone cell cultures by insulin-like growth factors.
Endocrinology
137:
4665-4670,
1996[Abstract].
12.
Edwards, D. R.,
P. Waterhouse,
M. L. Holman,
and
D. T. Denhardt.
A growth-responsive gene (16C8) in normal mouse fibroblasts homologous to a human collagenase inhibitor with erythroid-potentiating activity: evidence for inducible and constitutive transcripts.
Nucleic Acids Res.
14:
8863-8878,
1986[Abstract].
13.
Ernst, M.,
and
E. R. Froesch.
Triiodothyronine stimulates proliferation of osteoblast-like cells in serum-free culture.
FEBS Lett.
220:
163-166,
1987[Medline].
14.
Feinberg, A.,
and
B. Vogelstein.
A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.
Anal. Biochem.
137:
266-267,
1984[Medline].
15.
Fowlkes, J. L.,
K. M. Thrailkill,
D. M. Serra,
K. Suzuki,
and
H. Nagase.
Matrix metalloproteinases as insulin-like growth factor binding protein-degrading proteinases.
Prog. Growth Factor Res.
6:
255-263,
1995[Medline].
16.
Franchimont, N.,
D. Durant,
and
E. Canalis.
Interleukin-6 and its soluble receptor regulate the expression of insulin-like growth factor binding protein-5 in osteoblast cultures.
Endocrinology
138:
3380-3886,
1997[Abstract/Free Full Text].
17.
Franchimont, N.,
S. Rydziel,
A. M. Delany,
and
E. Canalis.
Interleukin-6 and its soluble receptor cause a marked induction of collagenase 3 expression in rat osteoblast cultures.
J. Biol. Chem.
272:
12144-12150,
1997[Abstract/Free Full Text].
18.
Franchimont, N.,
S. Rydziel,
and
E. Canalis.
Interleukin-6 is autoregulated by transcriptional mechanisms in cultures of rat osteoblastic cells.
J. Clin. Invest.
100:
1797-1803,
1997[Abstract/Free Full Text].
19.
Freije, J. M. P.,
I. Diez-Itza,
M. Balbin,
L. M. Sanchez,
R. Blasco,
J. Tolivia,
and
C. Lopez-Otin.
Molecular cloning and expression of collagenase 3, a novel human matrix metalloproteinase produced by breast carcinomas.
J. Biol. Chem.
269:
16766-16773,
1994[Abstract/Free Full Text].
20.
French, R. P.,
D. Warshawsky,
L. Tybor,
N. D. Mylniczenko,
and
L. Miller.
Upregulation of AP-2 in the skin of Xenopus laevis during thyroid hormone-induced metamorphosis.
Dev. Genet.
15:
356-365,
1994[Medline].
21.
Garnero, P.,
V. Vassy,
A. Bertholin,
J. P. Riou,
and
P. D. Delmas.
Markers of bone turnover in hyperthyroidism and the effects of treatment.
J. Clin. Endocrinol. Metab.
78:
955-959,
1994[Abstract].
22.
Grant, D. J.,
J. E. McMurdo,
P. A. Mole,
C. R. Paterson,
and
R. R. Davies.
Suppressed TSH levels secondary to thyroxine replacement therapy are not associated with osteoporosis.
Clin. Endocrinol. (Oxf.)
39:
529-533,
1993[Medline].
23.
Guo, C. Y.,
A. P. Weetman,
and
R. Eastell.
Longitudinal changes of bone mineral density and bone turnover in postmenopausal women on thyroxine.
Clin. Endocrinol. (Oxf.)
46:
301-307,
1997[Medline].
24.
Harvey, R. D.,
K. C. McHardy,
I. W. Reid,
F. Paterson,
P. D. Bewsher,
A. Duncan,
and
S. P. Robins.
Measurement of bone collagen degradation in hyperthyroidism and during thyroxine replacement therapy using pyridinium cross-links as specific urinary markers.
J. Clin. Endocrinol. Metab.
72:
1189-1194,
1991[Abstract].
25.
Hasling, C.,
E. R. Eriksen,
P. Charles,
and
L. Mosekilde.
Exogenous triiodothyronine activates bone remodeling.
Bone
8:
65-69,
1987[Medline].
26.
Hill, P. A.,
A. J. P. Docherty,
K. M. K. Bottomley,
J. P. O'Connell,
J. R. Murphy,
J. J. Reynolds,
and
M. C. Meikle.
Inhibition of bone resorption in vitro by selective inhibitors of gelatinase and collagenase.
Biochem. J.
308:
167-175,
1995[Medline].
27.
Holliday, L. S.,
H. G. Welgus,
C. J. Fliszar,
G. M. Veith,
J. J. Jeffrey,
and
S. L. Gluck.
Initiation of osteoclast bone resorption by interstitial collagenase.
J. Biol. Chem.
272:
22053-22058,
1997[Abstract/Free Full Text].
28.
Jeffrey, J. J.,
W. T. Roswit,
and
L. S. Ehlich.
Regulation of collagenase production by steroids in uterine smooth muscle cells: an enzymatic and immunologic study.
J. Cell. Physiol.
143:
396-403,
1990[Medline].
29.
Kassem, M.,
L. Mosekilde,
and
E. F. Eriksen.
Effects of triiodothyronine on DNA synthesis and differentiation markers of normal human osteoblast-like cells in vitro.
Biochem. Mol. Biol. Int.
30:
779-788,
1993[Medline].
30.
Katz, R. W.,
and
R. J. Koenig.
Nucleotide substitutions differentially affect direct repeat and palindromic thyroid hormone response elements.
J. Biol. Chem.
269:
9500-9505,
1994[Abstract/Free Full Text].
31.
Kawaguchi, H.,
C. C. Pilbeam,
F. N. Woodiel,
and
L. G. Raisz.
Comparison of the effects of 3,5,3'-triiodothyroacetic acid and triiodothyronine on bone resorption in cultured fetal rat long bones and neonatal mouse calvariae.
J. Bone Miner. Res.
9:
247-253,
1994[Medline].
32.
Knauper, V.,
C. Lopez-Otin,
B. Smith,
G. Knight,
and
G. Murphy.
Biochemical characterization of human collagenase-3.
J. Biol. Chem.
271:
1544-1550,
1996[Abstract/Free Full Text].
33.
Kusano, K.,
C. Miyaura,
M. Inada,
T. Tamura,
A. Ito,
H. H. Nagase,
K. Kamoi,
and
T. Suda.
Regulation of matrix metalloproteinases (MMP-2, -3, -9, and -13) by interleukin-1 and interleukin-6 in mouse calvaria: association of MMP induction with bone resorption.
Endocrinology
139:
1338-1345,
1998[Abstract/Free Full Text].
34.
Larsen, P. R.,
J. E. Silva,
and
M. M. Kaplan.
Relationships between circulating and intracellular thyroid hormones: physiological and clinical implications.
Endocr. Rev.
2:
87-102,
1981[Medline].
35.
Leco, K. J.,
L. J. Hayden,
R. R. Sharma,
H. Rocheleau,
A. H. Greenberg,
and
D. R. Edwards.
Differential regulation of TIMP-1 and TIMP-2 mRNA expression in normal and Ha-ras-transformed murine fibroblasts.
Gene
117:
209-217,
1992[Medline].
36.
Leco, K. J.,
R. Khokha,
N. Pavloff,
S. P. Hawkes,
and
D. R. Edwards.
Tissue inhibitor of metalloproteinases-3 (TIMP-3) is an extracellular matrix-associated protein with a distinctive pattern of expression in mouse cells and tissues.
J. Biol. Chem.
269:
9352-9360,
1994[Abstract/Free Full Text].
37.
Lehmke, J.,
U. Bogner,
D. Felsenberg,
H. Peters,
and
H. Schleusener.
Determination of bone mineral density by quantitative computed tomography and single photon absorptiometry in subclinical hyperthyroidism: a risk of early osteopaenia in post-menopausal women.
Clin. Endocrinol. (Oxf.)
36:
511-517,
1992[Medline].
38.
Lorenzo, J. A.,
C. C. Pilbeam,
J. F. Kalinowsk,
and
M. S. Hibbs.
Production of both 92- and 72-kDa gelatinases by bone cells.
Matrix
12:
282-290,
1992[Medline].
39.
Marcocci, C.,
F. Golia,
G. Bruno-Bossio,
E. Vignali,
and
A. Pinchera.
Carefully monitored levothyroxine suppressive therapy is not associated with bone loss in premenopausal women.
J. Clin. Endocrinol. Metab.
78:
818-823,
1994[Abstract].
40.
Masure, S.,
G. Nys,
P. Fiten,
J. Van Damme,
and
G. Opdenakker.
Mouse gelatinase B. cDNA cloning, regulation of expression and glycosylation in WEHI-3 macrophages and gene organisation.
Eur. J. Biochem.
218:
129-141,
1993[Abstract].
41.
Matrisian, L. M.,
and
B. L. M. Hogan.
Growth factor regulated proteases and extracellular matrix remodeling during mammalian development.
Curr. Top. Dev. Biol.
24:
219-259,
1990[Medline].
42.
Mauviel, A.
Cytokine regulation of metalloproteinase expression.
J. Cell. Biochem.
53:
288-295,
1993[Medline].
43.
Mitchell, P. G.,
H. A. Magna,
L. M. Reeves,
L. L. Lopresti-Morrow,
P. J. Rosner,
K. F. Geoghegan,
and
J. E. Hambor.
Cloning, expression, and type II collagenolytic activity of matrix metalloproteinase-13 from human osteoarthritic cartilage.
J. Clin. Invest.
97:
761-768,
1996[Abstract/Free Full Text].
44.
Morodomi, T.,
Y. Ogata,
Y. Sasaguri,
M. Morimatsu,
and
H. Nagase.
Purification and characterization of matrix metalloproteinase 9 from U937 monocytic leukaemia and HT1080 firbrosarcoma cells.
Biochem. J.
285:
603-611,
1992[Medline].
45.
Mundy, G. R.,
J. L. Shapiro,
J. G. Bandelin,
E. M. Canalis,
and
L. G. Raisz.
Direct stimulation of bone resorption by thyroid hormones.
J. Clin. Invest.
58:
529-534,
1976[Medline].
46.
Muscat, G. E.,
L. Mynett-Johnson,
D. Dowhan,
M. Downes,
and
R. Griggs.
Activation of myoD gene transcription by 3,5,3'-triiodo-L-thyronine: a direct role for the thyroid hormone and retinoid X receptors.
Nucleic Acids Res.
22:
583-591,
1994[Abstract].
47.
Ohishi, K.,
H. Ishida,
T. Nagata,
N. Yamauchi,
C. Tsurumi,
S. Nishikawa,
and
Y. Wakano.
Thyroid hormone suppresses the differentiation of osteoprogenitor cells to osteoblasts, but enhances functional activities of mature osteoblasts in cultured rat calvaria cells.
J. Cell. Physiol.
161:
544-552,
1994[Medline].
48.
Partridge, N. C.,
J. J. Jeffrey,
L. S. Ehlich,
S. L. Teitelbaum,
C. Fliszar,
H. G. Welgus,
and
A. J. Kahn.
Hormonal regulation of the production of collagenase and a collagenase inhibitor activity by rat osteogenic sarcoma cells.
Endocrinology
120:
1956-1962,
1987[Abstract].
49.
Pernasetti, F.,
L. Caccavelli,
C. Van de Weerdt,
J. A. Martial,
and
M. Muller.
Thyroid hormone inhibits the human prolactin gene promoter by interfering with activating protein-1 and estrogen stimulations.
Mol. Endocrinol.
11:
986-996,
1997[Abstract/Free Full Text].
50.
Quinn, C. O.,
D. K. Scott,
C. E. Brinckerhoff,
L. M. Matrisian,
J. J. Jeffrey,
and
N. C. Partridge.
Rat collagenase. Cloning, amino acid sequence comparison, and parathyroid hormone regulation in osteoblastic cells.
J. Biol. Chem.
265:
22342-22347,
1990[Abstract/Free Full Text].
51.
Rajakumar, R. A.,
and
C. O. Quinn.
Parathyroid hormone induction of rat interstitial collagenase mRNA in osteosarcoma cells is mediated through an AP-1-binding site.
Mol. Endocrinol.
10:
867-878,
1996[Abstract].
52.
Rifas, L.,
A. Fausto,
M. J. Scott,
L. V. Avioli,
and
H. G. Welgus.
Expression of metalloproteinases and tissue inhibitors of metalloproteinases in human osteoblast-like cells: differentiation is associated with repression of metalloproteinase biosynthesis.
Endocrinology
134:
213-221,
1994[Abstract].
53.
Roach, J.,
S. J. Choi,
R. L. Schaub,
R. J. Leach,
G. D. Roodman,
and
S. V. Reddy.
Further characterization of the murine collagenase (type IVB) gene promoter and analysis of mRNA expression in murine tissues.
Gene
208:
117-122,
1998[Medline].
54.
Rydziel, S.,
and
E. Canalis.
Analysis of hydroxyproline by high performance liquid chromatography and its application to collagen turnover studies in bone cultures.
Calcif. Tissue Int.
44:
421-424,
1989[Medline].
55.
Schmid, C.,
I. Schlapfer,
E. Futo,
M. Waldvogel,
J. Schwander,
J. Zapf,
and
E. R. Froesch.
Triiodothyronine (T3) stimulates insulin-like growth factor (IGF)-1 and IGF binding protein (IGFBP)-2 production by rat osteoblasts in vitro.
Acta Endocrinol.
126:
467-473,
1992[Medline].
56.
Sudo, H., H.-A. Kodama, Y. Amagai, S. Yamamoto, and S. Kasai.
In vitro differentiation and calcification in a new clonal
osteogenic cell line derived from newborn mouse calvaria.
J. Cell Biol. 96: 191-198, 1983.
58.
Tardif, G.,
J.-P. Pelletier,
M. Dupuis,
J. E. Hambor,
and
J. Martel-Pelletier.
Cloning, sequencing, and characterization of the 5'-flanking region of the human collagenase-3 gene.
Biochem. J.
323:
13-16,
1997[Medline].
59.
Tarjan, G.,
and
P. H. Stern.
Triiodothyronine potentiates the stimulatory effects of interleukin-1 beta on bone resorption and medium interleukin-6 content in fetal rat limb bone cultures.
J. Bone Miner. Res.
10:
1321-1326,
1995[Medline].
60.
Tso, J. Y.,
X. H. Sun,
T.-H. Kao,
K. S. Reece,
and
R. Wu.
Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene.
Nucleic Acids Res.
13:
2485-2502,
1985[Abstract].
61.
Uzzan, B.,
J. Campos,
M. Cucherat,
P. Nony,
J. P. Boissel,
and
G. Y. Perrett.
Effects on bone mass of long term treatment with thyroid hormones: a meta-analysis.
J. Clin. Endocrinol. Metab.
81:
4278-4289,
1996[Abstract].
62.
Varghese, S.,
A. M. Delany,
L. Liang,
B. Gabbitas,
J. J. Jeffrey,
and
E. Canalis.
Transcriptional and posttranscriptional regulation of interstitial collagenase by platelet-derived growth factor BB in bone cell cultures.
Endocrinology
137:
431-437,
1996[Abstract].
63.
Varghese, S.,
M. L. Ramsby,
J. J. Jeffrey,
and
E. Canalis.
Basic fibroblast growth factor stimulates expression of interstitial collagenase and inhibitors of metalloproteinases in rat bone cells.
Endocrinology
136:
2156-2162,
1995[Abstract].
64.
Wall, F. J.
Statistical Data Analysis Handbook (1st ed.). New York: McGraw-Hill, 1986.
65.
Williams, G. R.,
R. Bland,
and
M. C. Sheppard.
Characterization of thyroid hormone (T3) receptors in three osteosarcoma cell lines of distinct osteoblast phenotype: interactions among T3, vitamin D3, and retinoid signaling.
Endocrinology
135:
2375-2385,
1994[Abstract].
66.
Zandomeni, R.,
D. Bunick,
S. Ackerman,
B. Mittleman,
and
R. Weinmann.
Mechanism of action of DRB. Effect on specific in vitro initiation of transcription.
J. Mol. Biol.
167:
561-574,
1983[Medline].
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