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(Received for publication, December 14, 1994; and in revised form, June 30, 1995) From the
Glucocorticoids regulate both bone formation and bone
resorption. In osteoblasts, they inhibit type I collagen synthesis;
however, there is limited information about their effects on
interstitial collagenase, the enzyme that degrades type I collagen. We
used primary cultures of osteoblast-enriched cells from fetal rat
calvariae (Ob cells) to study the effects of cortisol on collagenase
expression. Northern blot analysis showed that cortisol increased
collagenase transcript levels in a dose- and time-dependent manner,
which was paralleled by an increase in immunoreactive metalloproteinase
in the culture medium. Cortisol increased the half-life of collagenase
mRNA from 6 to 12 h in transcription-arrested Ob cells. In contrast,
cortisol modestly decreased collagenase gene transcription after 24 h
of treatment. The up-regulation of collagenase by cortisol is
osteoblast-specific, since the glucocorticoid decreased phorbol
12-myristate 13-acetate-induced collagenase mRNA expression in rat
fibroblasts, a result that agrees with other studies of collagenase
gene regulation in fibroblastic cells. In conclusion, cortisol
increases interstitial collagenase transcript levels by
post-transcriptional mechanisms in osteoblastic cells. Our data
demonstrate that glucocorticoids regulate collagenase gene expression
in a novel tissue-specific manner, further highlighting the differences
in gene regulation between osteoblastic and fibroblastic cells.
Glucocorticoids have marked effects on bone metabolism,
regulating bone formation and bone resorption(1) . In vitro studies have shown that glucocorticoids have complex effects on
osteoblast gene expression, and these effects are dependent on the
stage of osteoblast growth and differentiation and on the cell model
and culture conditions used(2) . Glucocorticoids induce cells
of the osteoblastic lineage to differentiate into mature cells
expressing the osteoblastic phenotype (3, 4, 5) . However, their inhibitory actions
on multiple aspects of osteoblastic function have a major impact on
bone mass. Glucocorticoids inhibit cell replication, depleting a cell
population capable of synthesizing bone collagen, and they inhibit
Additionally, glucocorticoids regulate bone collagen degradation,
although the effects have varied with the models and culture conditions
used(1) . Recently, glucocorticoids were shown to increase
interstitial collagenase (matrix metalloproteinase 1) transcript levels
in osteoblast cultures(8) . This effect is observed only in
osteoblasts; indeed, glucocorticoids inhibit transcription of
interstitial collagenase in nonskeletal
fibroblasts(9, 10, 11, 12) . This
suggests novel tissue-specific regulation of collagenase by
glucocorticoids. Matrix metalloproteinases and their inhibitors are
considered active participants in the degradation of osteoid, and
interstitial collagenases are the only proteases known to initiate the
degradation of type I collagen at neutral pH(13) . Thus,
up-regulation of osteoblast collagenase may play a role in the bone
loss associated with pathological glucocorticoid excess. However, the
mechanisms by which glucocorticoids stimulate the expression of
collagenase in osteoblasts are unknown. Investigations of the
regulation of interstitial collagenase are critical for understanding
the role glucocorticoids play in bone remodeling. Since glucocorticoids
differentially regulate collagenase transcripts in osteoblasts and
fibroblasts, determining the mechanisms by which this occurs will
contribute to our knowledge of cell type-specific gene regulation. In
this study, we examined the mechanisms of action of cortisol on
interstitial collagenase synthesis in cultures of osteoblast-enriched
cells from fetal rat calvariae (Ob cells) (
Continuous treatment of Ob cells with 1 µM
cortisol caused a time-dependent increase in collagenase steady state
transcripts. Northern blot analysis showed that treatment with cortisol
increased collagenase transcripts 2-fold after 8 h, and 7-fold after 16
and 24 h (Fig. 1). This stimulatory effect on collagenase
transcripts was sustained for at least 48 h (not shown), and cortisol
treatment did not modify the abundance of the 1.9-kb
glyceraldehyde-3-phosphate dehydrogenase transcript. The basal
unstimulated level of collagenase transcripts decreased with time in
culture, possibly due to the inhibitory effect of endogenous
insulin-like growth factor I. (
Figure 1:
Effect of cortisol at 1 µM on collagenase mRNA expression in cultures of Ob cells treated for
8, 16, or 24 h. Total RNA from control (C) or glucocorticoid (GC) treated cultures was subjected to Northern blot analysis
and hybridized with a
Figure 2:
Effect of cortisol on collagenase mRNA
expression in cultures of Ob cells treated for 24 or 48 h. Total RNA
from control (C) or from cultures treated with cortisol at 0.01-1
µM was subjected to Northern blot analysis and hybridized
with a
Figure 3:
Effect of cortisol at 1 µM on
collagenase accumulation in the medium of Ob cells treated for 24 h.
Medium from control (C) or glucocorticoid (GC)
treated cultures was subjected to Western blot analysis, and
collagenase was detected using an anti-rat collagenase antibody and a
chemiluminescence detection system. The immunoreactive band with the
same mobility as purified rat uterine collagenase is indicated by the arrow. These results are representative of five
cultures.
To
determine if the effect of cortisol on collagenase transcripts was
dependent on protein synthesis, confluent cultures of Ob cells were
treated with cortisol in the presence or absence of 3.6 µM cycloheximide, a dose known to inhibit protein synthesis in Ob
cells by at least 85%(32) . After 24 h of treatment,
cycloheximide alone superinduced collagenase mRNA levels, suggesting
that it may stabilize the transcript(33, 34) .
Co-treatment with cortisol reduced the superinduction of collagenase by
cycloheximide (Fig. 4).
Figure 4:
Effect of cortisol at 1 µM,
in the presence or absence of cycloheximide (CX) at 3.6
µM, on collagenase mRNA expression in cultures of Ob cells
treated for 24 h. Total RNA from control (C) or glucocorticoid (GC) treated cultures was subjected to Northern blot analysis
and hybridized with a
To determine if cortisol modified the
stability of collagenase mRNA in Ob cells, the RNA polymerase
II-specific inhibitor DRB was used to arrest transcription, and the
decay of collagenase mRNA was monitored by Northern blot
analysis(35, 36) . Serum-deprived confluent cultures
of Ob cells were exposed to control medium or to 1 µM cortisol for 4 h and then treated with 72 µM DRB for
up to 12 h in the absence or presence of cortisol at 1 µM.
In transcription-arrested Ob cells, the half-life of collagenase mRNA
was approximately 6 h, and cortisol increased the half-life of the
transcript to approximately 12 h (Fig. 5, left panel).
A similar increase in collagenase transcript stability was observed in
Ob cells treated with cortisol for 12 h prior to the addition of DRB (Fig. 5, right panel). In both experiments, the slope
for the collagenase mRNA decay in the cortisol-treated cells was
significantly different from control(31) . The decay of
glyceraldehyde-3-phosphate dehydrogenase transcripts was the same in
control and cortisol treated cultures (not shown). To determine if
cortisol modified transcription of the collagenase gene, nuclear
run-off assays were performed on nuclei from Ob cells treated with 1
µM cortisol for 2, 6, or 24 h. The levels of
Figure 5:
Effect of cortisol at 1 µM on
collagenase mRNA half-life in transcription-arrested Ob cells.
Confluent cultures were serum-deprived and exposed to cortisol or to
control medium for 4 h (left panel) or 12 h (right
panel) prior to the addition of 72 µM DRB. At
selected times after the addition of DRB, total RNA from control
(
Figure 6:
Effect
of cortisol at 1 µM on collagenase gene transcription.
Nuclei were isolated from control (C) or glucocorticoid (GC) treated Ob cells. In one experiment, cells were treated
for 6 h, and in the other experiment they were treated for 24 h.
Nascent transcripts were labeled in vitro with
[
In human
and rabbit fibroblastic cells, glucocorticoids antagonize the induction
of collagenase by the protein kinase C agonist
PMA(9, 10, 11, 12) . To determine if
up-regulation of collagenase by cortisol in rat osteoblasts was a
species-specific or a cell type-specific phenomenon, rat skin
fibroblasts were treated for 6 and 24 h with 0.1 µM PMA in
the presence or absence of cortisol at 1 µM (Fig. 7). Collagenase transcripts in untreated cells were
almost undetectable, but treatment with PMA dramatically increased
collagenase mRNA after 6 h. Co-treatment with cortisol antagonized this
effect, and cortisol alone did not increase collagenase mRNA,
documenting the specific nature of the cortisol effect in Ob cells. The
ability of cortisol to augment or antagonize the PMA induction of
collagenase transcripts in Ob cells also was tested (Fig. 8).
After 2 h of treatment, PMA at 0.1 µM increased
collagenase transcripts, while cortisol at 1 µM was
modestly inhibitory. After 6 h, PMA increased collagenase mRNA levels
20-fold, and co-treatment with cortisol antagonized this effect by
approximately 40%. Treatment with PMA for 24 h down-regulated
osteoblast collagenase transcript levels to approximately 20% of the
untreated control. In contrast, cortisol increased collagenase mRNA by
5-fold at 24 h, and co-treatment with PMA decreased this effect by
60-80%.
Figure 7:
Effect of cortisol at 1 µM on
PMA-induced collagenase transcripts in fibroblasts. Rat skin
fibroblasts were treated with control medium (C) or with PMA (P) at 0.1 µM, and the glucocorticoid cortisol (GC) at 1 µM for 6 or 24 h. Total RNA was
isolated and subjected to Northern blot analysis with a
Figure 8:
Effect of cortisol at 1 µM on
PMA-induced collagenase transcripts in osteoblasts. Ob cells were
treated with control medium (C) or with PMA (P) at
0.1 µM and the glucocorticoid cortisol (GC) for
2, 6, or 24 h. Total RNA was isolated and subjected to Northern blot
analysis with a
To further characterize the effects of PMA and
cortisol on transcription of the collagenase gene in osteoblasts, Ob
cells were transiently transfected with a construct containing a 2.1-kb
fragment of the rat collagenase promoter driving expression of the
reporter gene luciferase. Treatment of transfected cells with cortisol
alone for 6 h caused a 25% decreased in luciferase activity (p < 0.05) (Fig. 9). Treatment of transfected cells with
0.1 µM PMA increased luciferase activity 2-fold, and
co-treatment with 1 µM cortisol antagonized this effect.
Figure 9:
Effect of cortisol at 1 µM and PMA at 0.1 µM on collagenase promoter activity in
transiently transfected Ob cells. A 2.1-kb fragment of the rat
collagenase promoter was used to drive expression of the luciferase
gene in the promoterless reporter plasmid pGL2-Basic. Ob cells were
transiently co-transfected with collagenase promoter-luciferase plasmid
and plasmid containing the cytomegalovirus promoter driving expression
of the
Glucocorticoids have significant effects on bone remodeling.
Previous work demonstrated that they inhibit Experiments using the RNA polymerase II inhibitor DRB
demonstrated that cortisol stabilized collagenase mRNA in
transcriptionally arrested Ob cells. In contrast, nuclear run-off
assays and transient transfection of a rat interstitial collagenase
promoter-reporter gene construct showed that collagenase gene
transcription was slightly decreased by cortisol. These results
indicate that cortisol increases collagenase expression by increasing
transcript stability. The rat collagenase mRNA has a 1.2-kilobase
3`-untranslated region that is AU-rich and contains three repeats of
the sequence AUUUA(17) . In short lived mRNAs, such AU-rich
sequences play a role in regulating transcript stability (reviewed in (37) ). The human and rabbit collagenase mRNAs contain repeats
of AUUUA, and mutation of these motifs in the human transcript
increases the stability of the RNA(38) . It is probable that
the glucocorticoid-mediated increase in Ob cell collagenase transcripts
involves proteins interacting with such AU-rich regions of the
mRNA(39) . Cortisol antagonized the induction of collagenase
promoter activity by PMA in Ob cells. These effects are similar to
those observed in fibroblasts(10, 11, 12) ,
suggesting that osteoblasts and fibroblasts share a common mechanism
for the regulation of collagenase transcription. Like the human
interstitial collagenase gene, which is transcriptionally regulated by
PMA and glucocorticoids, the rat collagenase promoter contains a
TPA-responsive
element(10, 11, 12, 40, 41) .
Components of the AP-1 complex can interact with the TPA-responsive
element, and glucocorticoid repression of collagenase gene
transcription in fibroblastic cells can be mediated through antagonism
of AP-1(10, 11, 12) . Collagenase gene
transcription and mRNA levels were down-regulated in Ob cells treated
for 24 h with PMA. A nuclear run-off assay showed that after 24 h
treatment with PMA, collagenase gene transcription was barely
detectable. ( The
expression of collagenase in osteoblasts is up-regulated by a number of
osteoresorptive agents, including parathyroid hormone, glucocorticoids
and prostaglandins(8, 40, 42) . The role of
osteoblast-derived interstitial collagenase in the bone compartment is
currently being explored, and there is increasing evidence for the
coupling of osteoblastic function with osteoclastic bone resorption
(reviewed in (43) ). Localized extracellular matrix degradation
by osteoblast collagenase may provide a means for activated osteoclasts
to adsorb to target bone surfaces. Collagenase may also play a role in
regulating the availability of bone growth factors. For example,
localized matrix degradation may release growth factors sequestered in
the matrix, which may stimulate or inhibit osteoblastic function or may
activate or be chemotactic for osteoclasts(44, 45) .
Insulin-like growth factors are among the most prevalent growth factors
secreted by bone cells, and they stimulate the differentiated function
of the osteoblast(45) . Their activity can be modulated by
insulin-like growth factor binding proteins, the abundance of which can
be regulated by proteases, including Ca In conclusion, cortisol
causes a cell type-specific increase in interstitial collagenase
expression in osteoblasts, which is mediated by post-transcriptional
mechanisms. These results further highlight the differences in gene
regulation between osteoblasts and fibroblasts, two cell types that
arise from a common precursor.
Volume 270,
Number 44,
Issue of November 3, 1995 pp. 26607-26612
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1(I) collagen expression by transcriptional and
post-transcriptional mechanisms(6, 7) . 
)
Culture Technique
The culture method used was
described in detail previously(14) . Parietal bones were
obtained from 22-day-old fetal rats immediately after the mothers were
sacrificed by blunt trauma to the nuchal area. This project was
approved by the Institutional Animal Care and Use Committee of Saint
Francis Hospital and Medical Center. Cells were obtained by five
sequential digestions of the parietal bone, using bacterial collagenase
(CLS II, Worthington Biochemical, Freehold, NJ). Cell populations
harvested from the third to the fifth digestions were cultured as a
pool at a density of 10,000 cells/cm
and have been
previously shown to have osteoblastic characteristics(14) . In
one set of experiments, fibroblastic cells derived from collagenase
digestion of skin from 22-day-old fetal rats were cultured and
tested(9) . Cells were cultured in Dulbecco's modified
Eagle's medium supplemented with nonessential amino acids (Life
Technologies, Inc.) and 10% fetal bovine serum (HyClone, Logan,
UT)(14) . Except for the nuclear run-off assay, primary
cultures of Ob cells were used in all experiments, whereas skin
fibroblasts were used after three or four passages. At confluence the
cells were rinsed and transferred to serum-free medium for 24 h and
then exposed to test or control medium in the absence of serum for
2-48 h. In experiments lasting longer than 24 h, the medium was
replaced with freshly prepared test and control solutions. Cortisol,
cycloheximide, phorbol 12-myristate 13-acetate (PMA) and
5,6-dichlorobenzimidazole riboside (DRB) (all from Sigma) were
dissolved in absolute ethanol, and at dilutions of <1:10,000 an
equal amount of ethanol was added to control cultures.Northern Blot Analysis
Total cellular RNA was
isolated with guanidine thiocyanate, at acid pH, followed by a
phenol-chloroform (Sigma) extraction as described(15) . Equal
amounts of RNA were denatured and subjected to electrophoresis through
formaldehyde-agarose gels(16) , and the RNA was blotted onto
Gene Screen Plus as directed by the manufacturer (DuPont). Restriction
fragments containing a 2.6-kilobase (kb) rat interstitial collagenase
cDNA (17) (kindly provided by C. Quinn, St. Louis, MO) and an
800-base pair rat glyceraldehyde-3-phosphate dehydrogenase
cDNA(18) , (kindly provided by R. Wu, Ithaca, NY) were labeled
with [-P]dCTP (specific activity 3,000
Ci/mmol; DuPont), by random-primed second strand synthesis
(Prime-A-Gene, Promega, Madison, WI)(19) . Hybridizations were
carried out at 42 °C in 50% formamide, 5
SSPE (750 mM sodium chloride, 50 mM sodium phosphate, 5 mM EDTA), 5 Denhardt's solution, and 0.4% SDS (Sigma).
Post-hybridization washes were performed at 65 °C in 1 SSC
(150 mM sodium chloride, 15 mM sodium citrate) and
0.1% SDS. Autoradiograms were analyzed by densitometry, and collagenase
RNA levels were normalized to those of glyceraldehyde-3-phosphate
dehydrogenase. Northern analyses shown are representative of two or
more cultures.Nuclear Run-off Assay
Subconfluent cultures of Ob
cells were treated with trypsin, harvested, subcultured at a 1:6
dilution, and allowed to grow to confluence as described
previously(20) . These first passage Ob cells retain the
osteoblastic phenotype and respond to cortisol in a manner similar to
that of primary cultures(7) . At confluence, cells were
serum-deprived and treated for 2-24 h, and nuclei were isolated
by Dounce homogenization in a Tris-Cl buffer containing 0.5% Nonidet
P-40. Nascent transcripts were labeled by incubation of nuclei in a
reaction buffer containing 500 µM each ATP, GTP, and CTP,
150 units of RNAsin (Promega), and 250 µCi of
[P]UTP (3000 Ci/mM, DuPont) (modified
from (21) ). RNA was isolated by treatment with DNase I and
proteinase K, followed by ethanol precipitation. Linearized plasmid DNA
containing
1 µg of cDNA was immobilized onto GeneScreen Plus
by slot blotting according to the manufacturer's directions
(DuPont). The plasmid vector pGL2-Basic (Promega) was used as a control
for nonspecific hybridization, and cDNA for rat
1(I) collagen was
used as a positive control(22) . Equal counts per minute of
[P]RNA from each sample were hybridized to cDNA
using the same conditions as for Northern blot analysis and were
visualized by autoradiography. The nuclear run-off assay shown is
representative of three experiments.
Transient Transfections and Reporter Gene Assays
A NotI/XhoI rat genomic DNA fragment containing 2.1 kb
of a recently cloned rat interstitial collagenase promoter ()was used to drive expression of the luciferase gene in the
vector pGL2-Basic. A construct containing the cytomegalovirus promoter
driven 
-galactosidase gene (pCMV-Gal, Clontech, Palo Alto,
CA) was used to control for transfection efficiency. Ob cells were
cultured to approximately 80% confluence and were transiently
co-transfected with the collagenase promoter-luciferase construct and
pCMV-Gal by calcium phosphate/DNA coprecipitation, followed by
glycerol shock as described(23, 24, 25) .
Cells were washed, serum-deprived, and treated for 6 h with control
medium or 1 µM cortisol. Cell lysates were made using 1
Reporter Lysis Buffer (Promega), and luciferase activity was
measured by injecting luciferase assay reagent (Promega) into a portion
of the cell lysate and counting photons using an Optocomp luminometer
(MGM Instruments, Hamden, CT) according to the manufacturer's
instructions. -galactosidase activity was measured by incubating
cell lysates with the chemiluminescent substrate for
-galactosidase
3-(4-methoxyspiro[1,2-dioxetane-3,2`-tricyclo[3.3.l.l.]decan]-yl)phenyl-
-D-galactopyranoside
(Galacton; Tropix, Bedford, MA) with the modifications
described(24) . Luciferase activity was normalized to
-galactosidase activity to control for slight variations in
transfection efficiency.Collagenase Immunoassay
Immunoreactive collagenase
was determined by an enzyme-linked immunosorbant assay, as described
previously(26, 27) . Briefly, samples were incubated
with antiserum to rat interstitial collagenase and added to the wells
of microtiter plates coated with 0.25 µg pure rat uterine
interstitial collagenase. The plates were incubated overnight at 4
°C and washed, and the surface-bound antibody was quantitated by
the addition of goat antirabbit -globulin conjugated with alkaline
phosphatase. The bound alkaline phosphatase-dependent formation of p-nitrophenol was then determined spectrophotometrically. This
assay detects collagenase at concentrations as low as 1 ng/ml of
culture medium.
Western Immunoblot Analysis
Ob cells were cultured
as described previously and media were stored at -80 °C after
the addition of polyoxyethylene sorbitan monolaurate (Tween 20, Pierce)
to a final concentration of 0.1%. Proteins were separated by
polyacrylamide gel electrophoresis under denaturing, nonreducing
conditions and transferred to Immobilon P membranes (Millipore,
Bedford, MA)(26) . After blocking with 2% bovine serum albumin,
the membranes were exposed to a 1:1000 dilution of rabbit antisera
raised against rat collagenase (27, 28) and then to
goat anti-rabbit IgG antisera conjugated to horseradish peroxidase. The
blots were washed and developed with a horseradish peroxidase
chemiluminescence detection reagent (DuPont), visualized by
autoradiography on DuPont Reflection film employing Reflection
intensifying screens, and analyzed by densitometry. The Western blot
shown is representative of five cultures.Statistical Analysis
Statistical differences were
calculated by analysis of variance, and post hoc examination was
performed by the Ryan-Einot-Gabriel-Welsh F
test(29, 30) . Slopes were analyzed by the method of
Sokal and Rohlf(31) .
)The cortisol-mediated
increase in collagenase mRNA levels was dose-dependent and was observed
at 0.1 and 1 µM cortisol (Fig. 2). In parallel with
its effects on collagenase transcripts, cortisol increased levels of
the protein in the culture medium of Ob cells treated for 24 h. The
concentration of immunoreactive metalloproteinase was below the limit
of detection in control cultures but was increased to 44 ± 6
ng/ml (mean ± S.E.; n = 4) in cultures treated
with 1 µM cortisol. Western blot analysis showed a 5-fold
increase in a 57-kDa immunoreactive protein in the medium of cells
treated with cortisol (Fig. 3). This band had the same mobility
as the rat uterine procollagenase standard, while the lower molecular
weight cross-reactive band, which was not regulated by cortisol
treatment, may represent another species of metalloproteinase.
P-labeled rat collagenase cDNA; the
blot was stripped and hybridized with a labeled rat
glyceraldehyde-3-phosphate dehydrogenase cDNA. Transcripts were
visualized by autoradiography, and collagenase mRNA is shown in the upper panel while glyceraldehyde-3-phosphate dehydrogenase
mRNA is shown below. These results are representative of three
cultures.
P-labeled rat collagenase cDNA; the blot was
stripped and hybridized with a labeled rat glyceraldehyde-3-phosphate
dehydrogenase cDNA. Transcripts were visualized by autoradiography, and
collagenase mRNA is shown in the upper panel while
glyceraldehyde-3-phosphate dehydrogenase mRNA is shown below.
These results are representative of three
cultures.
P-labeled rat collagenase cDNA; the
blot was stripped and hybridized with a labeled rat
glyceraldehyde-3-phosphate dehydrogenase cDNA. Transcripts were
visualized by autoradiography, and collagenase mRNA is shown in the upper panel, glyceraldehyde-3-phosphate dehydrogenase mRNA is
shown in the middle panel, and the ethidium bromide-stained
gel is shown below. These results are representative of six
cultures.
1(I)
collagen gene transcription were used as a control, since cortisol has
been shown to decrease transcription of this gene (7) .
Cortisol did not alter transcription of the collagenase gene after 2 h
(not shown) or 6 h (Fig. 6), although it did decrease 1(I)
collagen gene transcription. After 24 h of treatment, cortisol caused a
small decrease in transcription from the collagenase gene.
) or cortisol (
) treated cultures was subjected to Northern
blot analysis with P-labeled rat collagenase cDNA.
Collagenase mRNA was visualized by autoradiography and quantitated by
densitometry. Values are mean ± S.E. for three cultures. In the left panel, the slope for DRB = -0.052, and the
slope for DRB + cortisol = -0.022. These values were
significantly different, p < 0.01. In the right
panel, the slope for DRB = -0.054, and the slope for
DRB + cortisol = -0.029. These values were
significantly different, p <
0.05.
P]UTP, and the labeled RNA was hybridized to
immobilized cDNA for
1(I) collagen, glyceraldehyde-3-phosphate
dehydrogenase (GAPD), and rat collagenase. pGL2-Basic vector
DNA (Promega) was used as a control for nonspecific hybridization.
Transcripts were visualized by autoradiography. These results are
representative of three experiments.
P-labeled rat collagenase cDNA; the blot was stripped and
rehybridized with a labeled rat glyceraldehyde-3-phosphate
dehydrogenase cDNA. Transcripts were visualized by autoradiography, and
collagenase mRNA is shown in the upper panel while
glyceraldehyde-3-phosphate dehydrogenase mRNA is shown below.
These results are representative of two
cultures.
P-labeled rat collagenase cDNA; the blot
was stripped and rehybridized with a labeled rat
glyceraldehyde-3-phosphate dehydrogenase cDNA. Transcripts were
visualized by autoradiography, and collagenase mRNA is shown in the upper panel while glyceraldehyde-3-phosphate dehydrogenase
mRNA is shown below. These results are representative of five
cultures.
-galactosidase gene. Transfected cells were treated for 6 h
with control medium (C) or with medium containing
glucocorticoid (GC), PMA, or PMA plus glucocorticoid (PMA + GC). Luciferase activity was normalized to
-galactosidase activity to control for slight differences in
transfection efficiency. These results are representative of three
experiments. *, significantly different from C, p < 0.01;
**, significantly different from PMA, p <
0.01.
1(I) collagen
synthesis by transcriptional and post-transcriptional mechanisms, but
there is limited information about their effects on collagen
degradation and collagenase expression(1, 7) . In the
present study we demonstrated that cortisol causes a time- and
dose-dependent stimulation of interstitial collagenase transcripts in
cultures of Ob cells, which was paralleled by increased levels of
immunoreactive collagenase in the culture medium. These effects were
observed at doses of cortisol that modify parameters of osteoblastic
differentiated function and at concentrations that were only slightly
higher than physiological serum levels of
cortisol(3, 4, 5, 6) . The same
doses of cortisol modestly decreased transcripts for tissue inhibitor
of metalloproteinases (TIMP) 1 and did not affect the expression of
TIMPs 2 and 3, suggesting that up-regulation of collagenase
by glucocorticoids is important in increased extracellular matrix
degradation.
)This is most likely due to depletion of
protein kinase C activity following prolonged exposure to phorbol
esters. In cells treated with PMA and cortisol for 24 h, the level of
collagenase mRNA was intermediate between that of cortisol alone and
PMA alone. This suggests that the effects of cortisol and PMA may be
additive at this time point and that cortisol and PMA increase
collagenase transcripts in Ob cells by distinct mechanisms.-dependent
serine proteases and
metalloproteinases(46, 47, 48, 49) .
A study characterizing a parathyroid hormone-regulated receptor for
collagenase on a rat osteosarcoma cell line showed that this receptor
is responsible for clearance of collagenase from the cellular
environment (50) . This suggests that the osteoblast maintains
a tight control over collagenase activity and that promiscuous
expression of collagenase would be undesirable. The importance of
appropriately regulated osteoblast collagenase also is suggested by the
development of collagenase-expressing bone tumors in transgenic mice
overexpressing c-fos(51) .
))))
We thank Dr. Cheryl Quinn for the rat interstitial
collagenase cDNA clone, Dr. R. Wu for the rat
glyceraldehyde-3-phosphate dehydrogenase cDNA clone, and Drs. David
Rowe and Barbara Kream for providing the 1(I) procollagen cDNA. We
thank Cathy Boucher, Deena Kjeldsen, and Teresa Passaretti for expert
technical assistance and Beverly Faulds for secretarial help.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 A. M. Delany, D. Durant, and E. Canalis Glucocorticoid Suppression of IGF I Transcription in Osteoblasts Mol. Endocrinol., October 1, 2001; 15(10): 1781 - 1789. [Abstract] [Full Text] [PDF]
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 F. Blanquaert, R. C. Pereira, and E. Canalis Cortisol inhibits hepatocyte growth factor/scatter factor expression and induces c-met transcripts in osteoblasts Am J Physiol Endocrinol Metab, March 1, 2000; 278(3): E509 - E515. [Abstract] [Full Text] [PDF]
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E. Dennison, P. Hindmarsh, C. Fall, S. Kellingray, D. Barker, D. Phillips, and C. Cooper Profiles of Endogenous Circulating Cortisol and Bone Mineral Density in Healthy Elderly Men J. Clin. Endocrinol. Metab., September 1, 1999; 84(9): 3058 - 3063. [Abstract] [Full Text]
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A. M. Delany and E. Canalis Dual Regulation of Stromelysin-3 by Fibroblast Growth Factor-2 in Murine Osteoblasts J. Biol. Chem., June 26, 1998; 273(26): 16595 - 16600. [Abstract] [Full Text] [PDF]
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 J. Rubin, D. M. Biskobing, L. Jadhav, D. Fan, M. S. Nanes, S. Perkins, and X. Fan Dexamethasone Promotes Expression of Membrane-Bound Macrophage Colony-Stimulating Factor in Murine Osteoblast-Like Cells Endocrinology, March 1, 1998; 139(3): 1006 - 1012. [Abstract] [Full Text] [PDF]
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N. Franchimont, 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., May 2, 1997; 272(18): 12144 - 12150. [Abstract] [Full Text] [PDF]
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J. B. Lian, V. Shalhoub, F. Aslam, B. Frenkel, J. Green, M. Hamrah, G. S. Stein, and J. L. Stein Species-Specific Glucocorticoid and 1,25-Dihydroxyvitamin D Responsiveness in Mouse MC3T3-E1 Osteoblasts: Dexamethasone Inhibits Osteoblast Differentiation and Vitamin D Down-Regulates Osteocalcin Gene Expression Endocrinology, May 1, 1997; 138(5): 2117 - 2127. [Abstract] [Full Text] [PDF]
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B. Gabbitas, J. M. Pash, A. M. Delany, and E. Canalis Cortisol Inhibits the Synthesis of Insulin-like Growth Factor-binding Protein-5 in Bone Cell Cultures by Transcriptional Mechanisms J. Biol. Chem., April 12, 1996; 271(15): 9033 - 9038. [Abstract] [Full Text] [PDF]
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