(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
1(I) collagen expression by transcriptional and
post-transcriptional mechanisms(6, 7) .
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. ()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.
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 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.
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 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.
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 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.
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 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.
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
() 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.
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
[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.
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 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.
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 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.
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 -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.
Glucocorticoids have significant effects on bone remodeling.
Previous work demonstrated that they inhibit 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.
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. ()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.
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-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) .
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.