Cortisol inhibits hepatocyte growth factor/scatter factor
expression and induces c-met transcripts in
osteoblasts
Frederic
Blanquaert1,
Renata C.
Pereira1, and
Ernesto
Canalis1,2
1 Departments of Research and Medicine, Saint
Francis Hospital and Medical Center, Hartford 06105; and
2 The University of Connecticut School of
Medicine, Farmington, Connecticut 06030
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ABSTRACT |
Hepatocyte growth
factor/scatter factor (HGF/SF) is expressed by osteoblasts and has
important effects on repair and bone remodeling. Because
glucocorticoids regulate these two functions, we tested the effects of
cortisol on the expression of HGF/SF and c-met, the
protooncogene encoding the HGF/SF receptor, in cultures of
osteoblast-enriched cells from 22-day fetal rat calvariae (Ob cells).
Cortisol decreased HGF/SF mRNA levels and diminished the induction of
HGF/SF transcripts by fibroblast growth factor-2 (FGF-2) and
platelet-derived growth factor BB (PDGF BB). Cortisol also decreased
FGF-2 and PDGF BB-induced HGF/SF mRNA and polypeptide levels in MC3T3
cells. In contrast, cortisol enhanced the expression of c-met
transcripts in Ob cells. Cortisol did not modify the half-life of
HGF/SF or of c-met mRNA in transcriptionally arrested cells, and it
increased the rate of transcription of c-met. In conclusion, cortisol
decreases HGF/SF transcripts in Ob cells and enhances c-met expression
transcriptionally. The effects of cortisol on HGF/SF could be relevant
to its inhibitory actions on bone formation and repair.
skeletal tissue; glucocorticoids; wound healing; fractures; growth
factors
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INTRODUCTION |
GLUCOCORTICOIDS HAVE MARKED EFFECTS on the skeleton,
and prolonged exposure to excessive amounts of these corticosteroids results in osteoporosis (8, 13). Glucocorticoids have complex actions
on bone formation and resorption, although their inhibitory effects on
bone formation appear central to the bone loss observed after
glucocorticoid excess (8, 13). Glucocorticoids decrease the pool of
available osteoblasts and cause osteoblast apoptosis (44). In addition,
they have direct actions on specific genes expressed by the osteoblast
and regulate the synthesis and activity of locally produced growth
factors (8, 13). For example, glucocorticoids have opposite effects to
those of insulin-like growth factor (IGF) I on bone formation and
inhibit the transcription of the growth factor in osteoblasts (12, 24).
This would suggest a possible role of IGF-I and other growth factors in
mediating selected actions of glucocorticoids in bone.
Hepatocyte growth factor/scatter factor (HGF/SF) is a polypeptide
composed of a 69-kDa
-chain with four krinkle domains and a 34-kDa
-chain with a serum protease-like sequence linked by disulfide bonds
(31, 38). HGF/SF stimulates mitogenesis in hepatic and extrahepatic
cells, enhances angiogenesis, and plays a role in repair in liver and
kidney, and possibly in other tissues (26, 33, 36, 38, 42). HGF/SF
signals via the product of the protooncogene c-met, a tyrosine
kinase-activated receptor (5, 32). HGF/SF and c-met are expressed by
mesenchymal cells, osteoblasts, and osteoclasts (4, 21). HGF/SF is
mitogenic for cells of the osteoblastic and osteoclastic lineage, and
its synthesis by the osteoblast is enhanced by growth factors with a
role in wound and fracture repair (4, 21). Therefore, it was postulated
to have a function in bone remodeling and repair (21).
Glucocorticoids not only cause a decrease in bone formation, but they
also alter wound and possibly fracture healing (3). Although this may
be the result of direct actions of glucocorticoids on cellular events
at the wound or fracture site, it may involve alterations in the
production or activity of locally produced factors, such as HGF/SF. In
an initial effort to explore a possible role of HGF/SF as a mediator of
glucocorticoid action in bone, in the present study we examined the
effects of cortisol on the expression of HGF/SF and c-met in cultures
of osteoblast-enriched cells from 22-day fetal rat calvariae (Ob cells).
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MATERIALS AND METHODS |
Culture technique.
The culture method used was described in detail previously (29).
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. Cells were obtained by
five sequential digestions of the parietal bone by use of bacterial
collagenase (CLS II, Worthington Biochemical, Freehold, NJ). Cell
populations harvested from the third to the fifth digestions were
cultured as a pool and were previously shown to have osteoblastic
characteristics (26). Ob cells 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). Cells were cultured in DMEM (Life
Technologies, Grand Island, NY) supplemented with nonessential amino
acids, 20 mM HEPES, and 10% fetal bovine serum (FBS; Summit,
Biotechnology, Fort Collins, CO) and were grown to confluence. MC3T3
cells, an osteoblastic cell line created by Sudo et al. (39) and
derived from fetal mouse calvaria, were cultured in
-MEM (Life
Technologies) supplemented with 20 mM HEPES and 10% FBS under the same
conditions as Ob cells and grown to confluence (39). At confluence,
cells were transferred to serum-free medium for 20-24 h and
exposed to test or control medium in the absence of serum for 2-48
h, as indicated in the text and legends. In 48-h-treated cultures, the
medium was replaced after 24 h with fresh control or test solutions.
For nuclear run-on experiments, Ob cells were grown to subconfluence,
trypsinized, replated, grown to confluence, serum-deprived for
20-24 h, and exposed to test or control solutions for 2-24 h.
Fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor
BB (PDGF BB) (both from Austral, San Ramon, CA) were added directly to
the medium. Cortisol, cycloheximide, and 5,6-dichlorobenzimidazole riboside (DRB) (all from Sigma Chemical, St. Louis, MO) were dissolved in absolute ethanol and diluted 1:10,000, 1:1,000, and 1:200, respectively, in DMEM; all experimental groups were exposed to an equal
amount of ethanol. For RNA analysis, the cell layer was extracted with
guanidine thiocyanate at the end of the incubation and stored at
70°C. For nuclear run-on assays, nuclei were isolated by
Dounce homogenization. For protein levels, the culture medium was
collected in the presence of 0.1% polyoxyethylenesorbitan monolaureate
(Tween-20, Pierce Chemical, Rockford, IL), and assays were performed at
the completion of the culture.
Northern blot analysis.
Total cellular RNA was isolated using an RNeasy kit and following the
manufacturer's instructions (Qiagen, Chatsworth, CA). The RNA
recovered was quantitated by spectrometry, 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, documenting equal RNA
loading of the various experimental samples. The RNA was then blotted
onto Gene Screen Plus charged nylon (Du Pont, Wilmington, DE), and
uniformity of transfer was documented by revisualization of ribosomal
RNA. A 1.4-kb EcoR I restriction fragment of a rat HGF/SF cDNA
(kindly provided by T. Nakamura, Osaka, Japan) and a 4.0-kb Not
I restriction fragment of a mouse c-met cDNA (kindly provided by C. C. Lee, Bethesda, MD) were purified by agarose gel electrophoresis (25,
31). HGF/SF and c-met cDNAs were labeled with
[
-32P]deoxycytidine triphosphate (dCTP) and
[
-32P]deoxyadenosine triphosphate (dATP) (50 µCi each at a specific activity of 3,000 Ci/mmol; Du Pont) by use of
the random hexanucleotide primed second strand synthesis method (17).
Hybridizations were carried out at 42°C for 16-72 h, and
posthybridization washes were performed in 0.5× saline-sodium
citrate (SSC) at 65°C. The blots were stripped and rehybridized
with an
-32P-labeled 752-bp BamH I/Sph I
restriction fragment of the murine 18S cDNA (American Type Culture
Collection, Rockville, MD) under the conditions described, followed by
two posthybridization washes in 1× SSC at room temperature and
one wash in 0.1× SSC at 65°C. Unlabeled 18S cDNA was added in
excess to the
-32P-labeled probe before being added to
the hybridization mixture to ensure sufficient quantity of 18S cDNA to
bind to the 18S rRNA. The bound radioactive material was visualized by
autoradiography on Kodak X-AR5 film (Eastman Kodak, Rochester, NY)
employing Cronex Lightning Plus (Du Pont) or Biomax MS (Eastman Kodak)
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 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 adenosine, cytidine, and guanosine
triphosphates, 150 units RNasin (Promega, Madison, WI), and 250 µCi
[
32P]uridine triphosphate (UTP) (3,000 Ci/mM, Du Pont) (22). 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 each of cDNA
was immobilized onto GeneScreen Plus by slot blotting according to the
manufacturer's directions (Du Pont). Murine 18S cDNA was used to
estimate uniformity of counts applied to the membrane. Equal counts per
minute of [32P]RNA from each sample were
hybridized to cDNAs at 42°C for 72 h and washed in 1× SSC at
65°C for 30 min. Hybridized cDNAs were visualized by
autoradiography. Nuclear run-on assays were done twice.
HGF/SF immunoassay.
An enzyme immunoassay (EIA) detection kit (Institute of Immunology,
Tokyo, Japan) was used to measure immunoreactive rodent HGF/SF (2).
Medium samples were cleared by centrifugation, and a 50-µl aliquot of
the supernatant was dispensed in duplicate into a 96-well plate
precoated with anti-rat HGF/SF mouse monoclonal antibody. HGF/SF
standard solutions were provided by the manufacturer. After an
overnight incubation at room temperature, the plate was washed and
incubated with anti-rat HGF/SF rabbit polyclonal antibody followed by
the addition of peroxidase-labeled goat anti-rabbit immunoglobulin.
HGF/SF levels were detected by colorimetry after an enzymatic reaction
using the peroxidase substrate o-phenylenediamine and
measurement of the product with a microplate spectrophotometer at 490 nm. Data are expressed in nanograms of HGF/SF per milliliter of medium
or nanograms per milligram of protein, determined by use of a Bio-Rad
DC protein assay kit according to the manufacturer's instructions
(Bio-Rad, Hercules, CA).
Statistical analysis.
Data are expressed as means ± SE, and statistical differences for
immunoreactive HGF/SF levels were determined by ANOVA and post hoc
examination by Scheffé's test. Slopes of mRNA decay were
analyzed by the method of Sokal and Rohlf (37).
 |
RESULTS |
Northern blot analysis of total RNA extracted from Ob cells revealed
HGF/SF transcripts of 6.3, 3.7, and 3.1 kb (Fig.
1). There was a time-dependent increase in
HGF/SF mRNA levels in serum-deprived confluent Ob cells cultured over a
2- to 48-h period. This increase was noted after 24 and 48 h, and it
was prevented by cortisol at 1 µM so that cortisol decreased HGF/SF
mRNA levels from a respective 24-h control value of 1.00 to a value of
0.6 ± 0.05 (SE; n = 13), and from a respective 48-h cortisol
value of 1.00 to a value of 0.3 ± 0.04 (n = 3), as
determined by densitometry (Fig. 1). The inhibitory effect of cortisol
on HGF/SF mRNA was dose dependent, and continuous treatment of Ob cells
with cortisol at 100 nM and 1 µM for 24 h decreased HGF/SF
transcripts from a control value of 1.00 to values of 0.7 ± 0.04 (SE; n = 7) and 0.6 ± 0.05 (n = 13) (Fig.
2).

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Fig. 1.
Effect of glucocorticoid (GC) cortisol at 1 µM on hepatocyte growth
factor/scatter factor (HGF/SF) mRNA expression in cultures of Ob cells
treated for 2, 6, 24, or 48 h. Total RNA from control ( ) or
cortisol (+)-treated cultures was subjected to Northern blot analysis
and hybridized with an -32P-labeled HGF/SF cDNA. Blot
was stripped and rehybridized with labeled murine 18S cDNA. HGF/SF mRNA
was visualized by autoradiography and is shown above; 18S mRNA
is shown below.
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Fig. 2.
Effect of GC cortisol at 0.01-1 µM on HGF/SF mRNA expression in
cultures of Ob cells treated for 24 h. Total RNA from control (0) or
cortisol (GC)-treated cultures was subjected to Northern blot analysis
and hybridized with an -32P-labeled HGF/SF cDNA. Blot
was stripped and rehybridized with labeled murine 18S cDNA. HGF/SF mRNA
was visualized by autoradiography and is shown above;
18S mRNA is shown below.
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Cortisol inhibited control and growth factor-induced expression
of HGF/SF. Confirming previous observations in MC3T3 cells, FGF-2
at 2 nM and PDGF BB at 3.3 nM for 24 h increased HGF/SF mRNA levels in
Ob cells, and cortisol at 1 µM for 24 h decreased the induction of
HGF/SF mRNA levels by the two growth factors (Fig.
3) (4). The constitutive expression of
HGF/SF in serum-deprived MC3T3 cells is minimal; therefore, FGF-2 and
PDGF BB tend to cause a more pronounced relative stimulatory effect on
HGF/SF in MC3T3 than in Ob cells compared with control untreated
cultures (Figs. 3 and 4). The stimulatory
effect of FGF-2 at 2 nM and PDGF BB at 3.3 nM in MC3T3 cells also was
opposed by cortisol at 1 µM for 24 h (Fig. 4).

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Fig. 3.
Effect of fibroblast growth factor-2 (FGF-2) at 2 nM and
platelet-derived growth factor BB (PDGF BB) at 3.3 nM, in the absence
( ) and in the presence (+) of GC cortisol at 1 µM, on HGF/SF
mRNA expression in cultures of Ob cells treated for 24 h. Total RNA
from control, PDGF BB, FGF-2, and cortisol (GC)-treated cultures was
subjected to Northern blot analysis and hybridized with an
-32P-labeled HGF/SF cDNA. Blot was stripped and
rehybridized with labeled murine 18S cDNA. HGF/SF mRNA was visualized
by autoradiography and is shown above; 18S mRNA is shown
below.
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Fig. 4.
Effect of FGF-2 (FGF) at 2 nM and FDGF BB (BB) at 3.3 nM in absence
( ) and in presence (+) of GC cortisol at 1 µM on HGF/SF mRNA
expression in cultures of MC3T3 cells treated for 24 h. Total RNA from
control, FGF-2, PDGF BB, and cortisol (GC)-treated cultures was
subjected to Northern blot analysis and hybridized with an
-32P-labeled HGF/SF cDNA. Blot was stripped and
rehybridized with labeled murine 18S cDNA. HGF/SF mRNA was visualized
by autoradiography and is shown above; 18S mRNA is shown
below.
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The levels of immunoreactive HGF/SF in control untreated and
cortisol-treated Ob and MC3T3 cells were below the limit of detection of the assay, which is 0.4 ng/ml. Neither FGF-2 at 2 nM nor PDGF BB at
3.3 nM for 24 or 48 h caused a detectable increase in immunoreactive HGF/SF in Ob cells, so that the inhibitory effect of cortisol on
immunoreactive HGF/SF could not be tested in these cells. In contrast,
FGF-2 and PDGF BB increased HGF/SF polypeptide levels in MC3T3 cells
treated for 48 h, and the effect was opposed by cortisol (Table
1).
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Table 1.
Effect of FGF-2 and PDGF BB in the presence and absence of cortisol on
HGF/SF levels in cultures of MC3T3 cells
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Northern blot analysis of total RNA extracted from confluent cultures
of Ob cells revealed a predominant c-met transcript of 8.6 kb (Fig.
5). Continuous treatment of Ob cells with
cortisol caused a time-dependent increase in c-met steady-state mRNA
levels. The effect was first consistently observed after 6 h of
exposure to cortisol at 1 µM and was sustained for 48 h. Treatment
with cortisol increased c-met mRNA levels by 2.5 ± 0.3 (SE; n
= 6-9), 2.5 ± 0.2, and 2.5 ± 0.1 multiples of increase
after 6, 24, and 48 h, respectively, as determined by densitometry
(Fig. 5). The effect of cortisol was dose dependent, and continuous
treatment of Ob cells with cortisol for 24 h at 10 nM, 100 nM, and 1 µM increased c-met transcripts by 2.0 ± 0.4 (SE; n = 4),
3.3 ± 0.9, and 3.6 ± 0.6 multiples of increase,
respectively (Fig. 6).

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Fig. 5.
Effect of GC cortisol at 1 µM on c-met mRNA expression in cultures of
Ob cells treated for 2, 6, 24, or 48 h. Total RNA from control
( ) or cortisol (+)-treated cultures was subjected to Northern
blot analysis and hybridized with an -32P-labeled c-met
cDNA. Blot was stripped and rehybridized with labeled murine 18S cDNA.
c-met mRNA was visualized by autoradiography and is shown
above; 18S mRNA is shown below.
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Fig. 6.
Effect of GC cortisol at 0.01-1 µM on c-met mRNA expression in
cultures of Ob cells treated for 24 h. Total RNA from control (0) or
cortisol (GC)-treated cultures was subjected to Northern blot analysis
and hybridized with an -32P-labeled c-met cDNA. Blot was
stripped and rehybridized with labeled murine 18S cDNA. c-met mRNA was
visualized by autoradiography and is shown above; 18S mRNA is
shown below.
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To determine possible mechanisms involved in the regulation of HGF/SF
and c-met by glucocorticoids, we examined whether or not the effects
were protein synthesis dependent, and whether they occurred at the
transcriptional or posttranscriptional level. To determine whether the
effect of cortisol was dependent on protein synthesis, Ob cells were
treated with cortisol at 1 µM for 24 h in the presence or absence of
cycloheximide at 3.6 µM, a dose previously shown to block protein
synthesis in osteoblasts (11). Densitometric analysis revealed that, in
an experiment in which cortisol decreased HGF/SF mRNA levels from
control values of 1.00 to 0.6 ± 0.1 (SE; n = 3),
cycloheximide increased HGF/SF mRNA levels to 1.7 ± 0.2 in the
absence, and to 2.0 ± 0.2 in the presence of cortisol. Consequently,
the inhibitory effect of cortisol could not be detected in the presence
of cycloheximide, although the results are difficult to interpret
because of the accumulation of HGF/SF mRNA in cycloheximide-treated
cells. This accumulation or superinduction of transcripts in the
presence of protein synthesis inhibitors is usually attributed to the
inhibition of RNA-degrading enzymes (1, 6). The effect of cortisol on
c-met mRNA levels appeared to be independent of de novo protein
synthesis, because treatment with cycloheximide increased c-met mRNA
levels and enhanced the stimulatory effect of cortisol (Fig.
7). To determine whether cortisol decreased
HGF/SF or increased c-met mRNA levels by changing transcript stability,
cultures of Ob cells were exposed to cortisol at 1 µM for 1-4 h
and then treated with the RNA polymerase II inhibitor DRB for 30 min to
18 h (45). About 75% of Ob cells are viable in the presence of DRB for
24 h, as determined by trypan blue exclusion (Canalis, unpublished
observations). The half-lives of both HGF/SF and c-met mRNA in
transcriptionally arrested osteoblasts were ~3-4 h, and these
were not significantly altered by cortisol (Fig.
8). To determine the effect of cortisol
on the rate of transcription of the HGF/SF and c-met genes, nuclear
run-on assays were performed. Nuclei isolated from Ob cells exposed to
control medium or cortisol at 1 µM for 2, 6, or 24 h revealed that
cortisol did not cause a detectable change in the rate of HGF/SF
transcription but increased the rate of c-met transcription by about
twofold (Fig. 9).

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Fig. 7.
Effect of GC cortisol at 1 µM, in absence and in presence of
cycloheximide (Cx) at 3.6 µM, on c-met mRNA expression in cultures of
Ob cells treated for 24 h. Total RNA from control ( ) and
cortisol (GC)-, and cycloheximide (CX)-treated cultures was subjected
to Northern blot analysis and hybridized with an
-32P-labeled c-met cDNA. Blot was stripped and
rehybridized with labeled murine 18S cDNA. c-met mRNA was visualized by
autoradiography and is shown above; 18S mRNA is shown
below.
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Fig. 8.
Effect of GC cortisol at 1 µM on HGF/SF (A) or c-met
(B) mRNA decay in transcriptionally arrested Ob cells. Cultures
were treated with cortisol for 1-2 h (HGF/SF) or for 4 h (c-met)
before and 30 min to 18 h after addition of 5,6-dichlorobenzimidazole
riboside (DRB). RNA was subjected to Northern blot analysis, hybridized
with -32P-labeled HGF/SF (A) or c-met
(B) cDNAs, visualized by autoradiography, and quantitated by
densitometry. Ethidium bromide staining of ribosomal RNA was used to
check for uniform loading of the gels and transfer. Values are means ± SE for 3-6 cultures. Values were obtained by densitometric
scanning and are presented as percentage of HGF/SF or c-met mRNA levels
relative to time of DRB addition. Slopes were analyzed by the method of
Sokal and Rohlf and found to be statistically not different.
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Fig. 9.
Effect of GC cortisol at 1 µM on HGF/SF (HGF) and c-met transcription
rates in cultures of Ob cells treated for 2, 6, or 24 h. Nascent
transcripts from control ( ) and cortisol (+)-treated cultures
were labeled in vitro with [ 32P]UTP, and
labeled RNA was hybridized to immobilized cDNA for HGF/SF or c-met.
Murine 18S cDNA was used to demonstrate loading.
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DISCUSSION |
Recent studies have shown that glucocorticoids have significant effects
on the number and function of osteoblasts, acting through a variety of
mechanisms (8, 13, 44). The present investigation was undertaken to
determine whether cortisol regulates the expression of HGF/SF and c-met
in osteoblasts. There was a time-related increase in HGF/SF mRNA levels
in serum-deprived Ob cell cultures, and this increase was probably due
to an accumulation of endogenous growth factors, such as FGF and PDGF,
which are synthesized by skeletal cells and can enhance HGF/SF
synthesis in osteoblasts (4, 10, 35). Cortisol prevented the
time-related increase, causing a relative decrease in HGF/SF mRNA
levels in osteoblasts. Cortisol also decreased the induction of HGF/SF
transcripts by FGF-2 and PDGF BB in Ob and MC3T3 cells.
Cycloheximide superinduced HGF/SF transcripts in the presence and
absence of cortisol, suggesting the inhibition of HGF/SF mRNA-degrading
enzymes, which could be induced by cortisol and could be responsible
for the decrease in HGF/SF mRNA caused by this steroid (1, 6).
Glucocorticoids have been found to induce cytosolic proteins in
osteoblasts, which are responsible for changes in the stability of
other transcripts, such as those of collagenase 3 (15). Cytosolic
proteins are known to bind to AU-rich elements in the RNA, and these
sequences often modulate mRNA stability of other genes (20, 43).
Although cortisol may regulate RNA-binding proteins in osteoblasts, it
is not known whether or not they bind to AU-rich regions of HGF/SF RNA
and whether or not they play a role in the inhibitory effect of
cortisol on HGF/SF mRNA expression. Furthermore, experiments in
transcriptionally blocked Ob cells, by use of the RNA polymerase II
inhibitor DRB, revealed that cortisol did not destabilize HGF/SF
transcripts (45). It is possible that cortisol destabilizes HGF/SF
mRNA, but the effect was not detectable under conditions of
transcriptional arrest, which may have suppressed the expression of
genes coding for proteins required to regulate HGF/SF transcript
stability. Our data are not conclusive, because it was not possible to
demonstrate a decrease in the rate of HGF/SF transcription. This could
be due to lack of a transcriptional effect or lack of sufficient sensitivity for the detection of an inhibitory effect. Similar difficulties were encountered to prove a transcriptional effect of
FGF-2 on HGF/SF expression in osteoblasts and of various cytokines in
fibroblasts (4, 41).
The levels of immunoreactive HGF/SF in control and growth
factor-induced Ob cells were below the limit of detection with use of
currently available assays, so that we could not demonstrate a decrease
in HGF/SF levels by cortisol in Ob cells. However, detectable levels of
HGF/SF were achieved in MC3T3 cells after induction with FGF-2 and PDGF
BB. HGF/SF levels in MC3T3 cells were suppressed by cortisol, revealing
that this steroid has the capability to reduce HGF/SF synthesis in
osteoblasts. It is not clear why HGF/SF levels can be induced to a
greater extent in MC3T3 than in Ob cells, but differences in the level
of growth factor expression between Ob and MC3T3 cells are not uncommon (19).
In contrast to the inhibitory effects on HGF/SF expression, cortisol
caused a time- and dose-dependent increase in c-met mRNA levels in Ob
cells. Cycloheximide superinduced c-met transcripts and had an additive
effect to that of cortisol, suggesting the presence of c-met
mRNA-degrading enzymes in Ob cell cultures. The effect of cortisol on
c-met occurred by transcriptional mechanisms, because cortisol caused
no change in the half-life of the transcript in transcriptionally
arrested cells and increased the rate of transcription.
In our study, the effects of cortisol on HGF/SF and c-met expression
were observed at doses that modify other parameters of metabolic
function in Ob cells, suggesting that the effect is physiologically
relevant. Glucocorticoids have complex effects on bone remodeling and
have a major impact on bone formation. The inhibitory actions of
glucocorticoids on bone formation are secondary to a decrease in bone
cell replication, to a decrease in bone collagen synthesis, and to an
increase in collagenase 3 expression (7, 14, 15). In addition, some of
the actions of glucocorticoids are due to modifications in the
synthesis of growth factors produced by skeletal cells or alterations
in receptor binding or binding proteins (8, 12, 18, 34). The decrease in HGF/SF expression by cortisol may explain selected actions of
glucocorticoids in bone, and it may be particularly relevant to the
impaired healing of tissues exposed to glucocorticoids.
FGF-2 and PDGF BB stimulate the replication of cells of the
osteoblastic lineage and have been implicated in wound and fracture repair; the two growth factors induced HGF/SF expression, an effect attenuated by glucocorticoids (9, 16, 23, 28, 30). This, in conjunction
with the known effect of HGF/SF in tissue repair, would suggest a role
for HGF/SF in bone repair, which can be opposed by glucocorticoids (26,
38). This effect may serve, in part, to explain the inhibitory actions
of glucocorticoids on wound and fracture healing. The induction of
c-met by glucocorticoids may be a compensatory mechanism to maintain
HGF/SF function in bone. Whereas the induction of c-met by
glucocorticoids seems unique to osteoblasts, the decrease in HGF/SF
production also occurs in bone marrow stromal cells and fibroblasts
(27, 40).
In conclusion, cortisol decreases the synthesis of HGF/SF and increases
c-met expression in osteoblasts. These effects may play a role in the
inhibitory actions of glucocorticoids on bone formation and repair.
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ACKNOWLEDGEMENTS |
We thank Dr. T. Nakamura for the rat HGF/SF cDNA clone, Dr.
C. C. Lee for the mouse c-met cDNA, the Genetics Institute
for BMP-2, Sheila Rydziel for technical advice, Cathy Boucher and Deena
Durant for technical assistance, and Charlene Gobeli and Karen Berrelli
for secretarial help.
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FOOTNOTES |
This study was supported by Grant DK-45227 from the National Institute
of Diabetes and Digestive and Kidney Diseases.
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,
Department of Research, Saint Francis Hospital and Medical Center,
114 Woodland St., Hartford, CT 06105-1299
(E-mail:ecanalis{at}stfranciscare.org).
Received 13 July 1999; accepted in final form 22 October 1999.
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