(Received for publication, November 17, 1995; and in revised form, February 2, 1996 )
From the
Glucocorticoids inhibit the synthesis of insulin-like growth factor-binding protein-5 (IGFBP-5) in osteoblasts, but the mechanisms involved are unknown. IGFBP-5 stimulates bone cell growth, and its inhibition by glucocorticoids may be relevant to the action of this binding protein on bone formation. We tested the effects of cortisol on IGFBP-5 expression in cultures of osteoblast-enriched cells from fetal rat calvariae (Ob cells). Cortisol decreased IGFBP-5 polypeptide levels in the extracellular matrix and caused a time- and dose-dependent decrease in IGFBP-5 mRNA. IGFBP-5 transcripts were markedly decreased by cycloheximide, and further suppressive effects of cortisol could not be determined. Cortisol did not modify the decay of IGFBP-5 mRNA in transcriptionally arrested Ob cells. Cortisol decreased IGFBP-5 hnRNA, the rate of IGFBP-5 transcription, and the activity of the murine IGFBP-5 promoter by 35% in transient transfection experiments. Deletion analysis showed that the region responsive to cortisol is from base pairs -70 to +22, and E-box-binding proteins or c-Myb-related nuclear factors may be involved in its regulation. In conclusion, cortisol inhibits IGFBP-5 transcription in Ob cells through the Myb-binding domain. This effect may be partly responsible for the effect of glucocorticoids on bone formation.
Skeletal cells secrete insulin-like growth factors (IGFs) ()I and II as well as the six known IGF-binding proteins
(IGFBP)(1, 2, 3, 4, 5) .
IGF-I and IGF-II are among the most important local regulators of bone
cell function, and their activity is modified by
IGFBPs(6, 7, 8, 9, 10, 11, 12) .
Although the exact function of IGFBPs in bone is not known, IGFBP-5 is
unique in that it consistently increases bone cell growth and enhances
the anabolic actions of IGF-I in bone(12) . The regulation of
IGFBP-5 synthesis in bone cells is complex, and it is often coordinated
with the regulation of IGF-I and the state of cell differentiation.
Agents known to stimulate bone cell replication, such as transforming
growth factor
, fibroblast growth factor, and platelet-derived
growth factor, inhibit IGF-I and IGFBP-5
synthesis(13, 14) . In contrast, agents that induce
osteoblast cell differentiation, such as retinoic acid and IGF-I,
stimulate IGFBP-5 synthesis in skeletal
cells(11, 15) .
Glucocorticoids are known to have complex effects on bone formation and resorption(16) . Some of these effects are probably due to direct actions of glucocorticoids on specific genes expressed by the osteoblast, whereas others may be indirect(17) . Glucocorticoids inhibit DNA and collagen synthesis in bone cultures and decrease the synthesis of IGF-I and selected IGFBPs in osteoblasts(5, 16, 18) . These effects may play a critical role in the actions of glucocorticoids in bone. Recent studies demonstrated that glucocorticoids inhibit IGFBP-5 mRNA levels in cultured human osteoblasts. However, the mechanism of this effect was not explored, and it could involve transcriptional and post-transcriptional processes (5) . Since the mechanism of glucocorticoid action in bone has remained elusive, it is important to define possible levels of regulation of genes that appear essential to bone cell function.
This study was undertaken to examine the effects of cortisol on IGFBP-5 synthesis in cultures of osteoblast-enriched cells from fetal rat calvariae (Ob cells) and to determine the mechanism of action of cortisol on IGFBP-5 gene expression.
Northern blot analysis of total RNA extracted from confluent
cultures of Ob cells revealed a predominant IGFBP-5 transcript of 6.0
kilobases (Fig. 1). Continuous treatment of Ob cells with
cortisol caused a time-dependent decrease in IGFBP-5 steady-state mRNA
levels. Treatment of Ob cells with cortisol at 1 µM for 2
h had no effect on IGFBP-5 mRNA. However, 6 h of treatment had a small
effect, and 24 h of treatment caused a 51 ± 6% (n = 6) decrease in IGFBP-5 mRNA (Fig. 1). The effect
of cortisol was dose-dependent, and continuous treatment of Ob cells
with cortisol at 10 nM to 1 µM for 24 h inhibited
IGFBP-5 transcripts by 16 ± 13% (n = 4) to 51
± 6% (n = 6) as determined by densitometry (Fig. 2). Western immunoblot analysis of the extracellular
matrix of untreated Ob cells confirmed the presence of a major form of
immunoreactive IGFBP-5 with a molecular mass of 31 kDa, which
comigrated with an IGFBP-5 standard (Fig. 3). The identity of
this protein as an IGFBP was confirmed in previous studies in which the
immunoblot was stripped and the band visualized with I-labeled IGF-II as a ligand(14, 15) .
Cortisol at 1 µM for 24 h decreased the levels of
immunoreactive IGFBP-5 in the extracellular matrix by 44 ± 7% (n = 4). Because IGFBP-5 is found primarily in the
extracellular matrix of Ob cell cultures and its expression in the
medium is low, the detection of an inhibitory effect in the medium is
impractical(14) . To determine whether or not the effects
observed on IGFBP-5 mRNA levels were dependent on protein synthesis,
serum-deprived confluent cultures of Ob cells were treated with
cortisol in the presence or absence of cycloheximide at 3.6
µM. In earlier experiments, cycloheximide at doses of 2
µM and higher was found to inhibit protein synthesis in Ob
cell cultures by 80-85%(37) . Northern blot analysis
revealed that treatment with cycloheximide for 24 h caused a 93
± 8% (n = 4) decrease in IGFBP-5 transcript
levels, so further inhibitory effects of cortisol were difficult to
detect (Fig. 4).
Figure 1:
Effect of
the glucocorticoid cortisol at 1 µM on IGFBP-5 mRNA
expression in cultures of Ob cells treated for 2, 6, or 24 h. Total RNA
from control (C) or cortisol (glucocorticoid (GC))-treated cultures was subjected to Northern blot analysis
and hybridized with -
P-labeled rat IGFBP-5 cDNA.
IGFBP-5 mRNA was visualized by autoradiography and is shown in the upper panels, while 18 S rRNA is shown below. kb,
kilobases.
Figure 2:
Effect of the glucocorticoid cortisol from
10 nM to 1 µM on IGFBP-5 mRNA expression in
cultures of Ob cells treated for 24 h. Total RNA from control or
cortisol (glucocorticoid (CG))-treated cultures was subjected
to Northern blot analysis and hybridized with
-
P-labeled rat IGFBP-5 cDNA. IGFBP-5 mRNA was
visualized by autoradiography and is shown in the upper panel,
while 18 S rRNA is shown below. kb,
kilobases.
Figure 3: Effect of the glucocorticoid cortisol at 1 µM on IGFBP-5 polypeptide levels in cultures of Ob cells treated for 24 h. Extracellular matrix extracts from control (C) and cortisol (glucocorticoid (GC))-treated cultures were subjected to Western immunoblot analysis, and IGFBP-5 was detected using an anti-IGFBP-5 antibody and visualized using a chemiluminescent detection system.
Figure 4:
Effect of the glucocorticoid cortisol at 1
µM in the presence (+) or absence(-) of
cycloheximide at 3.6 µM on IGFBP-5 mRNA expression in
cultures of Ob cells treated for 24 h. Total RNA from control (C) or cortisol (glucocorticoid (GC))-treated
cultures was subjected to Northern blot analysis and hybridized with
-
P-labeled rat IGFBP-5 cDNA. IGFBP-5 mRNA was
visualized by autoradiography and is shown in the upper panel,
while 18 S rRNA is shown below. Cyhex, cycloheximide; kb, kilobases.
To examine whether or not the effect of cortisol on IGFBP-5 mRNA levels was due to changes in transcript stability, Ob cells were exposed to DMEM or cortisol for 60 min and then treated with the RNA polymerase II inhibitor DRB in the absence or presence of cortisol at 1 µM for 6, 16, or 24 h(24) . The half-life of IGFBP-5 mRNA in transcriptionally arrested Ob cells was estimated at 18 h (Fig. 5). Slope analysis indicated no significant difference between control (slope = -0.0154, n = 11) and cortisol-treated (slope = -0.0194, n = 12) cultures(25) . Treatment of Ob cells with cortisol for 6 and 24 h decreased IGFBP-5 hnRNA expression by 68 ± 8% (n = 3) and 23-34% (n = 2), respectively, as estimated by reverse transcription-PCR (Fig. 6). No signal of the hnRNA product was detected in any of the samples tested when the reverse transcription step was omitted prior to the PCR, eliminating the possibility of DNA contamination. To confirm whether cortisol modified the transcription of the IGFBP-5 gene, nuclear run-on assays were performed on nuclei from Ob cells treated with 1 µM cortisol for 2, 6, and 24 h. Although the effect was small at 2 h, cortisol inhibited the rate of IGFBP-5 transcription by 29 ± 7% (n = 3) at 6 h and by 54 ± 4% (n = 3) at 24 h (Fig. 7).
Figure 5:
Effect of cortisol at 1 µM on
IGFBP-5 mRNA decay in transcriptionally blocked Ob cells. Cultures were
treated with DMEM or cortisol 60 min before and 6, 16, or 24 h after
the addition of DRB. RNA was subjected to Northern blot analysis,
hybridized with -
P-labeled rat IGFBP-5 cDNA,
visualized by autoradiography, and quantitated by densitometry.
Ethidium bromide staining of ribosomal RNA was used to check uniform
loading of the gels and transfer. Values are means ± S.E. for
three cultures. Values were obtained by densitometric scanning and are
presented as percentage of IGFBP-5 mRNA levels relative to the time of
DRB addition. The graphs were generated by linear regression, and slope
analysis was performed, indicating no significant difference between
cultures treated with DRB (
) or DRB plus cortisol
(
).
Figure 6:
Effect of the glucocorticoid cortisol at 1
µM on IGFBP-5 hnRNA expression in cultures of Ob cells
treated for 6 or 24 h. Total RNA from control (C) or cortisol
(glucocorticoid (GC))-treated cultures was extracted, and 1
µg was subjected to reverse transcription-PCR in the presence of
IGFBP-5 exon 1- and intron 1-specific sense and antisense primers and
of 5 µCi of [-
P]dCTP. The reverse
transcription-PCR products were fractionated by polyacrylamide gel
electrophoresis and visualized by autoradiography. The sizes of the PCR
products were confirmed as 260 bp for IGFBP-5 hnRNA and 318 bp for the
internal standard using a radiolabeled DNA ladder. IGFBP-5 hnRNA (bottom) and the internal standard (top), prepared as
described under ``Materials and Methods,'' are both
shown.
Figure 7:
Effect of the glucocorticoid cortisol at 1
µM on IGFBP-5 transcription rates in cultures of Ob cells
treated for 2, 6, and 24 h. Nascent transcripts from control (C) or cortisol (glucocorticoid (GC))-treated
cultures were labeled in vitro with
[-
P]UTP, and the labeled RNA was hybridized
to immobilized cDNA for IGFBP-5. Rat glyceraldehyde-3-phosphate
dehydrogenase (GAPD) cDNA was used to demonstrate loading, and
pGL2-Basic vector DNA was used as a control for nonspecific
hybridization.
The ability of cortisol to
regulate putative promoter regions of the IGFBP-5 gene in Ob cells was
examined using transient transfections of luciferase constructs
containing IGFBP-5 promoter sequences spanning bp -2695 to
+120. Deletion constructs from bp -2695 to +120 to bp
-70 to +120 (Fig. 8, A and B)
showed a 35% decrease in IGFBP-5 promoter activity when treated with
cortisol at 1 µM for 6 h (Fig. 8C). The
reverse orientation of the largest construct, bp +120 to
-2695, yielded little luciferase activity and no inhibition by
cortisol. Site-directed mutations and a 3`-truncation of the bp
-70 to +120 deletion construct were generated by PCR and
used to further analyze the responsive regions of the IGFBP-5 promoter.
A putative CAAT motif was mutated near the 5`-end, and a truncation
from the 3`-end of the construct was made that eliminated a potential
binding site for a nuclear factor for interleukin-6 expression (NFIL-6)
(T(G/T)NNGNTT(G/T)) (Fig. 9, A and B). In
addition, a region that contains a putative CCAAT/enhancer-binding
protein binding motif (T(T/G)NNG(C/T)AA(T/G)) was selected for
mutation. In a representative experiment (n = 6), these
mutated constructs each showed a 40-50% (p < 0.05)
decrease in promoter activity in response to 1 µM cortisol
for 6 h (Fig. 9C). In contrast, mutation of a consensus
binding site for E-box proteins or c-Myb ((T/C)AAC(G/T)G) abrogated the
inhibitory effect of 1 µM cortisol on IGFBP-5 promoter
activity.
Figure 8:
A, murine IGFBP-5 promoter showing
restriction sites used for deletion constructs. B, IGFBP-5
promoter constructs made by successive deletions from the 5`-end with
restriction enzymes. C, effect of the glucocorticoid (GC) cortisol at 1 µM on IGFBP-5 promoter
activity in transiently transfected Ob cells. Cultures were transfected
with pGL2-Basic containing the deletion constructs shown in B and exposed to DMEM (white bars) or cortisol (striped
bars) for 6 h. Bars indicate luciferase units normalized
to -galactosidase (BGal) activity and represent means ±
S.E. (n = 6). *, significantly different from control (p < 0.05).
Figure 9:
A, murine IGFBP-5 promoter from bp
-70 to +120 showing enhancer elements, potential
transcription factor-binding sites, and two sites for the start of
transcription (indicated by arrows). B, wild-type (WT) IGFBP-5 promoter from bp -70 to +120 is shown
above; areas of interest are underlined, with mutated
sequences shown in boldface below. A 3`-truncation is
represented at the bottom. C, effect of the glucocorticoid (GC) cortisol at 1 µM on IGFBP-5 promoter
activity in transiently transfected Ob cells. Cultures were transfected
with pGL2-Basic containing the constructs shown in B and
exposed to DMEM (white bars) or cortisol (striped
bars) for 6 h. A larger construct in reverse orientation, bp
+120 to -2695, was used as a vector control (Rev). Bars indicate luciferase units normalized to
-galactosidase (BGal) activity and represent means
± S.E. (n = 6). *, significantly different from
control (p < 0.05). C/EBP
,
CCAAT/enhancer-binding protein
.
Recent studies have demonstrated that cortisol decreases the
synthesis of IGF-I and IGFBP-5 in skeletal cells, and this
investigation was undertaken to determine the mechanism by which
cortisol inhibits IGFBP-5 expression in calvaria-derived Ob cells. We
demonstrated that cortisol decreases IGFBP-5 mRNA levels in Ob cells in
a time- and dose-dependent manner. The basal expression of IGFBP-5
requires protein synthesis, and it was not possible to determine
whether the effect of cortisol on IGFBP-5 was protein
synthesis-dependent. Experiments in transcriptionally blocked Ob cells
revealed that cortisol did not modify IGFBP-5 mRNA
stability(24) . This, in conjunction with a decrease in hnRNA
levels and in rates of transcription, indicates that cortisol inhibits
IGFBP-5 expression at the transcriptional level. Although cortisol
inhibited both the levels of hnRNA and the rates of transcription, the
effect on hnRNA was more pronounced after 6 h, whereas the effect on
the rates of transcription was more evident after 24 h. Although
changes in hnRNA frequently match changes in the rate of transcription,
hnRNA levels also reflect RNA processing, which could account for the
differences observed. Cortisol also inhibited the activity of murine
IGFBP-5 promoter constructs driving a luciferase reporter gene in
transiently transfected Ob cells. The elements responsible for the
suppression of the IGFBP-5 promoter are located between bp -70
and +22, and the putative E-box or Myb motif is required for basal
transcription and cortisol-mediated transcriptional repression. The
Myb-binding site has been shown to be responsible for a major
gel-shifted band in the IGFBP-5 promoter, but its exact function has
yet to be determined(38) . The region between bp -70 and
+22 contains a CCAAT/enhancer-binding protein consensus
binding sequence and a CAAT motif, but mutations of these binding
sequences did not eliminate the cortisol response, and it is unlikely
that they play an important role in this process. There is also a
potential binding site for AP-2, which was not evaluated by specific
mutation, but constructs were not responsive to cortisol when the Myb
site alone was altered and the AP-2 site was left intact, indicating
that AP-2 is probably not involved in IGFBP-5 regulation by cortisol.
Although neither c-Myb- nor E-box-binding sites have been reported to
act as negative glucocorticoid-responsive elements, constitutive
expression of Myb increases IGF-I and IGF-I receptor mRNAs by
transcriptional mechanisms in fibroblasts(39) . Glucocorticoids
may alter the expression or activity of Myb in bone cells. Our results
indicate that Myb may have effects on the IGF-IGFBP axis that play an
important role in mediating the effects of glucocorticoids in the
skeletal system.
Intact IGFBP-5 is primarily present in the extracellular matrix of skeletal and nonskeletal cells, and cortisol decreased IGFBP-5 in this compartment(14, 35) . The amount of IGFBP-5 secreted to the culture medium of Ob cells under the described culture conditions is small, and peptide degradation is known to occur. Modification of IGFBP-5 protease concentration or activity is another level of regulation by which cortisol could modify IGFBP-5 polypeptide levels in bone cells. Recently, it was shown that IGFBP-5 is degraded by calcium-dependent serine proteases and by matrix metalloproteinases(40, 41) . Cortisol increases the levels of collagenase-3 mRNA by post-transcriptional mechanisms in osteoblasts and increases the synthesis of the enzyme(42) . Consequently, cortisol may also increase IGFBP-5 degradation. IGFBP-5 fragments were not detected in Western immunoblots of extracellular matrix proteins from cortisol-treated cells. Perhaps this is because the cortisol effect on collagenase-3 mRNA in osteoblasts is maximally observed after 24-48 h and the cells were studied only up to 24 h or because limited IGFBP-5 degradation occurs in the extracellular matrix and fragments are released to the medium.
The effects of cortisol on IGFBP-5 synthesis were observed at physiological doses, at doses that modify other parameters of metabolic function in Ob cells, and at doses that are known to inhibit IGF-I synthesis(16, 18) . This suggests that the inhibition of IGFBP-5 synthesis may be physiologically relevant. IGFBP-5 stimulates bone cell replication, and its expression is coordinated with stages of osteoblast cell growth and, to an extent, with IGF-I expression(18, 43) . Since cortisol inhibits multiple parameters of bone formation, including cell growth, it is possible that the inhibition of IGFBP-5 is mechanistically important to the actions of cortisol in bone. IGFBP-5 associated with the extracellular matrix of fibroblasts enhances IGF-I actions on cell growth(35) . This is also probably the case with osteoblasts since IGFBP-5 is known to enhance the effect of IGF-I on osteoblast cell replication, and the reduction of IGFBP-5 levels by cortisol in the extracellular matrix may be a mechanism by which cortisol decreases the skeletal effects of IGF-I. Although glucocorticoids have a number of actions on bone metabolism that are independent of their effects on the IGF-IGFBP axis, the inhibition of IGF-I and IGFBP-5 synthesis in osteoblasts may be relevant to the actions of cortisol on bone cell function.
In conclusion, this study demonstrates that cortisol inhibits IGFBP-5 mRNA and polypeptide levels in skeletal cells through mechanisms that involve diminished transcription. The gene elements responsible for this effect are located between bp -70 and +22 in the IGFBP-5 promoter, and E-box-binding proteins or c-Myb-related nuclear factors may be involved. The cortisol-reduced level of IGFBP-5 in the bone microenvironment may be relevant to its inhibitory actions on bone formation.