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(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) ( 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
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
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
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
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
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 [
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
[
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
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
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
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 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.
Volume 271,
Number 15,
Issue of April 12, 1996 pp. 9033-9038
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)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) .
Culture Technique
The culture method used has
been described in detail previously(19) . Parietal bones were
obtained from 22-day-old fetal rats immediately after the mothers were
killed 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). Cell populations harvested from the third to the fifth
digestions were cultured as a pool and were previously shown to have
osteoblastic characteristics. Ob cells were plated at a density of
8,000-12,000 cells/cm
and cultured in a humidified 5%
CO incubator at 37 °C until reaching confluence
(
50,000 cells/cm). For the nuclear run-on experiments,
first passage cultures were used. For transient transfections,
subconfluent primary cultures were used. Cells were cultured in
Dulbecco's modified Eagle's medium (DMEM) supplemented with
nonessential amino acids (Life Technologies, Inc.) and 10% fetal bovine
serum (Hyclone Laboratories, Logan, UT). At confluence (subconfluence
for transfection experiments), the cells were rinsed and transferred to
serum-free medium for 18-24 h, when they were again rinsed with
serum-free medium and exposed to test or control medium in the absence
of serum for 2-24 h. Cortisol (Sigma) was dissolved in ethanol
and diluted 1:10,000 or greater in DMEM; cycloheximide (Sigma) was
added directly to the medium. 5,6-Dichlorobenzimidazole riboside (DRB)
(Sigma) was dissolved in absolute ethanol and diluted 1:200 in DMEM,
and 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 -80
°C. For the nuclear run-on assay, nuclei were isolated by Dounce
homogenization. For protein analysis, the extracellular matrix was
extracted and processed for Western blots.Northern Blot Analysis
Total cellular RNA was
isolated with guanidine thiocyanate, at acid pH, followed by
phenol/chloroform (Sigma) extraction(20) . RNA was precipitated
with isopropyl alcohol, resuspended, and reprecipitated with ethanol.
The RNA recovered was quantitated by spectrometry, and equal amounts of
RNA from control or test samples were loaded on a formaldehyde-agarose
gel following 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 GeneScreen Plus charged nylon (DuPont NEN), and uniformity of
transfer was confirmed by revisualization of ribosomal RNA. A 300-base
pair HindIII restriction fragment of the rat IGFBP-5 cDNA
(kindly provided by Dr. S. Shimasaki, The Whittier Institute for
Diabetes and Endocrinology, La Jolla, CA) was purified by agarose gel
electrophoresis(21, 22) . IGFBP-5 cDNA was labeled
with [
-
P]dCTP and
[-P]dATP) (50 µCi each at a specific
activity of 3,000 Ci/mmol; DuPont NEN) using the random
hexanucleotide-primed second strand synthesis method(23) .
Hybridizations were carried out at 42 °C for 16-72 h, and
post-hybridization washes were performed at 65 °C in 0.1 
SSC. The blots were stripped and rehybridized with a 752-bp BamHI-SphI restriction fragment of the gene encoding
murine 18 S ribosomal RNA (American Type Culture Collection, Rockville,
MD) under the same conditions. The bound radioactive material was
visualized by autoradiography on Kodak X-AR5 film employing Cronex
Lightning Plus intensifying screens (DuPont NEN). Relative
hybridization levels were determined by densitometry. Northern analyses
shown are representative of three or more cultures.mRNA Stability
To determine the half-life of mRNA
and to examine changes in transcript stability, confluent cultures of
Ob cells were exposed to control or cortisol for 1 h and then to the
RNA polymerase II inhibitor DRB in the presence or absence of cortisol (24) . Cultures were harvested at intervals and processed for
Northern blotting and densitometry. The amount of mRNA present was
plotted using linear regression, and the slopes were compared by the
method of Sokal and Rolf(25) .hnRNA Analysis
To examine changes in hnRNA,
specific primers were designed to amplify DNA spanning the junction
between intron 1 and exon 1 of the IGFBP-5 gene, in accordance with
published sequences(21, 22, 26) . Based on
these sequences, a sense exon 1-specific amplimer
(5`-AAAGCTCTGTCCATGTGTC-3`) and an antisense intron 1-specific amplimer
(5`-AAACCCCAGTAGCGCTCAC-3`) were synthesized. To determine changes in
IGFBP-5 hnRNA, reverse transcription-polymerase chain reaction (PCR) (27, 28) was used. Total RNA from control and test
samples was prepared as described for Northern analysis. One µg of
RNA was treated with amplification-grade DNase I and
reverse-transcribed in the presence of the antisense intron 1-specific
amplimer at 42 °C for 30 min with Moloney murine leukemia virus
reverse transcriptase (Life Technologies, Inc.). The newly transcribed
cDNA was amplified by 20 PCR cycles of 94 °C/1 min, 56 °C/1
min, and 72 °C/1 min following the addition of the sense exon
1-specific amplimer, Taq DNA polymerase, and 5 µCi of
[-P]dCTP (3,000 Ci/mmol; DuPont
NEN)(27, 28) . The PCR products were fractionated by
electrophoresis on a 6% polyacrylamide denaturing gel, visualized by
autoradiography, and quantitated by densitometry. The PCR product
increased linearly with increasing amounts of RNA. Five fg of an
internal DNA standard were included in the PCR to correct for
variations in amplification. The standard was obtained by amplification
of SV40 promoter sequences in the pGL2-P plasmid DNA (Promega, Madison,
WI) using the composite sense primer
5`-AAAGCTCTGTCCATGTGTCattagtcagcaaccatagtc-3` and the composite
antisense primer 5`-AAACCCCAGTAGCGCTCACggttccatcctctagaggat-3` (the
upper-case letters indicate IGFBP-5 sequences, and the lower-case
letters represent SV40 sequences in the pGL2-P plasmid). The DNA was
gel-purified and used as an internal standard during the PCR for hnRNA.
To determine the variability of the procedure, Ob cell RNA was pooled;
independent aliquots were reversed-transcribed and amplified by PCR;
and IGFBP-5 hnRNA was quantitated by densitometry, which revealed a
coefficient of variation of 11% (n = 13) for the assay. Nuclear Run-on Assay
To examine changes in the
rate of transcription, nuclei were isolated by Dounce homogenization in
Tris buffer, pH 7.4, containing 0.5% Nonidet P-40. Nascent transcripts
were labeled by incubation of nuclei in reaction buffer containing 500
µM ATP, 500 µM CTP, 500 µM GTP,
150 units of RNasin (Promega), and 250 µCi of
[P]UTP (3,000 Ci/mmol; DuPont NEN)(29) .
RNA was isolated by treatment with DNase I and proteinase K, followed
by phenol/chloroform extraction and ethanol precipitation. Linearized
plasmid DNA containing 1 µg of cDNA was immobilized onto
GeneScreen Plus by slot blotting according to the manufacturer's
directions. The plasmid vector pGL2-basic (Promega) was used as a
control for nonspecific hybridization. Rat glyceraldehyde-3-phosphate
dehydrogenase cDNA (kindly provided by R. Wu, Cornell University,
Ithaca, NY) was used to confirm equal application of
[P]RNA, representing various experimental
conditions, to the membranes(30) . Equal counts/minute of
[P]RNA from each sample were hybridized to cDNAs
at 42 °C for 72 h and washed in 1 SSC at 65 °C for 20
min. Hybridized cDNAs were visualized by autoradiography.Deletion Constructs and Site-directed
Mutagenesis
To determine changes in promoter activity, deletion
constructs were made by digestion of the murine IGFBP-5 promoter
(kindly provided by P. Rotwein, Washington University School of
Medicine, St. Louis, MO) with restriction enzymes. Internal mutations
were prepared by PCR from the smallest deletion construct, bp -70
to +120, using oligonucleotide primers containing the various
mutations in the 5`-end. A 3`-truncation of the IGFBP-5 promoter was
also prepared by PCR through the generation of a new restriction site
at bp +22. Deletion constructs and PCR products were purified and
cloned into the luciferase construct pGL2-Basic(24) . All DNA
products generated by PCR were sequenced to confirm mutations and to
eliminate the possibility of unintended mutations or deletions.Transient Transfections
Ob cells were cultured to
70% confluence and transiently transfected with IGFBP-5 deletion
and mutation constructs by calcium phosphate-DNA coprecipitation as
described(31) . After 4 h, cells were exposed for 3 min to 10%
glycerol. Ob cells were allowed to recover in serum-containing DMEM for
20 h, serum-deprived for 18 h, and exposed to control or test medium as
described below and in the figure legends. Cells were washed with
phosphate-buffered saline and harvested in reporter lysis buffer
(Promega). To control for transfection efficiency, cells were
cotransfected with a construct containing the -galactosidase gene
driven by the cytomegalovirus promoter (pCMV-Gal, CLONTECH, Palo
Alto, CA). Luciferase and -galactosidase activities were measured
using a luciferase assay kit (Promega) and a -galactosidase assay
using Galacton reagent (Tropix Inc., Bedford, MA), both in accordance
with the manufacturers' instructions. Data are expressed as means
± S.E. of luciferase activity corrected for -galactosidase
activity. Statistical differences were determined by analysis of
variance and post hoc examination by Ryan-Einot-Gabriel-Welch
F test(32, 33) .Western Blot Analysis
Extracellular matrix was
prepared as described(34, 35) . Briefly, Ob cells were
rinsed in phosphate-buffered saline; cell membranes were removed with
0.5% Triton X-100 (Sigma), pH 7.4; and nuclei and cytoskeleton were
removed by incubation with 25 mM ammonium acetate, pH 9.0, for
5 min. The extracellular matrix was rinsed with phosphate-buffered
saline, extracted with Laemmli sample buffer containing 2% sodium
dodecyl sulfate, and fractionated by polyacrylamide gel electrophoresis
on a 12% denaturing gel(36) . Proteins were transferred to
Immobilon P membranes (Millipore Corp., Bedford, MA), blocked with 2%
bovine serum albumin, and exposed to a 1:500 dilution of rabbit
antiserum raised against native human IGFBP-5 (Upstate Biotechnology
Inc., Lake Placid, NY) in 1% bovine serum albumin overnight. Blots were
exposed to horseradish peroxidase-conjugated goat anti-rabbit IgG
antiserum, washed, and developed with a horseradish peroxidase
chemiluminescent detection reagent (DuPont NEN). The presence of
IGFBP-5 was confirmed by comigration with recombinant human IGFBP-5
(Austral, San Ramon, CA). Western immunoblots are representative of
four cultures.
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).
-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.
-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.
-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.
-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
(
).
-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.
-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.
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.
-galactosidase (BGal) activity and represent means ±
S.E. (n = 6). *, significantly different from control (p < 0.05).
-galactosidase (BGal) activity and represent means
± S.E. (n = 6). *, significantly different from
control (p < 0.05). C/EBP ,
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.
)
We thank Dr. S. Shimasaki for the rat IGFBP-5 cDNA
clone, Dr. R. Wu for the rat glyceraldehyde-3-phosphate dehydrogenase
cDNA clone, Dr. P. Rotwein for the IGFBP-5 promoter construct, and
Cathy Boucher and Deena Durant for technical assistance.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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