©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Cortisol Inhibits the Synthesis of Insulin-like Growth Factor-binding Protein-5 in Bone Cell Cultures by Transcriptional Mechanisms (*)

(Received for publication, November 17, 1995; and in revised form, February 2, 1996 )

Bari Gabbitas (1) James M. Pash (1) (2) Anne M. Delany (1) (2) Ernesto Canalis (1) (2)(§)

From the  (1)Departments of Research and Medicine, Saint Francis Hospital and Medical Center, Hartford, Connecticut 06105 and the (2)University of Connecticut School of Medicine, Farmington, Connecticut 06030

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Skeletal cells secrete insulin-like growth factors (IGFs) (^1)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 beta, 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.


MATERIALS AND METHODS

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^2 and cultured in a humidified 5% CO(2) incubator at 37 °C until reaching confluence (50,000 cells/cm^2). 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 [alpha-P]dCTP and [alpha-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 times 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 [alpha-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 times 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 beta-galactosidase gene driven by the cytomegalovirus promoter (pCMVbeta-Gal, CLONTECH, Palo Alto, CA). Luciferase and beta-galactosidase activities were measured using a luciferase assay kit (Promega) and a beta-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 beta-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.


RESULTS

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 alpha-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 alpha-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 alpha-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 alpha-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 (circle) or DRB plus cortisol (bullet).




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 [alpha-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 [alpha-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 alpha 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 beta-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 beta-galactosidase (BGal) activity and represent means ± S.E. (n = 6). *, significantly different from control (p < 0.05). C/EBP alpha, CCAAT/enhancer-binding protein alpha.




DISCUSSION

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 alpha 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.


FOOTNOTES

*
This work was supported by NIDDK Grant DK45227. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Research, Saint Francis Hospital and Medical Center, 114 Woodland St., Hartford, CT 06105-1299. Tel.: 203-548-4068; Fax: 203-548-5415.

(^1)
The abbreviations used are: IGFs, insulin-like growth factors; IGFBP, IGF-binding protein; DMEM, Dulbecco's modified Eagle's medium; DRB, 5,6-dichlorobenzimidazole riboside; bp, base pair(s); PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

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.


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