Cortisol inhibits the synthesis of insulin-like growth factor-binding protein-5 in bone cell cultures by transcriptional mechanisms.

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) 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 -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.

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 (ϳ50,000 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 Ϫ80°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 quan-titated 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 Gene-Screen 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 [␣-32 P]dCTP and [␣-32 P]dATP) (50 Ci 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 ϫ 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 (5Ј-AAAGCTCTGTCCATGTGTC-3Ј) and an antisense intron 1-specific amplimer (5Ј-AAACCCCAGTAGCGCTCAC-3Ј) were synthesized. To determine changes in IGFBP-5 hnRNA, reverse transcriptionpolymerase chain reaction (PCR) (27,28) was used. Total RNA from control and test samples was prepared as described for Northern analysis. One g 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 Ci of [␣-32 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 5Ј-AAAGCTCTGTCCATGTGT-Cattagtcagcaaccatagtc-3Ј and the composite antisense primer 5Ј-AAAC-CCCAGTAGCGCTCACggttccatcctctagaggat-3Ј (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 ϭ 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 M ATP, 500 M CTP, 500 M GTP, 150 units of RNasin (Promega), and 250 Ci of [ 32 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 ϳ1 g 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 [ 32 P]RNA, representing various experimen-tal conditions, to the membranes (30). Equal counts/minute of [ 32 P]RNA from each sample were hybridized to cDNAs at 42°C for 72 h and washed in 1 ϫ 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 Ϫ70 to ϩ120, using oligonucleotide primers containing the various mutations in the 5Ј-end. A 3Ј-truncation of the IGFBP-5 promoter was also prepared by PCR through the generation of a new restriction site at bp ϩ22. 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 ϳ70% 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 Ϯ 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 IG-FBP-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- Total RNA from control (C) or cortisol (glucocorticoid (GC))-treated cultures was subjected to Northern blot analysis and hybridized with ␣-32 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. state mRNA levels. Treatment of Ob cells with cortisol at 1 M 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 Ϯ 6% (n ϭ 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 M for 24 h inhibited IGFBP-5 transcripts by 16 Ϯ 13% (n ϭ 4) to 51 Ϯ 6% (n ϭ 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 IG-FBP-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 125 I-labeled IGF-II as a ligand (14,15). Cortisol at 1 M for 24 h decreased the levels of immunoreactive IGFBP-5 in the extracellular matrix by 44 Ϯ 7% (n ϭ 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 M. In earlier experiments, cycloheximide at doses of 2 M 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 Ϯ 8% (n ϭ 4) decrease in IGFBP-5 transcript levels, so further inhibitory effects of cortisol were difficult to detect (Fig. 4).
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 M 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 ϭ Ϫ0.0154, n ϭ 11) and cortisol-treated (slope ϭ Ϫ0.0194, n ϭ 12) cultures (25). Treatment of Ob cells with cortisol for 6 and 24 h decreased IGFBP-5 hnRNA expression by 68 Ϯ 8% (n ϭ 3) and 23-34% (n ϭ 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 M 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 Ϯ 7% (n ϭ 3) at 6 h and by 54 Ϯ 4% (n ϭ 3) at 24 h (Fig. 7).
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 Ϫ2695 to ϩ120. Deletion constructs from bp Ϫ2695 to ϩ120 to bp Ϫ70 to ϩ120 (Fig. 8, A and  B) showed a 35% decrease in IGFBP-5 promoter activity when 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 ␣-32 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 Ϯ 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 (E) or DRB plus cortisol (q). treated with cortisol at 1 M for 6 h (Fig. 8C). The reverse orientation of the largest construct, bp ϩ120 to Ϫ2695, yielded little luciferase activity and no inhibition by cortisol. Sitedirected mutations and a 3Ј-truncation of the bp Ϫ70 to ϩ120 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 5Ј-end, and a truncation from the 3Ј-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 ϭ 6), these mutated constructs each showed a 40 -50% (p Ͻ 0.05) decrease in promoter activity in response to 1 M 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 M cortisol on IGFBP-5 promoter activity. 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 Ϫ70 and ϩ22, 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 Ϫ70 and ϩ22 contains a CCAAT/enhancer-binding protein ␣ consensus binding sequence and a CAAT motif, but mutations of these binding sequences did not eliminate the cortisol re- sponse, 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 posttranscriptional 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 IG-FBP-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 Ϫ70 and ϩ22 in the IGFBP-5 promoter, and E-boxbinding 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.