Osteonectin/SPARC polymorphisms in Caucasian men with idiopathic osteoporosis.

UNLABELLED: Animal models suggest a role for osteonectin/SPARC in determination of bone mass. We found haplotypes consisting of three single nucleotide polymorphisms (SNPs) in the 3' untranslated region (UTR) of the osteonectin gene are associated with bone density in Caucasian men with idiopathic osteoporosis. INTRODUCTION: Osteonectin is a matricellular protein regulating matrix assembly, osteoblast differentiation, and survival. Animal studies indicate that osteonectin is essential for normal bone mass. The 3' UTR is a regulatory region controlling mRNA stability, trafficking, and translation, and we determined whether osteonectin 3' UTR haplotypes could be associated with bone mass and/or idiopathic osteoporosis. METHODS: Single strand conformation polymorphism and allele-specific PCR analysis were used to assess alleles at osteonectin cDNA bases 1046, 1599, and 1970, using genomic DNA from middle-aged Caucasian men with idiopathic, low turnover osteoporosis (n = 56) and matched controls (n = 59). Bone density was measured by DXA at spine, hip and radius. Allele and haplotype frequencies were analyzed by Chi square analysis and Fisher's exact test. RESULTS: Five common osteonectin 3' UTR haplotypes were identified. The frequency of one haplotype (1046C-1599C-1970T) was higher in controls compared with patients, and this haplotype was also associated with higher bone densities at multiple sites in patients. In contrast, a second haplotype (1046C-1599G-1970T) was associated with lower bone densities in patients at multiple sites. CONCLUSIONS: Osteonectin regulates skeletal remodeling and bone mass in animals, and haplotypes in the 3' UTR of this gene are associated with bone density in Caucasian men with idiopathic osteoporosis.

Effects of cortisol and bone morphogenetic protein-2 on stromal cell differentiation: correlation with CCAAT-enhancer binding protein expression.

Bone marrow stroma contain pluripotential cells with the potential to differentiate into various mesenchymal cell lineages. We compared the effect of cortisol and bone morphogenetic protein-2 (BMP-2) on the differentiation of murine ST-2 stromal cells into mature osteoblasts or adipocytes. ST-2 cells were cultured for 3-27 days in the presence of 10% fetal bovine serum, 100 microg/mL ascorbic acid, and 5 mmol/L beta-glycerolphosphate in the presence or absence of cortisol at 1 micromol/L or BMP-2 at 1 nmol/L. Untreated ST-2 cells expressed high levels of alkaline phosphatase activity (APA) 15 days after confluence, and this was followed by the appearance of mineralized nodules after 24 days. BMP-2 accelerated and intensified the appearance of cells expressing APA and the presence of mineralized nodules. In contrast, cortisol decreased APA, prevented the formation of mineralized nodules, and induced a cellular phenotype characteristic of adipocytes. Untreated stromal cells expressed osteocalcin, Cbfa1, type I collagen, and alkaline phosphatase mRNA. BMP-2 increased osteocalcin and alkaline phosphatase mRNA, whereas cortisol suppressed their expression, as well as Cbfa1 and type I collagen transcripts. Cortisol enhanced, and BMP-2 downregulated, peroxisome proliferator-activated receptor gamma 2 and adipsin transcripts. The C/EBP transcription factors regulate genes critical for adipocytic and osteoblastic differentiation. Cortisol increased the expression of C/EBP alpha, beta, delta, and gamma mRNA levels, whereas BMP-2 had minor effects on C/EBP expression. In conclusion, BMP-2 accelerates the differentiation of stromal cells toward an osteoblastic phenotype, whereas glucocorticoids induce their differentiation toward an adipocytic phenotype.

Glucocorticoid suppression of IGF I transcription in osteoblasts.

Glucocorticoids have profound effects on bone formation, decreasing IGF I transcription in osteoblasts, but the mechanisms involved are poorly understood. We previously showed that the bp +34 to +192 region of the rat IGF I exon 1 promoter was responsible for repression of IGF I transcription by cortisol in cultures of osteoblasts from fetal rat calvariae (Ob cells). Here, site-directed mutagenesis was used to show that a binding site for members of the CAAT/enhancer binding protein family of transcription factors, within the +132 to +158 region of the promoter, mediates this glucocorticoid effect. EMSAs demonstrated that cortisol increased binding of osteoblast nuclear proteins to the +132 to +158 region of the IGF I promoter. Supershift assays showed that CAAT/enhancer binding protein alpha, beta, and delta interact with this sequence, and binding of CAAT/enhancer binding protein delta, in particular, was increased in the presence of cortisol. Northern blot analysis showed that CAAT/enhancer binding protein delta and beta transcripts were increased by cortisol in Ob cells. Further, cortisol increased the transcription of these genes and increased the stability of CAAT/enhancer binding protein delta mRNA. In conclusion, cortisol represses IGF I transcription in osteoblasts, and CAAT/enhancer binding proteins appear to play a role in this effect.

Cortisol inhibits the differentiation and apoptosis of osteoblasts in culture.

Glucocorticoids decrease the replication of cells of the osteoblastic lineage and the function of the osteoblast. However, under certain conditions, they enhance the differentiation of osteoblastic cells, an effect that appears contradictory to their inhibitory actions on cell function. In this study we examine the effects of cortisol on the proliferation, differentiation, and fate of osteoblastic enriched cells from 22-day-old fetal rat calvariae (osteoblastic cells) in the absence and presence of beta-glycerophosphate. In the absence of beta-glycerophosphate, there was a progressive accumulation of DNA and cells, which was impaired by cortisol. In the presence of beta-glycerophosphate, there was an initial accumulation of DNA and cells followed by a marked decline that was prevented by cortisol. Despite the sustained number of cells, cortisol did not affect their mineralization, and inhibited Core binding factor a1 (Cbfa1), but not alkaline phosphatase, osteocalcin, or type I collagen transcripts. The decrease in cell number by cortisol observed in the absence of beta-glycerophosphate was due to a decrease in DNA synthesis, whereas the increase in cell number observed in the presence of beta-glycerophosphate was due to a relative increase in DNA synthesis and a decrease in apoptosis as determined by DNA fragmentation and acridine orange staining of the cells. This was correlated by a decrease in transcripts of proapoptotic genes and caspase 3 activity, and an increase of antiapoptotic genes. In conclusion, cortisol decreases the replication of cells of the osteoblastic lineage, but under conditions of differentiation/mineralization, cortisol prevents terminal differentiation of the cells and maintains an immature cell population.

The metastasis-associated metalloproteinase stromelysin-3 is induced by transforming growth factor-beta in osteoblasts and fibroblasts.

Bone matrix serves as a reservoir of growth factors important in growth and tissue remodeling, and transforming growth factor-beta (TGF-beta) is abundant in bone matrix. Normal processes, such as remodeling, and pathological processes, such as osteolytic metastasis, cause the release of growth factors from the matrix, allowing them to influence the behavior of cells within their microenvironment. Breast cancer metastases frequently establish themselves in the bone compartment, often causing localized osteolysis. Stromelysin-3 is a matrix metalloproteinase associated with tumor metastases. Its expression in host tissues favors the homing and survival of malignant epithelial cells in early tumorigenesis by releasing and/or activating growth factors sequestered in the extracellular matrix. Osteoblasts express stromelysin-3, and Northern and Western blot analysis show that its messenger RNA and protein levels are increased by TGF-beta. Nuclear run-off assays demonstrate activation of gene transcription, and experiments using transcription inhibitors demonstrate stabilization of stromelysin-3 messenger RNA by TGF-beta. Importantly, TGFbeta induces stromelysin-3 in fibroblasts by similar mechanisms, indicating that it is likely to stimulate stromelysin-3 expression in breast stroma. Stimulation of stromelysin-3 expression by TGF-beta in fibroblasts and osteoblasts could play a role in the metastasis of breast cancer cells and their homing and survival in bone.

Osteopenia and decreased bone formation in osteonectin-deficient mice.

Bone continuously remodels in response to mechanical and physiological stresses, allowing vertebrates to renew bone as adults. Bone remodeling consists of the cycled synthesis and resorption of collagenous and noncollagenous extracellular matrix proteins, and an imbalance in this process can lead to disease states such as osteoporosis, or more rarely, osteopetrosis. There is evidence that the extracellular matrix glycoprotein osteonectin or secreted protein acidic and rich in cysteine (BM-40) may be important in bone remodeling. Osteonectin is abundant in bone and is expressed in areas of active remodeling outside the skeleton. In vitro studies indicate that osteonectin can bind collagen and regulate angiogenesis, metalloproteinase expression, cell proliferation, and cell-matrix interactions. In some osteopenic states, such as osteogenesis imperfecta and selected animal models for bone fragility, osteonectin expression is decreased. To determine the function of osteonectin in bone, we used contact x-ray, histomorphometry, and Northern blot analysis to characterize the skeletal phenotype of osteonectin-null mice. We found that osteonectin-null mice have decreased bone formation and decreased osteoblast and osteoclast surface and number, leading to decreased bone remodeling with a negative bone balance and causing profound osteopenia. These data indicate that osteonectin supports bone remodeling and the maintenance of bone mass in vertebrates.

Fibroblast growth factor-2 induces hepatocyte growth factor/scatter factor expression in osteoblasts.

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional growth factor with a major role in tissue morphogenesis and repair. It stimulates the proliferation of cells of the osteoblast and osteoclast lineages. Mitogenic factors playing a role in fracture repair may act by regulating HGF/SF expression or activity in bone-forming cells. We investigated the effect of fibroblast growth factor-2 (FGF-2) on the expression of HGF/SF and its receptor, encoded by c-met, in the MC3T3-E1 osteoblastic cell line. MC3T3-E1 cells expressed low levels of HGF/SF messenger RNA (mRNA), which were markedly increased by FGF-2 in a dose- and time-dependent manner. FGF-2 also induced HGF/SF polypeptide synthesis. The stimulation of HGF/SF mRNA expression by FGF-2 was blocked by cycloheximide, a protein synthesis inhibitor, but not by DNA or prostaglandin synthesis inhibitors. FGF-2 increased the rate of HGF/SF gene transcription by approximately 2-fold, as determined by nuclear run-on assays, and did not modify the decay of HGF/SF mRNA in transcriptionally arrested cells. FGF-2 also caused a dose- and time-dependent stimulation of c-met mRNA. In conclusion, FGF-2 induces HGF/SF expression in osteoblasts and may promote HGF/SF activity by increasing the expression of its receptor. Through these mechanisms, HGF/SF could mediate FGF actions on bone repair.

Dual regulation of stromelysin-3 by fibroblast growth factor-2 in murine osteoblasts.

Osteoblasts express stromelysin-3, a matrix metalloproteinase associated with normal remodeling processes and with stromal fibroblasts surrounding many invasive carcinomas. Fibroblast growth factors (FGFs) play an important role in skeletal development, fracture repair, and osteoblast function. The osteoblastic cell line MC3T3 was used to study the regulation of stromelysin-3 by FGF-2. Acutely, FGF-2 decreased stromelysin-3 mRNA levels, whereas prolonged treatment caused an induction of stromelysin-3 mRNA. RNA stability studies and nuclear run-off assays indicated that acute treatment with FGF-2 decreased stromelysin-3 mRNA stability but did not alter gene transcription. However, the induction of stromelysin-3 after prolonged treatment with FGF-2 resulted from increased gene transcription, with no effect on RNA stability. The stimulatory effect was protein synthesis-dependent, whereas the inhibitory effect was not. This study demonstrates dual regulation of stromelysin-3 by FGF-2: acute destabilization of stromelysin-3 mRNA, followed by induction of gene transcription. This complex regulation may be important in the function of stromelysin-3 in bone and in remodeling processes, such as wound and fracture repair.

Basic fibroblast growth factor destabilizes osteonectin mRNA in osteoblasts.

Osteonectin (secreted protein acidic and rich in cysteine, 40-kDa basement membrane) is a glycoprotein abundantly expressed in bone and in other tissues undergoing active remodeling. Fibroblast growth factors (FGFs) are important in skeletal development and fracture repair, events associated with extracellular matrix remodeling. We used the murine osteoblastic cell line MC3T3 to determine whether basic FGF (bFGF) regulates osteonectin expression in bone. Northern blot analysis showed that bFGF decreased osteonectin transcripts in a dose- and time-dependent manner. This regulation was independent of the mitogenic effect of bFGF but was dependent on new protein synthesis. Immunoprecipitation of [35S]methionine-cysteine osteoblast-conditioned medium and cell layer proteins showed that bFGF decreased osteonectin synthesis. Nuclear runoff assays failed to reveal regulation of osteonectin gene transcription by bFGF. However, bFGF dramatically decreased the stability of osteonectin mRNA in transcriptionally arrested osteoblasts. This destabilization of osteonectin mRNA may be one means by which bFGF regulates extracellular matrix remodeling.

Insulin-like growth factor I inhibits the transcription of collagenase 3 in osteoblast cultures.

Insulin-like growth factor (IGF) I is an autocrine regulator of bone remodeling which inhibits bone collagen degradation and interstitial collagenase 3 mRNA levels. The mechanism of this inhibitory effect on collagenase 3 expression is not known. We tested the effects of IGF I on collagenase 3 gene expression in cultures of osteoblast-enriched cells from 22 day fetal rat calvariae (Ob cells) to determine whether transcriptional or posttranscriptional mechanisms were involved in the regulation of the collagenase 3 gene. IGF I at 10-100 nM caused a dose-dependent decrease in collagenase mRNA and protein levels. IGF I did not modify the half-life of collagenase 3 mRNA in transcriptionally arrested Ob cells, whereas it decreased the levels of interstitial collagenase 3 heterogeneous nuclear RNA. In addition, IGF I decreased the rates of transcription of the collagenase gene and the activity of a 2.1 kilobase collagenase 3 promoter construct transiently transfected into Ob cells. In conclusion, IGF I decreases the expression of collagenase 3 mRNA by transcriptional mechanisms.

Interleukin-6 and its soluble receptor cause a marked induction of collagenase 3 expression in rat osteoblast cultures.

Interleukin-6 (IL-6), a cytokine produced by skeletal cells, increases bone resorption, but its effects on collagenase expression are unknown. We tested the effects of IL-6 and its soluble receptor on collagenase 3 expression in osteoblast-enriched cells from fetal rat calvariae (Ob cells). IL-6 caused a small increase in collagenase mRNA levels, but in the presence of IL-6-soluble receptor (IL-6sR), IL-6 caused a marked increase in collagenase transcripts after 2-24 h. In addition, IL-6sR increased collagenase mRNA when tested alone. IL-6 and IL-6sR increased immunoreactive collagenase levels. Cycloheximide and indomethacin did not prevent the effect of IL-6 and IL-6sR on collagenase mRNA levels. IL-6 and IL-6sR did not alter the decay of collagenase mRNA in transcriptionally arrested Ob cells and increased the levels of collagenase heterogeneous nuclear RNA and the rate of collagenase gene transcription in Ob cells. IL-6 and IL-6sR increased collagenase 3 mRNA in MC3T3 cells but only modestly in skin fibroblasts. IL-6 and IL-6sR enhanced the expression of tissue inhibitor of metalloproteinases 1. In conclusion, IL-6, in the presence of IL-6sR, increases collagenase 3 synthesis in osteoblasts by transcriptional mechanisms. This effect may contribute to the action of IL-6 on bone matrix degradation and bone resorption.

Autocrine down-regulation of collagenase-3 in rat bone cell cultures by insulin-like growth factors.

Insulin-like growth factors (IGF)-I and -II are presumed to act as autocrine regulators of bone formation. Recently, we demonstrated that IGF-I and -II inhibit bone collagen degradation and collagenase-3 synthesis in osteoblast cultures. Therefore, we tested the autocrine role of IGFs in the endogenous expression of collagenase-3 in cultures of osteoblast-enriched cells from 22-day fetal rat calvariae (Ob cells). Steady-state messenger RNA (mRNA) levels were determined by Northern blot analysis and collagenase concentrations in the culture medium were determined by Western immunoblot. Basal level collagenase-3 transcripts decreased in Ob cell cultures, coinciding with an increase in IGF-I and -II protein levels. Removal of the conditioned medium modestly increased collagenase-3 mRNA levels and restored the ability of exogenously added IGF-I to repress collagenase-3 transcripts. IGF neutralizing antibodies and IGF binding proteins-2 and -3 in excess increased and sustained collagenase mRNA, heterogeneous nuclear RNA, and protease levels in Ob cell cultures. In conclusion, IGF-I and -II are autocrine repressors of collagenase-3 synthesis, and this effect may contribute to their actions on the maintenance of a normal bone collagen matrix.

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.

Transcriptional and posttranscriptional regulation of interstitial collagenase by platelet-derived growth factor BB in bone cell cultures.

Platelet-derived growth factor (PDGF), a bone cell mitogen, stimulates bone collagen degradation and does not enhance bone matrix apposition rates. The mechanism of the effect on collagen degradation is unknown, and it could involve changes in interstitial collagenase synthesis. We tested the effects of PDGF on interstitial collagenase expression in cultures of osteoblast-enriched cells from fetal rat calvariae (Ob cells). After 4-8 h of treatment, PDGF BB at 0.3 nM increased steady state collagenase messenger RNA (mRNA), whereas PDGF AA had no effect. The effect of PDGF BB on collagenase transcripts was dose dependent. PDGF BB increased the levels of immunoreactive collagenase after 6 h, whereas the levels were decreased after 16 h. Stimulation of collagenase mRNA by PDGF BB was dependent on de novo protein synthesis and activation of protein kinase C. PDGF BB prolonged the half-life of collagenase mRNA in transcriptionally arrested cells. PDGF BB initially increased and subsequently decreased the rate of collagenase gene transcription and the levels of collagenase heterogeneous nuclear RNA. In conclusion, PDGF BB regulates interstitial collagenase in Ob cells by transcriptional and posttranscriptional mechanisms, and this effect may contribute to its stimulatory actions on bone collagen degradation.

Cortisol increases interstitial collagenase expression in osteoblasts by post-transcriptional mechanisms.

Glucocorticoids regulate both bone formation and bone resorption. In osteoblasts, they inhibit type I collagen synthesis; however, there is limited information about their effects on interstitial collagenase, the enzyme that degrades type I collagen. We used primary cultures of osteoblast-enriched cells from fetal rat calvariae (Ob cells) to study the effects of cortisol on collagenase expression. Northern blot analysis showed that cortisol increased collagenase transcript levels in a dose- and time-dependent manner, which was paralleled by an increase in immunoreactive metalloproteinase in the culture medium. Cortisol increased the half-life of collagenase mRNA from 6 to 12 h in transcription-arrested Ob cells. In contrast, cortisol modestly decreased collagenase gene transcription after 24 h of treatment. The up-regulation of collagenase by cortisol is osteoblast-specific, since the glucocorticoid decreased phorbol 12-myristate 13-acetate-induced collagenase mRNA expression in rat fibroblasts, a result that agrees with other studies of collagenase gene regulation in fibroblastic cells. In conclusion, cortisol increases interstitial collagenase transcript levels by post-transcriptional mechanisms in osteoblastic cells. Our data demonstrate that glucocorticoids regulate collagenase gene expression in a novel tissue-specific manner, further highlighting the differences in gene regulation between osteoblastic and fibroblastic cells.

Transcriptional repression of insulin-like growth factor I by glucocorticoids in rat bone cells.

Insulin-like growth factor I (IGF-I) is an abundant autocrine and paracrine growth factor secreted by osteoblasts. It promotes osteoblast proliferation and expression of their differentiated phenotype. Glucocorticoids decrease IGF-I production by osteoblasts, which may mediate some actions of the steroid on bone in both normal and pathological states. The mechanisms by which the glucocorticoid cortisol down-regulates IGF-I transcripts were explored using cultures of osteoblast-enriched cells derived from fetal rat calvaria (Ob cells). Repression of IGF-I transcripts was apparent after 8 h of treatment, was sustained for at least 24 h, and was not altered by cotreatment with cycloheximide. Cortisol did not alter the stability of IGF-I messenger RNAs in transcriptionally arrested Ob cells. Cortisol decreased IGF-I heterogeneous nuclear RNA and gene transcription, as determined by reverse transcription-linked polymerase chain reaction and nuclear run-on assay, respectively. Transient transfection of Ob cells with constructs containing portions of the rat IGF-I exon 1 promoter and 5'-flanking DNA linked to the reporter gene luciferase were performed to determine glucocorticoid-responsive region of the rat IGF-I exon 1 promoter was localized to 34 to 192 relative to the first start site of transcription. In conclusion, cortisol inhibits the transcription of IGF-I in osteoblasts, an effect that may be relevant to the actions of cortisol in bone.

Insulin-like growth factors inhibit interstitial collagenase synthesis in bone cell cultures.

Insulin-like growth factor-I (IGF-I) and IGF-II are among the most prevalent growth factors secreted by bone cells and are presumed to act as autocrine regulators of bone formation. We recently demonstrated that IGFs inhibit bone collagen degradation, and we postulated that they may either inhibit the expression of interstitial collagenase or stimulate the synthesis of tissue inhibitors of metalloproteinase-1 (TIMP-1), -2, or -3. We tested the effects of IGF-I and -II on collagenase and TIMP-1, -2, and -3 expression in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells). Steady state messenger RNA (mRNA) levels were determined by Northern blot analysis, and collagenase concentrations were determined in the culture medium by a specific immunoassay. After 2-6 h of treatment, IGF-I and -II decreased collagenase transcripts by up to 80%. IGF-I was a more potent inhibitor than IGF-II, because it was active at doses as low as 10 nM, whereas a dose of 100 nM was required to observe the IGF-II effect. In addition, IGF-I and -II opposed the stimulatory effect of retinoic acid on collagenase transcripts. Immunoreactive collagenase levels were not detectable in control or IGF-treated cultures, but IGF-I and -II decreased the levels induced by retinoic acid by 70-90%. The protein synthesis inhibitor cycloheximide superinduced collagenase transcripts, and IGF-I or -II decreased this mRNA induction to levels similar to, but not lower than, those observed in control cultures. The effects of IGF-I and -II on collagenase transcripts were not modified by the DNA synthesis inhibitor hydroxyurea at 1 mM. Neither IGF-I nor IGF-II modified the expression of TIMP-1, -2, or -3 mRNA in Ob cells. TIMP protein levels were not determined, and our study does not exclude a translational or posttranslational effect of IGF. In conclusion, IGF-I and -II decrease interstitial collagenase transcripts as well as induced protease levels in Ob cells, and this effect may contribute to their inhibitory actions on bone collagen degradation.

Cortisol downregulates osteoblast alpha 1 (I) procollagen mRNA by transcriptional and posttranscriptional mechanisms.

Glucocorticoids decrease osteoblast proliferation and type I collagen production, and this may play a role in the development of glucocorticoid-induced osteoporosis. Osteoblast-enriched cultures derived from fetal rat calvaria were used to determine the mechanisms by which cortisol decreases alpha 1 (I) procollagen expression in bone cells. A 24 h treatment with cortisol decreased collagen synthesis in these cultures in a dose-dependent manner. Cortisol decreased alpha 1 (I) procollagen transcripts in a dose- and time-dependent manner as well. Repression of alpha 1 (I) procollagen transcripts was evident as early as 2 h of treatment and was maximal after 48 h of treatment. Nuclear run-off assays showed that cortisol downregulated transcription of the alpha 1 (I) procollagen gene. In addition, pretreatment with cortisol decreased the stability of alpha 1 (I) procollagen mRNA in transcription-arrested osteoblast cultures. The ability of cortisol to downregulate alpha 1 (I) procollagen transcripts was sensitive to cycloheximide treatment, suggesting that the gene is under "secondary control" by glucocorticoids. Since cortisol decreases alpha 1 (I) procollagen gene transcription in osteoblasts but does not affect alpha 1 (I) procollagen gene transcription in fibroblasts, we suggest that the mechanisms controlling glucocorticoid repression of collagen expression are cell-type specific.