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Endocrinology Vol. 142, No. 3 1341-1348
Copyright © 2001 by The Endocrine Society

ARTICLES

Cloning and in Vitro Characterization of {alpha}1(I)-Collagen 11{beta}-Hydroxysteroid Dehydrogenase Type 2 Transgenes as Models for Osteoblast-Selective Inactivation of Natural Glucocorticoids1

Henning W. Woitge, John R. Harrison, Ante Ivkosic, Zygmunt Krozowski and Barbara E. Kream

Department of Medicine, School of Medicine (H.W.W., A.I., B.E.K.), and Department of Orthodontics, School of Dental Medicine (J.R.H.), University of Connecticut Health Center, Farmington, Connecticut 06030; and Laboratory of Molecular Hypertension, Baker Medical Research Institute (Z.K.), Prahran 3181, Australia

Address all correspondence and requests for reprints to: Barbara E. Kream, Ph.D., Department of Medicine, MC-1850, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, Connecticut 06030. E-mail: kream{at}nso1.uchc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The NAD-dependent enzyme, 11{beta}-hydroxysteroid dehydrogenase type II (11{beta}HSD2), catalyzes the unidirectional conversion of biologically active glucocorticoids to inactive metabolites. In vivo, 11{beta}HSD2 protects the mineralocorticoid receptor from activation by glucocorticoids in mineralocorticoid target tissues such as kidney. The goal of the present study was to use targeted overexpression of 11{beta}HSD2 as a novel means of disrupting glucocorticoid signaling in osteoblastic cells. Rat 11{beta}HSD2 complementary DNA was cloned downstream of a 2.3- and 3.6-kb {alpha}1(I)-collagen (Col1a1) promoter fragment to produce the expression plasmids Col2.3-HSD2 and Col3.6-HSD2, respectively, which were transiently and/or stably transfected in osteoblastic ROS 17/2.8 and MC3T3-E1 cells. Transgene messenger RNA and protein were detected in transfected cells by Northern blot analysis and immunostaining, respectively. Transfection of 11{beta}HSD2 led to higher rates of conversion of [3H]corticosterone to [3H]dehydrocorticosterone and reduced glucocorticoid-dependent regulation of a mouse mammary tumor virus promoter-reporter construct, cell growth, and messenger RNA markers compared with transfection of a control vector. Expression of 11{beta}HSD2 under the control of Col1a1 promoter fragments may provide a novel model to study the role of glucocorticoid signaling in osteoblastic cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BY SIGNALING through nuclear receptors, steroid hormones elicit a wide range of responses in almost all organ systems (1, 2). Various experimental strategies, such as surgical or pharmacological intervention (3, 4, 5), antisense transgenes (6, 7, 8), and global (9, 10, 11) or tissue-specific (12) receptor knockouts, have been used to study steroid hormone function in vivo. Although these approaches have advanced our understanding of steroid hormone biology, they often have limitations. For example, surgical removal of an endocrine gland to eliminate hormone production or pharmacological treatment with a receptor agonist or antagonist may affect multiple organ systems and result in secondary effects on the tissue of interest (5, 13). Antisense transgenes to eliminate messenger RNA (mRNA) expression rarely lead to a complete abrogation of the gene product of interest (14, 15). Tissue-specific ablation of nuclear receptors can circumvent embryonic or perinatal lethality and infertility associated with global nuclear receptor knockout models (12). However, due to receptor redundancy in certain nuclear receptor families, the thyroid (16, 17) and retinoic acid receptors (18, 19) for example, the inactivation of a specific nuclear receptor isoform may still allow a ligand to signal through alternative pathways.

The goal of our laboratory is to develop in vivo models that will help us understand the role of glucocorticoids in bone development and remodeling. Previously, the function of glucocorticoid signaling has been studied in a variety of genetic mouse models (7, 8, 9, 12). Global knockout of the glucocorticoid receptor (GR) produces perinatal lethality in transgenic mice (9). These mice are characterized by impaired embryonic development, severely atelectatic lungs at birth, and adrenal hypertrophy due to impaired feedback regulation via the hypothalamic-pituitary-adrenal axis. Tissue-specific knockouts of the GR using Cre/loxP technology have been developed in specific target tissues such as the brain (12). Inactivation of GR, however, does not prevent glucocorticoids from signaling through alternative pathways, such as the mineralocorticoid receptor (MR). For example, in GR knockout mice, corticosterone repressed hippocampal expression of serotonin 1A receptor mRNA, which is predominantly mediated via the MR (20). These data indicated that a corticosterone-dependent MR-mediated suppression of gene expression can take place in the complete absence of the GR. Thus, it is possible that glucocorticoid function may remain partly intact in GR knockout mice.

A biological mechanism to inactivate glucocorticoid signaling in a target cell is ligand metabolism. The NADdependent enzyme, 11{beta}-hydroxysteroid dehydrogenase type 2 (11{beta}HSD2), catalyzes the unidirectional conversion of biologically active glucocorticoids to inactive metabolites: cortisol to cortisone in humans, and corticosterone to 11-dehydrocorticosterone in rodents (21, 22, 23). 11{beta}HSD2 protects the MR from activation by glucococorticoids in mineralocorticoid target tissues such as kidney (23, 24, 25). Moreover, 11{beta}HSD2 is abundant in human (26, 27) and rodent (28, 29, 30, 31) placenta, where it protects the fetus from high levels of maternal glucocorticoids. With this biological paradigm in mind, we reasoned that transgenic expression of 11{beta}HSD2 could be a way to abrogate all possible intracellular glucocorticoid signaling pathways in a target cell of interest.

11{beta}HSD2 is expressed in osteosarcoma cell lines (32) and human bone at low levels (33), although its biological role in bone metabolism is unknown. As type I collagen is highly expressed in bone, we cloned rat 11{beta}HSD2 complementary DNA (cDNA) downstream of two different rat {alpha}1(I)-collagen (Col1a1) promoter fragments that are expressed selectively in osteoblastic cells and then transfected the resulting constructs into osteoblastic ROS 17/2.8 and MC3T3-E1 cells. Col1a1-driven 11{beta}HSD2 was highly expressed in transiently and stably transfected cells and reduced glucocorticoiddependent induction of a mouse mammary tumor virus promoter-reporter construct. In contrast, the synthetic glucocorticoid dexamethasone, which is reversibly metabolized by 11{beta}HSD2, maintained partial activity in transfected cells. This study is the first step in developing an in vivo transgenic model in which glucocorticoid signaling pathways are blocked in osteoblasts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cloning of expression plasmids
The pBR327-based plasmid Col2.3-{Delta}TK-ClaPa, containing the rat Col1a1 gene from -2295 to +115 bp (34), served as the starting vector. A rat 11{beta}HSD2 cDNA was excised from pcDNA1 (22) with XhoI and HindIII and cloned into pBC/SK+/- (Stratagene, La Jolla, CA). The HindIII site in the pBC/SK+/- polylinker was replaced with an XhoI site, and the 1.3-kb 11{beta}HSD2 cDNA fragment was isolated with XhoI. {Delta}TK cDNA was excised from Col2.3-{Delta}TK-ClaPa with BamHI and replaced with an XhoI-containing adapter to produce Col2.3-XhoI-ClaPa (hereafter called Col2.3-Cont) that would accept the 11{beta}HSD2 cDNA insert. Col3.6-XhoI-ClaPa (hereafter called Col3.6-Cont) was obtained by cloning a 1.3-kb HindIII fragment of the Col1a1 promoter from -3518 to -2296 bp into Col2.3-XhoI-ClaPa directly upstream of bp -2995. Finally, the 1.3-kb 11{beta}HSD2 cDNA was inserted into the XhoI adapter of Col2.3-Cont and Col3.6-Cont to give the expression constructs called Col2.3-HSD2 and Col3.6-HSD2, respectively. The correct orientation of the 11{beta}HDS2 insert was verified with XbaI digestion. Figure 1Go shows the two Col1a1–11{beta}HSD2 expression plasmids. A cytomegalovirus-driven 11{beta}HSD2 expression construct was made by cloning 11{beta}HSD2 cDNA into pCR3.1-Uni (Invitrogen, Carlsbad, CA) using the HindIII and XhoI sites in the polylinker. Mouse mammary tumor virus (MMTV)-chloramphenicol acetyltransferase (CAT) (35) and MMTV-luciferase (36) reporter constructs were gifts from Dr. Gordon Hager (NIH, Bethesda, MD).



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  		Figure 1. Col1a1–11{beta}HSD2 expression plasmids. Schematic representation of Col2.3-HSD2 (upper panel) and Col3.6-HSD2 (lower panel) showing selected restriction sites. PCR was performed with p5' (forward primer) and p3' (reverse primer) to generate an 11{beta}HSD2 cDNA probe. bGH PA, Bovine GH polyadenylation sequence.

 
ROS 17/2.8 and MC3T3-E1 cell cultures
ROS 17/2.8 cells were cultured in F-12 medium containing 10% nonheat-inactivated FCS and a penicillin/streptomycin cocktail. MC3T3-E1 cells were cultured in DMEM containing 10% heat-inactivated FCS and a penicillin/streptomycin cocktail. For both cell lines, medium was changed every 3 days; cells were trypsinized and passaged at a 1:4 ratio every 6 days.

Transient and stable transfections of ROS 17/2.8 and MC3T3-E1 cells
For transient transfection, ROS 17/2.8 and MC3T3-E1 cells were plated in 35-mm wells at a density of 20,000–25,000 cells/cm2 24 h before transfection. Cells were incubated for 5 h at 37 C in 5% CO2 with 10 µl Lipofectamine reagent (Life Technologies, Inc., Grand Island, NY) and a total of 2 µg plasmid DNA/well in serum-free medium. The transfection medium was removed and replaced with 10% FCS medium (F-12 for ROS 17/2.8 and DMEM for MC3T3-E1). After 48 h, 10% FCS medium was replaced with serum-free medium (F-12 for ROS 17/2.8 and DMEM for MC3T3-E1), and experiments were performed 24 h later. For stable transfections, cells were plated in 35-mm wells at a density of 20,000–25,000 cells/cm2 24 h before transfection and then incubated for 5 h at 37 C in 5% CO2 with 10 µl Lipofectamine reagent. Expression plasmid DNA (a total of 2 µg) plus 0.1 µg of a selection plasmid carrying the resistance gene for hygromycin were used. Cells were grown in 10% FCS/F-12 medium for 48 h, then switched to F-12 medium containing 10% FCS, a penicillin/streptomycin cocktail, and hygromycin as the selection antibiotic. A similar protocol with DMEM was used for stable transfection of MC3T3-E1 cells. However, we were not able to generate MC3T3-E1 cell lines stably transfected with the Col1a1–11{beta}HSD2 constructs. Therefore, only experiments with stably transfected cells ROS 17/2.8 cells are reported.

Regulation of cell growth and endogenous gene expression in ROS 17/2.8 cells
For assessment of glucocorticoid-dependent regulation of cell growth, stably transfected ROS 17/2.8 cells were plated at 10,000 cells/well in 24-well culture dishes. Cells were grown in complete medium (F-12 containing 10% FCS, a penicillin/streptomycin cocktail, and hygromycin) for 48 h. Then, the medium was replaced with complete medium containing vehicle, 100 nM dexamethasone, 100 nM corticosterone, or 100 nM cortisol. Cell counts were determined after 24, 48, 72, and 96 h of treatment (n = 6/group). For assessment of glucocorticoid-dependent regulation of endogenous gene expression, stably transfected ROS 17/2.8 cells were plated at a density of 20,000–25,000 cells/cm2 in 35-mm wells and grown to confluence in complete medium. Then, the medium was replaced with complete medium containing vehicle, 100 nM dexamethasone, 100 nM corticosterone, or 100 nM cortisol for 48 h. RNA was extracted and analyzed by Northern blotting as described below.

RNA extraction and Northern blot analysis
Total RNA was prepared using TRIzol reagent (Life Technologies, Inc., Grand Island, NY) according to the manufacturer’s protocol. Northern blot analysis was performed as described previously (37) with 10 µg total RNA. After hybridization at 42 C for 20 h with 5–6 x 106 cpm/ml of a cDNA that was random primer labeled with [32P]deoxy-GTP (DuPont Merck Pharmaceutical Co., Wilmington, DE), membranes were washed, air-dried, and exposed to a phosphorimager for quantitation. Then, membranes were exposed to photographic film (BioMax MR-1 film, Eastman Kodak Co., Rochester, NY) with an enhancing screen at -80 C. A rat 11{beta}HSD2 probe, including part of the bovine GH polyadenylation sequence in the ClaPa construct and part of the 11{beta}HSD2 sequence, was generated by PCR using 5'-CTGACCTTAGCCCCGTTGTAG-3' (p5') and 5'-GCGAGGGGCAAAGAACAGATG-3' (p3'; Fig. 1Go). Membranes were also probed with osteocalcin (OC), bone sialoprotein (BSP), and Col1a1 cDNAs. The hybridization signal obtained with each cDNA was normalized to the signal obtained by hybridization with an 18S RNA probe.

Immunocytochemical studies
Transiently and/or stably transfected ROS 17/2.8 or MC3T3-E1 cells were plated at a density of 10,000 cells/chamber in an 8-chamber slide system (Lab-Tek Brand Products, Nalge Nunc International, Naperville, IL) and grown for 72 h in 10% FCS medium. The Vectastain ABC peroxidase kit (Vector Laboratories, Inc., Burlingame, CA) was used according to the manufacturer’s protocol. RAH23, an immunopurified rabbit polyclonal antirat 11{beta}HSD2 antibody (38), was used as the primary antibody at 1–2.5 µg/ml. Cells were counterstained with hematoxylin for 30 sec, mounted, and baked for 20 min at 70 C.

TLC
11{beta}HSD2 activity in transfected ROS 17/2.8 and MC3T3-E1 cells was determined by measuring the conversion of [3H]corticosterone to [3H]11-dehydrocorticosterone using TLC. Cells were plated in 35-mm wells at a density of 20,000–25,000 cells/cm2 and transfected after 24 h. Cells were allowed to grow in 10% FCS medium for 48 h and then serum-deprived for 22 h. Stably transfected cells were plated in 35-mm wells at the same density, allowed to grow in 10% FCS medium for 72 h, and serum-deprived for 22 h. Two hours before the end of culture, this medium was removed and replaced with 200 µl serum-free medium containing 5 nM [3H]corticosterone (91.0 Ci/mmol; Amersham Pharmacia Biotech, Arlington Heights, IL) and varying concentrations of unlabeled corticosterone. Cells were incubated for 2 h at 37 C, then the medium was extracted with 1 ml methylene chloride. After centrifugation for 10 min at room temperature, the aqueous phase was removed, and the organic phase was evaporated overnight at room temperature. Dried extracts were dissolved in 50 µl acetone, spotted on silica gel plates (J. T. Baker, Phillipsburg, NJ), and developed in chloroform/acetone (82:18, vol/vol) for 2 h. Silica plates were scraped into scintillation vials, each containing 1 ml isopropanol, and radioactivity was counted in a liquid scintillation counter.

Measurement of CAT and luciferase activities
CAT activity was measured with a fluor diffusion assay as previously described (39). Cells were rinsed twice with PBS, resuspended in 1 ml CAT scraping buffer, and centrifuged for 5 min. The pellet was resuspended in a 0.25-M Tris-HCl/0.5% Triton X-100 buffer, followed by three freeze-thaw cycles. The extracts were heated at 65 C for 15–20 min and centrifuged at 14,000 x g for 3 min. Up to 10 µl of each extract were used in 200 µl reaction mixture containing 1 mM chloramphenicol, 0.2 µCi [3H]acetyl-coenzyme A (200 mCi/mmol), and 0.025 M Tris-HCl. The mixture was layered beneath 5 ml of Econofluor-2-(NEN Life Science Products, Inc., Boston, MA)-based scintillation fluid. The vials were incubated at 37 C for up to 5 h, and radioactivity was counted every hour to determine the amount of acetylated chloramphenicol product. Luciferase activity was measured with a luciferase assay system (Promega Corp., Madison, WI). Cells were incubated with 100 µl/well of 1 x luciferase lysis buffer for 15 min at room temperature. The cell lysates were collected into 1.5-ml Eppendorf tubes and centrifuged for 1 min. After the addition of luciferase assay substrate, 10 µl of each cell lysate were assayed for luciferase activity in an LB 9501/16 luminometer (Berthold, Pittsburgh, PA). Both CAT and luciferase activities were normalized to protein content in the extract determined with the bicinchoninic acid assay (Pierce Chemical Co., Rockford, IL) as described previously (40).

Statistical analysis
Each value is presented as the mean ± SEM unless otherwise stated. Group differences were determined using repeated measures ANOVA. To correct for multiple comparisons, P values were adjusted according to the Bonferroni correction (significance level/number of tests). All statistical tests were two-tailed, and P < 0.01 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
11{beta}HSD2 mRNA expression in transiently and stably transfected ROS 17/2.8 cells
A PCR-generated probe was used to measure 11{beta}HSD2 expression in transfected ROS 17/2.8 cells. The probe was designed to include part of the bovine GH polyadenylation sequence in the ClaPa construct and part of the 11{beta}HSD2 sequence to allow distinction between endogenous and overexpressed 11{beta}HSD2 mRNA. A single transcript of about 1.9 kb was detected in transiently and stably transfected cells. Typically, 11{beta}HSD2 mRNA expression was lower in Col2.3-HSD2- than in Col3.6-HSD2-transfected cells (Fig. 2Go). This was expected because we previously showed that deletion of the Col1a1 promoter from -3.6 to -2.3 kb reduces promoter activity in ROS 17/2.8 cells and other osteoblastic cell lines (41). With our PCR-generated probe, we were not able to detect an endogenous 11{beta}HSD2 mRNA signal in transfected cells, even after prolonged exposure time (>1 week). However, this probe was able to detect a strong endogenous 11{beta}HSD2 signal, slightly larger than the 1.9-kb transgenically expressed 11{beta}HSD2 transcript, in kidneys of postnatal mice (Woitge, H., and B. Kream, unpublished results). This may indicate that either Northern analysis is not sensitive enough to detect low abundance 11{beta}HSD2 expression in ROS 17/2.8 cells, or 11{beta}HSD2 is not expressed in ROS 17/2.8 cells.



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  		Figure 2. 11{beta}HSD2 mRNA is expressed in transfected ROS 17/2.8 cells. Cells were transiently transfected with Col2.3-Cont, Col2.3-HSD2, Col3.6-Cont, and Col3.6-HSD2. RNA was extracted and analyzed for 11{beta}HSD2 and 18S RNA by Northern blot analysis. The figure shows results from one of six similar experiments.

 
11{beta}HSD2 protein expression in transiently and stably transfected ROS 17/2.8 cells
Transgenic 11{beta}HSD2 protein was detected in transiently and stably transfected ROS 17/2.8 cells by immunohistochemistry using the RAH23 antibody (38). ROS 17/2.8 cells stably transfected with control vectors (Col2.3-Cont and Col3.6-Cont) showed little background staining compared with 11{beta}HSD2-transfected cells (Fig. 3Go). Similar to the results for the transgene mRNA expression, immunohistochemical staining was stronger in Col3.6-HSD2- than in Col2.3-HSD2-transfected cells (Fig. 3Go). Transgenic 11{beta}HSD2 protein also was detected in MC3T3-E1 cells transiently transfected with Col2.3-HSD2 and Col3.6-HSD2.



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  		Figure 3. 11{beta}HSD2 protein is expressed in transfected ROS 17/2.8 cells. Cells were stably transfected with Col2.3-Cont, Col2.3-HSD2, Col3.6-Cont, and Col3.6-HSD2. Immunohistochemical staining of 11{beta}-HSD2 was performed using the RAH23 antibody. The figure shows results from one of six similar experiments. The number of RAH23-positive cells in this experiment was about 43% of that in Col2.3-HSD2 and 83% of that in Col3.6-HSD2 stably transfected cells.

 
Conversion of [3H]corticosterone to [3H]11-dehydrocorticosterone in transfected cells
To determine whether transgenic 11{beta}HSD2 had enzymatic activity, the conversion of [3H]corticosterone to [3H]11dehydrocorticosterone was measured using TLC. In ROS 17/2.8 cells transiently transfected with Col3.6-HSD2 and Col2.3-HSD2, conversion of 5 nM tracer [3H]corticosterone to [3H]11-dehydrocorticosterone in a 2-h incubation was 98 ± 0.3% and 93 ± 3%, respectively, compared with 17 ± 6% in untransfected cells (n = 3/group). In stably transfected cells, addition of 100 nM unlabeled corticosterone plus 5 nM tracer [3H]corticosterone resulted in a conversion rate of the tracer of 51 ± 2% in Col2.3-HSD2 cells and 85 ± 5% in Col3.6-HSD2 cells (Fig. 4Go).



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  		Figure 4. Overexpressed 11{beta}HSD2 has enzymatic activity in transfected ROS 17/2.8 cells. Cells were stably transfected with Col2.3-Cont, Col2.3-HSD2, Col3.6-Cont, and Col3.6-HSD2. The medium contained 5 nM tracer [3H]corticosterone and 100 nM unlabeled corticosterone. The conversion of [3H]corticosterone to [3H]11-dehydrocorticosterone was measured by TLC. For each experimental group, the value is the mean ± SEM of triplicate determinations. The blank value is the percentage of radioactivity in an aliquot of [3H]corticosterone tracer that migrated in the [3H]11-dehydrocorticosterone spot.

 
Glucocorticoid-dependent induction of MMTV-CAT in transfected cells
To determine whether 11{beta}HSD2 could block glucocorticoid function, cells were transiently cotransfected with MMTV-CAT or MMTV-Luc and either a control plasmid or an 11{beta}HSD2 plasmid, and then treated with glucocorticoids for 12 h. In ROS 17/2.8 cells transfected with a control plasmid, 100 nM dexamethasone, 100 nM corticosterone, and 100 nM cortisol caused 13.4-, 4.0-, and 3.7-fold inductions of MMTV-CAT, respectively (Fig. 5Go). In MC3T3-E1 cells transfected with Col3.6-Cont, a similar protocol led to 6.2-, 3.7-, and 3.8-fold inductions of MMTV-Luc, respectively. The differences in MMTV promoter induction may be due to differences in the biological potency of the glucocorticoids, with dexamethasone having a 7-fold higher affinity for the glucocorticoid receptor than cortisol. In ROS 17/2.8 cells transfected with Col3.6-HSD2, cortisol- and corticosterone-dependent induction of MMTV-CAT was completely blocked, whereas dexamethasone-dependent induction of MMTV-CAT was reduced by 63%. Similar results were observed in MC3T3-E1 cells (Fig. 5Go). In ROS 17/2.8 cells transiently cotransfected with Col2.3-HSD2, cortisol- and corticosterone-dependent inductions of MMTV-CAT were reduced by 40% and 41%, respectively, and dexamethasone-dependent induction of MMTV-CAT was reduced by 29%. Results were similar in MC3T3-E1 cells and when using stably transfected Col2.3-HSD2 and Col3.6-HSD2 ROS 17/2.8 cells and MMTV-reporter (data not shown).



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  		Figure 5. Overexpression of 11{beta}HSD2 blocks glucocorticoid-dependent induction of MMTV-CAT in transfected ROS 17/2.8 cells and of MMTV-Luc in transfected MC3T3-E1 cells. Cells were transiently cotransfected with MMTV-CAT or MMTV-Luc and the plasmid shown and treated for 12 h with vehicle, 100 nM dexamethasone, 100 nM corticosterone, or 100 nM cortisol. CAT and luciferase activities are expressed as fold induction (activity in hormone-treated cells relative to activity in vehicle-treated cells). Each value is the mean ± SEM of three determinations. *, P < 0.01 vs. the same treatment of cells cotransfected with MMTV-CAT or MMTV-Luc and Col3.6-Cont.

 
Effects of 11{beta}HSD2 expression on glucocorticoid-dependent regulation of cell growth and osteoblastic mRNA levels in ROS 17/2.8 cells
To determine whether 11{beta}HSD2 expression could reverse glucocorticoid-dependent inhibition of cell growth, stably transfected ROS 17/2.8 cells were treated with vehicle or glucocorticoids for 24–96 h. In cells stably transfected with Col2.3-Cont or Col3.6-Cont, treatment with 100 nM dexamethasone, 100 nM corticosterone, and 100 nM cortisol decreased cell number compared with treatment with vehicle after 24–96 h (Table 1Go). In contrast, in cells stably transfected with Col2.3-HSD2 or Col3.6-HSD2, decreases in cell number were observed only in dexamethasone-treated cultures (Table 1Go). Thus, glucocorticoid-dependent inhibition of ROS 17/2.8 cell growth was prevented by overexpression of Col2.3-HSD2 or Col3.6-HSD2.


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  		Table 1. Glucocorticoid-dependent inhibition of cell growth is prevented in ROS 17/2.8 cells stably transfected with Col2.3-HSD2 and Col3.6-HSD2

 
Likewise, to determine whether 11{beta}HSD2 expression could prevent glucocorticoid-dependent regulation of osteoblast mRNA markers, confluent stably transfected ROS 17/2.8 cells were treated with vehicle or glucocorticoids for 48 h. In cells stably transfected with Col3.6-Cont, 100 nM dexamethasone, 100 nM corticosterone, and 100 nM cortisol decreased OC and Col1a1 mRNA levels and increased BSP mRNA levels (Fig. 6Go). In contrast, in cells stably transfected with Col3.6-HSD2, there was a differential effect of the glucocorticoids; only dexamethasone decreased OC and Col1a1 mRNA levels and increased BSP mRNA levels, whereas corticosterone and cortisol were ineffective (Fig. 6Go). Similar results were obtained with one additional ROS 17/2.8 cell population stably transfected with Col3.6-HSD2 and, although less pronounced, with two ROS 17/2.8 cell populations stably transfected with Col2.3-HSD2 (data not shown). Thus, the expression of 11{beta}HSD2 was able to prevent glucocorticoid-dependent changes in the expression of osteoblastic marker genes.



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  		Figure 6. Glucocorticoid-dependent regulation of osteoblastic marker genes is blocked in ROS 17/2.8 cells stably transfected with Col3.6-HSD2. ROS 17/2.8 cells stably transfected with Col3.6-Cont or Col3.6-HSD2 were grown to confluence and then treated with vehicle, 100 nM dexamethasone, 100 nM corticosterone, or 100 nM cortisol for 48 h. RNA was extracted and analyzed for 11{beta}HSD2, OC, BSP, Col1a1, and 18S RNA by Northern blot analysis. Similar results were obtained with one additional ROS 17/2.8 cell population stably transfected with Col3.6-HSD2 and two ROS 17/2.8 cell populations stably transfected with Col2.3-HSD2.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Glucocorticoids have widespread and diverse effects on bone metabolism in vitro. In rodent calvarial organ and cell cultures, glucocorticoids at physiological levels enhance preosteoblast and osteoblast differentiation and act as permissive hormones for the actions of other osteotropic factors (42). Moreover, in rat, mouse, and human bone marrow stromal cell cultures and calvarial organ cultures, glucocorticoids induce osteoblast differentiation and bone nodule formation (43, 44, 45, 46). In contrast, glucocorticoids at pharmacological levels decrease cell proliferation and type I collagen synthesis and increase osteoblast apoptosis in in vitro models (47, 48, 49, 50). All of these pathways (decreased preosteoblast proliferation, decreased osteoblast function, and increased osteoblast apoptosis) are probably involved in the pathogenesis of glucocorticoid-induced osteoporosis (51, 52).

Our long-term goal is to develop an in vivo system that will allow us to study the physiological effects of glucocorticoids in bone. The present study describes a novel approach that can be used to disrupt glucocorticoid signaling in osteoblasts or any target cell of interest by overexpressing an enzyme that metabolizes endogenous glucocorticoids. In this way, it may be possible to determine the role of endogenous glucocorticoids on bone development and remodeling in a tissue-targeted manner. The NAD-dependent 11{beta}HSD2 is found primarily in mineralocorticoid target tissues such as kidney, sweat gland, salivary gland, the gastrointestinal tract, and placenta (22, 25). By catalyzing the unidirectional conversion of biological active glucocorticoids to inactive metabolites, 11{beta}HSD2 protects the nonselective MR from glucocorticoid activation or, in the case of placenta, protects the fetus from maternal glucocorticoids (22, 23, 24, 25). We cloned rat 11{beta}HSD2 cDNA downstream of a 2.3- or 3.6-kb Col1a1 promoter fragment to produce Col2.3-HSD2 and Col3.6-HSD2, respectively. Transfection of these constructs into osteoblastic ROS 17/2.8 and MC3T3-E1 cells produced strong 11{beta}HSD2 mRNA and protein expression, increased the percent conversion of [3H]corticosterone to [3H]dehydrocorticosterone, impaired glucocorticoid-dependent induction of MMTV-CAT, and prevented glucocorticoiddependent regulation of cell growth and osteoblastic marker genes. Taken together, these data demonstrate that Col2.3-HSD2 and Col3.6-HSD2 drove expression of enzymatically active 11{beta}HSD2 in transfected osteoblastic cells.

Not only were 11{beta}HSD2 mRNA and protein expression stronger in ROS 17/2.8 cells transfected with Col3.6-HSD2 compared with Col2.3-HSD2, the conversion of [3H]corticosterone to [3H]11-dehydrocorticosterone and the prevention of cortisol- and corticosterone-dependent MMTV-CAT induction were also greater in Col3.6–11HSD2 cells. These findings were not surprising, as we previously showed that deletion of the Col1a1 promoter from -3.6 to -2.3 kb reduces promoter activity in transfected osteoblastic cell lines (41). However, in transgenic mice, the 3.6- and 2.3-kb Col1a1 promoters have equivalent activity in calvariae (53). Thus, when transgenic mice are generated, we predict that the two 11{beta}HSD2 transgenes, Col3.6-HSD2 and Col2.3-HSD2, should have comparable activities in bone. The use of these two different Col1a1 promoter fragments should have the potential to disrupt glucocorticoid signaling at different developmental stages in vivo. In cultured murine bone marrow stromal cells and calvarial cells, the 3.6-kb promoter is expressed earlier during osteoblast lineage progression than the 2.3-kb promoter. Likewise, in vivo, the 3.6-kb promoter is expressed in a wider spectrum of osteoblast lineage cells (periosteal cells, preosteoblasts, and mature osteoblasts) and soft connective tissue cells than the 2.3-kb promoter, which is expressed primarily in mature osteoblasts and is more specific for bone (54, 55).

To demonstrate that transfected cell lines were protected from glucocorticoid signaling, we examined the induction of MMTV-CAT (35), which is activated by natural and synthetic glucocorticoids (56). Transfection of Col3.6-HSD2 completely prevented cortisol- and corticosterone-dependent MMTV induction, whereas transfection of Col2.3-HSD2 did not completely abrogate cortisol- and corticosterone-dependent MMTV induction, probably due to reduced promoter expression. However, another possibility is that ROS 17/2.8 cells have endogenous 11{beta}HSD1 activity that reconverts cortisone to cortisol, and 11-dehydrocorticosterone to corticosterone. With all 11{beta}HSD2 constructs, dexamethasonedependent induction of MMTV-CAT was partly maintained. This is consistent with the known reductase activity exhibited by 11{beta}HSD2 on 11-dehydrodexamethasone in contrast to its lack of reductase activity on naturally occurring 11-dehydro metabolites (57, 58). Thus, our finding that dexamethasone was still able to activate the MMTV promoter in 11{beta}HSD2-transfected cells could be explained by reconversion of 11-dehydrodexamethasone to dexamethasone (58).

We wanted to determine whether overexpression of 11{beta}HSD2 could block typical glucocorticoid-dependent responses in ROS 17/2.8 cells, namely the inhibition of proliferation (59), the inhibition of OC (60) and Col1a1 (59) expression, and the stimulation of BSP expression (61). All of the glucocorticoids tested (dexamethasone, corticosterone, and cortisol) decreased the growth of ROS 17/2.8 cells transfected with the Col1a1-Cont constructs. However, the inhibitory effect of corticosterone and cortisol on ROS 17/2.8 cell growth was prevented by overexpression of 11{beta}HSD2, whereas the effect of dexamethasone was not blocked. Also, expression of Col1a1–11{beta}HSD2 constructs in ROS 17/2.8 cells blocked corticosterone- and cortisol-dependent regulation of osteoblastic marker genes, whereas dexamethasone-dependent regulation persisted. These findings suggest that overexpression of 11{beta}HSD2 in osteoblastic cell lines can indeed prevent some hallmark biological responses to natural glucocorticoids, but not to dexamethasone.

A transgenic mouse model using ligand inactivation would have applicability to other nuclear receptor signaling pathways, provided that appropriate metabolic enzymes could be targeted in a tissue-specific manner to inactivate endogenous ligands. The model would be particularly attractive for use with ligands that can signal through multiple nuclear receptors. For example, estrogens, which signal through estrogen receptors {alpha} and {beta} (11), can be modified by estrogen sulfotransferase (62). In conclusion, steroid hormone-modifying enzymes, which are now gaining prominence as important components of physiological signal transduction pathways (63), could be used as tools to prevent steroid hormone signaling in target cells of interest.


   Footnotes
 
1 This work was supported by grants from the Deutsche Forschungsgemeinschaft (Wo 729/1–1 to H.W.W.) and from the NIH (P01-AR-38933 to B.E.K.). Back

Received July 31, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Beato M 1989 Gene regulation by steroid hormones. Cell 56:335–344[CrossRef][Medline]
  2. Granner DK 1995 Hormonal actions. In: Becker KL (ed) Principles and Practice of Endocrinology and Metabolism, Ed 2. Lippincott, Philadelphia, pp 20–34
  3. Lim SK, Won YJ, Lee HC, Huh KB, Park YS 1999 A PCR analysis of ER{alpha} and ER{beta} mRNA abundance in rats and the effect of ovariectomy. J Bone Miner Res 14:1189–1196[CrossRef][Medline]
  4. Zhang MZ, Harris RC, McKanna JA 1999 Regulation of cyclooxygenase-2 (COX-2) in rat renal cortex by adrenal glucocorticoids and mineralocorticoids. Proc Natl Acad Sci USA 96:15280–15285[Abstract/Free Full Text]
  5. Jacobson L 1999 Glucocorticoid replacement, but not corticotropin-releasing hormone deficiency, prevents adrenalectomy-induced anorexia in mice. Endocrinology 140:310–317[Abstract/Free Full Text]
  6. McCarthy MM, Schlenker EH, Pfaff DW 1993 Enduring consequences of neonatal treatment with antisense oligodeoxynucleotides to estrogen receptor messenger ribonucleic acid on sexual differentiation of rat brain. Endocrinology 133:433–439[Abstract/Free Full Text]
  7. Pepin MC, Pothier F, Barden N 1992 Impaired type II glucocorticoid-receptor function in mice bearing antisense RNA transgene. Nature 355:725–728[CrossRef][Medline]
  8. King LB, Vacchio MS, Dixon K, Hunziker R, Margulies DH, Ashwell JD 1995 A targeted glucocorticoid receptor antisense transgene increases thymocyte apoptosis and alters thymocyte development. Immunity 3:647–656[CrossRef][Medline]
  9. Cole TJ, Blendy JA, Monaghan AP, Krieglstein K, Schmid W, Aguzzi A, Fantuzzi G, Hummler E, Unsicker K, Schütz G 1995 Targeted disruption of the glucocorticoid receptor gene blocks adrenergic chromaffin cell development and severely retards lung maturation. Genes Dev 9:1608–1621[Abstract/Free Full Text]
  10. Karas RH, Hodgin JB, Kwoun M, Krege JH, Aronovitz M, Mackey W, Gustafsson JA, Korach KS, Smithies O, Mendelsohn ME 1999 Estrogen inhibits the vascular injury response in estrogen receptor {beta}-deficient female mice. Proc Natl Acad Sci USA 96:15133–15136[Abstract/Free Full Text]
  11. Windahl SH, Vidal O, Andersson G, Gustafsson JA, Ohlsson C 1999 Increased cortical bone mineral content but unchanged trabecular bone mineral density in female ER{beta}(-/-) mice. J Clin Invest 104:895–901[Medline]
  12. Tronche F, Kellendonk C, Kretz O, Gass P, Anlag K, Orban PC, Bock R, Klein R, Schutz G 1999 Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety. Nat Genet 23:99–103[CrossRef][Medline]
  13. Finch CE, Felicio LS, Mobbs CV, Nelson JF 1984 Ovarian and steroidal influences on neuroendocrine aging processes in female rodents. Endocr Rev 5:467–497[Abstract/Free Full Text]
  14. Campbell JW, Pollack IF 1997 Growth factors in gliomas: antisense and dominant negative mutant strategies. J Neurooncol 35:275–285[CrossRef][Medline]
  15. Stein CA 2000 Is irrelevant cleavage the price of antisense efficacy? Pharmacol Ther 85:231–236[CrossRef][Medline]
  16. Dellovade TL, Chan J, Vennstrom B, Forrest D, Pfaff DW 2000 The two thyroid hormone receptor genes have opposite effects on estrogen-stimulated sex behaviors. Nat Neurosci 3:472–475[CrossRef][Medline]
  17. Gauthier K, Chassande O, Plateroti M, Roux JP, Legrand C, Pain B, Rousset B, Weiss R, Trouillas J, Samarut J 1999 Different functions for the thyroid hormone receptors TR{alpha} and TR{beta} in the control of thyroid hormone production and post-natal development. EMBO J 18:623–631[CrossRef][Medline]
  18. Wendling O, Chambon P, Mark M 1999 Retinoid X receptors are essential for early mouse development and placentogenesis. Proc Natl Acad Sci USA 96:547–551[Abstract/Free Full Text]
  19. Johnson A, Chandraratna RA 1999 Novel retinoids with receptor selectivity and functional selectivity. Br J Dermatol 140:12–17
  20. Meijer OC, Cole TJ, Schmid W, Schutz G, Joels M, De Kloet ER 1997 Regulation of hippocampal 5-HT1A receptor mRNA and binding in transgenic mice with a targeted disruption of the glucocorticoid receptor. Brain Res Mol Brain Res 46:290–296[Medline]
  21. Albiston AL, Obeyesekere VR, Smith RE, Krozowski ZS 1994 Cloning and tissue distribution of the human 11{beta}-hydroxysteroid dehydrogenase type 2 enzyme. Mol Cell Endocrinol 105:R11–R17
  22. Zhou MY, Gomez-Sanchez EP, Cox DL, Cosby D, Gomez-Sanchez CE 1995 Cloning, expression, and tissue distribution of the rat nicotinamide adenine dinucleotide-dependent 11{beta}-hydroxysteroid dehydrogenase. Endocrinology 136:3729–3734[Abstract]
  23. Krozowski Z 1999 The 11{beta}-hydroxysteroid dehydrogenases: functions and physiological effects. Mol Cell Endocrinol 151:121–127[CrossRef][Medline]
  24. Funder JW, Pearce PT, Smith R, Smith AI 1988 Mineralocorticoid action: target tissue specificity is enzyme, not receptor, mediated. Science 242:583–585[Abstract/Free Full Text]
  25. Edwards CR, Stewart PM, Burt D, Brett L, McIntyre MA, Sutanto WS, de Kloet ER, Monder C 1988 Localisation of 11{beta}-hydroxysteroid dehydrogenase–tissue specific protector of the mineralocorticoid receptor. Lancet 2:986–989[CrossRef][Medline]
  26. Sun K, Adamson SL, Yang K, Challis JR 1999 Interconversion of cortisol and cortisone by 11{beta}-hydroxysteroid dehydrogenases type 1 and 2 in the perfused human placenta. Placenta 20:13–19[CrossRef][Medline]
  27. Shams M, Kilby MD, Somerset DA, Howie AJ, Gupta A, Wood PJ, Afnan M, Stewart PM 1998 11{beta}-Hydroxysteroid dehydrogenase type 2 in human pregnancy and reduced expression in intrauterine growth restriction. Hum Reprod 13:799–804[Abstract/Free Full Text]
  28. Saegusa H, Nakagawa Y, Liu YJ, Ohzeki T 1999 Influence of placental 11{beta}-hydroxysteroid dehydrogenase (11{beta}-HSD) inhibition on glucose metabolism and 11beta-HSD regulation in adult offspring of rats. Metabolism 48:1584–1588[CrossRef][Medline]
  29. Roland BL, Funder JW 1996 Localization of 11{beta}-hydroxysteroid dehydrogenase type 2 in rat tissues: in situ studies. Endocrinology 137:1123–1128[Abstract]
  30. Welberg LA, Seckl JR, Holmes MC 2000 Inhibition of 11{beta}-hydroxysteroid dehydrogenase, the foeto-placental barrier to maternal glucocorticoids, permanently programs amygdala GR mRNA expression and anxiety-like behaviour in the offspring. Eur J Neurosci 12:1047–1054[CrossRef][Medline]
  31. Waddell BJ, Benediktsson R, Brown RW, Seckl JR 1998 Tissue-specific messenger ribonucleic acid expression of 11{beta}-hydroxysteroid dehydrogenase types 1 and 2 and the glucocorticoid receptor within rat placenta suggests exquisite local control of glucocorticoid action. Endocrinology 139:1517–1523[Abstract/Free Full Text]
  32. Bland R, Worker CA, Noble BS, Eyre LJ, Bujalska IJ, Sheppard MC, Stewart PM, Hewison M 1999 Characterization of 11{beta}-hydroxysteroid dehydrogenase activity and corticosteroid receptor expression in human osteosarcoma cell lines. J Endocrinol 161:455–464[Abstract]
  33. Cooper MS, Walker EA, Bland R, Fraser WD, Hewison M, Stewart PM 2000 Expression and functional consequences of 11{beta}-hydroxysteroid dehydrogenase activity in human bone. Bone 27:375–381[Medline]
  34. Bedalov A, Breault DT, Sokolov BP, Lichtler AC, Bedalov I, Clark SH, Mack K, Khillan JS, Woody CO, Kream BE, Rowe DW 1994 Regulation of the {alpha}(I) collagen promoter in vascular smooth muscle cells. Comparison with other {alpha}1(I) collagen-producing cells in transgenic animals and cultured cells. J Biol Chem 269:4903–4909[Abstract/Free Full Text]
  35. Ostrowski MC, Huang AL, Kessel M, Wolford RG, Hager GL 1984 Modulation of enhancer activity by the hormone responsive regulatory element from mouse mammary tumor virus. EMBO J 3:1891–1899[Medline]
  36. Bresnick EH, John S, Berard DS, LeFebvre P, Hager GL 1990 Glucocorticoid receptor-dependent disruption of a specific nucleosome on the mouse mammary tumor virus promoter is prevented by sodium butyrate. Proc Natl Acad Sci USA 87:3977–3981[Abstract/Free Full Text]
  37. Woitge HW, Kream BE 2000 Calvariae from fetal mice with a disrupted Igf1 gene have reduced rates of collagen synthe-sis but maintain responsiveness to glucocorticoids. J Bone Miner Res 15:1956–1964[CrossRef][Medline]
  38. Smith RE, Li KX, Andrews RK, Krozowski Z 1997 Immunohistochemical and molecular characterization of the rat 11{beta}-hydroxysteroid dehydrogenase type II enzyme. Endocrinology 138:540–547[Abstract/Free Full Text]
  39. Purschke WG, Muller PK 1994 An improved fluor diffusion assay for chloramphenicol acetyltransferase gene expression. BioTechniques 16:264–269[Medline]
  40. Smith PK, Krohn RI, Hermanson GT, Mailia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC 1985 Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85[CrossRef][Medline]
  41. Pavlin D, Lichtler AC, Bedalov A, Kream BE, Harrison JR, Thomas HF, Gronowicz GA, Clark SH, Woody CO, Rowe DW 1992 Differential utilization of regulatory domains within the {alpha}1(I) collagen promoter in osseous and fibroblastic cells. J Cell Biol 116:227–236[Abstract/Free Full Text]
  42. Lukert BP, Kream BE 1996 Clinical and basic aspects of glucocorticoid action in bone. In: Bilezikian J, Raisz LG, Rodan GA (eds) Principles of Bone Biology. Academic Press, San Diego, vol 1:533–548
  43. Bellows CG, Heersche JNM, Aubin JE 1990 Determination of the capacity for proliferation and differentiation of osteoprogenitor cells in the presence and absence of dexamethasone. Dev Biol 140:132–138[CrossRef][Medline]
  44. Herbertson A, Aubin JE 1995 Dexamethasone alters the subpopulation make-up of rat bone marrow stromal cell cultures. J Bone Miner Res 10:285–294[Medline]
  45. Dietrich JW, Canalis EM, Maina DM, Raisz LG 1978 Effects of glucocorticoids on fetal rat bone collagen synthesis in vitro. Endocrinology 104:715–721[Abstract/Free Full Text]
  46. Kream BE, Petersen DN, Raisz LG 1990 Cortisol enhances the anabolic effects of insulin-like growth factor I on collagen synthesis and procollagen messenger ribonucleic acid levels in cultured 21-day fetal rat calvariae. Endocrinology 126:1576–1583[Abstract/Free Full Text]
  47. Lukert B, Mador A, Raisz LG, Kream BE 1991 The role of DNA synthesis in the responses of fetal rat calvariae to cortisol. J Bone Miner Res 6:453–460[Medline]
  48. Weinstein RS, Jilka RL, Parfitt AM, Manolagas SC 1998 Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. Potential mechanisms of their deleterious effects on bone. J Clin Invest 102:274–282[Medline]
  49. Kim HT, Chen TL 1989 1,25-Dihydroxyvitamin D3 interaction with dexamethasone and retinoic acid: effects on procollagen messenger ribonucleic acid levels in rat osteoblast-like cells. Mol Endocrinol 3:97–104[Abstract/Free Full Text]
  50. Ng KW, Manji SS, Young MF, Findlay DM 1989 Opposing influences of glucocorticoid and retinoic acid on transcriptional control in preosteoblasts. Mol Endocrinol 3:2079–2085[Abstract/Free Full Text]
  51. Manolagas SC 2000 Corticosteroids and fractures: a close encounter of the third cell kind. J Bone Miner Res 15:1001–1005[CrossRef][Medline]
  52. Lane NE, Lukert B 1998 The science and therapy of glucocorticoid-induced bone loss. Endocrinol Metab Clin North Am 27:465–483[CrossRef][Medline]
  53. Bogdanovic Z, Bedalov A, Krebsbach PH, Woody CO, Clark SH, Thomas HF, Rowe DW, Kream BE, Lichtler AC 1994 Upstream regulatory elements necessary for expression of the rat COL1A1 promoter in transgenic mice. J Bone Miner Res 9:285–292[Medline]
  54. Kalajzic I, Kaliterna M, Mallico E, Nahounou M, Kalajzic Z, Clark SH, Gronowicz G, Lichtler AC, Rowe DW 1999 Use of Col1A1GFP transgenes to identify osteoblastic lineage. J Bone Miner Res 14:S165 (Abstract)
  55. Rossert J, Eberspaecher H, de Crombrugghe B 1995 Separate cis-acting DNA elements of the mouse pro-{alpha}1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice. J Cell Biol 129:1421–1432[Abstract/Free Full Text]
  56. Warriar N, Page N, Govindan MV 1994 Transcription activation of mouse mammary tumor virus-chloramphenicol acetyltransferase: a model to study the metabolism of cortisol. Biochemistry 33:12837–12843[CrossRef][Medline]
  57. Penning TM 1997 Molecular endocrinology of hydroxysteroid dehydrogenases. Endocr Rev 18:281–305[Abstract/Free Full Text]
  58. Ferrari P, Smith RE, Funder JW, Krozowski ZS 1996 Substrate and inhibitor specificity of the cloned human 11{beta}-hydroxysteroid dehydrogenase type 2 isoform. Am J Physiol 270:E900–E904
  59. Hodge BO, Kream BE 1988 Variable effects of dexamethasone on protein synthesis in clonal rat osteosarcoma cells. Endocrinology 122:2127–2133[Abstract/Free Full Text]
  60. Aslam F, Shalhoub V, van Wijnen AJ, Banerjee C, Bortell R, Shakoori AR, Litwack G, Stein JL, Stein GS, Lian JB 1995 Contributions of distal and proximal promoter elements to glucocorticoid regulation of osteocalcin gene transcription. Mol Endocrinol 9:679–690[Abstract/Free Full Text]
  61. Ogata Y, Yamauchi M, Kim RH, Li JJ, Freedman LP, Sodek J 1995 Glucocorticoid regulation of bone sialoprotein (BSP) gene expression-Identification of a glucocorticoid response element in the bone sialoprotein gene promoter. Eur J Biochem 230:183–192[Medline]
  62. Strott CA 1996 Steroid sulfotransferases. Endocr Rev 17:670–697[Abstract/Free Full Text]
  63. Song WC, Melner MH 2000 Steroid transformation enzymes as critical regulators of steroid action in vivo. Endocrinology 141:1587–1589[Free Full Text]



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