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Endocrinology Vol. 138, No. 6 2291-2300
Copyright © 1997 by The Endocrine Society

ARTICLES

Transcriptional Up-Regulation of the Human Androgen Receptor by Androgen in Bone Cells1

Kristine M. Wiren, Xiaowei Zhang, Chawnshang Chang, Edward Keenan and Eric S. Orwoll

Bone and Mineral Research Unit, Veterans Affairs Medical Center, (K.M.W., X.Z., E.S.O.) Portland, Oregon 97201; the Departments of Behavioral Neuroscience (K.M.W.), Cell and Developmental Biology (K.M.W.), Medicine (K.M.W. and E.S.O.), Pharmacology (E.K.), and Surgery (E.K.), Oregon Health Sciences University, Portland, Oregon 97201; and the Department of Medicine, Comprehensive Cancer Center and Endocrinology-Reproductive Physiology Program, University of Wisconsin (C.C.), Madison, Wisconsin 53792

Address all correspondence and requests for reprints to: Kristine Wiren, Ph.D., Research Service 151Q, Veterans Administration Medical Center, 3710 SW Veterans Hospital Road, Portland, Oregon 97201. E-mail: wirenk{at}teleport.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Androgen regulation of androgen receptor (AR) expression has been observed in a variety of tissues, generally as inhibition, and is thought to attenuate cellular responses to androgen. AR is expressed in osteoblasts, the bone-forming cell, suggesting direct actions of androgens on bone. Here we characterized the effect of androgen exposure on AR gene expression in human osteoblastic SaOS-2 and U-2 OS cells.

Treatment of osteoblastic cells with the nonaromatizable androgen 5{alpha}-dihydrotestosterone increased AR steady state messenger RNA levels in a time- and dose-dependent fashion. Reporter assays with 2.3 kilobases of the proximal 5'-flanking region of the human AR promoter linked to the chloramphenicol acetyltransferase gene in transfected cultures showed that up-regulation of AR promoter activity by androgen was time and dose dependent. Treatment with other steroid hormones, including progesterone, 17ß-estradiol, and dexamethasone, was without effect. The antiandrogen hydroxyflutamide completely antagonized androgen up-regulation.

Thus, in contrast to many other androgen target tissues, androgen exposure increases steady state AR messenger RNA levels in osteoblasts. This regulation occurs at least partially at the level of transcription, is mediated by the 5'-promoter region of the AR gene, and is dependent on functional AR. These results suggest that physiological concentrations of androgens have significant effects on AR expression in skeletal tissue.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANDROGENS ARE important regulators of skeletal metabolism. Significant levels of testosterone circulate in both females and males. In men and experimental animals, orchidectomy results in a rapid increase in the rate of bone remodeling and progressive bone loss (1). Osteopenia has been described in association with a variety of hypogonadal states in men (2), and hypogonadism is present in as many as one third of men who present with osteoporosis (3), implicating androgen deficiency as an important risk factor for osteoporosis. Androgen replacement therapy prevents the development of bone loss in orchidectomized animals and has been reported to improve bone density in osteoporotic hypogonadal men (4). Androgens have also been suggested to play a role in bone physiology in women (5, 6, 7, 8) and have been shown to be effective treatment in the prevention of postmenopausal bone loss (9, 10). Finally, evidence suggests that estrogen and androgen in combination are more effective in the treatment of bone loss than estrogen replacement alone (11). Hence, the importance of androgens in normal bone physiology is clear.

Osteoblasts, the bone-forming cells, respond to androgens by changes in specific messenger RNA (mRNA) and/or protein synthesis. Osteoblastic differentiation is influenced by exposure to physiological concentrations of either testosterone or 5{alpha}-dihydrotestosterone (DHT), with increased calcium accumulation (12, 13), inhibition of PTH-stimulated PGE2 (14) and cAMP production (15), and increased alkaline phosphatase, collagen, and transforming growth factor-ß protein and/or mRNA expression reported (16, 17). DHT and/or testosterone have been shown to either stimulate (12, 18) or inhibit (16) osteoblastic cell proliferation. These in vitro studies, which include the use of nonaromatizable androgens such as DHT, clearly identify androgens (independent of estrogenic metabolites) as primary effectors of osteoblastic function and thus support the clinical evidence of an effect of androgens on bone biology. In addition, androgen receptor (AR) has been identified in normal human osteoblastic cells (19), human and rat osteoblastic osteosarcoma cell lines (16, 20), and immortalized mouse osteoblastic cells (18, 21). Steroid hormones are generally dependent on binding their specific intracellular/nuclear receptors, which act as ligand dependent trans-acting factors, to produce biological responses through changes in gene transcription (22). It is, therefore, likely that in bone, androgens act primarily by interacting with the AR, which subsequently regulates androgen-responsive gene transcription.

In some androgen target tissues, responses to androgens have been linked to AR concentrations (23), thus demonstrating the biological significance of AR regulation. Autoregulation of the AR by androgen has been reported. However, the mode of regulation of AR levels by androgen is both complex and tissue specific. In many tissues, AR transcription and/or steady state mRNA levels are down-regulated after androgen exposure (24, 25, 26, 27). The down-regulation of AR levels is thought to self-limit androgen responsiveness in these androgen target tissues and is a general phenomenon observed with a variety of steroid hormone receptors. On the other hand, androgen exposure has been shown to variably up-regulate AR expression in other cell types (28, 29, 30, 31, 32). Other investigators have not reproduced AR up-regulation in similar cells (24, 27, 33), so this observation remains somewhat controversial. No information is available about regulation of AR expression in bone, except one report showing increased AR levels after long term androgen exposure in the osteosarcoma cell line TE-85 (13). Thus, although homologous regulation of AR levels after androgen exposure has been described in other tissues, and androgens play an important role in skeletal physiology, little is known about the regulation of AR levels in the osteoblast.

In this study, we have evaluated the effects of androgen exposure on human AR gene expression in osteoblastic cells. Changes in steady state mRNA levels after androgen exposure were determined using a ribonuclease (RNase) protection assay. In addition, to gain a better understanding of the transcriptional regulation of AR in skeletal tissue, promoter analysis using chloramphenicol acetyltransferase (CAT) reporter assays was performed in transiently transfected cells with a construct containing 2.3 kilobases of the 5'-flanking region of the human AR promoter ligated into the promoter-less plasmid pBS-CAT (AR-CAT). The results show up-regulation of human AR gene expression in osteoblasts to be cell specific, hormone specific, time and androgen dose dependent, and mediated by functional AR protein. We conclude that androgen exposure increases steady state AR mRNA levels in osteoblasts, at least partially at the level of transcription, mediated by the 5'-promoter region of the AR gene and dependent on functional AR. These results suggest that up-regulation of AR by androgen in osteoblasts could amplify, rather than suppress, the response of osteoblasts to androgen. Therefore, physiological concentrations of androgens may have significant effects on AR expression and function in skeletal tissue.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and reagents
The isotopes [{alpha}-32P]UTP (800 Ci/mmol) and [3H]acetyl coenzyme A (200 mCi/mmol; CAT assay grade) were purchased from DuPont-New England Nuclear (Boston, MA). All media, buffers, supplements, and reagents for cell culture were obtained from Life Technologies (Grand Island, NY) and Sigma Chemical Co. (St. Louis, MO). Steroid hormones and other reagents were obtained from Sigma Chemical Co., and the AR antagonist hydroxyflutamide (HF) was provided by Schering Corp. (Bloomfield, NJ). MAXIscript in vitro transcription and RPA II RNase protection assay kits were obtained from Ambion (Austin, TX).

Cell culture
Human clonal osteoblastic osteosarcoma (SaOS-2, passage 55, and U-2 OS, passage 45) and human prostatic carcinoma LNCaP (passage 58) cell lines were obtained from the American Type Culture Collection (Rockville, MD). Monolayer cultures of SaOS-2 and U-2 OS cells were maintained in phenol red-free McCoy’s 5A medium containing 7% bovine calf serum (BCS) and 50 µg/ml each of penicillin and streptomycin at 37 C in 5% CO2. LNCaP cultures were maintained in RPMI 1640 medium with 10% BCS and 50 µg/ml each of penicillin and streptomycin at 37 C in 5% CO2. For all hormone treatments, cultures were grown with medium containing charcoal-dextran-stripped calf serum to reduce endogenous steroid levels. Within one experiment, all incubations were terminated at the same time for the control and hormone-treated samples.

Plasmid constructs
Human BS-AR-275 plasmid for RNase protection analysis of steady state AR mRNA levels was obtained from Dr. Marco Marcelli (34). Mouse ß-actin complementary DNA (cDNA) for RNase protection analysis was purchased from Ambion (Austin, TX). The androgen-responsive CAT construct pG29Gtk-CAT (tk, thymidine kinase) was obtained from Dr. Rainer Renkawitz (35), and the estrogen-responsive reporter construct EREtkCAT (ERE, estrogen response element) was provided by Dr. Ming-Jer Tsai (36). All plasmid preparations were purified by CsCl density centrifugation.

RNA isolation and RNase protection analysis
RNA was isolated by the single step acid guanidinium isothiocyanate-phenol-chloroform extraction method (37), except that ß-mercaptoethanol was omitted from the denaturing solution. Quantitation of RNA was performed by spectrophotometric determination at 260 nm. Fifty micrograms of total RNA from each of the osteoblastic osteosarcoma cell lines SaOS-2 and U-2 OS or 10 µg LNCaP RNA were hybridized to a 32P-labeled antisense complementary RNA (cRNA) AR probe generated against a 275-bp segment of the human AR cDNA corresponding to the coding region for nucleotides 890-1165 in exon 1 (34). The plasmid was linearized with HindIII and transcribed with T7 RNA polymerase in the presence of 50 µCi [{alpha}-32P]UTP to a specific activity of over 1 x 109 cpm/µg. For a loading control, 10 µg total RNA were hybridized to a 32P-labeled antisense cRNA probe generated from mouse ß-actin cDNA, linearized with EcoRI, and transcribed with SP6 RNA polymerase. Radiolabeled antisense cRNA probes were generated using the MAXIscript system (Ambion) and purified by gel filtration. Sample RNA was hybridized to 8 x 104 cpm probe overnight at 42 C in the manufacturer’s buffer. Unhybridized probe was then removed by treating the reaction with 200 µl of a 1:100 dilution of RNase A-RNaseT1 for 30 min at 37 C. For normalization to ß-actin, incubations were performed with RNaseT1 only. The samples were precipitated, resuspended in gel loading buffer, heated at 90 C for 5 min, loaded onto a 5% polyacrylamide-8 M urea sequencing gel, and electrophoresed at 250 V for 30 min in Tris-borate buffer. Gels were dried and exposed to Kodak X-AR5 autoradiographic film at -70 C for up to 5 days. Quantitation of films was performed by scanning densitometry (GS300, Hoefer Scientific, San Francisco, CA). Size markers were DNA marker V standards (Boehringer Mannheim, Indianapolis, IN) run in adjacent lanes.

Transient transfections
Twenty-four to 48 h before transfection, confluent SaOS-2 were split at a 1:4 ratio in phenol-red free McCoy’s containing 7% BCS such that they would be 50–75% confluent the next day. The cells were then harvested by trypsinization, centrifuged (1000 rpm for 5 min), and resuspended in a small volume of serum-free McCoy’s medium. The cell suspension was counted and diluted to 8000 cells/µl. Approximately 5 x 106 cells were mixed with 20 µg full-length AR-CAT DNA in a sterile gene-pulse chamber. Cells were exposed to a controlled electrical field of 960 microfarads at 200 V in a Bio-Rad Gene Pulser with Capacitance Extender (Bio-Rad Laboratories, Richmond, CA). Each electroporation also contained 20 µg of the ß-galactosidase (ß-gal) expression plasmid pSV-ß-gal vector (Promega, Madison, WI), which was used to control for transfection efficiency. Each electroporation was diluted in 20 ml serum-containing medium and divided into 10 35-mm dishes, used in various experimental conditions. Cells were incubated in normal medium for 18 h, then hormones were added for an additional 24–48 h before isolation for CAT, ß-gal, and protein determinations.

Assay of transcriptional activity
CAT activity was determined by the fluor diffusion method (38). Cultures were lysed in 250 µl reporter lysis buffer (Promega). CAT activity was measured in 50 µl cell extract after inactivation of endogenous acetylases by incubation at 65 C for 15 min. The extract was mixed with 200 µl chloramphenicol at 0.4 mg/ml and [3H]acetyl-coenzyme A at 2.5 µCi/ml in Tris-HCl, pH 7.8. The reaction mixture was carefully overlaid with 1 ml organic scintillation fluid (Econofluor, New England Nuclear) and counted repeatedly over a 2- to 3-h period in a liquid scintillation counter. CAT activity was determined from the linear portion of the slope. The ß-gal activity was determined colorimetrically as previously described (39), using 150 µl cell extract. The assay buffer contained 60 mM sodium phosphate (pH 7.5), 1 mM MgCl2, 0.67 mg/ml o-nitrophenyl-ß-D-galactopyranoside, and 40 mM ß-mercaptoethanol. A standard curve containing 2–100 µU ß-gal was run with each assay. Assay buffer was incubated with cell extract for 60 min at 37 C, and the reaction was stopped by the addition of Na2CO3 to a final concentration of 625 mM. Absorbance readings were taken at 420 nm. There was no endogenous ß-gal activity in any of the cell lines analyzed. Protein concentrations were determined in the samples by the Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, CA) following the manufacturer’s recommendations and using BSA as the standard. The data shown represent the mean ± SEM from triplicate samples generally and were performed as independent transfections two to five times. Plasmid DNAs derived from different preparations were generally used and showed 2- to 3-fold differences in basal expression. Values obtained for CAT activity were corrected to values for ß-gal activity expressed as counts per min/min·mU ß-gal to correct for differences in transfection efficiency, and then generally normalized to control values to characterize hormonal regulation.

Statistical analysis
Data for time-course and dose-response studies were analyzed using single factor ANOVA followed by two-tailed Student’s t test between treatment groups. The individual contrast between treatment groups was made with an unpaired two-tailed Student’s t test. Differences of P < 0.05 were considered significant. Results are presented as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of androgen exposure on AR steady state mRNA expression in osteoblastic and prostatic carcinoma cells
In many androgen target tissues, androgen exposure leads to down-regulation of AR gene expression. In this study, we have characterized the effect of androgen exposure on human AR gene expression in osteoblastic cells. Human SaOS-2 cells were chosen as an osteoblastic model because they express high levels of alkaline phosphatase activity and other phenotypic markers typical of osteoblasts (40) and, in addition, have been shown to be androgen responsive and to express AR protein (15, 20). For all hormone treatments, cultures were maintained with phenol red-free medium containing charcoal-dextran-stripped calf serum to reduce endogenous steroid levels.

AR mRNA abundance was characterized by RNase protection analysis in human osteoblast-like and human prostatic carcinoma lines using SaOS-2 cells and the androgen-responsive LNCaP cell line, respectively. We examined the effect of treatment with the nonaromatizable androgen DHT, which cannot be converted into estrogen, for increasing periods of time on AR mRNA abundance in SaOS-2 cells compared to that in similarly treated LNCaP cells. Confluent cultures of both cell types were treated with DHT at 10-8 M for 24, 48, and 72 h and subjected to RNase protection analysis (Fig. 1Go). An autoradiograph representative of four experiments is shown in Fig. 1Go. Changes in human AR steady state mRNA abundance were detected as early as 24 h after exposure in both cell lines. However, over the 3-day period, AR steady state mRNA levels declined by about 50% in LNCaP cells, whereas in SaOS-2 osteoblastic cells, AR mRNA levels increased by 2- to 3-fold over the same time period. The down-regulation of AR mRNA levels by androgen observed here with LNCaP cells is consistent with that previously reported (24, 25, 26, 27). These results again demonstrate that the mode of regulation of AR expression by androgen is tissue specific.



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  		Figure 1. Dichotomous regulation of AR mRNA levels in osteoblast-like and prostatic carcinoma cell lines after exposure to androgen. A, Time course of changes in AR mRNA abundance after DHT exposure in human SaOS-2 osteoblastic cells and human LNCaP prostatic carcinoma cells. To determine the effect of androgen exposure on hAR mRNA abundance, confluent cultures of either osteoblast-like cells (SaOS-2) or prostatic carcinoma cells (LNCaP) were treated with 10-8 M DHT for 0, 24, 48, or 72 h. Total RNA was then isolated and subjected to RNase protection analysis with 50 µg total cellular RNA from SaOS-2 osteoblastic cells and 10 µg total RNA from LNCaP cultures. An antisense riboprobe of a 275-bp fragment corresponding to nucleotides 890-1165 in exon 1 of the human AR cDNA (34) was hybridized to total mRNA isolated from these two cultures. The resultant RNA:RNA hybrids were digested with both ribonucleases A and T1, and the protected fragment was separated on a 5% denaturing polyacrylamide-urea gel. Total RNA (10 µg) was also probed by RNase protection for ß-actin to control for differences in loading. Yeast transfer RNA was added as a negative control. Dried gels were exposed to x-ray film, and the relative amounts of each band quantitated by densitometry. B, Densitometric analysis of AR mRNA steady state levels. The AR mRNA to ß-actin ratio is expressed as the mean ± SEM compared to the control value from three to five independent assessments.

 
To determine the dose-response relationship for androgen regulation of human AR gene expression in osteoblastic cells, confluent SaOS-2 cultures were treated for 48 h with vehicle or concentrations of DHT ranging from 10-14–10-6 M. Total RNA was isolated, and AR steady state mRNA abundance was determined by RNase protection assay (Fig. 2Go). The accumulation of AR mRNA was significantly up-regulated by DHT treatment in these cultures in a dose-dependent fashion (P < 0.05). Maximal 2- to 3-fold stimulation of steady state mRNA levels was observed with 10-8 M hormone, as shown in both the time-course and dose-response studies. These results are consistent with androgen binding to the AR, with a Kd near 10-9 M.



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  		Figure 2. Dose response of AR mRNA up-regulation by androgen in an osteoblast-like cell line. A, DHT dose response in SaOS-2 cells. To determine the dose-response relationship, confluent SaOS-2 cultures were treated for 48 h with vehicle or DHT concentrations ranging from 10-14-10-6 M. To quantitate the steady state level of AR mRNA, a RNase protection assay was performed by incubating 50 µg total cellular RNA from SaOS-2 osteoblastic cells with a radiolabeled antisense AR cRNA probe, as described in Fig. 1Go. Total RNA (10 µg) was also probed by RNase protection for ß-actin to control for differences in loading. Yeast transfer RNA was added as a negative control. Dried gels were exposed to x-ray film, and the relative amounts of each band were quantitated by densitometry. B, Densitometric analysis of AR mRNA abundance. The AR mRNA to ß-actin ratio is displayed expressed as the mean ± SEM compared to the control value from two to four independent assessments.

 
Up-regulation of AR-CAT activity by androgen in osteoblastic cells
To more fully characterize androgen up-regulation of AR gene expression in osteoblastic cells, we performed transient transfection experiments with a plasmid containing an AR promoter-reporter gene construct, again with the AR-containing and androgen-responsive SaOS-2 cells. The AR-CAT plasmid contains the proximal 2330 bp from the 5'-flanking region of the human AR promoter (-2330 to +573) driving expression of the bacterial CAT gene (41). Cultures were cotransfected with CAT and ß-gal expression constructs and grown for 48–72 h before isolation of cell lysates for CAT, ß-gal, and protein determinations. All CAT activity determinations were normalized to ß-gal activity to correct for differences in transfection efficiency. Changes observed in CAT activity were interpreted as changes in AR promoter activity.

Transcriptional regulation of AR expression in osteoblasts by the two major androgen metabolites was then characterized using AR-CAT. Transiently transfected SaOS-2 cells were treated either with testosterone at 10-9 or 10-8 M or with DHT at 10-8 M. Eighteen hours after transfection, hormone was added to the medium, and cells were grown for 24 h before harvest for CAT, ß-gal, and protein determinations. Both testosterone and DHT induce expression of CAT activity from the AR promoter in osteoblastic cells (Fig. 3AGo). The potency of stimulation by these two androgens is roughly equivalent and again corresponds to their respective receptor binding affinities. The specificity of regulation was characterized in cells transfected with pSV2-CAT, a plasmid with expression driven by an unselective promoter. There was no effect of DHT treatment on expression of CAT activity using the plasmid pSV2-CAT (Fig. 3BGo). There was also no measurable CAT activity in cultures transfected with the promoterless parent construct pBS-CAT, with or without DHT treatment (data not shown).



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  		Figure 3. Characterization of androgen regulation of AR promoter activity with AR-CAT in transiently transfected SaOS-2 osteoblastic osteosarcoma cultures. A, Treatment of transiently cotransfected SaOS-2 cells with different forms of androgen. SaOS-2 osteoblastic osteosarcoma cells were transiently cotransfected with 20 µg each of the AR-CAT reporter construct AR-CAT and the ß-gal expression vector. Cells were grown for 18 h, then treated with either testosterone at 10-9 M (-9 Test) or 10-8 M (-8 Test) or 10-8 M DHT (-8 DTH) as indicated for an additional 24 h before harvest for CAT, ß-gal, and protein determinations as described in Materials and Methods. All CAT activity determinations were normalized to ß-gal activity to correct for differences in transfection efficiency. Results are the mean ± SEM of at least three independent experiments. ***, P < 0.001 (statistically significant difference compared with the appropriate control group). B, Comparison between AR promoter activity and pSV2-CAT expression after DHT treatment. SaOS-2 osteoblastic osteosarcoma cells were transiently cotransfected with 20 µg each of the AR-CAT reporter construct AR-CAT or with pSV2-CAT, and the ß-gal expression vector. Cells were grown for 18 h, then treated with 10-8 M DTH for an additional 24 h before harvest. Results are the mean ± SEM of at least three independent experiments. *, P < 0.01 (statistically significant difference compared with the appropriate control group).

 
Androgen up-regulation of the AR promoter by androgen was examined in more extensive dose-response experiments. SaOS-2 cultures were treated after transfection with increasing concentrations of DHT, from 10-12–10-7 M for 24 h as described in Fig. 3Go. Concentrations as low as 10-12 M significantly induced the expression of CAT activity from the AR promoter (P < 0.05; Fig. 4AGo). Maximal stimulation was observed with 10-8 M DHT. These results are similar in magnitude to those seen with DHT regulation of AR steady state mRNA abundance shown in Fig. 2Go.



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  		Figure 4. AR-CAT dose response and time course after DHT treatment in SaOS-2 osteoblastic cells. SaOS-2 osteoblastic osteosarcoma cells were transiently cotransfected with 20 µg each of the AR-CAT reporter construct AR-CAT and the ß-gal expression vector, and CAT activity was determined as described in Fig. 3Go. A, DHT dose response for induction of AR-CAT activity in SaOS-2 osteoblastic cells. Data are shown as stimulation of activity by hormone above the control value. Results are the mean ± SEM of at least three independent experiments. *, P < 0.05; ***, P < 0.001 (statistically significant difference compared with the appropriate control group). B, Time course of induction of AR-CAT activity after DHT treatment in SaOS-2 osteoblastic cultures. SaOS-2 cells were grown for 18 h after transfection, then treated for 0, 1, 2, 4, 24, or 40 h with 10-8 M DHT before harvest for CAT, ß-gal, and protein determinations. Data are shown as stimulation of activity by hormone above the control level. Results are the mean ± SEM of at least two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (statistically significant difference compared with the appropriate control group).

 
We next examined the time course for androgen induction of AR promoter activity in osteoblastic cells. SaOS-2 cells were transfected with AR-CAT, and 18 h later, androgen treatments were initiated. Cultures were treated with 10-8 M DHT for 1, 2, 4, 24, and 40 h before harvest (Fig. 4BGo). Significant changes in CAT activity were seen as early as 2 h after treatment (P < 0.05). This rapid induction of CAT activity observed suggests direct regulation of the AR promoter after androgen treatment without the requirement for intermediate protein synthesis.

Androgen up-regulation of AR expression in another osteoblastic model system
SaOS-2 cells are a clonal line that represent one model of osteoblastic phenotypic expression. To determine the generalizability of androgen regulation of AR expression that we observed, a second independent clonal human osteoblastic cell line U-2 OS was used. These cultures were employed in transient transfection assays with AR-CAT. U-2 OS cells were transfected and 18 h later were treated with increasing concentrations of DHT (10-12, 10-10, or 10-8 M) for an additional 24 h before harvest. As shown in Fig. 5Go, dose-dependent induction of AR-CAT activity similar to that seen with SaOS-2 cells (Fig. 4AGo) was observed in U-2 OS cultures. In addition, treatment of U-2 OS cultures with DHT resulted in increased AR mRNA steady state abundance, as determined by RNase protection analysis (data not shown). These results indicate that up-regulation of AR expression by androgen occurs in a second, independently derived, clonal osteoblastic model system.



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  		Figure 5. Dose response for androgen regulation of AR-CAT activity in a second clonal human osteoblastic cell line, U-2 OS. U-2 OS cultures were transiently cotransfected with 20 µg each of the AR-CAT reporter construct AR-CAT and the ß-gal expression vector. Cells were grown for 18 h, then treated with increasing concentrations of DHT, from 10-12–10-8 M, for an additional 24 h before harvest for CAT, ß-gal, and protein determinations as described in Fig. 3Go. Data are shown as stimulation of activity by hormone above control. Each data point represents the mean ± SEM of three independent experiments. *, P < 0.05; ***, P < 0.001 (statistically significant difference compared with the appropriate control group).

 
Specificity of response to androgen
The specificity of hormonal regulation of the AR promoter by androgen was characterized after treatment of transfected cultures with other steroid hormones. SaOS-2 cells are responsive to and/or express receptors for glucocorticoid, estrogen, and progesterone steroid hormones (15, 20, 40). Transiently transfected SaOS-2 cells were treated with 10-8 M progesterone, 10-7 M of the synthetic glucocorticoid dexamethasone (dex), 10-8 M 17ß-estradiol, or 10-8 M DHT for 24 h before harvest for CAT activity determinations (Fig. 6Go). Although DHT exposure again resulted in up-regulation of AR-CAT expression in these experiments, none of the other steroid hormones tested significantly regulated CAT expression from the AR promoter in osteoblastic cells.



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  		Figure 6. Androgen specificity for regulation of AR promoter activity in osteoblastic cells seen after treatment with a variety of steroid hormones. SaOS-2 osteoblastic osteosarcoma cells were transiently cotransfected with the AR-CAT reporter construct AR-CAT and the ß-gal expression vector. Cells were grown for 18 h, then treated with 10-8 M DHT, 10-8 M 17ß-estradiol (E2), 10-7 M dex, or 10-8 M progesterone (prog) for 24 h before harvest. Data are shown as stimulation of activity by hormone above control. Each data point represents the mean ± SEM of three independent transfections. ***, P < 0.001 (statistically significant difference compared with the appropriate control group).

 
We next determined whether SaOS-2 cells were capable of demonstrating regulation of appropriate promoter sequences for these steroid hormones through their cognate receptor proteins. These studies used the glucocorticoid-responsive pG29GtkCAT (42), in which two synthetic copies of a glucocorticoid (GRE)/progesterone/androgen (ARE) response element are ligated to the CAT gene driven by the thymidine kinase promoter (35), or the estrogen-responsive reporter construct EREtkCAT (36), containing a synthetic copy of the vitellogenin A2 ERE sequence also ligated to the CAT gene driven by the thymidine kinase promoter. SaOS-2 cultures were transiently transfected with either pG29GtkCAT or EREtkCAT, treated for 24 h with the concentrations of steroids used for analysis of AR promoter regulation, and harvested for CAT determinations as described above (Fig. 7Go). There was significant stimulation of CAT activity at 10-8 M DHT (P < 0.001). Similar regulation of the GRE-containing promoter was seen with dex and progesterone treatment (P < 0.001). The estrogen-responsive reporter construct EREtkCAT was also significantly stimulated by treatment with 17ß-estradiol (P < 0.001). It is interesting to note that the induction of CAT activity by DHT in osteoblastic cells from the promoter containing two synthetic hormone response elements (HREs) is similar in magnitude to that seen with the full-length human AR promoter construct, as shown in Fig. 4AGo. Although glucocorticoid, progesterone, and estrogen treatment was ineffective in regulating CAT activity from the AR promoter in these osteoblastic cells (Fig. 6Go), exposure to the same concentrations of this variety of steroid hormones significantly stimulated CAT activity in SaOS-2 cells transfected with the appropriate reporter construct using either the GRE-containing pG29GtkCAT or the estrogen-responsive reporter construct EREtkCAT. These data suggest that, in striking contrast to the results obtained with these two steroid-responsive promoters, regulation of the AR promoter by DHT is mediated by sequences that are responsive specifically to androgen, but not to other steroid hormones tested in these cultures.



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  		Figure 7. SaOS-2 osteoblastic cells respond to estrogen, androgen, progesterone, and glucocorticoid hormones. Steroid regulation of CAT activity in SaOS-2 osteoblastic osteosarcoma cells transiently cotransfected with 20 µg each of either the androgen-responsive reporter construct pG29GtkCAT or the estrogen-responsive promoter EREtkCAT, and the ß-gal expression vector. Cells were grown for 18 h, then treated with vehicle or the appropriate hormone as follows: DHT at 10-8 M, dex at 10-7 M, progesterone (prog) at 10-8 M, and 17ß-estradiol (E2) at 10-8 M for 24 h before harvest. Data are shown as stimulation of activity by hormone above the control level. Each data point represents the mean ± SEM of two to four independent experiments. ***, P < 0.001 (statistically significant difference compared with the appropriate control group).

 
Regulation is dependent on functional AR
The transduction of steroid hormone signals into the nucleus that lead to changes in gene expression generally have been shown to involve the steroid hormone receptor protein. To determine whether functional AR was required for androgen regulation of the AR promoter in osteoblasts reported here, we characterized AR-CAT expression in osteoblastic cells treated with DHT in the presence of an AR antagonist. The nonsteroidal aromatic antiandrogen HF has been shown to antagonize androgen regulation of gene expression, while lacking any agonist activity (43). Because HF has a relatively low affinity for the AR (44, 45), a several hundred-fold molar excess over DHT is required for effective blockage of the DHT effect (46, 47).

SaOS-2 cells were transiently transfected with AR-CAT and incubated with DHT at 10-8 M, HF at 10 µM, or their combination for 24 h. Figure 8AGo again shows DHT up-regulation of AR-CAT activity. However, coincubation with HF completely abrogated the effect of DHT on the AR promoter. HF alone had no significant effect on CAT activity in SaOS-2 cells. Similar results were obtained in U-2 OS cells (data not shown). These results indicate that trans-activation of the AR promoter mediated by androgens requires the presence of functional AR protein.



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  		Figure 8. Regulation of AR-CAT activity by androgen is dependent on AR protein function. Androgen regulation of AR-CAT is blocked by coincubation with the AR antagonist HF. Cells were grown for 18 h, then treated with 10-8 M DHT or 10 µM HF, alone or in combination, for 24 h before harvest for CAT, ß-gal, and protein determinations. Data are shown as stimulation of activity by hormone above the control level. Each data point represents the mean ± SEM of at least two independent experiments. **, P < 0.01 (statistically significant differences compared with the appropriate control group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report here approximately 2-fold enhancement of both AR steady state mRNA levels and AR promoter activity after exposure to physiologically relevant concentrations of the nonaromatizable androgen DHT in osteoblastic cells. This response is coincident with a similar increase of 2- to 3-fold in endogenous AR protein concentrations, as determined by binding studies and Western analysis (Keenan, E., K. Wiren, and E. Orwoll, manuscript submitted). This report documents transcriptional up-regulation of the AR promoter by androgen. Previously, androgen induction of endogenous AR levels has been reported to be mediated by stabilization of AR protein (48, 49) and has not generally been documented to occur through correlated changes in mRNA transcription. Our results in osteoblasts, however, show increased AR 5'-promoter activity with elevated levels of AR mRNA after androgen exposure, that when translated would result in increased AR protein concentrations. These changes are in the 2- to 3-fold range and are consistent with specific transcriptional activation of AR gene expression by ligand-bound AR.

The sequences mediating androgen up-regulation of the AR promoter in osteoblasts are yet to be identified. There are several intriguing possibilities, including multiple consensus sequence ARE/GRE half-sites, activating protein-1, cAMP response element, and interleukin-6 consensus sequence sites that are present in the human AR promoter (41). There is a consensus ARE in the mouse AR promoter at -497/-483 (50) and the rat AR promoter at -501/-487, which is divergent in the human promoter sequence (41). Downstream sequences in the 5'-untranslated region of the human AR gene that are present in AR-CAT may also be important in mediating hormonal responses, as a functional cAMP response element has been reported to be located in that region (50). While this paper was in review, Dai and Burnstein (32) published data describing AR mRNA up-regulation in AR-transfected prostatic PC3 cells mediated by sequences far downstream in the AR-coding region not present in AR-CAT. Little is known about regulation of the AR 5'-promoter region by androgen in any cell type, except for one report that androgen suppressed activity characterized with a mouse AR promoter construct (-546/+971) linked to CAT after transfection into quail QT6 or mouse {alpha}T3–1 cells, but the putative ARE sequence was not involved (50). Additional studies will be required to identify and characterize the sequences involved in the homologous up-regulation of AR gene expression from the AR promoter observed here.

Our results suggest that the sequences mediating androgen up-regulation of human AR promoter expression are relatively specific for trans-activation by the ligand-bound AR and not by other members of the GR superfamily. AR promoter regulation is androgen specific; neither progesterone nor dex treatment was effective in regulating the AR promoter in osteoblastic cells, even though SaOS-2 cells are responsive to both hormones, and the pG29GtkCAT construct, containing two copies of a HRE, was up-regulated by progesterone, dex, and DHT treatment in these cells. Some simple HREs can be regulated nonspecifically by steroid receptor-ligand complexes that are in the GR superfamily class, such as the HREs in the mouse mammary tumor virus long terminal repeat, if the specific receptor is present (51). However, at so-called composite hormone response elements (52), receptor activation by ligand binding results in specific and distinct promoter regulation. DNA sequences other than the HRE may also bind nonreceptor factors that are critical in determining hormonal specificity (53). Finally, estrogen administration was ineffective in regulating the AR promoter.

Previous studies have shown AR regulation by androgen in a variety of cell types, but the mode of regulation is both complex and tissue specific, as noted above. AR steady state mRNA levels are down-regulated after androgen exposure in most tissues, including prostate (24, 25), LNCaP prostatic carcinoma cells (24, 25, 26, 27), and in several other androgen target tissues (24). On the other hand, androgen exposure has been reported to variably up-regulate AR expression in some tissues/cell lines by some investigators (28, 29, 30, 31, 32), but not others (24, 27, 33). Androgen exposure has been shown to stabilize AR protein (48, 49). Except for one report showing an increase in AR levels after chronic androgen exposure in an osteosarcoma cell line (13), little is known about the regulation of AR levels in bone. Our results thus differ significantly from those observed in prostatic and many other androgen-responsive tissues, as androgen exposure in these osteoblastic cells results in increased expression. It is important to note, however, that these osteosarcoma cell lines are used as models for studying osteoblastic action, but may not be entirely representative of normal cell behavior. As AR down-regulation may play a significant role in attenuating cellular responsiveness to hormone in other androgen target tissues, the up-regulation observed in these osteoblastic cells suggests a critical role for AR in general bone homeostasis. Lack of functional AR expression in males from birth, such as in the androgen-insensitive tfm rat, results in the development of a female-type bone structure (54). However, neither the effect of reductions in AR function or expression on bone dynamics in a normal male hormonal setting nor the effect of castration on AR expression in skeletal tissue has yet been described. Furthermore, these data again show that the mode of regulation of AR gene expression by androgen is tissue specific and, in fact, can be opposite in direction depending on the cell type. A possible mechanism for such dichotomous regulation involves control of gene expression by proteins/trans-acting factors that are expressed in a tissue-specific fashion. These proteins then influence the ability of ligand-bound receptor, itself a trans-acting factor, to specifically regulate gene expression (52, 53).

We have shown androgen up-regulation of the human AR promoter to be dependent on a functional AR receptor, as the antiandrogen hydroxyflutamide completely abrogates the androgen effect. The classic mechanism for steroid regulation of gene expression involves steroid hormone binding to specific intracellular/nuclear receptors, which act as ligand-dependent trans-acting factors, to produce biological responses through changes in gene transcription (for review, see 22 . The rapid induction of CAT activity suggests that transcription of the AR 5'-promoter is directly influenced by the androgen ligand-receptor complex, without the requirement for intermediate protein synthesis. Additional studies with protein synthesis inhibitors will be required to definitely answer this question. Nevertheless, these results are consistent with the hypothesis that androgen treatment directly influences transcription of the AR gene and may thus represent an important pathway for up-regulation of AR expression in osteoblasts.

Finally, as shown by the dose-response, time-course, and steroid specificity studies presented here, the AR promoter sequences confer induction of AR expression in a ligand-specific manner. Both testosterone and DHT were nearly equally effective at increasing AR-CAT expression at concentrations that correlated with the reported affinity for the AR. Although DHT is nonaromatizable, testosterone can be converted to nonandrogenic steroids, i.e. estrogen, but estrogen treatment has no effect on AR promoter activity. As both androgens were effective at regulating expression, this result suggests that osteoblastic cells do not metabolize testosterone to a significant degree. This is consistent with reports of the relative absence of aromatase activity in SaOS-2 (Wiren, K., and S. Plymate, University of Washington, unpublished observation) and in some other osteoblastic cells (21), but not in all bone cell preparations (55, 56, 57).

In summary, these studies document homologous up-regulation of AR gene expression by physiologically relevant concentrations of the nonaromatizable androgen DHT in clonal osteoblastic cells. We have shown up-regulation of AR gene expression in osteoblasts to be cell specific, hormone specific, and time and androgen dose dependent, and that it requires functional AR protein. We conclude that exposure of osteoblasts to androgens specifically results in increased expression of AR mRNA, that occurs at least partially at the level of transcription, and is mediated by the 5'-region of the AR gene. These results suggest that physiological concentrations of androgens may have important effects on AR expression in skeletal tissue. The biological significance of AR regulation in other tissues has been described (23), but the importance of such regulation for AR in skeletal tissues has yet to be documented. It is interesting to speculate that androgen up-regulation of AR would result in an amplified response, rather than the classic dampening of the response, to androgen in osteoblasts. Further characterization of the significance of AR regulation osteoblasts in the bone-forming cell should facilitate our understanding of the significant roles of both androgen and AR in bone homeostasis.


   Acknowledgments
 
We especially thank Drs. Roger Birnbaum and Stephen Plymate for helpful discussions and critical reading of the manuscript. We are also indebted to Kristina Fausti and Amy Malone for technical assistance throughout the course of these studies. The androgen-responsive reporter construct pG29Gtk-CAT was obtained from Dr. Rainer Renkawitz, the estrogen-responsive reporter construct EREtkCAT was provided by Dr. Ming-Jer Tsai, and the human AR cDNA plasmid for RNase protection analysis of steady state AR mRNA levels was kindly provided by Dr. Marco Marcelli.


   Footnotes
 
1 Presented in part at the 10th International Congress of Endocrinology, San Francisco, CA (Abstract 214). This work was supported by Grant DK-46668 (to E.O., E.K., and K.W.) from the NIDDK and the Veterans Affairs Merit Review system (to K.W.). Back

Received September 10, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Wakley GK, Schutte HD, Hannon KS, Turner RT 1991 Androgen treatment prevents loss of cancellous bone in the orchidectomized rat. J Bone Miner Res 6:325–330[Medline]
  2. Greenspan SL, Oppenheim DS, Klibanski A 1989 Importance of gonadal steroids to bone mass in men with hyperprolactinemic hypogonadism. Ann Intern Med 110:526–531
  3. Jackson JA, Kleerekoper M 1990 Osteoporosis in men: diagnosis, pathophysiology, and prevention. Medicine 69:137–152[Medline]
  4. Finkelstein JS, Klibanski A, Neer RM, Doppelt SH, Rosenthal DI, Segre GV, Crowley WF 1989 Increases in bone density during treatment of men with idiopathic hypogonadotropic hypogonadism. J Clin Endocrinol Metab 69:776–783[Abstract]
  5. Dhuper S, Warren MP, Brooks-Gunn J, Fox R 1990 Effects of hormonal status on bone density in adolescent girls. J Clin Endocrinol Metab 71:1083–1088[Abstract]
  6. Ott SM 1990 Editorial: attainment of peak bone mass. J Clin Endocrinol Metab 71:1082A–1082C
  7. Goulding A, Gold E 1993 Flutamide-mediated androgen blockade evokes osteopenia in the female rat. J Bone Miner Res 8:763–769[Medline]
  8. Gasperino J 1995 Androgenic regulation of bone mass in women. Clin Orthop 311:278–286
  9. Chesnutt CH, Ivey JL, Gruber HE, Matthews M, Nelp WE, Sismom K, Baylink D 1983 Stanozolol in postmenopausal osteoporosis: therapeutic efficacy and possible mechanisms of action. Metabolism 32:571–580[CrossRef][Medline]
  10. Need AG, Horowitz M, Bridges A, Morris HA, Nordin C 1989 Effects of nandrolone decanoate and antiresorptive therapy on vertebral density in osteoporotic postmenopausal women. Arch Intern Med 149:57–60[Abstract]
  11. Buchanan JR, Hospodar P, Myers C, Leuenberger P, Demers LM 1988 Effect of excess endogenous androgens on bone density in young women. J Clin Endocrinol Metab 67:937–943[Abstract]
  12. Kapur SP, Reddi AH 1989 Influence of testosterone and dihydrotestosterone on bone-matrix induced endochondral bone formation. Calcif Tissue Int 44:108–113[Medline]
  13. Takeuchi M, Kakushi H, Tohkin M 1994 Androgens directly stimulate mineralization and increase androgen receptors in human osteoblast-like osteosarcoma cells. Biochem Biophys Res Commun 204:905–911[CrossRef][Medline]
  14. Pilbeam CC, Raisz LG 1990 Effects of androgens on parathyroid hormone and interleukin-1-stimulated prostaglandin production in cultured neonatal mouse calvariae. J Bone Miner Res 5:1183–1188[Medline]
  15. Fukayama S, Tashjian AH 1989 Direct modulation by estradiol of the response of human bone cells (SaOS-2) to human parathyroid hormone (PTH) and PTH-related protein. Endocrinology 124:397–401[Abstract]
  16. Benz DJ, Haussler MR, Thomas MA, Speelman B, Komm BS 1991 High-affinity androgen binding and androgenic regulation of {alpha}1(I)-procollagen and transforming growth factor-ß steady state messenger ribonucleic acid levels in human osteoblast-like osteosarcoma cells. Endocrinology 128:2723–2730[Abstract]
  17. Gray C, Colston KW, Mackay AG, Taylor ML, Arnett TR 1992 Interaction of androgen and 1,25-dihydroxyvitamin D3: effects on normal rat bone cells. J Bone Miner Res 7:41–46[Medline]
  18. Masuyama A, Ouchi Y, Sato F, Hosoi T, Nakamura T, Orimo H 1992 Characteristics of steroid hormone receptors in cultured MC3T3–E1 osteoblastic cells and effect of steroid hormones on cell proliferation. Calcif Tissue Int 51:376–381[CrossRef][Medline]
  19. Colvard DS, Eriksen EF, Keeting PE, Wilson EM, Lubahn DB, French FS, Riggs BL, Spelsberg TC 1989 Identification of androgen receptors in normal human osteoblast-like cells. Proc Natl Acad Sci USA 86:854–857[Abstract/Free Full Text]
  20. Orwoll ES, Stribska L, Ramsey EE, Keenan E 1991 Androgen receptors in osteoblast-like cell lines. Calcif Tissue Int 49:183–187[Medline]
  21. Nakano Y, Morimoto I, Ishida O, Fujihira T, Mizokami A, Tanimoto A, Yanagihara N, Izumi F, Eto S 1994 The receptor, metabolism and effects of androgen in osteoblastic MC3T3–E1 cells. Bone Miner 26:245–259[Medline]
  22. Carson-Jurica MA, Schrader WT, O’Malley BW 1990 Steroid receptor family: structure and functions. Endocr Rev 11:201–220[Abstract]
  23. Song CS, Rao TR, Demyan WF, Mancini MA, Chatterjee B, Roy AK 1991 Androgen receptor messenger ribonucleic acid (mRNA) in the rat liver: changes in mRNA levels during maturation, aging, and calorie restriction. Endocrinology 128:349–356[Abstract]
  24. Quarmby VE, Yarbrough WG, Lubahn DB, French FS, Wilson EM 1990 Autologous down-regulation of androgen receptor messenger ribonucleic acid. Mol Endocrinol 4:22–28[Abstract]
  25. Trapman J, Ris-Stalpers C, Van der Korput JA, Kuiper GGJM, Faber PW, Romijn JC, Mulder E, Brinkman AO 1990 The androgen receptor: functional structure and expression in transplanted human prostate tumors and prostate tumor cell lines. J Steroid Biochem Mol Biol 37:837–842[CrossRef][Medline]
  26. Tilley WD, Marcelli M, McPhaul MJ 1990 Expression of the human androgen receptor gene utilizes a common promoter in diverse human tissues and cell lines. J Biol Chem 265:13776–13781[Abstract/Free Full Text]
  27. Wolf DA, Herzinger T, Hermeking H, Blaschke D, Horz W 1993 Transcriptional and posttranscriptional regulation of human androgen receptor expression by androgen. Mol Endocrinol 7:924–936[Abstract]
  28. Yu L, Nagasue N, Makino Y, Nakamura T 1995 Effect of androgens and their manipulation on cell growth and androgen receptor (AR) levels in AR-positive and -negative human hepatocellular carcinomas. J Hepatol 22:295–302[CrossRef][Medline]
  29. Gad YZ, Berkovitz GD, Migeon CJ, Brown TR 1988 Studies of up-regulation of androgen receptors in genital skin fibroblasts. Mol Cell Endocrinol 57:205–213[CrossRef][Medline]
  30. Takeda H, Nakamoto T, Kokontis J, Chodak GW, Chang C 1991 Autoregulation of androgen receptor expression in rodent prostate: immunocytochemical and in situ hybridization analysis. Biochem Biophys Res Commun 177:488–496[CrossRef][Medline]
  31. Kerr JE, Allore RJ, Beck SG, Handa RJ 1995 Distribution and hormonal regulation of androgen receptor (AR) and AR messenger ribonucleic acid in the rat hippocampus. Endocrinology 136:3213–3221[Abstract]
  32. Dai JL, Burnstein KL 1996 Two androgen response elements in the androgen receptor coding region are required for cell-specific up-regulation of receptor mRNA. Mol Endocrinol 10:1582–1594[Abstract]
  33. Shan LX, Rodriguez MC, Janne OA 1990 Regulation of androgen receptor protein and mRNA concentrations by androgens in rat ventral prostate and seminal vesicles and in human hepatoma cells. Mol Endocrinol 4:1636–1646[Abstract]
  34. Marcelli M, Haidacher SJ, Plymate SR, Birnbaum RS 1995 Altered growth and insulin-like growth factor-binding protein-3 production in PC3 prostate carcinoma cells stably transfected with a constitutively active androgen receptor complementary deoxyribonucleic acid. Endocrinology 136:1040–1048[Abstract]
  35. Schule R, Muller M, Kaltschmidt C, Renkawitz R 1988 Many transcription factors interact synergistically with steroid receptors. Science 242:1418–1420[Abstract/Free Full Text]
  36. Elliston JF, Tsai SY, O’Malley BW, Tsai M-J 1990 Superactive estrogen receptors. J Biol Chem 265:11517–11521[Abstract/Free Full Text]
  37. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  38. Neumann JR, Morency CA, Russian KO 1987 A novel rapid assay for chloramphenicol acetyltransferase gene expression. BioTechniques 5:444–447
  39. Rosenthal N 1987 Identification of regulatory elements of cloned genes with functional assays. Methods Enzymol 152:704–720[Medline]
  40. Rodan SB, Imai Y, Thiede MA, Wesolowski G, Thompson D, Bar-Shavit Z, Shull S, Mann K, Rodan GA 1987 Characterization of a human osteosarcoma cell line (Saos-2) with osteoblastic properties. Cancer Res 47:4961–4966[Abstract/Free Full Text]
  41. Mizokami A, Yeh S, Chang C 1994 Identification of 3',5'-cyclic adenosine monophosphate response element and other cis-acting elements in the human androgen receptor gene promoter. Mol Endocrinol 8:77–88[Abstract]
  42. Faber PW, King A, Rooij HV, Brinkmann AO, Both NJ, Trapman J 1991 The mouse androgen receptor: functional analysis of the protein and characterization of the gene. Biochem J 278:269–278
  43. Simard J, Luthy I, Guay J, Belanger A, Labrie F 1986 Characteristics of interaction of the antiandrogen flutamide with the androgen receptor in various target tissues. Mol Cell Endocrinol 44:261–270[CrossRef][Medline]
  44. Warriar N, Page N, Koutsilieris M, Govindan MV 1993 Interaction of antiandrogen-androgen receptor complexes with DNA and transcription activation. J Steroid Biochem Mol Biol 46:699–711[CrossRef][Medline]
  45. Wong C-I, Kelce WR, Sar M, Wilson EM 1995 Androgen receptor antagonist vs. agonist activities of the fungicide vinclozolin relative to hydroxyflutamide. J Biol Chem 270:19998–20003[Abstract/Free Full Text]
  46. Gorczynska E, Handelsman DJ 1995 Androgens rapidly increase the cytosolic calcium concentration in Sertoli cells. Endocrinology 136:2052–2059[Abstract]
  47. Kemppainen JA, Lane MV, Sar M, Wilson EM 1992 Androgen receptor phosphorylation, turnover, nuclear transport, and transcriptional activation. J Biol Chem 267:968–974[Abstract/Free Full Text]
  48. Blok LJ, Hoogerbrugge JW, Themmen APN, Baarends WM, Post M, Grootegoed JA 1992 Transient down-regulation of androgen receptor messenger ribonucleic acid (mRNA) expression in Sertoli cells by follicle-stimulating hormone is followed by up-regulation of androgen receptor mRNA and protein. Endocrinology 131:1343–1349[Abstract]
  49. Syms AJ, Norris JS, Panko WB, Smith RG 1985 Mechanism of androgen-receptor augmentation. J Biol Chem 260:455–461[Abstract/Free Full Text]
  50. Lindzey J, Grossmann M, Kumar VJ, Tindall DJ 1993 Regulation of the 5'-flanking region of the mouse androgen receptor gene by cAMP and androgen. Mol Endocrinol 7:1530–1540[Abstract]
  51. Gunzburg WH, Salmons B 1992 Factors controlling the expression of mouse mammary tumor virus. Biochem J 283:625–632
  52. Pearce D, Yamamoto KR 1993 Mineralocorticoid and glucocorticoid receptor activities distinguished by nonreceptor factors at a composite response element. Science 259:1161–1165[Abstract/Free Full Text]
  53. Scarlett CO, Robins DM 1995 In vivo footprinting of an androgen-dependent enhancer reveals an accessory element integral to hormonal response. Mol Endocrinol 9:413–423[Abstract]
  54. Vanderschueren D, Van Herck E, Suiker AMH, Visser WJ, Schot LPC, Chung K, Lucas RS, Einhorn TA, Bouillon R 1993 Bone and mineral metabolism in the androgen-resistant (testicular feminized) male rat. J Bone Miner Res 8:801–809[Medline]
  55. Schweikert H-U, Wolf L, Romalo G 1995 Oestrogen formation from androstenedione in human bone. Clin Endocrinol (Oxf) 43:37–42[Medline]
  56. Purohit A, Flanagan AM, Reed MJ 1992 Estrogen synthesis by osteoblast cell lines. Endocrinology 131:2027–2029[Abstract]
  57. Tanaka S, Haji Y, Yanase T, Takayanagi R, Nawata H 1993 Aromatase activity in human osteoblast-like osteosarcoma cell. Calcif Tissue Int 52:107–109[CrossRef][Medline]



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