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Endocrinology Vol. 140, No. 7 3114-3124
Copyright © 1999 by The Endocrine Society

ARTICLES

Homologous Androgen Receptor Up-Regulation in Osteoblastic Cells May Be Associated with Enhanced Functional Androgen Responsiveness1

Kristine Wiren, Edward Keenan, Xiaowei Zhang, Betsy Ramsey and Eric Orwoll

Bone and Mineral Research Unit, Veterans Affairs Medical Center, (K.M.W., X.Z., E.S.O.) Portland, Oregon 97201; and the Departments of Behavioral Neuroscience (K.M.W.), Cell and Developmental Biology (K.M.W.), Medicine (K.M.W. and E.S.O.), and Physiology and Pharmacology (E.K and B.R.), Oregon Health Sciences University, Portland, Oregon 97201

Address all correspondence and requests for reprints to: Dr. Kristine Wiren, Portland VA Medical Center P3R&D39, 3710 SW Veterans Hospital Road, Portland, Oregon 97201. E-mail: wirenk{at}ohsu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although androgens have myriad effects on the skeleton, the regulation of androgen action in bone is not well understood. Androgen receptors (ARs) are known to play an important role in mediating androgen action. We have examined the effects of androgens and other sex steroids on AR levels in osteoblastic cells in vitro using two clonal human cell lines, SaOS-2 and U-2 OS. AR protein levels were quantitated both by specific androgen binding studies and Western analyses, and AR messenger RNA was measured with RNase protection assays. Potential changes in AR functionality was assessed by reporter assays.

Treatment of osteoblastic cells with the nonaromatizable androgen 5{alpha}-dihydrotestosterone (DHT) increased specific androgen binding 2- to 4-fold. Similar increases in AR protein levels were documented by Western analysis in both cell lines. The androgen-mediated increase in receptor levels was time and dose dependent as well as androgen specific. Steady-state AR messenger RNA levels were also increased by DHT. When AR concentrations in osteoblastic cells were elevated with exogenous receptor, there was an enhancement of DHT responsiveness, measured by increased trans-activation of an androgen-responsive promoter.

Thus, androgen exposure increased androgen receptor protein levels and specific androgen binding in osteoblastic cells. Androgen action as measured by androgen-mediated transcriptional activation is enhanced in the presence of elevated AR levels. Consequently, these studies have revealed an additional means by which androgens may modulate skeletal metabolism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BONE METABOLISM is profoundly influenced by androgen action. In males, androgens are essential for significant skeletal changes during puberty (1, 2, 3) and likely also play a role in female development as well (4). Gonadal deficiency in adult men results in definitive bone loss (5) in conjunction with changes in biochemical indices, which suggest an increase in remodeling rates (6, 7). In addition, numerous studies suggest androgens also play a role in the remodeling of bone in women (8, 9, 10). In animals, orchiectomy causes immediate changes in the character of bone remodeling, and those alterations can be prevented by the administration of nonaromatizable androgens (11, 12). These effects are at least in part mediated through direct effects of androgens on osteoblasts, as testosterone or dihydrotestosterone (DHT) affect a host of cellular events in bone cells in vitro, including osteoblast proliferation, matrix protein production, cytokine regulation, amongst other actions (13, 14, 15, 16, 17, 18, 19, 20, 21). As expected in light of these effects, both androgen receptor (AR) messenger RNA (mRNA) and AR protein are present in osteoblastic cell lines as well as in primary cultures (22, 23, 24, 25). The molecular size and binding characteristics of the AR found in osteoblastic cells is similar to that found in other tissues, receptor concentrations are in a range associated with physiological relevance (22, 23, 24), and androgen action in vivo is blocked by AR antagonists (26). Taken together, these findings suggest that the AR is an important mediator of androgen action in the skeleton.

Because many of the physiological actions of steroids are mediated by their cognate steroid hormone receptors (27), regulation of receptor levels may influence the ultimate expression of steroid-mediated responses. Ligand-dependent homologous regulation of steroid receptor levels is one pathway of steroid receptor control (28, 29, 30), including AR. AR concentrations appear to be regulated by androgen in a variety of tissues including muscle, fat, liver, and sexual tissues including breast and prostate (31, 32, 33, 34, 35, 36, 37). Interestingly, the direction of modulation, i.e. up- or down-regulation, appears to be influenced by tissue and/or cell type (38, 39, 40, 41, 42, 43, 44). It is apparent that homologous AR regulation reflects an elaborate system of control that is potentially important in the determination of androgen action.

Although the AR is known to exist in bone remodeling cells, there has been only limited evaluation of the control of its expression by steroid hormones. In certain settings, down-regulation of AR levels by androgen exposure in osteoblastic cells has been shown (45). However, we and others have demonstrated homologous up-regulation of AR gene expression in osteoblastic cells in different models (25, 46). In this study, we have further evaluated the effects of androgen exposure on the protein and mRNA expression of human AR in osteoblastic cells and have begun to explore the functional consequences of changes in AR levels. Alterations in AR levels were determined both by radioreceptor binding studies and by Western analysis. Changes in steady-state mRNA abundance was determined by ribonuclease (RNase) protection analysis. The functional consequences of changes in AR abundance on androgen-mediated transcriptional activation were determined by reporter assays using a chloramphenicol acetyltransferase (CAT) reporter containing androgen-response elements (AREs) in transiently transfected cultures. To increase AR levels without the complication of continued steroid presence, we cotransfected human osteoblastic cells with the human AR expression construct, CMV-AR (47). The results show up-regulation of human AR protein levels to be androgen specific, time and dose dependent, and mediated by functional AR. Increased AR abundance leads to enhanced AR functionality, measured by stimulated transcriptional activation by functional AR. These results suggest that up-regulation of AR by androgen in osteoblasts could have significant effects on AR function and thus androgen action in skeletal tissues.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures
Human osteoblastic cell lines were obtained from the American Type Culture Collection (Manassas, VA). U-2 OS (passage 58–80) and SaOS-2 (passage 55–88) cells were grown in phenol-red free McCoy’s medium containing 7% charcoal-stripped bovine calf serum (BCS). Upon reaching confluence, growth medium was removed and replaced with serum-free McCoy’s. Following a period of 24 h, medium was removed and replaced with serum-free McCoy’s with either ethanol vehicle (0.01%) or various concentrations of steroids as indicated in the text. Treatment media were then removed, and monolayers were washed twice and washed for an additional 24 h with serum-free McCoy’s for cells in binding studies. After this experimental period, cells were rinsed and detached with 1 mM EDTA + 0.25% trypsin in PBS and pelleted. Pellets were either stored at -80 C for subsequent analysis or were immediately processed. Cells for Western and RNA analyses were treated with hormone as described above, except that the final 24 h treatment-free period was eliminated.

Preparation of cell extracts for binding assays
Cells for binding studies were detached with trypsin, pelleted, and stored briefly at -80 C as previously described. Frozen cell pellets (1–5 x 108 cells) were homogenized in sucrose Tris magnesium (TM) buffer [0.25 M sucrose, 10 mM Tris-HCl, pH 7.4, 1.0 mM MgCl2, 1.0 mM phenylmethylsulphonylfluoride (PMSF)] and centrifuged at 1,500 x g for 15 min to pellet nuclei (24). Cytosol fractions were separated from crude nuclear pellets and diluted with buffer to yield final concentrations of 50 mM Tris-HCl, pH 7.4, 1.5 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol, 0.2 M sucrose, 0.8 mM MgCl2, 20 mM Na2MoO4, and 1.0 mM PMSF. Diluted cytosols were poured onto dextran coated charcoal (DCC) and vortexed resulting in a 0.05% dextran, 0.5% charcoal suspension. Suspensions were incubated at 0-4 C for 30 min with periodic vortexing and centrifuged at 40,000 x g to remove residual steroids. Nuclear extracts were prepared by extraction of crude nuclear pellets (0-4 C for 30 min) with hypertonic buffer (0.6 M KCl, 50 mM Tris-HCl, pH 7.4, 1.5 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol) and centrifuged (40,000 x g for 30 min). Residual pellets were processed for DNA quantitation according to the method of Burton (48).

Specific androgen binding analysis
Nuclear ARs were radiolabeled by exchange assay (12 C for 20 h) using [3H]R1881 (methyltrienolone) at 0.20–2.0 nM and bound radioligand separated using hydroxyapatite. R1881 was chosen for the binding analyses because it cannot be metabolized by osteoblasts and has a higher affinity to the AR than physiologic steroids such as testosterone or DHT (49). ARs present in the cytosolic fraction were equilibrated (0-4 C for 16 h) with [3H]R1881 at 0.20–2.0 nM, and bound and free radioligand was separated using DCC. Specificity of [3H]R1881 binding to ARs was established by competitive displacement (100-fold excess unlabeled R1881). Supersaturating concentration of triamcinolone acetonide (500-fold excess) was added to all tubes to decrease cross-reactivity of R1881 to other classes of steroid receptors (22). Specific binding was calculated as the difference between total and nonspecific 3[H]R1881 binding, normalized to DNA content. The maximum binding capacity (Bmax) and the apparent dissociation constant (Kd) of binding were determined by Scatchard plot analysis (50), subjected to linear regression analysis of all data points. AR binding was expressed as the total specific 3[H]R1881 bound, which represented the sum of the specific binding in the nuclear and cytosolic fractions. Results are expressed as mean ± SEM.

Gel electrophoresis and Western blot analysis
Cultures were washed once with PBS containing 1.0 mM PMSF, scraped from the flask surface, and suspended in lysis buffer (10 mM Tris-HCl, pH 7.6, 1.2 M KCl, 150 mM EDTA, 10 nM DHT, and fresh 1.0 mM PMSF). Lysates were diluted 1:1 with reducing buffer (125 mM Tris-HCl, pH 6.8, 20% glycerol, 4% SDS 10% ß-mercaptoethanol and 0.2% bromophenol blue) and boiled for 5 min. Extracts (20–30 µg/well) were electrophoresed on a 7.5% SDS-polyacrylamide gel at 150 V for 45 min on mini-protean II system (Bio-Rad Laboratories, Inc., Richmond, CA). The gel was electrically transferred at 100 V for 1 h in a Trans-Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories, Inc., Richmond, CA) in transfer buffer [192 mM glycine, 25 mM Tris-HCl, pH 8.3, 50 mM NaCl and 20% (vol/vol) methanol] onto a 0.45 µM nitrocellulose membrane at 4 C. Membranes were blocked overnight in 13% nonfat powdered milk in Tris-buffered saline (TBS; 10 mM Tris-HCl, pH 7.5 and 150 mM NaCl), washed with T-TBS (TBS buffer, 0.05% Tween-20) and probed with primary anti-AR antibody (polyclonal rabbit clone PG-21, Affinity BioReagents, Inc., Nesharic Station, NH) at 0.4 µg/ml dilution for 1 h at room temperature. The membrane was washed 6 times for 5 min in T-TBS and probed with 0.2 µg/ml horseradish peroxidase-labeled goat antirabbit IgG (Bio-Rad Laboratories, Inc.) in TBS. The final wash was six times for 5 min with T-TBS. Bound antibodies were visualized by enhanced chemiluminescence (ECL) detection (Amersham Corp., Arlington Heights, IL) on Kodak X-AR5 autoradiographic film. Quantitation of films was performed in the linear response range of the film by scanning densitometry (GS300, Hoefer Scientific, San Francisco, CA).

RNA isolation and RNase protection analysis
RNA was isolated by the single-step acid guanidinium isothiocyanate-phenol-chloroform extraction method (51), except that ß-mercaptoethanol was omitted from the denaturing solution. Quantitation of RNA was performed by spectrophotometric determination at 260 nm. RNase protection analysis was performed as previously described (25). Briefly, 100 µg of total RNA from each of the osteoblastic osteosarcoma cell lines SaOS-2 and U-2 OS, or 10 µg LNCaP RNA was hybridized to a [32P]labeled antisense cRNA AR probe (52). 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 of total RNA was hybridized to a [32P]labeled antisense cRNA probe generated from mouse ß-actin complementary DNA (cDNA), linearized with XbaI and HindIII, and transcribed with SP6 RNA polymerase. Radiolabeled antisense complementary RNA (cRNA) probes were generated using the MAXIscript system (Ambion, Inc., Austin, TX). Sample RNA was hybridized overnight at 42 C. Unhybridized probe was digested with a 1:100 dilution of RNaseA/RNaseT1 for 30 min at 37 C. ß-actin mRNA levels were used for normalization, which we have shown not to be regulated under these conditions (data not shown). ß-actin mRNA incubations were with RNaseT1 only. The samples separated on a 5% polyacrylamide/8 M urea sequencing gel. Gels were dried and exposed to Kodak X-AR5 autoradiographic film at -70 C for 18–48 h. Quantitation of films was performed in the linear response range of the film by scanning densitometry (GS300, Hoefer Scientific, San Francisco, CA). Size markers were DNA marker V standards run in adjacent lanes.

Transient transfections
Transient transfections were performed on proliferating SaOS-2 cells as previously described (25). Briefly, approximately 5 x 106 cells were mixed with DNA in a sterile gene-pulse chamber. Each DNA sample contained 4 µg of G29Gtk-CAT, 6 µg of the ß-galactosidase expression plasmid pSV-ß-galactosidase vector (Promega Corp., Madison, WI) used to control for transfection efficiency, with 0–2 µg of the human AR expression construct CMV-AR. The total DNA concentration was brought to 20 µg per transfection using pBluescript (pBS) vector DNA (Stratagene, La Jolla, CA). Each electroporation was divided into 10 wells (35 mm) generally distributed among different experimental conditions. Cells were incubated in normal media for 3 h, then hormones were added in serum-free media for an additional 48 h before isolation for chloramphenicol acetyl transferase, ß-galactosidase, and protein determinations. DNA stocks from different plasmid isolations were used. A transfection efficiency 5–10% was observed under these conditions.

Assay of transcriptional activity
Chloramphenicol acetyl transferase (CAT) activity was determined by the fluor-diffusion method (53). Cultures were lysed in 250 µl of reporter lysis buffer (Promega Corp., Madison, WI). CAT activity was measured in 50 µl of 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-CoA at 2.5 µCi/ml in Tris-HCl, pH 7.8. The reaction mixture was overlaid with organic scintillation fluid (Econofluor, New England Nuclear, Boston, MA) 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-galactosidase (ß-gal) activity was determined colorimetrically as described (54), using 150 µl of 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. 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, Inc., Richmond, CA) following manufacturer’s recommendations with BSA as the standard. All CAT activity determinations were normalized to ß-gal activity to correct for variations in transfection efficiency. Values obtained for CAT activity were corrected to values for ß-gal activity expressed as cpm/min/mU ß-gal, and then generally normalized to control values. The data shown represent the mean ± SEM from triplicate samples generally, and were performed in independent transfections two to three times.

Chemicals and reagents
The isotopes [{alpha}-32P]UTP (800 Ci/mmol), [3H]acetyl coenzyme A (200 mCi/mmol, CAT assay grade) and [3H]R1881 (70–87 mCi/mmol) were purchased from DuPont NEN (Boston, MA). All the media, buffers, supplements and reagents for cell culture were obtained from Gibco BRL-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 active metabolite hydroxyflutamide ({alpha}, {alpha}, {alpha}-trifluoro-2-methyl-4'-nitro-m-lactotoluidide, SCH 16423) was kindly provided by Schering-Plough Corp. (Madison, NJ). Hydroxyflutamide was added to the cultures 30 min before hormone addition. MAXIscript in vitro transcription and RPA II ribonuclease protection assay kits were obtained from Ambion, Inc. (Austin, TX). Both the human AR-275 plasmid for RNase protection (52) and the CMV-AR expression construct (47) was obtained from Dr. Marco Marcelli.

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


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Up-regulation of AR binding by androgen in osteoblastic model systems
We have previously shown human AR mRNA levels are up-regulated after androgen exposure in osteoblastic cells in a time and dose-dependent manner (25). In this report, we have extended these studies to further characterize the effect of androgen exposure on the level of both specific binding and AR protein expression, and we have begun to examine the biologic consequences of such increases in AR abundance in terms of androgen responsiveness. Human clonal 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 (55). They are an appropriate model in which to examine potential androgen actions, as they have been shown to be both androgen responsive and to express AR mRNA and protein (15, 24). Here we examine a single androgen-dependent response by characterizing homologous regulation of androgen receptor.

To better characterize hormonal regulation of androgen receptor levels in osteoblastic cells, the effect of androgen exposure on binding capacity was determined. We analyzed whether the observed change in binding capacity was due to increased receptor number and/or to a change in receptor affinity. SaOS-2 cultures were treated with 10-8 M DHT for 72 h and binding capacity was determined after incubation with [3H]R1881. Binding isotherms after DHT treatment are shown in Fig. 1AGo, with Scatchard transformation of the data indicated in panel B. The levels of specific androgen binding increased nearly 3-fold after androgen exposure in SaOS-2 cultures.



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  		Figure 1. The effect of androgen exposure on specific androgen binding in osteoblastic cells. A, SaOS-2 cells were grown to confluence in the absence of androgen, then exposed to 10-8 M DHT for 72 h. A representative receptor binding analysis using 0.2 to 2.0 nM [3H]R1881 is shown for cytosolic extracts from control cells (open circles) or hormone-treated cells (closed circles). B, Scatchard transformation of binding capacity in this analysis is shown with Kd (dissociation constant) at 0.17 ± 0.3 nM, and Bmax (maximum binding capacity) at 149 ± 29 fmol/mg DNA. The experiment has been repeated six times with similar results.

 
We also characterized AR homologous up-regulation in a second well-characterized clonal human osteoblastic cell line, U-2 OS, to determine whether the observed up-regulation was generalizable to other osteoblastic models. Table 1Go shows results of saturation binding analyses from three to six independent experiments using [3H]R1881 to determine the maximum binding capacity (Bmax), binding affinity (Kd) and number of specific androgen binding sites (receptor number) after Scatchard transformation for cytosolic receptor in both SaOS-2 and U-2 OS cells. The AR characteristics are similar to those we and others have previously reported (20, 24, 56). Reports for AR receptor number varies depending on the source of osteoblastic cells and on method of analysis, i.e. whether numbers were determined as nuclear, cytosolic or whole-cell. Nevertheless, AR is present in physiologic concentrations in a range similar to that found in other androgen target tissues such as kidney. Furthermore, exposure to 10-8 M DHT for 72 h results in significant increases in maximum binding capacity of 2- to 3-fold in both SaOS-2 (P < 0.05) and U-2 OS (P < 0.01) cells. The increase in specific androgen binding capacity produced by DHT treatment was associated with a modest but statistically significant increase in Kd in U-2 OS cells (control Kd = 0.18 nM ± 0.02 vs. DHT Kd = 0.33 nM ± 0.05; P < 0.05), while a similar but nonsignificant increase in Kd was observed in SaOS-2 cells (control Kd = 0.17 nM ± 0.03 vs. DHT Kd = 0.30 nM ± 0.06). Therefore, specific high-affinity, saturable androgen binding was present in the extracts from both SaOS-2 and U-2 OS cultures, and both show homologous up-regulation after DHT exposure.


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  		Table 1. Characterization of total AR in two osteoblastic cell lines

 
We next established the time-course and dose-response characteristics of homologous AR regulation. We first determined the time-course of increased androgen binding in SaOS-2 cells after exposure to 10-8 M DHT for 2, 5, 10, 24, 48, or 72 h. Binding isotherms after DHT treatment are shown in Fig. 2AGo. An increase in specific androgen binding after androgen exposure was clearly apparent by 24 h. Androgen binding levels continued to increase up to 3-fold by 72 h following exposure (P < 0.01). These changes in androgen receptor concentrations reflect a similar time course for alterations in mRNA abundance after androgen exposure, as we have previously published (25). It should be noted that androgen receptor concentrations as reflected by RRA and Western blotting remained elevated for at least 48 h after withdrawal of DHT (data not shown).



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  		Figure 2. The time and dose dependence of the effect of androgen exposure on specific androgen binding in osteoblastic cells. A, Time course analysis for homologous regulation of androgen binding in SaOS-2 cells grown to confluence in the absence of androgen, then exposed to 10-8 M DHT for up to 72 h. Analysis is shown for extracts from hormone-treated cultures at 2, 5, 10, 24, 48, and 72 h using whole-cell binding with [3H]R1881 as described in Materials and Methods. Values are the mean ± SEM of two to three experiments. *, P < 0.05; **, P < 0.01 (statistically significant difference compared with appropriate control). B, Androgen dose-response analysis for SaOS-2 osteoblastic cells grown to confluence in the absence of androgen, then exposed to vehicle or DHT at 10-9 M, 10-8 M, or 10-7 M for 72 h. Analysis for total binding using [3H]R1881 is shown for extracts from hormone-treated cultures are the mean ± SEM of three experiments. *, P < 0.05 (statistically significant difference compared with control).

 
To determine the dose-response relationship between androgen concentration and androgen binding, SaOS-2 cultures were treated with vehicle or increasing concentrations of DHT from 10-9 M to 10-7 M for 72 h. As shown in Fig. 2BGo, DHT concentrations as low as 10-9 M were associated with an increase in androgen binding concentrations (P < 0.05). The apparent maximal effect was seen with 10-8 M DHT, which again is similar to greatest changes observed in mRNA abundance (25).

Increased AR protein abundance after androgen exposure in osteoblastic cells and role of functional AR
To determine whether the changes in AR concentrations shown by binding studies could be confirmed at the level of protein abundance, Western analysis of AR levels was performed. Again, a second independent human clonal osteoblastic cell line U-2 OS was also characterized to determine the generalizability of androgen regulation. Treatment of confluent cultures of both SaOS-2 and U-2 OS cells with 10-8 M DHT for 72 h resulted in an elevation in AR protein (Fig. 3AGo). Comigration of AR to the LNCaP prostatic carcinoma cell line standard is shown at the right. This increase of 2- to 4-fold (Fig. 3BGo) is near the magnitude of change that was seen in binding studies, and was significant for both cell lines in 9–11 independent experiments (P < 0.01 compared with control in SaOS-2; P < 0.001 compared with control in U-2 OS). The DHT-mediated increase in AR protein levels was dose-dependent, with elevations observed at 10-10 M and a maximal increase at 10-8 M (data not shown). Thus, the finding that androgen exposure in osteoblastic cells increases specific androgen binding by increased AR protein levels was confirmed by Western analysis.



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  		Figure 3. Western analysis of AR levels in two osteoblastic models showing androgen up-regulation and dependence on functional AR for regulation of AR using the receptor antagonist hydroxyflutamide (HF). A, SaOS-2 and U-2 OS cells were grown to confluence in the absence of androgen, then exposed to treatments for 72 h. Cells were treated with vehicle (con), 10-8 M DHT, the combination of 10-5 M HF + 10-8 M DHT (D+HF), or 10-5 M HF alone. Cell extracts (30 µg) were separated on 7.5% SDS-polyacrylamide gels and AR protein levels were analyzed by Western blotting using polyclonal anti-AR antibody (PG-21) as described in Materials and Methods. The typical AR from human LNCaP prostatic carcinoma cells is indicated by the arrow at 110 kDa. This experiment was repeated twice with similar results. B, Quantitative analysis of AR protein levels after androgen treatment is shown. The densitometric signal is expressed as mean ± SEM from nine to eleven independent assessments. **, P < 0.01; ***, P < 0.001 (statistically significant difference compared with control).

 
To determine whether the increase in AR levels was mediated by functional AR, SaOS-2 cultures were treated with DHT in the presence of an AR antagonist. The nonsteroidal aromatic antiandrogen hydroxyflutamide (HF) has been shown to antagonize androgen regulation of gene expression, while lacking any agonist activity (57). Due to the relatively lower affinity HF exhibits for the AR (58, 59), we have employed a 1,000-fold molar excess of HF relative to DHT for effective AR blockade (60, 61). As shown in Fig. 3AGo, the increase in AR protein concentrations after 10-8 M DHT treatment for 48 h was blocked by the addition of 10-5 M HF.

Specificity of response to androgen
To determine the steroid specificity of AR regulation, Western analysis was performed on protein isolated from SaOS-2 cultures treated with various steroids for 72 h. As shown in Fig. 4Go, the stimulation of androgen binding by DHT was androgen specific. The effect of testosterone (10-8 M) was essentially the same as the equivalent dose of DHT (lanes 2 and 3). However, progesterone (P, lane 4) 17ß-estradiol (E2, lane 5), and synthetic glucocorticoid triamcinolone acetonide (TA, lane 6) were ineffective in increasing androgen binding levels. Comigration to the LNCaP AR standard is shown in lane 7.



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  		Figure 4. Western analysis for characterization of steroid specificity of AR regulation in SaOS-2 and U-2 OS cells. A, Osteoblastic cells were grown to confluence in the absence of steroid, and were then exposed to treatments as described. Lane 1, vehicle control (con); Lane 2, DHT; Lane 3, testosterone (T); Lane 4, progesterone (P); Lane 5, 17ß-estradiol (E2); Lane 6, triamcinolone acetonide (TA) all at 10-8 M for 48 h. Cell extracts (30 µg protein) were separated on 7.5% polyacrylamide gels, and AR protein levels were then analyzed by Western blotting as described in Fig. 3Go. Typical AR is shown at 110 kDa from extracts from LNCaP cells in Lane 7.

 
Increased mRNA abundance after androgen exposure in osteoblastic cells
We next determined whether DHT treatment under these conditions resulted in changes in the steady-state abundance of AR mRNA. RNase protection analysis was performed using total RNA isolated from both SaOS-2 and U-2 OS cultures treated with 10-8 M DHT for 48 h. As shown in Fig. 5AGo, and rogen exposure resulted in increased AR mRNA levels in both cell lines. Equivalent loading of total RNA is shown from the ß-actin signal below. AR mRNA abundance was normalized to ß-actin levels, and expressed as fold stimulation over the mean of control. Quantitation revealed an elevation of nearly 1.5-fold in U-2 OS and 1.7-fold with SaOS-2 cultures after DHT treatment (Fig. 5BGo). This result did not reach statistical significance in U-2 OS cells, but was significant (P < 0.05) with SaOS-2 cultures. Therefore, in concert with increases in both specific androgen binding and AR protein concentrations following androgen treatment, AR steady-state mRNA levels increased as well. However, this elevation was not as robust as that observed at the level of protein abundance.



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  		Figure 5. The effect of androgen treatment on AR mRNA concentrations in both SaOS-2 and U-2 OS cells. A, Changes in AR mRNA abundance after DHT exposure in human SaOS-2 and U-2 OS osteoblastic cells. To determine the effect of androgen exposure on hAR mRNA abundance, confluent cultures of two independently isolated clonal osteoblast-like cells (SaOS-2 and U-2 OS) were treated with vehicle or 10-8 M DHT for 72 h. Total RNA was then isolated from treated and control cells, and from prostatic carcinoma cells (LNCaP) as a positive control for human AR mRNA, and subjected to RNase protection analysis with 100 µg of total cellular RNA from osteoblastic cells and 10 µg of 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 (52 ) was hybridized to total mRNA isolated from the cells as described. The resultant RNA: RNA hybrids were digested with both ribonuclease A and T1, and the protected fragment then separated on a 5% denaturing polyacrylamide/urea gel. Total RNA from all cell lines (10 µg) was also probed by RNase protection for ß-actin expression to control for potential differences in loading. Yeast tRNA (+RNase) was added as a negative control to document complete digestion. Full-length probe at approximately 342 bp (-RNase) is indicated on the right. B, Quantitative analysis of homologous regulation of AR mRNA steady-state levels. Dried gels were exposed to x-ray film, and the relative amounts of each band quantitated by densitometry. The AR mRNA to ß-actin ratio was determined from the densitometric signal in the linear response range of the film. The data are represented as fold over control, normalized to the mean of the control from two to five measurements. *, P < 0.05 (statistically significant difference compared with control).

 
Elevation of AR concentrations by cotransfection with AR cDNA
Elevation of AR concentrations in osteoblastic cells should lead to increased transcriptional activation by AR after androgen exposure. This presumption is difficult to test in the current experimental setting because androgen treatment to increase receptor level would complicate analysis and interpretation of subsequent androgen responsiveness. Thus, to avoid prolonged treatment of cultures with androgens to up-regulate receptor, SaOS-2 cells were transiently transfected with a cDNA expression plasmid encoding human AR, CMV-AR (52). Control cultures were transfected with the same concentration of DNA using pBS in place of CMV-AR as described in Materials and Methods.

We first characterized whether increased AR concentrations would lead to increased transcriptional activation by cotransfecting SaOS-2 cultures with the androgen-responsive G29Gtk-CAT reporter construct. This construct, kindly provided by Dr. Rainer Renkawitz, contains two synthetic copies of a glucocorticoid/progesterone/androgen hormone response element (HRE) are ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene driven by the thymidine kinase (tk) promoter (62). Cultures were cotransfected with increasing concentrations of CMV-AR (0 to 2 µg) and with the ß-galactosidase (ß-gal) expression construct to control for variations in transfection efficiency. Equal DNA concentrations were used for all transfections, using pBS to bring the total DNA concentration to 20 µg. Three hours after transfection, 10-8 M DHT was added to the serum-free medium and the cells were grown for an additional 48 h before harvest for CAT, ß-gal, and protein determinations. CAT activity was determined by fluor-diffusion assay (53), corrected for total protein and normalized to ß-gal activity to correct for differences in transfection efficiency. Changes observed in CAT activity were interpreted as changes in promoter activity at the HREs mediated by functional AR. As shown in Fig. 6AGo, increasing AR cDNA concentrations resulted in enhanced HRE/ARE-responsive promoter activity. As can be seen, even concentrations as low as 0.125 µg of CMV-AR lead to enhanced trans-activation by androgen that is statistically significant. Maximal stimulation of CAT activity occurred with 1 µg of CMV-AR cDNA (P < 0.01). Thus, increased AR levels in osteoblastic cells results in increased AR functionality as assessed by an ARE-containing promoter.



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  		Figure 6. Effect of increasing concentrations of CMV-AR on transcriptional activation by androgen in osteoblastic cells. A, Activation of an ARE-containing promoter by androgen. SaOS-2 cultures were cotransfected with increasing concentrations of the human AR cDNA expression construct CMV-AR (0, 0.125, 0.25, 0.5, 1.0, or 2.0 µg), the androgen-responsive reporter construct G29Gtk-CAT, the ß-gal expression construct pSV-ß-Galactosidase to control for transfection efficiency and variable amounts of pBS such that the total DNA concentration was 20 µg. Cells from each transfection were plated into ten 35-mm wells. After 3 h, the cells were switched to serum-free media with vehicle or 10-8 M DHT for 48 h. CAT activity was determined by fluor-diffusion assay (53 ), corrected for total protein and normalized to ß-gal activity. The data shown are expressed for DHT induction as fold above vehicle control. Data represent the mean ± SEM from triplicate samples. **, P < 0.01 (statistically significant difference compared with control). B, Western analysis of AR levels in SaOS-2 cultures after cotransfection with AR cDNA. Cultures were cotransfected without or with 1 µg CMV-AR with G29Gtk-CAT, ß-gal and pBS (20 µg total). Cells from each transfection were plated into four 60-mm wells. After 3 h, the cells were switched to serum-free media for 48 h without treatment. Cell extracts (20 µg protein) were prepared and AR abundance was analyzed by Western blotting as described in Fig. 3Go. Lane 1, LNCaP cells; Lane 2, control transfected SaOS-2 cultures (con); Lane 3, human AR cDNA transfected cultures (CMV-AR). The typical AR band migration is indicated by the arrow at 110 kDa.

 
To determine the increase in AR levels that result from CMV-AR expression, Western analysis was performed. Extracts were prepared from the control-transfected vs. the CMV-AR-transfected cultures using cells transfected with optimal concentration of 1 µg CMV-AR, and AR levels were characterized (Fig. 6BGo). As can be seen, cultures with exogenous AR derived from CMV-AR cDNA had higher levels of AR protein (compare control lane 2 to CMV-AR lane 3). The increase in AR abundance was approximately 4-fold. However, due to the efficiency of transient transfection, the average increase per cell would be higher but difficult to measure in this setting. The average abundance of AR in transiently transfected cultures is below that found in the androgen responsive prostatic cell line LNCaP (lane 1). Using this strategy, AR concentrations in the transfected cultures is elevated without the addition of hormone, and thus provides a model to analyze the functional consequences of increased receptor levels.

Functional consequences of increased AR levels in osteoblastic cultures
The transiently transfected osteoblastic cultures were used to assess whether the higher levels of AR obtained in SaOS-2 cultures were biologically meaningful by using androgen-responsive reporter analyses. To determine whether additional AR would lead to increased transcriptional activation from functional AR, cultures were cotransfected without or with CMV-AR as described above, again with G29Gtk-CAT as the androgen-responsive promoter reporter construct. The response to increasing concentrations of androgen was then determined by CAT assay as a measure of transcriptionally active functional AR.

We further characterized functional AR by determining the DHT dose-response in SaOS-2 cultures with basal AR expression using control transfected cultures lacking CMV-AR as described above. In SaOS-2 cultures with basal AR levels, DHT treatment with 10-12 to 10-8 M stimulated ARE-dependent reporter expression in a dose-dependent manner (Fig. 7AGo). Maximal stimulation near 1.5-fold occurred with 10-8 M DHT. These results were statistically significant by one-way ANOVA (P < 0.01), and are similar to what we have previously reported here using modified transfection and growth conditions (25). Thus, the basal level of AR expression in SaOS-2 cultures was sufficient to lead to a modest but significant ARE-dependent transcriptional activation in the presence of DHT.



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  		Figure 7. Enhanced promoter activation by androgen in osteoblastic cells with elevated AR levels. SaOS-2 cells were cotransfected with or without the androgen receptor expression construct CMV-AR by electroporation using G29Gtk-CAT, ß-gal and pBS such that the total DNA concentration was 20 µg in (A) cultures with basal AR expression, or (B) cultures with increased AR expression from cotransfection with human AR cDNA. Cells were plated into ten 35-mm wells. After 3 h, the cells were treated with increasing concentrations of DHT as indicated in serum-free media for 48 h. CAT activity was determined by fluor-diffusion assay (53 ), corrected for total protein and normalized to ß-gal activity. The data shown are expressed as relative increase above vehicle control. Data represent the mean ± SEM *, P < 0.05; **, P < 0.01 (statistically significant difference compared with control). The experiments were repeated two times with two to three wells per treatment per experiment.

 
We next determined whether additional AR expression would lead to enhanced transcriptional activation after androgen addition. Cultures were cotransfected with the same concentration of DNA but in the presence of CMV-AR for additional AR expression at the optimal concentration of 1 µg determined from the dose-response curve. The cotransfected cultures show much more robust transcriptional activation of the androgen-responsive promoter that is also androgen dose dependent (Fig. 7BGo). The stimulation of CAT activity was nearly 20-fold (P < 0.01) compared with 1.5-fold in cultures with basal AR expression. The combined results from Figs. 6AGo and 7BGo suggest that an increase in AR abundance would result in increased transcriptional activation from ARE-containing promoters after androgen exposure over a wide range of AR concentrations.

Increased transcriptional activation is dependent on functional AR
To determine whether the increased AR transcriptional activation observed in CMV-AR transfected cultures was due to activation through the receptor itself, and not from changes due to transfection, we again employed the specific androgen receptor antagonist HF. ARE-dependent activation of G29Gtk-CAT expression was determined in CMV-AR cotransfected osteoblastic cells treated with DHT in the presence of HF.

SaOS-2 cells were transiently cotransfected with G29Gtk-CAT and CMV-AR as described above, and incubated with either DHT at 10-8 M, HF at 10 µM or the combination for 48 h. Figure 8Go again shows robust DHT up-regulation of G29Gtk-CAT activity, again to nearly 20-fold (P < 0.0001). However, coincubation with HF significantly inhibits the effect of DHT on the ARE-containing promoter to 48% of the DHT-treated value (P < 0.001). HF alone had a slight agonist effect on CAT activity in SaOS-2 cells, stimulating only 3.4-fold above basal. These results indicate that the increased trans-activation of the androgen-responsive promoter mediated by androgens requires the presence of functional AR protein. Thus, it appears that introduction of exogenous AR produces a more robust transcriptional response through functional AR and not through a nonspecific response resulting from transfection.



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  		Figure 8. Enhanced promoter activation by androgen with exogenous AR is mediated by functional AR. SaOS-2 cultures were cotransfected with G29Gtk-CAT, CMV-AR, ß-gal, and pBS (20 µg total) as described in Fig. 7Go. Cells were plated into ten 35-mm wells. After 3 h, the cells were treated with vehicle, 10-8 M DHT, 10-5 M HF, or DHT plus HF in serum-free media for 48 h. CAT activity was determined by fluor-diffusion assay (53 ), corrected for total protein and normalized to ß-gal activity. The data shown are expressed as relative increase above vehicle control. Data represent the mean ± SEM ****, P < 0.0001 (statistically significant difference compared with control). ***, P < 0.001 (statistically significant difference compared with DHT treated). ###, P < 0.001 (statistically significant difference compared with control). **, P < 0.01 (statistically significant difference compared with control). The experiments were repeated two times with two to three wells per treatment per experiment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Androgens affect a variety of osteoblast functions, including changes in proliferation, growth factor and cytokine production, transcription factor control, and bone matrix protein production (collagen, osteocalcin, osteopontin) (13, 14, 15, 16, 17, 18, 19, 20, 21). Although recently the role of androgens in bone has been questioned, particularly in light of reports of osteopenia in men with estrogen receptor or aromatase deficiencies, there are compelling data to indicate a direct effect of androgens plays an important role in skeletal homeostasis. For instance, the abnormalities in bone remodeling induced in vivo by castration are prevented by treatment with nonaromatizable androgens, while specific AR antagonists induce osteopenia. Similarly, in vitro experiments have confirmed the effects of nonaromatizable androgens on osteoblastic function. Hence, there is an excellent foundation in basic research supporting the precept that androgens are directly active in bone, and some of these reported effects (e.g. changes in osteoblast proliferation, growth factor production, matrix synthesis) may be considered the precursors to an anabolic effect at the tissue level (63). However, as of yet there is not a coherent understanding of how seemingly disparate effects on bone cell function translate into the effects reported in association with androgen action in vivo. Here, we have characterized one androgen-dependent response in two osteoblastic osteosarcoma cell lines, i.e. homologous regulation of AR.

ARs are present in physiological relevant concentrations in bone. Early reports documented the AR in both primary and clonal osteoblastic cells (22, 23, 24), and demonstrated that they were characterized by specific, high affinity androgen binding typical of ARs in other tissues. Subsequent studies (56, 64, 65) confirmed these findings, and revealed a similar binding affinity for DHT and testosterone. In the current studies, we have extended these observations to both examine the binding characteristics of the osteoblastic AR and have further characterized homologous regulation of the AR in osteoblastic cells.

AR concentrations are clearly increased by androgen exposure. A 2- to 4-fold increase in androgen binding occurs following androgen exposure in two clonal osteoblastic cell lines (SaOS-2 and U-2 OS), a finding similar to those reported by Takushi et al. in the TE-85 osteoblastic cell line (46). We have demonstrated the time- and dose-dependence of the increase in binding to be similar to those expected for androgenic effects.

The increase in receptor protein concentrations following androgen exposure confirmed by Western analysis clearly affirms that the increase in androgen binding capacity is not merely the result of a significant alteration in receptor affinity or functional configuration. The increase in receptor concentrations was specific for androgens and was inhibited by the AR antagonist HF, indicating that the positive regulation is directly related to androgen action mediated via the intracellular AR and not through other nonreceptor mediated pathways (66). This pattern of regulation is mirrored by similar changes in mRNA abundance over time in SaOS-2 cells (25). In other tissues, androgen exposure has induced increases in receptor stability. For instance, Kemppainen et al. showed that androgen exposure increased AR half-life from 2 h to approximately 6 h in prostate tissue (61). Although we have not directly examined the rate of receptor degradation in these experiments, the persistence of the increase in binding capacity and receptor protein concentrations up to 48 h after androgen withdrawal makes it unlikely that changes in receptor stability induced by androgen binding is capable of explaining the up-regulation noted in these studies.

The mechanism responsible for the increase in receptor levels in osteoblastic cells may be at least partially transcriptional. This is suggested by the increase in steady-state AR mRNA levels following androgen exposure, and by increased hAR promoter activity after DHT treatment (25). Transcriptional regulation is consistent with studies in prostate cells in which androgen mediated regulation of AR levels has been related partially to transcriptional control (37). The human AR promoter does not contain classic androgen response elements but that in itself does not eliminate the possibility of a direct AR mediated regulation of transcription (67). In addition, there are other structural elements of the AR promoter that might serve to transduce an androgenic signal, including cAMP response elements and AP-1 sites (67). The elevation in mRNA steady-state abundance after androgen treatment was less than that observed for protein levels. This result suggests that at least part of the increase in AR abundance is the result of an increase in AR mRNA levels through changes in transcription and/or mRNA stability. A portion of the androgen dependent increase in AR levels may also occur through changes in protein stability.

Increasing AR cDNA concentrations over a wide range resulted in enhanced HRE/ARE-responsive promoter activity, as has been shown previously with transfected AR in other cell types (68) and for other members of the steroid receptor superfamily (69, 70). The nearly 1.5-fold increase in promoter activity after androgen treatment in osteoblastic cells with basal AR levels was significant and similar to what we have previously reported using modified transfection and growth conditions (25). This level of regulation is also consistent with DHT regulation of an ARE-containing promoter construct in stably transfected human fetal osteoblasts using a similar human AR expression construct driven from the CMV promoter, with a response in those cells of 2- to 3-fold (71). The number of AR binding sites in the stably transfected cells (~4,000) is higher than the basal level in SaOS-2 and U-2 OS cultures (~2,000), suggesting a relative correspondence between receptor abundance and trans-activation in these various osteoblastic models. Here we have demonstrated an enhanced androgen-mediated increase in activation from an ARE-containing promoter, suggesting that other ARE-containing promoters would be similarly influenced.

The increase in trans-activation was also dependent on functional AR because HF inhibited the activation by androgen from the ARE in G29Gtk-CAT. HF treatment did not completely abrogate the DHT-mediated induction, as has been shown previously in cotransfection studies with different cell types (58). HF alone had a slight agonist effect on CAT activity in SaOS-2 cells; HF has been previously shown to possess weak agonist activity in the absence of androgen (58).

There are numerous examples of homologous steroid hormone receptor regulation, and the AR has been noted to be negatively regulated in prostate, epididymus, uterus, submandibular gland, and other tissues, while at the same time is positively regulated in liver, muscle, and bone. The concentration of steroid hormone receptors has dramatic effects on hormone action in some instances, and functional consequences of AR regulation may have important effects on bone metabolism. At the time of puberty, there are dramatic changes in androgen concentrations, and concomitantly there is a rapid increase in bone mass. The increase in bone mass at this time has been positively linked to androgen concentration in both sexes, and the regulation of androgen action in bone during this time may be in part the result of AR up-regulation. Similarly, later in life the decline in bone mass in men has been speculated to be related to relative androgen deficiency, a process that could be accentuated by a reduction in AR concentrations in bone as a result of falling androgen levels. Recent studies of AR localization by immunocytochemical analysis in intact bone did not show dramatic differences in AR abundance between males and females (72), and only slight differences between cells from young and old patients at the level of mRNA abundance and AR binding have been observed (45). Nevertheless, we have shown regulation of receptor levels can affect trans-activation in these osteoblastic cells. Thus, from a therapeutic perspective, the regulation of the AR in bone, particularly in specific bone compartments, by androgens or with other approaches, may be a useful means of enhancing the anabolic effects of androgens in the skeleton. This is particularly intriguing because AR tends to be localized in osteoblasts at sites of bone formation (72).

In conclusion, we have demonstrated consistent homologous up-regulation of AR in two independent osteoblastic cell lines via a mechanism involving an AR dependent process that is androgen specific, and time and dose dependent. At least in part, the increase in AR levels is the result of an increase in steady-state AR mRNA levels. Increases in AR levels by transfection over a wide range of cDNA concentrations results in enhanced androgen responsiveness, and suggests that homologous up-regulation may play a role in bone modeling/remodeling. Thus it is interesting to speculate that homologous regulation of the AR could occur with changes in steroid levels, in specific bone compartments or at times during development when the response to androgens is most important. Additional studies will be required to more fully delineate the importance of AR regulation in vivo.


   Acknowledgments
 
The authors wish to thank Karen Perez for her invaluable technical assistance. The authors also acknowledge Dr. M. Marcelli for the AR RNase protection and AR expression constructs, Dr. Rainer Renkawitz for the androgen reporter plasmid and Schering-Plough Corp. for providing the hydroxyflutamide.


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

Received September 30, 1998.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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