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Molecular Endocrinology, doi:10.1210/me.2008-0160
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Molecular Endocrinology 22 (11): 2448-2465
Copyright © 2008 by The Endocrine Society

Steroidal Androgens and Nonsteroidal, Tissue-Selective Androgen Receptor Modulator, S-22, Regulate Androgen Receptor Function through Distinct Genomic and Nongenomic Signaling Pathways

Ramesh Narayanan, Christopher C. Coss, Muralimohan Yepuru, Jeffrey D. Kearbey, Duane D. Miller and James T. Dalton

Preclinical Research and Development, GTx, Inc., Memphis, Tennessee 38163

Address all correspondence and requests for reprints to: James T. Dalton, Preclinical Research and Development, GTx, Inc., 3 North Dunlap Street, Memphis, Tennessee 38163. E-mail: jdalton{at}gtxinc.com.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Androgen receptor (AR) ligands are important for the development and function of several tissues and organs. However, the poor oral bioavailability, pharmacokinetic properties, and receptor cross-reactivity of testosterone, coupled with side effects, place limits on its clinical use. Selective AR modulators (SARMs) elicit anabolic effects in muscle and bone, sparing reproductive organs like the prostate. However, molecular mechanisms underlying the tissue selectivity remain ambiguous. We performed a variety of in vitro studies to compare and define the molecular mechanisms of an aryl propionamide SARM, S-22, as compared with dihydrotestosterone (DHT). Studies indicated that S-22 increased levator ani muscle weight but decreased the size of prostate in rats. Analysis of the upstream intracellular signaling events indicated that S-22 and DHT mediated their actions through distinct pathways. Modulation of these pathways altered the recruitment of AR and its cofactors to the PSA enhancer in a ligand-dependent fashion. In addition, S-22 induced Xenopus laevis oocyte maturation and rapid phosphorylation of several kinases, through pathways distinct from steroids. These studies reveal novel differences in the molecular mechanisms by which S-22, a nonsteroidal SARM, and DHT mediate their pharmacological effects.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ANDROGEN RECEPTOR (AR), a member of the steroid receptor superfamily, and its ligands play important roles in several biological processes such as the development and maintenance of secondary sexual organs, bone, muscle, and others (1). These biological effects are typically categorized as androgenic (i.e. the effects on secondary sexual organs) or anabolic (i.e. the effects on muscle and bone). Separation of these two effects is desirable for expanded therapeutic application and success of androgens (2). Despite the role of androgens in physiological processes, the use of steroidal androgens for pharmacological applications is limited due to their poor oral bioavailability, pharmacokinetic profile, lack of tissue specificity, and eventually deleterious side effects. The discovery of tamoxifen, a selective estrogen receptor (ER) modulator (SERM), three decades ago and its therapeutic success spearheaded the discovery of tissue-selective ligands for AR [selective AR modulator (SARM)] that separate the androgenic activity from anabolic actions (3). These classes of orally bioavailable nonsteroidal receptor modulators (SERMs, SARMs, etc.), called collectively selective receptor modulators (SRMs), act as tissue-selective agonists and/or antagonists via their respective receptor and provide a unique and tissue-selective pharmacological approach to associated diseases (e.g. cancer, osteoporosis, and muscle wasting) (3).

The molecular mechanism that mediates the tissue selectivity of SARMs or any SRM is still speculative, despite widespread and elegant efforts to deduce the mechanism. To date, all of these studies were done with SERMs owing to their established clinical use, prolonged availability, and advances in ER knowledge. Not surprisingly, hypothesized molecular mechanisms of SARM action have been largely adopted from our knowledge of SERM and ER pharmacology. Considering the importance of androgens in the treatment of several diseases, it is imperative to understand the molecular mechanisms of SARMs in relation to their structure-activity relationships. The most commonly discussed and studied mechanism of SRM action is the tissue-selective expression or function of coactivators and corepressors (4). The cell-specific progesterone receptor agonist/antagonistic activity of mifepristone, RU486, is determined by the coactivator and corepressor ratio (5). Similarly, tamoxifen’s agonistic activity in uterus and antagonistic activity in breast are attributed to the recruitment of coactivators and corepressors, respectively, to estrogen-responsive promoters (6). However, the role of intracellular signaling pathways that mediates genomic and nongenomic effects pivotal for the posttranslational modification and function of receptors and coactivators have thus far been neglected.

Signal transduction is the fundamental biological process that conducts and converts various external stimuli into intracellular functions (7, 8, 9). Signaling cascades direct a wide variety of activities such as secretion of neurotransmitters, hormones, and intracellular calcium and numerous other cellular and tissue functions (10, 11, 12). From the entry of ligand into a cell to gene transcription and protein translation, the entire biological process is dictated by the particular pathway active in a given tissue and the pathway adopted by the ligand. Posttranslational modifications such as phosphorylation, acetylation, sumoylation, and ubiquitination of receptors and coactivators are unique to the signaling pathway taken by the ligand or the liganded receptor (13, 14, 15). DHT and R1881 (a synthetic steroidal androgen) activate c-src kinase and lead to interaction of the AR with the Src homology-3 domain of src in LNCaP prostate cancer cells (16). On the other hand, R1881 and dihydrotestosterone (DHT) activate the ERK pathway in osteocytes, indicating that the signaling cascade for androgens, like estrogens, is also tissue specific (17). Inhibition of these kinases prevented the androgen-induced cell cycle progression (16).

Although the traditional view has been that androgens and other steroids mediate their effects solely via transcription (genomic effects), recent evidence indicates the existence of effects that are independent of transcription (nongenomic effects) (18). However, controversy still shrouds the receptor that mediates the nongenomic effects of androgens. Evidence supporting that it is the intracellular receptor and the existence of membrane G protein-coupled receptors as mediators of the nongenomic effects provide an emerging but unclear vision of mechanisms underlying these effects (18, 19, 20). The nongenomic effects of androgens and other steroids are now appreciated as key mediators of several physiological effects and promising drug discovery targets for potential therapeutic intervention (17, 19, 20, 21, 22).

Here we sought to identify the kinases that mediate the genomic and nongenomic effects, important in AR function, in response to DHT and an aryl propionamide SARM, S-22. SARMs of this class were earlier demonstrated to elicit tissue-selective pharmacological activity (23, 24). We provide the first experimental evidence that the tissue selectivity of S-22 results from its activation or repression of genomic and nongenomic signal transduction pathways that are distinct from those used by DHT. The structure and pharmacokinetic profiles of S-22 were published earlier in detail (25). Moreover, S-22 and DHT adopt distinctive pathways to mediate AR-dependent transcription, which affects the recruitment of AR and cofactors. Chromatin immunoprecipitation (ChIP) studies show the importance of different kinases to the recruitment of AR and cofactors and the ligand-specific nature of these effects.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DHT and S-22 Elicit Different Pharmacological Effects
The transactivation potential of S-22 and DHT were compared at a wide range of concentrations (0.0001 nM to 10 µM) in HEK-293 cells transfected with AR, GRE-LUC, and CMV-renilla luciferase reporter. Figure 1AGo shows that DHT (filled squares) more potently and efficiently stimulated AR transactivation than S-22 (open squares), whereas the inactive S-22 R-isomer (filled triangle) that demonstrates 3000-fold lesser AR binding affinity failed to significantly induce AR transactivation. The ability of S-22 to induce AR-dependent transcription of an endogenous gene, PSA, in LNCaP cells was similar to that observed for DHT (Fig. 1BGo).


Figure 1
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  		Fig. 1. S-22 Is a Tissue-SARM

A, Transactivation of AR by DHT and S-22. HEK-293 cells were transfected with 0.25 µg GRE-LUC, 0.5 ng CMV-renilla-LUC, and 25 ng pCR3.1 rat AR and treated with titration of DHT ({blacksquare}), S-22 ({square}), or S-22-R-isomer ({blacktriangleup}) for 24 h. Luciferase activity was measured and normalized as indicated in Materials and Methods. RLU, Relative light units. B, DHT and S-22 enhance PSA gene expression. LNCaP cells were serum starved for 3 d and treated in triplicate with vehicle (white bars), DHT (black bars), or S-22 (striped bars) for 24 h. RNA was isolated, and PSA gene expression was measured and normalized to 18S using real-time quantitative RT-PCR primers and probe. *, Significance at P < 0.05 from vehicle-treated samples. C, Tissue-selective actions of S-22 in rats. Sprague Dawley rats (n = 5) were treated with vehicle, 1 mg/d DHT (black bars) or S-22 (striped bars) for 14 d. At the end of 14 d, the animals were euthanized, and the weights of prostate and levator ani muscle were measured and interpreted as percent change from vehicle-treated animals. *, Significance at P < 0.05 from vehicle-treated samples. D, Effect of S-22 on bone marrow cell differentiation. Bone marrow cells from rats were cultured in triplicate and differentiated toward osteoblasts (left panel) or osteoclasts (right panel) in the respective medium and treated with indicated concentrations of DHT ({square}) or S-22 ({blacksquare}). At the end of 11 d, the cells were fixed and stained for alkaline phosphatase (ALP)-positive colonies to determine the differentiation toward osteoblasts or TRAP to determine the differentiation toward osteoclasts. TRAP-positive multinucleated (MNC) cultures are represented as changes from receptor activator of nuclear factor-{kappa}B ligand- plus granulocyte-macrophage colony-stimulating factor-treated cultures. E, S-22 inhibits the growth of LNCaP cells. LNCaP cells cultured in 1% csFBS in triplicate were treated with the indicated concentrations of S-22 ({square}) or DHT ({blacksquare}) for 3 d. Cell viability was measured with WTS reagent and expressed as percent change with vehicle-treated cultures taken as 100%. Values are expressed as mean ± SE wherever applicable.

 
In vivo results with S-22 demonstrated that separation between anabolic and androgenic effects was achieved (Fig. 1CGo). A variety of aryl propionamide SARMs were shown to increase levator ani muscle mass (and bone mineral density) with minimal agonist to inhibitory effects on prostate and seminal vesicles (24, 25, 26, 27). Similar to other aryl propionamide SARMs, male rats treated for 14 d with 1 mg/d S-22 showed increased levator ani muscle weight but significantly reduced prostate weight, demonstrating the tissue selectivity of S-22 (Fig. 1CGo). However, in the same experiment, 1 mg/d DHT significantly increased the weights of both levator ani and prostate. Similar to the in vivo pharmacological effects in muscle, DHT and S-22 increased the differentiation of bone marrow cells toward fibroblasts (Fig. 1DGo, left panel) and inhibited differentiation toward the osteoclast lineage (Fig. 1DGo, right panel) with similar potency, demonstrating the ability of androgens to promote the differentiation of osteoblasts and inhibit the differentiation of osteoclasts via the AR.

Steroidal androgens increase LNCaP cell growth (28). Because in vivo studies with S-22 using intact male rats showed a reduction in prostate size, we speculated that it would inhibit LNCaP cell growth. Figure 1EGo shows that S-22 retarded LNCaP cell growth, indicating that S-22 is selectively anabolic, promoting bone cell and muscle growth but not prostate or prostate cancer cell growth. DHT, similar to the results shown earlier, increased the growth of the cells (29). Representative pictures of tartrate-resistant acid phosphatase (TRAP) staining are shown as a supplemental figure (published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org).

Because this is the first mechanistic study with aryl propionamide SARMs, we evaluated the recruitment of AR and its coactivators to the PSA enhancer in LNCaP cells in the presence of DHT and S-22. DHT as shown earlier (30) recruited all of the tested coactivators, SRC-1, SRC-2, SRC-3, and cAMP response element-binding protein-binding protein (CBP), to the PSA enhancer (Fig. 2CGo). However, S-22 selectively recruited SRC-1, SRC-3, and CBP, but not SRC-2, to the PSA enhancer (Fig. 2CGo). The difference in SRC-2 recruitment between DHT and S-22 is very subtle. Both S-22 and DHT robustly recruited AR to the PSA enhancer (Fig. 2AGo). The specificity of ChIP assays were confirmed either by immunoprecipitating proteins with IgG and PCR amplifying the PSA enhancer region or by immunoprecipitating with AR antibody and PCR amplifying a nonspecific region near PSA enhancer (Fig. 2BGo).


Figure 2
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  		Fig. 2. S-22 Recruits AR and Coactivators to PSA Enhancer

A, S-22 and DHT recruit AR identically to PSA enhancer. LNCaP cells maintained for 5 d in RPMI plus 1% csFBS were treated with vehicle, 100 nM DHT, or S-22 for 90 min. Formaldehyde cross-linked cells were subjected to ChIP assay with AR antibodies as indicated in Materials and Methods. The PSA enhancer region was amplified by real-time PCR primers and probe and the results expressed as percent input. B, Measurement of specificity of ChIP assay. LNCaP cells maintained for 5 d in RPMI plus 1% csFBS were treated with vehicle or 100 nM DHT for 90 min and ChIP assay performed with AR antibody or IgG as indicated in Materials and Methods. The PSA enhancer region (PSA Enh.) and a nonspecific region (N.S.) were amplified by real-time PCR primers and probe and the results expressed as percent input. C, Recruitment of coactivators by DHT and S-22. LNCaP cells maintained for 5 d in RPMI plus 1% csFBS were treated with vehicle, 100 nM DHT, or S-22 for 90 min. Formaldehyde cross-linked cells were subjected to ChIP assay with SRC-1, SRC-2, SRC-3, CBP, or IgG antibodies as indicated in Materials and Methods. The PSA enhancer region was amplified by real-time PCR primers and probe and the results expressed as percent input. Values are expressed as mean ± SD from n = 3 experiments.

 
Nongenomic Effects of S-22 in Xenopus Oocytes
Maturation of Xenopus oocytes by androgens, although ligand dependent and mediated by AR, does not require transcription and thus is a good physiological measure of a ligand’s ability to mediate nongenomic effects (31). Lutz et al. (18) showed that some androgens such as testosterone or DHT mediate Xenopus oocyte maturation, whereas others like R1881 not only failed to mediate maturation but actually inhibited it. There are no reports regarding the effects of SARMs or other nonsteroidal AR ligands on oocyte maturation. We incubated Xenopus oocytes in the presence of testosterone or S-22 and counted the number of mature oocytes as evidenced by germinal vesicle breakdown (GVBD). Progesterone was used as a positive control (32). S-22, similar to testosterone, significantly induced GVBD, indicating the activation of nongenomic signaling in oocytes (Fig. 3AGo).


Figure 3
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  		Fig. 3. S-22 Matures X. laevis Oocytes through Nongenomic Signaling

A, X. laevis oocytes were isolated, and 25 oocytes in triplicates were incubated in MBSH medium with 100 nM S-22 (striped bars), testosterone (Test, black bars), or progesterone (Prog, gray bar) for 12–16 h at 18°C. Number of oocytes matured as indicated by GVBD was counted under microscope and graphed as percent maturation. B, AR and p42/44 MAPK is required for S-22-induced oocyte maturation. Twenty-five oocytes in triplicates were pretreated with 30 µM MEK inhibitor, U0126, or 1 µM AR antagonist hydroxyflutamide (OH-Flut) for 2 h and then treated with 100 nM S-22 and incubated for 12–16 h at 18°C. Matured oocytes as indicated by GVBD were counted and expressed as indicated in A. The experiments were performed in triplicate and represented as mean ± SE.

 
To evaluate the pathway that mediates the nongenomic signaling of S-22 in oocytes and the requirement of AR for S-22-induced maturation, the oocytes were preincubated with the MAPK kinase (MEK) inhibitor U0126 before treatment with S-22. Earlier studies indicated the involvement of the ERK or p42/44 MAPK pathway in progesterone-induced oocyte maturation (33). As indicated in Fig. 3BGo, S-22-induced oocyte maturation was blocked by the MEK inhibitor U0126 and AR antagonist hydroxyflutamide, indicating the requirement of ERK pathway and AR in the nongenomic actions of S-22 in oocytes.

S-22 and DHT Mediate Rapid Phosphorylation of Different Kinases in LNCaP Cells
The ability of S-22 to induce oocyte maturation led us to evaluate the kinases that are rapidly phosphorylated by S-22 and DHT in LNCaP cells. Phosphorylation or activation of kinases by ligands leads to several posttranslational modifications that are critical for the recruitment and function of steroid receptors and coactivators (34, 35, 36, 37, 38, 39). For this purpose, a phospho-MAPK array, containing antibodies in duplicates, that detects 18 different phosphorylated kinases was chosen. LNCaP cells were serum starved to reduce the basal phosphorylation status of the kinases and then treated with S-22 or DHT for 45 min. As shown in Fig. 4AGo, S-22 rapidly phosphorylated the p38 family of kinases, T180/Y182 of p38-{alpha} and T183/Y185 of p38-{gamma} (C9 and C10 and B9 and B10, respectively, in the array blot), whereas DHT phosphorylated the ERK kinase (B3 and B4 in the array blot, but not seen in the blot due to representative light exposure). Densitometric evaluation (Fig. 4AGo, right panel) indicates the robust phosphorylation of the p38 family by S-22 and ERK by DHT. The results were confirmed with Western blots for phospho-p38 and phospho-ERK (Fig. 4BGo).


Figure 4
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  		Fig. 4. Rapid Phosphorylation of Distinct Kinases by DHT and S-22 in LNCaP Cells

S-22 robustly phosphorylates p38 MAPK, whereas DHT phosphorylates ERK. LNCaP cells were maintained in RPMI plus 1% csFBS for 5 d and treated with vehicle, 100 nM DHT, or S-22 for 45 min. Protein was extracted, and 250 µg was blotted on a phospho-MAPK array as indicated in Materials and Methods. The top panel shows the template of the array. The blots were quantified and represented in bar graphs to the right as fold change from vehicle (white bars) for DHT (black bars) and S-22 (striped bars). Four blots were quantified and represented. B, The experiment described in A was repeated, and the protein extracts were run on a SDS-PAGE and Western blotted with antibodies for phospho-p38 MAPK, phospho-ERK, and actin. A representative blot is shown.

 
In addition, S-22 but not DHT also minimally phosphorylated other kinases such as glycogen synthase kinase-3β in LNCaP cells (data not shown).

S-22 and DHT Phosphorylate p42 MAPK or ERK in U2OS Bone Cells
Differences in pathways adopted or the kinases activated by DHT and S-22 in LNCaP cells could explain the observed functional differences between the two ligands. Conversely, one would expect both ligands to activate an identical kinase cascade in a bone or a muscle cell line. To explore this phenomenon, a U2OS bone cell line stably transfected with AR (U2OS-AR) was generated. Transactivation and AR expression data for U2OS-AR cells are shown in Fig. 5AGo. Treatment of U2OS-AR cells with S-22 and DHT, under the same experimental conditions as used with LNCaP, resulted in similar phosphorylation of T185/Y187 of p42 MAPK or ERK (Fig. 5BGo), corroborating the hypothesis that differences in the kinase cascade activated by S-22 vs. testosterone/DHT in different tissues contribute to tissue-selective pharmacological action. Representative Western blot is shown in Fig. 5BGo, right panel.


Figure 5
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  		Fig. 5. Rapid Phosphorylation of p42 MAPK by DHT and S-22 in U2OS-AR Cells

A, Characterization of U2OS-AR cells. U2OS osteoblasts stably transfected with AR (U2OS-AR) were transfected with 0.25 µg GRE-LUC and 5 ng CMV-renilla-LUC and treated with vehicle (white bars) or 0.1, 1, or 10 nM DHT for 24 h. Luciferase activity was measured and normalized as indicated in Materials and Methods. The right panel shows a comparison of Western blots for AR performed with protein extracted from LNCaP and U2OS-AR cells and LNCaP and untransfected U2OS cells. B, S-22 and DHT rapidly phosphorylate p42 MAPK. U2OS-AR cells were maintained in RPMI plus 1% csFBS for 5 d and treated with vehicle, 100 nM DHT, or S-22 for 45 min. Protein was extracted and blotted on a phospho-MAPK array as indicated in Materials and Methods. Three experiments were performed, and a representative blot was quantified and represented as fold change from vehicle (white bars) for DHT (black bars) and S-22 (striped bars). In the right panel, the experiment described in the left panel was repeated, and the protein extracts were run on a SDS-PAGE and Western blotted with antibodies for phospho-ERK and actin. A representative blot is shown. C, LNCaP and U2OS-AR cells express different levels of phosphorylated kinases. Protein was extracted from untreated LNCaP and U2OS-AR cells, and 250 µg was blotted on a phospho-MAPK array as indicated in Materials and Methods. The top panel shows the template of the array.

 
As with the coactivators and corepressors, the level of intracellular expression of active kinases plays a pivotal role in the function of several proteins. To determine whether the basal levels of phosphorylated kinases differ between LNCaP and U2OS cells, a phospho-MAPK array was used. Figure 5CGo (middle panel) shows that LNCaP cells express higher basal levels of phosphorylated T180/Y182 of p38-{alpha} (C9 and C10), phosphorylated T183/Y185 of p38-{gamma} (B9 and B10), phosphorylated S473 of Akt1 (B17 and B18), phosphorylated S474 of Akt2 (B19 and B20), and phosphorylated S473/S474/S472 of Akt (C19 and C20) but not ERK (B3 and B4 and C3 and C4). On the other hand, robust basal phosphorylation of ERK (B3 and B4 and C3 and C4) is observed in U2OS-AR cells, whereas the remaining kinases including p38 and Akt are very minimally or not phosphorylated (Fig. 5CGo, lower panel).

Activation of p38 MAPK Inhibits Basal and Ligand-Dependent AR Transactivation
Activation of p38 MAPK or c-Jun N-terminal kinase (JNK) is known to result in phosphorylation of Ser650 in AR and inhibition of its transactivation (38). Similarly, phosphorylation of p38 MAPK by IL-1{alpha} inhibits glucocorticoid receptor (GR) function (40). We speculated that the lesser AR transactivation potential of S-22 is due to its greater activation of p38 MAPK as compared with DHT. To establish this, HEK-293 (left panel) or LNCaP (right panel) cells were transfected with backbone or constitutively active MEK kinase-7 (MEKK-7), which activates p38 MAPK, and treated with DHT or S-22. Overexpression of constitutively active MEKK-7 completely inhibited the basal and ligand-dependent (i.e. DHT- and S-22-mediated) AR transactivation (Fig. 6Go), showing that increased p38 MAPK activation contributes to lesser AR transactivation in transfected HEK-293 or LNCaP cells.


Figure 6
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  		Fig. 6. Activation of p38 MAPK/JNK Pathway Inhibits Basal and Ligand-Dependent AR Transactivation

Left panel, HEK-293 cells were transfected using Lipofectamine with 0.1 µg vector backbone or constitutively active MEKK-7, 25 ng pCR3.1 rat AR, 0.25 µg GRE-LUC, and 0.5 ng CMV-renilla-LUC. Right panel, LNCaP cells were transfected using AMAXA electroporator with 4 µg GRE-LUC, backbone, or MEKK-7. The cells were treated with vehicle (white bars), 1 nM DHT (black bars), or 10 nM S-22 (striped bars). Twenty-four hours after treatment, the cells were harvested and luciferase assay performed and normalized to renilla luciferase.

 
Activation or Inhibition of Various Signaling Pathways Modulates AR Function Ligand Dependently
Modulation of cell signaling pathways impairs hormonal response and affects several physiological processes (41, 42, 43). We used specific inhibitors and activators to compare the ability of S-22 to modulate the activity of three pathways known to influence steroid-dependent (i.e. DHT-dependent) AR transactivation as a means to understand their role, if any, in S-22 action.

Previous reports showed that inhibition of p42/44 MAPK and Src kinases or activation of the muscarinic receptor (M2R) pathway abrogates steroid-dependent AR function (16, 18, 44, 45, 46, 47). We observed identical results with DHT in our experiments, with MEK1/2 inhibitor, src kinase inhibitor, and M2R agonist all significantly reducing DHT-induced AR transactivation (Fig. 7AGo, top panel). Although inhibition of p42/44 MAPK and Src kinase (by U0126 and PP2, respectively) inhibited S-22-mediated AR transactivation to a similar extent as observed with DHT, activation of the M2R pathway with carbachol was not inhibitory to S-22-dependent AR function (Fig. 7AGo, lower panel). This indicates that a different pathway upstream of p42/44 MAPK and Src kinase modulates S-22-dependent AR function. The results obtained in transient transfection were reproduced in LNCaP cells with endogenous PSA gene expression (Fig. 7BGo) and transactivation (Fig. 7FGo).


Figure 7
Figure 7
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  		Fig. 7. DHT and S-22 Activate AR through Distinct Signaling Pathways

A, HEK-293 cells were transfected as indicated in Materials and Methods with 0.25 µg GRE-LUC, 25 ng pCR3.1 rat AR, and 0.5 ng CMV-renilla-LUC. Twenty-four hours after transfection, the cells were pretreated for 30 min with 30 µM U0126 (MEK1/2 inhibitor), 30 µM carbachol (M2R agonist), or 10 µM PP2 (Src kinase inhibitor) and then treated with vehicle, indicated concentrations of DHT (top panel and black bars), or S-22 (bottom panel and striped bars). After 24 h treatment, the cells were harvested and luciferase assay performed and normalized to renilla luciferase. B, S-22 and DHT mediate AR function in LNCaP cells through distinct pathways. LNCaP cells were maintained in RPMI plus 1% csFBS for 3 d and were pretreated with 30 µM U0126 or 30 µM carbachol. Thirty minutes later, the cells were treated with 10 nM DHT (black bars) or S-22 (striped bars). The cells were harvested 24 h after treatment, RNA was isolated, and PSA gene expression was measured by real-time RT-PCR and normalized to 18S. C, Inhibition of M2R pathway increases AR function. HEK-293 cells were transfected as indicated in Materials and Methods with 0.25 µg GRE-LUC, 25 ng pCR3.1 rat AR, and 0.5 ng CMV-renilla-LUC. Twenty-four hours after transfection, the cells were pretreated for 30 min with 10 µM gallamine (M2R antagonist) and then treated with vehicle, indicated concentrations of DHT (black bars), or S-22 (striped bars). After 24 h treatment, the cells were harvested and luciferase assay performed and normalized to renilla luciferase. All the experiments were performed in triplicates. D, Activation of IP3 second messenger inhibits DHT- but not S-22-dependent AR function. HEK-293 cells were transfected as indicated in C. The cells were pretreated for 30 min with indicated concentrations of D-myo-IP3 and then treated with vehicle (white bars), 1 nM DHT (left panel and black bars), or 10 nM S-22 (right panel and striped bars). After 24 h treatment, the cells were harvested and luciferase assay performed and normalized to renilla luciferase. E, Selective inhibition of PI3K pathway inhibits DHT- but not S-22-dependent AR function. HEK-293 cells were transfected as indicated for C. Twenty-four hours after transfection, the cells were pretreated for 30 min with indicated concentrations of LY294002 (PI3K inhibitor) and then with vehicle, 1 nM DHT (black bars), or 10 nM S-22 (striped bars). After 24 h treatment, the cells were harvested and luciferase assay performed. All the experiments were performed in triplicates. F, Effect of signal modulators on endogenous AR transactivation in LNCaP cells. LNCaP cells were transfected using AMAXA electroporator with GRE-LUC and treated as shown in the figure and as described for the panels above. Twenty-four hours after transfection and 48 h after transfection, luciferase assay was performed.

 
To understand whether opposite results can be obtained by inhibiting the M2R pathway, gallamine, which specifically inhibits the M2R pathway, was cotreated with DHT or S-22, and AR transactivation was evaluated (48). As shown in Fig. 7CGo, inhibition of the M2R pathway with gallamine activated DHT-dependent AR transactivation but did not affect S-22-dependent AR transactivation, corroborating the results obtained with carbachol.

An activator of the phosphatidyl inositol pathway [D-myo-inositol-1,4,5-trisphosphate (D-myo-IP3)] and an inhibitor of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway (LY294002) were used to determine the role of pathways downstream of M2R that mediate DHT- and S-22-dependent AR transactivation. DHT-dependent AR transactivation was blocked by D-myo-IP3 (Fig. 7DGo, left panel), whereas S-22-dependent AR activity was unaffected (Fig. 7DGo, right panel). Similarly, inhibition of the PI3K/Akt pathway with LY294002 blocked DHT-dependent but not S-22-dependent AR transactivation, indicating that S-22 and DHT adopt different signaling pathways to activate AR (Fig. 7EGo).

Earlier results indicated the negative role of activated Jun and Fos transcription factors on AR function (49). To confirm that both S-22- and DHT-dependent AR function are susceptible to Jun and Fos activation, PSA gene expression was measured in LNCaP cells pretreated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and then with DHT or S-22. As shown in Fig. 8AGo, TPA inhibited both DHT- and S-22-dependent PSA expression.


Figure 8
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  		Fig. 8. Activation of JNK Pathway Modulates DHT- and S-22-Dependent AR Transactivation

A, LNCaP cells were pretreated for 30 min with vehicle or 100 nM TPA and then treated with vehicle (white bars), 1 nM DHT (black bars), or 10 nM S-22 (striped bars). After 24 h treatment, RNA was isolated, and PSA gene expression was measured and normalized to 18S. B, Overexpression of c-Jun and c-fos inhibit AR transactivation. HEK-293 cells were transfected as indicated in Materials and Methods with 0.25 µg GRE-LUC, 25 ng pCR3.1 rat AR, 0.5 ng CMV-LUC, 100 ng backbone, c-jun (left panel), or c-fos (right panel). Twenty-four hours after transfection, the cells were treated with vehicle (white bars), 1 nM DHT (black bars), or 10 nM S-22 (striped bars). After 24 h treatment, the cells were harvested and luciferase assay performed.

 
To determine whether c-fos, c-jun, or both the components of the activation protein-1 pathway are responsible for inhibiting AR transactivation, HEK-293 cells were transfected with backbone, c-jun (Fig. 8BGo, left panel), or c-fos (Fig. 8BGo, right panel) and then treated with vehicle, DHT, or S-22. As observed previously (49), only c-jun robustly inhibited DHT-mediated AR transactivation, whereas both c-jun and c-fos both completely inhibited S-22-mediated AR transactivation.

Modulation of Signaling Pathways Alters Recruitment of AR and Its Coactivators
Inhibition of ligand-dependent transcription results from abrogation in the recruitment of receptor or its associated cofactors to AR-responsive gene promoters. AR and its coactivators are substrates of several kinases for phosphorylation and other posttranslational modifications (50). Earlier results showed that modulation of some of these pathways is detrimental to the phosphorylation of AR and coactivators. Using ChIP assays, we explored whether DHT and S-22 recruited AR, SRC-1, and CBP to the PSA enhancer through an identical signaling cascade. As shown in Fig. 9A1Go, inhibition of the PI3K pathway with LY294002 led to complete inhibition of DHT-induced AR recruitment to the PSA enhancer. Treatment with phorbol 12-myristate 13-acetate (PMA) (an activator of jun and fos) partially inhibited DHT-induced AR recruitment, whereas other inhibitors demonstrated minimal effects. Subsequently, the recruitment of SRC-1 by DHT (Fig. 9A2Go) in the presence of signaling modulators was assessed. Consistent with the results obtained with LY294002 on AR recruitment and due to the lack of AR on the promoter, SRC-1 recruitment by DHT was also abrogated in the presence of LY294002. Inhibition of MEK by U0126 and activation of jun and fos with PMA eliminated DHT-dependent SRC-1 recruitment. Finally, activation of M2R with carbachol and inhibition of src kinase with PP2 completely inhibited the DHT-dependent CBP recruitment (Fig. 9A3Go).


Figure 9
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  		Fig. 9. Modulation of Signaling Pathways Alters the Recruitment of AR and Coactivators

A, Modulation of DHT-dependent recruitment of AR, SRC-1, and CBP to PSA enhancer by signaling modulators. LNCaP cells maintained for 5 d in RPMI plus 1% csFBS were pretreated with the inhibitors at concentrations indicated in Materials and Methods for 2 h and treated with 10 nM DHT for 90 min. Formaldehyde cross-linked cells were subjected to ChIP assay with AR (panel 1), SRC-1 (panel 2), or CBP (panel 3) antibodies as indicated in Materials and Methods. The PSA enhancer region was amplified by real-time PCR primers and probe and expressed as IP/input. B, Modulation of S-22-dependent recruitment of AR, SRC-1, and CBP to PSA enhancer by signaling modulators. LNCaP cells maintained for 5 d in RPMI plus 1% csFBS were pretreated with the inhibitors at concentrations indicated in Materials and Methods for 2 h and treated with 100 nM S-22 for 90 min. Formaldehyde cross-linked cells were subjected to ChIP assay with AR (panel 1), SRC-1 (panel 2), or CBP (panel 3) antibodies as indicated in Materials and Methods. The PSA enhancer region was amplified by real-time PCR primers and probe and expressed as IP/input. A representative of n = 4 experiments is shown here.

 
Treatment of cells with S-22 induced the recruitment of AR (Fig. 9B1Go), SRC-1 (Fig. 9B2Go), and CBP (Fig. 9B3Go). Cotreatment of cells with U0126 and PP2 inhibited S-22- induced SRC-1 recruitment (Fig. 9B2Go). Interestingly, pretreatment with PMA did not inhibit AR and SRC-1 recruitment but inhibited the S-22-induced CBP recruitment (Fig. 9B3Go).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Intracellular signaling kinases that regulate the function of proteins are complex and tissue dependent. Steroid receptors and their coactivators are phosphorylated by a variety of these kinases, especially by proline-directed serine or threonine kinases such as cyclin-dependent kinases (Cdks) or MAPK (37, 51, 52). Mutations of these phosphorylation sites cause significant alterations in the phenotype of the proteins (52, 53). SRC-3, an important coactivator of AR and ER signaling, requires all of the six phosphorylation sites in its sequence to coactivate AR or ER but requires phosphorylation at only selective sites to coactivate nuclear factor-{kappa}B (54). This shows the existence of an additional level of transcriptional regulation, where specific modulation of phosphorylation integrates diverse signaling events in cells. Determining precisely how kinases regulate steroid receptor transactivation has been challenging due to cross talk between the numerous potential kinases and cross-reactivity of steroidal ligands (55).

The expanded therapeutic success of androgens and estrogens in diseases such as osteoporosis, cachexia, hot flashes, and cancer may ultimately rely on the ability of ligands to selectively stimulate AR or ER ({alpha} and β) function and thus separate beneficial from unwanted pharmacological effects. Discovering and applying these ligands to therapeutic use will undoubtedly require an in-depth understanding of the role of genomic and/or nongenomic signaling in varying cell types. Some of the recently identified therapeutic opportunities arising from selective activation of nongenomic signaling include treatment of osteoporosis, central nervous system disorders, vasodilation, and others (56, 57, 58). Hence, understanding the influence of SARMs and SERMs on endogenous kinase cascade regulation and modulation of nongenomic signaling is critical for the future discovery of novel SERMs and SARMs.

Ten years after the discovery of the first SARM and with several of them now in clinical trials, we began a comprehensive evaluation of various signaling pathways to SARM action, including nongenomic signaling (59). SARM elicited distinct pharmacological effects on prostate and bone or muscle (Fig. 1Go). Although minor differences were observed in the ability of SARM and DHT to promote the recruitment of SRC-1, SRC-2, SRC-3, and CBP (SRC-2 was better recruited to the AR in the presence of DHT), the most profound differences were observed during studies of the phosphorylation of different kinases in prostate and bone cells (Figs. 4Go and 5Go).

Several lines of evidence suggested that the antiproliferative effects of p38 MAPK phosphorylation are mediated through inhibition of transcriptional activation of steroid receptors or through antagonistic effects on proliferative p42/44 MAPK or ERK. Phosphorylation of AR, GR, and coactivators by activated ERK increases the function of the protein and subsequently proliferation of the cells (60, 61, 62). Mutation of these phosphorylation sites in the receptor or coactivator prevents their activation by ERK (60, 62). On the other hand, phosphorylation of AR or GR through activation of p38 MAPK inhibits receptor function, whereas mutation of these sites restores it (38, 40).

SARM and DHT had differing effects on p38 MAPK and ERK in LNCaP cells. SARM activated antiproliferative p38 MAPK in LNCaP cells, whereas DHT activated proliferative ERK (Fig. 4Go). The observed increase in p38 MAPK activation by SARM in LNCaP cells could potentially explain its observed effects on in vivo prostate growth, wherein p38 MAPK activation inhibited endogenous ERK activation by DHT. On the other hand, SARM and DHT similarly activated proliferative ERK in the U2OS bone cell line and had similar effects on osteoblast osteoclast function, supporting the idea of a shared mechanism of action in bone cells.

Other results indicate that the phosphorylation and suppression of AR activity by glycogen synthase kinase-3β, another kinase modestly and selectively activated by SARM in LNCaP cells but not in U2OS cells may have contributed to the observed selectivity (data not shown) (63). These results imply that SARM selectively activates AR inhibitory and antiproliferative kinases in the prostate while activating proliferative or AR-activating kinases and signaling in bone.

The mediation of nongenomic signaling by SARM in oocytes is also a striking phenomenon (Fig. 3Go). Several studies have shown that maturation of oocytes through nongenomic signaling is exclusive to steroidal scaffolds. In this study, SARM promoted oocyte maturation as robustly as steroids, but the maturation was inhibited by the MEK inhibitor U0126 (Fig. 3Go).

The rapid activation of these selective signaling events impinges on AR function. The tight integration of nongenomic and genomic actions of steroids is now being appreciated more clearly (56). Some classical actions of the hormones on cell cycle and cell division that were thought originally to arise from genomic regulation are also found to be regulated through recruitment of rapid signaling pathways (56). To summarize these findings, DHT mediated the AR activation through M2R inhibition and phosphatidylinositol, PI3K, Src, and ERK pathway activation, whereas SARM mediated AR function through activation of protein kinase C, Src kinase, p38 MAPK, or ERK kinase. As a variety of SARM scaffolds with varying pharmacological effects are now emerging (2, 64, 65), our data suggest that a wide variety of structurally modified SARMs with unique pharmacological profiles and ability to modulate signaling cascades will be identified.

Increase in weight-bearing muscle mass either by exercise or by therapeutic agents requires the activation of the PI3K/Akt pathway (66, 67, 68). Studies with transgenic mice have demonstrated that activation of the PI3K/Akt pathway for 3 wk is sufficient to cause a dramatic increase in muscle mass (69). Interestingly, Fig. 7EGo demonstrates the imperative requirement of PI3K/Akt pathway for DHT but not for SARM-dependent AR transactivation. In conditions of muscular dystrophy, cachexia, and muscle wasting due to extensive disuse, the PI3K/Akt pathway is completely impaired (70, 71). Because steroidal androgens require PI3K/Akt signaling to activate AR, the muscle mass in these disorders cannot be increased with steroidal androgens such as testosterone. However, because the SARM stimulated AR function is independent of the PI3K/Akt signaling pathway, the SARM might still retain its anabolic effects and could be the choice of treatment for the above mentioned disorders where this crucial pathway is impaired. These results need to be verified in muscle in vivo or in vitro.

The results obtained in Fig. 7Go, D and E, with the IP3 activator and the PI3K/Akt inhibitor suggest the complex regulation of AR biology by intracellular signaling pathways. Earlier results have shown that PI3K/Akt and its upstream activator IP3 are critical for AR function (72). On the other hand, although the phosphatidylinositol pathway (IP3) is an upstream activator of PI3K/Akt, it also mobilizes intracellular calcium (73), which in turn inhibits AR function (74). Hence, it is likely that depending on the tissue studied, IP3 might mobilize calcium and/or activate PI3K/Akt to regulate AR function.

Earlier studies showed that activation of the IL-8 pathway converted the AR antagonist bicalutamide to an agonist with the ability to promote LNCaP growth (75). This effect was mediated through recruitment of coactivators instead of corepressors. Similarly, activation of the Her2/neu pathway leads to tamoxifen resistance in breast cancer and converts tamoxifen to an agonist (76).

Because the upstream phosphorylation events are very important for the recruitment of receptors and coactivators, the recruitment pattern of AR, SRC-1, and CBP were studied. Results with progesterone showed elegantly that inhibition of cdk2 completely blocked SRC-1 recruitment and inhibited progesterone receptor function (37). This work also showed how phosphorylation of SRC-1 by cdk2 is critical for its interaction with progesterone receptor. Another model proposes that androgen increases the phosphorylation of AR by negatively regulating the phosphatase binding to the ligand-binding domain or by inducing a conformation that is resistant to phosphatases action (77).

Having two ligands (DHT and SARM) that mediate different signaling pathways is a great tool to analyze the recruitment pattern of AR and its cofactors and the effects of these diverse signaling events on the recruitment pattern. The most puzzling question here arises from the observation that SARM and DHT mediate the recruitment of the same protein (e.g. SRC-1 or CBP) through distinct pathways. Inhibition of src kinase with PP2 inhibited SARM-induced SRC-1 recruitment. However, treatment with PP2 had no effect on DHT-induced recruitment of SRC-1, but prevented CBP recruitment (Fig. 9Go). On the other hand, inhibition of AR function by PMA (a JNK activator) inhibited DHT-induced recruitment of SRC-1 and SARM-induced recruitment of CBP. Similar to earlier results, we show here that the recruitment of coactivators is more susceptible to inhibition of kinases than is the recruitment of receptors (37). A variety of possible mechanisms may explain this observation. First, coactivators contain a large number of phosphorylation sites that are important for their posttranslational modification and function. Second, the phosphorylation of different sites by different ligands occurs through the same kinase, as was shown earlier for ER (39). Lastly, different structural modifications in the presence of different ligands result in increased or decreased susceptibility to kinase phosphorylation and their inhibition (78). ER phosphorylation at Ser104, Ser106, and Ser118 occurs through MAPK or cdk2 in the presence of various ligands such as estradiol, tamoxifen, or ICI 182,780 (79). Earlier studies showed that the transcriptional activities of transcription factors such as activation protein-1 and nuclear factor-{kappa}B in response to hormones are regulated through MAPK- or Akt- dependent pathways. This suggests that a large set of genomic effects of hormones are not dependent directly on interactions of steroid receptors with consensus sequences in the DNA, but rather on the nuclear actions of other transcription factors that are modulated via nongenomic mechanisms. This might hinder the possibility of identifying a ligand with the ability to selectively modulate individual genes within the complex network but potentially explains the overlapping effects of steroid receptors on diverse sets of genes in the absence of obvious mechanisms (e.g. consensus sequences) for genomic cross talk.

This study and the model add yet another level of complexity in AR-mediated signaling. This study also opens up a new window to analyze signaling cascades to evaluate tissue-selective functions of SRMs. These results indicate a two-step model of androgenic signaling that integrates the rapid nongenomic as well as chronic genomic androgenic actions. Both these pathways are tightly integrated with significant modulatory effects on gene transcription and drug discovery.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents
AR (N-20), SRC-1 (rabbit polyclonal), and CBP (rabbit polyclonal) antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Protein A-Sepharose was obtained from Amersham Pharmacia (Piscataway, NJ). WST-1 reagent was purchased from Roche Applied Science (Nutley, NJ). U0126 was obtained from Promega (Madison, WI). LY294002, PP2, and D-myo-IP3 were procured from Calbiochem (San Diego, CA), whereas carbachol, TPA, and gallamine were from Sigma Chemical Co. (St. Louis, MO). A human phospho-MAPK array (ARY002) was obtained from R&D Systems (Minneapolis, MN). Phospho-ERK antibody was obtained from Upstate Biotechnology (Lake Placid, NY), phospho-p38 MAPK antibody was obtained from BD Biosciences (Bedford, MA), and actin antibody was procured from Chemicon International (Temecula, CA). All cell culture medium was obtained from Invitrogen (Carlsbad, CA), and the serum for cell culture was obtained from Atlanta Biologicals (Atlanta, GA). All other reagents used were analytical grade.

Cell Culture
LNCaP prostate cancer cells, U2OS human osteosarcoma cells, and HEK-293 cells were obtained from the American Type Culture Collection (Manassas, VA). U2OS cells stably transfected with AR (denoted as U2OS-AR) were kindly provided by Dr. Jeetendra Eswaraka (GTx Inc., Memphis, TN). LNCaP cells were grown in RPMI 1640 (containing 2 mM L-glutamine, 10 mM HEPES, and 1 mM sodium pyruvate, penicillin, and streptomycin) supplemented with 10% fetal bovine serum (FBS). U2OS cells were grown in McCoy’s 5a medium supplemented with 1.5 mM L-glutamine and 10% FBS, and HEK-293 cells were grown in DMEM supplemented with 10% FBS. For the ChIP and MAPK array assays, cells were plated in 150-mm dishes at 10 million cells per dish in medium supplemented with 1% charcoal stripped FBS (csFBS). The cells were maintained in 1% csFBS for 6 d to reduce basal occupancy of promoters with medium changed on d 1 and 3 and before treatment on d 6.

ChIP Assay
ChIP assays were performed as described previously (37). Proteins were cross-linked by incubation with 1% formaldehyde (final concentration) at 37 C for 10 min. The cells were washed with 1x PBS twice, scraped in 1 ml PBS containing protease inhibitors (1 mg each of aprotinin, leupeptin, antipain, benzamidine HCl, and pepstatin/ml; 0.2 mM phenylmethylsulfonyl fluoride; and 1 mM sodium vanadate), pelleted, and resuspended in sodium dodecyl sulfate (SDS) lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1). After lysis on ice for 10 min, the cell extract was sonicated (Branson sonifier 250) in a cold room eight times for 10 sec each at constant duty cycle, with an output of 3 and with incubation on ice after every sonication. The debris was pelleted at 13,000 rpm for 10 min at 4 C, and the supernatant was diluted 10-fold with ChIP dilution buffer [0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris HCl (pH 8.1), 167 mM NaCl]. The proteins were precleared with 50 µl 1:1 protein A-Sepharose beads in Tris-EDTA (TE). An aliquot (300 µl) was reserved as input, whereas the remaining solution was incubated with 5 µg AR, SRC-1, or CBP antibody and 2 µg of sheared salmon sperm DNA (Stratagene, La Jolla, CA) rotating overnight at 4 C. The protein-DNA-antibody complex was precipitated by incubating with 100 µl 1:1 protein A-Sepharose beads and 2 µg salmon sperm DNA at 4 C for 2 h. The beads were pelleted and washed three times with low-salt wash buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris HCl (pH 8.1), 0.15 M NaCl], and twice with 1x TE [10 mM Tris HCl, 1 mM EDTA (pH 8.0)]. DNA-protein complexes were obtained by extracting the beads with 50 µl freshly prepared extraction buffer (1% SDS, 0.1 M NaHCO3) three times. Cross-linking of the DNA protein complexes was reversed by incubating at 65 C for 6 h. The DNA was extracted with a QIAquick PCR purification kit (QIAGEN, Valencia, CA). in 25 µl final volume of TE.

Real-time PCR was performed on an ABI 7300 (Applied Biosystems, Foster City, CA) using TaqMan PCR master mix under universal conditions. The numbers on the y-axis of the ChIP assay results were obtained by dividing the arbitrary quantitative PCR numbers obtained for each sample by the respective input. The following PCR primers and probes for PSA enhancer region and nonspecific region were synthesized by Biosource International (Camarillo, CA): PSA enhancer forward primer 5'-GCCTGGATCTGAGAGA GATATCATC-3', reverse primer 5'-ACACCTTTTTTTTTCTGGATTGTTG-3', and probe FAM 5'-TGCAAGGATGCCTGCTTTACAAACATCC-3' TAMRA and nonspecific forward primer 5'-TCATCATGAATCGCACTGT-3', reverse primer 5'-GCCCAAGTGCCTTGGTAT-3', and probe FAM 5'-TGAATCATCTGGCACGGC-3' TAMRA.

Bone Marrow Culture
Cell culture materials were obtained from Invitrogen. All animal studies were conducted under the auspices of an animal protocol approved by the Institutional Laboratory Animal Care and Use Committee at the University of Tennessee Health Science Center. Femurs were removed from female, Sprague-Dawley rats weighing approximately 300 g under anesthesia and, immediately before killing, rinsed in 70% ethanol and then washed three times with 5 ml each of penicillin and streptomycin (10,000 U penicillin and 10 mg/ml streptomycin). Both ends of the femurs were snapped, and the bone marrow cells were flushed with 15 ml MEM with penicillin, streptomycin, and fungizone into a 50-ml conical tube and stored on ice. The bone marrow cells were pooled and centrifuged at 1000 rpm for 5 min in a clinical centrifuge. The cells were resuspended in phenol red-free MEM supplemented with 10% csFBS, penicillin, streptomycin, and fungizone. The cells were triturated through a 22-gauge needle, counted under a microscope, and plated at 1.5 million cells per well of a six-well plate in phenol red-free MEM supplemented with 15% csFBS, penicillin, streptomycin, 300 ng/ml fungizone, 0.28 mM ascorbic acid, and 10 mM β-glycerophosphate to differentiate toward the fibroblast/osteoblast lineage. In separate wells, 2.5 million cells per well were plated in 24-well plates in phenol red-free MEM supplemented with 10% csFBS, penicillin, streptomycin, and 300 ng/ml fungizone to differentiate toward the osteoclast lineage. The medium was changed on d 2, and the cells were treated with the compound of interest. Studies in osteoclast cultures were performed in the presence of receptor activator of nuclear factor-{kappa}B ligand (50 ng) and granulocyte-macrophage colony-stimulating factor (10 ng) to induce osteoclastogenesis. Medium was completely changed every third day for osteoclast cultures. For fibroblast cultures, half the culture medium was changed every third day to leave the growth factors secreted by the cells (80).

Staining of Cells
At the end of 12 d, the cells were fixed in 10% buffered formalin for fibroblast cultures and in 4% formaldehyde in PBS for osteoclast cultures. The fibroblasts were stained for alkaline phosphatase activity, and the OD at 405 nm was measured using a spectrophotometer as described earlier (80). The osteoclasts were stained for TRAP, and cells having two or more nuclei were counted under the microscope (80).

RNA Analysis and RT-PCR
LNCaP cells were plated at 700,000 cells per well of a six-well plate in RPMI supplemented with 1% csFBS or in full serum. The cells were maintained for 3 d and were treated with vehicle, DHT, or S-22. RNA was isolated using Trizol (Invitrogen) and the expression of various genes measured using TaqMan primer probe mix from Applied Biosystems using one-step RT-PCR master mix on an ABI 7300 real-time PCR machine. The expression of individual genes was normalized to 18S rRNA levels.

Growth Assay
LNCaP cells were plated at 10,000 cells per well of a 96-well plate in RPMI supplemented with 1% csFBS. The cells were treated for 72 h with the indicated concentrations of S-22. The cell viability at the end of 72 h was measured using WST-1 reagent.

Plasmid Constructs and Transient Transfection
Rat AR was cloned from rat prostate cDNA into pCR3.1 vector backbone. Sequencing was performed to determine the absence of any mutations. CMV-renilla-LUC was obtained from Promega, whereas pFC-MEKK-7, pfA2-cJUN, pfA2-cFOS, and backbone plasmid pfc2DBD were obtained from BD Biosciences (Palo Alto, CA). For transfection, cells were plated at 90,000 cells per well of a 24-well plate in DMEM plus 5% csFBS. The cells were transfected using Lipofectamine (Invitrogen) with 0.25 µg GRE-LUC, 0.02 µg CMV-LUC (renilla luciferase), and 25 ng rat AR. The cells were treated 24 h after transfection with S-22, DHT, or an inactive isomer of S-22 (S-22 R-isomer) and the luciferase assay performed 48 h after transfection.

Transfections of LNCaP cells with Amaxa electroporator (Amaxa Inc., Gaithersburg, MD) were performed using solution R and program T-009 according to the manufacturer’s protocol. Briefly, two million cells suspended in solution R were incubated with 4 µg GRE-LUC or backbone or MEKK-7 and electroporated with program T-009. Twenty-four hours after transfection, medium was changed to DMEM plus 5% csFBS, and the cells were treated for an additional 24 h and luciferase assay performed as indicated above.

Human Phospho-MAPK Array
Cells were plated and maintained in 1% csFBS as indicated above and in individual figures and were treated with vehicle, DHT, or S-22 for 45 min. The cells were washed, harvested, and blotted on a human phospho-MAPK array as instructed by the manufacturer.

Western Blotting
Cells were grown and treated as described above for the MAPK array. Protein extracts were prepared and Western blotted as described earlier (37).

Xenopus laevis Oocyte Maturation
Xenopus ovary was received from NASCO (Fort Atkinson, WI). The ovary was immediately removed from ice and washed in calcium- and magnesium-free Ringers solution and placed in 1 mg/ml collagenase A in Ca/Mg-free Ringers solution for 2–4 h at room temperature. Stage V and VI oocytes were separated and plated at 25 oocytes per well of a six-well plate in calcium-free MBSH (modified Barth) solution. The oocytes were treated with the indicated steroids, S-22, or a combination of S-22 and signaling or AR inhibitors and incubated for 16–18 h at 18 C. Mature oocytes were counted under the microscope and were plotted as percent oocytes matured.

Animal Experiment
Five male Sprague Dawley rats per group (300 g) from Harlan (Indianapolis, IN) were housed with three animals per cage and were allowed free access to tap water and commercial rat chow (Harlan Teklad 22/5 rodent diet 8640). During the course of the study, the animals were maintained on a 12-h light, 12-h dark cycle. This study was reviewed and approved by the Institutional Laboratory Care and Use Committee of The University of Tennessee. The animals were dosed daily for 14 d with 1 mg/d S-22 or DHT or vehicle (polyethylene glycol). Dosing solutions were prepared daily by dissolving drug in dimethylsulfoxide and diluting in polyethylene glycol 300. At the end of 14 d, the animals were killed and the weights of prostate and levator ani measured.

All in vitro experiments were performed at least in triplicate, and values are expressed as mean ± SE. Statistical analysis was performed by t test.


   FOOTNOTES
 
Disclosure Statement: All authors are employees of GTx, Inc. and hold stock options in the company. J.D.K., D.D.M., and J.T.D. are inventors on SARM patents and may receive invention royalties from the University of Tennessee Research Foundation or The Ohio State University Research Foundation.

First Published Online September 18, 2008

Abbreviations: AR, Androgen receptor; CBP, cAMP response element-binding protein-binding protein; Cdk, cyclin-dependent kinase; ChIP, chromatin immunoprecipitation; csFBS, charcoal-stripped FBS; DHT, dihydrotestosterone; ER, estrogen receptor; FBS, fetal bovine serum; GR, glucocorticoid receptor; GVBD, germinal vesicle breakdown; IP3, inositol-1,4,5-trisphosphate; JNK, c-Jun N-terminal kinase; MEK, MAPK kinase; MEKK-7, MEK kinase-7; M2R, muscarinic receptor; PI3K, phosphatidylinositol 3-kinase; PMA, phorbol 12-myristate 13-acetate; SARM, selective AR modulator; SERM, selective ER modulator; SDS, sodium dodecyl sulfate; SRM, selective receptor modulator; TE, Tris-EDTA; TPA, 12-O-tetradecanoylphorbol-13-acetate; TRAP, tartrate-resistant acid phosphatase.

Received for publication May 16, 2008. Accepted for publication September 8, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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NURSA Molecule Pages Link:

Nuclear Receptors:   AR
Coregulators:   CBP  |  SRC-1  |  GRIP1  |  AIB1
Ligands:   Dihydrotestosterone



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V. C. Jordan
A Century of Deciphering the Control Mechanisms of Sex Steroid Action in Breast and Prostate Cancer: The Origins of Targeted Therapy and Chemoprevention
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