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Endocrinology, doi:10.1210/en.2007-0530
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Endocrinology Vol. 149, No. 3 1103-1112
Copyright © 2008 by The Endocrine Society

Cell-Specific Activation of the Human Skeletal {alpha}-Actin by Androgens

Mei Hua Hong, Hong Sun, Cheng He Jin, Mark Chapman, Junlian Hu, William Chang, Kelven Burnett, Jon Rosen, Andres Negro-Vilar and Jeffrey N. Miner

Department of Research and Development, Ligand Pharmaceuticals Inc., San Diego, California 92121

Address all correspondence and requests for reprints to: Mei Hua Hong, Department of Molecular and Cell Biology, Ligand Pharmaceuticals Inc., 10275 Science Center Drive, San Diego, California 92121. E-mail: mhong{at}ligand.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although it is evident that androgens increase muscle mass and strength, little is known about the critical molecular targets of androgens in skeletal muscle. In rodents, the skeletal {alpha}-actin gene is a tissue-specific gene expressed only in the levator ani and other skeletal muscles but not in the prostate or preputial gland, the well-known androgen target tissue. We identified tissue-specific androgen-regulated genes in the skeletal muscle in rats after oral administration of androgens and focused on androgen-dependent up-regulation of the skeletal {alpha}-actin gene. To investigate the mechanism of action, an in vitro system with various cell lines and a series of deletion mutants of the {alpha}-actin promoter were used. The human skeletal {alpha}-actin promoter was activated by androgens in the muscle cell line C2C12 but not in the liver, prostate, or breast cancer cell lines in which exogenous human androgen receptor is expressed. The sequence of the promoter is sufficient for cell-specific androgen response, providing a model for the tissue specificity demonstrated in vivo. Using a series of deletion mutants, the androgen response can be maintained using just the proximal promoter region. The importance of androgen regulation of this small portion of the human skeletal {alpha}-actin promoter was demonstrated by the correlation between muscle and the {alpha}-actin promoter activity for an array of selective androgen receptor modulators (SARMs), including an orally active SARM LGD2226. Taken together, the results suggest that the regulation of skeletal {alpha}-actin by androgens/SARMs may represent an important model system for understanding androgen anabolic action in the muscle.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANDROGENS ARE ESSENTIAL for the development and maintenance of male and female reproductive tissues, libido, and muscle mass and strength (1). Clinical data clearly demonstrate that androgen supplementation increases muscle size and strength in both hypogonadal and normal men (2, 3). Although the biological function of androgens in the skeletal muscle is evident, physiology-related target genes representing the anabolic activity of the androgen are not identified, nor is the mechanism of androgen action in the muscle entirely clear.

Androgens typically exert their biological actions through activation of the androgen receptor (AR), a member of the nuclear receptor superfamily (4). Upon ligand binding, the AR activates gene transcription by binding to specific DNA sequences, termed androgen-responsive elements (AREs) and participates in the assembly of an active transcriptional complex (5). The ARE confers AR-mediated transcriptional activation on genes, such as prostate-specific antigen (6, 7) in human prostate, the kallikrein family (7, 8, 9), probasin (10), and cystatin-related proteins (11) in rat prostate. AR can also participate in the regulation of gene expression via protein-protein interactions as well. Indirect activation of the chicken skeletal {alpha}-actin promoter by androgens has been demonstrated using the chicken skeletal {alpha}-actin promoter and was hypothesized to occur through a direct interaction with serum response factor (SRF) at a serum response element within the chicken promoter (12). Additionally, androgens can also repress gene expression by indirect mechanisms as exemplified by the IL-6 gene (13). On the other hand, the {alpha}-actin promoter has been demonstrated to exhibit muscle-specific activity through the tissue-specific action of SRF and SRF-related proteins (14).

Although human skeletal {alpha}-actin is comprised of thin and thick filaments, the skeletal {alpha}-actin is the major component of the thin filament. There are six distinct actin isotypes differing in the amino terminal region highly conserved throughout mammals (15). There are two striated muscle isotypes, skeletal-{alpha} and cardiac-{alpha} (16), two smooth muscle isotypes (17), and β and {gamma}, parts of the complex network of filaments that make up the cytoarchitecture of every nonmuscle cell (18).

In this study, we investigated the androgen-mediated gene expression profile in muscle tissues in rats and tested the hypothesis that the mechanism of androgen action on the human skeletal {alpha}-actin gene was related to, and important for, anabolic activity in vivo. Effects of androgens on muscle tissue were determined in rat levator ani muscle, a sexually dimorphic skeletal muscle, which expresses high levels of the AR. This muscle enlarges significantly in castrated animals upon the administration of androgens. A variety of selective androgen receptor modulators (SARMs) including an orally active SARM, LGD2226 (19), were used to demonstrate a correlation between effects on skeletal {alpha}-actin gene expression in rat skeletal muscle and the promoter activity in transfected mouse myoblastic C2C12 cells. Skeletal {alpha}-actin regulated by androgens is exquisitely tissue and cell specific, occurring only with the AR in the muscle in vivo and in muscle cells in vitro but not in other cell types. This activity depends on a specific region of the promoter that does not contain the binding site for the well-characterized muscle regulatory factor SRF (12). Thus, the regulation of skeletal {alpha}-actin by androgens and SARMs may provide an important model system for detecting anabolic activity in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All procedures involving the animals were approved by Ligand’s Institutional Animal Care and Use Committee. Male adult (7 wk old) Sprague Dawley rats were purchased from Harlan (Indianapolis, IN).

Two-week rat study
After a 1-wk acclimation period, rats were orchidectomized and sorted into groups such that no statistically significant differences in mean body weights were observed. The afternoon of surgery, each rat was treated with either sc vehicle or 3 mg/kg·d dihydrotestosterone for 14 consecutive days. For SARM compounds, orchidectomized rats were treated using oral gavage immediately after surgery with vehicle, fluoxymeterone, or SARM compounds (1, 3, 10, 30, and 100 mg/kg·d). The day after the 14th dose, the rats were euthanized, and levator ani muscles, preputial gland, and ventral prostate were weighed, collected, and snap frozen for total RNA isolation.

Total RNA was isolated from each sample using TRIzol reagent (Invitrogen Corp., Carlsbad, CA) and then cleaned up using the RNeasy minikit (QIAGEN Inc., Valencia, CA). Ten micrograms of total RNA were used to prepare the target for hybridization according to protocols of Affymetrix (Santa Clara, CA). The labeled target was subsequently hybridized to U34A rat expression arrays in an Affymetrix hybridization oven 640. After being washed and stained in an Affymetrix Fluidic Station, the array was scanned with an Affymetrix GeneArray 2500 scanner. Affymetrix MAS 5 was used to generate signal intensity.

Cells and chemicals
Mouse myoblastic C2C12, the human hepatoma HepG2, human mammary grand MDA-453, and human prostate MDA-pca-2b cells were obtained from American Type Cell Culture (Manassas, VA). Cultural media for the cells were from Invitrogen. Dihydrotestosterone (DHT), testosterone, fluoxymesterone, and oxandrolone were purchased from Sigma-Aldrich (St. Louis, MO) and Steraloids (Wilton, NH), respectively. 2-Hydroxyflutamide (2-OH flutamide) was purchased from (Archemi, MO). Restriction and modification enzymes used were obtained from Promega (Madison, WI) and Roche Molecular Biochemicals (Indianapolis, IN).

Northern blot analysis
Human multitissue poly A+ blot and the human β-actin probe were obtained from CLONTECH (Palo Alto, CA). Human AR probe was prepared by PCR. The forward and reverse primers specific for the sites 2270–2290 and 3051–3071 of the human AR were 5'-TCTGAAACTACAGGAGGAAGG-3'and 5'-CACTTGCACAGAGATGATCTC-3', respectively. The 801-bp PCR fragment was subcloned into PCR TA cloning kit (Invitrogen) and was confirmed by sequencing (Sequenase; Amersham Life Science, Arlington Heights, IL).

The human multitissue poly A+ RNA blot was hybridized with human β-actin and human AR probes. Northern blot and hybridization were performed as described previously (20).

Cloning of human skeletal {alpha}-actin promoter and construction of reporter systems
The forward and reverse primers specific for human skeletal {alpha}-actin promoter region from –708 to +202 bp were designed to include BglII and NheI sites overhangs at 5'-site for forward and reverse primers, respectively. Sequences of the forward and the reverse primers for this region of the human skeletal {alpha}-actin promoter were 5'-TAATGCTAGCGCCCTCCGGCCGCCG-3' and 5'-GCAGAGATCTCAGGGCGAGGCCTGG-3', respectively. The region of human skeletal {alpha}-actin promoter was cloned by PCR using the human liver genomic DNA (Promega) and inserted into a pGL3 basic reporter plasmid (Promega) after digesting the PCR fragment with BglII and NheI to produce A-708 construct. The A-708 construct containing 910 bp amplified DNA was used as the template for further deletion mutants by PCR using 5' primers corresponding to –407, –207, –153, –99, –77, –47, and –34 of the skeletal {alpha}-actin genomic sequence in combination with the reverse primer used for the cloning described above. These reactions generate a series of skeletal {alpha}-actin promoter fragments that were further cloned into pGL3 basic reporter vector to produce A-407, A-207, A-153, A-99, A-77, A-47, and A-34, respectively. The sequence and orientation of the constructs were confirmed by sequencing.

Site-directed mutagenesis
Point mutations were introduced by site-directed mutagenesis using an in vitro mutagenesis system (Stratagene, La Jolla, CA) as recommended by the manufacturer’s instructions. The presence of the appropriate mutation was confirmed by sequencing (Sequenase; Amersham Life Science).

EMSA
EMSA was performed as recommended by the manufacturer’s instructions (LICOR Biosciences, Linclon, NE). The human AR protein was produced using the TnT coupled-reticulocyte lysate system (Promega). Proteins derived from the in vitro translation system were mixed with 50 nM probe, annealed 5'-IRDye-700-labeled oligonucleotides with or without 200-fold excess cold oligonucleotides in LICOR IRDye EMSA reagents for 30 min at room temperature. The sequences used for the probes as a consensus ARE and the oligonucleotides from the promoter proximal region, –77 to approximately –35 of the human skeletal {alpha}-actin (skARE) are 5'-CAGAACATCATGTTCTGAGCTAC-3' and 5'-CAGCGACATTCCTGCGGGGTGGCGCGGAGGGAATCGCCCGCGG-3', respectively. Oligonucleotides used in competitive study were the oligonucleotides with the same sequences to the ARE (cold ARE) and skARE (cold skARE) except of 5'-IRDye-700, respectively. Nonspecific oligonucleotide used was the interferon response element with a sequence of 5'-AGCATGTTTCAAGGATTTGAGATGTATTTCCCAGAAAAG-3'. After completing the incubation, the reaction mixtures were loaded onto 5% acrylamide gel and run at 250 V for 1.5 h at 4 C. Gels were scanned using the LICOR Odyssey infrared imager, and the fluorescent signal from bands captured was analyzed with the associated Odyssey software.

Transient transfection and luciferase activity assay
Transient transfection was performed with the FuGene 6 (Roche Molecular Biochemicals) following the manufacturer’s instructions with minor modifications as described previously (19). C2C12 cells were seeded 24 h before transfection in 24-well plates (2 x 104 cells/well). FuGene 6 reagent (0.3 µl/well) was mixed with 0.1 µg/well of total DNA. The reporter plasmid containing the human skeletal {alpha}-actin promoter region-fused to the firefly luciferase gene or reference constructs mouse mammary tumor virus-luciferase MMTV-luc or the parental plasmid without the skeletal {alpha}-actin promoter fragment were cotransfected with human AR expression plasmid driven by CMV promoter, β-galactosidase reporter, and pGem carrier plasmid at the ratio of 1:4:5:10. The mixture was added to the cells and incubated overnight. Cells were further incubated for 24 h with or without various concentrations of androgens as indicated in the figure legends. Cell extracts were prepared and assayed for the luciferase and β-galactosidase activities as described previously (21).

Statistical analysis
Data obtained were analyzed by one-way ANOVA. Data are presented as the group means ± SEM. The criterion of significance was P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Target genes regulated by androgens in skeletal muscle
During the process of identifying target genes regulated by androgens in the skeletal muscle, we identified a group of genes regulated in rat muscle within 48 h after administration of 10 mg/kg of the androgen fluoxymesterone to castrated rats using Affymetrix microarray of rat gene expression as described in Materials and Methods. Skeletal {alpha}-actin was stimulated 4.9-fold by fluoxymesterone treatment. To measure the tissue selectivity of the androgen response of this gene, we examined the androgen regulation of rat skeletal {alpha}-actin RNA across a number of different tissues extracted from orchidectomized rats treated for 2 wk with 3 mg/kg DHT (Fig. 1Go). As expected, DHT stimulated the growth of the muscle (levator ani), ventral prostate, and preputial gland in these animals (Fig. 1CGo). DHT selectively stimulated the {alpha}-actin gene expression in the levator ani skeletal muscle but not other androgen target tissues, such as the prostate or preputial gland (Fig. 1AGo). The DHT treatment did not affect the {alpha}-actin gene or the ornithine decarboxylase (ODC) gene expressions in the gastrocnemius, suggestive of little response to DHT in the gastrocnemius muscle (Fig. 1Go, A and B). In contrast, the ODC gene was regulated by androgens in these androgen target tissues, indicating sufficient receptor levels in these tissues to regulate transcription (Fig. 1BGo). The results indicate that androgens regulate the skeletal {alpha}-actin gene in a muscle-specific fashion.


Figure 1
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  		FIG. 1. Tissue- and gene-specific androgen regulation in prostate, muscle, and preputial gland. Castrated rats were administered 3 mg/kg·d of DHT for 2 wk, and the organs were dissected out for analysis of mRNA expression levels using Affymetrix microarray of rat gene expression as described in Materials and Methods. The relative RNA expression of skeletal {alpha}-actin (A) or ODC in response to DHT gene expression (B) in the levator ani, gastrinemic muscle, ventral prostate, and preputial gland was compared with the level from vehicle control (Veh). The data were summarized from two separate sets of experiments. C, Relative organ weights from more than four separate sets of experiments for levator ani, ventral prostate, and preputial gland. *, The statistical significance of differences (P < 0.05) between vehicle control and DHT treatment.

 
Expression of skeletal {alpha}-actin and AR mRNA in human multitissues
To ensure that the tissue specificity in rodents matched that seen in humans, we measured androgen regulation of the skeletal {alpha}-actin gene in human tissues, we first conducted Northern blot analysis for the skeletal {alpha}-actin and AR mRNA in human multitissue poly A+ RNA blot (Fig. 2Go). The skeletal {alpha}-actin mRNA was detected only in the heart and skeletal muscle. AR mRNA, on the other hand, expressed in the heart (lane 9) and skeletal muscle (lane 14) as well as in the prostate (lane 3), testis (lane 4), and uterus (lane 5). The same result was obtained using different probes specific for various regions of the AR or when hybridizing with other blots of the same tissues (data not shown). These results indicate that the AR was coexpressed with {alpha}-actin mRNA in the heart (lane 9) and skeletal muscle (lane 14) and despite sufficient levels of AR in prostate, no expression of {alpha}-actin was detected (lane 3). In contrast, β-actin was expressed ubiquitously.


Figure 2
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  		FIG. 2. Coexpression of human AR and skeletal {alpha}-actin mRNA in human muscle. A human multitissue poly A+ RNA blot was hybridized with human AR and β-actin probes and exposed with a phosphor imager as described in Materials and Methods. The specific mRNA species corresponding to the AR and actin isotypes were indicated by arrows as AR and actin-{alpha} and -β, respectively.

 
Identification of a DHT-responsive region of human skeletal {alpha}-actin promoter
To determine whether the tissue-specific androgen regulation seen in vivo was promoter dependent, we constructed a reporter system with various lengths of the proximal promoter region of human skeletal {alpha}-actin and transfected those into both muscle and nonmuscle cell lines. A 910-bp promoter region of the skeletal {alpha}-actin was cloned from human liver genomic DNA by PCR and then subcloned into the upstream of a firefly luciferase coding region (A-708, Fig. 3AGo). In addition, a series of deletions within the promoter were introduced to assess the importance of specific upstream sequences (Figs. 3Go and 4Go). Myoblastic C2C12 cells were cotransfected with the deletion constructs and a human AR expression plasmid. The entire 910-bp portion (A-708) and other deletion mutants were induced significantly by androgens except of A-34, the shortest construct tested (Fig. 3BGo). However, as shown in Fig. 3Go, B and C, the deletion of sequences between –7 and –99 resulted in an increase in the androgen-dependent activation. This apparently inhibitory region contains the binding sites for both the muscle activator SRF and a transcriptional repressor YY1 (22, 23). These two factors have been shown to antagonize one another in muscle cells. Further deletion of an additional 43 nucleotides (region from –77 to –34) diminished the activity significantly (Fig. 3Go, B and C). The deletion of this region removes sterol regulatory element-binding protein SREBP, a highly conserved specificity protein-1 (SP1), a partially conserved nuclear factor-{kappa}B, and activator protein binding sites (Fig. 3AGo and Table 1Go). Point mutations either in single or multiple points in this region show that multiple sites are important for the androgen activation (Fig. 4Go). Additional deletion constructs that removed extra 3' sequences (+6 to ~+202) consistently enhanced androgen response in a range of 4- to 6-fold, further supporting the androgen response of the sequence between –77 and +6 (data not shown). Nevertheless, as measured by androgen response, both suppressive and enhancing elements appear to be located in this promoter. To further assess whether the androgen action is due to a direction interaction of the AR and cis-binding sites of the promoter, we conducted EMSA. As shown in Fig. 5Go, no binding of AR to the fragment (–77 to –35) was detected although AR activity is evident with a consensus ARE under the same condition. No binding was observed with a longer fragment (–77 to +2) either (data not shown). The binding data with functional tests are consistent with the notion that the AR may indirectly activate the {alpha}-actin promoter through this region.


Figure 3
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  		FIG. 3. Identification of an androgen-responsive region of the skeletal {alpha}-actin promoter. Panel A, Schematic illustration of human skeletal {alpha}-actin promoter reporter constructs with transcription factor binding sites. The proximal region of human skeletal {alpha}-actin promoter (from –708 to +202 bp) was cloned out by PCR and subcloned into the upstream of a firefly luciferase gene of pGL-3 basic plasmid. Panels B and C, DHT’s effect on human skeletal {alpha}-actin promoter activity. Myoblastic C2C12 cells were seeded in 24-well plate for 24 h and cotransfected with various reporter plasmids and the human AR expression plasmid. The cells were then treated with or without DHT for 48 h. The cells were lysed and luciferase and β-galactosidase activities were measured. The promoter activity was indicated as normalized luciferase activity after normalization of the luciferase activity by β-galactosidase activity (panel B) or as fold induction (panel C), respectively. *, The statistical significance of differences (P < 0.05) between vehicle control (Veh) and DHT treatment. C, CArG box; S, SREBP; P, SP1; N, NF-{kappa}B; A, AP2; and M, Myo D.

 

Figure 4
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  		FIG. 4. Androgen effect on the skeletal {alpha}-actin promoter with single- and multiple-point mutations. Panel A, Schematic illustration of the short skeletal {alpha}-actin promoter reporter constructs with mutations introduced. The proximal region of human skeletal {alpha}-actin promoter (from –77 to +202 bp) was obtained as one of a serial deletion constructs from the pGL-3-based reporter plasmid as described in Fig. 3Go. Single- and multiple-point mutations were introduced by site-directed mutagenesis, and their mutants were designated as M1–7, in which M6 was triple-point mutants. Panel B, DHT’s effect on the activity of human skeletal {alpha}-actin promoter with mutations. Myoblastic C2C12 cells were cotransfected with various mutated reporter plasmids and the human AR expression plasmid and then treated with or without 10–6 M DHT. The promoter activity was indicated as fold induction for normalized Luc activity. *, The statistical significance of differences (P < 0.05) between vehicle control and DHT treatment. S, SREBP; P, SP1; N, NF-{kappa}B; A, AP2; and M, Myo D.

 

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  		TABLE 1. Homology of the proximal region of skeletal {alpha}-actin promoters

 

Figure 5
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  		FIG. 5. Recombinant AR does not directly bind to the proximal promoter region of skeletal {alpha}-actin by EMSA. In vitro-translated human AR was incubated with probes in the presence or absence of cold oligonucleotides (200-fold excess) as described in Materials and Methods. The DNA binding protein complex was separated from free probe by a native gel and detected by LICOR Odyssey infrared imager. NS, Nonspecific oligonucleotide derived from interferon-responsive element.

 
Androgen specificity in the human skeletal {alpha}-actin promoter
We used A-77 construct, the best to further assess the effect of androgens on this promoter. Androgen regulation of the skeletal {alpha}-actin promoter depends on the presence of transfected AR (Fig. 6AGo) and exhibits the expected dose response when a number of anabolic steroids are used (DHT, testosterone, fluoxymesterone, and oxandrolone) (Fig. 6BGo). The order and magnitude of the activity of androgens are consistent with their binding affinity to the AR. The androgen antagonist, 2-OH flutamide, was unable to activate the promoter activity in the agonist mode and inhibited the activity of DHT (5 x 10–10 M) in the antagonist mode (Fig. 6CGo). The response of this promoter is also highly receptor specific because other steroid receptors did not activate as well as the AR at this promoter. Estrogen and aldosterone showed no effect on the skeletal {alpha}-actin promoter when cells were transfected with their respective receptors, estrogen receptor and mineralocorticoid receptor (data not shown). In addition, glucocorticoid receptor (GR) was an extremely weak activator of the actin promoter. In contrast, GR is an extremely powerful activator, compared with AR, in the context of the mouse mammary tumor virus promoter luciferase construct (data not shown). The in vitro activation of the skeletal {alpha}-actin promoter together with the in vivo gene expression data in response to the castration and androgen supplementation supports the notion that the skeletal {alpha}-actin promoter is specifically activated by the AR.


Figure 6
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  		FIG. 6. Androgens are active at the human skeletal {alpha}-actin promoter. A, AR-dependent activity at the skeletal {alpha}-actin promoter. C2C12 cells were transfected with or without the human AR plasmid and the promoter reporter plasmid. Twenty-four hours after transfection, the cells were exposed to 10–8 M DHT for a further 48 h. B and C, Agonist and antagonist modes. For the agonist mode, the cells were exposed to various concentrations of androgens for 48 h. For the antagonist mode, various concentrations of 2-OH flutamide, an AR antagonist, was added to the well with 5 x 10–10 M DHT (EC75) and incubated for 48 h. The antagonist mode was assessed by the recovery of inhibition by various concentrations of the AR antagonist, in the presence of 5 x 10–10 M DHT. The promoter activity was described as the normalized luciferase activity. The data are from three independent experiments and shown as mean ± SEM.

 
Tissue specificity in the regulation of the human skeletal {alpha}-actin promoter
To explore whether the tissue-specific androgen regulation of the {alpha}-actin promoter can be detected in vitro, we tested the androgen responsiveness of the skeletal {alpha}-actin promoter in three additional cell lines representing nonmuscle tissue types. In addition to the myoblastic C2C12 cell, we tested liver (HepG2), mammary gland (MDA-mb-453), and prostate (MDA-PCa-2b) cells. As shown in Fig. 7Go, DHT dose-dependently enhanced the human skeletal {alpha}-actin promoter only in the myoblastic C2C12 cell line but not the other lines tested. In contrast, the MMTV promoter activity was strongly stimulated by DHT in all cell lines tested. These results in cells are consistent with those seen in vivo (Fig. 1Go). This further supports the notion that both expression and androgen regulation of the skeletal {alpha}-actin promoter are tissue specific.


Figure 7
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  		FIG. 7. Tissue-specific effect of androgens in skeletal {alpha}-actin promoter construct. C2C12 (muscle), HepG2 (liver), and MDA-Pca-2b (prostate) cells were transfected with both human AR expression plasmid and skeletal {alpha}-actin promoter reporter plasmid (upper panel) or MMTV-reporter plasmid (bottom panel). MDA-453 (breast) cell was transfected only with the reporter plasmid because the cell expresses endogenous AR. Cells were then exposed to various concentrations of DHT for 48 h and lysed for luciferase assay as described in Fig. 3Go. The data are from three independent experiments and shown as mean ± SEM. *, The statistical significance of differences (P < 0.05) between vehicle control and DHT treatment.

 
Correlation between androgen action in vitro and anabolic effect in rat muscle
To assess the biological relevance of the tissue-specific activity measured with this promoter, we evaluated the correlation of the skeletal {alpha}-actin assay and the in vivo muscle assay in which activities of a group of compounds synthesized over a period of 6 months were examined. These compounds represent a wide range of activities from weakly to fully potent and efficacious. One of our SARM compounds, LGD2226 (19), was also included. We included the fluoxymesterone for a reference on the graph. Figure 8Go shows a correlation of the maximum efficacy of androgens in the skeletal {alpha}-actin in vitro assay and that of anabolic effect in the rat levator ani muscle assay (Fig. 8AGo) and that of androgenic effect in rat prostate weight gain (Fig. 8BGo). The maximum efficacy of these compounds evaluated with the human skeletal {alpha}-actin in vitro assay appears to be correlated well with that from the in vivo muscle assay (R2 = 0.6275). In contrast, the correlation with a nonmuscle tissue, the ventral prostate is lower (R2 = 0.3957), suggesting that {alpha}-actin regulation is not as important for the growth of the prostate, compared with growth of muscle. This result emphasizes the biological relevance of the human {alpha}-actin promoter for the anabolic action of androgens in the muscle.


Figure 8
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  		FIG. 8. Correlation between an in vivo muscle assay and the skeletal {alpha}-actin promoter assay. A, Each point represents one compound tested in the {alpha}-actin assay and the effects on muscle in vivo. The maximal efficacy in each assay is plotted (R2 = 0.6275). B, Each point represents one compound tested in the {alpha}-actin assay and the effects of the compound on ventral prostate weight in vivo. The maximal efficacy in each assay is plotted (R2 = 0.3957). LA, Levator ani; VP, ventral prostate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We describe tissue-specific androgen regulation of skeletal {alpha}-actin gene by a non-tissue-specific transcription factor, AR. The AR is expressed widely in various human tissues in which its actions are typically anabolic in nature, and it regulates genes in response to the hormone testosterone. Treatment of castrated male rats with androgens results in an increase in the size of the muscle, prostate, and sebaceous preputial gland (24, 25, 26). Examining gene expression in these tissues resulted in the detection of tissue-specific regulation of the skeletal {alpha}-actin gene. We confirmed androgen regulation of the skeletal {alpha}-actin in the muscle by RT-PCR analysis (data not included). The results show that androgens regulate gene expression of skeletal {alpha}-actin gene only in the skeletal muscle but not other androgen-target organs, such as the prostate or preputial gland in vivo. This is consistent with a previous analysis of chicken skeletal {alpha}-actin muscle-specific activity, and it can now be extended to muscle-specific androgen regulation (12).

The anabolic effect of androgens has been evaluated in the skeletal muscle of rats as well as the rodent kidney (27). The end points commonly used are the change in target organ mass (increase) or the enhancement in ODC and glucuronidase gene expression or activity in the kidney (28). Other groups have reported androgen-specific binding in rat skeletal muscles using a radiolabeled ligand binding assay (29). We demonstrate that the receptor expression is coincident with the skeletal {alpha}-actin expression by Northern blot. These data suggest that androgens may play a direct role in anabolic actions on the skeletal muscle through the AR. Thyroid hormone response elements were identified on human {alpha}-actin promoter (30). They are located within the –273/–249 and –173/–149 regions of the human {alpha}-actin promoter, which are different from the androgen-regulatory region we identified here.

Androgens stimulate skeletal {alpha}-actin gene expression via a cis-element in the proximal region of the promoter in a dose-dependent manner. Androgen regulation of this promoter is AR-dependent and androgen-specific. Taken together, our data indicate that skeletal {alpha}-actin is a target of androgens in the skeletal muscle. The elements necessary for that tissue-specific androgen regulation reside within the promoter of the skeletal {alpha}-actin gene with the muscle cell providing necessary factors for enhanced transcription in response to the anabolic steroid.

Typically the stimulatory effect of androgens for its target genes involves the AR, activated by its cognate ligands, binding directly to AREs located in the proximal region of target promoters. Homology search of the 5' proximal region of the human skeletal {alpha}-actin gene reveals a number of potential ARE consensus elements (Fig. 3Go). However, these sequences do not appear to be involved in androgen regulation of {alpha}-actin promoter based on the cotransfection data (Fig. 3Go) or gel shift assay (data not shown).

We were able to further narrow down the androgen-responsive region within –77 to +6 of the promoter with a series of deletion and point mutations. It appears that multiple factors are required for this androgen regulation, but more analysis needs to be conducted to establish this. Notably, this region does not contain the CArG box, which binds the activator SRF, commonly associated with muscle-specific activity (22, 23). Thus, androgen regulation maps to a different location than the muscle-specific activation suggesting the involvement of different factors in the androgen response of the human promoter. Experiments conducted on the chicken promoter implicated the SRF binding site for androgen-mediated up-regulation (12). Mutation of the SRF binding site reduces the AR-mediated activity from the chicken promoter (12). We show that the tissue-specific androgen regulation of the endogenous gene could be recapitulated with just the proximal promoter region from the skeletal {alpha}-actin gene because androgen activation occurs only in a myoblastic cell background but not nonmuscle cells. In contrast, other ARE-driven reporter systems responded to androgens in a variety of cell backgrounds.

This promoter also exhibits receptor selectivity because other nuclear receptors do not activate this promoter in muscle cells, whereas these receptors are fully active at alternative promoters. These data suggest that the androgen action at the skeletal {alpha}-actin gene is mediated indirectly through a tissue-specific factor(s) in skeletal muscle cells rather than a direct binding of the activated AR to the skeletal {alpha}-actin promoter DNA. Other indirect mechanisms of androgen regulation have been described (13). Androgens are known to inhibit IL-6 gene expression via an indirect mechanism without direct interaction on DNA (13). In addition, other steroid receptors have been reported to activate transcription indirectly by binding to specific transcription factors such as GR and signal transducer and activator of transcription-5 (31), estrogen receptor, and activator protein-1 (32, 33). AR has been shown to interact indirectly with SP1 (34, 35) and cooperate with SP1 potentially via bridging activities of small nuclear ring finger protein (36, 37). SP1 binding sites are present within the proximal skeletal {alpha}-actin promoter, and we are investigating their role in selective regulation.

Genetics can be used to establish biologically relevant connections between different phenotypes. We argue that in much the same way mutational analysis can be used to detect interactions between processes, chemical genetics can be used to detect important relationships. We show a series of AR ligands with subtle alterations in structure that exhibit a range of activities on the AR. We used this compound set to establish a relationship between the androgen regulation of the skeletal {alpha}-gene and the androgen regulation of muscle growth in vivo. The presence of this relationship is significant because not all cell-based models exhibit this correlation with in vivo end points. Chemical genetics can also detect meaningful connections between biological responses (38).

Skeletal {alpha}-actin is the major component of the skeletal muscle; therefore, an anabolic androgen would be expected to enhance its expression in the muscle. Several pieces of evidence support this notion: the coexpression pattern of skeletal {alpha}-actin and the AR gene in the human skeletal muscle, the selective androgen action in the skeletal {alpha}-actin promoter-based reporter systems in muscle cells, and the correlation between the anabolic activity in levator ani in vivo assay and the reporter assay using chemical genetics. Our results demonstrate a clear link between the anabolic effects of androgens and a known structural component of muscle tissue and the utility of the muscle gene-based assay for the discovery of novel SARM.


   Acknowledgments
 
We thank Robin Chedester for help with manuscript preparation and Wen Luo, Andrew Gibbs, Manny R. Corpuz, Robert Hill, and Cesar Igno for help in carrying out these experiments. We thank the Department of Medicinal Chemistry of Ligand Pharmaceutical Inc. for help with the synthesis of SARM compounds. We also thank Keith Marschke for helpful comments and suggestions.


   Footnotes
 
Present address for C.H.J.: 3760 Torrey Hill Lane, San Diego, California 92130.

Disclosure Statement: C.H.J. has nothing to declare. M.H.H., H.S., M.C., J.H., W.C., K.B., J.R., A.N.-V., and J.N.M. have equity interest in Ligand Pharmaceuticals, Inc.

First Published Online December 6, 2007

Abbreviations: AR, Androgen receptor; ARE, androgen-responsive element; DHT, dihydrotestosterone; GR, glucocorticoid receptor; ODC, ornithine decarboxylase; 2-OH flutamide, 2-hydroxyflutamide; SARM, selective androgen receptor modulator; skARE, skeletal {alpha}-actin; SP1, specificity protein-1; SRF, serum response factor.

Received April 25, 2007.

Accepted for publication November 26, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Mooradian AD, Morley JE, Korenman SG 1987 Biological actions of androgens. Endocr Rev 8:1–28[Abstract/Free Full Text]
  2. Bhasin S, Tenover JS 1997 Age-associated sarcopenia—issues in the use of testosterone as an anabolic agent in older men. J Clin Endocrinol Metab 82:1659–1660[Free Full Text]
  3. Bhasin S, Storer TW, Berman N, Callegari C, Clevenger B, Phillips J, Bunnell TJ, Tricker R, Shirazi A, Casaburi R 1996 The effects of supraphysiologic doses of testosterone on muscle size and strength in normal men. N Engl J Med 335:1–7[Abstract/Free Full Text]
  4. Evans RM 1988 The steroid and thyroid hormone receptor superfamily. Science 240:889–895[Abstract/Free Full Text]
  5. Roche PJ, Hoare SA, Parker MG 1992 A consensus DNA-binding site for the androgen receptor. Mol Endocrinol 6:2229–2235[Abstract/Free Full Text]
  6. Luke MC, Coffey DS 1994 Human androgen receptor binding to the androgen response element of prostate specific antigen. J Androl 15:41–51[Abstract/Free Full Text]
  7. Young CY, Montgomery BT, Andrews PE, Qui SD, Bilhartz DL, Tindall DJ 1991 Hormonal regulation of prostate-specific antigen messenger RNA in human prostatic adenocarcinoma cell line LNCaP. Cancer Res 51:3748–3752[Abstract/Free Full Text]
  8. Winderickx J, Swinnen K, Van Dijck P, Verhoeven G, Heyns W 1989 Kallikrein-related protease in the rat ventral prostate: cDNA cloning and androgen regulation. Mol Cell Endocrinol 62:217–226[CrossRef][Medline]
  9. Ashley PL, MacDonald RJ 1985 Tissue-specific expression of kallikrein-related genes in the rat. Biochemistry 24:4520–4527[CrossRef][Medline]
  10. Parker MG, Scrace GT, Mainwaring WI 1978 Testosterone regulates the synthesis of major proteins in rat ventral prostate. Biochem J 170:115–121[Medline]
  11. Chamberlin LL, Mpanias OD, Wang TY 1983 Isolation, properties, and androgen regulation of a 20-kilodalton protein from rat ventral prostate. Biochemistry 22:3072–3077[CrossRef][Medline]
  12. Vlahopoulos S, Zimmer WE, Jenster G, Belaguli NS, Balk SP, Brinkmann AO, Lanz RB, Zoumpourlis VC, Schwartz RJ 2005 Recruitment of the androgen receptor via serum response factor facilitates expression of a myogenic gene. J Biol Chem 280:7786–7792[Abstract/Free Full Text]
  13. Keller ET, Chang C, Ershler WB 1996 Inhibition of NF{kappa}B activity through maintenance of I{kappa}B{alpha} levels contributes to dihydrotestosterone-mediated repression of the interleukin-6 promoter. J Biol Chem 271:26267–26275[Abstract/Free Full Text]
  14. Chang PS, Li L, McAnally J, Olson EN 2001 Muscle specificity encoded by specific serum response factor-binding sites. J Biol Chem 276:17206–17212[Abstract/Free Full Text]
  15. Vandekerckhove J, Weber K 1979 Amino-acid sequence analysis of the amino-terminal tryptic peptides of different actins from the same mammal. Arch Int Physiol Biochim 87:210–212[Medline]
  16. Vandekerckhove J, Weber K 1979 The complete amino acid sequence of actins from bovine aorta, bovine heart, bovine fast skeletal muscle, and rabbit slow skeletal muscle. A protein-chemical analysis of muscle actin differentiation. Differentiation 14:123–133[CrossRef][Medline]
  17. Vandekerckhove J, Weber K 1979 The amino acid sequence of actin from chicken skeletal muscle actin and chicken gizzard smooth muscle actin. FEBS Lett 102:219–222[CrossRef][Medline]
  18. Vandekerckhove J, Weber K 1978 Mammalian cytoplasmic actins are the products of at least two genes and differ in primary structure in at least 25 identified positions from skeletal muscle actins. Proc Natl Acad Sci USA 75:1106–1110[Abstract/Free Full Text]
  19. Miner JN, Chang W, Chapman MS, Finn PD, Hong MH, Lopez FJ, Marschke KB, Rosen J, Schrader W, Turner R, van Oeveren A, Viveros H, Zhi L, Negro-Vilar A 2006 An orally active selective androgen receptor modulator is efficacious on bone, muscle and sex function with reduced impact on prostate. Endocrinology 148:363–373[CrossRef][Medline]
  20. Jin CH, Miyaura C, Ishimi Y, Hong MH, Sato T, Abe E, Suda T 1990 Interleukin 1 regulates the expression of osteopontin mRNA by osteoblasts. Mol Cell Endocrinol 74:221–228[CrossRef][Medline]
  21. Guido EC, Delorme EO, Clemm DL, Stein RB, Rosen J, Miner JN 1996 Determinants of promoter-specific activity by glucocorticoid receptor. Mol Endocrinol 10:1178–1190[Abstract/Free Full Text]
  22. Gualberto A, LePage D, Pons G, Mader SL, Park K, Atchison ML, Walsh K 1992 Functional antagonism between YY1 and the serum response factor. Mol Cell Biol 12:4209–4214[Abstract/Free Full Text]
  23. Lee TC, Shi Y, Schwartz RJ 1992 Displacement of BrdUrd-induced YY1 by serum response factor activates skeletal {alpha}-actin transcription in embryonic myoblasts. Proc Natl Acad Sci USA 89:9814–9818[Abstract/Free Full Text]
  24. Huggins C, Parsons FM, Jensen EV 1955 Promotion of growth of preputial glands by steroids and the pituitary growth hormone. Endocrinology 57:25–32[Medline]
  25. Ebling FJ, Ebling E, McCaffery V, Skinner J 1973 The responses of the sebaceous glands of the hypophysectomized-castrated male rat to 5-androstanedione and 5-androstane-3,17-diol. J Invest Dermatol 60:183–187[CrossRef][Medline]
  26. Mesquita-Guimaraes J, Coimbra A 1981 The effect of sexual hormones on the lipid and proteinaceous secretion of the rat preputial sebaceous gland. Arch Dermatol Res 270:325–331[CrossRef][Medline]
  27. Kochakian CD 1977 Regulation of kidney growth by androgens. Adv Steroid Biochem Pharmacol 6:1–34[Medline]
  28. Pajunen AE, Isomaa VV, Janne OA, Bardin CW 1982 Androgenic regulation of ornithine decarboxylase activity in mouse kidney and its relationship to changes in cytosol and nuclear androgen receptor concentrations. J Biol Chem 257:8190–8198[Free Full Text]
  29. Hickson RC, Kurowski TT, Galassi TM, Daniels DG, Chatterton Jr RJ 1985 Androgen cytosol binding during compensatory overload-induced skeletal muscle hypertrophy. Can J Biochem Cell Biol 63:348–354[Medline]
  30. Muscat GE, Griggs R, Downes M, Emery J 1993 Characterization of the thyroid hormone response element in the skeletal {alpha}-actin gene: negative reg-ulation of T3 receptor binding by the retinoid X receptor. Cell Growth Differ 4:269–279[Abstract]
  31. Groner B 2002 Transcription factor regulation in mammary epithelial cells. Domest Anim Endocrinol 23:25–32[CrossRef][Medline]
  32. Safe S 2001 Transcriptional activation of genes by 17β-estradiol through estrogen receptor-Sp1 interactions. Vitam Horm 62:231–252[Medline]
  33. Janne OA, Moilanen AM, Poukka H, Rouleau N, Karvonen U, Kotaja N, Hakli M, Palvimo JJ 2000 Androgen-receptor-interacting nuclear proteins. Biochem Soc Trans 28:401–405[Medline]
  34. Darne CH, Morel L, Claessens F, Manin M, Fabre S, Veyssiere G, Rombauts W, Jean CL 1997 Ubiquitous transcription factors NF1 and Sp1 are involved in the androgen activation of the mouse vas deferens protein promoter. Mol Cell Endocrinol 132:13–23[CrossRef][Medline]
  35. Xiao F, Mirwald A, Papaioannou M, Baniahmad A, Klug J 2005 Secretoglobin 2A1 is under selective androgen control mediated by a peculiar binding site for Sp family transcription factors. Mol Endocrinol 19:2964–2978[Abstract/Free Full Text]
  36. Poukka H, Aarnisalo P, Santti H, Janne OA, Palvimo JJ 2000 Coregulator small nuclear RING finger protein (SNURF) enhances Sp1- and steroid receptor-mediated transcription by different mechanisms. J Biol Chem 275:571–579[Abstract/Free Full Text]
  37. Curtin D, Ferris HA, Hakli M, Gibson M, Janne OA, Palvimo JJ, Shupnik MA 2004 Small nuclear RING finger protein stimulates the rat luteinizing hormone-β promoter by interacting with Sp1 and steroidogenic factor-1 and protects from androgen suppression. Mol Endocrinol 18:1263–1276[Abstract/Free Full Text]
  38. Iannone MA, Simmons CA, Kadwell SH, Svoboda DL, Vanderwall DE, Deng SJ, Consler TG, Shearin J, Gray JG, Pearce KH 2004 Correlation between in vitro peptide binding profiles and cellular activities for estrogen receptor-modulating compounds. Mol Endocrinol 18:1064–1081[Abstract/Free Full Text]
  39. Fornwald JA, Kuncio G, Peng I, Ordahl CP 1982 The complete nucleotide sequence of the chick {alpha}-actin gene and its evolutionary relationship to the actin gene family. Nucleic Acids Res 10:3861–3876[Abstract/Free Full Text]



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