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Division of Endocrinology, Metabolism, and Molecular Medicine, Charles R. Drew University of Medicine and Science, Los Angeles, California 90059
Address all correspondence and requests for reprints to: Shalender Bhasin, M.D., Charles R. Drew University of Medicine and Science, 1731 East 120th Street, Los Angeles, California 90059. E-mail: sbhasin{at}ucla.edu.
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Abstract |
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Introduction |
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We recently demonstrated that androgenic steroids promote the commitment and differentiation of a pluripotent, mesenchymal cell line into myogenic lineage and inhibit its differentiation into adipogenic lineage (20). Testosterone effects on pluripotent cell commitment and differentiation are blocked by an AR antagonist, indicating that these effects are mediated through an AR pathway (20). These observations led us to hypothesize that ARs would be expressed in mesenchymal precursor cells within the human skeletal muscle. This hypothesis predicts that satellite cells and other CD34+ precursor cells within the skeletal muscle that are capable of myogenic commitment would express AR protein.
To test this hypothesis, we used immunohistochemical staining to detect AR protein and determine its cellular localization in human skeletal muscle obtained by percutaneous needle biopsies of the vastus lateralis. Visualization of AR expression was characterized further by confocal immunofluorescence microscopy. Different cell types within the skeletal muscle were identified by their anatomical location and by the expression of specific cellular markers. We confirmed the localization of AR in muscle satellite cells in culture by immunohistochemical staining, Western blot analysis, reverse transcription followed by real-time PCR, and sequencing of reverse-transcribed and PCR-amplified cDNA product. We also determined the effects of androgen treatment on AR expression in vivo and in vitro.
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Subjects and Methods |
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Percutaneous biopsies from the vastus lateralis muscle were obtained from six healthy, young men who were participating in a study of testosterone supplementation that had been approved by the institutional review boards at Drew University and Research and Education Institute (Los Angeles, CA) (9). All subjects provided written, informed consent. The participants in this study were healthy, young men, 18–35 yr old, who had normal serum testosterone levels. The detailed description of the study design and the participants has been published previously (9).
Muscle biopsies obtained at baseline were used for AR localization. Muscle biopsies obtained from six men before and after treatment with a long-acting GnRH agonist and 600 mg of testosterone enanthate weekly for 20 wk were used to evaluate androgen effects on AR expression and distribution.
Tissue fixation, light microscopy, and confocal microscopy
Muscle biopsies were fixed in 4% formaldehyde (Sigma-Aldrich, St. Louis, MO), embedded in paraffin blocks, cut into 5-µm sections, and treated for antigen retrieval in 0.1 M citrate buffer (pH 6) by using microwaves for 5 min in four pulses (21) with 5-min cooling in between pulses. Tissue sections were immunostained for cell type-specific markers to identify cell types that were present in vastus lateralis. Satellite cells were identified by their location outside the sarcolemma but inside the basilar lamina membranes that were stained by anti-syndecan-4 antibody (goat polyclonal, sc-9497; Santa Cruz Biotechnology, Santa Cruz, CA), and by anti-CD34 staining (mouse monoclonal, sc7324; Santa Cruz Biotechnology). Expression of CD34 characterizes mesenchymal stem cells in the muscle but is also observed in satellite cells. Syndecan-4 expression is restricted in adult skeletal muscle to cells retaining myogenic capacity (22). The percentage of AR-positive myonuclei and satellite cells was calculated by dividing the sum of AR-positive myonuclei and satellite cells by total number of myonuclei and satellite cells in 100 muscle fibers.
We used anti-platelet endothelial cell adhesion molecule (PECAM) antibodies (mouse monoclonal, ab7385; Research Diagnostics, Flanders, NJ) to identify endothelial cells, anti-smooth muscle 
-actin (mouse monoclonal, sc-8432; Santa Cruz Biotechnology) to identify smooth muscle cells in the blood vessel wall, and antivimentin antibody (mouse monoclonal, sc-6260; Santa Cruz Biotechnology) to identify fibroblasts. The mast cells were identified by toluidine blue staining of the tissue sections embedded in Epon.
After identification of individual cell types, we performed double immunolabeling using mouse monoclonal antibodies against cell type-specific markers and a polyclonal antibody against AR (rabbit polyclonal, N-20, sc-816; Santa Cruz Biotechnology). Staining was performed using appropriate secondary antibodies from goat (pk-6105), mouse (pk-6002), and rabbit (pk-6101) (Vector Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA). Detection kits included sk4800 Vector Novared used for staining CD34 and AR and Vector SG kit sk-4700 for staining syndecan-4. For laser confocal microscopy, we used the fl-2000 kit labeled with fluorescein (green) for detection of mouse monoclonal primary antibodies and the fl-1000 kit labeled with Texas red for rabbit polyclonal antibodies. A laser-scanning confocal microscope (Leica-TCS SP2; Leica, Wetzlar, Germany) with appropriate filters (Leica) was used for analysis. Entire tissue sections were evaluated systematically to identify the distribution of specific antigens inside the muscle fiber and in the interstitial space.
For quantitation of the number of interstitial cells that expressed CD34 and/or AR, tissue sections obtained at baseline and after 20 wk of testosterone treatment were double immunolabeled for CD34 and AR. AR was labeled with Texas Red, and CD34 was labeled with fluorescein. Five interstitial areas in each tissue section were photographed using both green and red filters on a RT Color Spot Digital Camera attached to a Leica LB Fluorescent Microscope. The interstitial cells expressing CD34, AR, or both were counted by using the point counting method (13, 14).
Immunoelectron microscopy
For immunoelectron microscopy, tissues were fixed in a mixture of 1% glutaraldehyde and 1% paraformaldehyde for 2 h and washed in phosphate buffer. Fixed tissue sections were passed sequentially through graded concentrations of ethanol and propylene oxide, infiltrated with Epon, and embedded overnight. We first scanned thick tissue sections by using toluidine blue staining to select appropriate areas for preparation of ultrathin sections that were mounted on nickel grids.
Immunogold labeling
We used immunogold labeling, using goat antirabbit IgG and gold conjugate (40 nm, 150 proteins/particle) obtained from Ted Pella, Inc. (Reading, CA; no. 15729). Antigen retrieval was performed by incubating a thin section (70 nm) of the tissue in sodium citrate buffer (pH 6) for 30 min at room temperature. Tissue sections were blocked in 10% normal horse serum in a humid chamber and incubated with primary antibody for 30 min at room temperature. After several washings, the sections were incubated with gold conjugate (1:10 dilution) for 30 min at room temperature. The unbound gold conjugates were removed by repeated washing, and tissue sections were poststained with 3% uranyl acetate and floated on lead nitrate for 4 min. The sections were dried and examined for regions of gold-conjugated protein aggregation or dense particle distribution.
For semiquantitative analysis of the distribution of AR in satellite cells, myonuclei, and muscle fiber cytoplasm, we counted immunogold particles in five replicates of 10-mm2 areas in each electron micrograph of gold-labeled tissue section. The counts were adjusted for magnification and expressed as number of immunogold particles per millimeter squared area.
Culture of satellite cells derived from human skeletal muscle
Enriched cultures of satellite cells from human skeletal muscle were purchased from Cambrex Laboratories (SkMC, Walkersville, MD) in cryopreserved vials, and cultured using the Cambrex protocol in SkGM culture medium that contained growth factors to prevent the cells from differentiating into myotubes. We added 2% fetal bovine serum to allow faster cell proliferation.
The satellite cells and myoblast cultures were plated in eight-well chambered slides (Nalge Nunc, Naperville, IL) for performing immunohistochemistry either at 30–50% confluence for single cell analysis or grown up to 100% confluence for evaluation of differentiated myotubes. To promote differentiation of satellite cells into myotubes, satellite cell cultures were grown to 95–100% confluence, and then the medium was changed to differentiating medium (Cambrex Inc.) that contained DMEM-F12 (50:50) and 2% horse serum and grown for another 7 to 14 d. Satellite cell cultures of subconfluent cells were stained with anticadherin or anti-C-met antibody (rabbit polyclonal, sc-8370; Santa Cruz Biotechnology) to determine the percentage of satellite cells.
Testosterone at 0 or 100 nM or dihydrotestosterone (DHT) at 0 or 30 nM was added to appropriate cultures of satellite cells and myoblasts. These concentrations were selected to approximate serum testosterone and DHT concentrations that were achieved in healthy men treated with 600 mg testosterone enanthate in our testosterone dose response studies (9). The androgen effects were investigated after incubation of cells with medium alone or with medium containing testosterone or DHT for 1, 3, 6, or 24 h, or for longer time periods, as indicated.
RT-PCR and real-time quantitative PCR for quantitation of AR mRNA
Total RNA was extracted from cells by using the Trizol reagent (Invitrogen, Carlsbad, CA). RT was performed by using 2 µg total RNA and Moloney murine leukemia virus reverse transcriptase at 42 C for 20 min (PerkinElmer, Norwalk, CT). Four-microliter aliquots of cDNA were amplified by PCR using different primer pairs for 36 cycles at 94 C for 30 sec, primer annealing at 58 C for 30 sec, and extension at 72 C for 1 min in a Gene Mate thermocycler (Intermountain Science, Kaysville, UT). The products of PCR were analyzed by electrophoresis in 2% agarose gels and stained with ethidium bromide. The primers for PCR and real-time PCR for human AR were as follows: 187 bp, forward primer Hu-AR-F at locus 2280–2299 on gb, M20132; reverse primer Hu-AR-R at locus 2464–2445; for glyceraldehyde phosphate dehydrogenase (GAPDH), 152 bp, forward primer GAPDH-F at locus 606–626 on µ BC023196; and reverse primer GAPDH-R at locus 758–738.
For real-time RT-PCR analysis of mRNA level, the Qiagen Syber-green PCR reagent set with HotStar Taq DNA polymerase was used (Qiagen, Valencia, CA) using the i-Cycler PCR thermocycler and fluorescent light detector lid (Bio-Rad, Hercules, CA). Samples of cDNA, reverse transcribed as above, were analyzed in quadruplicate wells containing 50-ng equivalents of total RNA template and in parallel wells for GAPDH control using 50 ng RNA template, as described (20). Standard curves for the respective primers were generated by log dilutions of skeletal muscle cell cDNA and pGAPDH plasmid, using serial plasmid DNA dilutions ranging from 1 fg to 1 ng. We used a protocol that included initial melting for 15 min at 95 C, followed by 40 cycles of PCR including melting for 15 sec at 95 C, annealing for 30 sec at 58 C, and elongation for 60 sec at 72 C, followed by a melting curve from 55 to 95 C. The inverse derivative of the melting curves showed sharp peaks for the AR product at 84 C and for GAPDH at 87 C, indicating the correct products. Standard curves were generated by using the i-Cycler (iQ, Bio-Rad) software that plotted cycle threshold vs. log of starting quantity of DNA standard for both AR and GAPDH templates, respectively. The starting quantities of mRNA were computed from these standard curves. The values for quadruplicate samples were averaged, and the ratio of AR to GAPDH average value was calculated and normalized using a value of 100% for the control (0 nM testosterone).
Western blot analysis of AR protein
Cell cultures were washed in PBS, dissolved in protein sample buffer containing 50 mM Tris-HCl (pH 7.8), 0.5% SDS, and a mixture of protease inhibitors. Protein concentrations were determined by the bicinchoninic acid protein assay, and 50-µg aliquots of total protein extract were analyzed by electrophoresis on 7.5% polyacrylamide SDS gels and blotted onto polyvinylidene diflouride membranes. Western immunodetection of proteins was performed in Tris-buffered saline plus Tween 20 (0.1%) buffer as described previously (20, 23). AR (110 kDa) protein was analyzed using rabbit polyclonal anti-AR antibody at 1:500 dilution (Santa Cruz Biotechnology) as our primary antibody and goat antirabbit IgG-HRP as the secondary antibody. The products were visualized using enhanced chemiluminescence reagent (Amersham, Chicago, IL) detected by hypersensitive film (Fuji Films, Tokyo, Japan).
Statistical analysis
Data are mean ± SEM. The distributional characteristics of the data were evaluated to assure that the assumptions of normalcy for parametric tests were met. The values at baseline and during wk 20 were compared by using Student’s t test for paired samples. For AR mRNA levels, values for control and androgen-treated wells were compared using Student’s t test for unpaired samples. P 
0.05 was considered statistically significant.
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Results |
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AR immunohistochemical staining
AR-positive myonuclei and satellite cells were detected by immunohistochemistry using anti-AR antibody (Fig. 1
). Satellite cells were identified by their anatomical location outside the sarcolemma but inside the basilar lamina and by anti-syndecan-4 and anti-CD34 staining. Myonuclei were distinguished from satellite cells by their location inside the sarcolemma. We also used anticadherin and anti-C-met staining in some sections to confirm the identity of satellite cells (not shown). With light microscopy, immunostaining for AR was found predominantly in satellite cells and in the myonuclei of the muscle fibers (Fig. 1A). Although almost all the satellite cells expressed AR protein, only about 50% of the myonuclei on average expressed AR protein. After testosterone administration, the average proportion of myonuclei that was positive for AR increased from 50% to 76% (Table 1). As discussed below, some cell types in the interstitial compartment also expressed AR (Fig. 1B).
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We used double immunostaining for AR and cell type-specific markers to verify the identity of the cell types that expressed AR protein. Thus, AR protein colocalized in nearly all the cells that were identified as satellite cells by immunostaining with CD34 and by their location (Fig. 2A-1). In separate sections, AR protein colocalized in cells that stained positive for syndecan-4 and were identified as satellite cells (not shown). A few cells in the interstitial tissue that stained positively for CD34 and are likely mesenchymal stem cells demonstrated AR expression (Fig. 2A-2). CD34 expression is characteristic of mesenchymal, pluripotent stem cells, although satellite cells also express CD34. In biopsies obtained at baseline, 17 ± 4% of cells in the interstitium were CD34+; approximately two thirds of CD34+ interstitial, mesenchymal, stem cells were AR positive. The percentage of interstitial CD34+ cells that expressed AR was significantly higher in biopsies obtained after testosterone treatment than at baseline (78 ± 2 vs. 62 ± 5% of CD34+ cells; P < 0.005).
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). Similarly, the smooth muscle cells in the blood vessels, identified by their expression of smooth muscle -actin, demonstrated immunostaining for AR protein (Fig. 2C).
Additional cell types within the interstitium of the muscle expressed AR protein. Some interstitial cells that stained positively for vimentin and thus identified as fibroblasts also expressed AR protein (Fig. 2D).
Immunoelectron microscopy
We performed immunogold labeling to determine the intracellular localization of the AR protein. The specificity of immunogold staining was established in part by observations that sections processed without primary antibody showed no immunogold particles (Fig. 3A). Although several cell types in the vastus lateralis muscle demonstrated AR expression to varying degrees, the satellite cells and the myonuclei had higher levels of AR expression (Figs. 3, B and C, and 4, A and B). The nucleoli of satellite cells and myonuclei had a greater degree of aggregation of immunogold particles than other cell types in the muscle. There was a significant increase in AR expression by immunogold staining in sections obtained after testosterone treatment; most of the nucleoli showed aggregation of immunogold particles in biopsy sections obtained after testosterone treatment (Fig. 4C; Table 2). The number of immunogold particles in satellite cells and myonuclei, a marker of AR expression, was significantly higher after testosterone treatment than at baseline (Table 2).
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, A and B).
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Satellite cells in enriched cultures of human skeletal muscle cells were identified by immunohistochemical staining for satellite cell markers such as C-met (Fig. 6A) and CD34 (not shown). Greater than 95% of the skeletal muscle cells in culture expressed C-met and CD34. The identity of the skeletal muscle cells in cultures as satellite cells was further confirmed by demonstrating that these cells when incubated in differentiating conditions formed multinucleated myotubes that expressed myosin heavy chain. Almost all the CD34 and C-met-positive satellite cells in skeletal muscle cell cultures stained strongly for AR protein. Under basal conditions, the immunostaining for AR was localized within the cytoplasm (Fig. 6B), but after testosterone treatment, AR immunostaining was also observed in the nuclei of some satellite cells (Fig. 6C).
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To detect the presence of AR mRNA in human satellite cell cultures, we performed RT-PCR analysis on total RNA extracted from muscle cell cultures enriched for satellite cells. The estimated size of the PCR product of human AR was consistent with the expected DNA product length of 187 bp. The identity of this PCR product as human AR was confirmed by its DNA sequencing; the nucleotide sequence of the reverse-transcribed PCR product was identical to the published cDNA sequence of the human AR (gb: M20132, 24–26). The AR mRNA levels, measured by real-time PCR and normalized to GAPDH, were higher in cells grown in the presence of either testosterone or DHT, which was significantly higher by 70% (P = 0.05), compared with cells treated with medium alone (Fig. 7B); however, the changes in AR mRNA levels were relatively small.
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The AR protein in total cell extracts was identified as a 110-kDa band by Western blot analysis. Treatment of skeletal muscle cells in culture with testosterone led to a 2- to 3-fold higher levels of AR expression than control wells treated with medium alone (Fig. 7A).
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Discussion |
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Several investigators have shown previously that extracts of human skeletal muscle have demonstrable expression of AR (15, 16, 17, 18, 19). Similarly, studies performed in rodents and other experimental animals have provided evidence of AR expression in the skeletal muscle (24, 25, 26, 27, 28, 29, 30, 31, 32, 33); these studies report significant differences in the intensity of AR expression in different muscle groups. In general, more androgen-responsive muscle groups, such as levator ani, express higher levels of AR than less responsive muscle groups, such as gastrocnemius (29, 30). AR expression in the skeletal muscle decreases after castration and is up-regulated by androgen administration (26, 28, 30). Doumit et al. (16) demonstrated the expression of AR in enriched cultures of porcine satellite cells. Others have reported AR expression in muscle fibroblasts and motor endplates (25), although a systematic evaluation of the AR expression in the skeletal muscle has not been performed previously. Our studies constitute the most comprehensive evaluation of AR expression in the human skeletal muscle and provide novel evidence of its expression in multiple mesenchymal precursor cells outside the skeletal muscle fiber. The analysis of the reverse-transcribed cDNA sequence shows that AR mRNA transcripts in vastus lateralis muscle are similar to those expressed in prostate and other human tissues (34, 35, 36). Our in vivo and in vitro data demonstrate that although a number of cell types express AR, muscle satellite cells are the predominant site of AR localization.
Testosterone supplementation increases fat-free mass in healthy, hypogonadal men (4, 5, 6), older men with low testosterone levels (10, 11), and men with chronic illness and low testosterone levels (37, 38). Testosterone supplementation induces hypertrophy of both types I and II skeletal muscle fibers (12). The mechanisms by which androgens induce skeletal muscle hypertrophy are not well understood (3). In pioneering studies, Kochakian (39) and Kenyon et al. (40) reported that testosterone supplementation promotes nitrogen retention in castrated males of many mammalian species, including eunuchoidal men. These early observations of androgen effects on nitrogen retention led to the hypothesis that androgens stimulate protein synthesis. Recent studies provided further evidence that testosterone promotes muscle anabolism by stimulating fractional muscle protein synthesis, inhibiting muscle protein degradation, and by increasing the efficiency of amino acid reuse by the skeletal muscle (4, 12, 41). However, the muscle protein synthesis hypothesis does not explain a number of observed effects of androgen administration on body composition; it cannot easily explain the reciprocal decrease in fat mass and the observed increase in the number of satellite cells and myonuclei during testosterone administration. Recently, we reported that testosterone promotes the commitment of a pluripotent, mesenchymal cell line into the myogenic lineage and inhibits the differentiation of these precursor cells into the adipogenic lineage (20). The hypothesis that androgens regulate body composition by modulating the commitment of pluripotent mesenchymal cells provides a unifying explanation for the reciprocal changes in fat and muscle mass during androgen administration (3); it is consistent with the observed increase in satellite cell number after androgen administration (3). Our observation that mesenchymal precursor cells within the skeletal muscle, including satellite cells, are the predominant site of AR expression supports the hypothesis that mesenchymal precursor cells resident within the skeletal muscle are the target of androgen action.
The demonstration of AR expression in multiple cell types, including fibroblasts, vascular endothelial and smooth muscle cells, and mast cells within the human skeletal muscle is intriguing. Fibroblasts in other tissues have been shown to express AR. Endoneurial fibroblasts of the sciatic nerve show prominent AR immunostaining (42). Although there were only a few mast cells in tissue sections, we noted dense distribution of AR on the metachromatic granules within the mast cells. Mast cells participate in a multitude of inflammatory and immune responses and stimulate local cell proliferation (43, 44); these mast cell reactions might be important in skeletal muscle repair and remodeling in response to injury or hypertrophic stimuli.
The aggregation of the immunogold particles within the nucleoli of satellite cells and myonuclei is a novel finding; testosterone administration was associated with increased nucleolar aggregation of immunogold particles. The nucleoli are the site of processing and partial assembly of the three major RNA classes. We do not know whether nucleolar aggregation of AR is related to androgen stimulation of protein synthesis.
There was a qualitative increase in the density of AR within satellite cells and myonuclei after administration of a supraphysiologic dose of testosterone enanthate. Similarly, in enriched satellite cell cultures, incubation with androgens increased the expression of AR protein, measured by Western blot analysis. The changes in AR mRNA levels, measured by real-time PCR, were very modest. These data are consistent with those of Sheffield-Moore et al. (12) and Kadi et al. (17, 18), who also reported an increase in AR expression in the skeletal muscle biopsies from men receiving androgenic steroids. These studies, including our own, used supraphysiologic doses of testosterone or other anabolic steroids; we do not know whether physiologic replacement doses of testosterone can produce similar up-regulation of AR in the skeletal muscle. In a mesenechymal, pluripotent cell line, C3H 10T1/2, testosterone and DHT up-regulate the expression of AR (20). Speculation that androgens might augment the responsiveness of the skeletal muscle to further androgen administration by up-regulating their own receptor needs further evaluation (12).
We only evaluated the expression of the classical AR; it has been speculated that some actions of androgens might be mediated through nongenomic, membrane binding sites (45, 46, 47). The precise nature of these nongenomic binding sites is not known, although it has been suggested that nongenomic effects might involve G protein signaling (47).
In summary, although multiple cell types within the human skeletal muscle express AR protein, satellite cells, and myonuclei are the predominant sites of AR expression. ARs aggregate within the nucleoli of satellite cells and myonuclei. Testosterone and DHT up-regulate AR expression in vivo and in vitro. These data are consistent with the proposal that androgens induce skeletal muscle hypertrophy by acting at multiple sites within the muscle through multiple mechanisms, including modulation of pluripotent stem cell commitment and differentiation and regulation of muscle protein synthesis; further studies are needed to elucidate the molecular basis of androgen action on human skeletal muscle.
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Footnotes |
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Abbreviations: AR, Androgen receptor; DHT, dihydrotestosterone; GAPDH, glyceraldehyde phosphate dehydrogenase; PECAM, platelet endothelial cell adhesion molecule.
Received January 16, 2004.
Accepted July 21, 2004.
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References |
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