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Anabolic responsiveness of skeletal muscles correlates with androgen receptor protein but not mRNA Douglas A. Monks, Will Kopachik, S. Marc Breedlove, and Cynthia L. Jordan
Abstract: Anabolic effects of androgens on skeletal muscle are well documented, but the physiological and biochemical bases of these effects are poorly understood. Skeletal muscles that differ in their androgen responsiveness can be used to examine these mechanisms. We compared androgen receptor mRNA and protein levels of the rat levator ani, a perineal skeletal muscle that depends on androgens for its normal maintenance and function with that of the rat extensor digitorum longus, a limb muscle that does not require androgens. Western immunoblotting indicated that androgen receptor protein is significantly elevated in the levator ani relative to the extensor digitorum longus. Surprisingly, steady state androgen receptor mRNA levels were equivalent in these muscles, as determined by Northern blot analysis and quantitative RT-PCR. These results suggest that androgen responsiveness of skeletal muscles is determined by the level of androgen receptor protein in a particular muscle and that androgen receptor protein content is regulated by translational or post-translational mechanisms. Key words: spinal nucleus of the bulbocavernosus, levator ani, extensor digitorum longus, testosterone, sexual dimorphism, anabolic steroids. Re´sume´ : Les effets anabolisants des androge`nes sur le muscle squelettique son bien documente´s, mais on comprend mal les bases physiologiques et biochimiques de ces effets. Les muscles squelettiques qui ont une re´activite´ diffe´rente aux androge`nes peuvent eˆtre utilise´s pour examiner ces me´canismes. Nous avons compare´ les niveaux de la prote´ine et de l’ARNm du re´cepteur des androge`nes du muscle releveur de l’anus du rat, un muscle squelettique du pe´rine´e qui de´pend des androge`nes pour son maintien et sa fonction, a` celui du muscle extenseur commun des orteils du rat, un muscle de la jambe qui ne ne´cessite pas d’androge`nes. Le transfert Western a montre´ un niveau e´leve´ de la prote´ine du RA dans le muscle e´le´vateur de l’anus par rapport a` celui du muscle extenseur commun des orteils. Fait surprenant, les niveaux d’ARNm du RA a` l’e´tat stationnaire ont e´te´ e´quivalents dans ces deux muscles, tel qu’indique´ par le transfert Northern et la RT-PCR quantitative. Ces re´sultats donnent a` penser que la re´activite´ des muscles squelettiques aux androge`nes est de´termine´e par le niveau de la prote´ine du re´cepteur des androge`nes dans un muscle particulier et que la teneur en prote´ines du re´cepteur des androge`nes est re´gule´e par des me´canismes translationnels et post-translationnels. Mots cle´s : noyau spinal du muscle bulbo-caverneux, muscle e´le´vateur de l’anus, muscle extenseur commun des orteils, testoste´rone, dimorphisme sexuel, ste´roı¨de anabolisant. [Traduit par la Re´daction]
Introduction Androgens are necessary for the maintenance and function of male-specific muscles that are involved in reproductive Received 9 March 2005. Published on the NRC Research Press Web site at http://cjpp.nrc.ca on 5 April 2006. D.A. Monks.1 Department of Psychology, University of Toronto at Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada. W. Kopachik. Department of Zoology, Michigan State University, East Lansing, MI 48824, USA. S.M. Breedlove and C.L. Jordan. Neuroscience Program and Department of Psychology, Michigan State University, East Lansing, MI 48824, USA. 1Corresponding
author (e-mail:
[email protected]).
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behavior. Motoneurons of the spinal nucleus of the bulbocavernosus (SNB; Breedlove and Arnold 1980) and their targets, the bulbocavernosus (BC) and levator ani (LA) muscles, mediate penile reflexes that are essential for male fertility (Hart 1983; O’Hanlon and Sachs 1986). The adult LA undergoes marked regression following castration, with decreases in wet mass, fiber size, and neuromuscular junction size, unlike the extensor digitorum longus (EDL; Lubischer and Bebinger 1999; Monks et al. 2004). The EDL and LA have similar oxidative and fiber twitch properties (Hanzlikova et al. 1970; Edgerton and Simpson 1971), suggesting that neither of these properties account for the observed differences in androgen responsiveness. We have recently reported that a higher proportion of myonuclei in the LA are androgen receptor-immunoreactive
doi:10.1139/Y05-157
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(AR-IR) than in the EDL (Monks et al. 2004), consistent with AR within skeletal muscles mediating the anabolic actions of androgens on this tissue. The present study tests whether these differences in AR-IR myonuclei are indicative of greater AR protein and mRNA in the LA muscle relative to the EDL.
documentation system (BioRad Laboratories, Hercules, Calif.). AR-IR optical density was normalized for possible loading and transfer differences against actin immunoreactivity for corresponding samples. Paired t tests were used to compare the normalized optical density of AR-IR of LA and EDL muscle protein samples.
Materials and methods
RNA isolation Total RNA was prepared from freshly dissected EDL and LA using Trizol reagent (Invitrogen, San Diego, Calif,) according to the manufacturer’s instructions. RNA concentration was determined by 280/260 nm UV absorption spectophotometry and the integrity of RNA was verified by fluorometric visualization on a denaturing 0.7% agarose gel containing ethidium bromide.
Animals All animals used in these studies were sexually naive Sprague–Dawley (Charles River, St. Constant, Que.) male rats 60–90 days of age group-housed under standard laboratory conditions with food and water available ad libitum. All animal procedures used in these studies were approved by the Michigan State University All University Committee on Animal Use and Care. The EDL and LA muscles were dissected from animals deeply anesthetized with sodium pentobarbital. These muscles were harvested from 15 animals: 6 for experiments using AR immunoblotting, 5 for experiments using quantitative reverse transcriptase polymerase chain reaction (Q RT-PCR), and 4 for experiments using Northern blot analysis. AR immunoblotting Protein was isolated from fresh dissected EDL and LA muscles by homogenizing on ice using a Polytron homogenizer in radioimmunoprecipitation buffer (RIPA: 50 mmol/L Tris–HCl pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA) in the presence of a protease inhibitor cocktail (final concentration 2 mmol/L 4-(2aminoethyl)-bezenesulfonylfluoride HCl (AEBSF), 1 mmol/L EDTA, 130 mmol/L bestatin, 14 mmol/L E-64, 1 mmol/L leupeptin, 0.3 mmol/L aprotinin, (Sigma–Aldrich, St. Louis, Mo.)). Protein concentration of samples was determined using the bicinchoninic acid (BCA) assay (Pierce Biotechnology, Rockford, Ill.). Twenty micrograms of protein from each sample was heat denatured at 60 8C for 10 min, electrophoresed on 8% acrylamide gels and transferred onto nitrocellulose membranes (Schleicher-Schuell Bioscience, Keene, N.H.) using the Novex system (Invitrogen, San Diego, Calif.). Membranes were washed in 0.1 mol/L Tris–HCL (pH 7.4) buffered saline solution (TBS) containing 0.05% Tween 20 and blocked for 1 h in TBS containing 5% dry milk powder, 1% bovine serum albumin (BSA) and 0.05% Tween 20 and then incubated overnight at 4 8C with 1:500 anti-AR polyclonal antiserum N-20 (Santa Cruz Biotechnology, Santa Cruz, Calif.) in TBS containing 1% dry milk powder, 1% BSA and 0.05% Tween 20. Following further rinsing in TBS containing 0.05% Tween 20, membranes were incubated with 1:500 horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Santa Cruz Biotechnology (SCB), Santa Cruz, Calif.) in TBS containing 1% dry milk powder, 1% BSA and 0.05% Tween 20 for 1 h. After washing with TBS, antibody binding was visualized using Luminol reagent (SCB) and XOMAT film (Kodak, Rochester, N.Y.). Blots were stripped and reprobed for actin using a HRP-conjugated anti-actin goat antibody (sc-1616-HRP (SCB); 1/10 000) and similarly visualized. Normalized optical density of the 112 kDa band corresponding to AR was determined using the Quantity One gel
Quantitative reverse transcriptase-polymerase chain reaction To minimize variability between samples, all reactions were carried out by addition of template nucleic acids to aliquots of identical solutions. For reverse transcription, 2 mg of DNase (DNaseI, Invitrogen, San Diego, Calif.)-treated total RNA was heat denatured at 95 8C for 5 min and incubated in a 20-mL reaction with superscript reverse transcriptase (RT – Invitrogen, San Diego, Calif.), poly d(T) primers, deoxyribonucleotides (dNTPs, 10 mmol/L each), first strand buffer (100 mmol/L Tris–HCl, 900 mmol/L KCl and 1 mmol/L MgCl2), and 2.5 mmol/L dithiothreitol. The RT reactions were carried out at 42 8C for 50 min, then heated to 95 8C for 10 min to denature the RT. The resulting cDNA (3.3 mL) was used as a template for PCR. Quantitative PCR was performed using an ABI Prism 7700 Sequence Detection System in conjunction with SYBR green dye to detect the accumulation of double stranded DNA (PCR product). PCR primers were designed using Primer Express software (Applied Biosystems, Foster City, Calif.). Primers for AR were F = 5’-GGCACCTTTCTCAAGAGTTTGG-3’ and R = 5’-GAAGAGTAGCAGTGCTTTCATGCA-3’. These sequences correspond to nucleotides 3305–3326 (F) and 3381–3358 (R) of a rat AR cDNA clone (GenBank acc. No. M20133). Primers for a actin were F = 5’-CTGGTG AAGGCTGGCTTTG-3’ and R = 5’-GGCGACCCACGAT GGA-3’. These sequences correspond to nucleotides 130– 148 (F) and 196–181 (R) of a rat a actin cDNA clone (GenBank acc. No. X80130.1). The specificity of primer sets was verified by visualization of quantitative PCR products on an agarose gel containing ethidium bromide, as well as by sequencing of quantitative PCR products. Each 50-mL quantitative PCR reaction contained the following components: 20 mmol/L Tris–HCl (pH 8.0), 50 mmol/L KCl, 3 mmol/L MgCl2, 1 mmol/L dNTPs (Invitrogen, San Diego, Calif.), SYBR green (1:80000, Sigma–Aldrich, St. Louis, Mo.), 6-ROX (25 ug/uL, Sigma–Aldrich), 5 U Platinum Taq polymerase (Invitrogen), 6.25 pmol/L of appropriate primers, 3 mL RT reaction. PCR conditions were as follows: following an initial denaturing step of 95 8C for 10 min, 40 cycles of 95 8C for 15 s followed by 60 8C for 1 min were performed. SYBR green fluorescence was measured at the end of every cycle. Other variables affecting fluorescence were controlled for by the inclusion of a passive dye reference (ROX-6, #
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Sigma–Aldrich). The threshold cycle number (CT) was defined as the cycle when SYBR green fluorescence was greater than 2 standard deviations from a baseline value established during cycles 5–15. Northern analysis Northern analysis of 20 ug of total RNA from EDL and LA muscles was performed essentially as previously described (Monks et al. 2001), with the following exceptions: an EcoRI/HindIII fragment corresponding to the steroid binding and DNA binding domains of the full-length rat AR cDNA (kind gift of E.Wilson, University of North Carolina, Chapel Hill, N.C.) was used in a PCR reaction to make an AR probe that included a[32P]dCTP (Amersham, Piscataway, N.J.) and the blots were washed in 2 SSC, 0.1% SDS for 20 min at 42 8C, then in 0.5 SSC, 0.1% SDS for 20 min at 42 8C, and finally in 0.1 SSC, 0.1% SDS for 20 min at 42 8C. Autoradiograms were photographed and the optical density of bands was determined using using a Gel Doc system equipped with Quantity One software (BioRad Laboratories, Hercules, Calif.).
275 Fig. 1. Western blot analysis indicates that androgen receptor protein is more abundant in the levator ani (LA) relative to the extensor digitorum longus (EDL). (a) Immunoblot of matched extensor digitorum longus (E) and levator ani (L) muscle samples from each of 6 rats (animal 1 through 6). The blot was probed for androgen receptor (AR, top) that was observed at an apparent molecular mass of 112 kDa then stripped and reprobed for actin (bottom), which was observed at an apparent molecular mass of 43 kDa. (b) Bar graph summarizing normalized measurements of AR immunoreactivity in the above immunoblot. Bars represent the mean + SE optical density for AR immunoreactivity for EDL and LA samples normalized to actin immunoreactivity. AR protein is more abundant in the LA relative to the EDL. *, p < 0.05 was calculated using a paired t test statistic comparing normalized optical density for AR immunoreactivity between the EDL and LA (n = 6 rats per muscle).
Results The LA has a higher concentration of AR protein than does the EDL A major band with a molecular mass of approximately 112 kDa corresponding to full-length AR was observed in both EDL and LA muscle (Fig. 1). Significantly more ARIR protein was detected in homogenates of LA muscle relative to homogenates of EDL muscle (Fig. 1, p < 0.0001). Note that the LA contained more AR than the EDL for all 6 animals, including even the 1 animal (No. 6) in which AR levels was considerably less overall. In no case did it appear based on actin-IR to reflect differences in protein loading (Fig. 1). This result is consistent with observed differences in AR-IR protein in LA and plantaris muscle homogenates (Antonio et al. 1999) and with differences in the proportion of AR-IR nuclei within the muscle fibers of EDL and LA (Monks et al. 2004). The androgen-responsive LA therefore appears to contain considerably more AR protein than other skeletal muscles that are relatively unresponsive to androgen. LA and EDL AR mRNA levels are equivalent Quantitative PCR was performed to estimate AR mRNA abundance relative to a-actin, an internal control. No difference in AR mRNA was observed between EDL and LA muscles (Fig. 2, p = 0.58). This result was unexpected in light of the greater AR protein content (Fig. 1), cytosolic androgen binding (Rance and Max 1984), and AR immunoreactivity (Monks et al. 2004) in the LA vs. the EDL. To further evaluate mRNA levels in these muscles, we performed Northern blot analysis using mRNA samples from LA and EDL muscles of different animals. Abundance of AR mRNA was estimated by measuring the intensity of the AR band and normalizing this to GAPDH band intensity (Fig. 3). Again, no statistically significant difference in normalized AR mRNA level was observed between EDL and LA muscles (Fig. 3, p = 0.47). As these 2 independent methods of estimating LA and EDL mRNA
abundance agree, we conclude that steady-state AR mRNA is equivalent between these 2 muscles.
Discussion Prior to cloning the AR gene (Tan et al. 1988), it was known that the rat LA accumulates radiolabeled androgen about 4-fold more than does the EDL (Rance and Max 1984). Development of specific antisera to AR (Prins et al. 1991) allowed measurement of AR expression in muscle based on Western blotting and immunohistochemistry. Immunohistochemical data indicate that whereas several cell #
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276 Fig. 2. Quantitative RT-PCR indicates equivalent androgen receptor mRNA abundance between the extensor digitorum longus (EDL) and levator ani (LA). The mean + SE cycles of PCR amplification required to increase the intensity of SYBR green fluorescence above threshold (CT). The CT for androgen receptor was normalized to the CT for a-actin to control for template RNA abundance and reverse transcription efficiency. We observed no statistically significant differences in androgen receptor mRNA abundance between the (EDL) and LA muscles using quantitative RT-PCR (n = 5 rats per group, p = 0.58).
types express ARs in both the LA and EDL including muscle fibers, fibroblasts and endothelial cells (Monks et al. 2004), the major difference in AR expression between these muscles appears to lie within the muscle fibers themselves. The percentage of AR-IR myonuclei in the LA is more than 10 times greater than in the EDL, whereas both muscles contain the same proportion (62%) of AR-IR fibroblasts. These data suggest that muscle fibers are likely the major contributor to overall differences in AR protein levels in the LA vs. the EDL muscle. The present data also show that AR mRNA levels are equivalent between the LA and EDL, suggesting that differences in the amount of AR protein between these muscles may involve differences in translational efficiency and (or) AR protein turnover. Non-coordinate regulation of AR protein and mRNA is not without precendence. For example, neonatal estrogen exposure decreases AR protein in the rat ventral prostate without decreasing AR mRNA levels (Woodham et al. 2003). This estrogen treatment also increases proteosome-mediated proteolysis of AR, suggesting that the selective reduction of AR protein occurs via this mechanism. Changes in AR protein may also result from differences in efficiency of translation of steady state mRNA. For example, androgens increase AR protein but not AR transcript in the prostate of castrates by inducing a rapid accumulation of AR mRNA in polyribosomes (Mora et al. 1996; Mora and Mahesh 1999). Finally, the AR, like other steroid receptors, is a target for numerous kinases that can also affect AR stability (Blok et al. 1996; Lin et al. 2002), raising the possibility that posttranslational modifications of AR protein that affect its stability could account for differences in AR protein between the EDL and LA. Given that the LA and EDL come from the same animal and therefore experience the same hormonal milieu, it seems reasonable to expect that there are inherent differences between these 2 muscles in key proteins that critically regulate the amount of AR protein post-
Can. J. Physiol. Pharmacol. Vol. 84, 2006 Fig. 3. Northern analysis indicates equivalent androgen receptor mRNA abundance between the extensor digitorum longus (EDL) and levator ani (LA). (a) Top panel: Northern analysis of total RNA isolated from matched EDL and LA muscles using a radiolabled rat androgen receptor (AR) cDNA probe. Middle panel: The membrane used for AR mRNA analysis was stripped and reprobed with a radiolabeled rat glyceraldehyde 3’ phosphate dehydrogenase (GAPDH) cDNA probe to estimate RNA loading. (b) The mean + SE optical density of AR hybridization normalized to the optical density of GAPDH hybridization to RNA from matched EDL and LA muscle samples. No differences in AR mRNA abundance were observed using Northern analysis (n = 4 rats per group; p = 0.47).
transcriptionally. Identifying which mechanisms contribute to such differences in AR protein will undoubtedly add to our understanding of the molecular basis of androgen responsiveness.
Acknowledgements The authors thank Laura L. Baum and David C. Jensen for assistance with the Western immunoblotting. These studies were funded by operating grant NIH R01 NS045195 (CJ), a postdoctoral fellowship (D.A.M.) from the Canadian #
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Institutes of Health Research (CIHR) and a Strategic Partnership Grant from the MSU Foundation.
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