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Endocrinology, doi:10.1210/en.2008-0664
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Endocrinology Vol. 149, No. 11 5449-5460
Copyright © 2008 by The Endocrine Society

Expression of the Muscle Atrophy Factor Muscle Atrophy F-Box Is Suppressed by Testosterone

Weidong Zhao, Jiangping Pan, Xiaolei Wang, Yong Wu, William A. Bauman and Christopher P. Cardozo

Center of Excellence for the Medical Consequences of SCI (W.Z., J.P., X.W., Y.W., W.A.B., C.P.C.), James J. Peters Veterans Affairs Medical Center, Bronx, New York 10468; and Department of Medicine (W.A.B., C.P.C.), Mount Sinai School of Medicine, New York, New York 10029

Address all correspondence and requests for reprints to: Dr. Christopher Cardozo, James J. Peters Veterans Affairs Medical Center, Room 1E-02, Bronx, New York 10468. E-mail: chris.cardozo{at}mssm.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The ubiquitin ligase muscle atrophy F-box (MAFbx; also called atrogin-1) is thought to play important roles in muscle loss. Conversely, testosterone reduces atrophy from glucocorticoids or denervation associated with repression of MAFbx. To characterize mechanisms of such repression, the effects of testosterone on MAFbx expression in C2C12 cells were tested. Testosterone reduced MAFbx mRNA levels as well as expression of a reporter gene under the control of 3.1 kb of the human MAFbx promoter. Repression required the androgen receptor (AR) as well as sequences within the first 208 bases upstream of the first codon of the MAFbx gene. This sequence is downstream of known forkhead transcription factor binding sites and testosterone did not alter Forkhead box O 3A phosphorylation. The AR associated with sequences conferring repression in a manner that was stimulated by testosterone and was independent of DNA binding. In gel shift studies, octamer binding transcription factor (Oct)-1 bound two predicted Oct-1 sites within these sequences. Deletion of Oct-1 sites from reporter genes prevented repression by testosterone. Gene knockdown of Oct-1 blocked repression of MAFbx reporter gene activity by testosterone and binding of AR to sequences conferring repression. In conclusion, testosterone represses MAFbx expression via interactions of the AR with Oct-1 that are associated with sequences within the 5' untranslated region of the MAFbx promotor located just upstream of the first codon. This action of testosterone may contribute to beneficial actions of testosterone on muscle.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MUSCLE ATROPHY CAN be caused by diverse medical conditions and results from accelerated catabolism of muscle proteins (1, 2, 3). The muscle-specific ubiquitin ligase muscle atrophy F-box (MAFbx; also called atrogin-1) is up-regulated in conditions associated with muscle loss (4, 5, 6). Overexpression of MAFbx reduces the size of cultured muscle cell lines, suggesting accelerated protein catabolism, whereas disruption of the MAFbx gene slows muscle loss caused by nerve transection (4). For these reasons, the expression of MAFbx is one factor that is believed to determine the extent of muscle loss.

The MAFbx gene is expressed exclusively in heart and skeletal muscle (4), apparently through the action of tissue-specific factors on the promotor (7). The human MAFbx promotor contains a transcriptional start site located 340 bp upstream of the first codon (7). Transcription of the human MAFbx is strongly increased by sequences located in the first 200 bp upstream of the first codon (7) and is greatly up-regulated by glucocorticoids (4).

One mechanism for up-regulation of MAFbx is activation of the forkhead family transcription factor Forkhead box O (FOXO)-3A (8). The human MAFbx promotor contains two binding sites for forkhead transcription factors that are located just downstream of the transcriptional start site, and full activation of MAFbx expression by glucocorticoids requires only the core promotor and downstream sequences that include these sites (7). Conversely, IGF-I represses dexamethasone-induced increases in expression of MAFbx by activation of the phosphatidylinositol 3-kinase/Akt pathway, resulting in phosphorylation and inactivation of forkhead family member FOXO1 (9, 10).

Administration of testosterone increases muscle mass in elderly men (11, 12) or men administered glucocorticoids (13, 14) as well as preserves muscle mass in burn patients (15). These studies have also suggested, through analyses using C13-labeled amino acids, that increases in muscle mass were attributable to reduced rates of protein catabolism (11, 12, 15). One possibility raised by these findings is that the effect of testosterone to reduce muscle breakdown could be attributable, in part, to suppression of the expression of genes encoding factors that control muscle protein catabolism, such as MAFbx. This possibility is supported by findings that testosterone prevents dexamethasone-induced muscle atrophy and up-regulation of MAFbx in rats and inhibits dexamethasone-induced protein catabolism and MAFbx expression in C2C12 myotubes (16). Additionally, the anabolic steroid nandrolone slows denervation atrophy associated with repression of MAFbx (17). Mechanisms underlying repression are not known.

Most actions of testosterone are mediated by binding of this hormone to the androgen receptor (AR), which is a member of the steroid hormone receptor subfamily of nuclear receptor superfamily (18, 19). The AR is a transcription factor that is activated by binding of hormone. It has a modular structure common to all steroid hormone receptors that includes a transactivating domain, a DNA-binding domain (DBD), and a ligand-binding domain (LBD) responsible for specific binding of the cognate hormones. After activation, the AR is capable of binding to specific DNA sequences termed androgen response elements (AREs) (18, 19). Once bound, the AR interacts with coactivators (20) and other transcription factors (21) to up-regulate transcription. Repression of gene expression by the AR, however, has thus far been found to be independent of binding of the AR to AREs and shown to involve interactions between the AR and other transcriptional regulators. The AR has been found to repress transcription via interactions with nuclear factor-{kappa}B, activator protein-1 (AP-1), octamer binding transcription factor (Oct)-1 and avian erythroblastosis virus E2 oncogene homolog 1 (Ets-1) as well as cause repression of transcription by competition for coactivators (22, 23, 24, 25, 26). The goal of the current studies was to characterize the molecular mechanisms by which testosterone represses MAFbx expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Testosterone and IGF-I were from Sigma-Aldrich (St. Louis, MO).

Plasmids
The reporter genes pMAF3.1, pMAF2.4, pMAF948, pMAF400, and pMAF400–241 express firefly luciferase under the control of proximal promotor regions of the human MAFbx gene and were described previously (7). The rARC562G vector, encoding rat AR with a C562->G mutation in the first zinc binding module that disrupts binding of AR to DNA (27), was a generous gift of Dr. Jorma Palvimo (University of Kuopio, Kuopio, Finland). pAR.4RKM (expressing AR with 4 R/K->M mutations in the nuclear localization sequence that block nuclear entry) and pAR.ABC (incorporating exons A–C of the human AR gene but lacking the LBD) were generous gifts of Dr. Elizabeth Wilson (University of North Carolina, Chapel, NC) (28). The pBabe.puro retroviral backbone (29) with a Not1 site introduced into the polylinker was a generous gift of Dr. Robert Kraus (Mount Sinai School of Medicine, New York, NY). The ARE reporter gene expressing firefly luciferase under control of the mouse mammary tumor virus (MMTV) hormone response element, and pSP72hAR-1, containing an insert of the full-length cDNA clone of the human AR, were gifts from Dr. Diane Robins (University of Michigan, Ann Arbor, MI). pRC/CMV Oct-1, which expresses a human full-lengthOct-1 protein, was from Dr. Neil Segil (Keck School of Medicine, University of Southern California, Los Angeles, CA) (30). pGEX 4T-1 hAR2, a vector expressing glutathione-S-transferase fused to the N terminus of the AR DBD (31), was a generous gift from Dr. Robert J. Matusik (Vanderbilt University Medical Center, Nashville, TN). pRL-CMV expressing Renilla luciferase was from Promega (Madison, WI).

Deletion of the two Oct-1 binding sites from pMAF400 was carried out using the Phusion site-direct mutagenesis kit (Promega) as recommended by the manufacturer with the following PCR conditions: initial denaturation 98 C of 30 sec x one cycle; amplification cycles of 98 C of 10 sec, 70 C of 30 sec, 72 C of 30 sec. The final extension was 72 C of 10 min x one cycle. The upstream Oct-1 site was deleted to yield pMAF400{Delta}1 using the following primers:

Forward primer was 5'-GTCGCGGGCCCCTGCACCCCGAGCATCC-3', reverse 5'-GAACGGCGCGGGGCACCCTGCGGGGTG-3'. The second (downstream) Oct-1 site was deleted from pMAF400{Delta}1 to yield pMAF400{Delta}1/2 by use of the following primers: forward, 5'-CGTCTCCCCATCCGTCTAGTC-3', reverse, 5'-GGGACGTGCCACCCGGGGCGGATGCTC-3'. Synthesis, purification, and 5' phosphorylation of primers was performed by IDT Inc. (Coralville, IA). Constructs were verified by DNA sequencing.

Cell culture and transfection
C2C12 cells (American Type Culture Collection, Manassas, VA) were maintained in DMEM containing 10% fetal bovine serum (FBS) and penicillin (100 U/ml)/streptomycin (100 µg/ml) (growth medium). Cells were incubated at 37 C in humidified air containing 10% CO2. 293T cells (American Type Culture Collection) were maintained in growth medium as above. Transfection was achieved using Lipofectamine-Plus (Invitrogen, Carlsbad, CA) using 100 ng total DNA per square centimeter of the growth plate or well.

Primary human skeletal muscle myoblasts
Primary cultures of human skeletal muscle myoblasts (Lonza, Walkersville, MD) were maintained following the manufacturer’s recommended procedures. Cells were passaged once and frozen. Frozen aliquots were thawed then seeded into 100-mm plates and then incubated for 5 d according to the manufacture’s recommended procedures, including addition of dexamethasone (1 µM) to the culture media. Cells (myoblasts) were then covered with fresh growth medium lacking dexamethasone and supplemented with testosterone (500 nM) and incubated overnight. Cells were then processed for chromatin immunoprecipitation.

Retrovirally transformed C2C12 cell lines
Assessment of repression of MAFbx expression by testosterone was dependent on the availability of muscle cell lines that express functional levels of the AR. The ability of testosterone to induce luciferase expression in C2C12 myoblasts transfected with a reporter gene containing the MMTV hormone response element was tested. Testosterone induced negligible increases in luciferase expression compared with ethanol (supplemental data Fig. 1A, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).

To facilitate gene transfer, a retroviral backbone for expressing human AR was constructed by ligating the BglII-XbaI fragment of pSP72hAR-1, including the complete coding region for the human AR, into the BamHI and HindIII sites of the pBabe.puro vector. This construct was cotransfected together with vectors expressing gag-pol and VSV-G into 293T cells. The supernatants were harvested and the cell debris was removed by centrifugation at 500 x g. The supernatant was used to infect the wild-type C2C12 cells after addition of polybrene (4 µg/ml). After 24 h, puromycin (1600 ng/ml) was added to the culture medium. The selected cells are referred to as C2C12.AR cells. To allow us to control for nonspecific effects of the retroviral transformation, an additional cell line was prepared by transforming C2C12 cells with virus assembled using the pBabe.puro backbone without insert and is referred to as C2C12.pBabe.

When C2C12.AR cells were transfected with the MMTV-Luc reporter, testosterone caused a marked increase in luciferase (supplemental data Fig. 1A). Analysis of total RNA from these cells by Northern blotting revealed the presence of human AR mRNA (supplemental data Fig. 1B), and Western blotting confirmed that AR protein is expressed in C2C12.AR cells but not wild-type C2C12 cells (supplemental data Fig. 1C).

Luciferase assays
Cells were seeded into wells of 24-well plates (5 x 105 cells/well) and then maintained overnight in DMEM with 10% charcoal-dextran-stripped (CDS)-FBS. C2C12 cells proliferate as a myoblast and can be induced to differentiate into multinucleated myotubes by incubation in media containing 2% horse serum (32). For experiments with C2C12 myoblasts (undifferentiated form), cells were cotransfected with reporter genes (see figures) and pRL-CMV. Cells were incubated in the transfection mixture overnight. Where expression in myoblasts was studied, cells were then covered with DMEM supplemented with 10% CDS-FBS and either ethanol (EtOH) or hormones dissolved in EtOH (see figure legends). Cells were maintained for an additional 24 h, at which time both firefly and renilla luciferase activity were determined. For experiments in which cells were differentiated to form myotubes, transfection was performed as above, followed by incubation of the cells in medium supplemented with 2% horse serum to induce differentiation for 48 h. Medium was then supplemented with testosterone or vehicle and cells were incubated overnight. Luciferase activity was then determined. Luciferase activity in cell lysates was measured using the dual luciferase assay (Promega) and a Berthold 96-well plate luminometer. EtOH concentrations were less than 0.1%. Solutions containing testosterone were prepared freshly on the day of the experiment because loss of activity was observed with storage of testosterone in EtOH solutions. Firefly luciferase activities were normalized relative to renilla luciferase. Luciferase activities were then normalized relative to mean values for EtOH-treated cells.

Effects of testosterone on mRNA levels and half-life
Incubation of cells and extraction of RNA.
Cells were seeded into wells of 6-well plates at 3 x 106 per well in DMEM supplemented with 10% CDS-FBS and incubated overnight in this medium. Cells were then incubated for 48 h in DMEM containing 2% horse serum to induce differentiation. To test effects of testosterone on mRNA levels, testosterone or EtOH was added and cells were incubated overnight. For determination of mRNA half-life, medium was supplemented with actinomycin (5 µg/ml) at various times before processing for isolation of total RNA. For RNA isolation, cells were disrupted with Qiashredder columns (QIAGEN, Valencia, CA). Total RNA was extracted from cultured cells using RNAeasy columns (QIAGEN) after digestion on the column of residual genomic DNA with ribonuclease-free deoxyribonuclease (DNase; QIAGEN) and eluted with water.

Real-time PCR (qPCR)
RNA was quantified by absorbance at 260 nm. One microgram of total RNA was used to prepare cDNA libraries using the high capacity cDNA archive kit (Applied Biosystems, Foster City, CA). Libraries were diluted 25-fold with water. Real-time PCR was performed using Taqman 2x PCR buffer (Applied Biosystems) and an Applied Biosystems 7500 thermocycler using inventoried Taqman Assay on Demand probes (Applied Biosystems). Data were normalized relative to that for 18S RNA. Real-time PCR assays were performed in triplicate, and mean values for the crossing point of these replicates were used for subsequent calculations. of relative expression level of MAFbx mRNA using the 2-{Delta}{Delta}Ct method (33) in which expression is presented as fold change relative to a control. For experiments testing effects, testosterone on MAFbx mRNA levels to the control group was the vehicle-treated cells. For mRNA half-life studies, of the control group was mRNA levels at time zero.

Western blotting
C2C12 cells (2 x 106) were seeded per plate in 100-mm tissue culture plates. The following day cells were transfected with the indicated expression vectors (1 µg/plate) as above. The following day, media were removed and cells were covered with 500 µl of cell lysis buffer (no. 9803; Cell Signaling, Danvers, MA) containing 5 µl/ml of protease inhibitor cocktail (no. 8340; Sigma-Aldrich). Cells were lysed by 20 passages through a 26-gauge needle. Lysates were cleared by centrifugation, and protein concentrations of the supernatant fractions were determined with the Bio-Rad protein assay (Bio-Rad, Hercules, CA) using BSA as a standard. Proteins (50 µg/lane) were resolved by SDS-PAGE on 8% gels and then transferred onto polyvinyl difluoride membranes. Membranes were blocked with 5% milk. Primary antibodies were 1:1000 dilutions of: anti-AR antibodies recognizing an epitope at the N terminus (no. sc-816; Santa Cruz Biotechnology Inc., Santa Cruz, CA); total FOXO3A (Upstate, Charlottesville, VA; catalog no. 06-951); antiphospho-FKHKL/FOXO3A Thr32 (Upstate; catalog no. 07-695); or anti-β-actin (Sigma Chemical Co., St. Louis, MO); the secondary antibody was a 1:5000 dilution of horseradish peroxidase-conjugated antirabbit IgG (MP Biomedical, Solon, OH). Immunostaining was visualized by enhanced chemiluminescence and captured on photographic film. The densities of protein bands were measured by using an AlphaImager 2200 Gel Doc system (Alpha Innotech, San Leandro, CA).

DNase I footprint assay
A human MAFbx promoter fragment, spanning –311 and –74 bp, was generated from pMAF400 by PMLI and HindIII digestion (New England Biolabs, Ipswitch, MA). The fragment was labeled by polynucleotide kinase (New England Biolabs) with {gamma}32P ATP (MP Biomedical) followed by removal of the 3' end by digestion with EcoRI. The gel purified probe (30,000 cpm) was incubated with either 50 µg of BSA or nuclear extract from C2C12.AR cells obtained using the NE-PER kit (Pierce Biotechnology, Rockford, IL) in a binding buffer [25 mM Tris (pH 8.0), 50 mM NaCl, 6 mM MgCl2, 1 mM dithiothreitol] on ice for 10 min. Samples were treated with 0.15 or 1.5 U RQ1-DNase I (Promega) in a total reaction volume of 100 µl, and the digestion was terminated after 1 min by adding 90 µl of 200 mM NaCl, 30 mM EDTA, 1% sodium dodecyl sulfate, and 100 µg/ml yeast RNA. DNA fragments were phenol/chloroform extracted, ethanol precipitated, and separated by electrophoresis on a 6% polyacrylamide sequencing gel in 1x Tris-borate EDTA buffer. The sequence of the protected region was determined by alignment with a sequencing reaction using a Sequenase version 2.0 DNA sequencing kit (United States Biochemical, Cleveland OH) and the control primer and DNA.

In vitro translation of Oct-1
pRC/CMV Oct-1, which expresses a human full-length Oct-1 protein, was linearized by digestion with ApaI. In vitro transcription was performed using T7 riboMax large-scale RNA production kit; in vitro translation was performed using rabbit reticulocyte lysates (both from Promega). These procedures were carried out according to the manufacturer’s recommendations.

EMSAs
Oligonucleotides were: Oct-1 site A forward, 5'-CCG CCG CCC CGC CGC CCC CGT CGC G-3', reverse, 5'-CGG GGT GCA GGG GCC CGC GAC GGG-3'; Oct-1 site B forward, 5'-GAG CCC ACC AGG CCG GCC CCG TCT CCC-3', reverse, 5'-GAC TAG ACG GAT GGG GAG ACG GGG-3'. Oligonucleotides were labeled with {alpha}32P-dCTP by filling in the ends using the Klenow fragment of Escherichia coli DNA polymerase (New England Biolabs) in the presence of deoxynucleotide triphosphates at 37 C for 30 min. Probes were purified using ProbeQuant G-50 microcolumns (Amersham Biosciences, Piscataway, NJ) and eluted from the columns with water. Protein-DNA complexes were formed by incubation of recombinant protein with 1 pmol of 32P-labeled probe, and 2 µg of polydeoxyinosine-deoxycytosine in binding buffer [5% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl (150 mM for Oct-1 site B, 50 mM for Oct-1 site B), 10 mM Tris-HCl (pH 7.5)] at 4 C for 30 min. Complexes were resolved by nondenaturing electrophoresis for 10 min at 350 V with cooling on 5% polyacrylamide minigels containing 0.5x Tris-glycine EDTA buffer and visualized by phosphor imaging.

Chromatin immunoprecipitation (ChIP)
C2C12.AR cells were seeded into 100-mm plates (5 x 106/plate). The following day, cells were transfected with reporter genes (either pMAF400 or pMAF400–241). One day later, medium was replaced with differentiation medium and cells were maintained in this medium for 48 h at which time testosterone (500 nM) was added. ChIP was performed after overnight incubation using the ChIP assay kit from Upstate according to the manufacturer’s instructions. Briefly, C2C12 or C2C12.AR cells were cultured in 100-mm tissue culture plates and transfected with pMAF400 or pMAF400–241. After 24 h, cells were cross-linked with 1% formaldehyde at 37 C for 10 min and washed with cold 1x PBS. Cells were covered with lysis buffer with 1% protease inhibitor cocktail (Sigma), sonicated, and centrifuged at 14,000 rpm for 10 min. Fifty microliters of the total cell lysate were used for the positive control DNA extraction (input DNA). The supernatant was diluted with ChIP dilution buffer and precleared with salmon sperm DNA/protein A agarose slurry for 30 min at 4 C. Immunoprecipitation was carried out by incubating precleared cell lysates with 5 µl of anti-AR or anti-Oct-1 (Santa Cruz Biotechnology) antiserum or with normal rabbit serum; incubations were continued overnight at 4 C. Immune complexes were precipitated with 60 µl of protein A-Sepharose for 1 h at 4 C and washed. Cross-linking was reversed by 4 h at 65 C in 200 mM NaCl. DNA was isolated from the immune complexes by proteinase K (Invitrogen) digestion, phenol-chloroform extraction, and ethanol precipitation and dissolved in 20 µl of water. Two microliters of DNA were subjected to PCR amplification. Primers corresponding to the promoter region of human MAFbx are: pair A (for pMAF 400–241), 5'-CAGCACCGCTTCAAGTTT-3' (upstream) and 5'-AGCTCTTTGTTGCCGGAAG-3; pair B (for all others), 5'-ACAAAGAGCTGCGGCCGGCT-3' (upstream) and 5'-GATGCTCGGGGTGCAGGG-3'. For PCR, 35 cycles were performed with an annealing temperature of 60 C with platinum Taq DNA polymerase (Invitrogen). Intensities of bands corresponding to PCR products was quantified using Imagequant TL (GE Life Sciences, Piscataway, NJ) and was normalized relative to that for the PCR product for input DNA.

Statistics
Data are expressed as mean values ± SEM. Statistical analysis was performed using Prism 4.0c software (GraphPad Software, San Diego, CA). Comparisons between two means were performed using a Student’s t test. Comparisons among multiple means were performed using one-way ANOVA with a Newman-Keuhls test post hoc to determine significance of differences between specific means. A P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To determine whether testosterone reduced MAFbx mRNA levels, C2C12.AR myotubes were incubated overnight with this hormone. Testosterone (50 nM) significantly reduced MAFbx mRNA levels (Fig. 1AGo). To assess possible effects of testosterone on MAFbx mRNA stability, the half-life of MAFbx mRNA was determined in C2C12 myoblasts (Fig. 1BGo). MAFbx mRNA half-life was not significantly altered by testosterone in C2C12 myoblasts. The half-lives for cells incubated with EtOH or testosterone were 0.86 and 1.46 h, respectively. These data suggest that testosterone reduced transcription of the MAFbx gene. To for this possibility, we determined whether testosterone inhibited activity of pMAF3.1, a reporter gene expressing firefly luciferase under the control of 3.1 kb of proximal promotor from the human MAFbx gene (Fig. 2AGo) (7). Testosterone significantly reduced reporter gene activity in C2C12.AR myoblasts (undifferentiated) and myotubes (differentiated) at concentrations from 5 to 500 nM (Fig. 2Go, B and C). The apparent increase in repression with higher testosterone concentrations was not significant. Testosterone had no effect on reporter gene activity in cells lacking the AR, specifically wild-type C2C12 cells or C2C12.pBabe, which are cells transduced with retrovirus lacking an insert (Fig. 2Go, D–G).


Figure 1
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  		FIG. 1. Testosterone reduces MAFbx mRNA levels in C2C12 cells through transcriptional repression. A, C2C12.AR myotubes were incubated overnight with testosterone (50 nM) or EtOH, at which time MAFbx mRNA expression was assessed by qPCR. Data are mean values ± SEM for seven different experiments, each performed in duplicate. *, P < 0.0001, t test. B, C2C12.AR myoblasts were incubated overnight in growth medium supplemented with testosterone (Test) or vehicle (EtOH). Actinomycin was added to media and cells were harvested at indicated times thereafter for determination of MAFbx mRNA levels by qPCR. Levels of MAFbx mRNA are expressed as a percentage of those at time zero on a logarithmic scale. Data are for three separate experiments. Each determination was performed in triplicate. Half-life of MAFbx mRNA in cells incubated with EtOH and testosterone did not differ (P = 0.22, F test).

 

Figure 2
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  		FIG. 2. Testosterone reduces activity of the pMAF3.1 reporter gene in a mechanism dependent on the AR. A, Map of the pMAF3.1 reporter gene (further described in Materials and Methods). The arrowhead indicates the transcriptional start site. B–G, Effects of testosterone on reporter gene activity in C2C12 line transduced with a retrovirus expressing the AR (C2C12.AR), with an empty retrovirus (C2C12.pBabe), or in uninfected C2C12 cells (C2C12 WT). Myoblast lines were transfected with pMAF3.1. Transfected myoblasts were incubated overnight with testosterone at the concentrations indicated or differentiated for 48 h and then incubated overnight with this hormone. Data are normalized relative to mean luciferase values for cells incubated with EtOH. Results are means ± SEM for at least three different experiments, each in at least triplicate. *, P < 0.05 vs. EtOH (ANOVA).

 
To gain further insight into the mechanism of repression, experiments were repeated in wild-type C2C12 cells transiently transfected with the pMAF3.1 reporter and vectors expressing wild-type AR or mutant ARs deficient in specific AR functions, the structures of which are shown in Fig. 3AGo. Western blotting confirmed that each of the mutants was expressed in C2C12 cells (Fig. 3BGo). Testosterone significantly depressed reporter gene activity in C2C12 myoblasts that were transiently transfected with wild-type AR (Fig. 3CGo). No repression was observed in cells transfected with an AR defective in nuclear entry (4RKM) (28). Repression was also absent in cells expressing a mutant AR lacking the LBD but able to enter the nucleus and bind chromatin (28). Significant repression was also observed when cells were transfected with an AR containing a mutation in the DBD that is unable to bind chromatin (C562G) (27), although the magnitude of repression was reduced, compared with that for wild-type AR (P < 0.005, t test).


Figure 3
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  		FIG. 3. Suppression of MAFbx promotor activity by testosterone does not require DNA binding. A, Map of mutants used. TAD, Transactivating domain; NLS, nuclear localization signal. B, Wild-type C2C12 cells were transiently transfected with vectors expressing rat AR, β-galactosidase (β-gal), or mutants and then incubated overnight with testosterone (500 nM). Cell lysates (20 µg/lane) were then subjected to Western blotting with anti-AR antibodies. Membranes were then reprobed with anti-β-actin. Western blots show results for two separate wells for each mutant and are representative of findings from two separate experiments. C, C2C12 Wild-type myoblasts were cotransfected with pMAF3.1, pRL-CMV, and a vector expressing either wild-type AR or an AR mutant and then incubated overnight with vehicle (EtOH) or testosterone (500 nM). Data are normalized relative to that for ethanol-treated cells transfected with the same version of AR. Data are means ± SEM for at least three experiments of six replicates each. *, P < 0.01 vs. EtOH, t test.

 
These data provide evidence that binding of the AR to DNA is not necessary for repression, indicating that regulation of MAFbx transcription is regulated by other, indirect mechanisms. Because one mechanism of up-regulation MAFbx expression is activation of FOXO3A by its dephosphorylation (8), we tested whether testosterone might decrease FOXO3A activity reflected as increased phosphorylation (Fig. 4Go). Exposure of C2C12.AR myotubes to testosterone did not significantly increase FOXO3A phosphorylation. Consistent with prior reports (8, 9), dexamethasone significantly reduced FOXO3A phosphorylation, whereas IGF-1 significantly increased FOXO3A phosphorylation. Testosterone did not reverse this effect of dexamethasone. These findings do not support a role for changes in FOXO3A activity in mechanisms of repression of MAFbx expression by testosterone.


Figure 4
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  		FIG. 4. A, Testosterone does not alter FOXO3A phosphorylation status. A, C2C12.AR myotubes were incubated for 6 h with EtOH, testosterone (500 nM), dexamethasone (Dex; 100 nM), dexamethasone plus testosterone (Dex + Test), or IGF-I (10 ng/ml). Results of densitometry scanning analysis of Western blots are shown. Data are expressed as the ratio of phospho-FOXO3A to total FOXO3A normalized relative to ratios for cells treated with EtOH. Data are means ± SEM from at least two experiments, each performed in quadruplicate. a, P < 0.05 vs. EtOH (ANOVA); b, not significantly different from EtOH; c, not significantly different from dexamethasone. B, Representative Western blots for the experiments presented in A. TS, Testosterone; DEX, dexamethasone.

 
To determine whether specific sequences within the MAFbx promotor were necessary for repression, experiments were repeated using reporter genes having progressive 5' or 3' deletions. Removal of virtually all sequence 5' to the start site did not significantly reduce repression by testosterone (Fig. 5AGo). By contrast, a 3' deletion of 208 bases (pMAF400–241) encoding the 3' portion of the 5' untranslated region (UTR) abolished suppression by testosterone.


Figure 5
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  		FIG. 5. Repression by testosterone requires sequences coding for the 3' region of the MAFbx mRNA 5' UTR with which the AR associates. A, Left panel, Map of reporter genes used. Luc, luciferase. Arrow, Transcriptional start site (–340 relative to the first codon). Right panel, C2C12.AR myoblasts were transfected with the indicated reporter genes and pRL-CMV and then incubated overnight with either EtOH or testosterone (500 nM). Data are means ± SEM and are normalized relative to values for cells transfected with pMAF3.1 and incubated with EtOH. Data are from three different experiments each with at least three replicates. *, P < 0.05 vs. EtOH for the same reporter gene (t test). B, Human primary myoblasts were incubated overnight with testosterone (500 nM) after which association of the AR or Oct-1 to sequences conferring repression was tested by ChIP using PCR primers that amplified the first 400 bp upstream of the first codon. NRS, Normal rabbit serum. C, C2C12.AR cells were transfected with pMAF400, differentiated into myotubes, and incubated overnight with EtOH or testosterone. Association of AR with DNA sequences necessary for repression was assessed by ChIP using antibodies against AR and PCR primers that amplified the first 241 bases upstream of ATG.

 
To determine whether AR was recruited to these sequences in endogenous DNA, human primary myoblasts were incubated overnight with testosterone followed by ChIP. Anti-AR antibodies immunoprecipitated chromatin containing the sequences conferring repression by testosterone (Fig. 5BGo). To test whether AR associates with this promotor sequence in the reporter gene, ChIP experiments were performed using C2C12.AR myotubes transfected with pMAF400. Antibodies against the AR immunoprecipitated the 208 bp sequence necessary for repression (Fig. 5CGo). Association was stimulated by testosterone.

Additional studies were performed in C2C12 cells transfected with reporter genes and mutant versions of the AR to define the specificity of the interaction of AR with these DNA sequences as assessed by chromatin immunoprecipitation. No chromatin was immunoprecipitated by anti-AR antibodies when cells were transfected with pMAF400–241, which is not repressed by testosterone (Fig. 6AGo, lane 4). ChIP experiments indicated that testosterone-induced recruitment of C562G DBD mutant to sequences conferring repression (Fig. 6BGo), whereas the ABC mutant, lacking the LBD and ineffective in mediating repression, did not associate with this sequence (Fig. 6CGo). The findings that the AR C562G DBD mutant was associated with chromatin at a region necessary for repression suggested that this interaction was indirect.


Figure 6
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  		FIG. 6. AR and Oct-1 AR selectively associate with the 208-bp segment necessary for repression. C2C12. A, AR cells were transfected with either pMAF400 or pMAF400–241, differentiated for 48 h, and incubated overnight with testosterone (500 nM) followed by ChIP. Primers used amplified either the sequence spanning 241 bases upstream of ATG (for pMAF400) or a sequence from –241 to –400 bases upstream of ATG (for pMAF400–241). B, C2C12 cells were cotransfected with pMAF400 and a vector expressing either wild-type AR or the C562G mutant. Cells were differentiated for 48 h and then incubated overnight with testosterone (500 nM). ChIP was performed using anti-AR antibodies and primers amplifying the first 241 bases upstream of ATG. C, C2C12 cells were cotransfected with pMAF400 and a vector expressing truncated AR lacking the LBD, differentiated, and incubated overnight with testosterone (500 nM). ChIP was then performed using anti-AR antibodies and primers amplifying the first 241 bases upstream of ATG.

 
A potential mechanism for repression of MAFbx by testosterone is recruitment of AR to specific transcription factors bound to regulatory elements within the first 208 bases upstream of the first codon in the MAFbx gene. A computer-assisted search of this sequence revealed two potential binding sites for Oct-1 (Fig. 7AGo). A DNase I protection assay revealed protection of a this 208-bp sequence at multiple sites along its length, including sequences corresponding to each of the putative Oct-1 binding sites (Fig. 7BGo).


Figure 7
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  		FIG. 7. Protection by nuclear proteins of predicted Oct-1 binding sites within the 3' portion of sequences encoding the 5' UTR. A, Sequence of the 3' portion of the segment coding the 5'UTR of the human MAFbx gene. Putative Oct-1 sites are underlined. Sites were identified by a search of the Transfac database, version 4.0A using the Transcription Element Search System (44 ). B, A probe consisting of the 3' portion of the segment coding for the 5'UTR was 3' labeled with P32 and then incubated with nuclear extract from C2C12 cells and digested with DNase I, followed by resolution of the fragments by PAGE and visualization of fragments by phosphor imaging. For reactions shown in lanes 1 and 3, the probe was incubated with 50 µg BSA, whereas for those shown in lanes 2 and 4, the probe was incubated with 50 µg nuclear extract from C2C12 myotubes. Incubations contained 0.15 (lanes 1 and 2) or 1.5 U (lanes 3 and 4) DNase I. Lanes labeled with C, A, T, and G indicate corresponding nucleotides in DNA sequencing reactions. Numbers to the left of the gel indicate sizes of the fragments/sequencing products.

 
In ChIP experiments, antibodies against Oct-1 immunoprecipitated sequences conferring repression from chromatin within primary human myoblasts that had been incubated with testosterone (Fig. 5BGo). Similarly, anti-Oct-1 antibodies immunoprecipitated sequences conferring repression by testosterone from C2C12.AR myotubes transfected with pMAF400 and then incubated with testosterone (Fig. 6AGo, lane 10) but not from myotubes transfected with a reporter lacking sequences conferring repression (Fig. 6AGo, lane 5).

As a further test of whether Oct-1 bound the putative Oct-1 sites, interactions of recombinant human Oct-1 expressed in rabbit reticulocyte lysates with oligonucleotides having sequences incorporating either site were examined. For each of the two sites, a complex was formed when probes were incubated with lysates containing recombinant Oct-1 but not those containing recombinant luciferase (a negative control) (Fig. 8Go); complexes disappeared on addition of excess unlabeled Oct-1 consensus sequence probes but not when excess probe with an AP-1 consensus sequence was added.


Figure 8
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  		FIG. 8. Binding of in vitro-translated Oct-1 to putative Oct-1 sites. A, A radiolabeled probe containing the putative Oct-1 site A was incubated without (lane 1) or with (lanes 2–9) rabbit reticulocyte lysate in which Oct-1 (lanes 4–9) or luciferase (lane 3) had been in vitro translated and then resolved by nondenaturing PAGE. Excess unlabeled probe was included in some incubations, e.g. AP-1 in lane 5 and Oct-1 consensus sequence in lanes 6 and 7. The arrow indicates the location of complexes formed with in vitro-translated Oct-1. IVT, in vitro translated. B, As in A except that a radiolabeled probe including the putative Oct-1 site B was used. The arrow indicates the location of the Oct-1/DNA complex.

 
The involvement of binding of AR to Oct-1 in repression of MAFbx by testosterone was studied in additional experiments in which expression of Oct-1 was knocked down by transfection of cells with small interfering RNA (siRNA). Transfection of C2C12.AR cells with siRNA against Oct-1 blocked repression by testosterone of pMAF400 reporter gene expression (Fig. 9AGo), whereas repression occurred in cells transfected with this reporter and a scrambled siRNA. Parallel experiments assessed the effect of gene knockdown on association of AR and Oct-1 with MAFbx promotor sequences (Fig. 9Go, B and C). When intensities of bands for PCR products were quantified and normalized relative to those for input DNA, siRNA against Oct-1 significantly reduced chromatin binding of Oct-1 to the 208-bp sequence necessary for repression. Binding of AR to this sequence was also significantly reduced in cells transfected with anti-Oct-1 siRNA (Fig. 9Go, B and C).


Figure 9
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  		FIG. 9. Repression of MAFbx by testosterone requires Oct-1. A, C2C12.AR myoblasts were cotransfected with siRNA against Oct-1 or scrambled siRNA, as indicated, together with pMAF400, and pCMV-RL and then differentiated for 48 h and incubated overnight with testosterone (500 nM) or EtOH. Luciferase activity was determined the following day. Data are mean ± SEM for three experiments, each with six replicates. *, P < 0.05 vs. the mean for percentage repression by testosterone for cells transfected with scrambled siRNA (t test). B, C2C12.AR myoblasts were cotransfected with pMAF400 together with either scrambled siRNA or siRNA against Oct-1, followed by differentiation for 48 h. Testosterone (500 nM) was added and, after overnight incubation, ChIP was performed using PCR primers that amplified a 241-bp sequence just upstream of ATG. Intensities of PCR products are expressed after normalization relative to the intensity of the band for input DNA. Data are mean values for three to four experiments. *, P < 0.05 vs. scrambled siRNA, two-way ANOVA. C, Representative gel for experiments shown in B. NRS, Normal rabbit serum; IP, immunoprecipitation.

 
To determine more specifically whether these Oct-1 binding sites identified in the MAFbx promotor were necessary for repression of MAFbx expression by testosterone, a mutant version of pMAF400 was constructed in which the two sites were deleted (Fig. 10AGo). This deletion completely prevented repression of reporter gene activity by testosterone on C2C12.AR myotubes (Fig. 10BGo).


Figure 10
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  		FIG. 10. Repression by testosterone of MAFbx expression requires Oct-1 binding sites within the promotor sequences conferring repression. A, Map of the structure of the pMAF{Delta}1/2 reporter gene showing the location of the two sequences that were deleted. B, C2C12.AR myoblasts were transfected with the indicated reporter gene and differentiated and incubated overnight with testosterone. Data are expressed as a percentage of luciferase activity for pMAF400 for cells incubated with EtOH. *, P < 0.05 vs. EtOH, t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Testosterone suppressed activity of the human MAFbx promotor in C2C12 cells at testosterone concentrations as low as 5 nM, just below the normal range of circulating levels in humans of 10–35 nM (34). Repression was mediated by a sequence extending approximately 200 bases upstream of the first codon. This sequence codes for the 3' portion of the 5' UTR of the MAFbx mRNA.

Transcriptional repression by AR may occur through several mechanisms. One of these is recruitment of transcriptional corepressors including mothers against decapentaplegic-3 (SMAD) (35), dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome (DAX) (36), and silencing mediator of retinoid and thyroid receptors (SMRT) (37). Repression has also been ascribed to interactions of the AR with other transcription factors that reduce their transcriptional activity. The AR has been found to negatively affect transcriptional activation by nuclear factor-{kappa}B, AP-1, Ets-1, Oct-1, and cAMP response element-binding protein (CREB) (22, 23, 24, 25, 26). Although FOXO3A has been implicated in up-regulation of MAFbx in response to glucocorticoids (8), two findings from our study argue against modulation by AR of FOXO family transcription factors as a mechanism of repression. Testosterone did not alter phosphorylation of FOXO3A (a surrogate measure of the activity of this transcription factor), and repression was blocked by a 3' truncation of the MAFbx promotor that did not remove the FOXO3A sites.

Testosterone did, however, stimulate the physical association of the AR with the promotor region responsible for repression. Repression was not attributable to direct binding of the AR to DNA, as indicated by findings that disruption of the AR DBD did not block repression and that a DBD mutant of the AR associated with these sequences. Several types of evidence support the conclusions that repression involved interactions of AR with Oct-1. Oct-1 associated with the DNA sequences containing the postulated Oct-1 binding sites in ChIP studies. Finally, siRNA against Oct-1 reduced association of Oct-1 with sequences containing Oct-1 binding sites associated with loss of repression by testosterone and association of AR with such sequences, and deletion of the Oct-1 sites prevented repression by testosterone.

Our findings suggest that Oct-1 bound directly to DNA within the sequence conferring repression. The predicted Oct-1 sites within this sequence were protected by nuclear proteins in DNase protection studies and that each of the predicted Oct-1 sites bound to Oct-1 in gel shift studies. Deletion of these Oct-1 binding sites prevented repression by testosterone.

The conclusion that AR represses MAFbx expression via interactions with Oct-1 is consistent with other findings indicating that interactions between the steroid hormone receptors and Oct-1 are important in transcriptional regulation of target genes for steroid hormones. Deletion of Oct-1 sites abrogates repression of the dehydroepiandrosterone sulfotransferase gene by the AR (23). Binding of Oct-1 to sites in the promotor for the mouse Slp gene results in interactions of Oct-1 with AR or glucocorticoid receptor (GR) bound to steroid hormone response element that confer selectivity for transactivation for AR over GR (21, 38). Binding of the AR to Oct-1 has been reported based on findings that Oct-1 is associated with the AR in immunoprecipitation studies that have used cell lysates as well as in vitro-translated AR or GR and recombinant Oct-1 or fragments thereof (21, 38, 39).

It is intriguing that the sequences necessary for repression were located within the last 200 bases coding for the 5' UTR of MAFbx mRNA. Transcriptional repression mediated by sequences coding for the 5' UTR has been described in several other genes, although limited information has been published as to specific molecular mechanisms mediating repression at these sites. A motif located approximately 50 bp upstream of the first codon of the T-cadherin gene was found to be responsible for repression of this gene by aryl hydrocarbons (40). Conversely, mutation of either an E-box or any of four specificity protein-1 (SP1) sites from the MAPK3 promotor significantly reduced transcriptional activity (41). In this context, it appears likely that testosterone acts on the MAFbx promotor by blocking transcriptional activation attributable to Oct-1 bound to sequences in the 3' 200 bases encoding the 5'UTR. An intriguing aspect of this regulatory mechanism is the likely dynamic nature of such chromatin-protein complexes, which might be predicted to dissociate and reform with each passage of RNA polymerase.

Considerable work implicates MAFbx in muscle atrophy, the most compelling being findings that loss of the MAFbx gene slows denervation atrophy and that its overexpression reduced the size of cultured muscle cell lines (4). Insights into potential mechanisms by which MAFbx promotes hypertrophy include findings that this ubiquitin ligase regulates ubiquitin-proteasome-dependent degradation of the nuclear myogenic factor myoD (42) and calcineurin, a key regulator of muscle fiber size (43). Studies in rats have shown that testosterone blocks dexamethasone-induced increases in MAFbx expression and glucocorticoid-induced atrophy (16). Additionally, the androgenic steroid nandrolone was shown to reduce denervation atrophy and MAFbx expression during the subacute phase of denervation atrophy (17).

In conclusion, our findings raise the possibility that the effect of testosterone to reduce MAFbx expression may be one mechanism by which androgenic steroid hormones slow muscle catabolism such as that observed in men with reduced blood testosterone levels (11) or burn victims (15).


   Footnotes
 
This work was supported by Grants B4162C, B3347K, and B4616R from the Department of Veterans Affairs Rehabilitation Research and Development Service.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 3, 2008

Abbreviations: AP-1, Activator protein-1; AR, androgen receptor; ARE, androgen response element; CDS, charcoal dextran stripped; ChIP, chromatin immunoprecipitation; DBD, DNA-binding domain; DNase, deoxyribonuclease; EtOH, ethanol; FBS, fetal bovine serum; FOXO, Forkhead box O; GR, glucocorticoid receptor; LBD, ligand-binding domain; MAFbx, muscle atrophy F-box; MMTV, mouse mammary tumor virus; Oct, octamer binding transcription factor; qPCR, real-time PCR; siRNA, small interfering RNA; UTR, untranslated region.

Received May 7, 2008.

Accepted for publication June 25, 2008.


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