Skip to main content
PMC home page
Endocrinology logoLink to Endocrinology
. 2009 Dec 23;151(3):1050–1059. doi: 10.1210/en.2009-0530

REDD1 Is a Major Target of Testosterone Action in Preventing Dexamethasone-Induced Muscle Loss

Yong Wu 1, Weidong Zhao 1, Jingbo Zhao 1, Yuanfei Zhang 1, Weiping Qin 1, Jiangping Pan 1, William A Bauman 1, Robert D Blitzer 1, Christopher Cardozo 1
PMCID: PMC2840688  PMID: 20032058

Abstract

Glucocorticoids are a well-recognized and common cause of muscle atrophy that can be prevented by testosterone. However, the molecular mechanisms underlying such protection have not been described. Thus, the global effects of testosterone on dexamethasone-induced changes in gene expression were evaluated in rat gastrocnemius muscle using DNA microarrays. Gene expression was analyzed after 7-d administration of dexamethasone, dexamethasone plus testosterone, or vehicle. Dexamethasone changed expression of 876 probe sets by at least 2-fold. Among these, 474 probe sets were changed by at least 2-fold in the opposite direction in the dexamethasone plus testosterone group (genes in opposition). Major biological themes represented by genes in opposition included IGF-I signaling, myogenesis and muscle development, and cell cycle progression. Testosterone completely prevented the 22-fold increase in expression of the mammalian target of rapamycin (mTOR) inhibitor regulated in development and DNA damage responses 1 (REDD1), and attenuated dexamethasone induced increased expression of eIF4E binding protein 1, Forkhead box O1, and the p85 regulatory subunit of the IGF-I receptor but prevented decreased expression of IRS-1. Testosterone attenuated increases in REDD1 protein in skeletal muscle and L6 myoblasts and prevented dephosphorylation of p70S6 kinase at the mTOR-dependent site Thr389 in L6 myoblast cells. Effects of testosterone on REDD1 mRNA levels occurred within 1 h, required the androgen receptor, were blocked by bicalutamide, and were due to inhibition of transcriptional activation of REDD1 by dexamethasone. These data suggest that testosterone blocks dexamethasone-induced changes in expression of REDD1 and other genes that collectively would otherwise down-regulate mTOR activity and hence also down-regulate protein synthesis.

In a model of dexamethasone-induced atrophy of skeletal muscle, testosterone prevents up-regulation by glucocorticoids of REDD1, an inhibitor of the master controller of cell size and protein synthesis, mTOR.

Glucocorticoid-induced muscle atrophy is associated with both increased catabolism (1) and reduced synthesis (2) of muscle proteins. The ubiquitin-proteosome pathway has been linked to much of the increase in muscle protein catabolism that occurs during atrophy (3,4). In this pathway, proteins are marked by ubiquitin ligases for degradation by covalent modification with polyubiquitin chains (5). Two muscle-specific ubiquitin E3 ligases, muscle atrophy F-box (MAFbx; also called atrogin-1) and muscle ring finger 1 (MuRF1) play important roles in muscle atrophy. In genetic studies in mice, loss of either of these genes slowed muscle atrophy due to nerve transection (6,7), and loss of MuRF1 prevented dexamethasone-induced degradation of myofibrillar components (8).

Glucocorticoids reduce initiation of translation in skeletal muscle and protein synthesis within hours of administration (9). The rate-limiting step in cap-dependent translation initiation is the binding of eIF4G to eIF4E. This is inhibited by dexamethasone through increased availability of dephosphorylated eIF4E binding protein 1 (4EBP1; Ref. 10), which competes with eIF4G for binding to eIF4E to form an inactive 4EBP1:eIF4E complex. Glucocorticoids also rapidly reduce activity of p70S6 kinase (9), a determinant of cell size and ribosome biogenesis. One mechanism for these actions of glucocorticoids is up-regulation of regulated in development and DNA damage responses1 (REDD1; Ref. 11), an inhibitor of the protein kinase mammalian target of rapamycin (mTOR). mTOR is a master positive regulator of protein synthesis which integrates signals from growth factors, nutrients, hypoxia, and cellular stress. By phosphorylating and inactivating 4EBP1, mTOR promotes assembly of eIF4G/eIF4E complexes and cap-dependent translation initiation; phosphorylation and activation by mTOR of p70S6 kinase increases ribosomogenesis, and is an important determinant of cell and skeletal muscle size (12,13,14).

Skeletal muscle atrophy programs have been linked to activation of the Forkhead transcription factors Forkhead box O1 (FOXO1) and FOXO3A, which are dephosphorylated in response to glucocorticoids, resulting in nuclear import and transcriptional activity (15). Dephosphorylation is attributable, in part at least, to reduced Akt activity (15), which may be related to reduced coupling of IGF-I receptors to downstream kinases (16). There is a growing list of atrophy-related genes that have been shown to be up-regulated by FOXO1 and FOXO3A, including MAFbx (15), MuRF1 (17), 4EBP1 (18), and REDD1 (19).

The male hormone testosterone has anabolic actions that include increasing the size and increasing the strength of skeletal muscle (20,21). Testosterone and its synthetic derivatives, the anabolic steroids, reduce muscle atrophy caused by burns, disuse, HIV infection, denervation, or glucocorticoid administration (22,23,24). Testosterone also prevents glucocorticoid-induced atrophy in rats and blocks glucocorticoid-induced degradation of proteins in cultured muscle cell lines (25). Very little is known about the molecular mechanisms by which testosterone blocks or reverses the adverse effects of glucocorticoids on muscle. To gain insight into mechanisms that underly this functional antagonism between glucocorticoid and testosterone signaling we examined the effects of testosterone on dexamethasone-induced changes in gene expression across the transcriptome using DNA microarrays and confirmed selected changes in gene expression by real-time PCR (qPCR) and Western blotting. The experiments used an animal model of dexamethasone-induced muscle atrophy in which we have previously shown that supraphysiological doses of testosterone prevent atrophy of the gastrocnemius muscle (25). In this model, animals were administered a high (pharmacological) dose of dexamethasone and a supraphysiological dose of testosterone. The dose of testosterone corresponded to approximately 10 times the dose needed to achieve high-normal levels in rats with hormone replacement therapy.

Materials and Methods

Animals

These studies were approved by the Institutional Animal Care and Use Committee of the James J. Peters VA Medical Center (Bronx, NY). Male Wistar rats weighing 250 g (Taconic Farms, Hudson, NY) underwent implantation of Alzet pumps that infused vehicle (propylene glycol; Sigma, St. Louis, MO), dexamethasone (0.7 mg/kg·d, Sigma), or dexamethasone (0.7 mg/kg·d) plus testosterone (28 mg/kg·d; Spectrum Chemical Co., Gardena, CA). Seven days later, animals were euthanized by inhalation of carbon dioxide, and gastrocnemius muscles were excised, weighed, and flash-frozen using liquid nitrogen. Gastrocnemius muscle total RNA used in the microarray analyses was from animals for which effects on muscle weights and expression of MAFbx and MuRF1 of dexamethasone alone or with testosterone were previously reported (25). Tissue lysates extracted from gastrocnemius muscle for Western blotting were from a second group of animals subjected to identical procedures. This second group of animals demonstrated gastrocnemius muscle weight changes that were similar to those we have previously reported.

Microarray analysis

Total RNA was extracted from gastrocnemius muscle homogenates with phenol-chloroform (26) then further enriched using RNeasy minicolumns (Qiagen, Valencia, CA). Samples selected for microarray analysis yielded RNA integrities of more than 8.0 using an Agilent Bioanalyzer (Santa Clara, CA). Microarray analysis employed Affymetrix (Santa Clara, CA) rat genome 230 2.0 arrays following the manufacturers recommended procedures. Verification of RNA integrity and microarray analysis were performed by the Microarray Shared Resource Facility at the Mount Sinai School of Medicine (New York, NY). A total of nine microarrays were analyzed (one array per animal, three animals per group, where groups were vehicle, dexamethasone, and dexamethasone plus testosterone). The data presented herein have been deposited in NCBI’s Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo; Ref. 27) and are accessible through GEO Series accession no. GSE12296.

Data filtering and mining

Differentially expressed genes were identified using BRB Array Tools version 3.6.2 (BRB, Biometric Research Branch, National Cancer Institute, Bethesda, MD), developed by Dr. Richard Simon and Amy Peng Lam of the National Cancer Institute (Bethesda, MD) with the following parameters for normalization and filtering. Data for each array were normalized using the median for the entire array; expression values were set to 10 if below this value and excluded from further analysis if more than 20% of the values were at least 1.5-fold greater or smaller than the median for that probe set. Significance of differences in expression among the three groups was determined using an F test with significance set at P < 0.05. Tests for enrichment of biological themes were performed using GoMiner (National Institutes of Health, Bethesda, MD) with a minimal significance of P < 0.05. To limit the number of gene ontology categories, only categories with at least three and less than 100 genes were considered. Biological functions of differentially expressed genes were determined using NIH Database for Annotation, Visualization, and Integrated Discovery (DAVID; Ref. 28) and GeneCards at www.genecards.org (29).

Cell culture and L6.AR cells

L6 rat myoblasts (American Type Culture Collection, Manassas, VA) were maintained at 37 C in humidified air containing 10% carbon dioxide in DMEM containing 10% fetal bovine serum and antibiotics (growth medium). Because this cell line does not express androgen receptor (AR) at levels sufficient to regulate reporter gene expression, we have generated L6.AR cells that express the human AR after infection with a retrovirus generated using the pBabe backbone and selection with puromycin by using methods we have previously described for C2C12 myoblasts (30). L6.AR cells stably express the AR (supplemental Fig. 1, which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). L6.AR cells are maintained in growth medium containing puromycin (1.6 μg/ml) to ensure against loss of the transgene. L6.AR cells were seeded into wells of 6-well plates (1 × 106 cells per well). After overnight incubation, cells were covered with differentiation medium for which FBS was replaced with 2% horse serum. After 48 h either dexamethasone, alone or in combination with testosterone, or vehicle (ethanol) was added, and cells were incubated for the indicated periods. mRNA stability studies were performed by following decay of REDD1 mRNA. This was done after adding actinomycin as described (30), using L6.AR cells that had been differentiated then incubated overnight with dexamethasone alone or combined with testosterone. Half-life was determined assuming a one-phase exponential decay. Significance of difference in half-lives was determined using an F test (30).

qPCR

Total RNA (1 μg) was used to prepare a cDNA library by reverse transcription (High Capacity cDNA Archive Kit, Applied Biosystems, Foster City, CA). qPCR was performed in triplicate, and the mean for the crossing points of triplicates was used in subsequent calculations. Data were normalized relative to expression of 18S RNA (31,32). Levels of gene expression were expressed as fold induction relative to controls using the 2−ΔΔCt method (33).

Western blotting

Gastrocnemius muscle (35 mg) was homogenized in 300 μl of ice-cold homogenization buffer, which contained Cell Lysis Buffer (Cell Signaling, Danvers, MA), Protease Inhibitor Cocktail Complete (Sigma, St. Louis, MO), and Phosphatase Inhibitor Cocktail 1 (Roche, Indianapolis, IN). Homogenates were cleared by centrifugation at 4 C in a microcentrifuge. Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA) with BSA as the standard. Proteins (30 μg) were resolved by SDS-PAGE then electrophoretically transferred to polyvinyl fluoride membranes. The primary antibodies used were: REDD1 (1:1000; group no. 10638-1-AP; Proteintech, Chicago, IL), total 4EBP1 (1:1000; no. 9644; Cell Signaling, Boston, MA), p70S6 kinase (phospho Thr389 and total; Cell Signaling), and anti-ß-tubulin(1:2000; AB6046; Abcam, Cambridge, MA). Labeled bands were detected using enhanced chemiluminescence and captured on photographic film with subsequent digitization of images. Intensity of bands on digitized images was quantified using Imagequant TL (GE Life Sciences, Piscataway, NJ) and normalized relative to ß-tubulin. Western blotting of proteins from cultured cells followed the same procedures, except cells were covered with 100 μl of homogenization buffer, detached using a rubber policeman in the same buffer in situ in the tissue culture plate, and then disrupted by sonication.

Results

Data filtering

A three-way comparison of gene expression levels between groups of animals that were administered vehicle, dexamethasone, or dexamethasone combined with testosterone revealed significant differences in expression for 1658 Affymetrix probe sets. To identify effects of dexamethasone that were mitigated by testosterone, we used a filter that included only genes for which expression was changed 2-fold by dexamethasone compared with vehicle and at least 2-fold in the opposite direction in the dexamethasone plus testosterone group compared with the dexamethasone group. By use of these criteria, dexamethasone changed expression of 876 probe sets, of which 474 probe sets were changed by at least 2-fold in the opposite direction in the dexamethasone plus testosterone group. This group of genes is referred to as genes regulated in opposition.

Enriched biological themes

Among the 474 probe sets regulated in opposition, there were 235 transcripts with names assigned. This subset of genes was analyzed for enrichment in biological themes (supplemental Table 1). Significant enrichment was found for categories related to muscle atrophy and hypertrophy, including insulin and IGF signaling and regulation of myoblast differentiation, cell cycle arrest, and cell death. Also enriched were genes encoding extracellular matrix constituents, cell adhesion molecules, and metabolism.

Genes regulated in opposition

Genes regulated in opposition are listed in Table 1 and supplemental Tables 2–9. Because of findings that glucocorticoids reduce protein synthesis (9) and activity of PI3 kinase (16) and hence Akt and FOXO1, we focused our subsequent analysis on genes involved in regulating translation or IGF-I signaling. Two other genes known to be involved in muscle atrophy were also included.

Table 1.

Genes for IGF-I pathways and translation

GO category Description Gene symbol Probe set P value Dex/Veh DexTs/Dex DexTs/Veh
IGF-I binding IGF binding protein 3 IGFBP3 1386881_at 0.0006 3.31 0.40 1.32
IGF binding protein 5 IGFBP5 1370960_at 0.015 0.19 3.87 0.74
IGF-I receptor signal transduction pathway Forkhead box O1A FOXO1 1396965_at 0.031 1.88 0.36 0.68
Insulin receptor substrate 1 IRS1 1369771_at 0.002 0.26 3.17 0.83
Phosphatidylinositol 3-kinase, regulatory subunit, polypeptide 1 PIK3R1 1371776_at 0.004 2.77 0.36 0.99
NF-κB signaling pathway B-cell leukemia/lymphoma 3 (predicted) Bcl3 (predicted) 1385627_at 0.006 0.25 2.64 0.67
Nuclear factor of κ-light-chain-gene-enhancer in B-cells inhibitor, α Nfkbia 1389538_at 0.008 3.02 0.43 1.3
Response to stress DNA-damage-inducible transcript 4 REDD1/Ddit4/RTP801 1368025_at 0.001 10.64 0.16 1.73
Translational initiation Eukaryotic translation initiation factor 4E binding protein 1 4EBP1 1386888_at 0.002 6.06 0.30 1.86
Eukaryotic translation initiation factor 4γ, 3 (predicted) Eif4g3 (predicted) 1373364_at 0.005 3.08 0.37 1.15
Open in a new tab

Data shown are microarray data representing mean values for three arrays for each of three groups: Veh, vehicle; Dex, dexamethasone; DexTs, dexamethasone combined with testosterone; Dex/Veh, the ratio obtained when dividing the mean expression for Dex by that for Veh; DexTs/Dex, the ratio obtained by dividing mean expression for DexTs by that for Dex. Probe set refers to the Affymetrix identifier for the probe used for the corresponding mRNA. P values are those for the F-test among the three groups (Veh, Dex, DexTs). GO, Gene ontology. 

Regulation of translation

Among the largest dexamethasone-induced increases in gene expression was that for REDD1 (Table 1). Up-regulation of REDD1 expression was nearly completely prevented by testosterone. Expression of the translation initiation factor eIF4G3 was also increased by dexamethasone, as was expression of 4EBP1, an inhibitor of translation initiation (12). When coadministered with dexamethasone, testosterone normalized eIF4G and 4EBP1 mRNA levels.

IGF-I signaling

Dexamethasone and testosterone exerted opposing effects on expression of several genes involved in IGF-I binding and signaling (Table 1). Expression of IGF binding protein-3 (IGFBP-3) was increased by dexamethasone, whereas that of IFGBP-5 was repressed. Dexamethasone decreased IRS-1 expression and increased expression of PI3 kinase p85 regulatory subunit (PI3KR1) and of FOXO1. Testosterone reduced FOXO1 expression below baseline levels, normalized p85 expression, and attenuated effects of dexamethasone on IRS1 and the IGFBPs.

Protein catabolic and muscle atrophy pathways

Dexamethasone altered expression of several genes shown to participate in muscle atrophy, specifically the NF-κB inhibitor Bcl3 (Table 1; Ref. 34), and, consistent with prior findings, MuRF1/Trim63 (supplemental Table 8; Ref. 25). MAFbx was excluded during data filtering. Dexamethasone reduced expression of Bcl3 but increased that of MuRF1; testosterone blocked these changes.

Androgen and glucocorticoid receptors (GRs)

Levels for mRNA for the AR and GR were not significantly altered in the dexamethasone (Dex) or Dex/testosterone (Ts) groups compared with vehicle (F test). Compared with vehicle (Veh), AR mRNA levels were unchanged in Dex and Dex/Ts groups with values for Dex/Veh and Dex/Ts/Veh of 1.06 and 0.95, respectively. Compared with vehicle, GR mRNA levels tended to be reduced in both the Dex and Dex/Ts groups (values for Dex/Veh and Dex/Ts/Veh were 0.58 and 0.26, respectively).

qPCR validation

Levels of expression of selected genes were verified by qPCR (Table 2). Consistent with findings from the microarray data, dexamethasone significantly reduced expression of IGFBP-5 and IRS-1 but significantly increased expression of IGFBP-3, p85 regulatory subunit of PI3 kinase (PI3KR1), and 4EBP1. Coadministration of testosterone significantly increased expression of IRS-1 and significantly reduced expression of p85 and 4EBP1. The marked up-regulation of REDD1 was also significant and was abolished by coadministration of testosterone.

Table 2.

qPCR. Quantitation of expression levels of selected genes. Gene expression is normalized relative to that for animals administered vehicle

Mean expression level
Gene Vehicle Dex DexTs
IGF-I binding
 IGFBP-3 1.17 ± 0.30 (5) 4.45 ± 0.99 (5)a 1.11 ± 0.21 (4)
 IGFBP-5 1.12 ± 0.25 (5) 0.24 ± 0.07 (5)a 0.59 ± 0.13 (4)
IGF-I signaling
 IRS-1 1.02 ± 0.06 (9) 0.36 ± 0.07 (9)a,b 0.85 ± 0.10 (8)
 PIK3R1 1.07 ± 0.14 (9) 7.18 ± 1.11 (9)a,b 2.62 ± 0.52 (8)
Translation
 eIF4EBP1 1.02 ± 0.08 (9) 7.16 ± 0.93 (9)a,b 1.56 ± 0.24 (8)
 REDD1 1.02 ± 0.06 (13) 22.6 ± 2.0 (13)a,b 1.35 ± 0.22 (11)
Open in a new tab

Data are mean values ± sem. Values in parentheses represent n. Dex, Dexamethasone, DexTs, dexamethasone combined with testosterone. 

a

P < 0.05 vs. vehicle. 

b

P < 0.05 vs. DexTs (ANOVA). 

REDD1 and 4EBP1 protein in skeletal muscle

Effects of testosterone on dexamethasone-induced expression of REDD1 and 4EBP1 were assessed by Western blotting after 7 d administration of dexamethasone alone or together with testosterone. Dexamethasone markedly increased REDD1 protein levels with more modest though significant increases in 4EBP1 expression (Fig. 1); these increases were prevented by coadministration of testosterone.

Figure 1.

Figure 1

Open in a new tab

Testosterone blocks glucocorticoid-induced increases in REDD1 and 4EBP1 protein levels in gastrocnemius muscle. A (Inset), Representative Western blot showing REDD1 levels. Graph, Band intensities for REDD1 in gastrocnemius muscle from rats treated for 7 d with vehicle, dexamethasone (DEX), or dexamethasone plus testosterone (DEX/TS) as determined by densitometry scanning. Values were first normalized relative to corresponding values for ß-tubulin then normalized relative to the average value for controls. Data are for one experiment with seven to eight animals per group and are representative of findings from three different experiments. B, Data for Western blotting for 4EBP1, otherwise as in A. *, P < 0.05, one-way ANOVA with Tukey’s post hoc test.

Because overexpression of FOXO1 reduced levels of mTOR in cultured myoblasts (35) and FOXO1 expression was up-regulated by dexamethasone (Table 1), we also determined levels of mTOR and p70S6 kinase protein in gastrocnemius muscle. This analysis showed no change in skeletal muscle levels of mTOR or p70S6 kinase in either the dexamethasone or dexamethasone plus testosterone groups (supplemental Fig. 2).

Testosterone blocks dexamethasone-induced dephosphorylation of p70s6 kinase

Incubation of L6 cells with dexamethasone reduced phosphorylation of p70S6 kinase through the REDD1-dependent inhibition of mTOR (11). Because testosterone opposed the effect of dexamethasone on REDD1 levels, we tested whether testosterone could also normalize mTOR activity assessed by determining phosphorylation of p70S6 kinase at the mTOR-specific site Thr389. For this, we conducted additional experiments in which L6 cells stably expressing the AR were incubated overnight with dexamethasone alone or with testosterone. Overnight incubation with dexamethasone significantly increased REDD1 protein levels, and this effect was largely prevented by coadministration of testosterone (Fig. 2A). Dexamethasone also up-regulated REDD1 mRNA levels, an effect which was significantly reduced by 10 nm tes-tosterone and nearly completely abolished by 100 and 500 nm testosterone (Fig. 2B). Overnight incubation with dexamethasone significantly reduced phosphorylation of the mTOR-dependent site of p70S6 kinase at Thr389 (Fig. 2C) without altering total p70S6 kinase levels (Fig. 2D), indicative of reduced mTOR activity. Coadministration of testosterone prevented dexamethasone-induced reductions in p70S6 kinase phosphorylation.

Figure 2.

Figure 2

Open in a new tab

Testosterone prevents dexamethasone-induced mTOR inhibition in L6.AR cells. A, L6.AR cells were differentiated for 48 h then incubated overnight with or without dexamethasone (Dx, 100 nm) alone or combined with testosterone (D/T, 500 nm). Levels of REDD1 protein were determined by Western blotting. Membranes were stripped and reprobed for ß-tubulin. Values were first normalized relative to corresponding values for ß-tubulin then normalized relative to the average value for controls. Values are means for six determinations in total from two experiments. B, L6.AR cells were differentiated for 48 h, incubated overnight with Dex or testosterone as indicated, and then assessed for REDD1 mRNA levels. Data are means for two experiments each with three separate determinations per condition. C, As in A except that membranes were probed for p70S6 kinase phosphoThr389. Arrow indicates the band corresponding to phosphorylated p70S6 kinase. D, Membranes from B were reprobed for total p70S6 kinase then reprobed for ß-tubulin. *, P < 0.05, **, P < 0.01, ***, P < 0.001 for the indicated comparison (one-way ANOVA with Tukey’s post hoc test). EtOH (E), Ethanol; NS, not significant.

Binding of testosterone to the AR is required to block up-regulation of REDD1

To determine whether the AR was necessary for testosterone to block up-regulation of REDD1, experiments were conducted using L6 cells lacking the human AR transgene; these cells lack detectable AR by Western blot (supplemental Fig. 1) and are not androgen responsive in assays with reporter genes containing androgen response elements (data not shown). Dexamethasone significantly increased REDD1 expression (Fig. 3A), whereas testosterone had no effect on this action of dexamethasone.

Figure 3.

Figure 3

Open in a new tab

Up-regulation of REDD1 mRNA is blocked by testosterone through transcriptional regulation by the AR. A, L6 cells lacking exogenous AR were differentiated for 48 h then incubated overnight with dexamethasone (Dex; 100 nm) or testosterone (500 nm), after which REDD1 mRNA levels were determined. B, L6.AR cells were differentiated for 48 h then incubated overnight with Dex, testosterone, or bicalutamide (Bical) as indicated, after which REDD1 mRNA levels were determined. C, L6.AR cells were differentiated for 48 h then incubated for 1 h with Dex (100 nm) or testosterone (500 nm), after which REDD1 mRNA levels were measured. D, REDD1 mRNA half-life was determined in L6.AR cells that had been differentiated for 48 h then incubated overnight with ethanol (EtOH) or with Dex (100 nm) alone or in combination with testosterone (Dex/Ts; 500 nm). Levels of REDD1 mRNA were then determined by qPCR at different times after adding actinomycin. Half-lives were 113, 42, and 58 min for EtOH, Dex, and Dex/Ts, respectively; values for R2 were 0.90, 0.96, and 0.94 for EtOH, Dex, and Dex/Ts, respectively. There were significant differences in half-life among the three groups, but no difference was found when comparing Dex and Dex/Ts. A–C, ***, P < 0.001, using one-way ANOVA with a Tukey’s post hoc test. D, Half-lives were determined by curve fits assuming exponential decay, and differences in half-life were tested for significance by an F test. Data are means for six to nine separate determinations in total from at least two experiments. NS, Not significant.

To test whether binding of testosterone to the AR ligand binding domain was necessary, the effect of the AR antagonist bicalutamide was examined in additional experiments using L6.AR cells. These experiments used a reduced testosterone concentration to ensure that bicalutamide was able to compete with testosterone for binding to AR. Testosterone completely blocked up-regulation of REDD1 by dexamethasone in a fashion that was antagonized by bicalutamide at 20 μm (Fig. 3B). Bicalutamide had no significant effect on dexamethasone-induced expression of REDD1 in the absence of testosterone (Fig. 3B). Consistent with a prior report (36), up-regulation of REDD1 expression by dexamethasone was blocked by the partial GR agonist RU486 (supplemental Fig. 3).

Effects of testosterone on REDD1 mRNA level are rapid and unrelated to mRNA half-life

The data indicate that testosterone blocks up-regulation by dexamethasone of REDD1 in L6.AR cells after overnight incubation. To determine whether this effect was rapid or delayed, experiments were performed in which effects on REDD1 mRNA levels were examined after incubation for 1 h with dexamethasone alone or with testosterone. Dexamethasone significantly increased REDD1 mRNA levels, whereas testosterone significantly reduced this dexamethasone effect (Fig. 3C). To test whether changes in mRNA stability could explain these alterations in REDD1 mRNA levels, L6.AR cells were incubated overnight with dexamethasone alone or together with testosterone then treated with actinomycin and assessed for the rate of decay of REDD1 mRNA. Dexamethasone more than halved the half-life of REDD1 mRNA from 113 to 42 min (Fig. 3D). When incubated with dexamethasone combined with testosterone (58 min), half-life did not change from what it was for dexamethasone alone (Fig. 3D).

Discussion

Testosterone blocks dexamethasone-induced expression of inhibitors of protein synthesis

The major conclusion supported by our studies is that testosterone blocked dexamethasone-induced gene expression alterations that have been previously shown to reduce mTOR activity and protein synthesis. Testosterone blocked up-regulation of REDD1 and 4EBP1 and attenuated the reductions in phosphorylation of p70S6 kinase at the mTOR-dependent site Thr389. Several lines of evidence directly link REDD1 to reductions in mTOR activity, protein synthesis, and cell size. REDD1 was originally described as a transcript that was up-regulated by glucocorticoids, hypoxia, or stress (36,37,38). REDD1 was later found to inhibit activity of mTOR, a master regulator of protein synthesis, cell size, and cell proliferation (37,38,39). A linkage between REDD1 levels, mTOR activity, and cell size has been supported by findings indicating that overexpression of REDD1 diminished cell size (37), whereas gene knockdown using siRNA against REDD1 increased cell size in an mTOR-dependent manner (37). There is strong evidence that up-regulation of REDD1 in L6 cells treated with dexamethasone plays a causal role in reducing muscle protein synthesis. Specifically, dephosphorylation of 4EBP1 and p70S6 kinase caused by dexamethasone was prevented by siRNA against REDD1 mRNA or by overexpression of RheB, a small GTPase which activates mTOR and acts downstream of REDD1 (11).

Studies using genetic approaches and mTOR inhibitors consistently demonstrate a tight linkage between mTOR, p70S6 kinase, and the mass and fiber cross-sectional area of skeletal muscle. Inhibition of mTOR with rapamycin prevents muscle hypertrophy due to overload and prevents recovery of muscle weight after atrophy due to hind limb suspension (40). Conversely, in mice lacking p70S6 kinase muscle size and fiber cross-sectional area were reduced without any change in fiber number or fiber type (41).

Consistent with prior reports in lymphocytes (36), our finding that up-regulation of REDD1 by dexamethasone was blocked by the GR antagonist RU486 indicates a requirement for the binding of the ligand to this receptor for up-regulation of REDD1. Our findings that dexamethasone did not prolong REDD1 mRNA half-life and even shortened it suggest that dexamethasone acts to increase transcription of the REDD1 gene.

The REDD1 promoter contains a FOXO response element and is transcriptionally activated by FOXO3A (19), suggesting that dexamethasone-related activation of FOXO1 and FOXO3A (15) represents one mechanism underlying increased expression of REDD1. Our findings do not permit conclusions regarding possible roles for hypoxia inducible factor (HIF)-1α and AMPK, which have been shown to be required for up-regulation of REDD1 by hypoxia (42). Expression levels for these factors were not significantly altered by dexamethasone in our study, and dexamethasone enhanced transcriptional activity of HIF-1α in some studies but repressed it in others (43,44).

The effect of testosterone to block up-regulation of REDD1 by dexamethasone required binding of ligand to the AR, was rapid, and was not mediated by increased degradation of REDD1 mRNA. These findings indicate that the AR blocks transcriptional activation of the REDD1 gene by dexamethasone. Our findings do not permit conclusions about mechanisms by which this occurs, although several have been proposed. These include antagonism by androgens of the GR (45), formation of AR:GR heterodimers acting as transcriptional repressors (46), and binding of AR to other transcriptional regulators to modify their activity (30).

Testosterone blocks other changes in gene expression that may amplify REDD1 effects

Inhibitory effects of REDD1 on mTOR activity may be amplified by other changes in gene expression stimulated by dexamethasone and blocked by testosterone, which may act both upstream and downstream of mTOR. One of these is increased levels of 4EBP1 mRNA and protein. When dephosphorylated, 4EBP1 competes with eIF4G for binding to eIF4E thus diminishing levels of the eIF4E/eIF4G complex, which is rate limiting for initiation of cap-dependent translation. Phosphorylation of 4EBP1 by mTOR prevents its binding to eIF4E and stimulates protein synthesis (12,13,14), whereas dexamethasone reduces 4EBP1 phosphorylation (11).

A surprising finding of our study was that dexamethasone up-regulated both 4EBP1 and eIF4G3 and that these changes were attenuated by testosterone. It has been shown that FOXO1 up-regulates 4EBP1 expression and that dexamethasone activates FOXO1 (15,18), suggesting that up-regulation of 4EBP1 may be a direct effect of dexamethasone action. Mechanisms by which eIF4G is regulated are poorly understood, and it is unknown why eIF4G is up-regulated together with 4EBP1. One possible explanation for our findings is that the decrease in protein synthesis that results from up-regulation of 4EBP1 stimulates expression of eIF4G as a compensatory response.

Dexamethasone has also been reported to reduce activity of PI3 kinase/Akt signaling (15,47). Activity of mTOR is increased by activation of Akt in response to insulin, IGF-I, or other growth factors. Some mechanisms by which dexamethasone reduces Akt activity have been elucidated. In cultured cells, dexamethasone increased p85 expression and phosphorylation of Ser307 on IRS-1, resulting in reduced IRS-1-associated PI3 kinase activity (16). These deficits in IGF-I receptor signaling may be exacerbated by glucocorticoid-induced repression of IGF-I expression (48,49). Although IGF-I receptor signaling is impaired, it appears to remain functional after dexamethasone treatment, because exogenous IGF-I blocks activation of FOXO1 and atrophy of cultured muscle cells (47). It is noteworthy that testosterone prevented dexamethasone-induced reductions in IRS-1 and increases in p85 regulatory subunit for PI3 kinase. These actions of testosterone may improve coupling of the IGF-I receptor to PI3-kinase, Akt, and mTOR.

Comparison with testosterone effects in other studies of skeletal muscle

Several studies have examined effects of testosterone on the transcriptome in skeletal muscle. One compared gene expression in the gastrocnemius muscle of wild-type and AR knockout mice (50). In agreement with our findings, loss of the AR resulted in altered expression of genes linked to myoblast differentiation, muscle contraction, myogenesis, transcriptional regulation, signal transduction, cell cycle regulation, and RNA splicing. There was minimal overlap, however, between the genes regulated by knockout of the AR and genes regulated in opposition in our study, with only approximately 10% of genes being regulated by testosterone in both cases. Of interest, and in contrast to the findings in our study, loss of the AR had no effect on expression of FOXO1.

Microarray analysis was also used to characterize the effects of 14 d of treatment with testosterone on gene expression in the vastus lateralis muscle of HIV-infected men who were losing weight (51). Consistent with our findings, among genes regulated by testosterone in these individuals, enriched biological themes included IGF-I signaling, muscle development, transcriptional regulation, cycle control, and apoptosis. In contrast to our findings, in HIV-infected men, no effect of testosterone was observed on REDD1, FOXO1, the p85 subunit of PI3 kinase, or IRS-1.

We suggest that the differences in results between these studies with respect to genes affected by testosterone reflect the very different disease contexts in which testosterone was studied. Specifically and in contrast to the other two studies, we examined effects of testosterone under conditions where levels of glucocorticoids were high in the absence of infection or inflammation. Direct evidence that the physiological state of muscle determines its response to androgens comes from findings that nandrolone reduced denervation atrophy and expression of MAFbx and MuRF1 within 7 d when begun 28 d after nerve transection, but this agent had no effect on these parameters over the 14 d when begun coincident with nerve transection (25). Muscle atrophy due to glucocorticoids, infection or inflammation, or disuse is linked to activation of final common pathways converging on FOXO1 and is dependent upon MAFbx and MuRF1 (6,8,52). However, the initial upstream signals that initiate such atrophy are diverse and likely to activate or inhibit additional pathways acting on or in parallel with FOXO1 (e.g. TNF receptors, NF-κB, and/or p38 MAPK).

Conclusions

The ability of androgens to increase muscle size and strength and to prevent or reverse atrophy due to disuse, HIV infection, burns, and glucocorticoids has led to the clinical use of these agents in muscle loss and to the realization that development of derivatives that lack adverse effects on prostate and other tissues should be pursued. Our findings suggest that in muscle loss states, androgens regulate key genes controlling the activity of the intertwined signaling, degradative, and transcriptional networks that determine levels of mTOR activity, protein synthetic rate, and turnover of myofibrillar proteins, and thereby regulate muscle size. Our findings indicate a key role for repression by testosterone of REDD1 in mitigating negative effects of glucocorticoids on mTOR activity and protein synthesis, thereby suggesting that these testosterone actions are involved in protection against glucocorticoid-induced muscle loss. In dexamethasone-induced atrophy, testosterone may act through additional genes, which include FOXO1, and determinants of IGF-I receptor coupling to downstream kinases. The differences in skeletal muscle genes regulated by androgens in conditions of muscle atrophy point to additional levels of regulation, which are likely to be largely transcriptional, and determine the effects of testosterone on gene expression in muscle atrophy states.

Supplementary Material

[Supplemental Data]
en.2009-0530_index.html (730B, html)

Acknowledgments

We thank the Microarray Shared Research Facility for their assistance with the microarray studies reported here.

Footnotes

This work was supported by the Veterans Health Administration, Rehabilitation Research and Development Service (Grant B4162C to W.A.B., and B3347K and B3522R to C.C.). R.D.B. was supported by National Institutes of Health Grant GM054508.

Disclosure Summary: W.A.B. and C.C. received grant support from the Veterans Health Administration Rehabilitation Research and Development Service. Y.W., W.Z., J.Z., Y.Z., W.Q., J.P., and R.D.B. have nothing to disclose.

First Published Online December 23, 2009

Abbreviations: AR, Androgen receptor; 4EBP1, eIF4E binding protein 1; FOXO1, Forkhead box O1; GR, glucocorticoid receptor; IGFBP-3, IGF binding protein-3; MAFbx, muscle atrophy F-box; mTOR, mammalian target of rapamycin; MuRF1, muscle ring finger 1; qPCR, real-time PCR.

References

  1. Kayali AG, Young VR, Goodman MN 1987 Sensitivity of myofibrillar proteins to glucocorticoid-induced muscle proteolysis. Am J Physiol Endocrinol Metab 252(5 Pt 1):E621–E626 [DOI] [PubMed] [Google Scholar]
  2. Millward DJ, Garlick PJ, Nnanyelugo DO, Waterlow JC 1976 The relative importance of muscle protein synthesis and breakdown in the regulation of muscle mass. Biochem J 156:185–188 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Auclair D, Garrel DR, Chaouki Zerouala A, Ferland LH 1997 Activation of the ubiquitin pathway in rat skeletal muscle by catabolic doses of glucocorticoids. Am J Physiol 272:C1007–C1016 [DOI] [PubMed] [Google Scholar]
  4. Jackman RW, Kandarian SC 2004 The molecular basis of skeletal muscle atrophy. Am J Physiol Cell Physiol 287:C834–C843 [DOI] [PubMed] [Google Scholar]
  5. Pickart CM 2001 Mechanisms underlying ubiquitination. Annu Rev Biochem 70:503–533 [DOI] [PubMed] [Google Scholar]
  6. Bodine SC, Latres E, Baumhueter S, Lai VK, Nunez L, Clarke BA, Poueymirou WT, Panaro FJ, Na E, Dharmarajan K, Pan ZQ, Valenzuela DM, DeChiara TM, Stitt TN, Yancopoulos GD, Glass DJ 2001 Identification of ubiquitin ligases required for skeletal muscle atrophy. Science 294:1704–1708 [DOI] [PubMed] [Google Scholar]
  7. Lecker SH, Jagoe RT, Gilbert A, Gomes M, Baracos V, Bailey J, Price SR, Mitch WE, Goldberg AL 2004 Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression. FASEB J 18:39–51 [DOI] [PubMed] [Google Scholar]
  8. Clarke BA, Drujan D, Willis MS, Murphy LO, Corpina RA, Burova E, Rakhilin SV, Stitt TN, Patterson C, Latres E, Glass DJ 2007 The E3 Ligase MuRF1 degrades myosin heavy chain protein in dexamethasone-treated skeletal muscle. Cell Metab 6:376–385 [DOI] [PubMed] [Google Scholar]
  9. Shah OJ, Kimball SR, Jefferson LS 2000 Acute attenuation of translation initiation and protein synthesis by glucocorticoids in skeletal muscle. Am J Physiol Endocrinol Metab 278:E76–E82 [DOI] [PubMed] [Google Scholar]
  10. Shah OJ, Kimball SR, Jefferson LS 2000 Glucocorticoids abate p70(S6k) and eIF4E function in L6 skeletal myoblasts. Am J Physiol Endocrinol Metab 279:E74–E82 [DOI] [PubMed] [Google Scholar]
  11. Wang H, Kubica N, Ellisen LW, Jefferson LS, Kimball SR 2006 Dexamethasone represses signaling through the mammalian target of rapamycin in muscle cells by enhancing expression of REDD1. J Biol Chem 281:39128–39134 [DOI] [PubMed] [Google Scholar]
  12. Proud CG 2007 Signalling to translation: how signal transduction pathways control the protein synthetic machinery. Biochem J 403:217–234 [DOI] [PubMed] [Google Scholar]
  13. Lang CH, Frost RA, Vary TC 2007 Regulation of muscle protein synthesis during sepsis and inflammation. Am J Physiol Endocrinol Metab 293:E453–E459 [DOI] [PubMed] [Google Scholar]
  14. Reiling JH, Sabatini DM 2006 Stress and mTORture signaling. Oncogene 25:6373–6383 [DOI] [PubMed] [Google Scholar]
  15. Sandri M, Sandri C, Gilbert A, Skurk C, Calabria E, Picard A, Walsh K, Schiaffino S, Lecker SH, Goldberg AL 2004 Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy. Cell 117:399–412 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Wang X, Hu J, Price SR 2007 Inhibition of PI3-kinase signaling by glucocorticoids results in increased branched-chain amino acid degradation in renal epithelial cells. Am J Physiol Cell Physiol 292:C1874–C1879 [DOI] [PubMed] [Google Scholar]
  17. Waddell DS, Baehr LM, van den Brandt J, Johnsen SA, Reichardt HM, Furlow JD, Bodine SC 2008 The glucocorticoid receptor and Foxo1 synergistically activate the skeletal muscle atrophy-associated Murf1 gene. Am J Physiol Endocrinol Metab 295:E785–E797 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Southgate RJ, Neill B, Prelovsek O, El-Osta A, Kamei Y, Miura S, Ezaki O, McLoughlin TJ, Zhang W, Unterman TG, Febbraio MA 2007 FOXO1 regulates the expression of 4E-BP1 and inhibits mTOR signaling in mammalian skeletal muscle. J Biol Chem 282:21176–21186 [DOI] [PubMed] [Google Scholar]
  19. Harvey KF, Mattila J, Sofer A, Bennett FC, Ramsey MR, Ellisen LW, Puig O, Hariharan IK 2008 FOXO-regulated transcription restricts overgrowth of Tsc mutant organs. J Cell Biol 180:691–696 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Urban RJ, Bodenburg YH, Gilkison C, Foxworth J, Coggan AR, Wolfe RR, Ferrando A 1995 Testosterone administration to elderly men increases skeletal muscle strength and protein synthesis. Am J Physiol 269:E820–E826 [DOI] [PubMed] [Google Scholar]
  21. Bhasin S, Woodhouse L, Casaburi R, Singh AB, Bhasin D, Berman N, Chen X, Yarasheski KE, Magliano L, Dzekov C, Dzekov J, Bross R, Phillips J, Sinha-Hikim I, Shen R, Storer TW 2001 Testosterone dose-response relationships in healthy young men. Am J Physiol Endocrinol Metab 281:E1172–E1181 [DOI] [PubMed] [Google Scholar]
  22. Crawford BA, Liu PY, Kean MT, Bleasel JF, Handelsman DJ 2003 Randomized placebo-controlled trial of androgen effects on muscle and bone in men requiring long-term systemic glucocorticoid treatment. J Clin Endocrinol Metab 88:3167–3176 [DOI] [PubMed] [Google Scholar]
  23. van Balkom RH, Dekhuijzen PN, van der Heijden HF, Folgering HT, Fransen JA, van Herwaarden CL 1999 Effects of anabolic steroids on diaphragm impairment induced by methylprednisolone in emphysematous hamsters. Eur Respir J 13:1062–1069 [DOI] [PubMed] [Google Scholar]
  24. Van Balkom RH, Dekhuijzen PN, Folgering HT, Veerkamp JH, Van Moerkerk HT, Fransen JA, Van Herwaarden CL 1998 Anabolic steroids in part reverse glucocorticoid-induced alterations in rat diaphragm. J Appl Physiol 84:1492–1499 [DOI] [PubMed] [Google Scholar]
  25. Zhao W, Pan J, Zhao Z, Wu Y, Bauman WA, Cardozo CP 2008 Testosterone protects against dexamethasone-induced muscle atrophy, protein degradation, and MAFbx up-regulation. J Steroid Biochem Mol Biol 110:125–129 [DOI] [PubMed] [Google Scholar]
  26. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159 [DOI] [PubMed] [Google Scholar]
  27. Barrett T, Troup DB, Wilhite SE, Ledoux P, Rudnev D, Evangelista C, Kim IF, Soboleva A, Tomashevsky M, Edgar R 2007 NCBI GEO: mining tens of millions of expression profiles—database and tools update. Nucleic Acids Res 35:D760–D765 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Dennis Jr G, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA 2003 DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol 4:P3 [PubMed] [Google Scholar]
  29. Safran M, Chalifa-Caspi V, Shmueli O, Olender T, Lapidot M, Rosen N, Shmoish M, Peter Y, Glusman G, Feldmesser E, Adato A, Peter I, Khen M, Atarot T, Groner Y, Lancet D 2003 Human gene-centric databases at the Weizmann Institute of Science: GeneCards, UDB, CroW 21 and HORDE. Nucleic Acids Res 31:142–146 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Zhao W, Pan J, Wang X, Wu Y, Bauman WA, Cardozo CP 2008 Expression of the muscle atrophy factor MAFbx is suppressed by testosterone. Endocrinology 149:5449–5460 [DOI] [PubMed] [Google Scholar]
  31. Haddad F, Roy RR, Zhong H, Edgerton VR, Baldwin KM 2003 Atrophy responses to muscle inactivity. II. Molecular markers of protein deficits. J Appl Physiol 95:791–802 [DOI] [PubMed] [Google Scholar]
  32. Hyatt JP, Roy RR, Baldwin KM, Edgerton VR 2003 Nerve activity-independent regulation of skeletal muscle atrophy: role of MyoD and myogenin in satellite cells and myonuclei. Am J Physiol Cell Physiol 285:C1161–C1173 [DOI] [PubMed] [Google Scholar]
  33. Livak KJ, Schmittgen TD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(-ΔΔC(T)) method. Methods 25:402–408 [DOI] [PubMed] [Google Scholar]
  34. Hunter RB, Kandarian SC 2004 Disruption of either the Nfkb1 or the Bcl3 gene inhibits skeletal muscle atrophy. J Clin Invest 114:1504–1511 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wu AL, Kim JH, Zhang C, Unterman TG, Chen J 2008 Forkhead box protein O1 negatively regulates skeletal myocyte differentiation through degradation of mammalian target of rapamycin pathway components. Endocrinology 149:1407–1414 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wang Z, Malone MH, Thomenius MJ, Zhong F, Xu F, Distelhorst CW 2003 Dexamethasone-induced gene 2 (dig2) is a novel pro-survival stress gene induced rapidly by diverse apoptotic signals. J Biol Chem 278:27053–27058 [DOI] [PubMed] [Google Scholar]
  37. Sofer A, Lei K, Johannessen CM, Ellisen LW 2005 Regulation of mTOR and cell growth in response to energy stress by REDD1. Mol Cell Biol 25:5834–5845 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Brugarolas J, Lei K, Hurley RL, Manning BD, Reiling JH, Hafen E, Witters LA, Ellisen LW, Kaelin Jr WG 2004 Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex. Genes Dev 18:2893–2904 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Corradetti MN, Inoki K, Guan KL 2005 The stress-inducted proteins RTP801 and RTP801L are negative regulators of the mammalian target of rapamycin pathway. J Biol Chem 280:9769–9772 [DOI] [PubMed] [Google Scholar]
  40. Bodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL, Bauerlein R, Zlotchenko E, Scrimgeour A, Lawrence JC, Glass DJ, Yancopoulos GD 2001 Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat Cell Biol 3:1014–1019 [DOI] [PubMed] [Google Scholar]
  41. Ohanna M, Sobering AK, Lapointe T, Lorenzo L, Praud C, Petroulakis E, Sonenberg N, Kelly PA, Sotiropoulos A, Pende M 2005 Atrophy of S6K1(−/−) skeletal muscle cells reveals distinct mTOR effectors for cell cycle and size control. Nat Cell Biol 7:286–294 [DOI] [PubMed] [Google Scholar]
  42. Pistollato F, Rampazzo E, Abbadi S, Della Puppa A, Scienza R, D'Avella D, Denaro L, Te Kronnie G, Panchision DM, Basso G 2009 Molecular mechanisms of HIF-1α modulation induced by oxygen tension and BMP2 in glioblastoma derived cells. PLOS One 4:e6206 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Burt-Pichat B, Lafage-Proust MH, Duboeuf F, Laroche N, Itzstein C, Vico L, Delmas PD, Chenu C 2005 Dramatic decrease of innervation density in bone after ovariectomy. Endocrinology 146:503–510 [DOI] [PubMed] [Google Scholar]
  44. Sun L, Chang J, Kirchhoff SR, Knowlton AA 2000 Activation of HSF and selective increase in heat-shock proteins by acute dexamethasone treatment. Am J Physiol Heart Circ Physiol 278:H1091–H1097 [DOI] [PubMed] [Google Scholar]
  45. Danhaive PA, Rousseau GG 1988 Evidence for sex-dependent anabolic response to androgenic steroids mediated by muscle glucocorticoid receptors in the rat. J Steroid Biochem 29:575–581 [DOI] [PubMed] [Google Scholar]
  46. Zhao J, Bauman WA, Huang R, Caplan AJ, Cardozo C 2004 Oxandrolone blocks glucocorticoid signaling in an androgen receptor-dependent manner. Steroids 69:357–366 [DOI] [PubMed] [Google Scholar]
  47. Stitt TN, Drujan D, Clarke BA, Panaro F, Timofeyva Y, Kline WO, Gonzalez M, Yancopoulos GD, Glass DJ 2004 The IGF-1/PI3K/Akt pathway prevents expression of muscle atrophy-induced ubiquitin ligases by inhibiting FOXO transcription factors. Mol Cell 14:395–403 [DOI] [PubMed] [Google Scholar]
  48. Kritsch KR, Murali S, Adamo ML, Ney DM 2002 Dexamethasone decreases serum and liver IGF-I and maintains liver IGF-I mRNA in parenterally fed rats. Am J Physiol Regul Integr Comp Physiol 282:R528–R536 [DOI] [PubMed] [Google Scholar]
  49. Delany AM, Canalis E 1995 Transcriptional repression of insulin-like growth factor I by glucocorticoids in rat bone cells. Endocrinology 136:4776–4781 [DOI] [PubMed] [Google Scholar]
  50. MacLean HE, Chiu WS, Notini AJ, Axell AM, Davey RA, McManus JF, Ma C, Plant DR, Lynch GS, Zajac JD 2008 Impaired skeletal muscle development and function in male, but not female, genomic androgen receptor knockout mice. FASEB J 22:2676–2689 [DOI] [PubMed] [Google Scholar]
  51. Montano M, Flanagan JN, Jiang L, Sebastiani P, Rarick M, LeBrasseur NK, Morris CA, Jasuja R, Bhasin S 2007 Transcriptional profiling of testosterone-regulated genes in the skeletal muscle of human immunodeficiency virus-infected men experiencing weight loss. J Clin Endocrinol Metab 92:2793–2802 [DOI] [PubMed] [Google Scholar]
  52. Sacheck JM, Hyatt JP, Raffaello A, Jagoe RT, Roy RR, Edgerton VR, Lecker SH, Goldberg AL 2007 Rapid disuse and denervation atrophy involve transcriptional changes similar to those of muscle wasting during systemic diseases. FASEB J 21:140–155 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

[Supplemental Data]
en.2009-0530_index.html (730B, html)
en.2009-0530_1.pdf (445.6KB, pdf)

Articles from Endocrinology are provided here courtesy of The Endocrine Society

RESOURCES