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J. Biol. Chem., Vol. 281, Issue 51, 39128-39134, December 22, 2006
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1
From the
Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, Massachusetts 02116
Received for publication, October 25, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Previous studies have shown that the down-regulation of protein synthesis caused by dexamethasone administration to rats in vivo (15) or the addition of the drug to the culture medium of L6 myoblasts (16, 17) temporally correlates with reduced phosphorylation of the mTOR substrates 4E-BP1 and S6K1, suggesting that glucocorticoids might decrease protein synthesis through repression of mTOR. The dexamethasone-induced reduction in 4E-BP1 and S6K1 phosphorylation is attenuated both by inhibitors of glucocorticoid receptor function and inhibitors of transcription and translation (16). Moreover, the glucocorticoid receptor is both necessary and sufficient for the dexamethasone-induced dephosphorylation of S6K1 (18). Mutational analysis of the glucocorticoid receptor reveals that the DNA binding and transcriptional activation functions, but not the transcriptional repression function, of the receptor are required for S6K1 regulation (18). Overall, the available evidence strongly suggests that the repression of mTOR signaling in response to glucocorticoids is a result of enhanced transcription of a gene that encodes a repressor of mTOR signaling. However, the identity of the putative mTOR repressor is unknown.
Recent studies have identified two novel repressors of mTOR signaling; proteins referred to as REDD1 and REDD2 (regulated in development and DNA damage responses; also known as RTP801 and RTP801l, respectively). REDD1 and REDD2 were originally identified as genes that are transcriptionally up-regulated in response to a variety of cellular stresses, including hypoxia (19–22) and exposure to arsenite (23), or by agents that cause DNA damage (24). More recent studies show that REDD1-induced repression of mTOR signaling requires Tuberin, the product of the TSC2 gene (19, 20, 25). Tuberin in a complex with Hamartin, the product of the TSC1 gene, functions as a GTPase-activating protein (GAP) toward a small G protein referred to as Rheb (Ras homologue enriched in brain), a positive upstream effector of mTOR (26). Binding of Rheb·GTP to mTOR enhances, whereas binding of Rheb·GDP inhibits, mTOR activity (27, 28). Thus, activation of Tuberin suppresses mTOR signaling by enhancing Rheb GTPase activity, resulting in an increase in the proportion of Rheb in an inhibitory complex with GDP (29–32). However, although it is clear that REDD1 requires Tuberin to repress mTOR signaling, the mechanism through which REDD1 might act to enhance Tuberin function is as yet undefined.
A recent study (33) reported that dexamethasone induces the expression of a gene referred to as dig2 (dexamethasone-induced gene-2). Sequencing of the dig2 mRNA revealed that it is identical to REDD1 (33), suggesting a possible link between induction of Dig2 expression and repression of signaling through mTOR. However, in that study, dexamethasone was found to induce REDD1 expression only in lymphoid cells and not in nonlymphoid cells, bringing into question the role of REDD1 in mediating the actions of glucocorticoids on mTOR signaling in other types of cells or tissues.
In the present study, the hypothesis that glucocorticoids repress mTOR signaling in skeletal muscle through induction of REDD1 and/or REDD2 was examined. In contrast to an earlier study (33) suggesting a lymphoid-specific up-regulation of REDD1 by dexamethasone, our results show that expression of REDD1 is enhanced by dexamethasone both in skeletal muscle in vivo and L6 myoblasts in culture. Moreover, REDD2 expression is repressed by dexamethasone in skeletal muscle and L6 myoblasts. Importantly, dexamethasone promotes the assembly of the active Tuberin·Hamartin complex, and complex assembly requires increased REDD1 expression. Finally, overexpression of Rheb attenuates the dexamethasone-induced repression of mTOR signaling. Overall, the results are consistent with a model wherein dexamethasone up-regulates REDD1 expression in muscle and REDD1 subsequently activates the Tuberin·Hamartin complex, resulting in repression of mTOR signaling.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Animal Protocol—The animal experimental protocol used for the studies described herein was reviewed and approved by the Institutional Animal Care and Use Committee of The Pennsylvania State University College of Medicine. Male Sprague-Dawley rats (200–300 g) were maintained on a 12 h light-12 h dark cycle with food and water provided ad libitum. Rats were injected intraperitoneally with dexamethasone-sodium phosphate (100 µg/100 mg of body wt) in sterile saline (0.15 M NaCl) (15). The control group received an equal volume of saline. Three hours later, half of the animals in each group were administered leucine (1.35 mg/kg of body weight) by oral gavage (35). Four hours after drug administration, rats were killed by decapitation. The left gastrocnemius and plantaris were rapidly excised as a unit and frozen between aluminum blocks precooled to the temperature of liquid nitrogen. The contralateral muscles were similarly excised, weighed, and homogenized in 7 volumes of ice-cold lysis buffer containing 20 mM HEPES, pH 7.4, 2 mM EGTA, 50 mM NaF, 100 mM KCl, 0.2 mM EDTA, 50 mM 
-glycerophosphate, 1 mM dithiothreitol, 1 mM benzamidine, 0.5 mM sodium vanadate, and 0.1 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged immediately at 1000 x g for 3 min at 4 °C, and the supernatant was collected for analysis.
Cell Culture, RNA Interference, and Transfection—L6 myoblasts were grown in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum (Atlas) and 1% penicillin/streptomycin (Invitrogen) at 37 °C in a humidified atmosphere containing 5% CO2. Cells (1 x 106) were electroporated using a NucleofectorTM device (Amaxa Biosystems) in the presence of 100 µl of Nucleofector Solution V and a mixture of three siRNAs (each at 1 µM) directed against REDD1. 48 h later, cells were deprived of serum for 
18 h and then treated with dexamethasone (1 µM) for 4 h prior to harvesting. Rat2 fibroblasts also were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% heat-inactivated fetal bovine serum (Atlas). Transfections were performed using LipofectamineTM 2000 (Invitrogen) according to the manufacturer's instructions.
Western Blot Analysis and Immunoprecipitation—The supernatants derived from muscle homogenates and cell lysates were combined with an equal volume of SDS sample buffer and boiled at 100 °C for 5 min. The samples were then subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted with primary antibody as indicated in the figure legends. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL) using a GeneGnome HR bioimaging system (Syngene). Immunoprecipitations were performed by incubating 1000 x g supernatants of muscle homogenate or cell lysate overnight with anti-eIF4E antibody or anti-Tuberin antibody followed by isolation of the immune complexes using BioMag IgG beads. The immunoprecipitates were washed 3 times with immunoprecipitation buffer (40 mM HEPES, pH 7.5, 120 mM NaCl, 1 mM EDTA, 10 mM sodium pyrophosphate, 10 mM -glycerophosphate, 50 mM NaF, 0.3% CHAPS, 1 mM dithiothreitol, 1 mM benzamidine, 0.5 mM sodium vanadate, and 0.1 mM phenylmethylsulfonyl fluoride), and proteins were eluted by boiling in 1x SDS sample buffer. Samples were then subjected to Western blot analysis as described previously (36, 37).
Isolation of total RNA and Reverse Transcription—RNA was extracted from muscle and cells using Trizol reagent according to the manufacture's protocol (Invitrogen). RNA concentration and quality were assessed spectrophotometrically using a Beckman Coulter DU 640 (Fullerton, CA). RNA (1 µg) was reversed transcribed using 50 units of Superscript II RNase H– reverse transcriptase (Invitrogen) in a 21-µl reaction volume containing 0.5 µg/µl oligo(dT), 10 mM dNTP mix, 10x RT buffer (25 mM MgCl2, 0.1 M dithiothreitol), and RNaseOUT recombinant RNase inhibitor (Invitrogen). The mixture of RNA and oligo(dT) was heated at 65 °C for 5 min and then chilled on ice; then all other reagents were added, and RT-PCR was conducted at 42 °C for 50 min and 70 °C for 15 min. The reaction mixture was then diluted 1:8 before analysis by Real-time PCR as described below.
Real-time PCR Analysis of GAPDH, REDD1, and REDD2 mRNA—Quantitative Real-time RT-PCR was conducted using an Applied Biosystems Prism 7300 Real Time PCR system using a QuantiTech SYBR Green PCR kit (Qiagen) according to the manufacturer's instructions. The primers used were as follows: GAPDH forward primer, 5'-GGGCTGCCTTCTCTTGTGA-3'; reverse primer, 5'-TGAACTTGCCGTGGGTAGA-3'; REDD1 forward primer, 5'-TAGTGCCCACCTTTCAGTTG-3'; reverse primer, 5'-GTCAGGGACTGGCTGTAACC-3'; REDD2 forward primer, 5'-CCAGCCTCAAGGACTTCTTC-3'; reverse primer, 5'-TCCTCAATGACTGTCGTTCC-3'. All PCR reactions were performed in 96-well optical plates (Applied Biosystems) containing each specific forward and reverse primer at 0.4 µM, 5 µl of diluted cDNA, and 12.5 µl of SYBR Green I in a total reaction volume of 25 µl. Each run included a buffer blank and no-template control to test for contamination of assay reagents. Gene expression levels were calculated and normalized to GAPDH mRNA expression.
Statistical Analysis—The data are expressed as mean ± S.E. One-way analysis of variance and Student's t test were used to compare differences among groups. p < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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To further explore the regulation of REDD1 and REDD2 mRNA expression and mTOR signaling by dexamethasone, the effect of the drug in L6 myoblasts was examined. As shown in Fig. 3, mTOR signaling was repressed in L6 myoblasts 4 h after dexamethasone administration, as assessed by a shift of both S6K1 (Fig. 3A) and 4E-BP1 (Fig. 3B) into less phosphorylated, i.e. more rapidly migrating, forms. As observed in skeletal muscle from dexamethasone-treated rats, dexamethasone enhanced the expression of REDD1 mRNA (Fig. 3C) and simultaneously repressed REDD2 mRNA expression in L6 myoblasts (Fig. 3D). Moreover, dexamethasone treatment resulted in a 5-fold increase in REDD1 protein content compared with controls (Fig. 2B).
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50% of the value observed in cells that had been administered a control siRNA. Moreover, administration of REDD1 siRNA prior to dexamethasone dramatically attenuated the drug-induced expression of the mRNA. As shown in Fig. 4B, the control siRNA had no effect on the dexamethasone-induced decrease in S6K1 phosphorylation. However, in cells transfected with the REDD1 siRNA, dexamethasone had little or no effect on S6K1 phosphorylation, suggesting that the drug repressed mTOR signaling through enhanced expression of REDD1. Further support for this suggestion was provided by the observation that the dexamethasone-induced dephosphorylation of 4E-BP1 was also attenuated in cells transfected with REDD1 siRNA (Fig. 4C). An important mechanism through which mTOR signaling is regulated involves the Tuberin·Hamartin complex. As shown in Fig. 4D, dexamethasone enhanced the amount of Hamartin associated with Tuberin in L6 myoblasts transfected with control siRNA. However, the drug had no effect on assembly of the Tuberin·Hamartin complex in cells transfected with REDD1 siRNA, suggesting that REDD1 may function upstream of Tuberin·Hamartin in regulating mTOR signaling.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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A number of recent studies have suggested a possible link between induction of REDD1 expression and repression of signaling through mTOR. Studies in both Drosophila (20) and mammalian cells (39) show that exogenous overexpression of either REDD1 or REDD2 represses mTOR signaling, whereas decreased expression of either protein increases signaling through mTOR. Moreover, in mouse embryo fibroblasts lacking REDD1, conditions that normally promote expression of the protein have little effect on the phosphorylation state of S6K1 or 4E-BP1 (25).
One mechanism through which mTOR is regulated involves its association with Rheb. Rheb binds to mTOR near the amino-terminal end of its catalytic domain (27). When it is associated with GTP, Rheb acts to enhance mTOR activity, but when it is associated with GDP, it is a repressor. The finding in the present study, that overexpression of Rheb in L6 myoblasts attenuates the down-regulation of mTOR signaling caused by dexamethasone, suggests that the drug may act to repress mTOR signaling by enhancing the proportion of Rheb present in the GDP-bound form. Such a suggestion is also supported by the observation that overexpression of Rheb in mouse embryo fibroblasts reverses the effect of exogenous expression of REDD1 on mTOR signaling (25).
Further evidence supporting the suggestion that dexamethasone may act to increase the amount of Rheb in the GDP-bound form is the novel observation that dexamethasone enhances the assembly of the Tuberin·Hamartin complex (Fig. 4). Moreover, REDD1 knockdown using siRNA prevented dexamethasone-induced assembly of the complex, suggesting that REDD1 acts to repress mTOR by signaling to Tuberin and Hamartin. Although Tuberin by itself exhibits GAP activity toward Rheb in in vitro assays, co-expression of both Tuberin and Hamartin is required for effective repression of mTOR signaling in cells in culture (40). Moreover, mutations in Tuberin that interfere with its binding to Hamartin repress the growth inhibitory effects of the protein in COS1 cells (41). That REDD1 functions through the Tuberin·Hamartin complex to modulate mTOR function is further supported by recent studies showing that REDD1 signaling to mTOR requires Tuberin. For example, in cells in which Tuberin expression is reduced using siRNA, induction of REDD1 expression has little or no effect on S6K1 phosphorylation (19, 42). Moreover, in contrast to wild type cells, exogenous expression of REDD1 in mouse embryo fibroblasts lacking Tuberin has no effect on S6K1 phosphorylation (25). However, to our knowledge, the effect of exogenous expression of REDD1 on assembly of the Tuberin·Hamartin complex has not been examined previously.
Although the mechanism through which REDD1 might regulate Tuberin function is unknown, one possibility involves changes in Tuberin phosphorylation. In this regard, Tuberin is regulated through phosphorylation by a number of protein kinases. For example, phosphorylation of Tuberin on multiple residues by protein kinase B (PKB; also known as Akt) is associated with inhibition of its GAP activity toward Rheb, resulting in enhanced signaling through mTOR (reviewed in Ref. 43). However, in mammalian cells, overexpression of REDD1 has no effect on PKB phosphorylation (39), and in Drosophila REDD1 functions downstream of PKB (20) suggesting that REDD1 does not regulate Tuberin through a PKB-dependent process. Tuberin function is also regulated through phosphorylation by the extracellular-signal regulated kinase (ERK) (44), but the finding that dexamethasone does not activate ERK in L6 myoblasts (15) suggests that ERK is not downstream of REDD1. The AMP-activated protein kinase (AMPK) also phosphorylates Tuberin, and in contrast to phosphorylation by PKB or ERK, phosphorylation by AMPK enhances Tuberin activity, resulting in repressed mTOR signaling (45). However, although REDD1 does not require AMPK to repress mTOR signaling (19, 25), AMPK requires REDD1 to modulate mTOR activity (25). Overall, the available evidence suggests that REDD1 modulates Tuberin·Hamartin assembly through an as yet undiscovered mechanism.
Of relevance to the mechanism through which REDD1 might modulate Tuberin function is the observation that oral administration of leucine to fasted rats has no effect on either basal or dexamethasone-induced REDD1 expression. In contrast, the magnitude of the leucine-induced increase in 4E-BP1 and S6K1 phosphorylation is unaffected by dexamethasone (38), suggesting that leucine acts downstream of REDD1 to activate mTOR. Amino acids have little (28, 46) or no effect (47) on the amount of GTP bound to Rheb. Moreover, in cells lacking Tuberin, amino acid deprivation represses mTOR signaling, and the readdition of amino acids to deprived cells restores phosphorylation of 4E-BP1 and S6K1 (28, 46), suggesting that amino acid regulation of mTOR can occur through a Tuberin-independent mechanism. Although the putative Tuberin-independent signaling pathway regulated by amino acids has not been delineated, a recent study suggests that amino acids promote the association of Rheb with mTOR and thereby enhance mTOR protein kinase activity (27). Therefore, in part, amino acids may overcome the repressive effects of glucocorticoids on mTOR signaling by stimulating the binding of Rheb to mTOR. A second possibility is that amino acids might promote the activation of a guanine nucleotide exchange protein (GEF) for Rheb. Although a Rheb GEF has yet to be identified, activation of such a protein might overwhelm the repressive effect of REDD1-mediated Tuberin activation and thereby enhance mTOR signaling. Such an idea will need to be addressed in future studies.
In conclusion, the present work demonstrates a pivotal role for REDD1 in down-regulating mTOR function in response to dexamethasone treatment. The results of the present study, in combination with previously published reports, support a model wherein dexamethasone binds to the glucocorticoid receptor, thereby enhancing REDD1 gene transcription. Through an as yet unidentified mechanism, REDD1 promotes the association of Hamartin with Tuberin (41) resulting in enhanced GAP activity toward Rheb. The resulting increase in Rheb complexed with GDP represses mTOR function, leading to decreased phosphorylation of both S6K1 and 4E-BP1 and subsequent alterations in mRNA translation and protein synthesis.
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FOOTNOTES |
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1 To whom correspondence should be addressed: Dept. of Cellular and Molecular Physiology, H166, The Pennsylvania State University College of Medicine, 500 University Dr., Hershey, PA 17033. Tel.: 717-531-8970; Fax: 717-531-7667; E-mail: skimball{at}psu.edu.
2 The abbreviations used are: mTOR, mammalian target of rapamycin; siRNA, short interfering RNA; eIF, eukaryotic initiation factor; 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcription; S6K1, ribosomal protein S6 kinase 1; GAP, GTPase-activating protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PKB, protein kinase B; ERK, extracellular-signal regulated kinase; AMPK, AMP-activated protein kinase.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, p < 0.001 versus leucine; **, p < 0.05 versus control; 
, 
, and 
forms of the proteins are denoted to the right of the respective blots. The results are representative of four studies performed. Within each study, three dishes of cells were analyzed individually. Representative blots are shown. RNA was isolated from L6 myoblasts and analyzed for the amount of REDD1 (C), REDD2 (D), and GAPDH mRNAs (not shown) present in the sample by quantitative RT-PCR as described under "Experimental Procedures."
