Dependence of dexamethasone-induced Akt/FOXO1 signaling,upregulation of MAFbx,and protein catabolism upon the glucocorticoid receptor

The muscle ubiquitin ligases MAFbx and MuRF1 are upregulated in and promote muscle atrophy. Upregulation of MAFbx and MuRF1 by glucocorticoids has been linked to activation of FOXO1 and FOXO3A resulting from reduced Akt activity. We determined the requirements for the glucocorticoid receptor (GR) in these biological responses in C2C12 cells in which GR expression was knocked down by stable expression of an shRNA. Loss of GR prevented dexamethasone-induced increases in protein catabolism. Loss of GR, or inhibition of ligand binding to GR with RU486, prevented upregulation of MAFbx and MuRF1 by dexamethasone. Loss of GR also prevented dexamethasone-induced decreases in Akt phosphorylation, and increases in the fraction of FOXO1 that was unphosphorylated. The findings establish a requirement for the GR in activating molecular signals that promote muscle protein catabolism.     

Glucocorticoid-induced skeletal muscle atrophy is associated with upregulation of myostatin gene expression

The mechanisms by which excessive glucocorticoids cause muscular atrophy remain unclear. We previously demonstrated that dexamethasone increases the expression of myostatin, a negative regulator of skeletal muscle mass, in vitro. In the present study, we tested the hypothesis that dexamethasone-induced muscle loss is associated with increased myostatin expression in vivo. Daily administration (60, 600, 1,200 micro g/kg body wt) of dexamethasone for 5 days resulted in rapid, dose-dependent loss of body weight (-4.0, -13.4, -17.2%, respectively, P < 0.05 for each comparison), and muscle atrophy (6.3, 15.0, 16.6% below controls, respectively). These changes were associated with dose-dependent, marked induction of intramuscular myostatin mRNA (66.3, 450, 527.6% increase above controls, P < 0.05 for each comparison) and protein expression (0.0, 260.5, 318.4% increase above controls, P < 0.05). We found that the effect of dexamethasone on body weight and muscle loss and upregulation of intramuscular myostatin expression was time dependent. When dexamethasone treatment (600 micro g. kg-1. day-1) was extended from 5 to 10 days, the rate of body weight loss was markedly reduced to approximately 2% within this extended period. The concentrations of intramuscular myosin heavy chain type II in dexamethasone-treated rats were significantly lower (-43% after 5-day treatment, -14% after 10-day treatment) than their respective corresponding controls. The intramuscular myostatin concentration in rats treated with dexamethasone for 10 days returned to basal level. Concurrent treatment with RU-486 blocked dexamethasone-induced myostatin expression and significantly attenuated body loss and muscle atrophy. We propose that dexamethasone-induced muscle loss is mediated, at least in part, by the upregulation of myostatin expression through a glucocorticoid receptor-mediated pathway.     

β-Hydroxy-β-methylbutyrate (HMB) prevents dexamethasone-induced myotube atrophy

High levels of glucocorticoids result in muscle wasting and weakness. β-hydroxy-β-methylbutyrate (HMB) attenuates the loss of muscle mass in various catabolic conditions but the influence of HMB on glucocorticoid-induced muscle atrophy is not known. We tested the hypothesis that HMB prevents dexamethasone-induced atrophy in cultured myotubes. Treatment of cultured L6 myotubes with dexamethasone resulted in increased protein degradation and expression of atrogin-1 and MuRF1, decreased protein synthesis and reduced myotube size. All of these effects of dexamethasone were attenuated by HMB. Additional experiments provided evidence that the inhibitory effects of HMB on dexamethasone-induced increase in protein degradation and decrease in protein synthesis were regulated by p38/MAPK- and PI3K/Akt-dependent cell signaling, respectively. The present results suggest that glucocorticoid-induced muscle wasting can be prevented by HMB.     

Dosing schedule-dependent attenuation of dexamethasone-induced muscle atrophy in mice

Many inflammatory and autoimmune diseases are treated using synthetic glucocorticoids. However, excessive glucocorticoid can often cause unpredictable effects including muscle atrophy. Endogenous glucocorticoid levels robustly fluctuate in a circadian manner and peak just before the onset of the active phase in both humans and nocturnal rodents. The present study determines whether muscle atrophy induced by exogenous glucocorticoid can be avoided by optimizing dosing times. We administered single daily doses of the glucocorticoid analog dexamethasone (Dex) to mice for 10 days at the times of day corresponding to peak (early night) or trough (early morning) endogenous glucocorticoid levels. Administration at the acrophase of endogenous glucocorticoids significantly attenuated Dex-induced wasting of the gastrocnemius (Ga) and tibialis anterior (TA) muscles that comprise mostly fast-twitch muscle fibers. Real-time RT-PCR revealed that the Dex-induced mRNA expression of genes encoding the atrophy-related ubiquitin ligases Muscle Atrophy F-box (Fbxo32, also known as MAFbx/Atrogin-1) and Muscle RING finger 1 (Trim63, also known as MuRF1) in the Ga and TA muscles was significantly attenuated by Dex when administered during the early night. Dex negligibly affected the weight of the soleus (So) muscle that mostly comprises slow-twitch muscle fibers, but significantly and similarly decreased the weight of the spleen at both dosing times. These results suggest that glucocorticoid-induced muscle atrophy can be attenuated by optimizing the dosing schedule.     

Identification of essential sequences for cellular localization in the muscle-specific ubiquitin E3 ligase MAFbx/Atrogin 1

In skeletal muscle atrophy, upregulation and nuclear accumulation of the Ubiquitin E3 ligase MAFbx is essential for accelerated muscle protein loss, but the nuclear/cytoplasmic shuttling of MAFbx is undefined. Here we found that MAFbx contains two functional nuclear localization signals (NLS). Mutation or deletion of only one NLS induced cytoplasmic localization of MAFbx. We identified a non-classical NES located in the leucine charged domain (LCD) of MAFbx, which is leptomycin B insensitive. We demonstrated that mutation (L169Q) in LLXXL motif of LCD suppressed cytoplasmic retention of MAFbx. Nucleocytoplasmic shuttling of MAFbx represents a novel mechanism for targeting its substrates and its cytosolic partners in muscle atrophy.     

Dexamethasone-induced protein degradation in cultured myotubes is p300/HAT dependent

Muscle proteolysis during sepsis and other catabolic conditions is, at least in part, regulated by glucocorticoids. Dexamethasone-treated myotubes are a commonly used in vitro model of muscle wasting. We reported recently that treatment of cultured L6 myotubes with dexamethasone resulted in increased gene and protein expression of the nuclear cofactor p300 but it is not known whether glucocorticoids upregulate p300 histone acetyl transferase (HAT) activity in muscle and whether p300/HAT activity regulates glucocorticoid-induced muscle proteolysis. Here, we found that treatment of cultured L6 myotubes with dexamethasone resulted in increased nuclear p300/HAT activity. Treatment of myotubes with p300 siRNA or transfection of muscle cells with a plasmid expressing p300 that was mutated in its HAT activity domain blocked the dexamethasone-induced increase in protein degradation, supporting a role of p300/HAT in glucocorticoid-induced muscle proteolysis. In addition to increased HAT activity, treatment of the myotubes with dexamethasone resulted in reduced nuclear expression and activity of histone deacetylases (HDACs) 3 and 6. When myotubes were treated with the HDAC inhibitor trichostatin A, protein degradation increased to the same degree as in dexamethasone-treated myotubes. The results suggest that glucocorticoids increase HAT and decrease HDAC activities in muscle, changes that both favor hyperacetylation. The results also provide evidence that dexamethasone-induced protein degradation in cultured myotubes is, at least in part, regulated by p300/HAT activity.     

Glycogen synthase kinase-3β is required for the induction of skeletal muscle atrophy

Skeletal muscle atrophy commonly occurs in acute and chronic disease. The expression of the muscle-specific E3 ligases atrogin-1 (MAFbx) and muscle RING finger 1 (MuRF1) is induced by atrophy stimuli such as glucocorticoids or absence of IGF-I/insulin and subsequent Akt signaling. We investigated whether glycogen synthase kinase-3β (GSK-3β), a downstream molecule in IGF-I/Akt signaling, is required for basal and atrophy stimulus-induced expression of atrogin-1 and MuRF1, and myofibrillar protein loss in C(2)C(12) skeletal myotubes. Abrogation of basal IGF-I signaling, using LY294002, resulted in a prominent induction of atrogin-1 and MuRF1 mRNA and was accompanied by a loss of myosin heavy chain fast (MyHC-f) and myosin light chains 1 (MyLC-1) and -3 (MyLC-3). The synthetic glucocorticoid dexamethasone (Dex) also induced the expression of both atrogenes and likewise resulted in the loss of myosin protein abundance. Genetic ablation of GSK-3β using small interfering RNA resulted in specific sparing of MyHC-f, MyLC-1, and MyLC-3 protein levels after Dex treatment or impaired IGF-I/Akt signaling. Interestingly, loss of endogenous GSK-3β suppressed both basal and atrophy stimulus-induced atrogin-1 and MuRF1 expression, whereas pharmacological GSK-3β inhibition, using CHIR99021 or LiCl, only reduced atrogin-1 mRNA levels in response to LY294002 or Dex. In conclusion, our data reveal that myotube atrophy and myofibrillar protein loss are GSK-3β dependent, and demonstrate for the first time that basal and atrophy stimulus-induced atrogin-1 mRNA expression requires GSK-3β enzymatic activity, whereas MuRF1 expression depends solely on the physical presence of GSK-3β.     

Translational suppression of atrophic regulators by microRNA-23a integrates resistance to skeletal muscle atrophy

Muscle atrophy is caused by accelerated protein degradation and occurs in many pathological states. Two muscle-specific ubiquitin ligases, MAFbx/atrogin-1 and muscle RING-finger 1 (MuRF1), are prominently induced during muscle atrophy and mediate atrophy-associated protein degradation. Blocking the expression of these two ubiquitin ligases provides protection against muscle atrophy. Here we report that miR-23a suppresses the translation of both MAFbx/atrogin-1 and MuRF1 in a 3'-UTR-dependent manner. Ectopic expression of miR-23a is sufficient to protect muscles from atrophy in vitro and in vivo. Furthermore, miR-23a transgenic mice showed resistance against glucocorticoid-induced skeletal muscle atrophy. These data suggest that suppression of multiple regulators by a single miRNA can have significant consequences in adult tissues.     

A cell-autonomous role for the glucocorticoid receptor in skeletal muscle atrophy induced by systemic glucocorticoid exposure

Glucocorticoids (GCs) are important regulators of skeletal muscle mass, and prolonged exposure will induce significant muscle atrophy. To better understand the mechanism of skeletal muscle atrophy induced by elevated GC levels, we examined three different models: exogenous synthetic GC treatment [dexamethasone (DEX)], nutritional deprivation, and denervation. Specifically, we tested the direct contribution of the glucocorticoid receptor (GR) in skeletal muscle atrophy by creating a muscle-specific GR-knockout mouse line (MGR(e3)KO) using Cre-lox technology. In MGR(e3)KO mice, we found that the GR is essential for muscle atrophy in response to high-dose DEX treatment. In addition, DEX regulation of multiple genes, including two important atrophy markers, MuRF1 and MAFbx, is eliminated completely in the MGR(e3)KO mice. In a condition where endogenous GCs are elevated, such as nutritional deprivation, induction of MuRF1 and MAFbx was inhibited, but not completely blocked, in MGR(e3)KO mice. In response to sciatic nerve lesion and hindlimb muscle denervation, muscle atrophy and upregulation of MuRF1 and MAFbx occurred to the same extent in both wild-type and MGR(e3)KO mice, indicating that a functional GR is not required to induce atrophy under these conditions. Therefore, we demonstrate conclusively that the GR is an important mediator of skeletal muscle atrophy and associated gene expression in response to exogenous synthetic GCs in vivo and that the MGR(e3)KO mouse is a useful model for studying the role of the GR and its target genes in multiple skeletal muscle atrophy models.     

Cytokine-Induced Loss of Glucocorticoid Function: Effect of Kinase Inhibitors,Long-Acting β2-Adrenoceptor Agonist and Glucocorticoid Receptor Ligands

Acting on the glucocorticoid receptor (NR3C1), glucocorticoids are widely used to treat inflammatory diseases. However, glucocorticoid resistance often leads to suboptimal asthma control. Since glucocorticoid-induced gene expression contributes to glucocorticoid activity, the aim of this study was to use a 2×glucocorticoid response element (GRE) reporter and glucocorticoid-induced gene expression to investigate approaches to combat cytokine-induced glucocorticoid resistance. Pre-treatment with tumor necrosis factor-α (TNF) or interleukin-1β inhibited dexamethasone-induced mRNA expression of the putative anti-inflammatory genes RGS2 and TSC22D3, or just TSC22D3, in primary human airway epithelial and smooth muscle cells, respectively. Dexamethasone-induced DUSP1 mRNA was unaffected. In human bronchial epithelial BEAS-2B cells, dexamethasone-induced TSC22D3 and CDKN1C expression (at 6 h) was reduced by TNF pre-treatment, whereas DUSP1 and RGS2 mRNAs were unaffected. TNF pre-treatment also reduced dexamethasone-dependent 2×GRE reporter activation. This was partially reversed by PS-1145 and c-jun N-terminal kinase (JNK) inhibitor VIII, inhibitors of IKK2 and JNK, respectively. However, neither inhibitor affected TNF-dependent loss of dexamethasone-induced CDKN1C or TSC22D3 mRNA. Similarly, inhibitors of the extracellular signal-regulated kinase, p38, phosphoinositide 3-kinase or protein kinase C pathways failed to attenuate TNF-dependent repression of the 2×GRE reporter. Fluticasone furoate, fluticasone propionate and budesonide were full agonists relative to dexamethasone, while GSK9027, RU24858, des-ciclesonide and GW870086X were partial agonists on the 2×GRE reporter. TNF reduced reporter activity in proportion with agonist efficacy. Full and partial agonists showed various degrees of agonism on RGS2 and TSC22D3 expression, but were equally effective at inducing CDKN1C and DUSP1, and did not affect the repression of CDKN1C or TSC22D3 expression by TNF. Finally, formoterol-enhanced 2×GRE reporter activity was also proportional to agonist efficacy and functionally reversed repression by TNF. As similar effects were apparent on glucocorticoid-induced gene expression, the most effective strategy to overcome glucocorticoid resistance in this model was addition of formoterol to high efficacy NR3C1 agonists.     

Resveratrol prevents dexamethasone-induced expression of the muscle atrophy-related ubiquitin ligases atrogin-1 and MuRF1 in cultured myotubes through a SIRT1-dependent mechanism

Resveratrol (3,5,4'-trihydroxystilbene) has been ascribed multiple beneficial biological effects but the influence of resveratrol on glucocorticoid-induced muscle atrophy is not known. We examined the effects of resveratrol on dexamethasone-induced atrogin-1 and MuRF1 expression, FOXO1 acetylation, protein degradation and atrophy in cultured L6 myotubes. In addition, the role of the deacetylase SIRT1 in the effects of resveratrol was determined by transfecting myotubes with SIRT1 siRNA. The catabolic effects of dexamethasone were prevented by resveratrol and the protective effects of resveratrol on dexamethasone-induced atrogin-1 and MuRF1 expression were abolished in myotubes transfected with SIRT1 siRNA. Results suggest that resveratrol can prevent glucocorticoid-induced muscle wasting and that this effect is at least in part SIRT1-dependent.     

Hindlimb casting decreases muscle mass in part by proteasome-dependent proteolysis but independent of protein synthesis

The hypothesis of the present study was that rats subjected to short-term unilateral hindlimb immobilization would incur skeletal muscle wasting and concomitant alterations in protein synthesis, controllers of translation, and indexes of protein degradation. Rats were unilaterally casted for 1, 3, or 5 days to avoid complications associated with other disuse models. In the casted limb, gastrocnemius wet weight decreased 12% after 3 days and thereafter remained constant. In contrast, the contralateral control leg displayed a steady growth rate over time. The rate of protein synthesis and translational efficiency were unchanged in the immobilized muscle at day 5. The total amount and phosphorylation state of regulators of translational initiation and elongation were unaltered. The mRNA contents of polyubiquitin and the ubiquitin ligases muscle atrophy F-box (MAFbx)/Atrogin-1 and muscle RING finger 1 (MuRF1) were elevated in immobilized muscle at all time points, with peak expression occurring at day 3. Daily injection of the type II glucocorticoid receptor antagonist RU-486 did not prevent decreases in gastrocnemius wet weight nor increases in mRNA for MAFbx/Atrogin-1 and MuRF1. However, in vivo administration of the proteasome inhibitor Velcade prevented 53% of wet weight loss associated with 3 days of immobilization. These data suggest that the loss of skeletal muscle mass in this model of disuse appears to be glucocorticoid independent, can be partially rescued with a potent proteasome inhibitor, and is associated with enhanced mRNA expression of multiple factors that contribute to ubiquitin- proteasome-dependent degradation and are likely to control the remodeling of immobilized skeletal muscle during atrophy.