Analysis of glucocorticoid regulated gene expression and skeletal muscle atrophy in glucocorticoid receptor mutant mice

The muscle specific ubiquitin E3 ligase MuRF1 has been implicated as a key regulator of muscle atrophy under a variety of conditions, such as during synthetic glucocorticoid treatment. FOXO class transcription factors have been proposed as important regulators of MuRF1 expression, but its regulation by glucocorticoids is not well understood. The MuRF1 promoter contains a near-perfect palindromic glucocorticoid response element (GRE) 200 base pairs upstream of the transcription start site. The GRE is highly conserved in the mouse, rat, and human genes along with a directly adjacent FOXO binding element (FBE). Transient transfection assays in HepG2 cells and C2C12 myotubes demonstrate that the MuRF1 promoter is responsive to both the dexamethasone (DEX)-activated glucocorticoid receptor (GR) and FOXO1, whereas coexpression of GR and FOXO1 leads to a dramatic synergistic increase in reporter gene activity. Mutation of either the GRE or the FBE significantly impairs activation of the MuRF1 promoter. Consistent with these findings, DEX-induced upregulation of MuRF1 is significantly attenuated in mice expressing a homodimerization-deficient GR despite no effect on the degree of muscle loss in these mice vs. their wild-type counterparts. Finally, chromatin immunoprecipitation analysis reveals that both GR and FOXO1 bind to the endogenous MuRF1 promoter in C2C12 myotubes, and IGF-I inhibition of DEX-induced MuRF1 expression correlates with the loss of FOXO1 binding. These findings present new insights into the role of the GR and FOXO family of transcription factors in the transcriptional regulation of the MuRF1 gene, a direct target of the GR in skeletal muscle.

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The Role of Passive Stretch and Repetitive Electrical Stimulation in Preventing Skeletal Muscle Atrophy While Reprogramming Gene Expression to Improve Fatigue Resistance

Journal of Cardiac Surgery ◽

10.1111/jocs.1991.6.1s.218 ◽

1991 ◽

Vol 6 (1S) ◽

pp. 218-224 ◽

Cited By ~ 29

Author(s):

David F. Goldspink ◽

Juliet Easton ◽

Susan K. Winterburn ◽

Pamela E. Williams ◽

Geoffrey E. Goldspink

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Electrical Stimulation ◽

Fatigue Resistance ◽

Muscle Atrophy ◽

Skeletal Muscle Atrophy ◽

Repetitive Electrical Stimulation ◽

Passive Stretch ◽

Improve Fatigue

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RNA sequencing (RNA-seq) analysis of gene expression provides new insights into hindlimb unloading-induced skeletal muscle atrophy

Annals of Translational Medicine ◽

10.21037/atm-20-7400 ◽

2020 ◽

Vol 8 (23) ◽

pp. 1595-1595

Author(s):

Qihao Cui ◽

Hua Yang ◽

Yuming Gu ◽

Chenyu Zong ◽

Xin Chen ◽

...

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Rna Sequencing ◽

Muscle Atrophy ◽

Hindlimb Unloading ◽

Skeletal Muscle Atrophy ◽

Rna Seq

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UBR5 is a Novel E3 Ubiquitin Ligase involved in Skeletal Muscle Hypertrophy and Recovery from Atrophy

10.1101/592980 ◽

2019 ◽

Author(s):

RA Seaborne ◽

DC Hughes ◽

DC Turner ◽

DJ Owens ◽

LM Baehr ◽

...

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Muscle Atrophy ◽

Ubiquitin Ligase ◽

Protein Level ◽

E3 Ubiquitin Ligase ◽

Skeletal Muscle Atrophy ◽

Muscle Fibres ◽

E3 Ligases ◽

Atrophy And Hypertrophy

AbstractWe aimed to investigate a novel and uncharacterised E3 ubiquitin ligase in skeletal muscle atrophy, recovery from atrophy/injury, anabolism and hypertrophy. We demonstrated an alternate gene expression profile for UBR5 versus well characterised E3-ligases, MuRF1/MAFbx, where after atrophy evoked by continuous-low-frequency electrical-stimulation in rats, MuRF1/MAFbx were both elevated yet UBR5 was unchanged. Furthermore, after recovery of muscle mass post tetrodotoxin (TTX) induced-atrophy in rats, UBR5 was hypomethylated and increased at the gene expression level, while a suppression of MuRF1/MAFbx was observed. At the protein level, we also demonstrated a significant increase in UBR5 after recovery of muscle mass from hindlimb unloading in both adult and aged rats, and after recovery from atrophy evoked by nerve crush injury in mice. During anabolism and hypertrophy, UBR5 gene expression increased following acute loading in three-dimensional bioengineered mouse muscle in-vitro, and after chronic electrical-stimulation-induced hypertrophy in rats in-vivo, without increases in MuRF1/MAFbx. Additionally, UBR5 protein abundance increased following functional overload-induced hypertrophy of the plantaris muscle in mice and during differentiation of primary human muscle cells. Finally, in humans, genetic association studies (>700,000 SNPs) demonstrated that the A alleles of rs10505025 and rs4734621 SNPs in the UBR5 gene were strongly associated with larger cross-sectional area of fast-twitch muscle fibres and favoured strength/power versus endurance/untrained phenotypes. Overall, we suggest that UBR5 is a novel E3 ubiquitin ligase that is inversely regulated to MuRF1/MAFbx, is epigenetically regulated, and is elevated at both the gene expression and protein level during recovery from skeletal muscle atrophy and hypertrophy.Key PointsWe have recently identified that a HECT domain E3 ubiquitin ligase, named UBR5, is altered epigenetically (via DNA methylation) after human skeletal muscle hypertrophy, where its gene expression is positively correlated with increasing lean leg mass after training and retraining.In the present study we extensively investigate this novel and uncharacterised E3 ubiquitin ligase (UBR5) in skeletal muscle atrophy, recovery from atrophy and injury, anabolism and hypertrophy.We demonstrated that UBR5 was epigenetically via altered DNA methylation during recovery from atrophy.We also determined that UBR5 was alternatively regulated versus well characterised E3 ligases, MuRF1/MAFbx, at the gene expression level during atrophy, recovery from atrophy and hypertrophy.UBR5 also increased at the protein level during recovery from atrophy and injury, hypertrophy and during human muscle cell differentiation.Finally, in humans, genetic variations of the UBR5 gene were strongly associated with larger fast-twitch muscle fibres and strength/power performance versus endurance/untrained phenotypes.

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

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00512.2011 ◽

2012 ◽

Vol 302 (10) ◽

pp. E1210-E1220 ◽

Cited By ~ 57

Author(s):

Monica L. Watson ◽

Leslie M. Baehr ◽

Holger M. Reichardt ◽

Jan P. Tuckermann ◽

Sue C. Bodine ◽

...

Keyword(s):

Skeletal Muscle ◽

Glucocorticoid Receptor ◽

Muscle Atrophy ◽

Target Genes ◽

Prolonged Exposure ◽

Skeletal Muscle Atrophy ◽

High Dose ◽

Nerve Lesion ◽

Nutritional Deprivation

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 (MGRe3KO) using Cre-lox technology. In MGRe3KO 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 MGRe3KO 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 MGRe3KO 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 MGRe3KO 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 MGRe3KO mouse is a useful model for studying the role of the GR and its target genes in multiple skeletal muscle atrophy models.

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Dual-specificity phosphatase 29 is induced during neurogenic skeletal muscle atrophy and attenuates glucocorticoid receptor activity in muscle cell culture

AJP Cell Physiology ◽

10.1152/ajpcell.00200.2020 ◽

2020 ◽

Vol 319 (2) ◽

pp. C441-C454

Author(s):

Lisa M. Cooper ◽

Rita C. West ◽

Caleb S. Hayes ◽

David S. Waddell

Keyword(s):

Skeletal Muscle ◽

Glucocorticoid Receptor ◽

Reporter Gene ◽

Muscle Cell ◽

Muscle Atrophy ◽

Muscle Cells ◽

Proximal Promoter ◽

Skeletal Muscle Atrophy ◽

Dual Specificity Phosphatase ◽

Dual Specificity

Skeletal muscle atrophy is caused by a decrease in muscle size and strength and results from a range of physiological conditions, including denervation, immobilization, corticosteroid exposure and aging. Newly named dual-specificity phosphatase 29 ( Dusp29) has been identified as a novel neurogenic atrophy-induced gene in skeletal muscle. Quantitative PCR analysis revealed that Dusp29 expression is significantly higher in differentiated myotubes compared with proliferating myoblasts. To determine how Dusp29 is transcriptionally regulated in skeletal muscle, fragments of the promoter region of Dusp29 were cloned, fused to a reporter gene, and found to be highly inducible in response to ectopic expression of the myogenic regulatory factors (MRF), MyoD and myogenin. Furthermore, site-directed mutagenesis of conserved E-box elements within the proximal promoter of Dusp29 rendered a Dusp29 reporter gene unresponsive to MRF overexpression. Dusp29, an atypical Dusp also known as Dupd1/Dusp27, was found to attenuate the ERK1/2 branch of the MAP kinase signaling pathway in muscle cells and inhibit muscle cell differentiation when ectopically expressed in proliferating myoblasts. Interestingly, Dusp29 was also found to destabilize AMPK protein while simultaneously enriching the phosphorylated pool of AMPK in muscle cells. Additionally, Dusp29 overexpression resulted in a significant increase in the glucocorticoid receptor (GR) protein and elevation in GR phosphorylation. Finally, Dusp29 was found to significantly impair the ability of the glucocorticoid receptor to function as a transcriptional activator in muscle cells treated with dexamethasone. Identifying and characterizing the function of Dusp29 in muscle provides novel insights into the molecular and cellular mechanisms for skeletal muscle atrophy.

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The Glucocorticoid Receptor and Its Direct Target Gene MuRF1 Are Required for Dexamethasone-Induced Skeletal Muscle Atrophy.

10.1210/endo-meetings.2010.part1.p1.p1-39 ◽

2010 ◽

pp. P1-39-P1-39

Author(s):

JD Furlow ◽

ML Watson ◽

LM Baehr ◽

E Karbassi ◽

HM Reichardt ◽

...

Keyword(s):

Skeletal Muscle ◽

Glucocorticoid Receptor ◽

Muscle Atrophy ◽

Target Gene ◽

Skeletal Muscle Atrophy ◽

Direct Target Gene ◽

Direct Target

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Spermine oxidase maintains basal skeletal muscle gene expression and fiber size and is strongly repressed by conditions that cause skeletal muscle atrophy

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00472.2014 ◽

2015 ◽

Vol 308 (2) ◽

pp. E144-E158 ◽

Cited By ~ 23

Author(s):

Kale S. Bongers ◽

Daniel K. Fox ◽

Steven D. Kunkel ◽

Larissa V. Stebounova ◽

Daryl J. Murry ◽

...

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Muscle Atrophy ◽

Muscle Fiber ◽

Skeletal Muscle Atrophy ◽

Spermine Oxidase ◽

Muscle Gene Expression ◽

Fiber Size ◽

Muscle Fiber Atrophy ◽

Muscle Gene

Skeletal muscle atrophy is a common and debilitating condition that remains poorly understood at the molecular level. To better understand the mechanisms of muscle atrophy, we used mouse models to search for a skeletal muscle protein that helps to maintain muscle mass and is specifically lost during muscle atrophy. We discovered that diverse causes of muscle atrophy (limb immobilization, fasting, muscle denervation, and aging) strongly reduced expression of the enzyme spermine oxidase. Importantly, a reduction in spermine oxidase was sufficient to induce muscle fiber atrophy. Conversely, forced expression of spermine oxidase increased muscle fiber size in multiple models of muscle atrophy (immobilization, fasting, and denervation). Interestingly, the reduction of spermine oxidase during muscle atrophy was mediated by p21, a protein that is highly induced during muscle atrophy and actively promotes muscle atrophy. In addition, we found that spermine oxidase decreased skeletal muscle mRNAs that promote muscle atrophy (e.g., myogenin) and increased mRNAs that help to maintain muscle mass (e.g., mitofusin-2). Thus, in healthy skeletal muscle, a relatively low level of p21 permits expression of spermine oxidase, which helps to maintain basal muscle gene expression and fiber size; conversely, during conditions that cause muscle atrophy, p21 expression rises, leading to reduced spermine oxidase expression, disruption of basal muscle gene expression, and muscle fiber atrophy. Collectively, these results identify spermine oxidase as an important positive regulator of muscle gene expression and fiber size, and elucidate p21-mediated repression of spermine oxidase as a key step in the pathogenesis of skeletal muscle atrophy.

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