ABSTRACT
The development and prevalence of diseases associated with aging presents a global health burden on society. One hallmark of aging is the loss of proteostasis which is caused in part by alterations to the ubiquitin-proteasome system (UPS) and lysosome-autophagy system leading to impaired function and maintenance of mass in tissues such as skeletal muscle. In the instance of skeletal muscle, the impairment of function occurs early in the aging process and is dependent on proteostatic mechanisms. The UPS plays a pivotal role in degradation of misfolded and aggregated proteins. For the purpose of this review, we will discuss the role of the UPS system in the context of age-related loss of muscle mass and function. We highlight the significant role that E3 ubiquitin ligases play in the turnover of key components (e.g., mitochondria and neuromuscular junction) essential to skeletal muscle function and the influence of aging. In addition, we will briefly discuss the contribution of the UPS system to lifespan. By understanding the UPS system as part of the proteostasis network in age-related diseases and disorders such as sarcopenia, new discoveries can be made and new interventions can be developed which will preserve muscle function and maintain quality of life with advancing age.
Subject(s)
Longevity , Ubiquitin , Muscle, Skeletal/metabolism , Proteasome Endopeptidase Complex/metabolism , Quality of Life , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
The strategy of gene delivery into skeletal muscles has provided exciting avenues in identifying new potential therapeutics toward muscular disorders and addressing basic research questions in muscle physiology through overexpression and knockdown studies. In vivo electroporation methodology offers a simple, rapidly effective technique for the delivery of plasmid DNA into postmitotic skeletal muscle fibers and the ability to easily explore the molecular mechanisms of skeletal muscle plasticity. The purpose of this review is to describe how to robustly electroporate plasmid DNA into different hindlimb muscles of rodent models. Furthermore, key parameters (e.g., voltage, hyaluronidase, and plasmid concentration) that contribute to the successful introduction of plasmid DNA into skeletal muscle fibers will be discussed. In addition, details on processing tissue for immunohistochemistry and fiber cross-sectional area (CSA) analysis will be outlined. The overall goal of this review is to provide the basic and necessary information needed for successful implementation of in vivo electroporation of plasmid DNA and thus open new avenues of discovery research in skeletal muscle physiology.
Subject(s)
Electroporation , Gene Transfer Techniques , Animals , DNA , Electroporation/methods , Genetic Therapy , Mice , Muscle, Skeletal , Plasmids/geneticsABSTRACT
Skeletal muscle size is highly plastic and sensitive to a variety of stimuli. Muscle atrophy occurs as the result of changes in multiple signaling pathways that regulate both protein synthesis and degradation. The signaling pathways that are activated or inhibited depend on the specific stimuli that are altered. To view this SnapShot, open of download the PDF.
Subject(s)
Muscle, Skeletal , Muscular Atrophy , Humans , Muscle, Skeletal/metabolism , Muscular Atrophy/metabolism , Muscular Atrophy/pathology , Signal Transduction/physiologyABSTRACT
Glucocorticoids are released in response to acute aerobic exercise. The objective was to define changes in the expression of glucocorticoid target genes in skeletal muscle in response to acute aerobic exercise at different times of day. We identified glucocorticoid target genes altered in skeletal muscle by acute exercise by comparing data sets from rodents subjected to acute aerobic exercise in the light or dark cycles to data sets from C2C12 myotubes treated with glucocorticoids. The role of glucocorticoid receptor signaling and REDD1 protein in mediating gene expression was assessed in exercised mice. Changes to expression of glucocorticoid genes were greater when exercise occurred in the dark cycle. REDD1 was required for the induction of genes induced at both times of day. In all, the time of day at which aerobic exercise is conducted dictates changes to the expression of glucocorticoid target genes in skeletal muscle with REDD1 contributing to those changes.
Subject(s)
Glucocorticoids , Muscle, Skeletal , Physical Conditioning, Animal , Animals , Circadian Rhythm , Glucocorticoids/genetics , Glucocorticoids/metabolism , Mice , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/metabolism , Physical Conditioning, Animal/physiology , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Skeletal muscle atrophy can result from a range of physiological conditions, including denervation, immobilization, hindlimb unweighting, and aging. To better characterize the molecular genetic events of atrophy, a microarray analysis revealed that FGGY carbohydrate kinase domain containing (Fggy) is expressed in skeletal muscle and is induced in response to denervation. Bioinformatic analysis of the Fggy gene locus revealed two validated isoforms with alternative transcription initiation sites that we have designated Fggy-L-552 and Fggy-S-387. Additionally, we cloned two novel alternative splice variants, designated Fggy-L-482 and Fggy-S-344, from cultured muscle cells suggesting that at least four Fggy splice variants are expressed in skeletal muscle. Quantitative RT-PCR was performed using RNA isolated from muscle cells and primers designed to distinguish the four alternative Fggy transcripts and found that the Fggy-L transcripts are more highly expressed during myoblast differentiation, while the Fggy-S transcripts show relatively stable expression in proliferating myoblasts and differentiated myotubes. Confocal fluorescent microscopy revealed that the Fggy-L variants appear to localize evenly throughout the cytoplasm, while the Fggy-S variants produce a more punctuate cytoplasmic localization pattern in proliferating muscle cells. Finally, ectopic expression of Fggy-L-552 and Fggy-S-387 resulted in inhibition of muscle cell differentiation and attenuation of the MAP kinase and Akt signaling pathways. The identification and characterization of novel genes such as Fggy helps to improve our understanding of the molecular and cellular events that lead to atrophy and may eventually result in the identification of new therapeutic targets for the treatment of muscle wasting.
Subject(s)
Muscle, Skeletal/enzymology , Muscular Atrophy/genetics , Phosphotransferases/genetics , Phosphotransferases/metabolism , RNA Splice Sites , Animals , Cell Differentiation/genetics , Cells, Cultured , Cytoplasm/enzymology , Gene Expression Regulation, Enzymologic , MAP Kinase Signaling System/genetics , Mice , Myoblasts/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal TransductionABSTRACT
MuRF1 (TRIM63) is a muscle-specific E3 ubiquitin ligase and component of the ubiquitin proteasome system. MuRF1 is transcriptionally upregulated under conditions that cause muscle loss, in both rodents and humans, and is a recognized marker of muscle atrophy. In this study, we used in vivo electroporation to determine whether MuRF1 overexpression alone can cause muscle atrophy and, in combination with ubiquitin proteomics, identify the endogenous MuRF1 substrates in skeletal muscle. Overexpression of MuRF1 in adult mice increases ubiquitination of myofibrillar and sarcoplasmic proteins, increases expression of genes associated with neuromuscular junction instability, and causes muscle atrophy. A total of 169 ubiquitination sites on 56 proteins were found to be regulated by MuRF1. MuRF1-mediated ubiquitination targeted both thick and thin filament contractile proteins, as well as, glycolytic enzymes, deubiquitinases, p62, and VCP. These data reveal a potential role for MuRF1 in not only the breakdown of the sarcomere but also the regulation of metabolism and other proteolytic pathways in skeletal muscle.
Subject(s)
Muscle Proteins , Muscle, Skeletal , Proteomics , Tripartite Motif Proteins , Ubiquitin-Protein Ligases , Animals , Humans , Mice , Muscle, Skeletal/metabolism , Muscular Atrophy/genetics , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics , Muscle Proteins/genetics , Tripartite Motif Proteins/geneticsABSTRACT
Muscle-specific E3 ubiquitin ligases have been identified in muscle atrophy-inducing conditions. The purpose of the current study was to explore the functional role of F-box and leucine-rich protein 22 (Fbxl22), and a newly identified splice variant (Fbxl22-193), in skeletal muscle homeostasis and neurogenic muscle atrophy. In mouse C2C12 muscle cells, promoter fragments of the Fbxl22 gene were cloned and fused with the secreted alkaline phosphatase reporter gene to assess the transcriptional regulation of Fbxl22. The tibialis anterior muscles of male C57/BL6 mice (12-16 wk old) were electroporated with expression plasmids containing the cDNA of two Fbxl22 splice variants and tissues collected after 7, 14, and 28 days. Gastrocnemius muscles of wild-type and muscle-specific RING finger 1 knockout (MuRF1 KO) mice were electroporated with an Fbxl22 RNAi or empty plasmid and denervated 3 days posttransfection, and tissues were collected 7 days postdenervation. The full-length gene and novel splice variant are transcriptionally induced early (after 3 days) during neurogenic muscle atrophy. In vivo overexpression of Fbxl22 isoforms in mouse skeletal muscle leads to evidence of myopathy/atrophy, suggesting that both are involved in the process of neurogenic muscle atrophy. Knockdown of Fbxl22 in the muscles of MuRF1 KO mice resulted in significant additive muscle sparing 7 days after denervation. Targeting two E3 ubiquitin ligases appears to have a strong additive effect on protecting muscle mass loss with denervation, and these findings have important implications in the development of therapeutic strategies to treat muscle atrophy.
Subject(s)
F-Box Proteins/genetics , Muscle Proteins/genetics , Muscular Atrophy/genetics , Tripartite Motif Proteins/genetics , Ubiquitin-Protein Ligases/genetics , Animals , Gene Expression Regulation, Developmental/genetics , Humans , Mice , Mice, Knockout , Muscle Cells/metabolism , Muscle Cells/pathology , Muscle, Skeletal/innervation , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Muscular Atrophy/physiopathology , TransfectionABSTRACT
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.
Subject(s)
Dual-Specificity Phosphatases/genetics , Muscular Atrophy/genetics , MyoD Protein/genetics , Myogenin/genetics , Animals , Cell Differentiation/genetics , Cell Line , Cell Proliferation/genetics , Gene Expression Regulation/genetics , Humans , MAP Kinase Signaling System/genetics , Muscle Cells/metabolism , Muscle Cells/pathology , Muscular Atrophy/pathology , Myoblasts/metabolism , Phosphorylation/genetics , Receptors, Glucocorticoid/genetics , Signal Transduction , Transcriptional Activation/geneticsABSTRACT
Protein phosphatase methylesterase 1 has been identified as a novel gene in skeletal muscle that is upregulated in response to neurogenic atrophy in mice. Western blot analysis confirms that Ppme1 is expressed during both muscle cell proliferation and differentiation. Additionally, the Ppme1 promoter is active in muscle cells, while mutation of a conserved E-box element prevents full induction of the Ppme1 reporter gene, suggesting that Ppme1 is transcriptionally regulated by myogenic regulatory factors. Interestingly, immunofluorescence analysis indicates that Ppme1 is localized to both the cytoplasm and the nucleus, while cell fractionation shows that Ppme1 is found only in the cytoplasm. Functional studies reveal that inhibition of Ppme1 using ABL127 or AMZ30 attenuates muscle cell differentiation. Interestingly, inhibition of Ppme1 by ABL127 led to a significant increase in AP-1 reporter activity, as well as, increases in ERK1/2, c-Jun, Ppme1, and PP2A protein levels in differentiating muscle cells. In contrast, AMZ30 treated cells showed a significant decrease in AP-1 reporter activity and a decrease in ERK1/2 and p38 phosphorylation levels. Finally, co-immunoprecipitation studies show that ABL127, but not AMZ30, causes disruption of the endogenous interaction between Ppme1 and PP2A. The data in this study show for the first time that Ppme1 is expressed in skeletal muscle and is upregulated in response to neurogenic atrophy. Furthermore, these findings suggest that Ppme1 may act as a sentinel of the MAP kinase signaling pathway and may indirectly regulate the ERK1/2 and p38 branches via a non-canonical mechanism leading to inhibition of muscle cell differentiation.
Subject(s)
Carboxylic Ester Hydrolases/metabolism , Cell Differentiation , MAP Kinase Signaling System/physiology , Animals , Carboxylic Ester Hydrolases/antagonists & inhibitors , Carboxylic Ester Hydrolases/genetics , Cell Line , Genes, Reporter , MAP Kinase Signaling System/genetics , Mice , Muscle, Skeletal/physiology , Myoblasts/physiology , Phosphorylation , Up-RegulationABSTRACT
Skeletal muscle atrophy is a serious health condition that can arise due to aging, cancer, corticosteroid exposure, and denervation. Previous work comparing gene expression profiles in control and denervated muscle tissue revealed for the first time that Fam83d is expressed in skeletal muscle and is significantly induced in response to denervation. Quantitative PCR and Western blot analysis found that Fam83d is more highly expressed in proliferating myoblasts compared to differentiated myotubes. Characterization of the transcriptional regulation of Fam83d showed that ectopic expression of myogenic regulatory factors inhibits Fam83d reporter gene activity. To assess where Fam83d is localized in the cell, Fam83d was fused with green fluorescent protein, expressed in C2C12 cells, and found to localize in a punctate manner to the cytoplasm of muscle cells. To assess function, Fam83d was ectopically expressed in cultured muscle cells and markers of muscle cell differentiation, the MAP Kinase signaling pathway, and the AKT signaling pathway were analyzed. Fam83d overexpression resulted in significant repression of myosin heavy chain and myogenin expression, while phosphorylated ERK and AKT were also significantly repressed. Interestingly, inhibition of the 26S proteasome and the MAP kinase signaling pathway both resulted in stabilization of Fam83d during muscle cell differentiation. Finally, Fam83d has a putative phospholipase D-like domain that appears to be necessary for destabilizing casein kinase Iα and inhibiting ERK phosphorylation in cultured myoblasts. The discovery that Fam83d is expressed in skeletal muscle combined with the observation that Fam83d, a potential modulator of MAP kinase and AKT signaling, is induced in response to neurogenic atrophy helps further our understanding of the molecular and cellular events of skeletal muscle wasting.
Subject(s)
Cell Cycle Proteins/physiology , Microtubule-Associated Proteins/physiology , Muscle, Skeletal , Muscular Atrophy/metabolism , Myoblasts , Signal Transduction , Animals , Cell Line , Gene Expression Regulation , Humans , Mice , Mitogen-Activated Protein Kinases/metabolism , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Myoblasts/metabolism , Myoblasts/pathology , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
Skeletal muscle atrophy is a debilitating condition that can arise due to aging, cancer, corticosteroid use, and denervation. To better characterize the molecular genetic events of neurogenic atrophy, a previous study analyzed gene expression patterns in gastrocnemius muscle following sciatic nerve transection and found for the first time that Zinc Finger Protein 593 (Zfp593) is expressed in skeletal muscle and is induced in response to denervation. Quantitative PCR and Western blot analyses confirmed that Zfp593 is expressed in both proliferating myoblasts and differentiated myotubes. To assess sub-cellular location, GFP-tagged Zfp593 was expressed in C2C12â¯cells and found to localize to the nucleus. The Zfp593 protein possesses a putative zinc finger domain and is believed to function as a modulator of the Oct-2 transcription factor. Interestingly, ectopic expression of Zfp593 did not affect the ability of Oct-1 or Oct-2 to inhibit an Oct reporter gene in muscle cells. Finally, Zfp593 overexpression in cultured muscle cells resulted in significant repression of muscle cell differentiation and attenuation of ERK1/2 and p38 phosphorylation, but did not vitiate protein synthesis. The discovery that Zfp593 is expressed in skeletal muscle combined with the observation that it is induced in response to neurogenic atrophy furthers our understanding of the molecular genetic events of muscle wasting.
Subject(s)
Cell Differentiation/genetics , DNA-Binding Proteins/genetics , Muscle Development/genetics , Muscular Atrophy/genetics , Myoblasts/physiology , Transcription Factors/genetics , Animals , Cells, Cultured , MAP Kinase Signaling System/genetics , Mice , Muscle, Skeletal/pathology , Muscle, Skeletal/physiology , Muscular Atrophy/metabolism , Muscular Atrophy/pathology , Up-Regulation/genetics , Zinc Fingers/genetics , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Tetratricopeptide repeat domain containing 39c (Ttc39c) is expressed in skeletal muscle and is transcriptionally activated in response to neurogenic atrophy in mice. Expression analysis using quantitative polymerase chain reaction and Western blots revealed that Ttc39c is expressed in both proliferating and differentiated muscle cells, peaking during early differentiation and then decreasing as cells progress further through the differentiation process. To further analyze the transcriptional regulation of Ttc39c, promoter fragments of the gene were cloned and fused with the secreted alkaline phosphatase reporter gene. The Ttc39c reporter plasmids were then transfected into cultured mouse muscle cells and found to have transcriptional activity. Furthermore, overexpression of MyoD and myogenin resulted in significant transcriptional repression of the Ttc39c reporter genes. To determine subcellular localization, an expression plasmid with the Ttc39c complementary DNA fused with green fluorescent protein was transfected into muscle cells and analyzed by confocal fluorescent microscopy showing that Tct39c localizes exclusively to the cytoplasm of cultured cells. To assess potential function in muscle, Ttc39c was overexpressed leading to vitiated muscle cell differentiation, impaired ERK1/2 MAP Kinase and Hedgehog signaling, and increased expression of IFT144 and IFT43, which are part of the IFT-A complex involved in retrograde transport in primary cilia. Interestingly, Ttc39c knockdown also resulted in inhibition of muscle cell differentiation and impaired Hedgehog and MAP Kinase signaling but did not affect IFT144 or IFT433 expression. The results of this study demonstrate that muscle cell differentiation is sensitive to abnormal Ttc39c expression and that normal Ttc39c expression appears to be necessary for proper MAP Kinase and Hedgehog signal transduction in developing muscle cells.
Subject(s)
Extracellular Signal-Regulated MAP Kinases/metabolism , Hedgehog Proteins/metabolism , Muscle Proteins/biosynthesis , Muscle, Skeletal/pathology , Muscular Atrophy/pathology , Neoplasm Proteins/biosynthesis , Animals , Cell Line , MAP Kinase Signaling System/physiology , Mice , Muscle Proteins/genetics , Neoplasm Proteins/genetics , RNA Interference , RNA, Small Interfering/genetics , Transcriptional Activation/geneticsABSTRACT
Skeletal muscle atrophy results from disparate physiological conditions, including denervation, corticosteroid treatment, and aging. The purpose of this study was to describe and characterize the function of dual-specificity phosphatase 4 (Dusp4) in skeletal muscle after it was found to be induced in response to neurogenic atrophy. Quantitative PCR and Western blot analysis revealed that Dusp4 is expressed during myoblast proliferation but rapidly disappears as muscle cells differentiate. The Dusp4 regulatory region was cloned and found to contain a conserved E-box element that negatively regulates Dusp4 reporter gene activity in response to myogenic regulatory factor expression. In addition, the proximal 3'-untranslated region of Dusp4 acts in an inhibitory manner to repress reporter gene activity as muscle cells progress through the differentiation process. To determine potential function, Dusp4 was fused with green fluorescent protein, expressed in C2C12 cells, and found to localize to the nucleus of proliferating myoblasts. Furthermore, Dusp4 overexpression delayed C2C12 muscle cell differentiation and resulted in repression of a MAP kinase signaling pathway reporter gene. Ectopic expression of a Dusp4 dominant negative mutant blocked muscle cell differentiation and attenuated MAP kinase signaling by preferentially targeting the ERK1/2 branch, but not the p38 branch, of the MAP kinase signaling cascade in skeletal muscle cells. The findings presented in this study provide the first description of Dusp4 in skeletal muscle and suggest that Dusp4 may play an important role in the regulation of muscle cell differentiation by regulating MAP kinase signaling.
Subject(s)
Extracellular Signal-Regulated MAP Kinases/biosynthesis , Muscle, Skeletal/metabolism , Protein Tyrosine Phosphatases/biosynthesis , Up-Regulation/physiology , Animals , Atrophy , Base Sequence , Cell Differentiation/physiology , Extracellular Signal-Regulated MAP Kinases/genetics , HEK293 Cells , Humans , Muscle, Skeletal/pathology , Protein Tyrosine Phosphatases/geneticsABSTRACT
Muscle atrophy results from a range of physiological conditions, including immobilization, spinal cord damage, inflammation and aging. In this study we describe two genes, NEFA-interacting nuclear protein 30 (Nip30) and RING Finger and SPRY domain containing 1 (Rspry1), which have not previously been characterized or shown to be expressed in skeletal muscle. Furthermore, Nip30 and Rspry1 were transcriptionally induced in response to neurogenic muscle wasting in mice and were also found to be expressed endogenously at the RNA and protein level in C2C12 mouse muscle cells. Interestingly, during analysis of Nip30 and Rspry1 it was observed that these genes share a 230 base pair common regulatory region that contains several putative transcription regulatory elements. In order to assess the transcriptional activity of the Nip30 and Rspry1 regulatory regions, a fragment of the promoter of each gene was cloned, fused to a reporter gene, and transfected into cells. The Nip30 and Rspry1 reporters were both found to have significant transcriptional activity in cultured cells. Furthermore, the Nip30-Rspry1 common regulatory region contains a conserved E-box enhancer, which is an element bound by myogenic regulatory factors that function in the regulation of muscle-specific gene expression. Therefore, in order to determine if the predicted E-box was functional, Nip30 and Rspry1 reporters were transfected into cells ectopically expressing the myogenic regulatory factor, MyoD1, resulting in significant induction of both reporter genes. In addition, mutation of the conserved E-box element eliminated MyoD1 activation of the Nip30 and Rspry1 reporters. Finally, GFP-tagged Nip30 was found to localize to the nucleus, while GFP-tagged Rspry1 was found to localize to the cytoplasm of muscle cells.
Subject(s)
DNA-Binding Proteins/genetics , Muscle, Skeletal/physiology , Nuclear Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Conserved Sequence , Cytoplasm/metabolism , DNA-Binding Proteins/metabolism , E-Box Elements , Gene Expression Regulation , HEK293 Cells , Humans , Mice , Molecular Sequence Data , Muscle, Skeletal/cytology , Muscular Atrophy/genetics , Muscular Atrophy/physiopathology , MyoD Protein/genetics , MyoD Protein/metabolism , Nuclear Proteins/metabolism , Protein Structure, TertiaryABSTRACT
Muscle atrophy can result from inactivity or unloading on one hand or the induction of a catabolic state on the other. Muscle-specific ring finger 1 (MuRF1), a member of the tripartite motif family of E3 ubiquitin ligases, is an essential mediator of multiple conditions inducing muscle atrophy. While most studies have focused on the role of MuRF1 in protein degradation, the protein may have other roles in regulating skeletal muscle mass and metabolism. We therefore systematically evaluated the effect of MuRF1 on gene expression during denervation and dexamethasone-induced atrophy. We find that the lack of MuRF1 leads to few differences in control animals, but there were several significant differences in specific sets of genes upon denervation- and dexamethasone-induced atrophy. For example, during denervation, MuRF1 knockout mice showed delayed repression of metabolic and structural genes and blunted induction of genes associated with the neuromuscular junction. In the latter case, this pattern correlates with blunted HDAC4 and myogenin upregulation. Lack of MuRF1 caused fewer changes in the dexamethasone-induced atrophy program, but certain genes involved in fat metabolism and intracellular signaling were affected. Our results demonstrate a new role for MuRF1 in influencing gene expression in two important models of muscle atrophy.
Subject(s)
Denervation/veterinary , Dexamethasone/adverse effects , Gene Expression Regulation/genetics , Muscle Proteins/metabolism , Muscular Atrophy/chemically induced , Muscular Atrophy/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Blotting, Western , DNA Primers/genetics , Mice , Mice, Knockout , Microarray Analysis , Muscle Proteins/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tripartite Motif Proteins , Ubiquitin-Protein Ligases/geneticsABSTRACT
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 C(2)C(12) 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 C(2)C(12) 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.
Subject(s)
Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/physiology , Muscle Proteins/physiology , Muscular Atrophy/genetics , Muscular Atrophy/pathology , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/physiology , Ubiquitin-Protein Ligases/physiology , Animals , Blotting, Northern , Cells, Cultured , Chromatin/metabolism , Dexamethasone/pharmacology , Forkhead Box Protein O1 , Genes, Reporter/genetics , Humans , Immunoprecipitation , Luciferases/genetics , Mice , Mice, Inbred BALB C , Muscle Proteins/genetics , Plasmids/genetics , Tripartite Motif Proteins , Ubiquitin-Protein Ligases/geneticsABSTRACT
The broad range of biological responses elicited by transforming growth factor-beta (TGF-beta) in various types of tissues and cells is mainly determined by the expression level and activity of the effector proteins Smad2 and Smad3. It is not fully understood how the baseline properties of Smad3 are regulated, although this molecule is in complex with many other proteins at the steady state. Here we show that nonactivated Smad3, but not Smad2, undergoes proteasome-dependent degradation due to the concerted action of the scaffolding protein Axin and its associated kinase, glycogen synthase kinase 3-beta (GSK3-beta). Smad3 physically interacts with Axin and GSK3-beta only in the absence of TGF-beta. Reduction in the expression or activity of Axin/GSK3-beta leads to increased Smad3 stability and transcriptional activity without affecting TGF-beta receptors or Smad2, whereas overexpression of these proteins promotes Smad3 basal degradation and desensitizes cells to TGF-beta. Mechanistically, Axin facilitates GSK3-beta-mediated phosphorylation of Smad3 at Thr66, which triggers Smad3 ubiquitination and degradation. Thr66 mutants of Smad3 show altered protein stability and hence transcriptional activity. These results indicate that the steady-state stability of Smad3 is an important determinant of cellular sensitivity to TGF-beta, and suggest a new function of the Axin/GSK3-beta complex in modulating critical TGF-beta/Smad3-regulated processes during development and tumor progression.
Subject(s)
Glycogen Synthase Kinase 3/metabolism , Repressor Proteins/metabolism , Signal Transduction , Smad3 Protein/metabolism , Transforming Growth Factor beta/metabolism , Axin Protein , Cells, Cultured , Humans , Phosphorylation , Polyubiquitin/metabolism , Proteasome Endopeptidase Complex/metabolism , Smad3 Protein/genetics , Threonine/metabolism , Ubiquitin-Protein Ligases/metabolism , Wnt Proteins/metabolismABSTRACT
Neural activity actively regulates muscle gene expression. This regulation is crucial for specifying muscle functionality and synaptic protein expression. How neural activity is relayed into nuclei and connected to the muscle transcriptional machinery, however, is not known. Here we identify the histone deacetylase HDAC4 as the critical linker connecting neural activity to muscle transcription. We found that HDAC4 is normally concentrated at the neuromuscular junction (NMJ), where nerve innervates muscle. Remarkably, reduced neural input by surgical denervation or neuromuscular diseases dissociates HDAC4 from the NMJ and dramatically induces its expression, leading to robust HDAC4 nuclear accumulation. We present evidence that nuclear accumulated HDAC4 is responsible for the coordinated induction of synaptic genes upon denervation. Inactivation of HDAC4 prevents denervation-induced synaptic acetyl-choline receptor (nAChR) and MUSK transcription whereas forced expression of HDAC4 mimics denervation and activates ectopic nAChR transcription throughout myofibers. We determined that HDAC4 executes activity-dependent transcription by regulating the Dach2-myogenin transcriptional cascade where inhibition of the repressor Dach2 by HDAC4 permits the induction of the transcription factor myogenin, which in turn activates synaptic gene expression. Our findings establish HDAC4 as a neural activity-regulated deacetylase and a key signaling component that relays neural activity to the muscle transcriptional machinery.
Subject(s)
Gene Expression Regulation , Histone Deacetylases/physiology , Myogenin/metabolism , Neurons/metabolism , Nuclear Proteins/metabolism , Receptors, Nicotinic/metabolism , Transcription, Genetic , Animals , Cell Nucleus/metabolism , DNA-Binding Proteins , Histone Deacetylases/metabolism , Mice , Mice, Inbred C57BL , Models, Biological , Muscle, Skeletal/metabolism , Neuromuscular Junction , Signal Transduction , Time Factors , Transcription FactorsABSTRACT
The filtration coefficient (Kfc) is a sensitive measure of microvascular hydraulic conductivity and has been reported for the alveolar lungs of many mammalian species, but not for the parabronchial avian lung. This study reports the Kfc in the isolated lungs of normal chickens and in the lungs of chickens given the edemogenic agents oleic acid (OA) or dimethyl amiloride (DMA). The control Kfc =0.04+/-0.01 ml min(-1) kPa(-1) g(-1). This parameter increased significantly following the administration of both OA (0.12+/-0.02 ml min(-1) kPa(-1) g(-1)) and DMA (0.07+/-0.01 ml min kPa(-1) g(-1)). As endothelial cadherins are thought to play a role in the dynamic response to acute lung injury, we utilized Western blot analysis to assess lung cadherin content and Northern blot analysis to assess pulmonary vascular endothelial (VE) cadherin expression following drug administration. Lung cadherin content decreases markedly following DMA, but not OA administration. VE cadherin expression increases as a result of DMA treatment, but is unchanged following OA. Our results suggest that the permeability characteristics of the avian lung are more closely consistent with those of the mammalian rather than the reptilian lung, and, that cadherins may play a significant role in the response to acute increases in avian pulmonary microvascular permeability.