Transcription Factors and Nuclear Cofactors in Muscle Wasting

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in Muscle Wasting

P.-O. Hasselgren

Introduction

Muscle wasting is commonly seen in patients with sepsis, severe injury, and cancer [1, 2]. The loss of muscle mass in these conditions mainly reflects ubiquitin-proteasome-dependent degradation of myofibrillar proteins although other proteolytic mechanisms may be involved as well [3]. Muscle atrophy is regulated by multiple factors, including glucocorticoids [4], the pro-inflammatory cytokines, interleukin (IL)-1q and tumor necrosis factor (TNF)- [ [5, 6], and myostatin [7]. In addition to these catabolic factors, a lack of anabolic signals, such as insulin-like growth factor (IGF)-1 and insulin, is probably also important for the development of muscle wasting in various catabolic conditions.

Although it may be argued that the increased peripheral release of amino acids that is associated with muscle wasting may be beneficial to the organism by providing energy and substrates to various organs and tissues, including the liver, gut mucosa, and cells in the immune system, during prolonged and severe catabolic conditions, the negative effects of muscle atrophy clearly outweigh any potentially beneficial effects.

Thus, muscle wasting results in weakness and fatigue that in turn delays ambulation of bed-ridden patients, increasing the risk for thromboembolic and pulmonary complications. The risk for pulmonary complications is further increased when the muscle wasting process affects respiratory muscles [8]. In patients with cancer, the accompanying muscle wasting can significantly impair quality of life and may even reduce the effec- tiveness of chemotherapy [9]. It has been estimated that approximately 20 % of deaths in cancer patients can be attributed to the catabolic response in skeletal muscle [10].

Thus, it is obvious that muscle wasting is a significant clinical problem with sometimes devastating consequences. Despite substantial progress during the past decade in our understanding of mechanisms regulating the development of muscle atrophy, we still do not have any effective treatment by which muscle wasting can be prevented or reversed in critically ill patients. Continued efforts to understand the molecular mechanisms behind the loss of muscle mass in these patients will hope- fully help in the development of therapeutic strategies for subjects with this debili- tating condition. In this chapter, novel insights into the role of various transcription factors and nuclear cofactors in the development of muscle atrophy are discussed.

Gene Transcription in Muscle Wasting

In previous studies, we and others found evidence that mRNA levels for various genes in the ubiquitin-proteasome pathway are increased in muscle wasting condi-

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tions. For example, in research from our laboratory, the gene expression for ubiquitin as well as different components of the 26S proteasome was increased in muscle from septic [11, 12] and burned rats [13] and in muscle from patients with sepsis [14] or cancer [15]. Results in other studies suggested that the increased mRNA levels for various components of the ubiquitin-proteaosme pathway reflected upregulated transcription of the genes rather than increased stability of the mRNA [16].

In more recent studies, Lecker et al. [17] reported that multiple catabolic conditions (uremia, fasting, muscle inactivity and denervation) were characterized by a common set of genes that were significantly upregulated. They called these genes

“atrogins” and found that the mRNA levels for two ubiquitin ligases, atrogin-1 and muscle ring finger 1 (MuRF1), were particularly increased [18]. The dramatic increase in the expression of these genes in atrophying muscle has been confirmed by others as well [19] and in studies in our laboratory, atrogin-1 and MuRF1 mRNA levels were increased almost 20-fold in skeletal muscle during sepsis [20]. Other studies provided evidence that the atrogin-1 and MuRF1 gene products regulate the development of muscle atrophy caused by various catabolic conditions and the mRNA levels for atrogin-1 and MuRF1 are frequently used as ‘molecular markers’ of muscle wasting.

Because the transcription of multiple genes is upregulated in atrophying muscle [17] it is not surprising that much attention has been given to the role of various transcription factors in the regulation of muscle mass. Here, the potential roles of the transcription factors C/EBPq and · , nuclear factor-kappa B (NF-κB) and Foxo1 and 3a in the development of muscle wasting are discussed. In addition, recent observations in our laboratory [21, 22] indicating an important role of the nuclear cofactor p300 and its histone actetyl transferase (HAT) activity in the regulation of muscle mass are described.

C/EBP Transcription Factors in Atrophying Muscle

The C/EBP (CCAAT/enhancer binding protein) family of transcription factors consists of at least six members: C/EBP[ , q , * , · , 5 , and C/EBP-homologous protein-10 (CHOP-10), also called Gadd 153 [23]. The different isoforms form homo- or hetero- dimers that influence the transcription of multiple genes in various organs and tissues. Among the different C/EBP family members, there is evidence that C/EBPq and · are particularly important for the inflammatory response [24].

We found that the expression of C/EBPq and · was upregulated in the nuclear fraction of muscles from septic rats [25]. This finding was accompanied by increased C/EBPq and · DNA binding activity determined by electrophoretic mobility shift assay (EMSA) with supershift analysis. Interestingly, the sepsis-induced increase in the expression and activity of C/EBPq and · was inhibited by the glucocorticoid receptor antagonist RU38486, supporting the role of glucocorticoids as an important mediator of sepsis-induced muscle wasting [4, 25]. Although we did not provide direct evidence that C/EBPq and · participate in the regulation of sepsis-induced muscle proteolysis, a sequence analysis demonstrated multiple putative binding sites for C/EBP in the promoter regions of genes that are upregulated in atrophying muscle, including genes for calpains and various components of the ubiquitin-proteasome proteolytic pathway [25].

Further support for a role of glucocorticoids in the upregulation of C/EBP transcription factors in atrophying skeletal muscle was found in experiments in which

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we treated cultured myotubes in vitro or rats in vivo with dexamethasone [26]. In those experiments, treatment with dexamethasone resulted in increased protein and mRNA expression and DNA binding activity of C/EBPq and · . Because dexamethasone treatment did not influence the expression of C/EBP[ , * , or 5 , it is possible that C/EBPq and · play a specific role among the C/EBP family members in atrophying muscle. The increase in C/EBPq and · mRNA levels in dexamtheasone-treated myotubes was not affected by the protein synthesis inhibitor, cycloheximide, indicating that the expression of the transcription factors was regulated directly by dexamethasone. This differed from the dexamethasone-induced upregulation of atrogin-1 mRNA levels that was blocked by cycloheximide, consistent with a model in which the atrogin-1 gene is activated in skeletal muscle secondary to the upregulation of another gene or genes, possibly C/EBPq and · [26].

Thus, our recent observations suggest that C/EBPq and · may be involved in the development of sepsis- and glucocorticoid-induced muscle protein breakdown and atrophy. Interestingly, other members of the C/EBP family may regulate lipid metabolism in atrophying muscle. Thus, in a recent study, muscle atrophy caused by denervation was associated with fatty degeneration and upregulated C/EBP[ expression in the interstitium of the muscles, suggesting a role of C/EBP[ in lipid metabolism in atrophying muscle [27]. This observation supports previous reports that C/

EBP[ is a transcription factor involved in the regulation of lipid metabolism in various cell types, including muscle cells [28].

NF- ␬B Plays a Role in Muscle Wasting

NF-κB is probably the most extensively studied transcription factor in the field of inflammation. It is beyond the scope of this chapter to review the mechanisms that regulate NF-κB activation. Recent extensive review articles describe the molecular biology of this important transcription factor, its activation, and the mechanisms by which it upregulates gene transcription [29, 30].

Although the exact mechanisms by which NF-κB regulates muscle protein breakdown are not completely understood at present, there is strong pharmacological and genetic evidence that NF-κB is involved in the development of muscle wasting in various catabolic conditions. We reported that NF-κB DNA binding activity was upregulated in skeletal muscle during early sepsis but was subsequently inhibited during the later course of an experimental model of sepsis in rats [31]. Interestingly, a biphasic response of NF-κB activity was observed in TNF- [ /interferon (IFN) * - treated myotubes as well [32]. Although the mechanisms for this biphasic response of NF-κB, and the biological implications with regards to which genes are activated or repressed, are not known at present, it is possible that the early activation and the late inhibition of NF-κB in septic muscle [31] reflects the role of different mediators involved in muscle wasting. For example, the early increase in NF-κB activity may reflect the influence of pro-inflammatory cytokines (most notably TNF-[ ) and the subsequent downregulation of NF-κB activity may reflect the effect of glucocorticoids. We and others have reported several lines of evidence that both cytokines and glucocorticoids are important mediators of muscle wasting [4 – 6]. Other studies have shown that NF-κB activity is upregulated by pro-inflammatory cytokines, including TNF-[ [32, 33] and IL-1 q [34] in skeletal muscle cells and that NF-κB activity may be inhibited by glucocorticoids in myocytes [35]. This may seem para- doxical because treatment of cultured myotubes with either cytokines or catabolic

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concentrations of dexamethasone results in increased protein degradation, activation of the ubiquitin-proteaosme proteolytic pathway, and muscle atrophy [32, 33, 36, 37].

An additional potential mechanism to explain some of these apparently conflict- ing observations may be that NF-κB influences individual muscle wasting-related genes differentially. For example, there is evidence that NF-κB activates the expression of the ubiquitin ligase, MuRF1, [38] but may act as a repressor of some of the proteaosme subunit genes [39]. An alternative explanation why the role of NF-κB may seem confusing (having a biphasic response; being activated or inhibited by mediators that all induce muscle atrophy) is that different pathways may be involved in the activation of NF-κB. The classic pathway, activated by pro-inflammatory cytokines, involves the activation of a p50/p65(RelA) heterodimer by ubiquitin-proteasome-dependent degradation of NF-κB inhibitor (IκB), triggered by its phosphorylation by the IκB kinase, IKK q . An alternative pathway for NF-κB activation is the IKK[ -mediated phosphorylation and proteolytic processing of p100, resulting in activation of the non-canonical NF-κB pathway involving p52/RelB heterodimer [29]. The exact role of the different pathways activating NF-κB and the influence of cytokines and glucocorticoids on the regulation of these pathways in atrophying muscle are areas for future study.

Although important questions remain to be addressed with regards to the role of NF-κB in muscle wasting, in recent studies we have found strong molecular evidence that NF-κB activation results in muscle wasting [38]. In those experiments, transgenic mice created in the laboratory of Dr Steven Shoelson were used. Mice with a muscle-specific overexpression of activated IKKq displayed upregulated NF-κB activity, increased expression of MuRF1 (but, interestingly enough, not atrogin-1), atrophy of muscle fibers, and a substantial loss of muscle mass. In other experiments in the same study, muscle-specific expression of an IκB [ superrepressor blocked NF-κB activation and prevented denervation- and tumor-induced muscle loss. A similar prevention of muscle atrophy was seen in mice treated with pharmacological inhibitors of NF-κB further supporting a role of NF-κB in the development of muscle wasting. In recent (unpublished) experiments in our laboratory we found that treatment of rats with the NF-κB inhibitor, curcumin, prevented sepsis-induced muscle proteolysis, suggesting that inhibition of NF-κB may prevent muscle wasting in different conditions characterized by muscle cachexia.

It should be noted that although there is evidence that NF-κB is activated in cata- bolic muscle in vivo [31] and in cytokine-treated muscle cells in vitro [32 – 34], and that muscle-specific activation of NF-κB in transgenic mice results in muscle atrophy [38], several important questions remain to be answered. First, it will be important in future studies to determine the mechanisms and biological consequences of the biphasic response of NF-κB in catabolic muscle and in cytokine-treated myotubes.

Second, the apparent contradiction between stimulation of protein degradation by both glucocorticoids and cytokines on the one hand, and the inhibition of NF-κB by glucocorticoids and activation of NF-κB by cytokines on the other hand, needs to be resolved. Third, it will be important to determine in greater detail which muscle wasting-related genes are upregulated and inhibited by NF-κB. Finally, the role of an interaction between glucocorticoids and cytokines, both at a systemic and a cellular level, in the regulation of NF-κB activity needs to be determined.

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Activation of Foxo Transcription Factors Results in Muscle Atrophy

Recent studies support an important role of some of the Foxo (Forkhead box O) transcription factors in the development of muscle atrophy [40, 41]. The Foxo sub- family of the forkhead transcription factors consists of three members: Foxo1, 3a, and 4. These transcription factors are downstream targets of Akt and are inactivated by Akt-mediated phosphorylation. Phosphorylated Foxo transcription factors are retained in the cytoplasm in their inactive form. Dephosphorylation results in transport into the nucleus of the activated Foxo transcription factors. In recent studies in cultured myotubes, treatment with dexamethasone activated Foxo1 and 3a and upregulated the atrogin-1 and MuRF1 genes, resulting in myotube atrophy [40, 41].

These observations are in line with in vivo observations of an association between Foxo transcription factor activation and muscle atrophy in diabetes, fasting, cachexia, and aging [reviewed in 42]. The influence of sepsis, severe injury, and cancer on Foxo transcription factors in skeletal muscle remains to be determined but considering the common response of multiple atrogins in different catabolic conditions, it is probably safe to predict that the Foxo transcription factors are involved in muscle wasting seen in those conditions as well. It should be noted that Foxo transcription factors do not regulate the catabolic response only in skeletal muscle but may also activate an atrogene transcriptional program in cardiomyocytes [43].

p300/HAT Expression and Activity Regulate Protein Degradation in Muscle Cells

In recent years, it has become increasingly clear that in addition to transcription factors, so called nuclear cofactors participate in the regulation of gene activation [44].

Among nuclear cofactors, p300 has attracted much attention [45]. p300 exerts some of its effects through its HAT activity. Although it was initially believed that the major mechanism by which HAT activity regulates gene activation was by increasing the acetylation of histones, disrupting chromatin and enhancing the accessibility of transcription factors to their DNA binding sites, there is evidence that p300 acety- lates other proteins as well, including transcription factors and other nuclear cofactors. Another important mechanism by which nuclear cofactors influence transcriptional activity is protein-protein interactions with transcription factors, nuclear cofactors, and other nuclear proteins that are components of the basal transcription machinery. The interaction with other proteins can result in the acetylation of those proteins but may also serve as a mechanism by which other proteins are recruited for regulation of gene transcription.

It should be noted that protein acetylation is determined not only by HAT activity but by histone deacetylase (HDAC) activity as well [46, 47], and the degree of acetylation is the result of the balance between ongoing acetylation and deacetylation of any given protein (analogous to the phosphorylation of proteins that is regulated by the balance between kinase and phosphatase activities).

Recent studies in our laboratory have focused on the potential role of p300 in the development of muscle wasting. In several of those studies we used glucocorticoid- treated cultured L6 myotubes, a rat skeletal muscle cell line. Glucocorticoid-treated myotubes have been used in a large number of studies, in our and other laborato- ries, as an in vitro model of muscle wasting characterized by increased ubiquitin- proteaosme-dependent proteolysis and reflecting the important role of glucocorti-

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coids as a mediator of muscle wasting [36, 37, 40, 41]. In initial experiments, we found that p300 protein and mRNA levels were increased in a time- and dose-dependent manner in dexamethasone-treated myotubes [21]. In the same study, co-immu- noprecipitation experiments provided evidence for a protein-protein interaction between p300 and C/EBPq . Because there was no interaction between p300 and C/

EBP· , it is possible that the interaction with C/EBP q is specific for this transcription factor among the C/EBP family members. In more recent experiments, we have found evidence that C/EBPq is hyperacetylated in dexamethasone-treated myotubes (unpublished observations), suggesting that the p300-C/EBPq interaction may influence gene transcription at least in part by hyperacetylation.

In subsequent experiments, we observed that p300-associated HAT activity was increased and HDAC3 and 6 expression and activity were reduced after treatment of the myotubes with dexamethasone [22]. In the same study, treatment of the myotubes with the HDAC inhibitor, trichostatin A (TSA), resulted in increased protein degradation, further supporting the role of hyperacetylation as a regulator of muscle protein breakdown. Interestingly, the increase in protein degradation caused by TSA was similar to the increase caused by dexamethasone and the combined treatment of the myotubes with dexamethasone and TSA gave rise to the same stimulation of proteolysis as was noticed after treatment with either drug alone, suggesting (but not proving) that they share a common mechanism in their actions.

Additional experiments in the same study [22] provided strong genetic evidence for a role of p300/HAT activity in the regulation of glucocorticoid-induced muscle protein degradation. In those experiments, silencing of p300 expression using a small interfering RNA (siRNA) technique blocked the dexamethasone-induced increase in protein degradation and a similar inhibition of dexamethasone-induced proteolysis was seen when muscle cells were transfected with a plasmid expressing p300 that had been mutated in its HAT activity domain and therefore lacked HAT activity.

Thus, our recent studies provide evidence for a role of hyperacetylation, mediated by increased HAT and decreased HDAC3 and 6 activities, in the regulation of glucocorticoid-induced muscle proteolysis. It will be important in future experiments to test whether similar mechanisms are involved in the in vivo regulation of muscle wasting seen in catabolic conditions such as sepsis, severe injury, and cancer. It will also be important to determine the exact role of C/EBPq acetylation in the development of muscle atrophy and to test whether other transcription factors (or other nuclear proteins) are acetylated in catabolic muscle. In that respect, it is interesting to note that studies suggest that the activities of both Foxo transcription factors [48]

and NF-κB [30] may be regulated by acetylation/deacetylation. For example, recent studies have provided evidence that the NF-κB p65 subunit is acetylated by p300 after its transport into the nucleus. This acetylation, which occurs on lysine residues 218, 221, and 310, results in increased NF-κB DNA binding and upregulated transcription of NF-κB target genes [30]. p65 is subsequently deacetylated by HDACs, in particular HDAC3, which facilitates the binding of p65 to the inhibitory IκB resulting in nuclear export of p65 and inhibition of NF-κB activity. Thus, the acetylation and deacetylation of p65 seem to act as an important molecular switch regulating NF-κB activity. Ongoing studies in our laboratories are designed to test whether acetylation of p65 (in addition to acetylation of C/EBPq ) plays a role in sepsis- and glucocorticoid-induced muscle wasting. It should be noticed that in other recent experiments we did not find evidence for acetylation of Foxo1 or 3a in dexamethasone-treated myotubes (unpublished observations) suggesting that transcription factors involved in muscle wasting may be differentially regulated by p300/HAT activity.

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Fig 1. Schematic illustration of the role of tran- scription factors and nuclear cofactors in the development of glucocorticoid- and cytokine-regulated muscle wasting in sepsis, severe injury and cancer. Although the roles of CCAAT/enhancer binding protein (C/EBP) transcription factors, nuclear factor-kappa B (NF-κB), forkhead box O (Foxo) 1 and 3a, p300, and histone deacetylases (HDACs) are discussed in this chapter, it is likely that other transcription factors and nuclear cofactors also participate in the regulation of protein degradation in atrophying muscle.

Conclusion

Muscle wasting in a number of catabolic conditions, such as sepsis, severe injury, and cancer, is associated with increased transcription of multiple genes, including (but not limited to) genes in the ubiquitin-proteasome proteolyitc pathway. There is increasing evidence that the expression and activity of transcription factors and nuclear cofactors play an important role in the regulation of muscle wasting-associated genes. Among transcription factors, C/EBPq and · , NF-κB, and Foxo1 and 3a seem to be particularly important for the development of muscle atrophy. Recent studies suggest that the expression and HAT activity of the nuclear cofactor, p300, are essential for the regulation of muscle protein breakdown, possibly by acetylating C/EBPq and the NF-κB p65 subunit. The roles of transcription factors and nuclear cofactors in the regulation of muscle proteolysis and the development of muscle wasting are summarized in Fig 1. An increased understanding of the molecular regulation of catabolic events in skeletal muscle is important for the development of novel and targeted therapeutic strategies for the care of patients with muscle wasting.

Acknowledgment: Studies performed in the author’s laboratory and described in this chapter were supported in part by NIH grants R01 DK37908 and R01 NR8545.

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