Articles in PresS. Am J Physiol Endocrinol Metab (August 5, 2014). doi:10.1152/ajpendo.00204.2014
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Skeletal Muscle Atrophy and the E3 Ubiquitin Ligases, MuRF1 and MAFbx/Atrogin-1
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and Physiology and Membrane Biology2
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University of California, Davis, Davis, CA, 95616.
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Northern California VA Health Systems3
Sue C. Bodine and 1,2,3 and Leslie M. Baehr2 Departments of Neurobiology, Physiology, and Behavior1;
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Running Head: E3 Ubiquitin Ligases and Sparing of Muscle Mass
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Keywords: Muscle Sparing, Ubiquitin Proteasome System, Atrogenes, Protein Quality Control
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Corresponding author:
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Sue C. Bodine, Ph.D.
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Department of Neurobiology, Physiology, and Behavior, 196 Briggs Hall
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University of California, Davis
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One Shields Avenue, Davis, California. 95616
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Phone: (530) 752-0694
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Fax: (530) 752-5582
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Email:
[email protected]
1 Copyright © 2014 by the American Physiological Society.
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Abstract
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over ten years ago as two muscle specific E3 ubiquitin ligases that are transcriptionally increased
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in skeletal muscle under atrophy-inducing conditions, making them excellent markers of muscle
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atrophy. In the past ten years much has been published about MuRF1 and MAFbx with respect
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to their mRNA expression patterns under atrophy-inducing conditions, their transcriptional
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regulation, and their putative substrates. However, much remains to be learned about the
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physiological role of both genes in the regulation of mass and other cellular functions in striated
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muscle. While both MuRF1 and MAFbx are enriched in skeletal, cardiac, and smooth muscle,
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this review will focus on the current understanding of MuRF1 and MAFbx in skeletal muscle,
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highlighting the critical questions that remain to be answered.
Muscle RING Finger 1 (MuRF1) and Muscle Atrophy F-Box (MAFbx)/atrogin-1 were identified
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37 38
Skeletal muscle is a dynamic, multicellular tissue capable of adaptation throughout life in
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response to a variety of signals including neural activation, mechanical loading,
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hormones/growth factors, cytokines, and nutritional status. Muscle size, i.e. fiber cross-sectional
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area, is a particularly adaptive and important property of skeletal muscle that undergoes
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continuous modification in order to regulate muscle strength and metabolism. Increases in fiber
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cross-sectional area occur in response to hormones/growth factors and increased loading
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depending on the life stage of the animal ((34, 110, 132) for review). Under many clinical
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conditions and chronic diseases (such as limb immobilization, bed rest, mechanical ventilation,
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sepsis, cancer cachexia, diabetes, congestive heart failure, neurodegeneration, muscular
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dystrophies, and aging) skeletal muscle mass is lost leading to muscle weakness, inactivity, and
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increased mortality. The loss of muscle mass, i.e. muscle atrophy, is a complex process that
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occurs as a consequence of a variety of stressors including neural inactivity, mechanical
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unloading, inflammation, metabolic stress, and elevated glucocorticoids. The molecules and
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cellular pathways regulating skeletal muscle atrophy are still being discovered, however,
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tremendous progress has been made in the last decade to identify key transcription factors,
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signaling pathways, and cellular processes involved in the initiation and sustained breakdown of
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muscle mass under a variety of conditions.
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The focus of this review is on the specific role of Muscle RING Finger 1 (MuRF1) and Muscle
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Atrophy F-Box (MAFbx) in the regulation of skeletal muscle atrophy, and is not meant to be a
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comprehensive analysis of all of the mechanisms involved in the loss of muscle mass (for review
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see (17, 64, 151). Further, while MuRF1 and MAFbx are expressed in all muscle tissues
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(skeletal, cardiac and smooth), this review will concentrate on the role of these E3 ligases in
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skeletal muscle. Readers are directed to recent reviews for additional information on the role of
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MuRF1 and MAFbx in the regulation of cardiac mass (139, 181).
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Identification of MuRF1 and MAFbx/Atrogin-1 in Skeletal Muscle
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Muscle atrophy occurs as the result of changes in the balance between anabolic and catabolic
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processes with the result being a loss of muscle mass when protein breakdown exceeds protein
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synthesis. At the end of the 20th century, a primary driver of muscle atrophy, under most
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conditions, was thought to be an increase in protein degradation (108). In 1969, Goldberg
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demonstrated that an increase in protein degradation contributed to the loss of muscle mass and
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myofibrillar proteins following denervation and glucocorticoid-treatment (49). Subsequently, it
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was shown that increases in proteolysis occur in a variety of muscle wasting conditions including
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sepsis, cancer, burns, diabetes and starvation, and that the primary pathway responsible for the
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increase in muscle proteolysis under these catabolic conditions was the ATP-dependent,
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ubiquitin-proteasome pathway (109, 140, 183). What remained a mystery, however, were the
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molecular mediators of protein degradation and the atrophy process. Moreover, it was unknown
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as to whether muscle atrophy, caused by different stressors, was regulated by a common
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signaling pathway.
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In 2001, two reports were published that identified two novel E3 ubiquitin ligases as key
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regulators of muscle atrophy (15, 51). In Bodine et al. (2001), a differential display approach
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was used to identify MuRF1 (Trim63) and MAFbx (FBX032) as genes that are similarly altered
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under disparate atrophy conditions including: immobilization, denervation, hindlimb unloading,
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dexamethasone treatment, and interleukin-1 induced cachexia. The approach taken was designed
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to identify a common trigger for atrophy and measured differentially expressed genes in the rat
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medial gastrocnemius after three days of ankle-immobilization, and then determined if a subset
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of genes were universally modified in other types of muscle atrophy. This approach revealed two
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genes whose expression was relatively low in resting skeletal muscle, but increased significantly
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within 24 hours of inactivity or decreased loading. Sequence analysis of the genes revealed that
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one gene was a RING finger protein previously identified as MuRF1 in cardiac tissue, but not
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previously associated with skeletal muscle atrophy (24). The other gene was novel and
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contained an F-box domain, which was characteristic of a family of E3 ligases that function as
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one component of a SCF (skp1, cullin, F-box protein) ubiquitin ligase complex (159, 160). This
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novel gene was shown to bind to both Skip and Cullin and was subsequently named MAFbx
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(Muscle Atrophy F-box).
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The FBX032 gene was simultaneously discovered by another group using a similar approach but
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different atrophy models, and given the name atrogin-1 (51). In pursuit of key factors that
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regulate muscle proteolysis under catabolic states, Gomes et al. (2001) used an Affymetrix
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microarray to identify differentially expressed genes from mouse gastrocnemius muscle
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following two days of food deprivation (51). The most highly up-regulated gene in their analysis
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was a novel F-box protein that they named atrogin-1, which turned out to be identical to MAFbx.
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Northern blot analysis of additional catabolic models showed that MAFbx/atrogin-1 was
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significantly increased in models of diabetes, cancer cachexia and renal failure (51). Subsequent
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analysis using Northern blots (since the MuRF1 gene was not represented on the microarray)
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revealed that expression of MuRF1 was also highly increased in all of these catabolic models
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(87). The E3 ubiquitin ligase, FBX032, is referred to in the literature as both MAFbx and atrogin-
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1. For the remainder of this review, we will use MAFbx to refer to the FBX032 gene.
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MuRF1 and MAFbx are E3 Ubiquitin Ligases
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Muscle RING Finger 1 (MuRF1) was first identified as a novel muscle-specific RING finger
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protein that bound to the kinase domain of the giant sarcomeric protein titin (24). Upon
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identification of MuRF1, additional yeast two hybrid screens were performed using heart cDNA
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libraries and MuRF1 as bait, leading to the identification of two additional RING finger proteins
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with high homology to MuRF1: MuRF2 having 62% homology to MuRF1 and MuRF3 having
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77% homology to MuRF1 (24). MuRF1, 2, and 3 are encoded by distinct genes and represent a
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subgroup of the TRIM superfamily of multidomain ubiquitin E3 ligases characterized by the
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presence of the N-terminal tripartite motif: RING domain, zinc-finger B-box domain, and
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leucine-rich coiled-coil domain (Figure 1) (24, 104, 165). Sequence comparison of the N-
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terminal region (140 residues) encoding the RING domain, a Zn-binding B-box motif, and a
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MuRF family-specific conserved box (MFC) show 82-85% homology between MuRF1, 2, and 3
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(24). Within the muscle fiber, MuRF1, MuRF2, and MuRF3 have been shown to localize at the
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M line of the sarcomere. Additionally, MuRF1 and MuRF3 have been found at the Z lines (24,
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104), while both MuRF1 and MuRF2 have been detected in the nucleus (86, 104, 135). The first
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MuRF protein to be characterized was MuRF3, which was discovered to be essential for muscle
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differentiation during development and microtubule stability (165). Further studies have
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revealed that all three MuRFs are tightly regulated during development, with MuRF2 acting
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predominantly at embryonic stages, whereas MuRF1 and MuRF3 expression increases after birth
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(131). Similar to MuRF3, MuRF2 has been shown to be important for microtubule stability
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along with other M-line proteins (105, 135). MuRF1 is the only family member shown to be
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associated with muscle atrophy and to result in the attenuation of muscle loss when deleted (7,
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15, 43, 82).
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Similar to MuRF1, MAFbx expression is selective to striated muscle. The identification of an F-
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box domain within its sequence suggested that MAFbx might function in a SCF ubiquitin ligase-
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complex (15, 51). Futher study using yeast two hybrids and full length MAFbx as bait revealed
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that MAFbx can bind both Skp1 and Cullin1, and that this interaction is dependent on the F-box
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domain (15). The F-box protein provides specificity for substrate binding, and through
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interactions with other subunits, delivers those substrates to an E2 enzyme for ubiquitination
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(21). In mammals, there are three classes of F-box proteins based on the structure of the
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substrate-binding domain. MAFbx is part of the FBX class because it lacks WD40 repeats and
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leucine-rich regions, which are common structural motifs found in the other two classes.
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Instead, MAFbx contains a PDZ motif and a cytochrome C family heme-binding site in the
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carboxyl terminus (Figure 1) (51). Additional characterization of MAFbx has revealed that it
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contains a leucine zipper domain and a leucine-charged residue-rich domain near its amino
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terminus which increases the number of substrates that MAFbx can potentially recognize (171).
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Interestingly, MAFbx also contains two nuclear localization signals, suggesting it may target
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transcription factors or other nuclear proteins for ubiquitination.
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E3 Ligases and the Ubiquitin Proteasome Pathway
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The many functions of MuRF1 and MAFbx in skeletal muscle continue to be investigated, but
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they are thought to involve the binding of selective substrates for ubiquitination and subsequent
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degradation by the 26S proteasome. Thus, increased expression of MuRF1 and MAFbx
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following an atrophy-inducing stressor is thought to be responsible for the shift in protein
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balance from net synthesis to net degradation, thus inducing a loss of muscle mass. The RING
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finger E3s, of which there are over 600 encoded in the human genome, regulate the addition of
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ubiquitin (monoubiquitylation, multi- monoubiquitylation, and polyubiquitylation) to specific
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proteins thus directing a variety of fates and function including: degradation by the 26S
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proteasome, altered localization with the cell, modification of protein interactions, regulation of
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transcription activity, and propagation of transmembrane signaling (38). Consequently,
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perturbations in the ubiquitin protein system can affect a variety of physiological functions and
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has been linked to aging, as well as, cancer, neurodegeneration, and muscular dystrophy (153).
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In the case of skeletal muscle, increases in the ubiquitin proteasome system are associated with
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the loss of muscle mass.
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The ubiquitylation process includes a series of reactions involving three classes of proteins:
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ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s, also referred to as
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ubiquitin carrier proteins (UBC)), and ubiquitin-protein ligases (E3s) (107) (Figure 2). The
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process begins with the ATP-dependent activation of ubiquitin (Ub) by an E1, which results in a
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high energy thioester linkage between the C-terminus of ubiquitin and the active site cysteine of
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the E1. The activated ubiquitin is then transferred to an E2 (of which there are ~40 in the
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human genome), again forming a thioester bond. The final step is the transfer of the ubiquitin
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from the E2 to a substrate via an E3 ligase (117, 127). Ubiquitin is usually transferred to a
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lysine, or on occasion to the N-terminus, of a substrate or another ubiquitin molecule (163, 164).
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Ubiquitin, a 76 amino acid protein with a molecular weight of 8.5 kDa, can be added to a protein
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as a single entity (monoubiquitin) or as a chain of variable length (polyubiquitin). Ubiquitin
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contains seven lysines (K6, K11, K27, K29, K33, K48, and K63) and thus ubiquitin molecules
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can be linked through any one of these seven lysines (77). Ubiquitin chains can be comprised of
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a single type of linkage (homotypic) or a mixture of linkages (heterotypic). Further, chains can
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be unbranched or branched (forked), the latter being the result of two or more ubiquitin
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molecules being attached to a single ubiquitin (77). Generally, it is thought that proteins with
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homotypic polyubiquitin chains consisting of K48 and K29 linkages are targeted to the 26S
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proteasome for degradation (127). MuRF1 and MAFbx can form K48 and K63 linkages in vitro,
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however, the specific linkages formed with protein substrates in vivo are still unclear, and likely
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depend on the specific E2 pairing (116).
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In mammals, there are two major classes of E3 ubiquitin ligases: the HECT (Homologous to the
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E6AP Carboxyl Terminus) E3 ligases and the RING (Really Interesting New Gene) E3 ubiquitin
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ligases (38). The two classes differ in that the HECT domain E3s are presumed to have
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enzymatic activity and directly catalyze the covalent attachment of ubiquitin to the substrate
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protein, while the RING/RING-like domain E3s do not contain a catalytic domain and instead
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function as a scaffold that bring the E2 and the protein substrate together (38, 107). The
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majority of E3 ligases belong to the RING E3 ligases, which can exist as monomers, dimers, or
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multi-subunit complexes. MuRF1 is an example of a simple RING E3 ligase, which can exist as
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a monomer or dimer with itself or MuRF2 and MuRF3 (24, 114). In contrast, MAFbx belongs to
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the cullin–RING E3 ligase (CRL) superfamily, and more specifically is a multi-subunit complex
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consisting of: SKP1 and a F-box protein which serve as the substrate adaptor module, a cullin
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protein (CUL1) that acts as a scaffold, and a RING finger protein (RBX1 or RBX2) that
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associates with CUL1 and recruits the ubiquitin charged E2s (21, 134). Approximately 70 F-box
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protein have been identified in the human genome (67).
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For the RING E3s, the specific E2-E3 pairing is very important and can influence the type of
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ubiquitin linkage and length of the ubiquitin chain (38). Moreover, a given RING E3 can pair
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with multiple E2s. For example, the E3 BRCA1 can interact with 10 different E2s (28). MuRF1
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has also been shown to interact with multiple E2s in vitro, with different E2-MuRF1 pairings
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leading to different ubiquitin chains. For example, MuRF1 paired with the dimeric E2,
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UbcH13/Uev1a, produces K63-chains, whereas MuRF1 paired with UbcH1 produces K48-
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chains on the substrate (72). An important unanswered question is: what E2s pair with MuRF1
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and MAFbx in skeletal muscle under conditions that induce muscle loss? It is quite possible that
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different E2s are upregulated in response to different stressors, which would affect the E2-E3
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pairings and substrate binding and ubiquitin linkages under divergent atrophy-inducing
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conditions.
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MuRF1 and MAFbx are Atrophy-Associated Genes
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The discovery of MuRF1 and MAFbx yielded two genes having characteristics of key regulators
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of skeletal muscle atrophy: (1) both genes are selectively expressed in striated muscle, (2) both
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genes are expressed at relatively low levels in resting skeletal muscle, and (3) the expression of
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both genes increases rapidly upon the onset of a variety of stressors and prior to the onset of
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muscle loss. Since their initial discovery, MuRF1 and MAFbx mRNA expression has been
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reported to be elevated in a wide range of atrophy-inducing conditions, and thus the two genes
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have become recognized as key markers of muscle atrophy (Table 1). One major gap in our
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understanding of MuRF1 and MAFbx, however, is the lack of reliable protein expression data.
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While we know much about the time course of mRNA expression of both genes under a variety
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of atrophy conditions, we know almost nothing about the kinetics of protein translation or decay
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of these ligases. In this regard, a major limitation has been the availability of selective antibodies
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that do not cross-react with other proteins.
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Demonstrations that MuRF1 and MAFbx are expressed in human muscle and are elevated during
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conditions that elicit muscle atrophy were critical findings that opened up the possibility that
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both genes could be targets for drug development (Table 1). However, instances have been
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found in humans when expression of MuRF1 and MAFbx was not found to be elevated during
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muscle atrophy (69, 122, 147). For the most part, these discrepancies have come from human
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biopsy samples taken during disuse muscle atrophy induced by limb immobilization (69) or bed
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rest (122, 147), and often the muscle samples were taken after an extended period of time (14-60
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days) following the initial unloading. The mRNA expression pattern of MuRF1 and MAFbx in
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rodent models of unloading and inactivity (i.e., immobilization, hindlimb suspension and
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denervation) is typically a rapid rise within the first 48 hours following the trigger, followed by
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sustained elevation for 7-10 days and then a gradual decrease to baseline by around 14 days (15,
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58, 95, 193). In humans, no time course studies comparable to the rodent experiments have been
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done. One study did report an increase in MuRF1, but not MAFbx after 10 days of
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immobilization in the vastus lateralis, followed by a decrease in expression of both genes from
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10 to 21 days (35), which is similar to what happens in rodents. Other human immobilization
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studies found no increase in MuRF1 or MAFbx after 48 hours (173), but significant increases in
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both genes at 14 days in the vastus lateralis (69). The inconsistencies between human and rodent
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immobilization studies may relate to the degree of neural inactivity and joint restriction between
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the models, which would result in varying degrees of muscle atrophy. For example, Bodine et
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al. (2001) induced immobilization by pinning the ankle joint, which caused complete joint
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restriction, some neural inactivity and rapid muscle loss (~10% loss at 3 days and 30% at 7
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days). In contrast, muscle loss in human immobilization studies often occurs at a significantly
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slower rate (5-10 % over 7 days), which could explain the slower time course and lower
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expression levels of MuRF1 and MAFbx in human muscle. In contrast to immobilization,
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mRNA expression of MuRF1 and MAFbx increases significantly within 48 hours following
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spinal cord injury in humans (172), and is at resting levels or reduced following chronic spinal
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cord injury (2 months to 30 years, (90)), which is an expression profile that closely parallels what
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is observed following denervation (13) and spinal cord injury (146, 193) in rodents.
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The animal and human data suggest that under conditions of disuse, MuRF1 and MAFbx RNA
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expression rapidly increases for a relatively short period of time; and thus the inability to
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measure elevated levels of MuRF1 and MAFbx after extended periods of unloading should not
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be interpreted to mean that these genes have not had a significant impact on the atrophy process.
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In addition to disuse conditions (i.e. unloading and inactivity), MuRF1 and MAFbx expression is
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elevated by inflammation, metabolic stress, and glucocorticoids (Table 1). To date, MuRF1 and
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MAFbx mRNA expression have been shown to be elevated at some point during every condition
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that induces skeletal muscle atrophy. In addition, MuRF1 and MAFbx are transiently expressed
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at the onset of reloading following disuse atrophy (161) and following eccentric contractions
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(101, 190), suggesting that they could be involved in the remodeling process. In this regard,
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caution should be used when referring to the genes as “atrogenes”, a term that is commonly used
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to describe the set of genes that are upregulated and responsible for the atrophy process.
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Identification of MuRF1 and MAFbx Targets
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Identification of the cellular targets of MuRF1 and MAFbx has been a difficult task and remains
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an active area of research. The literature suggests that a given E3 can pair with different E2s
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depending on the tissue (e.g. skeletal versus cardiac) and the cellular environment making it
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possible that the substrates targeted for ubiquitylation by MuRF1 and MAFbx vary as a function
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of the atrophy-inducing conditions (eg. denervation vs. unloading vs. glucocorticoids). The
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putative substrates for MuRF1 and MAFbx have primarily been identified through binding
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studies and in vitro ubiquitin ligase assays (71, 83, 94, 171, 184), and many have not been
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validated in vivo. The targets identified to date have varied depending on whether in vitro or in
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vivo systems have been used, the tissues examined (skeletal or cardiac), and the methods used to
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increase the expression of the E3 ligases (29, 31, 68, 145).
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In skeletal muscle, potential targets of MuRF1 have been based primarily on yeast two hybrid
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screens using MuRF1 as bait or in vitro ubiquitination assays using different E2 partners. Using
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a yeast two-hybrid screen of a skeletal muscle cDNA library, Witt et. al. (2005) identified two
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major classes of proteins as potential MuRF1 targets: proteins involved in ATP generation and
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myofibrillar proteins. A number of the myofibrillar proteins that were found to interact with
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MuRF1 (i.e., nebulin, titin, MLC-2, cTNI) were determined not to be primary MuRF1 targets
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since they had similar expression and ubiquitination levels in WT and MuRF1 KO mice (184).
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Examination of protein expression in mice selectively overexpressing MuRF1 in skeletal muscle
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has also suggested that the myofibrillar proteins are not the primary targets of MuRF1 since the
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muscles of the transgenic mice are not atrophied and the expression levels of myofibrillar
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proteins are not decreased relative to WT mice (60, 103). Instead, a comparison of the
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proteasomes and transcriptomes of MuRF1-Tg and WT mice revealed significant differences in
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enzymes involved in ATP generation, especially those involved in glycolysis, suggesting that
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MuRF1 may play a role in metabolic regulation (60).
299 300
A primary role for MuRF1 in the degradation of myofibrillar proteins has been suggested by
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studies using the MuRF1 KO mice and knock down of MuRF1 in C2C12 myotubes (29). Cohen
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et al. (2009) compared the atrophy response in wild type mice and mice expressing a RING-
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deletion mutant MuRF1, and reported that following denervation and fasting the loss of thick
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filament proteins, such as myosin-binding protein C (MyBP-C) and myosin light chains 1 and 2
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(MyLC1 and MyLC2), occurred prior to the loss of myosin heavy chain (MyHC), and was
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dependent on MuRF1. Further, it was concluded that upon disassembly of the myofibrils the
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ultimate ubiquitination and degradation of MyHC, but not actin or other thin filament proteins,
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was MuRF1-dependent. The selective degradation of thick filament proteins by MuRF1 is based
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on the observation that the content of MyLC1, MyLC2, MyBP-C and MyHC was higher in the
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MuRF1 mutant mice than WT mice after 14-days of denervation, while the content of actin and
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other thin filament proteins was decreased to a similar extent in both mutant and WT mice. Of
312
note, is that this selective sparing was only observed after 14 days of denervation, and not at
14
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earlier time points. Furthermore, it is curious that under resting and growth conditions the
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turnover of myofilament proteins appears to be normal in mice with a null deletion of MuRF1
315
which suggests the involvement of other ligases in the ubiquitination and turnover of both thick
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and thin filament proteins, at least under resting conditions. With respect to actin degradation,
317
multiple E3 ligases have been implicated in its ubiquitination and targeted destruction (32, 79,
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137). At this time it is clear that myofibrillar proteins can be ubiquitinylated by MuRF1 in vitro,
319
however, under in vivo conditions of atrophy it is not clear whether the myofibrillar proteins are
320
the primary targets of MuRF1-dependent ubiquitination and degradation, or are spared as a
321
consequence of changes in other signaling pathways.
322 323
The two most widely acknowledged targets of MAFbx in skeletal muscle are MyoD, a myogenic
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regulatory factor, and eukaryotic translation initiation factor 3 subunit f (eIF3-f) (83, 171). More
325
recently, MyHC and other sarcomeric proteins, such as the intermediate filament proteins
326
vimentin and desmin, have been identified as potential MAFbx substrates (97, 99). All of the
327
putative targets of MAFbx have been identified using C2C12 cells and require further in vivo
328
validation. In developing C2C12 cells, there is strong support for a direct link between MyoD and
329
MAFbx. First, MyoD degradation is mediated by the ubiquitin proteasome system and MAFbx
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was found to interact with MyoD and mediate its ubiquitination in vitro through a lysine-
331
dependent pathway (171). Second, MAFbx and MyoD expression was inversely related during
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the course of C2C12 differentiation, and over-expression of MAFbx suppressed MyoD-induced
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differentiation and inhibited myotube formation (171). Additional support for the conclusion
334
that high levels of MAFbx are incompatible with proper myoblast fusion and differentiation
335
comes from a study by Nicolas et al. who found elevated levels of MAFbx, satellite cell
15
336
deregulation, and failure of postnatal growth in transgenic mice expressing a truncated dominant-
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negative form of PW1 (118). However, other ligases must be able to mediate MyoD degradation
338
since myogenesis and postnatal growth is normal in mice with a null deletion of MAFbx.
339 340
The identification of MyoD and eIF3-f as MAFbx targets has led many to suggest that MAFbx
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controls protein synthesis, while MuRF1 controls protein degradation. While over-expression of
342
eIF3-f can induce hypertrophy and inhibition can cause atrophy in myotubes, there is no direct
343
data to show that eIF3-f expression levels in adult muscle fibers is controlled by MAFbx
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expression. Further, the extent and importance of MAFbx-mediated MyoD degradation in adult
345
muscle fibers during atrophy is not clear. In fact, mice with genetic ablation of MAFbx do not
346
have larger than normal myofibers, nor do they hypertrophy to a greater extent than WT mice in
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response to functional overload (8). To the contrary, mice with a null deletion of MuRF1 have
348
been shown to have elevated levels of protein synthesis under certain atrophy conditions (7, 76),
349
and to maintain the ability to hypertrophy in response to loading as they age (62). To date, both
350
MuRF1 and MAFbx have been reported to have the ability to bind MyHC and other myofibrillar
351
proteins and to mediate the ubiquitination of these proteins in vitro. It is possible that the two E3
352
ligases have overlapping sets of substrates, however, it is more likely the case that they have
353
distinct sets of substrates especially given their different phenotypes when challenged in vivo
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(Table 2). Clearly, additional studies are required to identify the primary in vivo substrates of
355
these atrophy-associated muscle specific E3 ligases, which could vary depending on the atrophy
356
conditions.
357 358
Transcriptional Regulation of MuRF1 and MAFbx
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359 360
The finding that MuRF1 and MAFbx are transcriptionally upregulated together under most
361
atrophy-inducing conditions suggests that the two E3 ligases are under the regulation of similar
362
sets of transcription factors. The first transcription factors shown to regulate transcription of
363
both E3 ligases were the class O type forkhead transcription factors (FOXO), which include
364
FOXO1, FOXO3a and FOXO4. All of the FOXO transcription factors are expressed in skeletal
365
muscle and expression of FOXO1 and FOXO3a increases during certain forms of atrophy (7, 27,
366
47, 87, 146, 148, 154). In 2004, two reports were published demonstrating the ability of the
367
FOXO transcription factors to activate MuRF1 and/or MAFbx (148, 167). In Stitt et al. it was
368
revealed that activated FOXO1 was necessary, but not sufficient to increase MuRF1 and MAFbx
369
gene expression in cultured myotubes, and that IGF-1 was able to block the transcriptional
370
activation of both genes in response to dexamethasome (167). In contrast, Sandri et al.
371
concentrated their investigation on FOXO3a and demonstrated that constitutively activate
372
FOXO3a was able to activate the MAFbx promoter in vitro and in vivo, and induce muscle
373
atrophy (148). Following these reports, Waddell et al. provided further evidence that the FOXO
374
transcription factors can directly bind to the MuRF1 and MAFbx promoter, however, it was
375
observed that not all FOXO family members equally activate the FOXO binding motif in the
376
promoters (177). Moreover, it was demonstrated that the MuRF1 promoter has a perfect
377
palindromic glucocorticoid response element (GRE) in the proximal region of the promoter that
378
directly binds the homodimerized glucocorticoid receptor (177). While expression of both
379
MuRF1 and MAFbx increase in response to the synthetic glucocorticoid dexamethasone, the
380
activation of MAFbx is indirect since the promoter activity is not increased by an activated
381
glucocorticoid receptor alone, and thus increased expression of MAFbx in response to
17
382
glucocorticoids is likely related to increased expression of other glucocorticoid responsive
383
transcription factors such as the FOXO transcription factors (7) or KLF-15 (Kruppel-Like
384
Factor-15) (157). While the induction of MuRF1 and MAFbx expression in response to
385
exogenous glucocorticoids is dependent on the glucocorticoid receptor, other atrophy-inducing
386
stimuli, such as denervation, do not require an activated glucocorticoid receptor to increase
387
MuRF1 and MAFbx expression (180).
388 389
The transcription factor, KLF-15, is a glucocorticoid responsive gene that is upregulated in
390
skeletal muscle by dexamethasone. Furthermore, forced overexpression of KLF-15 in muscle is
391
capable of increasing the expression of both MuRF1 and MAFbx (157). Examination of the
392
MuRF1 and MAFbx proximal promoter regions revealed that both genes have multiple KLF-15
393
binding sites, some of which are in close proximity to the FOXO binding sites and the GRE
394
(157). Interestingly, KLF-15 also increases the expression of FOXO1 and FOXO3a, and thus
395
KLF-15 has the ability to activate transcription of MuRF1 and MAFbx directly and through the
396
induction of the FOXO transcription factors. Indeed, activation of the MuRF1 and MAFbx
397
promoters in L6 myoblasts and in the tibialis anterior was significantly greater in response to a
398
combination of FOXO1 and KLF-15 as compared to the individual effects. This was a second
399
demonstration of cooperative activation of the MuRF1 promoter since Waddell et al.
400
demonstrated synergy between FOXO1 and the activated glucocorticoid receptor (177). In this
401
instance, it was shown that the GR-FOXO1 synergy was dependent on the interaction between
402
the GRE and the adjacent FOXO binding element. Of potential physiological relevance was the
403
finding that this interaction enabled low doses of glucocorticoids to fully activate the MuRF1
404
promoter in the presence of FOXO1.
18
405 406
The MuRF1 and MAFbx promoters have putative binding sites for other transcription factors
407
such as the NF-κB transcription factors, C/EBPβ, and Smad3, which can activate either MuRF1
408
or MAFbx individually or in combination with other factors. Numerous studies have
409
demonstrated a role for the NF-κB transcription factors (p65, c-Rel, RelB, p52, p50) in the
410
induction of muscle atrophy under conditions such as disuse (70, 174), denervation (112), aging
411
(12) and cachexia (20, 144). Muscle atrophy induced by elevated levels of the NF-κB
412
transcription factors is often associated with increased expression of the E3 ligases, MuRF1 and
413
MAFbx (20, 186); and in recent reports it was demonstrated that both genes are direct targets of
414
p50 and Bcl-3 and have conserved κB sites either upstream or downstream of the transcriptional
415
start site (65, 186).
416 417
Activation of MAFbx, but not MuRF1, also appears to be under the control of the C/EBP
418
transcription factor (194). Tumor necrosis factor- (TNF-) increases MAFbx expression both
419
in vitro and in vivo via a mechanism dependent on FOXO4 expression, but not FOXO1/3a, and
420
activation of p38/MAPK (96, 113). More recently it was demonstrated that increased p38
421
MAPK activity in myotubes lead to phosphorylation and activation of C/EBP, which was
422
capable of binding to and activating the MAFbx promoter (195).
423 424
Another example of diversity in the activation of MuRF1 and MAFbx is through myostatin, a
425
TGF family member, which when elevated in vitro results in an increase in the expression of
426
MAFbx, and to a lesser extent MuRF1 (97, 99). Activation of the TGF pathway in vivo through
427
overexpression of constitutively active activin receptor-like kinase 5 was shown to induce
19
428
muscle atrophy through a mechanism that involved the increased expression of MAFbx, but not
429
MuRF1 (149). More recently it was shown that overexpression of Smad3, a transcription factor
430
activated by myostatin, in myofibers resulted in 1.8 fold induction of MAFbx promoter activity,
431
and no change in MuRF1 activity, suggesting a mechanism by which myostatin could induce
432
MAFbx expression (54). However, in this report it was unclear as to whether Smad3 activation
433
of MAFbx was direct or indirect through the induction of another factor such as the transcription
434
factor FOX03a. Recently, Bollinger et al. examined the ability of Smad3 and FOXO3a to
435
increase both MuRF1 and MAFbx expression (16). It was found that coexpression of Smad3 and
436
FOXO3a (or FOXO1) increased MuRF1 and MAFbx transcriptional activity to a greater extent
437
than FOXO expression alone (16). This report differed from previous publications in that they
438
found that Smad3 overexpression alone did not increase the expression of either MuRF1 or
439
MAFbx. Examination of the proximal promoter regions of both MuRF1 and MAFbx revealed
440
multiple conserved FoxO responsive elements adjacent to SMAD-binding elements in the
441
MuRF1 promoter. Mutation of the proximal SMAD-binding site eliminated the synergistic
442
activation of the MuRF1 promoter by FOXO3a and Smad3 (16). These authors propose a
443
mechanism by which Smad3 regulates MuRF1 expression by increasing binding of the FOXO
444
transcription factors to the proximal promoter region and also by increasing FOXO3a protein
445
content through an increase in FOXO3a transcription. As noted earlier, cooperative regulation of
446
the MuRF1 promoter by the FOXO transcription factors and other factors such as the
447
glucocorticoid receptor (177) and KLF-15 (157) have been observed.
448 449
The literature suggests that transcriptional control of MuRF1 and MAFbx is complex. The
450
discovery that multiple transcription factors can activate MuRF1 and MAFbx may explain the
20
451
fact that these genes are activated under such a variety of atrophy conditions. Further, the ability
452
of multiple transcription factors to cooperate in the activation of these genes may explain the
453
variable expression patterns observed under different atrophy conditions.
454 455
The Role of MuRF1 and MAFbx in the Regulation of Muscle Mass
456 457
The generation of mice with a null deletion of either MuRF1 (Trim63) or MAFbx (Fbxo32) has
458
allowed for the examination of the physiological function and importance of these two E3 ligases
459
in the regulation of skeletal muscle mass (15). Table 2 describes the phenotype of each knock
460
out mouse under resting conditions and in response to various challenges. Interestingly, at birth
461
and during the first two months of postnatal development, neither mouse strain exhibits a
462
phenotype that differs from WT mice. With age, the two strains begin to show different
463
phenotypes, particularly with respect to the heart, suggesting that MuRF1 and MAFbx control
464
different sets of substrates (63, 192).
465 466
The first atrophy model to be tested using the knock out mice was denervation, where it was
467
demonstrated that deletion of each gene individually resulted in significant sparing of mass in
468
both the tibialis anterior and gastrocnemius muscles; MAFbx KO showing sparing at both 7 and
469
14 days and the MuRF1 KO showing sparing at 14 days (15). In follow-up studies with an
470
extended period of denervation (28 days) it was shown that the mass of both the gastrocnemius
471
and tibialis anterior muscles continued to be spared in MuRF1 and MAFbx KO mice (50).
472
Histological analysis, however, revealed a significant difference in the response of muscles from
473
MuRF1 and MAFbx KO mice to denervation, with muscles in the MAFbx KO mice showing an
21
474
increase in the percentage of vacuole-containing fibers and a greater incidence of muscle fiber
475
necrosis (50). The phenotype observed in the MAFbx KO mice following extended denervation
476
is reminiscent of the phenotype observed following denervation in mice with a muscle specific
477
deletion of Runx1 (runt-related transcription factor 1) (178), and might suggest that some level
478
of MAFbx expression is required to prevent rapid degeneration of muscle fibers following the
479
loss of innervation. It could be that preventing the ubiquitination of MAFbx targets prevents
480
their degradation, leading to their accumulation, which ultimately initiates fiber necrosis. This
481
theory requires further investigation, however, what is known is that complete suppression of
482
MAFbx is not required to achieve muscle sparing since MAFbx expression levels are not
483
suppressed, but maintained at elevated levels in the MuRF1 KO mice following denervation
484
(50).
485 486
Besides denervation, the deletion of MuRF1 has been shown to lead to functional sparing of
487
muscle mass following hindlimb unloading (82), treatment with excess synthetic glucocorticoids
488
(7), and acute lung injury (43). In contrast, deletion of MAFbx does not lead to muscle sparing
489
following glucocorticoid treatment or acute lung injury. Further, neither deletion of MuRF1 nor
490
MAFbx results in the sparing of muscle mass in response to nutritional deprivation (7). The role
491
of MuRF1 and MAFbx in age-related muscle loss has been controversial since no change (48),
492
decreased expression (42), and increased expression (30) of both E3 ligases has been reported in
493
aging rodents. We recently examined the expression of both MuRF1 and MAFbx in aging WT
494
(C57BL6 background) and MuRF1 KO mice, and found no significant increase in either gene in
495
WT mice up to 24 months of age; however, we did find a significant increase in MAFbx
496
expression in the MuRF1 KO mice (62). Interestingly, we found that deletion of MuRF1
22
497
resulted in the maintenance of muscle mass with age and in a maintained ability to hypertrophy
498
in response to increased loading with age (62). Age-related muscle loss was not examined in the
499
MAFbx KO mice since these mice develop congestive heart failure and die prematurely between
500
16-18 months of age (102, 192).
501 502
In general, the experiments on the MuRF1 and MAFbx knock out mice suggest that MuRF1
503
would be a better candidate than MAFbx for drug development targeted to treat muscle atrophy
504
because the deletion of MuRF1 results in muscle sparing under more conditions than the deletion
505
of MAFbx. Moreover, the mass that is spared in response to MuRF1 deletion appears to be
506
functional in that force output is proportional to muscle mass. The only instance where MuRF1
507
deletion did not lead to functional sparing was in aged (24 month old) mice, however further
508
studies are needed to rule out changes in innervation or synaptic instability in the MuRF1 KO
509
mice with age. In the case of MAFbx, it may be that suppression (as opposed to complete
510
inhibition) of MAFbx, along with complete inhibition of MuRF1 could be a good strategy for
511
many forms of muscle atrophy. A question that remains to be answered, and will likely require
512
pharmaceutical agents to block MuRF1 activity is: when and for how long does MuRF1 need to
513
be inhibited? The timing and duration of inhibition likely depends on the type of atrophy. For
514
example, in response to inactivity or unloading, MuRF1 expression increases rapidly and then
515
returns to baseline within two weeks. Interestingly, the muscle sparing effect in the MuRF1 null
516
mice does not occur until after the first week of inactivity when MuRF1 expression is on its
517
decline (15). Given this observation, one might propose that MuRF1 inhibition does not need to
518
occur until after the first week of inactivity; however, that may be too late since the critical
519
interactions between MuRF1 and its primary targets may occur early, soon after the onset of the
23
520
atrophy stimulus, setting off a cascade of events having significant ramifications at later time
521
points. While targeting MuRF1 early may be necessary for inactivity models of atrophy, it may
522
be that under chronic metabolic or inflammatory diseases suppression of MuRF1 would be
523
advantageous at any time during the course of the disease. In this regard, a better understanding
524
of the kinetics of MuRF1 and MAFbx protein expression is needed.
525 526
The ability of MuRF1 inhibition to produce functional muscle sparing in a variety of atrophy
527
models suggests that the deletion of MuRF1 may target key regulatory proteins involved in the
528
activation of critical signaling cascades that drive muscle atrophy. It is still unclear whether the
529
primary pathways affected by upregulation of MuRF1 expression are those involved in protein
530
degradation, protein synthesis, or energy production. Comparisons of wild type and MuRF1 KO
531
mice under a variety of challenges have revealed that different pathways are affected by the
532
deletion of MuRF1 depending on the model under study (Figure 3), however the suppression of
533
cellular stress may be a common theme in a number of the models which requires further
534
investigation.
535 536
We recently compared the effect of denervation on the activation of a number of proteolytic
537
pathways in WT and MuRF1 KO mice, and found that deletion of MuRF1 did result in
538
suppression of some components of the ubiquitin proteasome pathway such as Nedd4, USP19,
539
USP14, UCHL1, but did not suppress the overall capacity for proteolysis in that the activity of
540
calcium-dependent calpain I and II proteases and the lysosomal enzyme, cathepsin L, were
541
similar between WT and KO mice (46, 50). Moreover, it was found that both 20S and 26S
542
proteasome activities were elevated to a greater extent in KO compared to WT mice following
24
543
denervation, and that MAFbx expression remained elevated at 14 days in the MuRF1 KO mice
544
(50). Interestingly, we found that the fractional synthesis rate was elevated in both WT and KO
545
mice at 14 days of denervation (50); however, what remains to be determined is whether the
546
same set of proteins are being translated in the WT and MuRF1 KO mice following denervation.
547 548
Differential regulation of protein synthesis in MuRF1 KO relative to WT mice has been observed
549
in response to glucocorticoid treatment (7) and during load-induced growth in aged mice (62). In
550
response to glucocorticoids, muscle sparing in the MuRF1 KO mice was related primarily to the
551
early maintenance of protein synthesis and the overall suppression of FOXO1 expression (7).
552
More recently, we also observed that the deletion of MuRF1 prevents, or at least delays, the
553
development of anabolic resistance in older mice. Muscle growth in response to functional
554
overload is significantly reduced in old (18 months) relative to young (6 months) WT mice (62).
555
In contrast, there is no decrease in load-induced growth in old MuRF1 KO mice. The maintained
556
growth response in MuRF1 KO mice was associated with less ER stress and greater activation of
557
the Akt/mTOR signaling pathways, suggesting an increase in protein synthesis (62). One
558
mechanism by which the deletion of MuRF1 results in muscle sparing under a variety of
559
disparate conditions may be related to an increased ability to decrease cellular stress and
560
maintain global protein synthesis. This could be mediated in part through the ubiquitin
561
proteasome system and the removal of misfolded or modified proteins. Overall, we have found
562
no evidence that MuRF1 and MAFbx directly control other proteolytic pathways such as the
563
calcium-dependent calpains, lysosomal cathepsins, caspase-3, or autophagy.
564
25
565
The possibility exists that some of the targets of MuRF1-dependent ubiquitination are
566
transcription factors. MuRF1 has been reported to be localized at the M-line of the sarcomere
567
(24), but also to translocate to the nucleus of muscle fibers following inactivity and unloading
568
(121). Further, in vitro, MuRF1 has been reported to interact with known transcription factors
569
such as SRF (182) and GMEB-1 (104). The ability of MuRF1 to influence gene expression was
570
recently examined and compared in two models of atrophy, denervation and glucocorticoid
571
treatment (46). Evaluation of gene expression patterns in WT and MuRF1 KO mice following 3
572
and 14 days of denervation or dexamethasone-treatment revealed that MuRF1 influenced distinct
573
gene networks in these two distinct atrophy models. Overall, denervation resulted in the
574
differential expression of a much larger and different set of genes than dexamethasone treatment,
575
with only a small set of regulated genes being common to both models: MuRF1, MAFbx,
576
FOXO1, EIF4EBP1, ANKRD1 (mCARP), CDKN1A (p21) and TNFRSF12A (TWEAK) (46).
577
Following denervation, mice lacking MuRF1 showed suppression of genes associated with the
578
neuromuscular junction, which correlated with blunted HDAC4 upregulation (46).
579 580
Examination of muscle atrophy in MuRF1 KO mice, and to a lesser degree MAFbx KO mice,
581
have demonstrated the importance of these E3 ubiquitin ligases in mediating muscle atrophy in
582
response to divergent triggers. Sparing of muscle mass has been achieved through the
583
manipulation of other molecules such as the FOXO and NF-B transcription factors; however, in
584
the majority of these studies suppression of both MuRF1 and MAFbx has been reported to occur
585
and may be playing a role in the muscle sparing effect. For example, direct inhibition of FOXO
586
transcriptional activity leads to the attenuation of cachexia and disuse-induced muscle atrophy
587
through a mechanism that likely includes the suppression of MuRF1 and MAFbx expression
26
588
(143). Near complete sparing of immobilization-induced atrophy was found with over-
589
expression of HSP70, which suppressed the transcriptional activities of both FOXO3a and NF-
590
B and reduced the expression of MuRF1 and MAFbx (155). Muscle sparing and suppression of
591
MuRF1 and MAFbx expression has also been reported following hindlimb unloading in mice
592
lacking the Nfkb1 gene (that encodes the NFkB transcription factor, p50) or Bcl-3 (186). A role
593
for MuRF1 in NF-κB mediated loss of muscle mass was demonstrated in Cai et al. (2004), where
594
NF-B signaling was activated through muscle-specific expression of activated IB kinase
595
(MIKK mice) and shown to induce muscle atrophy (20). Increased expression of MuRF1, but
596
not MAFbx, was observed in the MIKK mice, and subsequent breeding of MIKK x MuRF1-/-
597
mice resulted in an intermediate phenotype with approximately a 50% increase in body weight in
598
the MIKK x MuRF-/- mice relative to the MIKK mice.
599 600
Several pharmaceutical treatments have been shown to result in muscle sparing with variable
601
effects on MuRF1 and MAFbx expression. Clenbuterol, a 2 adrenergic agonist, has been
602
shown to induce muscle hypertrophy in young and old rodents in an mTOR-dependent manner
603
and to attenuate the loss of muscle mass following denervation and hindlimb unloading through
604
both mTOR-dependent and independent mechanisms (59, 74, 162). Interesting, under multiple
605
conditions (resting, growth, and atrophy-inducing conditions) clenbuterol has been shown to
606
suppress the expression levels of both MuRF1 and MAFbx (52, 53, 74). Other compounds that
607
mediate their anti-atrophy responses through G-protein coupled receptors are acetylated and
608
unacetylated ghrelin (138). Recently both acetylated and unacetylated ghrelin have been shown
609
to spare muscle mass under conditions of fasting and denervation, through a mechanism that
610
appears to involve the suppression of MAFbx and possibly MuRF1 (138). Furthermore,
27
611
exogenous ghrelin has been shown to inhibit protein breakdown and significantly suppress the
612
upregulation of both MuRF1 and MAFbx mRNA and inflammatory cytokine expression in lower
613
limb muscles following a third-degree burn injury to the back affecting 30% of the total body
614
surface area (10).
615 616
These studies provide additional support for the conclusion that MuRF1 and MAFbx are critical
617
downstream regulators of the atrophy process, but they highlight the fact that other signaling
618
pathways are involved. As noted in the original publication of the MuRF1 and MAFbx KO mice
619
(15), deletion of each gene individually attenuates muscle atrophy, but does not prevent muscle
620
atrophy. Further, the sparing of muscle mass does not always occur immediately in either knock
621
out mouse. Of particular note is the fact that forced overexpression of MuRF1 or MAFbx alone
622
does not induce muscle atrophy. This may suggest that the substrates for MuRF1 and MAFbx
623
are not expressed in resting muscle and/or that MuRF1 and MAFbx require the interaction with
624
other signaling pathways to induce muscle loss.
625 626
Signaling pathways, which are independent of MuRF1 and MAFbx expression, are clearly
627
involved in muscle atrophy. Potential pathways that are involved in muscle atrophy and
628
independent of MuRF1/MAFbx expression are those regulated by ATF4 (activating transcription
629
factor 4) and the tumor suppressor p53 (18, 41, 44). Both transcription factors are upregulated
630
under a variety of atrophy conditions, and forced over expression of each gene induces muscle
631
loss (41, 44). Interestingly, mice with a muscle specific ATF4 deletion are protected from
632
immobilization-induced atrophy without the suppression of MuRF1 or MAFbx; however, the
633
sparing of muscle mass occurs only during the first four days of disuse and is not evident after
28
634
seven days (41). Furthermore, muscle specific ATF KO mice do not exhibit muscle sparing
635
following denervation (44). Muscle atrophy induced by p53 appears to be independent of both
636
ATF4 and MuRF1/MAFbx, and mice with muscle specific deletion of p53 are partially protected
637
from immobilization-induced atrophy (44). Of particular interest is the observation that muscle
638
specific deletion of both ATF4 and p53 results in complete sparing of muscle fiber cross-
639
sectional area following immobilization, at least for 3 days (44). These recent data, coupled with
640
the findings from the MuRF1 KO mice, show that multiple signaling pathways are involved in
641
muscle atrophy; and suggest that different signaling pathways may be responsible for muscle
642
loss at specific time points throughout the process. If this is the case, then multiple therapeutic
643
agents may be required to target multiple proteolytic pathways in order to prevent muscle
644
atrophy.
645 646
Other E3 ligases in muscle
647 648
In addition to MuRF1 and MAFbx, a number of other E3 ubiquitin ligases have been identified
649
in skeletal muscle and associated with the regulation of skeletal muscle under atrophy-inducing
650
conditions. A new F-box protein (Fbxo30), belonging to the SCF complex family of E3 ubiquitin
651
ligases, was recently identified in skeletal muscle and shown to be upregulated following
652
denervation and to undergo autoubiquitination, similar to other E3 ligases. Fbox30, named
653
MUSA1 (muscle ubiquitin ligase of SCF complex in atrophy-1), appears to be inhibited by the
654
BMP pathway under normal conditions based on the findings that MUSA1 is more highly
655
expressed in denervated muscles of Smad4-/- mice than WT mice and is elevated in muscles
656
overexpressing the bone morphogenetic protein (BMP) inhibitor, noggin (150). Knock down of
29
657
MUSA1 protein expression in the tibialis anterior using in vivo electroporation of shRNA
658
resulted in sparing of muscle mass following denervation and prevented the excessive
659
denervation-induced atrophy observed in Smad4-/- mice (150). These data suggest that MUSA1
660
is an important regulator of skeletal muscle mass, especially under extended durations of
661
inactivity. Interestingly, MuRF1 and MAFbx expression were not affected by manipulation of
662
BMP signaling (150).
663 664
Nedd4-1 (Neural precursor cell expressed developmentally down-regulated protein 4) is a HECT
665
domain ubiquitin ligase that shows sustained long-term increased expression in skeletal muscle
666
following denervation (13, 75), hindlimb unloading (75), and severe chronic obstructive
667
pulmonary disease (136). In a recent study, a skeletal muscle specific Nedd-4 knock out mouse
668
was generated and shown to have muscle sparing following both 7 and 14 days of denervation,
669
which differs from the MuRF1 KO that only shows sparing after 7 days of denervation (115).
670
Interestingly, Nedd-4 expression is selectively suppressed in the MuRF1 KO mice following
671
denervation (50). It is unknown whether MuRF1 and/or MAFbx expression were affected in the
672
Nedd-4 KO mice since their expression was not reported.
673 674
Trim32 is another E3 ubiquitin ligase belonging to the tripartite motif family, like MuRF1
675
(Trim63). Unlike MuRF1, Trim32 is ubiquitously expressed with expression levels being 100-
676
fold higher in brain than in skeletal muscle (80). Mutations in Trim32 have been linked to a mild
677
form of dystrophy, limb girdle muscular dystrophy type 2H, and sarcotubular myopathy (79,
678
152). Mice with a null deletion of Trim32 develop a mild myopathy and premature aging; yet
679
demonstrate a normal rate and amount of muscle loss in response to fasting and hindlimb
30
680
unloading (80). The observation that deletion of Trim32 does not affect muscle atrophy
681
contrasts with another study that reports that suppression of Trim32 expression, using in vivo
682
electroporation of shRNA, selectively blocked the loss of thin filament proteins and α-actinin
683
under fasting conditions (32). Cohen et al. (2012) proposed that fasting leads to the
684
phosphorylation of desmin, which leads to its ubiquitylation by Trim32 and subsequent
685
degradation. The loss of desmin subsequently leads to the degradation of α-actinin and the
686
loosening of the thin filaments, making them susceptible to ubiquitylation by Trim32 (32). This
687
model differs greatly from that proposed by Kudryashova et al. (2009) who suggested that
688
Trim32 plays an important role in muscle growth through the regulation of satellite cell
689
proliferation and differentiation since Trim32 KO mice were found to have impaired growth
690
following disuse induced by hindlimb unloading and demonstrate premature aging of skeletal
691
muscle. Trim32 is not upregulated under all forms of atrophy and its precise role in the
692
regulation of skeletal muscle size, especially muscle loss, remains to be determined.
693 694
TNF receptor adaptor protein 6 (TRAF6) is a member of the TRAF family of conserved adaptor
695
proteins that has been shown to be involved in the activation of various signaling cascades
696
including NF-κB, MAPK and PI3K/Akt (84, 129, 189). TRAF6 is distinct among the TRAF
697
family members in that it has been shown to have E3 ubiquitin ligase activity and to be
698
upregulated in skeletal muscle in response to denervation, starvation, and cancer cachexia (128,
699
129). Skeletal muscle specific deletion of TRAF6 in mice results in partial sparing of muscle
700
mass following denervation (129) and starvation (128), and prevention of cancer cachexia
701
induced by Lewis Lung Carcinoma cells (129). The protective effects of TRAF6 deletion were
702
related to suppression of NF-κB, AMPK, JNK and p38 MAPK activation, as well as a
31
703
suppression of MuRF1 and MAFbx expression (129). Interestingly, there was little suppression
704
of MuRF1 and MAFbx expression in the TRAF6 KO mice following starvation; however, this is
705
consistent with the finding that muscle loss in response to nutritional deprivation is not spared in
706
either the MuRF1 or MAFbx KO mice (7, 129). In TRAF6 KO mice, muscle sparing is much
707
greater in the cancer cachexia than denervation, which also correlates to the level of suppression
708
of MuRF1 (129). The data suggest that TRAF6 is upstream of MuRF1 and MAFbx and is an
709
important regulator of muscle mass under conditions involving the activation of pro-
710
inflammatory pathways (81).
711 712
Conclusions
713
MuRF1 and MAFbx represent just two of many E3 ubiquitin ligases that are expressed in
714
skeletal muscle. They are significant, however, because they are primarily expressed in muscle
715
tissues and their expression level in skeletal muscle is significantly increased in response to
716
multiple stressors that induce skeletal muscle atrophy. More importantly, suppression or
717
complete inhibition of their expression results in muscle sparing in multiple atrophy models. In
718
the case of MAFbx, complete inhibition of its expression during atrophy conditions appears to be
719
deleterious. Because the expression of MuRF1 and MAFbx increases during multiple atrophy-
720
inducing conditions they are often referred to as “atrogenes”. However, the expression of
721
MuRF1 and MAFbx is also transiently increased in response to mechanical load, and thus could
722
be associated with the remodeling process.
723 724
The distinct phenotypes of the MuRF1 and MAFbx knock out mice suggest that these two E3
725
ligases have distinct sets of substrates. However, the mechanisms by which suppression of
32
726
MuRF1, in particular, leads to sparing of muscle atrophy remain unclear. The primary substrates
727
for MuRF1 have been suggested to be proteins associated with the thick myofilament; however,
728
the broad effects of MuRF1 deletion on multiple cellular pathways suggest that the primary in
729
vivo substrates of MuRF1 have yet to be identified. One pathway that appears to be affected by
730
MuRF1 deletion in multiple atrophy models is endoplasmic reticulum stress, but further research
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is needed to determine the mechanism by which MuRF1 regulates cellular stress within the
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myofiber. In conclusion, MuRF1 and MAFbx are important muscle specific E3 ligases,
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however, much remains to be understood about their function in regulating muscle mass and
734
other cellular functions in skeletal muscle.
735 736
Acknowledgements
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We thank our colleagues at the University of California, Davis who have contributed to the
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published work presented in this review. We thank Regeneron Pharmaceuticals for providing the
739
MuRF1 and MAFbx KO mice and for supporting the research that went into the initial discovery
740
of these novel muscle-atrophy associated genes. This research was supported by grants to SB
741
from the Muscular Dystrophy Association, National Institute of Health (NIDDK) and the
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Veterans Administration (RR&D).
743
33
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Figure Legends
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Figure 1: Regulation of MuRF1 and MAFbx Expression in Skeletal Muscle.
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Skeletal muscle atrophy is induced by a number of stressors as illustrated in this figure. These
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stressors can lead to the increase in the expression of a number of transcription factors, including
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the forkhead transcription factors (FOXO1 and FOX03a), NF-κB transcription factors (p65, c-
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Rel, RelB, p52, p50), C/EBP, KLF-15 and/or activation of the glucocorticoid receptor. These
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transcriptional mediators can bind to the promoter regions of either the MuRF1 or MAFbx genes
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leading to an increase in their expression levels within the muscle. A schematic of the putative
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domain structure of each protein is shown. MuRF1 contains a RING-Finger domain (RING), a
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MuRF1 family conserved domain MFC), a B-box domain (B-Box), coiled coil domains (CC),
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and an acidic tail region (AR). MAFbx contains two nuclear localization signals (NLS), a
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leucine-zipper domain (LZ), a leucine-charged residue rich domain (LCD), an F-box domain (F-
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box), and a PDZ domain (PDZ).
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Figure 2: The Ubiquitin-Proteasome Pathway.
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The process of ubiquitination is controlled by the ubiquitin-activating enzymes (E1), ubiquitin-
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conjugating enzymes (E2) and ubiquitin-protein ligases (E3). MuRF1 and MAFbx are both E3
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ubiquitin ligases that control the ubiquitination of specific substrates. Ubiquitin can be added to a
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substrate as a single monomer on one (monoubiquitination) or more lysines (multiubiquitination)
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or as a chain of ubiquitins of variable length on a single lysine (polyubiquitination). The model
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of ubiquitination can lead to different substrate fates. Polyubiquitination of a substrate with a
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ubiquitin chain using K11 or K48 linkages generally results in proteasomal degradation, while,
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ubiquitin chains using K63 linkages can lead to alterations in signaling or endocytosis.
1410 1411
Figure 3: Phenotype of MuRF1 Null (KO) Mice.
1412
The major differences in the response of the MuRF1 KO mice relative to wild type mice are
1413
highlighted for glucocorticoid treatment, denervation, and aging (18-24 months old). A common
1414
phenotype of all of the models is muscle sparing.
1415 1416 1417 1418 1419 1420 1421 1422
49
1423 1424 1425
50
1426
Table 1. Atrophy models that have shown MuRF1 and MAFbx to be upregulated
Model
Gene
Species
Reference
Fasting
MAFbx MuRF1 and MAFbx
Mouse Mouse
(36, 51, 66) (3, 87, 111)
Glucocorticoids
MuRF1 and MAFbx MuRF1 MAFbx MuRF1 and MAFbx
Mouse Rat Rat Rat
(7, 15, 176) (4, 187) (188) (196) (191)
Denervation
MuRF1 and MAFbx MuRF1 and MAFbx
Mouse Rat
(15, 111, 146) (193)
MuRF1 and MAFbx MuRF1 and MAFbx MuRF1 and MAFbx
Mouse Rat Human
(146) (193) (172)
Hindlimb Suspension
MuRF1 and MAFbx MuRF1 and MAFbx
Mouse Rat
(15, 58) (100)
Immobilization
MuRF1 and MAFbx MuRF1 MAFbx MuRF1 and MAFbx MuRF1 MAFbx MuRF1 and MAFbx
Mouse Rat Rat Rat Human Human Human
(15, 22, 123) (73) (166) (11, 78) (35) (69) (25, 56)
Bedrest
MAFbx
Human
(122)
ICU mimic
MuRF1 and MAFbx
Rat
(120)
MuRF1 and MAFbx MAFbx MuRF1 and MAFbx
Mouse Rabbit Human
(168) (197) (93)
MuRF1 and MAFbx
Mouse
(87)
Spinal Cord Transection
Mechanical Ventilation
Chronic Kidney Disease
51
Diabetes
MAFbx MuRF1 and MAFbx MAFbx
Mouse Mouse Rat
(26, 36, 61) (87, 179) (89)
Sepsis
MuRF1, MAFbx
Rat
(45, 185)
LPS Injection
MuRF1 and MAFbx MuRF1 and MAFbx
Mouse Rat
(40) (37)
Cachexia
MAFbx MuRF1 and MAFbx MuRF1 and MAFbx
Mouse Mouse Rat
(130) (6, 87, 98, 158) (33)
HIV
MAFbx
Rat
(124, 141)
COPD
MAFbx MuRF1 and MAFbx
Human Human
(92, 126, 136, 169) (39)
Aging
MuRF1 MuRF1 and MAFbx MuRF1
Rat Rat Human
(5) (30) (142)
ALS
MAFbx
Human
(91)
Alcohol
MuRF1 and MAFbx
Rat
(125, 175)
Spinal Muscular Atrophy
MuRF1 and MAFbx
Mouse and Human
(19)
Heart Failure
MuRF1 and MAFbx
Rat
(23)
Space Flight
MAFbx MuRF1
Mouse Rat
(2) (119)
Thermal Injury
MuRF1 and MAFbx
Rat
(85, 156)
MuRF1 MAFbx MuRF1 and MAFbx
Mouse Mouse Mouse
(1) (96, 106) (15)
MAFbx
Human
(133)
Inflammatory Cytokines
Smoking
52
1427 1428
List of
1429
atrophy
1430
models in
1431
which
1432
MuRF1
1433
and/or
1434
Myositis
MAFbx
Human
(88)
Acute Lung Injury
MuRF1, MAFbx
Mouse
(43)
Statins
MAFbx
Humans
(57)
Arthritis
MuRF1, MAFbx
Rat
(55)
Pulmonary Arterial Hypertension
MuRF1, MAFbx
Human
(14)
Hypoxia
MuRF1, MAFbx
Mouse
(170)
Knee Arthroplasty
MuRF1, MAFbx
Human
(9)
MAFbx
1435
have been
1436
shown to be upregulated.
1437 1438
53
1439 1440
Table 2: Phenotype of MuRF1 and MAFbx Knock Out Mice
Postnatal (0 to 2 months) ADULT (2 to 18 months)
Aging (> 18 months)
MuRF1 KO
MAFbx KO
Normal Growth (15)
Normal Growth (15)
Elevated HIF-1 Increased capillary density Elevated glycolytic enzymes (GS, PFK, PDH) Reduced ER Stress (62)
Normal Life Span (62)
Die at 16-18 months of congestive heart failure (102, 192)
Normal growth response to Increased Loading (62)
1441 1442 1443 1444
Atrophy ModelsMuscle Sparing
Denervation (15, 50) Unloading (82) Glucocorticoids (7) Acute Lung Injury Aging (43)
Denervation (15, 50) Immobilization (unpublished)
Load-Induced Hypertrophy
Normal Growth (8, 63)
Attenuated Growth (8)
Characteristics of skeletal muscle in MuRF1 and MAFbx knock out mice at various life stages and in response to various atrophy and hypertrophy challenges.
54
Neural Inactivity
Stressors
Malnutrition
Unloading Oxidative Stress
Glucocorticoids Inflammation/Cytokines
FOXO1/FOXO3a, NF-ΚB, C/EBP β, KLF15, Smad 3, Glucocorticoid Receptor
Transcriptional Mediators Triggers
SCF E3 Ligase
Muscle Atrophy
PDZ
NLS
F-‐Box
LCD
NLS
AR
CC
CC
B-‐Box
MFC
RING
Monomeric E3 Ligase
LZ
MAFbx
MuRF1
E1 Ub + ATP
AMP + PPi Substrate
E2
Substrate
Skp1
Ub
RING
F box
Cul1
MuRF1
MAFbx
RBX
Substrate
Substrate
Skp1
RING
Ub
Cul1
E2
F box E2
RBX
Ub
Ub
Substrate Ub Ub Substrate
Ub
Substrate Monoubiquitination
Ub
Ub
Substrate
Ub
Polyubiquitination
Multimonoubiquitination
DNA Repair Gene Expression Endocytosis
Ub
K48 K29
K63 Receptor Endocytosis Signal Transduction Kinase Activation Endocytosis
Protein degraded into peptides
26S Proteasome
Glucocor'coid Treatment
Denervation Suppression of Selective Genes: USP14, USP19, Nedd4, HDAC4, embryonic AchR and other neuromuscular junction associated genes
No suppression of MAFbx Suppression of FOXO1/3a Expression Suppression of Selective Genes involved in Fat Storage, Insulin Action
Muscle Sparing Lower ER Stress
Increased Proteasome Activity Upregulation of MAFbx
Maintenance of Protein Synthesis
Prevention of Anabolic Resistance
Aging Figure 3
