Anabolic and catabolic signals: Edited by Vickie E. Baracos and Didier Attaix
This issue of ‘anabolic and catabolic signals’ mainly contains challenging reviews on the control of muscle mass. Two papers in this issue are devoted to recent progress in the elucidation of signaling pathways that control muscle size (Glass, pp. 225–229; McCarthy and Esser, pp. 230–235). Accordingly, the authors review the different mechanisms regulating the expression of two ubiquitin–protein ligases (E3) called MAFbx/Atrogin-1 and MuRF-1. These E3 figure prominently in recent research, as early work led to the hope that they might serve as sensitive biomarkers related to muscle wasting and to the specific rate of muscle protein degradation, an important process but one that is dauntingly difficult to measure in vivo. However, recent findings concerning MAFbx/Atrogin-1 also reveal unexpected problems of interpretation.
MAFbx/Atrogin-1 and MuRF1 were identified in 2001 in the laboratories of both Glass  and Goldberg . These studies nicely demonstrated that the two E3 were muscle-specific, their expression was increased by at least 6–10 times in several catabolic conditions (muscle disuse, hind limb suspension, denervation, and glucocorticoid or interleukin-1 treatment , fasting, cancer cachexia, diabetes, and renal failure ), and knockout mice for either E3 were partially resistant to muscle wasting .
At present, it has been already established that the ubiquitin–proteasome system (UPS) played a major role in muscle wasting and more particularly in the breakdown of myofibrillar proteins. The two identified E3 were critical components of the UPS (E3 recognize specific substrates for polyubiquitination that are subsequently targeted for breakdown by the 26S proteasome), were muscle-specific, and played a key role in muscle wasting. Accordingly, investigators were quick to use increased expression of either gene as a convenient marker of enhanced muscle protein breakdown. These studies confirmed that both MAFbx/Atrogin-1 and MuRF1 are overexpressed in numerous catabolic conditions. Moreover, significant advances have been made in elucidating the signaling pathways that control the expression of the two E3 and result in muscle atrophy [see Glass (pp. 225–229) and [3,4]]. Such studies are of importance for proposing new strategies to prevent or limit muscle wasting.
Subsequently, MAFbx/Atrogin-1 was the subject of reports by different laboratories [5–7] showing no correlation between the expression of MAFbx/Atrogin-1 and rates of protein breakdown both in rat muscles [5,6] and C2C12 myotubes . Numerous papers now are pointing out this discrepancy, including human studies [for recent reviews, see Murton and Greenhaff (pp. 249–254) and ]. That a more serious issue was raised when the MyoD transcription factor was reported to be a MAFbx/Atrogin-1 substrate  is important, as proteolysis of this transcription factor for muscle proteins would be expected to influence muscle differentiation and protein synthesis.
In 2007, the group of David J. Glass provided evidence that myosin heavy chain (MHC), a major myofibrillar protein, is recognized by MuRF-1 in muscle cells . More recent in-vivo experiments have demonstrated that not only MHC but also other myofibrillar proteins are targeted for breakdown by the proteasome in a MuRF-1-dependent fashion . These findings are in perfect agreement with a major role of MuRF-1 in skeletal muscle proteolysis. Interestingly, however, a role for MuRF-1 in protein synthesis has also been recently reported [12,13].
Where are we with the MAFbx/Atrogin-1 substrates? The only identified substrates of this E3 have been characterized by the group of Serge Leibovitch and to date include MyoD [9,14] and the eukaryotic initiation factor of protein synthesis eIF3-f [15–17]. These two proteins play key roles in the control of protein synthesis and not in the breakdown of muscle proteolysis. Leibovitch and coworkers recently provided strong further evidence that MAFbx/Atrogin-1 modulated muscle size by regulating protein synthesis. Firstly, in cultured myotubes undergoing atrophy, MAFbx/Atrogin-1 expression increased, leading to a cytoplasmic-nuclear shuttling of E3 and to selective MyoD suppression. Conversely, transfection of myotubes with sh-RNA-mediated MAFbx/Atrogin-1 gene silencing (shRNAi) inhibited atrophy-dependent MyoD breakdown. Overexpression of a MyoD mutant lacking MAFbx/Atrogin-1-mediated ubiquitination prevented atrophy of both mouse primary myotubes and skeletal muscle fibers in vivo . Secondly, in MAFbx/Atrogin-1-induced atrophy, the degradation of eIF3-f suppressed S6K1 activation by mTOR . By contrast, an eIF3-f mutant insensitive to MAFbx/Atrogin-1 polyubiquitination exhibited persistent phosphorylation of S6K1 and rpS6. A conserved motif in eIF3-f connected the mTOR/raptor complex, which phosphorylates S6K1 and regulates downstream effectors of mTOR and Cap-dependent translation initiation in terminal muscle differentiation. The authors concluded that the eIF3-f substrate of MAFbx/Atrogin-1 played a major role for proper activity of mTORC1 to regulate skeletal muscle size by modulating protein synthesis . Finally, and as also pointed out by McCarthy and Esser (pp. 230–235), recent and totally independent findings clearly indicated that the expression of MAFbx/Atrogin-1 and MuRF-1 is regulated by distinct mechanisms [18,19].
These recent data suggest that the two muscle-specific E3 play distinct roles in wasting. MuRF-1 is mostly clearly involved in the breakdown of myofibrillar proteins. By contrast, and so far, MAFbx/Atrogin-1 has a demonstrated role in only the control of protein synthesis. As muscle wasting results from both enhanced proteolysis and depressed protein synthesis in most catabolic states, the two E3 contribute to the depletion of muscle mass in a co-ordinate manner.
Although future research will continue to clarify the regulation of muscle protein turnover, it is presently evident that MuRF-1 is relatively unambiguously associated with muscle proteolysis, whereas MAFbx/Atrogin-1 seems more clearly related to downregulation of muscle protein synthesis and is not a correlate of rates of skeletal muscle protein breakdown [5–9,14–19]. This distinction may be particularly important in clinical studies, where we lack a reliable technique to measure skeletal muscle proteolysis in vivo. Human muscle tissue of sufficient quantity for RT-PCR analysis can be obtained by biopsy and this analysis is rapid and precise. This approach may be informative in different instances of muscle atrophy, provided that interpretation of the results takes into account the respective roles of MuRF-1 in proteolysis and of MAFbx/Atrogin-1 in protein synthesis.
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