Identification of atrogin-1-targeted proteins during the myostatin-induced skeletal muscle wasting.

Atrogin-1, a muscle-specific E3 ligase, targets MyoD for degradation through the ubiquitin-proteasome-mediated system. Myostatin, a member of the transforming growth factor-ß superfamily, potently inhibits myogenesis by lowering MyoD levels. While atrogin-1 is upregulated by myostatin, it is currently unknown whether atrogin-1 plays a role in mediating myostatin signaling to regulate myogenesis. In this report, we have confirmed that atrogin-1 increasingly interacts with MyoD upon recombinant human myostatin (hMstn) treatment. The absence of atrogin-1, however, led to elevated MyoD levels and permitted the differentiation of atrogin-1(-/-) primary myoblast cultures despite the presence of exogenous myostatin. Furthermore, inactivation of atrogin-1 rescued myoblasts from growth inhibition by hMstn. Therefore, these results highlight the central role of atrogin-1 in regulating myostatin signaling during myogenesis. Currently, there are only two known targets of atrogin-1. Thus, we next characterized the associated proteins of atrogin-1 in control and hMstn-treated C2C12 cell cultures by stably expressing tagged atrogin-1 in myoblasts and myotubes, and sequencing the coimmunoprecipitated proteome. We found that atrogin-1 putatively interacts with sarcomeric proteins, transcriptional factors, metabolic enzymes, components of translation, and spliceosome formation. In addition, we also identified that desmin and vimentin, two components of the intermediate filament in muscle, directly interacted with and were degraded by atrogin-1 in response to hMstn. In summary, the muscle wasting effects of the myostatin-atrogin-1 axis are not only limited to the degradation of MyoD and eukaryotic translation initiation factor 3 subunit f, but also encompass several proteins that are involved in a wide variety of cellular activities in the muscle.

Myostatin is a novel tumoral factor that induces cancer cachexia.

Humoral and tumoral factors collectively promote cancer-induced skeletal muscle wasting by increasing protein degradation. Although several humoral proteins, namely TNFα (tumour necrosis factor α) and IL (interleukin)-6, have been shown to induce skeletal muscle wasting, there is a lack of information regarding the tumoral factors that contribute to the atrophy of muscle during cancer cachexia. Therefore, in the present study, we have characterized the secretome of C26 colon cancer cells to identify the tumoral factors involved in cancer-induced skeletal muscle wasting. In the present study, we show that myostatin, a procachectic TGFß (transforming growth factor ß) superfamily member, is abundantly secreted by C26 cells. Consistent with myostatin signalling during cachexia, treating differentiated C2C12 myotubes with C26 CM (conditioned medium) resulted in myotubular atrophy due to the up-regulation of muscle-specific E3 ligases, atrogin-1 and MuRF1 (muscle RING-finger protein 1), and enhanced activity of the ubiquitin-proteasome pathway. Furthermore, the C26 CM also activated ActRIIB (activin receptor type II B)/Smad and NF-κB (nuclear factor κB) signalling, and reduced the activity of the IGF-I (insulin-like growth factor 1)/PI3K (phosphoinositide 3-kinase)/Akt pathway, three salient molecular features of myostatin action in skeletal muscles. Antagonists to myostatin prevented C26 CM-induced wasting in muscle cell cultures, further confirming that tumoral myostatin may be a key contributor in the pathogenesis of cancer cachexia. Finally, we show that treatment with C26 CM induced the autophagy-lysosome pathway and reduced the number of mitochondria in myotubes. These two previously unreported observations were recapitulated in skeletal muscles collected from C26 tumour-bearing mice.