Reactive oxygen species upregulate expression of muscle atrophy-associated ubiquitin ligase Cbl-b in rat L6 skeletal muscle cells.

Unloading-mediated muscle atrophy is associated with increased reactive oxygen species (ROS) production. We previously demonstrated that elevated ubiquitin ligase casitas B-lineage lymphoma-b (Cbl-b) resulted in the loss of muscle volume (Nakao R, Hirasaka K, Goto J, Ishidoh K, Yamada C, Ohno A, Okumura Y, Nonaka I, Yasutomo K, Baldwin KM, Kominami E, Higashibata A, Nagano K, Tanaka K, Yasui N, Mills EM, Takeda S, Nikawa T. Mol Cell Biol 29: 4798-4811, 2009). However, the pathological role of ROS production associated with unloading-mediated muscle atrophy still remains unknown. Here, we showed that the ROS-mediated signal transduction caused by microgravity or its simulation contributes to Cbl-b expression. In L6 myotubes, the assessment of redox status revealed that oxidized glutathione was increased under microgravity conditions, and simulated microgravity caused a burst of ROS, implicating ROS as a critical upstream mediator linking to downstream atrophic signaling. ROS generation activated the ERK1/2 early-growth response protein (Egr)1/2-Cbl-b signaling pathway, an established contributing pathway to muscle volume loss. Interestingly, antioxidant treatments such as N-acetylcysteine and TEMPOL, but not catalase, blocked the clinorotation-mediated activation of ERK1/2. The increased ROS induced transcriptional activity of Egr1 and/or Egr2 to stimulate Cbl-b expression through the ERK1/2 pathway in L6 myoblasts, since treatment with Egr1/2 siRNA and an ERK1/2 inhibitor significantly suppressed clinorotation-induced Cbl-b and Egr expression, respectively. Promoter and gel mobility shift assays revealed that Cbl-b was upregulated via an Egr consensus oxidative responsive element at -110 to -60 bp of the Cbl-b promoter. Together, this indicates that under microgravity conditions, elevated ROS may be a crucial mechanotransducer in skeletal muscle cells, regulating muscle mass through Cbl-b expression activated by the ERK-Egr signaling pathway.

[Plasticity of skeletal muscle against unloading stress.]

Recently, muscle atrophy caused by unloading, such as spaceflight and bed rest has been becoming a social problem in Japan. However, the effective countermeasures against these disuse atrophy have not been developed. We have reviewed the mechanisms of disuse atrophy and its possible countermeasures.

N-myristoylated ubiquitin ligase Cbl-b inhibitor prevents on glucocorticoid-induced atrophy in mouse skeletal muscle.

A DGpYMP peptide mimetic of tyrosine(608)-phosphorylated insulin receptor substrate-1 (IRS-1), named Cblin, was previously shown to significantly inhibit Cbl-b-mediated IRS-1 ubiquitination. In the present study, we developed N-myristoylated Cblin and investigated whether it was effective in preventing glucocorticoid-induced muscle atrophy. Using HEK293 cells overexpressing Cbl-b, IRS-1 and ubiquitin, we showed that the 50% inhibitory concentrations of Cbl-b-mediated IRS-1 ubiquitination by N-myristoylated Cblin and Cblin were 30 and 120 µM, respectively. Regarding the DEX-induced atrophy of C2C12 myotubes, N-myristoylated Cblin was more effective than Cblin for inhibiting the DEX-induced decreases in C2C12 myotube diameter and IRS-1 degradation. The inhibitory efficacy of N-myristoylated Cblin on IRS-1 ubiquitination in C2C12 myotubes was approximately fourfold larger than that of Cblin. Furthermore, N-myristoylation increased the incorporation of Cblin into HEK293 cells approximately 10-folds. Finally, we demonstrated that N-myristoylated Cblin prevented the wet weight loss, IRS-1 degradation, and MAFbx/atrogin-1 and MuRF-1 expression in gastrocnemius muscle of DEX-treated mice approximately fourfold more effectively than Cblin. Taken together, these results suggest that N-myristoylated Cblin prevents DEX-induced skeletal muscle atrophy in vitro and in vivo, and that N-myristoylated Cblin more effectively prevents muscle atrophy than unmodified Cblin.

Additional effects of simultaneous treatment with C14-Cblin and celastrol on the clinorotation-induced rat L6 myotube atrophy.

Two novel reagents, N-myristoylated Cbl-b inhibitory peptide (C14-Cblin) and celastrol, a quinone methide triterpene, are reported to be effective in preventing myotube atrophy. The combined effects of C14-Cblin and celastrol on rat L6 myotubes atrophy induced by 3D-clinorotation, a simulated microgravity model, was investigated in the present study. We first examined their effects on expression in atrogenes. Increase in MAFbx1/atrogin-1 and MuRF-1 by 3D-clinorotation was significantly suppressed by treatment with C14-Cblin or celastrol, but there was no additive effect of simultaneous treatment. However, celastrol significantly suppressed the upregulation of Cbl-b and HSP70 by 3D-clinorotation. Whereas 3D-clinorotation decreased the protein level of IRS-1 in L6 myotubes, C14-Cblin and celastrol inhibited the degradation of IRS-1. C14-Cblin and celastrol promoted the phosphorylation of FOXO3a even in microgravity condition. Simultaneous administration of C14-Cblin and celastrol had shown little additive effect in reversing the impairment of IGF-1 signaling by 3D-clinorotation. While 3D-clinorotation-induced marked oxidative stress in L6 myotubes, celastrol suppressed 3D-clinorotation-induced ROS production. Finally, the C14-Cblin and celastrol-treated groups were inhibited decrease in L6 myotube diameter and increased the protein content of slow-twitch MyHC cultured under 3D-clinorotation. The simultaneous treatment of C14-Cblin and celastrol additively prevented 3D-clinorotation-induced myotube atrophy than single treatment. J. Med. Invest. 69 : 127-134, February, 2022.