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, Oku...

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Published in:	American Journal of Physiology: Cell Physiology Vol. 314; no. 6; p. C721
Main Authors:	Uchida, Takayuki, Sakashita, Yoshihiro, Kitahata, Kanako, Yamashita, Yui, Tomida, Chisato, Kimori, Yuki, Komatsu, Akio, Hirasaka, Katsuya, Ohno, Ayako, Nakao, Reiko, Higashitani, Atsushi, Higashibata, Akira, Ishioka, Noriaki, Shimazu, Toru, Kobayashi, Takeshi, Okumura, Yuushi, Choi, Inho, Oarada, Motoko, Mills, Edward M, Teshima-Kondo, Shigetada, Takeda, Shin'ichi, Tanaka, Eiji, Tanaka, Keiji, Sokabe, Masahiro, Nikawa, Takeshi
Format:	Journal Article
Language:	English
Published:	United States 01-06-2018
Subjects:	Adaptor Proteins, Signal Transducing - genetics Adaptor Proteins, Signal Transducing - metabolism Animals Antioxidants - pharmacology Cercopithecus aethiops COS Cells Early Growth Response Transcription Factors - genetics Early Growth Response Transcription Factors - metabolism Extracellular Signal-Regulated MAP Kinases - metabolism Glutathione - metabolism Mechanotransduction, Cellular Muscular Atrophy - enzymology Muscular Atrophy - genetics Muscular Atrophy - pathology Muscular Atrophy - prevention & control Myoblasts, Skeletal - drug effects Myoblasts, Skeletal - enzymology Myoblasts, Skeletal - pathology Oxidation-Reduction Oxidative Stress - drug effects Phosphorylation Promoter Regions, Genetic Proto-Oncogene Proteins c-cbl - genetics Proto-Oncogene Proteins c-cbl - metabolism Rats Reactive Oxygen Species - metabolism Space Flight Time Factors Up-Regulation Weightlessness Weightlessness Simulation Egr unloading-mediated muscle atrophy ubiquitin ligase Cbl-b rat L6 cells ROS
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Summary:	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.
ISSN:	1522-1563
DOI:	10.1152/ajpcell.00184.2017