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

Takayuki Uchida, Yoshihiro Sakashita, Kanako Kitahata, Yui Yamashita, Chisato Tomida, Yuki Kimori, Akio Komatsu, Katsuya Hirasaka, Ayako Ohno, Reiko Nakao, Atsushi Higashitani, Akira Higashibata, Noriaki Ishioka, Toru Shimazu, Takeshi Kobayashi, Yuushi Okumura, Inho Choi, Motoko Oarada, Edward M. Mills, Shigetada Teshima-KondoShin’ichi Takeda, Eiji Tanaka, Keiji Tanaka, Masahiro Sokabe, Takeshi Nikawa

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

Abstract

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-acetyl-cysteine and TEMPOL, but not catalase, blocked the clinorotation-mediated activation of ERK1/2. The increased ROS induced transcrip-tional 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 mechano-transducer in skeletal muscle cells, regulating muscle mass through Cbl-b expression activated by the ERK-Egr signaling pathway.

Original languageEnglish
Pages (from-to)C721-C731
JournalAmerican Journal of Physiology - Cell Physiology
Volume314
Issue number6
DOIs
Publication statusPublished - 2018 Jun

Keywords

  • Egr
  • ROS
  • Rat L6 cells
  • Ubiquitin ligase Cbl-b
  • Unloading-mediated muscle atrophy

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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