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

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-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.

tBHQ Induces a Hormetic Response That Protects L6 Myoblasts against the Toxic Effect of Palmitate

“…The L6 myoblast cell line is commonly used to study damage and differentiation in response to redox modifications [54][55][56]. Previous work has clearly demonstrated the involvement of oxidative stress as one of the main factors in the induction of cell death by palmitate [57][58][59]; this was confirmed by our experiments where L6 myoblasts were incubated with palmitate for 24 h, resulting in a dramatic decrease in the cell survival and correlating with a significant decrease in the GSH/GSSG ratio.…”

High-density lipoprotein cholesterol is associated with multiple sclerosis fatigue: A fatigue-metabolism nexus?

“…40 ROS induce the NF-kB and other pathways, [41][42][43] which can directly promote protein catabolism that lead to muscle weakness and atrophy. 44,45 HDL-C has antiinflammatory effects and its antioxidant enzyme PON1 rapidly hydrolyzes lipid peroxides and protects lipoproteins from oxidative modifications. Higher HDL-C levels are associated with lower levels of BBB breakdown and gray matter atrophy.…”

Reduction of stearoyl-CoA desaturase (SCD) contributes muscle atrophy through the excess endoplasmic reticulum stress in chronic kidney disease

“…Myotube diameters was determined as previously reported. ( 21 ) We took 5 photos per cell-culture well at the high-power field on a fluorescence microscopy BIOREVO BZ-9000 (Keyence). We measured the diameter at the middle portion of the myotube with the built-in software BZ-II analyzer.…”