Hydrogen peroxide stimulates ubiquitin-conjugating activity and expression of genes for specific E2 and E3 proteins in skeletal muscle myotubes

Department of Medicine, Baylor College of Medicine, Houston, Texas 77030 Submitted 3 April 2003 ; accepted in final form 24 May 2003 Reactive oxygen species (ROS) are thought to promote muscle atrophy in chronic wasting diseases, but the underlying mechanism has not been determined. Here we show that H 2 O 2 stimulates ubiquitin conjugation to muscle proteins through transcriptional regulation of the enzymes (E2 and E3 proteins) that conjugate ubiquitin to muscle proteins. Incubation of C 2 C 12 myotubes with 100 µM H 2 O 2 increased the rate of 125 I-labeled ubiquitin conjugation to muscle proteins in whole cell extracts. This response required at least 4-h exposure to H 2 O 2 and persisted for at least 24 h. Preincubating myotubes with cycloheximide or actinomycin D blocked H 2 O 2 stimulation of ubiquitin-conjugating activity, suggesting that gene transcription is required. Northern blot analyses revealed that H 2 O 2 upregulates expression of specific E3 and E2 proteins that are thought to regulate muscle catabolism, including atrogin1/MAFbx, MuRF1, and E2 14k . These results suggest that ROS stimulate protein catabolism in skeletal muscle by upregulating the ubiquitin conjugation system. cachexia; proteolysis; reactive oxygen species; free radicals; aging Address for reprint requests and other correspondence: M. B. Reid, Dept. of Medicine, Baylor College of Medicine, One Baylor Plaza, Suite 520B, Houston, TX 77030 (E-mail: reid{at}bcm.tmc.edu ).