Treatment of cultured myotubes with the proteasome inhibitor beta-lactone increases the expression of the transcription factor C/EBPbeta.
The role of the proteasome in the regulation of cellular levels of the transcription factor CCAAT/enhancer-binding protein beta (C/EBPbeta) is poorly understood. We tested the hypothesis that C/EBPbeta levels in cultured myotubes are regulated, at least in part, by proteasome activity. Treatment of cultured L6 myotubes, a rat skeletal muscle cell line, with the specific proteasome inhibitor beta-lactone resulted in increased nuclear levels of C/EBPbeta as determined by Western blotting and immunofluorescent detection. This effect of beta-lactone reflected inhibited degradation of C/EBPbeta. Surprisingly, the increased C/EBPbeta levels in beta-lactone-treated myotubes did not result in increased DNA-binding activity. In additional experiments, treatment of the myotubes with beta-lactone resulted in increased nuclear levels of growth arrest DNA damage/C/EBP homologous protein (Gadd153/CHOP), a dominant-negative member of the C/EBP family that can form heterodimers with other members of the C/EBP family and block DNA binding. Coimmunoprecipitation and immunofluorescent detection provided evidence that C/EBPbeta and Gadd153/CHOP interacted and colocalized in the nuclei of the beta-lactone-treated myotubes. When Gadd153/CHOP expression was downregulated by transfection of myotubes with siRNA targeting Gadd153/CHOP, C/EBPbeta DNA-binding activity was restored in beta-lactone-treated myotubes. The results suggest that C/EBPbeta is degraded by a proteasome-dependent mechanism in skeletal muscle cells and that Gadd153/CHOP can interact with C/EBPbeta and block its DNA-binding activity. The observations are important because they increase the understanding of the complex regulation of the expression and activity of C/EBPbeta in skeletal muscle.
Wei
Wei
W
Department of Surgery, Beth Israel Deaconess Medical Center, 330 Brookline Avenue ST 919, Boston, MA 02215, USA.
Yang
Hongmei
H
Menconi
Michael
M
Cao
Peirang
P
Chamberlain
Chester E
CE
Hasselgren
Per-Olof
PO
D000818:Animals; D015153:Blotting, Western; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects; D015536:Down-Regulation; D005455:Fluorescent Antibody Technique; D007783:Lactones / Q000494:pharmacology*; D018485:Muscle Fibers, Skeletal / Q000187:drug effects* / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000502:physiology*; D061988:Proteasome Inhibitors; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D014018:Tissue Distribution; D051742:Transcription Factor CHOP / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D022782:CCAAT-Enhancer-Binding Protein-beta; D007783:Lactones; D061988:Proteasome Inhibitors; D034741:RNA, Small Interfering; D051742:Transcription Factor CHOP; C098869:clasto-lactacystin beta-lactone; D004247:DNA; D046988:Proteasome Endopeptidase Complex