Evidence for the participation of the proteasome and calpain in early phases of muscle cell differentiation.
Objectives were to investigate the role of the proteasome and m-calpain to muscle cell differentiation. Accordingly, we investigated the effects of lactacystin, a proteasome inhibitor, and calpain inhibitor-II (CI-II) on L8 muscle cell differentiation and assessed concentrations of proteasomal and calpain subunit mRNAs during differentiation. L8 myoblasts were induced to differentiate by culturing in mitogen-depleted medium. To assess the importance of the proteasome and calpain to differentiation, we examined effects of lactacystin and CI-II on creatine kinase (CK) activity. In the absence of inhibitor, CK activity was detectable within 48 h of mitogen depletion and myotubes were formed. Addition of lactacystin or CI-II to cultures drastically reduced CK activity and prevented formation of myotubes. Hence, proteasome and calpain are both necessary for differentiation. In order to identify which proteasomal subunits were regulated during differentiation, we examined the concentrations of two 20S core subunits (C8 and C9) and three 22S ATPases (MSS1, S4 and TBP1) during differentiation. Concentrations of m-calpain and beta-tubulin mRNAs were also assessed. Differentiation was associated with slight increases (ca. 30%) in concentrations of mRNAs encoding the proteasomal 20S core subunits (C8 and C9) and with large increases (approximately 2-fold) in mRNAs encoding the regulatory subunit ATPases. m-calpain mRNA concentration also increased two-fold following mitogen depletion. beta-Tubulin mRNA concentration remained unchanged early in the differentiation process and thereafter declined. Of interest, changes in proteasomal and m-calpain mRNAs occurred within 6-24 h of mitogen depletion (i.e., at least 24-36 h prior to detectable changes in creatine kinase activity). These results indicate that changes in expression of proteasome and calpains subunits occur early in the differentiation process. These changes may be required for the normal course of differentiation to proceed. Differentiation is associated with larger changes in proteasomal ATPase mRNAs than in 20S core particle mRNAs indicating that either turnover rates of the 22S ATPase subunits are more rapid in differentiating cells than of the 20S core particles or that functions of the regulatory subunits become more important during muscle cell differentiation.
Department of Animal Sciences, Oregon State University, Corvallis 97331-6702, USA.
Y
Y
Ueda
M C
MC
Wang
B R
BR
Ou
J
J
Huang
J
J
Elce
K
K
Tanaka
A
A
Ichihara
N E
NE
Forsberg
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000235:genetics; D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors*; D002352:Carrier Proteins / Q000235:genetics; D002454:Cell Differentiation; D002460:Cell Line; D003402:Creatine Kinase / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D033862:Iron-Binding Proteins; D009097:Multienzyme Complexes / Q000378:metabolism*; D009132:Muscles; D018008:Myogenin / Q000235:genetics; D009842:Oligopeptides / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D033863:Transferrin-Binding Proteins
D002352:Carrier Proteins; D015853:Cysteine Proteinase Inhibitors; D033862:Iron-Binding Proteins; D009097:Multienzyme Complexes; C503803:Myog protein, rat; D018008:Myogenin; D009842:Oligopeptides; D033863:Transferrin-Binding Proteins; C069861:calpain inhibitor 2; C067713:lactacystin; D003402:Creatine Kinase; D002154:Calpain; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases; D000111:Acetylcysteine