Regulation of calpain and calpastatin in differentiating myoblasts: mRNA levels, protein synthesis and stability.
Calpain (Ca(2+)-dependent intracellular protease)-induced proteolysis has been considered to play a role in myoblast fusion to myotubes. We found previously that calpastatin (the endogenous inhibitor of calpain) diminishes transiently during myoblast differentiation. To gain information about the regulation of calpain and calpastatin in differentiating myoblasts, we evaluated the stability and synthesis of calpain and calpastatin, and measured their mRNA levels in L8 myoblasts. We show here that mu-calpain and m-calpain are stable, long-lived proteins in both dividing and differentiating L8 myoblasts. Calpain is synthesized in differentiating myoblasts, and calpain mRNA levels do not change during differentiation. In contrast, calpastatin (though also a long-lived protein in myoblasts), is less stable in differentiating myoblasts than in the dividing cells, and its synthesis is inhibited upon initiation of differentiation. Inhibition of calpastatin synthesis is followed by a diminution in calpastatin mRNA levels. A similar calpastatin mRNA diminution is observed upon drug-induced inhibition of protein translation. On the other hand, transforming growth factor beta (which inhibits differentiation) allows calpastatin synthesis and prevents the diminution in calpastatin mRNA. The overall results suggest that at the onset of myoblast differentiation, calpastatin is regulated mainly at the level of translation and that an inhibition of calpastatin synthesis leads to the decrease in its mRNA stability. The existing calpastatin then diminishes, resulting in decreased calpastatin activity in the fusing myoblasts, allowing calpain activation and protein degradation required for fusion.
Barnoy
S
S
Department of Human Genetics and Molecular Medicine, Sackler Scool of Medicine, Tel-Aviv University, Ramat-Aviv, Tel Aviv 69978, Israel.
Supino-Rosin
L
L
Kosower
N S
NS
D000818:Animals; D015152:Blotting, Northern; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D015151:Immunoblotting; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism*; D011233:Precipitin Tests; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000378:metabolism
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D011691:Puromycin; C033668:calpastatin; D003513:Cycloheximide; D002154:Calpain