Brefeldin A inhibits muscle-specific gene expression during differentiation in C2C12 myoblasts

In analyzing regulatory mechanisms underlying differentiation-dependent expression of muscle proteins, we have found that mouse C2C12 myoblast cells treated with Brefeldin A (BFA), which specifically blocks the intracellular transport of protein from endoplasmic reticulum (ER) to the Golgi apparatus, failed to differentiate. BFA reversibly inhibited myotube formation at a threshold concentration of 1.0 micrograms/ml. The same concentration of BFA completely blocked accumulation of secretory proteins, e.g., fibronectin into the culture medium, probably due to retention in ER. Interestingly, the induction of muscle creatine phosphokinase (MCK) activity was sensitive to BFA, although a constitutive expression of lactate dehydrogenase, one of the cytosolic housekeeping enzymes, was resistant to BFA. In the kinetic analysis of MCK induction, translation of existing MCK mRNA was not inhibited by BFA since MCK activity became resistant to BFA once levels of MCK transcript accumulated. The differentiation-dependent accumulation of MCK transcript was also suppressed by BFA treatment to approximately 30% of control, although neither general transcriptional activity in vivo nor stability of existing mRNAs was affected even in the presence of BFA. Moreover, induction of muscle regulatory genes, MyoD1 and myogenin, which are upstream of the MCK gene, were inhibited at their transcription level by BFA. Such an effect on muscle regulatory gene transcription by BFA suggests that transport of some factor(s) to the cell surface may be a prerequisite for the muscle-specific gene expression upon differentiation of C2C12 myoblasts.