Knockdown of Hypoxia-Inducible Factor-1a by siRNA Inhibits C2C12 Myoblast Differentiation

Knockdown of Hypoxia-Inducible Factor-1a by siRNA Inhibits C2C12 Myoblast Differentiation

Knockdown of Hypoxia-Inducible Factor-1a by siRNA Inhibits C2C12 Myoblast Differentiation

Abstract

We analyzed the role of Hypoxia-inducible factor (HIF)-1a in myoblast differentiation by examining the expression and regulation of HIF-1ain proliferating and differentiating C2C12 myoblast, and by knocking down HIF-1aof C2C12 myoblasts with small interfering RNA (siRNA), given that HIF-1ahas been shown to be involved in differentiative
process in non-muscle tissues. Although HIF-1amRNA was constantly expressed in C2C12 myoblasts both under growth and differentiating phase, HIF-1a protein was hardly detectable in the growth phase but became detectable only during myogenic differentiation even under normoxia. During early stage of C2C12 myogenesis, HIF-1a accumulated in the nuclei of myogenin-positive myoblasts. The inhibition of proteasome in the growth phase led to HIF-1a protein
accumulation, whereas in the differentiation phase the inhibition of Hsp90, which stabilizes HIF-1a, suppressed HIF-1a
accumulation. Therefore, we suggest that the level of HIF-1a protein expression is regulated by a proteasome-and
chaperon-dependent pathway in C2C12 myoblast. Knockdown of HIF-1a effectively blocked myotube formation and
myosin heavy chain (MHC) expression. Finally, HIF-1aexpression in vivo was confirmed in the regenerative muscle tissue of mice after eccentric exercise. We conclude that HIF-1ais required for C2C12 myogenesis in vitro, and suggest that HIF-1a may have an essential role in regenerative muscle tissue in vivo. J. Cell. Biochem. 98: 642–649, 2006.
 2006 Wiley-Liss, Inc.
Key words: HIF-1a; HSP90; myogenesis Muscle development is a multi

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