Knockdown of hypoxia-inducible factor-1alpha by siRNA inhibits C2C12 myoblast differentiation.
We analyzed the role of Hypoxia-inducible factor (HIF)-1alpha in myoblast differentiation by examining the expression and regulation of HIF-1alpha in proliferating and differentiating C2C12 myoblast, and by knocking down HIF-1alpha of C2C12 myoblasts with small interfering RNA (siRNA), given that HIF-1alpha has been shown to be involved in differentiative process in non-muscle tissues. Although HIF-1alpha mRNA was constantly expressed in C2C12 myoblasts both under growth and differentiating phase, HIF-1alpha 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-1alpha accumulated in the nuclei of myogenin-positive myoblasts. The inhibition of proteasome in the growth phase led to HIF-1alpha protein accumulation, whereas in the differentiation phase the inhibition of Hsp90, which stabilizes HIF-1alpha, suppressed HIF-1alpha accumulation. Therefore, we suggest that the level of HIF-1alpha protein expression is regulated by a proteasome-and chaperon-dependent pathway in C2C12 myoblast. Knockdown of HIF-1alpha effectively blocked myotube formation and myosin heavy chain (MHC) expression. Finally, HIF-1alpha expression in vivo was confirmed in the regenerative muscle tissue of mice after eccentric exercise. We conclude that HIF-1alpha is required for C2C12 myogenesis in vitro, and suggest that HIF-1alpha may have an essential role in regenerative muscle tissue in vivo.
Ono
Yusuke
Y
Department of Medicine & Science in Sports & Exercise, Tohoku University Graduate School of Medicine, Sendai, Japan.
Sensui
Hiroomi
H
Sakamoto
Yuzuru
Y
Nagatomi
Ryoichi
R
D000818:Animals; D002454:Cell Differentiation; D005786:Gene Expression Regulation; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000172:deficiency* / Q000235:genetics*; D051379:Mice; D024510:Muscle Development / Q000235:genetics; D009132:Muscles / Q000166:cytology / Q000502:physiology; D032446:Myoblasts / Q000166:cytology* / Q000378:metabolism*; D061988:Proteasome Inhibitors; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D012038:Regeneration
D018841:HSP90 Heat-Shock Proteins; C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D061988:Proteasome Inhibitors; D012333:RNA, Messenger; D034741:RNA, Small Interfering