Mechanism Of Inhibiting Myoblast Proliferation By Clenbuterol Through P27 Pathway

Skeletal muscle plays an important role in the body. The mechanisms of growth and regeneration of skeletal muscle are critical for animal breeding and human health. Clenbuterol (CLB), a well known synthetic β2-adrenoceptors agonist, was previously showed to induce hypertrophy in skeletal muscle and repair the muscular damage. But the effects of CLB on myogenesis is not clear enough. C2C12 myoblast cell is a popular model to study myogenesis in vitro. Thus our study is to investigate the influence of CLB on proliferation during myogenesis process with C2C12 myoblast cell.Adding CLB to the cell culture medium, we observed a delay of cell cycle progression in C2C12 cells. Meanwhile, by BrdU incorporation experiment, DNA synthesis was inhibited after CLB administration. Besides, coupling with p27 level increasing, the protein levels of cyclin D, cyclin E and Cdk2 were decreased in C2C12 cells treated by CLB. All data indicated that CLB arrests the cell cycle of C2C12 cells.The gene transcription of p27 was not altered by CLB administration, according to the results of Real-time PCR and luciferase experiments. The degradation of p27 protein was inhibited by CLB administration, shown by adding the Cycloheximide (CHX) to the cell culture medium to inhibit the proteinsynthesis. Later we analyzed the cellular localization of p27 by immunocytochemistry and Western blot. We found an accumulation of p27 protein in the nucleus, coupling with a decrease in cytosolic. Skp2 protein has been implicated in the ubiqutin-mediated degradation of p27 protein. In our study, Skp2 protein rather than its mRNA was down-regulated by CLB, preventing p27 protein degradation. The results indicated that CLB increases p27 protein level by modulating its cellular localization.A dynamic change of cAMP level was occurred after CLB treatment, which was increased followed by a decrease. Consistent with these results was that β2AR was internalization and retention in the cytoplasm after CLB treatment for a long time. The cell cycle delay by CLB was rescued when β2AR was knocked down by siRNA, indicating that β2AR was involved in the cell cycle regulation by CLB.After CLB treatment, ERK1/2 phosphorylation was suppressed both in nucleus and cytoplasm. Furthermore, inhibiting ERK1/2 activation through U0126, induced p27 nuclear accumulation in C2C12 cells. Thus it was speculated that CLB inhibits P-ERK1/2 to lead a increase in p27 protein level. Previous studies have showed that muscular hypertrophy induced by CLB was inhibited by PKA inhibitor H-89 or β2-adrenoceptors antagonist ICI. However, the nucleus accumulation of p27 regulated by CLB was not suppressed by adding H-89 or ICI, suggesting the regulation of p27 by CLB was quite different from previous reports of hypertrophic signaling pathway.Our study showed that CLB inactivated ERK1/2 and down-regulated Skp2, contributing to decreased p27 protein degradation, thereby inhibiting cell proliferation. The regulation of p27 by CLB was Gs/cAMP/PKA independent. These findings will promote the understanding of the effect of CLB on skeletal myogenesis, and may be instructive for improving skeletal muscle development and repair.