高表达FoxO1抑制猪骨骼肌成肌细胞的分化

FoxO1(Forkhead box O1)是调控肌肉生长、代谢和细胞分化的重要转录因子,但其在成肌细胞分化中的作用还不甚清楚。为了研究FoxO1对哺乳动物成肌细胞分化的影响,以原代培养的长白仔猪成肌细胞作为实验材料,用2%马血清诱导分化,采用实时荧光定量PCR、Western blotting和脂质体转染等方法检测FoxO1及早期和晚期生肌调节因子MyoD和myogenin在猪成肌细胞分化过程中的表达变化。结果显示,在猪成肌细胞分化过程中,FoxO1mRNA表达量显著增加,但总蛋白量变化不显著,其磷酸化水平显著上调。同时,高表达FoxO1的猪成肌细胞中,生肌调节因子MyoD和myogenin mRNA表达受到显著抑制,而MyoD蛋白变化不显著,myogenin却显著下调(P0.05)。以上结果表明,FoxO1能够推迟猪成肌细胞的分化时间并抑制分化;同时推测,FoxO1可能通过抑制生肌调节因子的表达控制骨骼肌纤维类型的终末分化。     

RhoA GTPase regulates M-cadherin activity and myoblast fusion

The Rho family of GTP-binding proteins plays critical roles during myogenesis induction. To elucidate their role later during myogenesis, we have analyzed RhoA function during myoblast fusion into myotubes. We find that RhoA activity is rapidly and transiently increased when cells are shifted into differentiation medium and then is decreased until myoblast fusion. RhoA activity must be down-regulated to allow fusion, because expression of a constitutively active form of RhoA (RhoAV14) inhibits this process. RhoAV14 perturbs the expression and localization of M-cadherin, a member of the Ca2+-dependent cell-cell adhesion molecule family that has an essential role in skeletal muscle cell differentiation. This mutant does not affect N-cadherin and other proteins involved in myoblast fusion, beta1-integrin and ADAM12. Active RhoA induces the entry of M-cadherin into a degradative pathway and thus decreases its stability in correlation with the monoubiquitination of M-cadherin. Moreover, p120 catenin association with M-cadherin is decreased in RhoAV14-expressing cells, which is partially reverted by the inhibition of the RhoA effector Rho-associated kinase ROCK. ROCK inhibition also restores M-cadherin accumulation at the cell-cell contact sites. We propose that the sustained activation of the RhoA pathway inhibits myoblast fusion through the regulation of p120 activity, which controls cadherin internalization and degradation.     

ERK1/2 is required for myoblast proliferation but is dispensable for muscle gene expression and cell fusion

Skeletal muscle satellite cells, which are found between the muscle fiber and the basal lamina, remain quiescent and undifferentiated unless stimulated to remodel skeletal muscle or repair injured skeletal muscle tissue. Quiescent satellite cells express c-met and fibroblast growth factor receptors (FGFR) 1 and 4, suggesting these receptors are involved in maintaining the undifferentiated quiescent state or involved in satellite cell activation. Although the signaling pathways involved are poorly understood, the mitogen activated protein kinase (MAPK) cascade has been implicated in the regulation of skeletal muscle growth and differentiation by FGFs. In this study, we investigated if activation of the Raf-MKK1/2-ERK1/2 signaling cascade plays a role in FGF-dependent repression of differentiation and proliferation of MM14 cells, a skeletal muscle satellite cell line. Inactivation ofthe Raf-MKK1/2-ERK1/2 pathway in myoblasts through the overexpression of dominant negative mutants of Raf-1 blocks ERK1/2 activity and prevents myoblast proliferation. Additionally, inhibition of MKK1/2 by treatment with pharmacological inhibitors also blocks FGF-mediated stimulation of ERK1/2 and blocks the G1 to S phase transition of myoblasts. Unexpectedly, we found that inactivation of the Raf-ERK pathway does not activate a muscle reporter, nor does inactivation of this pathway promote myogenic differentiation. We conclude that FGF-stimulated ERK1/2 signaling is required during the G1 phase of the cell cycle for commitment of myoblasts to DNA synthesis but is not required for mitosis once cells have entered the S-phase. Moreover, ERK1/2 signaling is not required either to repress differentiation, to promote skeletal muscle gene expression, or to promote myoblast fusion.     

Notch Pathway Activation Contributes to Inhibition of C2C12 Myoblast Differentiation by Ethanol

The loss of muscle mass in alcoholic myopathy may reflect alcohol inhibition of myogenic cell differentiation into myotubes. Here, using a high content imaging system we show that ethanol inhibits C2C12 myoblast differentiation by reducing myogenic fusion, creating smaller and less complex myotubes compared with controls. Ethanol administration during C2C12 differentiation reduced MyoD and myogenin expression, and microarray analysis identified ethanol activation of the Notch signaling pathway target genes Hes1 and Hey1. A reporter plasmid regulated by the Hes1 proximal promoter was activated by alcohol treatment in C2C12 cells. Treatment of differentiating C2C12 cells with a gamma secretase inhibitor (GSI) abrogated induction of Hes1. On a morphological level GSI treatment completely rescued myogenic fusion defects and partially restored other myotube parameters in response to alcohol. We conclude that alcohol inhibits C2C12 myoblast differentiation and the inhibition of myogenic fusion is mediated by Notch pathway activation.     

干扰Sirt2促进C2C12成肌细胞分化

Sirt2是组蛋白去乙酰化酶(HDAC III)家族成员之一, 对细胞周期、自噬、脂肪细胞分化、神经细胞存活等生物学过程的调节发挥重要作用. 目前,Sirt2在肌肉发育过程中的研究尚未见报道.本文通过构建Sirt2慢病毒干扰载体,侵染C2C12成肌细胞,并用细胞免疫荧光化学、real-time PCR 和Western印迹方法,检测其对成肌分化标志基因及相关信号通路因子的影响. 结果显示,干扰质粒shRNA 663处理C2C12细胞后,Sirt2 mRNA及蛋白质表达水平与对照相比显著下调(P<0.01);C2C12细胞分化第4 d,MyoD,MyoG,MyHC mRNA及蛋白质表达均显著增加(P<0.01); PI3K,AKT,FoxO1磷酸化水平明显升高. 结果表明,Sirt2可通过PI3K/AKT/FOXO1信号通路来促进成肌细胞分化,是肌生成的一个潜在调节因子.