PDF(Pubmed)
Abstract：
During embryogenesis, basic fibroblast growth factor (bFGF) is released from neural tube and myotome to promote myogenic fate in the somite, and is routinely used for the culture of adult skeletal muscle (SKM) stem cells (MuSC, called satellite cells). However, the mechanism employed by bFGF to promote SKM lineage and MuSC proliferation has not been analyzed in detail. Furthermore, the question of if the post-translational modification (PTM) of bFGF is important to its stemness-promoting effect has not been answered. In this study, GST-bFGF was expressed and purified from E.coli, which lacks the PTM system in eukaryotes. We found that both GST-bFGF and commercially available bFGF activated the Akt-Erk pathway and had strong cell proliferation effect on C2C12 myoblasts and MuSC. GST-bFGF reversibly compromised the myogenesis of C2C12 myoblasts and MuSC, and it increased the expression of Myf5, Pax3/7, and Cyclin D1 but strongly repressed that of MyoD, suggesting the maintenance of myogenic stemness amid repressed MyoD expression. The proliferation effect of GST-bFGF was conserved in C2C12 over-expressed with MyoD (C2C12-tTA-MyoD), implying its independence of the down-regulation of MyoD. In addition, the repressive effect of GST-bFGF on myogenic differentiation was almost totally rescued by the over-expression of MyoD. Together, these evidences suggest that (1) GST-bFGF and bFGF have similar effects on myogenic cell proliferation and differentiation, and (2) GST-bFGF can promote MuSC stemness and proliferation by differentially regulating MRFs and Pax3/7, (3) MyoD repression by GST-bFGF is reversible and independent of the proliferation effect, and (4) GST-bFGF can be a good substitute for bFGF in sustaining MuSC stemness and proliferation.
摘要：
在胚胎发生期间,碱性成纤维细胞生长因子（bFGF）从神经管和肌体释放，以促进肌体中的肌源性命运，通常用于成年骨骼肌（SKM）干细胞的培养（MuSC，称为卫星细胞）。然而,尚未详细分析bFGF促进SKM谱系和MuSC增殖的机制.此外，尚未回答bFGF的翻译后修饰（PTM）是否对其促进茎效应很重要的问题。在这项研究中,从大肠杆菌中表达并纯化GST-bFGF,在真核生物中缺乏PTM系统。我们发现GST-bFGF和市售bFGF均激活Akt-Erk途径，并对C2C12成肌细胞和MuSC具有很强的细胞增殖作用。GST-bFGF可逆地损害了C2C12成肌细胞和MuSC的成肌作用，它增加了Myf5，Pax3/7和CyclinD1的表达，但强烈抑制了MyoD的表达，表明在抑制的MyoD表达中维持了肌源性干性。在用MyoD过表达的C2C12（C2C12-tTA-MyoD）中，GST-bFGF的增殖作用是保守的，暗示其对MyoD下调的独立性。此外,GST-bFGF对肌源性分化的抑制作用几乎完全由MyoD的过度表达所挽救。一起,这些证据表明（1）GST-bFGF和bFGF对成肌细胞增殖和分化具有相似的作用，和（2）GST-bFGF可以通过差异调节MRFs和Pax3/7促进MuSC的干性和增殖，（3）GST-bFGF对MyoD的抑制是可逆的，并且与增殖作用无关，（4）GST-bFGF在维持MuSC的干性和增殖方面可以很好地替代bFGF。
