Department of Internal Medicine I, University of Ulm, Ulm, Germany.
PLoS One. 2011 Jan 27;6(1):e14599. doi: 10.1371/journal.pone.0014599.
Muscle differentiation is a highly conserved process that occurs through the activation of quiescent satellite cells whose progeny proliferate, differentiate, and fuse to generate new myofibers. A defined pattern of myogenic transcription factors is orchestrated during this process and is regulated via distinct signaling cascades involving various intracellular signaling pathways, including members of the protein kinase C (PKC) family. The protein kinase D (PKD) isoenzymes PKD1, -2, and -3, are prominent downstream targets of PKCs and phospholipase D in various biological systems including mouse and could hence play a role in muscle differentiation. In the present study, we used a mouse myoblast cell line (C2C12) as an in vitro model to investigate the role of PKDs, in particular PKD2, in muscle stem cell differentiation. We show that C2C12 cells express all PKD isoforms with PKD2 being highly expressed. Furthermore, we demonstrate that PKD2 is specifically phosphorylated/activated during the initiation of mouse myoblast differentiation. Selective inhibition of PKCs or PKDs by pharmacological inhibitors blocked myotube formation. Depletion of PKD2 by shRNAs resulted in a marked inhibition of myoblast cell fusion. PKD2-depleted cells exhibit impaired regulation of muscle development-associated genes while the proliferative capacity remains unaltered. Vice versa forced expression of PKD2 increases myoblast differentiation. These findings were confirmed in primary mouse satellite cells where myotube fusion was also decreased upon inhibition of PKDs. Active PKD2 induced transcriptional activation of myocyte enhancer factor 2D and repression of Pax3 transcriptional activity. In conclusion, we identify PKDs, in particular PKD2, as a major mediator of muscle cell differentiation in vitro and thereby as a potential novel target for the modulation of muscle regeneration.
肌肉分化是一个高度保守的过程，通过激活静止的卫星细胞来实现，卫星细胞的后代增殖、分化并融合以产生新的肌纤维。在这个过程中，协调了一个明确的肌生成转录因子模式，并通过涉及各种细胞内信号通路的不同信号级联进行调节，包括蛋白激酶 C（PKC）家族的成员。蛋白激酶 D（PKD）同工酶 PKD1、-2 和 -3 是 PKC 和磷脂酶 D 在包括小鼠在内的各种生物系统中的重要下游靶标，因此可能在肌肉分化中发挥作用。在本研究中，我们使用小鼠成肌细胞系（C2C12）作为体外模型，研究 PKDs（特别是 PKD2）在肌肉干细胞分化中的作用。我们表明 C2C12 细胞表达所有 PKD 同工型，PKD2 高度表达。此外，我们证明 PKD2 在小鼠成肌细胞分化起始时特异性磷酸化/激活。PKC 或 PKD 的选择性抑制通过药理学抑制剂阻断肌管形成。PKD2 的 shRNA 耗竭导致成肌细胞融合明显抑制。PKD2 耗竭的细胞表现出肌肉发育相关基因的调节受损，而增殖能力保持不变。相反，PKD2 的强制表达增加了成肌细胞分化。这些发现在原代小鼠卫星细胞中得到了证实，其中 PKDs 的抑制也导致肌管融合减少。活性 PKD2 诱导肌细胞增强因子 2D 的转录激活和 Pax3 转录活性的抑制。总之，我们确定 PKDs（特别是 PKD2）是体外肌肉细胞分化的主要介质，因此是调节肌肉再生的潜在新靶点。
