Protein Kinase D2 Is an Essential Regulator of Murine Myoblast Differentiation

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

Die Proteinkinase D2 als kritischer Parameter während der Skelettmuskeldifferenzierung

The developement of every multicellular organism occurs through the highly conserved mechanism of genetic programs orchestrated by genes that coordinate cell growth, proliferation, differentiation, migration and apoptosis. This is regulated by signal transduction cascades mediated by kinases such as protein kinases or phospholipases. Muscle cell differentiation is regulated by the activation of a defined pattern of myogenic transcription factors involving members of the protein kinase C (PKC), Protein kinase D (PKD) or phospholipase D families. The protein kinase D (PKD) isoenzymes PKD1, -2, and -3 are prominent downstream targets of PKCs and PLDs in various biological systems. Recent data suggest that PKDs and PKCs also play an important role in muscle cell differentiation. Hence the influence of PKDs and PKCs was examined by establishing the model of C2C12 cells as a known in vitro model for muscle cell differentiation. PKD expression could be demonstrated in C2C12 cells with PKD2 being highly expressed. Furthermore the activation of PKD2 at the beginning of muscle cell differentiation could be observed and inihibited by using pharmacological inhibitors. Depletion of PKD2 by shRNA resulted in a marked inhibition of satellite cell fusion. Furthermore, an activation of myocyte enhancer factor 2D, a key transcription factor for muscle cell differentiation in satellite cells by PKD2 could be observed. In conclusion, the PKD family, especially PKD2 can be seen as a major mediator of muscle cell differentiation in vitro and thereby as a potential novel target for the modulation of muscle regeneration