Muscle differentiation is regulated by an orchestrated network of transcription factors that basically constitute two families; the MyoD group bHLH muscle regulatory factors (MRFs) and the MEF2 group of MADS-box regulators. It is a tightly controlled process and can be induced in vitro
by serum deprivation, activation of RTKs, such as the insulin- or the IGF-1 receptor, or by ligands of heptahelical receptors, such as S1P. At the signaling level, various signaling pathways, including PLCβ1 and PLCγ1, have been implicated as mediators of insulin-induced C2C12 myoblast differentiation 
. Recently it has been demonstrated that PKCε is up-regulated and its activity increases during muscle differentiation. Furthermore, PKCε and PLCγ1 were shown to form a complex and were found to be crucial for insulin-induced myogenic differentiation. In addition, both proteins co-localized with the Golgi marker 58-K in C2C12 cells 
. PLC enzymatic activity results in the production of inositol 1,4,5-trisphosphate and diacylglycerol (DAG). DAG activates not only DAG-sensitive PKC isoforms including PKCε, but also directly PKDs including PKD2 
. In addition, PKDs are prominent downstream targets of PKCs, in particular novel PKCη and ε. On the other hand, the highly expressed class II HDACs in skeletal muscle were known to repress the expression of MEF2-dependent genes by directly binding to MEF2. The loss of HDAC-MEF2 binding followed by the export of HDACs from the nucleus to the cytoplasm is mediated by the members of CaMK family. However, later studies have shown that PKDs were the crucial players in this process by serving the role of downstream effector kinases of PKCs and by stimulating the nuclear export of class II HDACs 
. Considering the critical role of HDACs in regulating myocyte differentiation and remodeling and PKCs in the signal transduction pathways of muscle differentiation, PKDs could play an important role in muscle differentiation. Our data show that C2C12 myoblasts express all PKD isoforms with PKD2 being the predominant isoform expressed during the entire course of differentiation: PKD2 was phosphorylated/activated during the initiation of C2C12 myoblast differentiation. Selective inhibition of PKCs or PKDs as well as depletion of PKD2 by specific shRNA constructs resulted in a marked inhibition of the expression of muscle differentiation markers, such as MyoD, Myogenin, Mef2D, Pax7 and MHC and myotube formation, respectively. This statement was further supported by data wherein the differentiation of primary satellite cells was inhibited by treatment of either PKC or PKD inhibitor. Thus, PKD2 appears to be required for differentiation of C2C12 myoblasts.
The obtained expressions with our two independent PKD2 knock down cells are from particular interest. In fact it is noteworthy that the alteration in myogenic genes during differentiation was dose-dependent in terms of PKD2 expression. The knock down in cell line #PKD2-2 was less efficient than in cell line #PKD2-4 while the loss of the myogenic markers was greater in cell line #PKD2-4. However, our expression data clearly show that the myogenic program was not entirely disrupted since e.g. myogenin and nicotinic cholinergic receptor were still up-regulated. However, this is not surprising. The myogenic program is highly conserved and tightly regulated 
. The four key factors-MyoD, myogenin, Myf5 and MRF4 act at multiple points in the skeletal muscle lineage to establish the skeletal muscle phenotype 
and thereby serve as the most crucial players during skeletal muscle myogenesis. However, both knock out mice of MyoD and Myf5 lack any myogenic phenotype 
. Given the fact that even MyoD and Myf5 are functionally redundant it is not surprising that myoblast differentiation is not finally governed by just one kinase.
On the other hand, we observed a differential expression pattern for the other two PKD isoforms. While PKD1 was expressed at very low levels during the entire course of differentiation, PKD3 was significantly upregulated at the terminal stage of differentiation. In accordance, knockdown of PKD1 and PKD3 did not display any significant effect on differentiation as judged by the expression of marker gene-MyoD with the exception being the effect of PKD3 knockdown on the expression of a late differentiation maker gene, mAch. However, this is explained by the fact that PKD3 is up regulated at the terminal stages of differentiation and therefore could play a role at this stage.
PKDs are major regulators of vesicle fission at the trans
-Golgi network (TGN) in many cells 
and are targeted to the Golgi by class I ARF proteins 
where they are activated by local DAG and βγ subunits 
. In C2C12 cells, PKD2 could be activated via two ways, indirectly via PKCε 
, which is also active during myogenic differentiation 
or directly via PLC-mediated production of DAG. We identified S1P as a potential physiological activator of PKD2 in C2C12 cell myogenic differentiation.
Nevertheless, it is noteworthy that PKD2 depletion strongly alters myogenic differentiation as shown at the level of morphology, gene expression and finally on protein level. Vice versa, we found increased myogenic differentiation upon overexpression of PKD2 in C2C12 myoblasts. PKD2 activates Mef2Ds. It has been shown previously that MEF2 factors and the myogenic bHLH factors such as MyoD each regulate the expression of a number of contractile protein genes. Thereby interaction between Mef2 proteins and the myogenic bHLH proteins is important in directing muscle gene expression 
. Furthermore, we show, for the first time, that PKD2 negatively regulates Pax3 transcriptional activity. Pax3 has been shown to prevent differentiation and increase self-renewal of C2C12 myoblasts in a variety of studies 
. Thereby, we propose that PKD2 functions in a dual mode of myoblast differentiation (i) repression of self-renewal and (ii) induction of differentiation. Taken together, these data identify PKDs, in particular PKD2 as a novel regulator of muscle differentiation downstream of PKCε and PLCγ and the S1P receptor and thereby, as a potential novel target for the modulation of muscle regeneration in myoblasts.