DGK is also involved in cell differentiation
DGKδ is also involved in cell differentiation. Previously, to investigate the physiological roles of DGKδ, DGKδ-deficient mice have been generated. On the basis of the analysis of DGKδ-deficient mice, Crotty et al. reported that DGKδ regulates the epidermal growth factor receptor (EGFR) pathway by attenuating cnPKC activities for the epithelial cell differentiation of the lungs and skin . In addition to being expressed in skeletal muscle cells , we have recently reported that DGKδ is highly expressed in the mouse GSK2292767 , and DGKδ-deficiency increases the number of long axons/neurites in the primary cultured cortical neurons, suggesting that DGKδ regulates neuronal differentiation in the brain . Furthermore, it has been reported that DGKδ is highly expressed in adipose tissue and is involved in adipocyte differentiation . These studies strongly indicate that DGKδ plays an important role in cell differentiation in the tissues expressing DGKδ. Therefore, we hypothesize that DGKδ, in addition to glucose uptake, regulates skeletal muscle differentiation, because DGKδ is abundantly expressed in skeletal muscle .
Materials and methods
Discussion Based on the analysis of DGKδ-deficient mice, Crotty et al. reported that DGKδ regulates the epithelial cell differentiation of lungs and skin . Moreover, our recent studies suggest that DGKδ controls the formation of long axons/neurites in mouse brains [28,29]. However, although DGKδ is highly expressed in skeletal muscle , the role of DGKδ in myogenic differentiation is still unknown. The present study demonstrated that the knockdown of DGKδ inhibited the expression of myogenin (Fig. 2 and Suppl. Fig. 1) and decreased the percentage of C2C12 cells expressing MHC (Fig. 3). It is known that myogenin is responsible for terminal myogenic differentiation [, , ] and MHC is a late-stage differentiation marker . Therefore, our results indicate that DGKδ regulates C2C12 myogenic differentiation. In addition to myogenic differentiation, previous studies suggested that DGKδ, which is also expressed in adipose tissue, is involved in not only adipocyte differentiation  but also its lipogenesis . We reported that DGKδ regulates the formation of long axons/neurites (differentiation) in primary cultured neurons . From these studies, it is possible that, in DGKδ-expressing tissues, DGKδ regulates cell differentiation and tissue-specific functions, e.g., myogenesis and glucose uptake in skeletal muscle or adipocyte differentiation and lipogenesis in adipose tissue. DGKδ was highly expressed in C2C12 cells for 24 h after initiation of differentiation and the expression levels was then decreased (Fig. 1). If the down-regulation of DGKδ is important for C2C12 differentiation, it is presumed that the knock-down of DGKδ accelerates the induction of myogenin and, conversely, the overexpression of DGKδ inhibits the induction of myogenin. On the other hand, if keeping high levels of DGKδ in the early stage is essential for C2C12 myogenic differentiation, it is supposed that the knock-down of DGKδ attenuates the induction of myogenin and the overexpression of DGKδ does not affect the induction of myogenin. As shown in Fig. 2 and Suppl. Figs. 1 and 3, the knock-down of DGKδ inhibited C2C12 differentiation and DGKδ overexpression did not affect myogenin expression during myogenic differentiation. Therefore, a possible explanation of these findings is that keeping high levels of DGKδ in the early stage is important for C2C12 myogenic differentiation and that the decrease of DGKδ expression is not essential for controlling C2C12 differentiation. In contrast, the suppression of DGKδ did not affect cyclin D1 expression in C2C12 myoblasts before differentiation (0 h of differentiation) (Fig. 2, Fig. 6 and Suppl. Fig. 1). Moreover, although DGKδ suppression slightly induced cyclin D1 expression during differentiation, it did not affect the number of C2C12 cells (Suppl. Fig. 2), suggesting that DGKδ does not strongly enhance the proliferation of C2C12 cells. Taken together, these results suggest that DGKδ is involved in the down-regulation of cyclin D1 expression in the early stage of C2C12 myogenic differentiation but not in myoblast proliferation. In fact, the levels of DGKδ expression were decreased later than the suppression of cyclin D1 expression during differentiation (Fig. 1), supporting that DGKδ acts upstream of cyclin D1.