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  • Cyclin D inhibits the transcriptional activity of the

    2020-09-10

    Cyclin D1 inhibits the transcriptional activity of the transcription factors myogenin and MEF2 [39,40]. In addition, myogenin activates MEF2 expression, which feeds back to amplify the myogenin promoter [38]. In this study, DGKδ suppression, in addition to the increase of cyclin D1, inhibited myogenin and MEF2 expression during differentiation (Fig. 2, Fig. 4, Fig. 6, and Suppl. Fig. 1). These results provide two possible mechanisms. The first is that cyclin D1, down-regulated by DGKδ during C2C12 differentiation, promotes myogenin expression through MEF2, and the second is that the cyclin D1 down-regulation directly induces myogenin expression (Suppl. Fig. 4). On the other hand, forced expression studies have indicated that cyclin D1 also inhibits the transcriptional activity of MyoD [40,47,48]. However, we did not observe the effect of DGKδ suppression on MyoD expression (Fig. 4). It is possible that cyclin D1 regulates MyoD expression in a differentiation stage-dependent manner and/or an upstream factor (e.g., DGKδ)-dependent manner. DGK metabolizes DG, which enhances PKC activity [10,11]. DGKδ regulates the EGFR pathway for the epithelial cell differentiation of lungs and skin [27] and controls insulin receptor signaling for glucose uptake in skeletal muscle [20] by attenuating cnPKC activities. In this study, we found that the suppression of DGKδ increased the levels of phospho-cnPKCs at 48 h of C2C12 myogenic differentiation (Fig. 5). In addition, the expression levels of cyclin D1 and cnPKCs were increased by DGKδ suppression, even at 24 h of differentiation (Fig. 6). These results suggest that the consumption of DG by DGKδ attenuates cnPKC activities for the down-regulation of cyclin D1 during C2C12 myogenesis (Suppl. Fig. 4). Previously, it has been reported that PKCε up-regulates cyclin D1 via an ERK and NF-κB/cyclic AMP-response element-mediated transcriptional mechanism in intestinal epithelial L-Adrenaline [49]. Therefore, PKCε may be a candidate for the cnPKC regulation by DGKδ. However, PKCε is up-regulated during C2C12 myogenic differentiation and is involved in the up-regulation of cyclin D3 and myogenin expression during myogenic differentiation [50]. Thus, the roles of PKCε on the DGKδ-dependent regulation of cyclin D1 expression and C2C12 differentiation are still elusive. In C2C12 cells, various cnPKC isoforms are expressed [50]. Therefore, it is important to investigate in detail the relationship between DGKδ and each cnPKC isoform for clarification of the molecular mechanism of myogenic differentiation regulation by DGKδ. To gain insight into the molecular mechanism regulated by DGKδ for C2C12 myogenic differentiation, we investigated the alteration of DG/PA species using liquid chromatography/mass spectrometry methods [51,52], However, although several DG/PA species increased at 24 h of differentiation, we were not able to find DG/PA species significantly influenced by the suppression of DGKδ using DGKδ-specific siRNA (data not shown). It is possible that DG/PA conversion by DGKδ is relatively minor in C2C12 differentiation when compared with background amounts of DG/PA species. Evangelisti et al. reported that DGKζ, a type IV DGK isozyme, is localized to the nucleus and the cytoplasm, and that nuclear DGKζ is up-regulated during C2C12 myogenic differentiation [53,54]. In addition, DGKζ-knockdown markedly impairs differentiation and results in a higher percentage of cells being in the S an G2/M phases of the cell cycle, demonstrating that DGKζ plays an important role in the cell cycle withdrawal [53,54]. Furthermore, DGKζ co-localizes and interacts with PI-specific PLCβ1 in C2C12 cells, indicating that the phosphoinositide signaling pathway regulated by DGKζ is important for C2C12 differentiation [53]. In the present study, we showed that DGKδ was down-regulated during C2C12 differentiation in contrast to DGKζ. DGKδ2, which is one of alternatively spliced DGKδ products [14] and is predominantly expressed in C2C12 myoblasts and myotubes [22,24], is located in punctate vesicles but not the nucleus [31,53]. Furthermore, our previous study showed that DGKδ preferentially metabolizes palmitic acid-containing DG species derived from the PC-PLC pathway, but not arachidonic acid-containing DG species derived from the PI-PLCβ1 pathway, in high glucose-stimulated C2C12 myoblasts [22,23]. These studies imply that DGKδ and DGKζ control C2C12 myogenesis through different signaling pathways. The other DGK isozymes (e.g., DGKα and DGKε) are also expressed in C2C12 myotubes [55]. Therefore, it is important to comprehensively understand each DGK function in C2C12 myoblasts and myotubes for the elucidation of myogenic differentiation.