C(16)-Ceramide-induced F-actin regulation stimulates mouse embryonic stem cell migration: involvement of N-WASP/Cdc42/Arp2/3 complex and cofilin-1/α-actinin.

Ceramide, a major structural element in the cellular membrane, is a key regulatory factor in various cellular behaviors that are dependent on ceramide-induced association of specific proteins. However, molecular mechanisms that regulate ceramide-induced embryonic stem cell (ESC) migration are still not well understood. Thus, we investigated the effect of ceramide on migration and its related signal pathways in mouse ESCs. Among ceramide species with different fatty acid chain lengths, C(16)-Cer increased migration of mouse ESCs in a dose- (≥1μM) and time-dependent (≥8h) manners, as determined by the cell migration assay. C(16)-Cer (10μM) increased protein-kinase C (PKC) phosphorylation. Subsequently, C(16)-Cer increased focal adhesion kinase (FAK) and Paxillin phosphorylation, which were inhibited by PKC inhibitor Bisindolylmaleimide I (1μM). When we examined for the downstream signaling molecules, C(16)-Cer activated small G protein (Cdc42) and increased the formation of complex with Neural Wiskott-Aldrich Syndrome Protein (N-WASP)/Cdc42/Actin-Related Protein 2/3 (Arp2/3). This complex formation was disrupted by FAK- and Paxillin-specific siRNAs. Furthermore, C(16)-Cer-induced increase of filamentous actin (F-actin) expression was inhibited by Cdc42-, N-WASP-, and Arp2/3-specific siRNAs, respectively. Indeed, C(16)-Cer increased cofilin-1/F-actin interaction or F-actin/α-actinin-1 and α-actinin-4 interactions in the cytoskeleton compartment, which was reversed by Cdc42-specific siRNA. Finally, C(16)-Cer-induced increase of cell migration was inhibited by knocking down each signal pathway-related molecules with siRNA or inhibitors. In conclusion, C(16)-Cer enhances mouse ESC migration through the regulation of PKC and FAK/Paxillin-dependent N-WASP/Cdc42/Arp2/3 complex formation as well as through promoting the interaction between cofilin-1 or α-actinin-1/-4 and F-actin.