P2Y6 regulates cytoskeleton reorganization and cell migration of C2C12 myoblasts via ROCK pathway

Migration of skeletal muscle precursor cells is required for limb muscle development and skeletal muscle repair. This study aimed to examine the role of P2Y6 receptor in C2C12 myoblasts migration. C2C12 myoblasts were treated with P2Y6 agonist UDP, P2Y6 antagonist MRS2578, Ca²⁺ channel blocker BTP2, or ROCK inhibitor GSK269962 or Y27632, and the migration ability of C2C12 cells was assessed by wound healing assay. The cellular Ca²⁺ content was analyzed with fluo-4 probe and the activation of ROCK (phosphorlyation of LIMK and cofilin) was assayed by western blot. The cytoskeleton was labeled with Actin-Tracker Green and Tubulin-Tracker-Red. Silencing P2Y6 expression in C2C12 myoblasts reduced intracellular Ca²⁺ content and cell motility. Whereas UDP increased cellular Ca²⁺ content, actin filaments, and cell migration, MRS2578 had the opposite effects. The effects of UDP were abrogated by BTP2 and GSK269962 (and Y27632). Disruption of P2Y6 signaling pathway caused C2C12 myoblasts to have an elongated morphology. These results demonstrated that P2Y6 signaled through Ca²⁺ influx and RhoA/ROCK to reorganize cytoskeleton and promote migration in myoblasts.