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

Abstract

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, Ca channel blocker BTP2, or ROCK inhibitor GSK269962 or Y27632, and the migration ability of C2C12 cells was assessed by wound healing assay. The cellular Ca 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 Ca content and cell motility. Whereas UDP increased cellular Ca 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 Ca influx and RhoA/ROCK to reorganize cytoskeleton and promote migration in myoblasts.

The P2Y6 receptor signals through Gαq/Ca2+/PKCα and Gα13/ROCK pathways to drive the formation of membrane protrusions and dictate cell migration

“…Values were expressed as the mean ± SEM from three to four independent experiments performed in duplicate. Statistical significance was determined by a two-way ANOVA test with multiple comparisons followed by a Tukey posttest myoblasts (Wang et al, 2018). Here, we inhibited ROCK activity using 1 μM Y-27632 before the stimulation of P2Y 6 with 5 μM MRS2693 (Figure 3).…”

“…These messengers modulate the activity of different effectors, such as protein kinases C (PKC) and MAP kinases (Bellefeuille, Molle, & Gendron, 2019). Recently, it was reported that P2Y 6 stimulated the Rho-associated protein kinase (ROCK) pathway to favor the migration of C2C12 myoblasts through the reorganization of the cytoskeleton following the phosphorylation of LIM kinases (LIMK) and cofilin (Wang et al, 2018). Interestingly, the modulation of C2C12 migration was not associated with any G-protein activity, although a previous study reported the potential association of P2Y 6 to Gα 12/13 proteins in cardiomyocytes (Nishida et al, 2008).…”

Knockdown of CNN3 Impairs Myoblast Proliferation, Differentiation, and Protein Synthesis via the mTOR Pathway

“…Proliferation and migration of myoblasts are essential for muscle development, as they facilitate cellular alignment in preparation for differentiation and fusion ( Wang et al, 2018 ). CNN3 plays a pivotal role in promoting the migration of cancer cells, skin cells, and trophoblast invasion ( Shibukawa et al, 2010 ; Daimon et al, 2013 ; Kim et al, 2018 ; Nair et al, 2019 ; Xia et al, 2020 ).…”

Generation and Characterization of Skeletal Muscle Cell based Model for GNE Myopathy: Implications in Actin Dynamics

“…Co lin also acts downstream of RhoA to regulate actin dynamics that may affect muscle cell migration and regeneration. The C2C12 myoblast migration is regulated by RhoA/ROCK signaling pathway where Co lin levels and activity is important 59 . Increased phosphorylation of Co lin inhibits its actin severing activity.…”