SCF/c-kit Transactivates CXCR4 Through GRK6 And Regulates Cardiac Stem Cell Migration

It has been reported that the adult human and mouse heart harbors Lin(-) c-kit(+) stem/progenitor cells. They are primitive and undifferentiated cells, also known as cardiac stem cells (CSCs). CSCs distribute throughout the heart and contribute to the homostasis of cardiomyocytes turnover. In our previous study, after myocardial infarction (MI), stem cell factor (SCF) levels can be significantly increased in the left ventricle myocardium, especially in peri-infarcted areas. Because of the chemoattractive effect of SCF, CSCs can migrate from remote areas into peri-infarcted areas. However, inhibition of p38 MAPK, which is the downstream signaling of SCF/c-kit, can not fully inhibit SCF-induced CSC migration. Consequently, although p38 MAPK is involved in the migration process, this mechanism can not solely explain the migration of CSCs. C-X-C chemokine receptor type 4 (CXCR4) is a typical member of the seven transmembrane-spanning G protein-coupled receptor (GPCR) and is also expressed in CSCs. As reported, CXCR4 could be transactivated by other receptor signaling even in the absence of its ligand SDF-1. However, whether CXCR4 could be transacivated by SCF/c-kit signaling and then regulate CSC migration is still unknown. Our study aims to reveal the role of transactivation of CXCR4 by SCF/c-kit signaling in CSCs migration, which includes three parts:Part one:The isolation and phenotypic characterization of cardiac stem cell. CSCs were obtained from the explants culture of C57BL/6 mouse hearts and expanded in vitro. The isolated cells were characterized by flow cytometry and immunocytochemistry staining, the results showed that cells were both positive for c-kit and CXCR4. In the meantime, CSCs were induced in the differentiation medium and gave rise to myocytes, smooth muscle, and endothelial cells. It indicated that CSCs were multipotent.Part two:The role of transactivation of CXCR4 in SCF/c-kit signaling induced CSC migration. First, CSCs were stimulated with SCF to determine the effect of SCF on CSC migration and MAPK activation through Transwell and western blotting. Then, we suppressed the expression of CXCR4 by using small interfering RNA to analyse whether knockdowning of CXCR4 could impair CSC migration. As reported that CXCR4-srine 339 phosphorylation played an important role in cell migration and mobilization. Therefore, both dose- and time-dependent assays were performed to analyse the effect of SCF on CXCR4-serine 339 transactivation. Next, we engineered a model with lacking CXCR4 serine 339 site to better understand the role of CXCR4-serine 339 phosphorylation in SCF/c-kit signalling. The results showed that SCF can promote CSCs migration and increase CXCR4-serine 339, p38 MAPK and ERK1/2 phosphorylation. Knockdowning the expression of CXCR4 impair SCF/c-kit signaling induced CSC migration and downregulate p38 MAPK and ERK1/2 phosphorylation. In HEK293 cell line, lacking serine 339 point in CXCR4 impair SCF/c-kit signaling induced cell migration.Part three:G protein-coupled receptor kinase 6 (GRK6) regulates CXCR4-serine 339 phosphorylation and SCF/c-kit signaling induced CSC migration. As reported, GRK6 participated in the process of CXCR4-serine 339 phosphorylation. Therefore, we used either GRK6 overexpression plasmid or siRNA to determine whether the up-or downregulation of GRK6 could affect SCF/c-kit signaling induced CXCR4-serine 339 phosphorylation and CSCs migration. Then we performed the experiment in vivo with GRK6-siRNA to investigate what extent role of GRK6 could be played in the process of CSCs migration during MI. The results showed that suppression the expression of GRK6 by siRNA reduced SCF/c-kit induced CXCR4-serine 339 phosphorylation, CSCs migration and MAPK activation. However, overexpression of GRK6 brought the adverse results. Furthermore, downregulation of GRK6 also impaired the CSCs migration ability in vivo during MI.In conclusion, we found that a potential cross-talk between c-kit and CXCR4 exists in CSCs. SCF/c-kit signaling could transactivate CXCR4-serine 339 phosphorylation through GRK6 and regulate cardiac stem cell migration and downstream MAPK activation. Because the signaling from c-kit and CXCR4 is associated with cardiac repair, the mechanism involved in the cross-talk would provide more strategies and evidences for the purpose- "motivating CSCs to regenerate the heart".