Background The G protein-coupled receptor CXCR4 and its ligand stromal–cell derived factor 1 (SDF-1) play an important role in directing progenitor cells (PC) homing to ischaemic tissue. The Src family protein kinases (SFK) can be activated by, and serve as effectors of, G proteins. However, whether SFK play a role in SDF-1/CXCR4-mediated PC homing is unknown.
Methods and results To investigate whether SDF-1–CXCR4 signalling activates SFK, we isolated mouse bone marrow mononuclear cells (BM MNCs) and applied onto VCAM1-coated plates, followed by addition of CXCR4 agonist SDF-1 and/or antagonist AMD3100. SDF-1 rapidly (in 2 min) and dose-dependently increased phosphorylation (activation) of Lyn, a major SFK in the BM; AMD3100 attenuated the SDF-1–induced Lyn phosphorylation. Notably, SDF-1 treatment did not increase Lyn phosphorylation in the BM MNCs isolated from Mx1-cre+CXCR4fl/fl mice in which the CXCR4 gene had been deleted. To investigate whether SFK play a role in SDF-1/CXCR4-mediated chemotaxis, we performed Boyden chamber migration assay; SU6656, a SFK inhibitor, significantly inhibited BM-MNC migration towards SDF-1 (p<0.001, n=4). To investigate whether SFK play a role in SDF-1/CXCR4-mediated BM-MNC homing to ischaemic heart tissue, we isolated BM MNCs from CXCR4BAC:eGFP transgenic mice and injected 1×106 cells into WT and SDF-1BAC:SDF1-RFP transgenic mice (in which the expression of SDF1-RFP fusion protein is driven by the SDF-1 genomic regulatory sequence and the level of total SDF-1 protein is doubled) that had undergone surgical myocardial infarction 8 h earlier. Some recipient mice also received two i.p. injections of SU6656 (6 mg/kg) at the time of cell injection and again 4 h later. We found a significantly greater amount of eGFP+ cells (1.6-fold, p<0.01, n=5) and eGFP+c-kit+ cells (1.9-fold, p<0.01, n=5) recruited in the infarct border area of the SDF-1BAC: SDF1-RFP recipients than in WT recipients. SU6656 treatments significantly reduced the amount of eGFP+ cells and eGFP+c-kit+ cells (p<0.01, n=5) in both WT and SDF-1BAC: RFP recipients and abrogate the difference between the two groups.
Conclusions SFK play a critical role in SDF-1/CXCR4–mediated BM PC homing to the ischaemic cardiac tissue thus may provide a target for modulation of tissue repair.