Crosstalk between SDF-1/CXCR4 and SDF-1/CXCR7 in cardiac stem cell migration

Abstract

Stromal cell-derived factor 1 (SDF-1) is a chemokine that can be expressed in injured cardiomyocytes after myocardial infarction (MI). By combining with its receptor CXCR4, SDF-1 induced stem and progenitor cells migration. CXCR7, a novel receptor for SDF-1, has been identified recently. We aimed to explore the roles of SDF-1/CXCR4 and SDF-1/CXCR7 pathway and their crosstalk in CSCs migration. In the present study, CXCR4 and CXCR7 expression were identified in CSCs. Transwell assay showed that SDF-1 caused CSCs migration in a dose- and time-dependent manner, which could be significantly suppressed by CXCR4 or CXCR7 siRNA. Phospho-ERK, phospho-Akt and Raf-1 significantly elevated in CSCs with SDF-1 stimulation. Knockdown of CXCR4 or CXCR7 significantly decreased phospho-ERK or phospho-Akt, respectively, and eventually resulted in the inhibition of CSCs migration. Moreover, western blot showed that MK2206 (Akt inhibitor) increased the expression of phospho-ERK and Raf-1, whereas PD98059 (ERK inhibitor) had no effect on phospho-Akt and Raf-1. GW5074 (Raf-1 inhibitor) upregulated the expression of phospho-ERK, but had no effect on phospho-Akt. The present study indicated that SDF-1/CXCR7/Akt and SDF-1/CXCR4/ERK pathway played important roles in CSCs migration. Akt phosphorylation inhibited Raf-1 activity, which in turn dephosphorylated ERK and negatively regulated CSCs migration.

Figure 1. Identification of CSCs and detection of CXCR4 and CXCR7 in CSCs.

(A) Identification of c-kit⁺ CSCs with a purity of 93.6% by flow cytometry. (B) RT-PCR analysis of CXCR4 and CXCR7 mRNA in CSCs. 4T1 cells were used as a positive control. (C) Western blot analysis of CXCR4 and CXCR7 protein in CSCs. 4T1 cells were used as a positive control. (D) RT-PCR analysis of CXCR4 mRNA in CSCs with CXCR4 siRNA. (E) RT-PCR analysis of CXCR7 mRNA in CSCs with CXCR7 siRNA. (F) Western blot analysis of CXCR4 and CXCR7 protein in CSCs with CXCR4 siRNA and CXCR7 siRNA. *P < 0.05 versus control.

Figure 2. Effects of SDF-1, CXCR4 and CXCR7 on CSCs migration in vitro.

(A) CSCs migration induced by different concentration of SDF-1 for 12 h was detected with transwell migration assay. (B) CSCs migration induced by 100 ng/mL SDF-1 for different time was detected with transwell migration assay. (C) Representative images of migrated CSCs induced by 100 ng/mL SDF-1 with or without CXCR4 siRNA by transwell migration assay. 1: Medium alone group; 2: 100 ng/mL SDF-1 group; 3: CXCR4 siRNA group; 4: SDF-1 + CXCR4 siRNA group. (D) Representative images of migrated CSCs induced by 100 ng/mL SDF-1 with or without CXCR7 siRNA by transwell migration assay. 1: Medium alone group; 2: 100 ng/mL SDF-1 group; 3: CXCR7 siRNA group; 4: SDF-1 + CXCR7 siRNA group. Original magnification, ×100. Results were depicted as means ± SEM. *P < 0.05 versus control.

Figure 3. Effects of SDF-1, CXCR4 and CXCR7 on Akt, ERK and Raf-1 expression in CSCs.

(A) Effect of SDF-1 on phospho-Akt, phospho-ERK and Raf-1 expression in CSCs was detected with western blot analysis. (B) Effect of CXCR4 siRNA on SDF-1-induced phospho-Akt, phospho-ERK and Raf-1 expression in CSCs was detected with western blot analysis. (C) Effect of CXCR7 siRNA on SDF-1-induced phospho-Akt, phospho-ERK and Raf-1 expression in CSCs was detected with western blot analysis. *P < 0.05 versus control.

Figure 4. Crosstalk between Akt and ERK in CSCs migration.

(A) CSCs migration induced by SDF-1 with different inhibitors was detected by transwell migration assay. *P < 0.05 versus control. **P < 0.05 versus SDF-1 group. (B) With ERK inhibitor PD98059, Akt, phospho-Akt, ERK, phospho-ERK and Raf-1 expression were detected by western blot. (C) With Akt inhibitor MK2206, Akt, phospho-Akt, ERK, phospho-ERK and Raf-1 expression were detected by western blot. (D) With RAF-1 inhibitor GW5074, Akt, phospho-Akt, ERK, phospho-ERK and Raf-1 expression were detected by western blot. *P < 0.05 versus SDF-1 group.