Epigenetic regulation of cardiac progenitor cells marker c-kit by stromal cell derived factor-1α

Abstract

Background: Cardiac progenitor cells (CPCs) have been proven suitable for stem cell therapy after myocardial infarction, especially c-kit(+)CPCs. CPCs marker c-kit and its ligand, the stem cell factor (SCF), are linked as c-kit/SCF axis, which is associated with the functions of proliferation and differentiation. In our previous study, we found that stromal cell-derived factor-1α (SDF-1α) could enhance the expression of c-kit. However, the mechanism is unknown.

Methods and results: CPCs were isolated from adult mouse hearts, c-kit(+) and c-kit(-) CPCs were separated by magnetic beads. The cells were cultured with SDF-1α and CXCR4-selective antagonist AMD3100, and c-kit expression was measured by qPCR and Western blotting. Results showed that SDF-1α could enhance c-kit expression of c-kit(+)CPCs, made c-kit(-)CPCs expressing c-kit, and AMD3100 could inhibit the function of SDF-1α. After the intervention of SDF-1α and AMD3100, proliferation and migration of CPCs were measured by CCK-8 and transwell assay. Results showed that SDF-1α could enhance the proliferation and migration of both c-kit(+) and c-kit(-) CPCs, and AMD3100 could inhibit these functions. DNA methyltransferase (DNMT) mRNA were measured by qPCR, DNMT activity was measured using the DNMT activity assay kit, and DNA methylation was analyzed using Sequenom's MassARRAY platform, after the CPCs were cultured with SDF-1α. The results showed that SDF-1α stimulation inhibited the expression of DNMT1 and DNMT3β, which are critical for the maintenance of regional DNA methylation. Global DNMT activity was also inhibited by SDF-1α. Lastly, SDF-1α treatment led to significant demethylation in both c-kit(+) and c-kit(-) CPCs.

Conclusions: SDF-1α combined with CXCR4 could up-regulate c-kit expression of c-kit(+)CPCs and make c-kit(-)CPCs expressing c-kit, which result in the CPCs proliferation and migration ability improvement, through the inhibition of DNMT1 and DNMT3β expression and global DNMT activity, as well as the subsequent demethylation of the c-kit gene.

Figure 1. Characterization of cultured CPCs.

(A) Cells (small, round, and phase-bright) migrated from the cardiac explants, and aggregated and proliferated on the fibroblast layer after 10 days of culture (×100 magnification). (B) Representative clone generated by CPCs (×100 magnification). (C) and (D) Representative flow cytometric analyses of c-kit(+)CPCs and c-kit(−)CPCs for the expression of the cell surface markers, namely, c-kit, and Sca-1. The Figure 1 panels A and B are excluded from this article's CC-BY license. See the accompanying retraction notice for more information.

Figure 2. SDF-1α up-regulation on the expression of c-kit.

(A) CPCs were stimulated with 100 ng/ml SDF-1α and 5 μg/ml AMD3100 for 48 h. Western blotting was performed to analyze the protein expressions of c-kit and GAPDH. (B) qPCR was performed to analyze the mRNA expression of c-kit. Data were obtained from three independent experiments and expressed as mean ± SD. n = 3. *P<0.05 versus the control group.

Figure 3. SDF-1α enhances the proliferation and migration of.

(A) CPCs were stimulated with 100 ng/ml SDF-1α and 5 μg/ml AMD3100 for 48 hours. And CCK-8 assay was used to determine the proliferation. (B) Quantitative analysis of migrated cells. (C) Representative migrated CPCs (stained with crystal violet) are shown (×200 magnification). Data were obtained from three independent experiments and are expressed as mean ± SD.n = 3.rol group.n of cit and GAPHD.0.Western blot wa c-kit at both protein and mRNA level *P<0.05 vs. control group. The Figure 3C panels are excluded from this article's CC-BY license. See the accompanying retraction notice for more information.

Figure 4. SDF-1α inhibition on the expression and activity of DNMT.

(A) CPCs were stimulated with 100 ng/ml SDF-1α and 5 μg/ml AMD3100 for 48 h. qPCR was used to determine the relative DNMT1 mRNA levels. (B) qPCR was used to determine the relative DNMT3α mRNA levels. (C) qPCR was used to determine relative DNMT3β mRNA levels. (D) EpiQuik DNMT activity assay kit was used to analyze the global DNMT activity. Data were obtained from three independent experiments and expressed as mean ± SD. n = 3.rol group.n of cit and GAPHD.0.Western blot wa c-kit at both protein and mRNA level *P<0.05 versus the control group.

Figure 5. Induction of SDF-1α on demethylation of the c-kit promoter in CPCs.

(A) Profiling of the site-specific methylation of CpG sites in the c-kit promoter region. Each line represents a CpG methylation profile of the c-kit promoter region from the control (PC1 to PC3) and the SDF-1 (PS1 to PS3) samples. The colors of each circle represent the methylation level of each corresponding CpG unit. The white circles represent the missing data at a given CpG site. (B) CPCs were stimulated with 100 ng/ml SDF-1α for 48 h. Genomic DNA was extracted and subjected to Bisulfite sequencing analysis. The data represent the percentage of methylation at corresponding CpG sites, with CpG site number corresponding to the sites identified in the schematic diagram. Data were obtained from three independent experiments and are expressed as mean ± SD. n = 3.rol group.n of cit and GAPHD.0.Western blot wa c-kit at both protein and mRNA level *P<0.05 versus the control group.