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

Chen Z, Pan X, Yao Y, Yan F, Chen L, Huang R, Ma G - PLoS ONE (2013)

Bottom Line: The results showed that SDF-1α stimulation inhibited the expression of DNMT1 and DNMT3β, which are critical for the maintenance of regional DNA methylation.Lastly, SDF-1α treatment led to significant demethylation in both c-kit(+) and c-kit(-) CPCs.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Zhongda Hospital, Medical School of Southeast University, Nanjing, Jiangsu, China.

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.

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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.
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pone-0069134-g003: 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.

Mentions: To determine whether SDF-1α will influence CPCs proliferation and migration toward SCF, we performed an in vitro CCK-8 assay and migration assay, and CPCs were placed under SDF-1α with or without the CXCR4 specific antagonist AMD3100. The results indicated that c-kit(+)CPCs proliferation rates of SDF-1α group (0.162±0.008 OD) increase significantly, compared with that of control group (0.114±0.002 OD) and SDF-1α+AMD3100group (0.125±0.003 OD), and c-kit(−) CPCs proliferation rates of SDF-1α group (0.135±0.004 OD) increase significantly, compared with that of control group (0.063±0.004 OD) and SDF-1α+AMD3100group (0.080±0.006 OD) (Figure 3A). And c-kit(+)CPCs migration rates of SDF-1α group (SCF+SDF-1α) (602.3±20.0 cells) also increase significantly, compared with that of control group (with SCF, without SDF-1α) (85.0±11.8 cells), and inhibition group (with SCF+SDF-1α+AMD3100) (138.7±14.6 cells), and c-kit(-)CPCs migration rates of SDF-1α group (SCF+SDF-1α) (272.0±50.7 cells) also increase signicantly, compared with that of control group (with SCF, without SDF-1α) (37.0±5.0 cells), and inhibition group (with SCF+SDF-1α+AMD3100) (67.3±11.4 cells) (Figure 3B, and 3C). (n = 3, mean±SD, P<0.05).


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

Chen Z, Pan X, Yao Y, Yan F, Chen L, Huang R, Ma G - PLoS ONE (2013)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3722185&req=5

pone-0069134-g003: 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.
Mentions: To determine whether SDF-1α will influence CPCs proliferation and migration toward SCF, we performed an in vitro CCK-8 assay and migration assay, and CPCs were placed under SDF-1α with or without the CXCR4 specific antagonist AMD3100. The results indicated that c-kit(+)CPCs proliferation rates of SDF-1α group (0.162±0.008 OD) increase significantly, compared with that of control group (0.114±0.002 OD) and SDF-1α+AMD3100group (0.125±0.003 OD), and c-kit(−) CPCs proliferation rates of SDF-1α group (0.135±0.004 OD) increase significantly, compared with that of control group (0.063±0.004 OD) and SDF-1α+AMD3100group (0.080±0.006 OD) (Figure 3A). And c-kit(+)CPCs migration rates of SDF-1α group (SCF+SDF-1α) (602.3±20.0 cells) also increase significantly, compared with that of control group (with SCF, without SDF-1α) (85.0±11.8 cells), and inhibition group (with SCF+SDF-1α+AMD3100) (138.7±14.6 cells), and c-kit(-)CPCs migration rates of SDF-1α group (SCF+SDF-1α) (272.0±50.7 cells) also increase signicantly, compared with that of control group (with SCF, without SDF-1α) (37.0±5.0 cells), and inhibition group (with SCF+SDF-1α+AMD3100) (67.3±11.4 cells) (Figure 3B, and 3C). (n = 3, mean±SD, P<0.05).

Bottom Line: The results showed that SDF-1α stimulation inhibited the expression of DNMT1 and DNMT3β, which are critical for the maintenance of regional DNA methylation.Lastly, SDF-1α treatment led to significant demethylation in both c-kit(+) and c-kit(-) CPCs.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Zhongda Hospital, Medical School of Southeast University, Nanjing, Jiangsu, China.

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.

Show MeSH
Related in: MedlinePlus