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C-Kit Promotes Growth and Migration of Human Cardiac Progenitor Cells via the PI3K-AKT and MEK-ERK Pathways.

Vajravelu BN, Hong KU, Al-Maqtari T, Cao P, Keith MC, Wysoczynski M, Zhao J, Moore JB, Bolli R - PLoS ONE (2015)

Bottom Line: SCF treatment led to a significant increase in cell survival and a reduction in cell death under serum depletion conditions.In addition, SCF significantly promoted CPC migration in vitro.These results imply that the efficiency of CPC homing to the injury site as well as their survival after transplantation may be improved by modulating the activity of c-kit.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Cardiology, Department of Medicine, University of Louisville, Louisville, KY 40202, United States of America.

ABSTRACT
A recent phase I clinical trial (SCIPIO) has shown that autologous c-kit+ cardiac progenitor cells (CPCs) improve cardiac function and quality of life when transplanted into patients with ischemic heart disease. Although c-kit is widely used as a marker of resident CPCs, its role in the regulation of the cellular characteristics of CPCs remains unknown. We hypothesized that c-kit plays a role in the survival, growth, and migration of CPCs. To test this hypothesis, human CPCs were grown under stress conditions in the presence or absence of SCF, and the effects of SCF-mediated activation of c-kit on CPC survival/growth and migration were measured. SCF treatment led to a significant increase in cell survival and a reduction in cell death under serum depletion conditions. In addition, SCF significantly promoted CPC migration in vitro. Furthermore, the pro-survival and pro-migratory effects of SCF were augmented by c-kit overexpression and abrogated by c-kit inhibition with imatinib. Mechanistically, c-kit activation in CPCs led to activation of the PI3K and the MAPK pathways. With the use of specific inhibitors, we confirmed that the SCF/c-kit-dependent survival and chemotaxis of CPCs are dependent on both pathways. Taken together, our findings suggest that c-kit promotes the survival/growth and migration of human CPCs cultured ex vivo via the activation of PI3K and MAPK pathways. These results imply that the efficiency of CPC homing to the injury site as well as their survival after transplantation may be improved by modulating the activity of c-kit.

No MeSH data available.


Related in: MedlinePlus

The effects of c-kit activation on CPCs are mediated via the PI3K-AKT and the MEK-ERK pathways.A, Human c-kit+ CPCs were either untreated (- SCF) or treated with 100 ng/ml SCF for the indicated duration. Levels of the indicated proteins at each time point were analyzed by Western blot. p-, phosphorylated (i.e., activated) form. B, CPCs were pre-treated with the indicated inhibitors for 2 hr followed by 20 min of SCF treatment. Levels of the indicated proteins were analyzed by Western blot. Imatinib, a c-kit inhibitor; Wortmannin, a PI3K inhibitor; and PD98059, a MEK inhibitor. C, Cell viability assay. CPCs were cultured in serum-free medium for 3 days in the presence (+) of absence (-) of SCF and the indicated inhibitors. Cell viability was assessed using PrestoBlueTM. D, Boyden chamber assay for cell migration. CPCs were plated in the transwells. After 24 hr, they were pre-treated with the inhibitors for 2 hr, and SCF was then added to the bottom chamber. Migrated cells were fixed, stained with propidium iodide, and counted. All values are normalized to the DMSO (vehicle) control. Values presented are mean ± SEM. *, p<0.05 compared to the DMSO control. #, p<0.05 compared to the SCF only treatment group.
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pone.0140798.g005: The effects of c-kit activation on CPCs are mediated via the PI3K-AKT and the MEK-ERK pathways.A, Human c-kit+ CPCs were either untreated (- SCF) or treated with 100 ng/ml SCF for the indicated duration. Levels of the indicated proteins at each time point were analyzed by Western blot. p-, phosphorylated (i.e., activated) form. B, CPCs were pre-treated with the indicated inhibitors for 2 hr followed by 20 min of SCF treatment. Levels of the indicated proteins were analyzed by Western blot. Imatinib, a c-kit inhibitor; Wortmannin, a PI3K inhibitor; and PD98059, a MEK inhibitor. C, Cell viability assay. CPCs were cultured in serum-free medium for 3 days in the presence (+) of absence (-) of SCF and the indicated inhibitors. Cell viability was assessed using PrestoBlueTM. D, Boyden chamber assay for cell migration. CPCs were plated in the transwells. After 24 hr, they were pre-treated with the inhibitors for 2 hr, and SCF was then added to the bottom chamber. Migrated cells were fixed, stained with propidium iodide, and counted. All values are normalized to the DMSO (vehicle) control. Values presented are mean ± SEM. *, p<0.05 compared to the DMSO control. #, p<0.05 compared to the SCF only treatment group.

Mentions: Activation of c-kit leads to subsequent activation of different downstream pathways, including the PI3K-AKT [52, 53], p38MAPK [54, 55], MEK-ERK [56, 57], SFK [56], phospholipase C [58] and JAK/STAT [59] pathways. Among these, the PI3K-AKT and the MEK-ERK pathways represent the major pathway activated upon c-kit stimulation in a wide variety of cell types [29, 60]. Therefore, we tested if these two pathways also mediate the pro-survival and pro-migratory effects of c-kit activation in CPCs. First, we examined if these two pathways are subsequently activated upon c-kit activation. We treated CPCs with SCF for varying durations ranging from 5 to 60 minutes, and analyzed the changes in the phosphorylated (i.e., activated) AKT and phosphorylated ERK levels by Western blot. A significant increase in the level of phosphorylated AKT was observed within 10 min of SCF treatment, while the phosphorylated ERK levels increased as early as 5 min of SCF treatment (Fig 5A). These observations suggest that both the PI3K-AKT and MEK-ERK pathways are indeed activated upon c-kit activation in CPCs. Next, we investigated the contribution of each pathway to the biological effects of SCF/c-kit signaling on CPCs. For this, we utilized a PI3K inhibitor, Wortmannin, and a MEK inhibitor, PD98059. Western blot analysis confirmed that both inhibitors were effective in down-regulating their corresponding pathways, which was indicated by a significant reduction in the levels of activated AKT or activated ERK in cells co-treated with SCF and the inhibitors (Fig 5B). Interestingly, inhibition of the MEK-ERK pathway also inhibited the PI3K-AKT pathway but not vice versa (Fig 5B), suggesting that the MEK-ERK pathway operates upstream of the PI3K-AKT pathway. Pre-treating CPCs with these inhibitors (i.e., Wortmannin and PD98059) either individually or in combination abolished the pro-survival effect of SCF when the CPCs were cultured under serum starvation, while treating the cells with inhibitors alone did not affect their cell numbers (Fig 5C). Next, we tested if the pro-migratory effect of SCF is also dependent on PI3K-AKT and/or MEK-ERK pathways. While SCF alone stimulated cell migration (as shown earlier), pre-treatment of the cells with either one of the inhibitors prior to the SCF treatment prevented CPCs from migrating towards the SCF gradient (Fig 5D). These results indicate that the PI3K-AKT and the MEK-ERK pathways are the major pathways that regulate both pro-survival and -migratory effects of c-kit activation in human CPCs.


C-Kit Promotes Growth and Migration of Human Cardiac Progenitor Cells via the PI3K-AKT and MEK-ERK Pathways.

Vajravelu BN, Hong KU, Al-Maqtari T, Cao P, Keith MC, Wysoczynski M, Zhao J, Moore JB, Bolli R - PLoS ONE (2015)

The effects of c-kit activation on CPCs are mediated via the PI3K-AKT and the MEK-ERK pathways.A, Human c-kit+ CPCs were either untreated (- SCF) or treated with 100 ng/ml SCF for the indicated duration. Levels of the indicated proteins at each time point were analyzed by Western blot. p-, phosphorylated (i.e., activated) form. B, CPCs were pre-treated with the indicated inhibitors for 2 hr followed by 20 min of SCF treatment. Levels of the indicated proteins were analyzed by Western blot. Imatinib, a c-kit inhibitor; Wortmannin, a PI3K inhibitor; and PD98059, a MEK inhibitor. C, Cell viability assay. CPCs were cultured in serum-free medium for 3 days in the presence (+) of absence (-) of SCF and the indicated inhibitors. Cell viability was assessed using PrestoBlueTM. D, Boyden chamber assay for cell migration. CPCs were plated in the transwells. After 24 hr, they were pre-treated with the inhibitors for 2 hr, and SCF was then added to the bottom chamber. Migrated cells were fixed, stained with propidium iodide, and counted. All values are normalized to the DMSO (vehicle) control. Values presented are mean ± SEM. *, p<0.05 compared to the DMSO control. #, p<0.05 compared to the SCF only treatment group.
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Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608800&req=5

pone.0140798.g005: The effects of c-kit activation on CPCs are mediated via the PI3K-AKT and the MEK-ERK pathways.A, Human c-kit+ CPCs were either untreated (- SCF) or treated with 100 ng/ml SCF for the indicated duration. Levels of the indicated proteins at each time point were analyzed by Western blot. p-, phosphorylated (i.e., activated) form. B, CPCs were pre-treated with the indicated inhibitors for 2 hr followed by 20 min of SCF treatment. Levels of the indicated proteins were analyzed by Western blot. Imatinib, a c-kit inhibitor; Wortmannin, a PI3K inhibitor; and PD98059, a MEK inhibitor. C, Cell viability assay. CPCs were cultured in serum-free medium for 3 days in the presence (+) of absence (-) of SCF and the indicated inhibitors. Cell viability was assessed using PrestoBlueTM. D, Boyden chamber assay for cell migration. CPCs were plated in the transwells. After 24 hr, they were pre-treated with the inhibitors for 2 hr, and SCF was then added to the bottom chamber. Migrated cells were fixed, stained with propidium iodide, and counted. All values are normalized to the DMSO (vehicle) control. Values presented are mean ± SEM. *, p<0.05 compared to the DMSO control. #, p<0.05 compared to the SCF only treatment group.
Mentions: Activation of c-kit leads to subsequent activation of different downstream pathways, including the PI3K-AKT [52, 53], p38MAPK [54, 55], MEK-ERK [56, 57], SFK [56], phospholipase C [58] and JAK/STAT [59] pathways. Among these, the PI3K-AKT and the MEK-ERK pathways represent the major pathway activated upon c-kit stimulation in a wide variety of cell types [29, 60]. Therefore, we tested if these two pathways also mediate the pro-survival and pro-migratory effects of c-kit activation in CPCs. First, we examined if these two pathways are subsequently activated upon c-kit activation. We treated CPCs with SCF for varying durations ranging from 5 to 60 minutes, and analyzed the changes in the phosphorylated (i.e., activated) AKT and phosphorylated ERK levels by Western blot. A significant increase in the level of phosphorylated AKT was observed within 10 min of SCF treatment, while the phosphorylated ERK levels increased as early as 5 min of SCF treatment (Fig 5A). These observations suggest that both the PI3K-AKT and MEK-ERK pathways are indeed activated upon c-kit activation in CPCs. Next, we investigated the contribution of each pathway to the biological effects of SCF/c-kit signaling on CPCs. For this, we utilized a PI3K inhibitor, Wortmannin, and a MEK inhibitor, PD98059. Western blot analysis confirmed that both inhibitors were effective in down-regulating their corresponding pathways, which was indicated by a significant reduction in the levels of activated AKT or activated ERK in cells co-treated with SCF and the inhibitors (Fig 5B). Interestingly, inhibition of the MEK-ERK pathway also inhibited the PI3K-AKT pathway but not vice versa (Fig 5B), suggesting that the MEK-ERK pathway operates upstream of the PI3K-AKT pathway. Pre-treating CPCs with these inhibitors (i.e., Wortmannin and PD98059) either individually or in combination abolished the pro-survival effect of SCF when the CPCs were cultured under serum starvation, while treating the cells with inhibitors alone did not affect their cell numbers (Fig 5C). Next, we tested if the pro-migratory effect of SCF is also dependent on PI3K-AKT and/or MEK-ERK pathways. While SCF alone stimulated cell migration (as shown earlier), pre-treatment of the cells with either one of the inhibitors prior to the SCF treatment prevented CPCs from migrating towards the SCF gradient (Fig 5D). These results indicate that the PI3K-AKT and the MEK-ERK pathways are the major pathways that regulate both pro-survival and -migratory effects of c-kit activation in human CPCs.

Bottom Line: SCF treatment led to a significant increase in cell survival and a reduction in cell death under serum depletion conditions.In addition, SCF significantly promoted CPC migration in vitro.These results imply that the efficiency of CPC homing to the injury site as well as their survival after transplantation may be improved by modulating the activity of c-kit.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Cardiology, Department of Medicine, University of Louisville, Louisville, KY 40202, United States of America.

ABSTRACT
A recent phase I clinical trial (SCIPIO) has shown that autologous c-kit+ cardiac progenitor cells (CPCs) improve cardiac function and quality of life when transplanted into patients with ischemic heart disease. Although c-kit is widely used as a marker of resident CPCs, its role in the regulation of the cellular characteristics of CPCs remains unknown. We hypothesized that c-kit plays a role in the survival, growth, and migration of CPCs. To test this hypothesis, human CPCs were grown under stress conditions in the presence or absence of SCF, and the effects of SCF-mediated activation of c-kit on CPC survival/growth and migration were measured. SCF treatment led to a significant increase in cell survival and a reduction in cell death under serum depletion conditions. In addition, SCF significantly promoted CPC migration in vitro. Furthermore, the pro-survival and pro-migratory effects of SCF were augmented by c-kit overexpression and abrogated by c-kit inhibition with imatinib. Mechanistically, c-kit activation in CPCs led to activation of the PI3K and the MAPK pathways. With the use of specific inhibitors, we confirmed that the SCF/c-kit-dependent survival and chemotaxis of CPCs are dependent on both pathways. Taken together, our findings suggest that c-kit promotes the survival/growth and migration of human CPCs cultured ex vivo via the activation of PI3K and MAPK pathways. These results imply that the efficiency of CPC homing to the injury site as well as their survival after transplantation may be improved by modulating the activity of c-kit.

No MeSH data available.


Related in: MedlinePlus