Bradykinin-mediated Ca2+ signalling regulates cell growth and mobility in human cardiac c-Kit+ progenitor cells

Abstract

Our recent study showed that bradykinin increases cell cycling progression and migration of human cardiac c-Kit⁺ progenitor cells by activating pAkt and pERK1/2 signals. This study investigated whether bradykinin-mediated Ca²⁺ signalling participates in regulating cellular functions in cultured human cardiac c-Kit⁺ progenitor cells using laser scanning confocal microscopy and biochemical approaches. It was found that bradykinin increased cytosolic free Ca²⁺ ( ${\mathrm{Ca}}_i^{2+}$ ) by triggering a transient Ca²⁺ release from ER IP3Rs followed by sustained Ca²⁺ influx through store-operated Ca²⁺ entry (SOCE) channel. Blockade of B2 receptor with HOE140 or IP3Rs with araguspongin B or silencing IP3R3 with siRNA abolished both Ca²⁺ release and Ca²⁺ influx. It is interesting to note that the bradykinin-induced cell cycle progression and migration were not observed in cells with siRNA-silenced IP3R3 or the SOCE component TRPC1, Orai1 or STIM1. Also the bradykinin-induced increase in pAkt and pERK1/2 as well as cyclin D1 was reduced in these cells. These results demonstrate for the first time that bradykinin-mediated increase in free ${\mathrm{Ca}}_i^{2+}$ via ER-IP3R3 Ca²⁺ release followed by Ca²⁺ influx through SOCE channel plays a crucial role in regulating cell growth and migration via activating pAkt, pERK1/2 and cyclin D1 in human cardiac c-Kit⁺ progenitor cells.

Keywords: bradykinin; cell cycle progression; human cardiac c-Kit+ progenitor cells; inositol 1,4,5-triphosphate receptor; migration; store-operated Ca2+ entry.

Figure 1

Increase in ${\mathrm{Ca}}_{\mathrm{i}}^{2+}$ by bradykinin (BK) in human cardiac c‐kit⁺ progenitor cells. A, Pseudocolour images (upper panel) showing changes in fluorescence intensity (ie ${\mathrm{Ca}}_{\mathrm{i}}^{2+}$) induced by 10 nmol L⁻¹ bradykinin at different time points indicated in the lower panel in human cardiac c‐Kit⁺ progenitor cells. The F/F₀ represents ${\mathrm{Ca}}_{\mathrm{i}}^{2+}$ level, where the F is the changes in cell fluorescence intensity of Fluo‐4 AM, and the F₀ is the initial level of fluorescence intensity. B, ${\mathrm{Ca}}_{\mathrm{i}}^{2+}$ increase induced by bradykinin was prevented in cells pre‐treated with 30 nmol L⁻¹ HOE140 (B2R inhibitor) (n = 80 cells of 5 experiments), but not the B1R inhibitor R715 (300 nmol L⁻¹) (n = 80 cells of 5 experiments). C, Only a transient ${\mathrm{Ca}}_{\mathrm{i}}^{2+}$ increase induced by bradykinin was observed in cells exposure to Ca²⁺‐free Tyrode's solution with 1 mmol L⁻¹ EGTA (n = 96 cells of 6 experiments). D, Bradykinin‐induced Ca²⁺ increase was absent in cells pre‐treated with the PLC blocker U73122 (10 μmol L⁻¹) (n = 80 cells of 5 experiments). E, ${\mathrm{Ca}}_{\mathrm{i}}^{2+}$ increase induced by bradykinin was abolished by the IP3R blocker araguspongin B (4 μmol L⁻¹) (n = 80 cells of 5 experiments), but not by the RyR inhibitor ryanodine (10 μmol L⁻¹) (n = 96 cells of 6 experiments). Traces are shown as mean ± SEM in corresponding experiments

Figure 2

Effects of membrane channel blockers on ${\mathrm{Ca}}_{\mathrm{i}}^{2+}$ increase by bradykinin (BK) in human cardiac c‐Kit⁺ progenitor cells. A, ${\mathrm{Ca}}_{\mathrm{i}}^{2+}$ increase was not affected by the L‐type Ca²⁺ channel inhibitor nifedipine (10 μmol L⁻¹) (n = 96 cells of 6 experiments) or NCX inhibitor KB‐R7943 (10 μmol L⁻¹) (n = 96 cells of 6 experiments). B, ${\mathrm{Ca}}_{\mathrm{i}}^{2+}$ increase was not affected by the TRPV2 channel blocker ruthenium red (10 μmol L⁻¹) (n = 96 cells of 6 experiments) or the TRPV4 blocker RN1734 (10 μmol L⁻¹) (n = 96 cells of 6 experiments). C, General TRP channel blocker La³⁺ (100 μmol L⁻¹) (n = 96 cells of 6 experiments) or the non‐selective TRPC channel blocker SKF96365 (50 μmol L⁻¹) (n = 80 cells of 5 experiments) inhibited the sustained Ca²⁺ influx, but not Ca²⁺ transient release induced by bradykinin. D, Selective SOCE channel blocker (La³⁺, 10 μmol L⁻¹), but not selective TRPC3 blocker (Pyr3, 10 μmol L⁻¹) (n = 96 cells of 6 experiments) or selective TRPC4 channel blocker (ML204, 10 μmol L⁻¹) (n = 96 cells of 6 experiments), inhibited the sustained Ca²⁺ influx, but not Ca²⁺ transient release induced by bradykinin. Summarized traces are shown as mean ± SEM in corresponding experiments

Figure 3

Effects of silencing related genes with corresponding siRNAs on ${\mathrm{Ca}}_{\mathrm{i}}^{2+}$ increase induced by bradykinin (BK). A, Bradykinin did not induce ${\mathrm{Ca}}_{\mathrm{i}}^{2+}$ increase in cells with silenced B2R (n = 80 cells of 5 experiments). B, Silencing IP3R3, but not IP3R1 or IP3R2, prevented the ${\mathrm{Ca}}_{\mathrm{i}}^{2+}$ increase induced by bradykinin (n = 80 cells of 5 experiments). C, Silencing TRPC1, but TRPC3 or TRPC4, prevented the Ca²⁺ influx without affecting transient Ca²⁺ release induced by bradykinin (n = 96 cells of 6 experiments). D, Silencing Orai1 or STIM1 prevented the Ca²⁺ influx without affecting transient Ca²⁺ release induced by bradykinin (n = 96 cells of 6 experiments). Summarized traces are shown as mean ± SEM in corresponding experiments

Figure 4

Effects of silencing IP3Rs or components of SOCE channel on cell growth and migration induced by bradykinin (BK). A, Cell proliferation determined with MTT assay in cells transfected with 40 nmol L⁻¹ siRNAs targeting IP3R1, IP3R2, IP3R3, TRPC1, TRPC3, TRPC4, Orai1 or STIM1 in the absence or presence of 10 nmol L⁻¹ bradykinin. B, Images of BrdU incorporation in cells transfected with 40 nmol L⁻¹ siRNAs targeting IP3R1, IP3R2, IP3R3, TRPC1, TRPC3, TRPC4, Orai1 or STIM1 in the absence or presence of 10 nmol L⁻¹ bradykinin. The proliferative cells show yellow colour from the merging of BrdU green staining with Propidium Iodide (PI) red nuclei staining. C, Percentage values of BrdU incorporation in cells transfected with 40 nmol L⁻¹ siRNAs targeting IP3R1, IP3R2, IP3R3, TRPC1, TRPC3, TRPC4, Orai1 or STIM1 in the absence or presence of 10 nmol L⁻¹ bradykinin. N = 5 experiments, **P < .01 vs control siRNA, ^## P < .01 vs control siRNA with 10 nmol L⁻¹ bradykinin

Figure 5

Effects of silencing IP3Rs or components of SOCE channel on cell cycling progression induced by bradykinin (BK). A, Representative flow cytometry graphs in cells transfected with control, IP3R3 or TRPC1 siRNA (40 nmol L⁻¹) in the absence or presence of 10 nmol L⁻¹ bradykinin. B, Percentage values of cell population at different cycling phases in cells transfected with control, IP3R1, IP3R2 or IP3R3 siRNA (40 nmol L⁻¹) in the absence or presence of 10 nmol L⁻¹ bradykinin. C, Percentage values of cell population at different cycle phases in cells transfected with control, TRPC1, TRPC3 or TRPC4 siRNA (40 nmol L⁻¹) in the absence or presence of 10 nmol L⁻¹ bradykinin. D, Percentage values of cell population at different cycle phases in cells transfected with control, Orai1 or STIM1 siRNA (40 nmol L⁻¹) in the absence or presence of 10 nmol L⁻¹ bradykinin treatment. N = 5, experiments *P < .05, **P < .01 vs control siRNA

Figure 6

Effects of silencing IP3Rs or components of SOCE channel on migration induced by bradykinin (BK). A, Representative images of wound‐healing assay in cells transfected with control, IP3R3, TRPC1, Orai1 or STIM1 siRNA in the absence or presence of 10 nmol L⁻¹ bradykinin treatment for 8 h. B, Mean values of cell number migrated into the acellular area in cells transfected corresponding siRNAs in the absence and presence of 10 nmol L⁻¹ bradykinin. C, Images of migrated cells on the lower surface membrane in transwell assay in cells transfected with control, IP3R3, TRPC1, Orai1 or STIM1 siRNA in the absence or presence of 10 nmol L⁻¹ bradykinin treatment for 8 h. D, Mean number of migrated cells on the lower surface membrane in cells transfected corresponding siRNAs in the absence or presence of 10 nmol L⁻¹ bradykinin. N = 6 experiments, **P < .01 vs control siRNA, ^## P < .01 vs control siRNA with 10 nmol L⁻¹ bradykinin

Figure 7

Effects of silencing IP3Rs or components of SOCE channel on increase of pAkt, pERK1/2 and cyclin D1 by bradykinin (BK). A, Western blots show that bradykinin (10 nmol L⁻¹) increased pAkt, pERK1/2 or cyclin D1 expression in cells transfected with control, IP3R1 and IP3R2 siRNA, but not with IP3R3 siRNA. B, Relative protein levels of pAkt, pERK1/2 and cyclin D1 in cells transfected with corresponding IP3R siRNAs. C, Western blots show that bradykinin (10 nmol L⁻¹) increased pAkt, pERK1/2 and cyclin D1 level in cells transfected with control, TRPC3 or TRPC4 siRNA, but not with TRPC1 siRNA. D, Relative protein levels of pAkt, pERK1/2 and cyclin D1 in cells transfected with control, TRPC1, TRPC3 or TRPC4 siRNA. E, Western blots show that bradykinin (10 nmol L⁻¹) increased pAkt, pERK1/2 and cyclin D1 level in cells transfected with control siRNA, but not with Orai1 or STIM1 siRNA. F, Relative protein levels of pAkt, pERK1/2 and cyclin D1 in cells transfected with control, Orai1 or STIM1 siRNA. N = 5, **P < .01 vs control siRNA treated with vehicle, ^## P < .01 vs control siRNA with 10 nmol L⁻¹ bradykinin