PDGF-BB activated Rac 1 in regulation of inner and outer nuclear distribution of rat aortic smooth muscle cell β-catenin

Objective: In this study, we observed the interaction between JNK and Rac1 and its effect on PDGF-BB induced ß-catenin inner and outer nuclear distribution, cell proliferation and migration. Methods: SD rats’ thoracic aortas were taken and rats’ aortic smooth muscle cells were cultured by explant method. Cell morphology and immunohistochemistry were relied on to identify the cell. Three pairs of Rac1siRNA were designed. Western blotting method was used for screening, and the most effective one was applied to the experiment. CCK8 kits and Transwell chambers were used to detect the effects of different concentrations of Rac1 inhibitor NSC23766 (25, 50, 100 μmol/L) and Rac1siRNA (50 nmol/L) on vascular smooth muscle cells’ proliferation and migration which was induced by PDGF-BB (50 μg/L). Results: The vascular smooth muscle cells grew out 5-7 days after explant method culture. The cell morphology and alpha -actin immunohistochemistry identified them as vascular smooth muscle cells. After 24 h PDGF-BB stimulation, vascular smooth muscle cell cultured CCK8 was significantly increased (OD, group NSC23766: 0.796+0.083 VS 0.371+0.055, P<0.01; group Rac1siRNA: 0.948+0.107 VS 0.443+0.042, P<0.01). But if given different concentrations of NSC23766 (25, 50, 100 mol/L) before treatment, the concentration of CCK8 decreased dependently (OD: 0.796+0.083 VS 0.559+0.064, P<0.05; 0.490+0.027, 0.370+0.025, P<0.01). CCK8 value was also decreased significantly (0.948+0.107 VS 0.670+0.072, P<0.01) after transfection of Rac1siRNA (50 nmol/L). Migrations of vascular smooth muscle cells were significantly increased after PDGF-BB stimulation (crystal violet OD, group NSC23766: 0.429+0.0400 VS 0.260+0.023, P<0.01; group Rac1siRNA: 0.439+0.042 VS 0.300+0.042, P<0.01). Cell migration was inhibited obviously (OD: 0.429+0.040 VS 0.401+0.015, P>0.05, 0.324+0.035, 0.290+0.013 and 0.439+0.042, P<0.01 VS 0.345+0.030, P<0.05) if they were cultured with different concentrations of NSC23766 (25, 50, 100 mol/L) and Rac1siRNA (50 nmol/L). Rac1 activity, expression of pi-JNK and cell nucleus beta -catenin were gradually increased after stimulation of PDGF-BB and reached its peak in 5 min, 15 min and 60 min. With different concentrations of NSC23766 (25, 50, 100 mol/L), SP600125 (10, 20, 40 g/L) and Rac1siRNA (50 nmol/L) treatment and the same time of PDGF-BB stimulation, the expression activity of Rac1, pi-JNK and nucleus beta -catenin were inhibited obviously and showed concentration dependent. Immunofluorescence results showed: After 60min PDGF-BB stimulation on vascular smooth muscle cell, Beta -catenin nuclear accumulation was obvious. When they were given NSC23766 (100 mol/L), SP600125 (40 g/L) and Rac1siRNA (50 nmol/L) treatment, this aggregation was inhibited. Conclusion: The interaction existed between the Rac1 activation and JNK phosphorylation, and it played a key regulatory role in PDGF-BB induced vascular smooth cell beta -catenin nuclear cohesive concentration. Thereby it regulated vascular smooth muscle cells’ proliferation and migration which were induced by PDGF-BB.