The Regulation Of Cyclophilin A On Extracellular Signal-regulated Kinases 1/2 Activated By Oxidative Stress In Vascular Smooth Muscle Cells

Aim Phosphorylation and nuclear translocation of extracellular signal-regulated kinases are most important for proliferation of oxidative-stressed vascular smooth muscle cells and Cyclophilin A is involved in this process. We investigate whether Cyclophilin A participated in extracellular signal-regulated kinases 1/2 pathway as a molecular chaperon.Methods Cell culture technique was used in vitro. Exposure vascular smooth muscle cells to LY83583 (1μM) for different time, and the effect on the proliferation of vascular smooth muscle cells was observed by measuring MTT metabolism and cell count, then Cyclophilin A, extracellular signal-regulated kinases 1/2 and phospho- extracellular signal-regulated kinases 1/2 in cell lysates were measured by western blot. Vascular smooth muscle cells were divided into 5 groups, control, LY83583 (1μM LY83583 for 120 min), DMSO+LY83583 (pretreated with DMSO for 30 min, then treated with 1μM LY83583 for 120 min). CyclosporinA+LY83583 (pretreated with CyclosporinA, a special inhibitor of Cyclophilin A, for 30 min, then treated with 1μM LY83583 for 120 min), LY83583+CyclosporinA (pretreated with 1μM LY83583 for 120 min, then treated with CyclosporinA for 30 min), flow cytometry was performed to track cell cycle progression.. Then the total, soluble, insoluble and nuclear cell lysates were collected, Cyclophilin A binding with extracellular signal-regulated kinases 1/2 and phospho- extracellular signal-regulated kinases 1/2 were quatified by immunoprecipitation and western blot. Immunofluorescence also was used to observe the nuclear translocation of phospho-extracellular signal-regulated kinases 1/2.Results The metablism of MTT and cell number by vascular smooth muscle cells are increased markedly in a time-dependent manner. Cyclophilin A got the peak at 120 min which corresponded to the second peak of phospho-extracellular signal-regulated kinases 1/2. Immunoprecipitation showed that LY83583 increased the complex of Cyclophilin A-phospho-extracellular signal-regulated kinases 1/2, and western blot analysis showed that LY83583 increased the total, soluble and nuclear phospho-extracellular signal-regulated kinases 1/2 and while the insoluble phospho-extracellular signal-regulated kinases 1/2 decreased in LY83583. Flow cytometry analysis indicated that the S-Phase fraction ratios is higher and G0/G1-Phase fraction ratios is lower. The immunofluorescence results suggested that plasmid and nuclear phospho-extracellular signal-regulated kinases 1/2 increased in LY83583. CyclosporinA attenuated these effects of LY83583.Conclusions Cyclophilin A bound with phospho-extracellular signal-regulated kinases 1/2 and promoted their nuclear translocation in oxidative-stressed vascular smooth muscle cells. Cyclophilin A is a key chaperon in oxidative induced phospho-extracellular signal-regulated kinases 1/2.