Hsp90 Increasing Reactive Oxygen Species Induced Activated ERK1/2 Function In Rat VSMC

AIM To investigate the effects of Hsp90 on p-ERKl/2 in oxidative-stressed rat VSMC.METHODS VSMC were treated with 1 μM LY83583, a generator of reactive oxygen species, for different time (control, 5 min, 10 min, 30 min, 60 min, 90 min, 120 min, 180 min) and blotted with Hsp90, ERK1/2 and p-ERK1/2. To investigate the interaction of Hsp90 and p-ERKl/2, VSMC were divided into 4 groups: control (PBS 120 min), LY (1 μM LY83583 for 120 min), Gel+DMSO+LY (pretreated with Geldanamycin, a special inhabitor of Hsp90, for 30 min, then treated with 1 μM LY83583 for 120 min), DMSO+LY (pretreated with DMSO, the vehicle of Geldanamycin, for 30 min, then treated with 1 μM LY83583 for 120 min). The total cell lysates were harvested and immunoprecipitated with Hsp90 and blotted with p-ERK1/2. Then the total, soluble, insoluble and nuclear cell lysates were collected and blotted with p-ERK1/2. Immunofluorescence also was used to observe the nuclear translocation of p-ERK1/2.We also used western-blot to observe the temporal effect of LY83583 on CyP A and CAV-1 expression. Then VSMC were divided into 4 groups: control, LY, Gel+DMSO+LY and DMSO+LY, and immunoprecipitated with p-ERKl/2 and then blotted with CyP A and CAV-1.RESULTS The expression of Hsp90 was time-dependent increased during 0~120 min. Hsp90 binded with p-ERK1/2 in LY and Geldanamycin attenuated LY83583-induced Hsp90- p-ERK1/2 complex in Gel+DMSO+LY. LY83583 increased the total, soluble and nuclear p-ERK1/2 and Geldanamycin attenuated LY83583-induced the increase of...