论文部分内容阅读
Aim:To investigate whether cytosolic heat shock protein 90 (HSP90) acts as amolecular chaperone on the activated extracellular signal-regulated kinase 1/2(ERK1/2) and cell proliferation stimulated by reactive oxygen species (ROS) in ratvascular smooth muscle cells (VSMC).Methods:VSMC were exposed to 1 μmol/LLY83583 (6-anilinoquinoline-5,8-quinolinedione,producer of ROS) for 120 min inthe presence or absence of 5 μmol/L geldanamycin,a specific inhibitor of HSP90.Then the total,soluble,and insoluble proteins of the cells were collected.HSP90,ERK1/2,and phosphor-ERK1/2 in the cell lysate were measured by Western blotting.The interaction of HSP90 and phosphor-ERK1/2 was analyzed by immunoprecipi-tation assay,and the nuclear phosphor-ERK1/2 was measured by Western blot-ting and immunofluorescence.Cell proliferation was tested by cell counting and3-(4,5-dimethylthiazol-2-yl)-3,5-di-phenyltetrazolium bromide (MTT).Results:Thecytosolic HSP90 of VSMC was upregulated by LY83583 in a time-dependent man-ner with the peak at 120 min,which is consistent with the late peak of phosphor-ERK1/2.Immunoprecipitation and Western blotting analyses showed that LY83583increased the interaction of HSP90 with phosphor-ERK 1/2,the phosphor-ERK 1/2level,and the soluble phosphor-ERK1/2 level by 1.8-,2.5-,and 2.9-fold,respectively.In contrast,the insoluble phosphor-ERK1/2 of VSMC was decreased.Interestingly,LY83583 treatment promoted the nuclear phosphor-ERK1/2 by 7.6-fold as con-firmed by Western blotting and immunofluorescence assays.Furthermore,cellcounting and the MTT assay showed that LY83583 stimulated VSMC prolifera-tion with the increased expression of HSP90 and levels of soluble and nuclearphosphor-ERK1/2.Pretreatment of geldanamycin antagonized the effect ofLY83583.Conclusion:HSP90 could mediate the oxidative stress-stimulated,late-phase activation of ERK1/2 and VSMC proliferation by promoting the ERK1/2phosphorylation,the association of itself with phosphor-ERK1/2,and the solubil-ity and nuclear translocation of phosphor-ERK 1/2.
Aim: To investigate whether cytosolic heat shock protein 90 (HSP90) acts as amolecular chaperone on the activated extracellular signal-regulated kinase 1/2 (ERK1 / 2) and cell proliferation stimulated by reactive oxygen species (ROS) in rat vascular smooth muscle cells VSMC). Methods: VSMC were exposed to 1 μmol / LLY83583 (6-anilinoquinoline-5,8-quinolinedione, producer of ROS) for 120 min in presence or absence of 5 μmol / L geldanamycin, a specific inhibitor of HSP90. Total, soluble, and insoluble proteins of the cells were collected. HSP90, ERK1 / 2, and phosphor-ERK1 / 2 in the cell lysate were measured by Western blotting. The interaction of HSP90 and phosphor- ERK1 / 2 was analyzed by immunoprecipi- tation assay, and the nuclear phosphor-ERK1 / 2 was measured by Western blot-ting and immunofluorescence. Cell proliferation was tested by cell counting and 3- (4,5-dimethylthiazol-2-yl) -3,5-di-phenyltetrazolium bromide (MTT). Results: Thecytosolic HSP90 of VSMC was upregulated by LY83583 in a time-depen dent man-ner with the peak at 120 min, which is consistent with the late peak of phosphor-ERK1 / 2. Immunoprecipitation and Western blotting analyzes showed that LY83583 was created by interaction with HSP90 with phosphor-ERK 1/2, the phosphor-ERK 1 / 2level, and the soluble phosphor-ERK1 / 2 level by 1.8-, 2.5-, and 2.9-fold, respectively.In contrast, the insoluble phosphor- ERK1 / 2 of VSMC was decreased.Interestingly, LY83583 treatment promoted the nuclear phosphor- ERK1 / 2 by 7.6-fold as con-firmed by Western blotting and immunofluorescence assays. Fluorrthermore, cellcounting and the MTT assay showed that LY83583 stimulated VSMC prolifera tion with the increased expression of HSP90 and levels of soluble and nuclear phosphory-ERK1 / 2. Pretreatment of geldanamycin antagonized the effect of LY83583.Conclusion: HSP90 could mediate the oxidative stress-stimulated, late-phase activation of ERK1 / 2 and VSMC proliferation by promoting the ERK1 / 2 phosphorylation, the association of itself with phosphor-ERK1 / 2, and the solubil-it yand nuclear translocation of phosphor-ERK 1/2.