Heat shock protein 90 acts as a molecular chaperone in late-phase activation of extracellular signal-regulated kinase 1/2 stimulated by oxidative stress in vascular smooth muscle cells

Abstract

Aim: To investigate whether cytosolic heat shock protein 90 (HSP90) acts as a molecular 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/L LY83583 (6-anilinoquinoline-5,8-quinolinedione, producer of ROS) for 120 min in the 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 blotting and immunofluorescence. Cell proliferation was tested by cell counting and 3-(4,5-dimethylthiazol-2-y1)-3,5-di- phenyltetrazolium bromide (MTT). Results: The cytosolic HSP90 of VSMC was upregulated by LY83583 in a time-dependent manner with the peak at 120 min, which is consistent with the late peak of phosphor-ERK1/2. Immunoprecipitation and Western blotting analyses showed that LY83583 increased the interaction of HSP90 with phosphor-ERK1/2, the phosphor-ERK1/2 level, 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 confirmed by Western blotting and immunofluorescence assays. Furthermore, cell counting and the MTT assay showed that LY83583 stimulated VSMC proliferation with the increased expression of HSP90 and levels of soluble and nuclear phosphor-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 solubility and nuclear translocation of phosphor-ERK1/2. © 2007 CPS and SIMM.