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

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 pmol/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-ERK 1/2 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-phenyltetrazoliumbromide （MTT）. Results： The cytosolic 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 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 con- firmed by Western blotting and immunofluorescence assays. Furthermore, cell counting and the MTT assay showed that LY83583 stimulated VSMC prolifera- tion 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 solubil- ity and nuclear translocation of phosphor-ERK 1/2.