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.
OBJECTIVE
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 micromol/L LY83583 (6-anilinoquinoline-5,8-quinolinedione, producer of ROS) for 120 min in the presence or absence of 5 micromol/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 immunoprecipitation 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.


CONCLUSIONS
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.
Liu
Dai-hua
DH
Division of Pharmacoproteomics, Institute of Pharmacy and Pharmacology, School of Life Science and Technology, University of South China, Hengyang 421001, China.
Yuan
Hao-yu
HY
Cao
Chun-ya
CY
Gao
Zhi-ping
ZP
Zhu
Bing-yang
BY
Huang
Hong-lin
HL
Liao
Duan-fang
DF
D000818:Animals; D049109:Cell Proliferation; D018841:HSP90 Heat-Shock Proteins / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D018832:Molecular Chaperones / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D018384:Oxidative Stress; D051381:Rats; D017207:Rats, Sprague-Dawley
D018841:HSP90 Heat-Shock Proteins; D018832:Molecular Chaperones; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3