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.
Similar content being viewed by others
Article PDF
References
Liao DF, Jin ZG, Bass AS, Daum G, Gygi SP, Aebersold R, et al. Purification and identification of secreted oxidative stress-induced factors from vascular smooth muscle cells. J Biol Chem 2000; 275: 189–96.
Jin ZG, Melaragno MG, Liao DF, Yan C, Haendeler J, Suh YA, et al. Cyclophilin A is a secreted growth factor induced by oxidative stress. Circ Res 2000; 87: 789–96.
Bamberger CM, Wald M, Bamberger AM, Schulte HM . Inhibition of mineralocorticoid and glucocorticoid receptor function by the heat shock protein 90-binding agent geldanamycin. Mol Cell Endocrinol 1997; 131: 233–40.
Grammatikakis N, Lin JH, Grammatikakis A, Tsichlis PN, Cochran BH . p50(cdc37) acting in concert with HSP90 is required for Raf-1 function. Mol Cell Biol 1999; 19: 1661–72.
Miyata Y, Ikawa Y, Shibuya M, Nishida E . Specific association of a set of molecular chaperons including HSP90 and Cdc37 with MOK, a member of the mitogen-activated protein kinase superfamily. J Biol Chem 2001; 276: 21 841–8.
Stancato LF, Silverstein AM, Owens-Grillo JK, Chow YH, Jove R, Pratt WB . The HSP90-binding antibiotic geldanamycin decreases Raf levels and epidermal growth factor signaling without disrupting formation of signaling complexes or reducing the specific enzymatic activity of Raf kinase. J Biol Chem 1997; 272: 4013–20.
Akagami M, Morrison P, Welch WJ . Benzoquinoid ansamycins (herbimycin A and geldanamycin) interfere with the maturation of growth factor receptor tyrosine kinases. Cell Stress Chaper-ones 1999; 4: 19–28.
Prima V, Depoix C, Masselot B, Formstecher P, Lefebvre P . Alteration of the glucocorticoid receptor subcellular localization by non steroidal compounds. J Steroid Biochem Mol Biol 2000; 72: 1–12.
Schulte TW, Blagosklonny MV, Ingui C, Neckers L . Disruption of the Raf-1-HSP90 molecular complex results in destabilization of Raf-1 and loss of Raf-1-Ras association. J Biol Chem 1995; 270: 24 585–8.
Stancato LF, Chow YH, Hutchison KA, Perdew GH, Jove R, Pratt WB . Raf exists in a native heterocomplex with HSP90 and p50 that can be reconstituted in a cell-free system. J Biol Chem 1993; 268: 21 711–6.
Cobb MH, Goldsmith EJ . How MAP kinases are regulated. J Biol Chem 1995; 270: 14 843–6.
Khokhlatchev AV, Canagarajah B, Wilsbacher J, Robinson M, Atkinson M, Goldsmith E, et al. Phosphorylation of the MAP kinase ERK2 promotes its homodimerization and nuclear translocation. Cell 1998; 93: 605–15.
Marshall CJ . Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 1995; 80: 179–85.
Campbell JH, Campbell GR . Culture techniques and their applications to studies of vascular smooth muscle. Clin Sci 1993; 85: 501–12.
Setalo G Jr, Singh M, Guan X, Toran-Allerand CD . Estradiol-induced phosphorylation of ERK1/2 in explants of the mouse cerebral cortex: the roles of heat shock protein 90 (HSP90) and MEK2. J Neurobiol 2002; 50: 1–12.
Backlund M, Johansson I, Mkrtchian S, Sundberg MI . Signal transduction-mediated activation of the aryl hydrocarbon receptor in rat hepatoma H4IIE cells. J Biol Chem 1997; 272: 31 755–63.
Teruel T, Hernandez R, Lorenzo M . Ceramide mediates insulin resistance by tumor necrosis factor-alpha in brown adipocytes by maintaining Akt in an inactive dephosphorylated state. Diabetes 2001; 50: 2563–71.
Wu LX, Xu JH, Huang XW, Zhang KZ, Wen CX, Chen YZ . Down-regulation of p210bcr/abl by curcumin involves disrupting molecular chaperone functions of HSP90. Acta Pharmacol Sin 2006; 27: 694–9.
Perdew GH, Wiegand H, Vanden Heuvel JP, Mitchell C, Singh SS . A 50 kilodalton protein associated with raf and pp60(v-src) protein kinases is a mammalian homolog of the cell cycle control protein cdc37. Biochemistry 1997; 36: 3600–7.
Stancato LF, Chow YH, Hutchison KA, Perdew GH, Jove R, Pratt WB . Raf exists in a native heterocomplex with HSP90 and p50 that can be reconstituted in a cell-free system. J Biol Chem 1993; 268: 21 711–6.
Lamphere L, Fiore F, Xu X, Brizuela L, Keezer S, Sardet C, et al. Interaction between Cdc37 and Cdk4 in human cells. Oncogene 1997; 14: 1999–2004.
Kimura Y, Rutherford SL, Miyata Y, Yahara I, Freeman BC, Yue L, et al. Cdc37 is a molecular chaperone with specific functions in signal transduction. Genes Dev 1997; 11: 1775–85.
Whitesell L, Mimnaugh EG, De Costa B, Myers CE, Neckers LM . Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation. Proc Natl Acad Sci USA 1994; 91: 8324–8.
Segnitz B, Gehring U . The function of steroid hormone receptors is inhibited by the HSP90-specific compound geldanamycin. J Biol Chem 1997; 272: 18 694–701.
Author information
Authors and Affiliations
Corresponding authors
Additional information
Project supported by the National Major Basic Research Program of China 973 (No 2006CB503808), the National Natural Science Foundation of China (No 30470719 and 30600249), and the Education Department of Hu-nan Province (No 99B11).
Rights and permissions
About this article
Cite this article
Liu, Dh., Yuan, Hy., Cao, Cy. et al. 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. Acta Pharmacol Sin 28, 1907–1913 (2007). https://doi.org/10.1111/j.1745-7254.2007.00702.x
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1111/j.1745-7254.2007.00702.x
Keywords
This article is cited by
-
Gene expression analysis of ovine prepubertal testicular tissue vitrified with a novel cryodevice (E.Vit)
Journal of Assisted Reproduction and Genetics (2019)
-
Erythroid Differentiation Regulator 1, an Interleukin 18-Regulated Gene, Acts as a Metastasis Suppressor in Melanoma
Journal of Investigative Dermatology (2011)
-
Heat-shock protein 90 alpha (HSP90α) modulates signaling pathways towards tolerance of oxidative stress and enhanced survival of hepatocytes of Mugil cephalus
Cell Stress and Chaperones (2011)
-
Genes associated with long-chain omega-3 fatty acids in bovine skeletal muscle
Journal of Applied Genetics (2010)