Purification and Identification of Secreted Oxidative Stress-induced Factors from Vascular Smooth Muscle Cells

doi:10.1074/jbc.275.1.189

Purification and Identification of Secreted Oxidative Stress-induced Factors from Vascular Smooth Muscle Cells*

From the ^tObCenter for Cardiovascular Research, University of Rochester Medical Center, Rochester, New York 14642 and the Departments of ^tOdMedicine, ^tOfSurgery, and ^tOgMolecular Biotechnology, University of Washington, Seattle, Washington 98195

Abstract

Reactive oxygen species have been implicated in the pathogenesis of atherosclerosis and hypertension, in part by promoting vascular smooth muscle cell (VSMC) growth. We have previously shown that LY83583, a generator of O⨪₂, activated extracellular signal-regulated kinases (ERK1/2) with early (10 min) and late (2 h) peaks and stimulated VSMC growth. To investigate whether secreted oxidative stress-induced factors (termed SOXF) from VSMC were responsible for late ERK1/2 activation in response to LY83583, we purified putative SOXF proteins from conditioned medium (2 h of LY83583 exposure) by sequential chromatography based on activation of ERK1/2. Proteins identified by capillary chromatography, electrospray ionization tandem mass spectrometry, and data base searching included heat shock protein 90-α (HSP90-α) and cyclophilin B. Western blot analysis of conditioned medium showed specific secretion of HSP90-α but not HSP90-β. Immunodepletion of HSP90-α from conditioned medium significantly inhibited conditioned medium-induced ERK1/2 activation. Human recombinant HSP90-α reproduced the effect of conditioned medium on ERK1/2 activation. These results show that brief oxidative stress causes sustained release of protein factors from VSMC that can stimulate ERK1/2. These factors may be important mediators for the effects of reactive oxygen species on vascular function.

Footnotes

↵* This work was supported by National Institutes of Health Grants HL18645 and HL49192 and an American Heart Association National Grant-in-Aid (to B. C. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵FNa These authors contributed equally to this study.
↵FNc Present address: Dept. of Pharmacology, Hengyang Medical College, Hengyang 421001, Hunan, People's Republic of China.
↵FNe Supported by an individual National Research Service Award Fellowship F32 HL09027 from the National Institutes of Health.
↵FNh Supported by National Institutes of Health Grant T32 HG00035.
↵i Supported by a grant from the National Science Foundation Science and Technology Center for Molecular Biotechnology.
↵FNj To whom correspondence should be addressed: Center for Cardiovascular Research, Box 679, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-273-1946; Fax: 716-473-1573; E-mail: bradford_berk@urmc.rochester.edu.
↵2 Z.-G. Jin and B. C. Berk, unpublished results.
Abbreviations:

ROS

reactive oxygen species

SOXF

secreted oxidative stress-induced factors

VSMC

vascular smooth muscle cell

ERK

extracellular signal-regulated kinases

FGF

fibroblast growth factor

PDGF

platelet-derived growth factor

EGF

epidermal growth factor

TGF

transforming growth factor

MAPK

mitogen-activated protein kinase

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

HBSS

Hanks' balanced salt solution

PAGE

polyacrylamide gel electrophoresis

PKC

protein kinase C
- Received August 30, 1999.
- Revision received October 22, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.