title: Purification and identification of secreted oxidative stress-induced factors from vascular smooth muscle cells.
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-alpha (HSP90-alpha) and cyclophilin B. Western blot analysis of conditioned medium showed specific secretion of HSP90-alpha but not HSP90-beta. Immunodepletion of HSP90-alpha from conditioned medium significantly inhibited conditioned medium-induced ERK1/2 activation. Human recombinant HSP90-alpha 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.
authors.affiliation: Center for Cardiovascular Research, University of Rochester Medical Center, Rochester, New York 14642, USA.
authors.forename: D F
authors.initials: DF
authors.lastname: Liao
authors.forename: Z G
authors.initials: ZG
authors.lastname: Jin
authors.forename: A S
authors.initials: AS
authors.lastname: Baas
authors.forename: G
authors.initials: G
authors.lastname: Daum
authors.forename: S P
authors.initials: SP
authors.lastname: Gygi
authors.forename: R
authors.initials: R
authors.lastname: Aebersold
authors.forename: B C
authors.initials: BC
authors.lastname: Berk
mesh_terms: D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt / Q000494:pharmacology; D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D019898:Autocrine Communication; D017077:Culture Media, Conditioned; D021983:Cyclophilins; D018841:HSP90 Heat-Shock Proteins / Q000302:isolation & purification* / Q000378:metabolism; D020104:Immunophilins / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000502:physiology*; D018384:Oxidative Stress / Q000502:physiology*; D019696:Peptidylprolyl Isomerase; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
chemical_list: D000634:Aminoquinolines; D017077:Culture Media, Conditioned; D018841:HSP90 Heat-Shock Proteins; D017382:Reactive Oxygen Species; C081786:cyclophilin B; D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; C041715:6-anilino-5,8-quinolinedione; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D014357:Trypsin; D021983:Cyclophilins; D020104:Immunophilins; D019696:Peptidylprolyl Isomerase