Purification and identification of secreted oxidative stress-induced factors from vascular smooth muscle cells

Reactive oxygen species have been implicated in the pathogenesis of atheros clerosis 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 (1 0 min) and late (2 h) peaks and stimulated VSMC growth. To investigate whet her 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 identifi ed by capillary chromatography, electrospray ionization tandem mass spectro metry, and data base searching included heat shock protein 90-alpha: (HSP90 -alpha) and cyclophilin B, Western blot analysis of conditioned medium show ed specific secretion of HSP90-alpha but not HSP90-beta, Immunodepletion of HSP90-alpha from conditioned medium significantly inhibited conditioned me dium-induced ERK1/2 activation. Human recombinant HSP90-alpha reproduced th e effect of conditioned medium on ERK1/2 activation. These results show tha t brief oxidative stress causes sustained release of protein factors from V SMC that can stimulate ERK1/2. These factors may be important mediators for the effects of reactive oxygen species on vascular function.