DIFFERENTIAL ACTIVATION OF MITOGEN-ACTIVATED PROTEIN-KINASES BY H2O2 AND O-2(-) IN VASCULAR SMOOTH-MUSCLE CELLS

Increased generation of active oxygen species such as H2O2 and O-2(-) may be important in vascular smooth muscle cell growth associated with atherosclerosis and restenosis. In previous work, we showed that H2O2 stimulated vascular smooth muscle cell growth and proto-oncogene expression. In the present study, we compared the effects of H2O2 and O-2(-) on cultured rat aortic vascular smooth muscle cell growth and signal transduction. O-2(-) was generated in a concentration-dependent manner by the naphthoquinolinedione LY83583. Vascular smooth muscle cell growth, as measured by [H-3]thymidine incorporation, was stimulated by 200 mu mol/L H2O2 (110% increase versus 0.1% serum) and 1 mu mol/L LY83583 (175% increase) to levels comparable to 10 mu g/mL platelet-derived growth factor (210% increase). Since activation of mitogen-activated protein kinase (MAP kinase) is one of the earliest growth factor signal events, the activity of MAP kinase was measured by changes in mobility on Western blot and by phosphorylation of myelin basic protein. There was a concentration-dependent increase in MAP kinase activity by LY83583 (maximum, 10 mu mol/L) but not by H2O2. The time course for activation of MAP kinase by LYS3583 showed a maximum at 5 to 10 minutes with return to baseline by 20 minutes. Activation of MAP kinase by LY83583 was protein kinase C dependent. Expression of MAP kinase phosphatase-1 (MKP-1), a transcriptionally regulated redox-sensitive protein tyrosine/threonine phosphatase, was also measured. Although H2O2 induced MKP-1 mRNA to a greater extent than did LY83583, the increased MKP-1 expression could not explain the inability of H2O2 to stimulate MAP kinase, because mRNA levels were not detected until 60 minutes. The findings that both O-2(-) and H2O2 stimulate vascular smooth muscle cell growth but only O-2(-) rapidly activates MAP kinase suggest that additional signal events are required for mitogenic effects of H2O2.