Differential Activation of Mitogen-Activated Protein Kinases by H 2 O 2 and O 2 − in Vascular Smooth Muscle Cells

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Abstract
Increased generation of active oxygen species such as H 2 O 2 and O 2 − may be important in vascular smooth muscle cell growth associated with atherosclerosis and restenosis. In previous work, we showed that H 2 O 2 stimulated vascular smooth muscle cell growth and proto-oncogene expression. In the present study, we compared the effects of H 2 O 2 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 [ 3 H]thymidine incorporation, was stimulated by 200 μmol/L H 2 O 2 (110% increase versus 0.1% serum) and 1 μmol/L LY83583 (175% increase) to levels comparable to 10 ng/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 μmol/L) but not by H 2 O 2 . The time course for activation of MAP kinase by LY83583 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 H 2 O 2 induced MKP-1 mRNA to a greater extent than did LY83583, the increased MKP-1 expression could not explain the inability of H 2 O 2 to stimulate MAP kinase, because mRNA levels were not detected until 60 minutes. The findings that both O 2 − and H 2 O 2 stimulate vascular smooth muscle cell growth but only O 2 − rapidly activates MAP kinase suggest that additional signal events are required for the mitogenic effects of H 2 O 2 .