OXIDANT-INDUCED ACTIVATION OF PROTEIN-KINASE-C IN UC11MG CELLS

Free radical formation and subsequent lipid peroxidation may participa te in the pathogenesis of tissue injury, including the brain injury in duced by hypoxia or trauma and cardiac injury arising from ischemia an d reperfusion. However, the exact cellular mechanisms by which the ini tial oxidative insult leads to the ultimate tissue damage are not know n. A number of reports have indicated that protein kinase C (PKC) may be activated following oxidative stress and that this enzyme may play an important role in the steps leading to cellular damage. In this wor k, we have examined in a cell model whether PKC is activated following oxidative exposure. UC11MG cells, a human astrocytoma cell line, were treated with H2O2. Incubation with 0.5 mM H2O2 increased malondialdeh yde levels by as early as 15 minutes. To assess the effects of H2O2 tr eatment on PKC activation, we measured phosphorylation of an endogenou s PKC substrate, the MARCKS (myristoylated alanine-rich C kinase subst rate) protein. Treatment of cells with 0.2-1.0 mM H2O2 resulted in a r apid increase in MARCKS phosphorylation. Phosphorylation was stimulate d approximately 2.5-fold following treatment with 0.5 mM H2O2 for ten minutes. Treatment with phorbol 12-myristate 13-acetate, a PKC activat or, increased MARCKS phosphorylation approximately 4-fold. The H2O2-in duced MARCKS phosphorylation was inhibited by the addition of the kina se inhibitors H-7 and staurosporine. Furthermore, specific down-regula tion of PKC by phorbol ester also inhibited H2O2-induced MARCKS phosph orylation. These results indicate that PKC is rapidly activated in cel ls following an oxidative exposure and that this cell system may be a good model to further investigate the role of PKC in regulating oxidat ive damage in the cell.