A Direct Redox Regulation of Protein Kinase C Isoenzymes Mediates Oxidant-induced Neuritogenesis in PC12 Cells

Abstract

In this study, we have used the PC12 cell model to elucidate the mechanisms by which sublethal doses of oxidants induce neuritogenesis. The xanthine/xanthine oxidase (X/XO) system was used for the steady state generation of superoxide, and CoCl 2 was used as a representative transition metal redox catalyst. Upon treatment of purified protein kinase C (PKC) with these oxidants, there was an increase in its cofactor-independent activation. Redox-active cobalt competed with the redoxinert zinc present in the zinc-thiolates of the PKC regulatory domain and induced the oxidation of these cysteine-rich regions. Both CoCl 2 and X/XO induced neurite outgrowth in PC12 cells, as determined by an overexpression of neuronal marker genes. Furthermore, these oxidants induced a translocation of PKC from cytosol to membrane and subsequent conversion of PKC to a cofactor-independent form. Isoenzyme-specific PKC inhibitors demonstrated that PKC⑀ plays a crucial role in neuritogenesis. Moreover, oxidant-induced neurite outgrowth was increased with a conditional overexpression of PKC⑀ and decreased with its knock-out by small interfering RNA. Parallel with PKC activation, an increase in phosphorylation of the growth-associated neuronal protein GAP-43 at Ser 41 was observed. Additionally, there was a sustained activation of extracellular signal-regulated kinases 1 and 2, which was correlated with activating phosphorylation (Ser 133 ) of cAMP-responsive element-binding protein. All of these signaling events that are causally linked to neuritogenesis were blocked by antioxidant N-acetylcysteine (both L and D-forms) and by a variety of PKC-specific inhibitors. Taken together, these results strongly suggest that sublethal doses of oxidants induce neuritogenesis via a direct redox activation of PKC⑀.Understanding the signaling mechanisms involved in neuritogenesis resulting from a compensatory response to injury is crucial to the development of therapeutic agents for recovery after spinal cord and traumatic brain injuries (1, 2). The study of neuritogenesis requires the identification of molecular targets using a suitable experimental model. One of the best characterized cellular models for studying the neuronal pathways involved in neuritogenesis is the rat pheochromocytoma cell line PC12 (3). This cell line continues to be an important model system for the study of cell signaling mechanisms induced by a variety of stimuli, including neurotrophins, hormones, and oxidants (4, 5).In addition to damage from mechanical forces, spinal cord and traumatic brain injuries may result from secondary mechanisms involving ischemia, excitotoxicity, cytokines, and an infiltration of neutrophils at the site of injury (6, 7). The inflammatory response results in the generation of reactive oxygen species (ROS), 2 including superoxide and hydrogen peroxide, which can cause oxidative damage to tissues and lead to cell death (8). Paradoxically, sublethal doses of oxidants can also induce a variety of cellular processes, including cell growth, adhesion, inva...

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“…Western Immunoblotting for PKC Isoenzymes-Cell extracts were prepared and subjected to SDS-polyacrylamide gel electrophoresis as described previously (56). Electrophoretically separated proteins were transferred to a polyvinylidene fluoride membrane.…”

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