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 PKCepsilon plays a crucial role in neuritogenesis. Moreover, oxidant-induced neurite outgrowth was increased with a conditional overexpression of PKCepsilon 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 PKCepsilon.

Highlights

  • 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]

  • Role of Redox Activation of protein kinase C (PKC)⑀ in nerve growth factor (NGF)-induced Neurite Outgrowth—In order to assess the biological significance of the neuritogenesis induced via oxidative activation of PKC⑀ by exogenous oxidants, we examined whether the small amounts of reactive oxygen species (ROS) generated during NGF signaling induce oxidative activation of PKC⑀ [13,14,15]

  • ROS are produced as part of the inflammatory response to spinal cord and traumatic brain injuries, and the enzymes directly and influenced by them are molecular targets for new therapies to promote neuritogenesis

Read more

Summary

Introduction

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]. 14430 JOURNAL OF BIOLOGICAL CHEMISTRY 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]. TrkA activation is coupled to phospholipase C␥ activation, which in turn leads to protein kinase C (PKC) activation [18] This is relevant, since PKC contributes to the activation of ERKs via Raf. Many different stimuli act on PC12 cells to induce signaling pathways that may converge in the sustained activation of the MAPK pathway. Since PKC is a family of more than 11 phospholipid-dependent serine/threonine protein kinases with variation in structure (26 – 28), there may be a difference in susceptibility to oxidants among the various isoenzymes

Objectives
Results
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call