A direct redox regulation of protein kinase C isoenzymes mediates oxidant-induced neuritogenesis in PC12 cells.
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.
Gopalakrishna
Rayudu
R
Department of Cell and Neurobiology, University of Southern California, Los Angeles, CA 90089, USA. rgopalak@usc.edu
Gundimeda
Usha
U
Schiffman
Jason Eric
JE
McNeill
Thomas H
TH
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects*; D002614:Chelating Agents / Q000494:pharmacology; D003035:Cobalt / Q000494:pharmacology; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004789:Enzyme Activation; D005978:Glutathione / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D020935:MAP Kinase Signaling System; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D016501:Neurites / Q000187:drug effects / Q000201:enzymology; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D016877:Oxidants / Q000494:pharmacology*; D010084:Oxidation-Reduction; D016716:PC12 Cells; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology
D000975:Antioxidants; D002614:Chelating Agents; D017362:Cyclic AMP Response Element-Binding Protein; D007527:Isoenzymes; D016877:Oxidants; D047428:Protein Kinase Inhibitors; D013438:Sulfhydryl Compounds; D003035:Cobalt; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C018021:cobaltous chloride; D005978:Glutathione; D015032:Zinc