Immediate early gene expression in PC12 cells exposed to lead: Requirement for protein kinase C

We previously demonstrated induction of c-fos mRNA in PC12 cells exposed to lead that was dependent on new transcription. In the current work, we examined two signal transduction mechanisms that are activated by lead and have been shown to mediate induction of c-fos mRNA. One mechanism involves protein kinase C, and the other requires calmodulin-dependent protein kinase II. Significant increases in the levels of c-fos, c-jun, and egr-1 but not NGFIB mRNA were observed in PC12 cells exposed to lead or phorbol 12- myristate 13-acetate. In contrast, PC12 cells depolarized with 56 mM K⁺ displayed an increase in c-fos, egr-1, and NGFIB but not c-jun mRNA. Similar to other activators of protein kinase C, lead increased AP-1 and Egr-1 DNA binding activity. Additionally, lead increased luciferase activity in cerebellar granule cells transfected with an AP-1 luciferase reporter construct. Lead did not increase c-fos mRNA in PC12 cells that were depleted of protein kinase C by a 24-h treatment with phorbol 12,13-dibutyrate or incubated with the protein kinase C inhibitor H-7. In contrast, an inhibitor of calmodulin-dependent protein kinase, KN62, and an inhibitor of calmodulin, W-7, did not block the induction of c-fos mRNA by lead. An increase in serum- response element DNA-binding activity was observed in nuclear extracts from PC12 cells exposed to lead. It is interesting that lead activated protein kinase C isoforms δ and ε, but not isoforms α and β. In conclusion, lead appears to induce the expression of immediate early genes by a mechanism that requires protein kinase C.