Inorganic lead activates the mitogen-activated protein kinase kinase-mitogen-activated protein kinase-p90^RSK signaling pathway in human astrocytoma cells via a protein kinase C-dependent mechanism

We have previously reported that lead acetate activates protein kinase Cα (PKCα) and induces DNA synthesis in human 1321 N1 astrocytoma cells. In this study, we investigated the ability of lead to activate the mitogen-activated protein kinase (MAPK) cascade. We found that exposure to lead acetate (1-50 μM) resulted in concentration- and time-dependent activation of MAPK (extracellular signal responsive kinase 1/2), as shown by increased phosphorylation and increased kinase activity. This effect was significantly reduced by the PKC-specific inhibitor bisindolylmaleimide (GF109203X), by down-regulation of PKC with 12-O-tetradecanoyl-phorbol 13-acetate, by a pseudosubstrate to PKCα, and by selective down-regulation of PKCα by prior lead exposure. Lead was also shown to activate MAPK kinase (MEK1/2), and this effect was mediated by PKC. Two MEK inhibitors, 2-(2′-amino-3′-methoxyphenol)-oxanaphthalen-4-one (PD98059) and 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (UO126), blocked lead-induced MAPK activation and inhibited lead-induced DNA synthesis, as measured by incorporation of [methyl-³H]thymidine into cell DNA. The 90 kDa ribosomal S6 protein kinase, p90^RSK, a substrate of MAPK, was also found to be activated by lead in a PKC- and MAPK-dependent manner. Stimulation of DNA synthesis by lead in astrocytoma cells may be of interest in light of the observed association between exposure to lead and an increased risk of astrocytomas.