NERVE GROWTH FACTOR-INDUCED DIFFERENTIATION OF PC12 CELLS EMPLOYS THEPMA-INSENSITIVE PROTEIN-KINASE C-ZETA ISOFORM

To elucidate the role of protein kinase C (PKC) in nerve growth factor (NGF)-induced differentiation, PMA downregulation of pheochromocytoma (PC12) cells was undertaken. Prolonged treatment (2 d) of PC12 cells with PMA (1 mu M) resulted in depleting the cells of alpha, beta, delt a, and epsilon-PKC isoforms, but had no effect on the expression of th e atypical PKC isoform xi. PC12 cells, which expressed only PKC xi, we re evaluated for their responses to NGF. Removal of the PMA-sensitive PKC isoforms enhanced the ability of NGF to promote neurite extension. Both the percentage cells with neurites and length of neurites were i ncreased in the PMA-treated cells, whereas no effect was observed on t he number of neurites per cell or branching of individual neurites. In addition, PMA downregulation resulted in an increase in the incorpora tion of H-3-thymidine without any significant effect on the expression of c-fos. Addition of NGF to PC12 cells depleted of the PMA-sensitive PKC isoforms resulted in the activation of PKC xi (Wooten et al., 199 4). To test whether the transient activation of PKC xi is a necessary component of the neuritogenetic pathway, antisense oligonucleotide str ategy was utilized to remove this particular PKC isoform. The addition of a 20-bp antisense oligonucleotide directed against the 5' coding s equence of PKC xi attenuated NGF-induced neurite outgrowth in PC12 cel ls lacking PMA-sensitive PKC isoforms. Sense oligonucleotide directed at the same site was without effect on NGF responses. These data indic ate that PKC xi comprises a portion of the NGF pathway and underscores the importance of this isoform in neuronal differentiation. Moreover, these findings demonstrate that the PMA-insensitive pathway, which wa s previously characterized as PKC-independent, and the neurite inducti on pathway are synonymous and mediated by PKC xi.