Participation of protein kinase C α isoform and extracellular signal-regulated kinase in neurite outgrowth of GT1 hypothalamic neurons

Abstract

In the present study, we investigated the selective role of protein kinase C (PKC) isoforms on neurite outgrowth of the GT1 hypothalamic neurons using several PKC isoform-selective inhibitors and transfection-based expression of enhanced green fluorescence protein (EGFP)-fused PKC isoforms. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced neurite outgrowth and growth cone formation, effects that were blocked by GF 109203X (a PKC inhibitor), safingol™ (a PKCα-selective inhibitor), but not by rottlerin™ (a PKCδ-selective inhibitor), indicating that PKCα may be selectively involved in neurite outgrowth and cytoskeletal changes of filamentous actin and β-tubulin. To define the differential localization of PKC isoforms, EGFP-tagged PKCα PKCγ, and PKCδ were transfected into GT1 neuronal cells. TPA treatment induced relocalization of PKCα-EGFP to growth cones and cell-cell adhesion sites, PKCγ-EGFP to the nucleus, and PKCδ-EGFP to the membrane ruffle, respectively. An EGFP chimera of the catalytic domain of PKCα (PKCα-Cat-EGFP), the expression of which was inducible by doxycycline, was employed to directly ascertain the effect of PKCα enzymatic activity on neurite outgrowth of GT1 cells. Transient transfection of PKCα-Cat-EGFP alone increased the neurite-outgrowth and doxycycline treatment further augmented the number of neurite-containing cells. We also examined the involvement of the extracellular signal-regulated kinase (ERK) MAP kinase in TPA-induced neurite outgrowth. TPA treatment increased phosphorylated ERK MAP kinase, but not p38 MAP kinase. Specific inhibition of PKCα with safingol blocked the phosphorylation of ERK induced by TPA. More importantly, both neurite outgrowth and phosphorylation of ERK by TPA were blocked by PD 098059, a specific inhibitor of MEK (MAP kinase/ERK kinase-1), but not by SB203580, a specific inhibitor of p38 MAP kinase. These results demonstrate that PKCα isoform-specific activation is involved in neurite outgrowth of GT1 hypothalamic neuronal cells via ERK, but not the p38 MAP kinase signal pathway.