Protein kinase C-epsilon is implicated in neurite outgrowth in differentiating human neuroblastoma cells.
A combination of basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I) or 16 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) and serum induces human SH-SY5Y neuroblastoma cells to undergo differentiation and acquire a neuronal phenotype. Nerve growth factor (NGF) added to SH-SY5Y cells stably transfected with the NGF-receptor TRK-A (SH-SY5Y/trk) induces a similar differentiated phenotype. SH-SY5Y cells express protein kinase C (PKC)-alpha, PKC-beta I, PKC-epsilon, and PKC-zeta protein, and phorbol ester- or growth factor-induced differentiation results in a sustained activation of PKC. The specific PKC inhibitor GF 109203X blocked TPA- and bFGF-IGF-I-induced neurite outgrowth in wild-type SH-SY5Y cells and NGF-induced neurite outgrowth in SH-SY5Y/trk cells. When added to differentiated cells, GF 109203X caused rapid retraction of growth cone filopodia. In TPA- and bFGF-IGF-I-treated cells, addition of GF 109203X also blocked induced expression of growth associated protein-43 and neuropeptide tyrosine while the increase in expression of these two genes was only slightly affected by the inhibitor in NGF-treated SH-SY5Y/trk cells. Thus, a portion of the NGF-induced phenotypic changes appears not to be mediated via PKC-dependent signaling. A high concentration of TPA (1.6 microM) down regulated PKC-alpha and PKC-beta I almost completely and PKC-epsilon partially in wild-type SH-SY5Y and SH-SY5Y/trk cells. Cells with down-regulated PKC-alpha and PKC-beta I after 1.6 microM TPA treatment still differentiated with growth factors. In these cells, the PKC-epsilon level was restored, and the PKC-epsilon protein was enriched in the growth cones. The 1.6 microM TPA-induced down-regulation of PKC-epsilon was counteracted by bFGF and NGF but not by platelet-derived growth factor or IGF-I. These data indicate that PKC activity is vital for neurite formation, and that the cells can differentiate under conditions when PKC-alpha and PKC-beta I are extensively down regulated. The close correlation between differentiation and presence of PKC-epsilon protein suggests an important function for this isoform during this process.
Fagerström
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Department of Pathology, University Hospital, Uppsala, Sweden.
Påhlman
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Gestblom
C
C
Nånberg
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E
D002454:Cell Differentiation / Q000502:physiology; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000502:physiology*; D008301:Maleimides / Q000494:pharmacology; D016501:Neurites / Q000187:drug effects*; D009447:Neuroblastoma; D009474:Neurons / Q000166:cytology / Q000201:enzymology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects
D004791:Enzyme Inhibitors; D006133:Growth Substances; D007211:Indoles; D007527:Isoenzymes; D008301:Maleimides; C496628:PRKCA protein, human; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate