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Protein kinase inhibitor H-7 differentially affects early and delayed nerve growth factor responses in PC12 cells
P Kahle1, M Mangold, T Kuwahara
1Pharma Division, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
Abstract:
The effects of the protein kinase inhibitor H-7 on early and delayed responses to nerve growth factor (NGF) were investigated in PC12 cells. H-7 reduced the NGF-induced expression of c-Fos in a dose-dependent manner without affecting the time course of c-Fos appearance. Conversely, H-7 potentiated delayed NGF effects, i.e., neurite outgrowth and Ca2+/phospholipid-dependent protein kinase (PKC) induction, but not choline acetyltransferase induction. Long-term treatment with NGF resulted in an increase of at least four tyrosine-phosphorylated protein bands with molecular masses between 39 and 48 kDa, which was also potentiated by H-7. In the absence of NGF, H-7 had no significant effect on c-Fos expression, tyrosine phosphorylation of the 45 kDa protein, or choline acetyltransferase activity. However, 4 days of exposure to H-7 alone induced PKC activity and tyrosine phosphorylation of the 39-kDa protein. The action of H-7 derivatives on neurite outgrowth did not correlate with their inhibition profile of cyclic nucleotide-dependent protein kinases. Down-regulation of PKC activity by prolonged exposure to phorbol ester did not completely abolish the effects of NGF and H-7 on induction of c-Fos, choline acetyltransferase activity, and neurite outgrowth, indicating that PKC-independent pathways contribute to these actions. These results suggest that additional pathway(s) sensitive to H-7 may exist, which induce immediate early gene expression and suppress neuronal differentiation of PC12 cells.
Insights
The protein kinase inhibitor H-7 affects nerve growth factor (NGF) responses in PC12 cells. H-7 inhibits early c-Fos expression but enhances delayed neurite outgrowth, suggesting complex signaling pathways.
Area of Science:
- Neuroscience
- Cell Biology
- Molecular Biology
Background:
- Nerve growth factor (NGF) is crucial for neuronal development and differentiation.
- PC12 cells are a widely used model system for studying NGF signaling.
- Protein kinase inhibitors offer insights into cellular signaling pathways.
Purpose of the Study:
- To investigate the effects of the protein kinase inhibitor H-7 on early and delayed responses to NGF in PC12 cells.
- To elucidate the role of H-7 in NGF-induced gene expression, protein phosphorylation, and neurite outgrowth.
- To explore the involvement of protein kinase C (PKC)-dependent and -independent pathways in these cellular processes.
Main Methods:
- PC12 cells were treated with NGF and/or H-7.
- Expression of c-Fos was analyzed.
- Neurite outgrowth was measured.
- Protein kinase C (PKC) activity and tyrosine phosphorylation were assessed.
- Choline acetyltransferase activity was determined.
Main Results:
- H-7 reduced NGF-induced c-Fos expression in a dose-dependent manner.
- H-7 potentiated delayed NGF effects, including neurite outgrowth and PKC induction.
- NGF treatment increased tyrosine phosphorylation of specific proteins, an effect potentiated by H-7.
- H-7 alone induced PKC activity and tyrosine phosphorylation.
- PKC-independent pathways contribute to NGF and H-7 actions.
Conclusions:
- H-7 differentially affects early and delayed NGF responses in PC12 cells.
- PKC-independent pathways play a significant role in NGF-mediated cellular processes.
- Additional H-7-sensitive pathways may regulate immediate early gene expression and neuronal differentiation.