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Effect of a dominant inhibitory Ha-ras mutation on neuronal differentiation of PC12 cells
J Szeberényi1, H Cai, G M Cooper
1Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115.
Abstract:
A dominant inhibitory mutation of Ha-ras which changes Ser-17 to Asn-17 in the gene product p21 [p21 (Asn-17)Ha-ras] has been used to investigate the role of ras in neuronal differentiation of PC12 cells. The growth of PC12 cells, in contrast to NIH 3T3 cells, was not inhibited by p21(Asn-17)Ha-ras expression. However, PC12 cells expressing the mutant Ha-ras protein showed a marked inhibition of morphological differentiation induced by nerve growth factor (NGF) or fibroblast growth factor (FGF). These cells, however, were still able to respond with neurite outgrowth to dibutyryl cyclic AMP and 12-O-tetradecanoylphorbol-13-acetate (TPA). Induction of early-response genes (fos, jun, and zif268) by NGF and FGF but not by TPA was also inhibited by high levels of p21(Asn-17)Ha-ras. However, lower levels of p21(Asn-17) expression were sufficient to block neuronal differentiation without inhibiting induction of these early-response genes. Induction of the secondary-response genes SCG10 and transin by NGF, like morphological differentiation, was inhibited by low levels of p21(Asn-17) whether or not induction of early-response genes was blocked. Therefore, although inhibition of ras function can inhibit early-response gene induction, this is not required to block morphological differentiation or secondary-response gene expression. These results suggest that ras proteins are involved in at least two different pathways of signal transduction from the NGF receptor, which can be distinguished by differential sensitivity to p21(Asn-17)Ha-ras. In addition, ras and protein kinase C can apparently induce early-response gene expression by independent pathways in PC12 cells.
Insights
A dominant inhibitory mutation of Ha-ras (p21 Asn-17) blocked neuronal differentiation in PC12 cells without affecting growth. Ras proteins mediate distinct NGF signaling pathways, separable by p21 Asn-17 sensitivity.
Area of Science:
- Molecular Biology
- Cell Signaling
- Neuroscience
Background:
- Ras proteins are key regulators of cell signaling pathways.
- Neuronal differentiation in PC12 cells is a well-established model for studying signal transduction.
- The role of Ras in NGF-induced differentiation requires further elucidation.
Purpose of the Study:
- To investigate the role of Ras signaling in neuronal differentiation of PC12 cells using a dominant inhibitory Ha-ras mutant.
- To determine if Ras inhibition affects PC12 cell growth and differentiation induced by various stimuli.
- To dissect the involvement of Ras in early and secondary gene responses during NGF signaling.
Main Methods:
- Expression of a dominant inhibitory Ha-ras mutant (p21 Asn-17) in PC12 cells.
- Assessment of cell proliferation and morphological differentiation.
- Analysis of gene induction by nerve growth factor (NGF), fibroblast growth factor (FGF), dibutyryl cyclic AMP, and 12-O-tetradecanoylphorbol-13-acetate (TPA).
Main Results:
- PC12 cell growth was not inhibited by p21 Asn-17, unlike NIH 3T3 cells.
- p21 Asn-17 expression markedly inhibited NGF- and FGF-induced morphological differentiation.
- Neurite outgrowth induced by cyclic AMP and TPA remained unaffected.
- NGF/FGF-induced early-response gene (fos, jun, zif268) induction was inhibited by high p21 Asn-17 levels.
- Lower p21 Asn-17 levels blocked differentiation without affecting early-response genes.
- Secondary-response gene (SCG10, transin) induction by NGF was inhibited by low p21 Asn-17 levels, independent of early-response gene induction.
Conclusions:
- Ras proteins are involved in distinct NGF receptor signal transduction pathways with differential sensitivity to p21 Asn-17.
- Inhibition of Ras is not required for blocking morphological differentiation or secondary-response gene expression.
- Ras and protein kinase C can independently induce early-response gene expression in PC12 cells.