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A novel, nerve growth factor-activated pathway involving nitric oxide, p53, and p21WAF1 regulates neuronal
W Poluha1, C M Schonhoff, K S Harrington
1Worcester Foundation for Biomedical Research, Shrewsbury, Massachusetts 01545, USA.
Abstract:
During development, neuronal differentiation is closely coupled with cessation of proliferation. We use nerve growth factor (NGF)-induced differentiation of PC12 pheochromocytoma cells as a model and find a novel signal transduction pathway that blocks cell proliferation. Treatment of PC12 cells with NGF leads to induction of nitric oxide synthase (NOS) (Peunova, N., and Enikolopov, G. (1995) Nature 375, 68-73). The resulting nitric oxide (NO) acts as a second messenger, activating the p21(WAF1) promoter and inducing expression of p21(WAF1) cyclin-dependent kinase inhibitor. NO activates the p21(WAF1) promoter by p53-dependent and p53-independent mechanisms. Blocking production of NO with an inhibitor of NOS reduces accumulation of p53, activation of the p21(WAF1) promoter, expression of neuronal markers, and neurite extension. To determine whether p21(WAF1) is required for neurite extension, we prepared a PC12 line with an inducible p21(WAF1) expression vector. Blocking NOS with an inhibitor decreases neurite extension, but induction of p21(WAF1) with isopropyl-1-thio-beta-D-galactopyranoside restored this response. Levels of p21(WAF1) induced by isopropyl-1-thio-beta-D-galactopyranoside were similar to those induced by NGF. Therefore, we have identified a signal transduction pathway that is activated by NGF; proceeds through NOS, p53, and p21(WAF1) to block cell proliferation; and is required for neuronal differentiation by PC12 cells.
Insights
Nerve growth factor (NGF) triggers a pathway involving nitric oxide (NO), p53, and p21(WAF1) to halt cell division and promote neuronal differentiation in PC12 cells.
Area of Science:
- Neuroscience
- Cell Biology
- Molecular Biology
Background:
- Neuronal differentiation typically involves the cessation of cell proliferation.
- PC12 pheochromocytoma cells are a common model for studying neuronal differentiation.
- Nerve growth factor (NGF) is a key regulator of neuronal development.
Purpose of the Study:
- To elucidate the novel signal transduction pathway linking NGF-induced differentiation with proliferation arrest in PC12 cells.
- To investigate the role of nitric oxide (NO) and p21(WAF1) in this process.
Main Methods:
- NGF treatment of PC12 cells.
- Inhibition of nitric oxide synthase (NOS) to block NO production.
- Analysis of p53 accumulation and p21(WAF1) promoter activation.
- Assessment of neuronal marker expression and neurite extension.
- Use of a PC12 cell line with an inducible p21(WAF1) expression vector.
Main Results:
- NGF induces NOS, leading to nitric oxide (NO) production.
- NO activates the p21(WAF1) promoter via both p53-dependent and p53-independent mechanisms.
- Inhibition of NOS reduces p53 levels, p21(WAF1) activation, neuronal marker expression, and neurite outgrowth.
- Induction of p21(WAF1) in the absence of NGF can restore neurite extension, suggesting its necessity.
Conclusions:
- A novel NGF-activated signaling pathway involving NOS, p53, and p21(WAF1) has been identified.
- This pathway is crucial for blocking proliferation and enabling neuronal differentiation in PC12 cells.
- p21(WAF1) acts as a key mediator in NGF-induced neuronal differentiation.