A Novel, Nerve Growth Factor-activated Pathway Involving Nitric Oxide, p53, and p21WAF1 Regulates Neuronal Differentiation of PC12 Cells

Wojciech Poluha,Christopher M Schonhoff,Kimberly S Harrington,Mahesh B Lachyankar,Nancy E Crosbie,Dylan A Bulseco,Alonzo H Ross

doi:10.1074/jbc.272.38.24002

Wojciech Poluha, Christopher M Schonhoff + Show 5 more

Open Access

https://doi.org/10.1074/jbc.272.38.24002

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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.

Highlights

Suppressor [4], and the p21WAF1 cyclin-dependent kinase inhibitor [5,6,7]
We found that the nitric oxide synthase (NOS) inhibitor, L-NAME, but not the inactive enantiomer D-NAME, inhibits nerve growth factor (NGF)-induced neurite extension by 73 Ϯ 15%, in agreement with Peunova and Enikolopov [3]
NGF binds to its receptors and induces expression of NOS, perhaps by activating mitogen-activated protein kinases [49] and the transcription factor, nuclear factor-␬B [50, 51], which are associated with NOS expression [52,53,54]

Summary

Introduction

Suppressor [4], and the p21WAF1 cyclin-dependent kinase inhibitor [5,6,7]. the relationship among these signaling events, arrest, and differentiation is poorly understood. NO is required for NGF-induced expression of two markers associated with neuronal differentiation. Using a PC12 line bearing an inducible expression vector for p21WAF1, we find that induction of recombinant p21WAF1 restores NGF-induced differentiation for cells treated with a NOS inhibitor.

Results

Conclusion