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

doi:10.1074/jbc.272.38.24002

A Novel, Nerve Growth Factor-activated Pathway Involving Nitric Oxide, p53, and p21^WAF1 Regulates Neuronal Differentiation of PC12 Cells*

From the Worcester Foundation for Biomedical Research, Shrewsbury, Massachusetts 01545

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-β-d-galactopyranoside restored this response. Levels of p21^WAF1 induced by isopropyl-1-thio-β-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.

Footnotes

↵* This work was supported in part by National Institutes of Health Grants NS-21716 and NS-28760 and by the Stork Foundation and the Fortin Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ Postdoctoral Fellow for National Institutes of Health Training Grant NS-07366.
↵§ To whom correspondence should be addressed: Worcester Foundation for Biomedical Research, 222 Maple Ave., Shrewsbury, MA 01545. Tel.: 508-842-8921; Fax: 508-842-9632; E-mail: Ross{at}sci.wfbr.edu.
↵1 The abbreviations used are: NGF, nerve growth factor; NO, nitric oxide; NOS, nitric oxide synthase; nNOS, eNOS, iNOS, neuronal, endothelial, and inducible nitric oxide synthase, respectively; l-NAME,N-nitro-l-arginine methyl ester; IPTG, isopropyl 1-thio-β-d-galactopyranoside; nAChR, nicotinic acetylcholine receptor; MAP1B, microtubule-associated protein 1B; CDTA,trans-1,2-diaminocyclohexaneN,N,N′,N′-tetraacetic acid; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
↵2 D. A. Bulseco and A. H. Ross, unpublished data.
- Received February 12, 1997.
- Revision received June 18, 1997.