A novel, nerve growth factor-activated pathway involving nitric oxide, p53, and p21 super(WAF1) regulates neuronal differentiation of PC12 cells

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). The resulting nitric oxide (NO) acts as a second messenger, activating the p21 super(WAF1) promoter and inducing expression of p21 super(WAF1) cyclin-dependent kinase inhibitor. NO activates the p21 super(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 super(WAF1) promoter, expression of neuronal markers, and neurite extension. To determine whether p21 super(WAF1) is required for neurite extension, we prepared a PC12 line with an inducible p21 super(WAF1) expression vector. Blocking NOS with an inhibitor decreases neurite extension, but induction of p21 super(WAF1) with isopropyl-1-thio- beta -D-galactopyranoside restored this response. Levels of p21 super(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 super(WAF1) to block cell proliferation; and is required for neuronal differentiation by PC12 cells.