The Regulation of nNOS During Neuronal Differentiation and the Effect of Nitric Oxide on Hdm2-p53 Binding: a Dissertation

Abstract

Nitrc oxide is a ubiquitous signaling molecule with both physiological and pathological functions in biological systems. Formed by the enzmatic conversion of arginine to citrlline, NO, has known roles in circulatory, imune and nerous tissues. In the nerous system nitrc oxide has been implicated in long-ter potentiation neurotranmitter release, chanel function, neuronal protection and neuronal degeneration. Much of our work has focused on yet another role for nitrc oxide in cells namely, neuronal differentiation. Dung development, neuronal differentiation is closely coupled with cessation of proliferation. We use nere growth factor (NGF)-induced differentiation ofPC12 pheochromocytoma cells as a model and fid a novel signal tranduction pathway that blocks cell proliferation. Treatment ofPC12 cells with NGF leads to induction of nitric oxide synthase (NOS). The resulting nitrc oxide (NO) acs as a second messenger activating the p21(WAFl) promoter and inducing expression ofp21(WAFl) cyclindependent kiase inibitor. NO acivates the p21 (W AFl) promoter by p53-dependent and p53-independent mechanisms. Blocking production of NO with an inhibitor of NOS reduces accumulation ofp53 , activation ofthe p21(WAFl) promoter, expression of neuronal marker, and neurte extenion. To detere whether p2 1 (WAF 1) is required for neurite extension, we prepared a PC 12 line with an inducible p21 (W AFl) expression vector. Blocking NOS with an inhibitor decreases neurite extension, but induction of p21 (W AFl) with isopropylthio-betaD-galactopyranoside restored this response. Levels of p21 (W AFl) induced by isopropylthio-betaD-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 (W AFl) to block cell proliferation; and is required for neuronal differentiation by PC12 cells. In fuher studies of this pathway, we have examed the role of MA kinase pathways in neuronal nitric oxide synthase (nNOS) induction during the differentiation of PC12 cells. In NGF-treated PC12 cells, we find that nNOS is induced at RNA and protein levels, resulting in increased NOS activity. We note that neither nNOS mRA nNOS protein nor NOS activity is induced by NGF treatment in cells that have been infected with a dominant negative Ra adenovis. We have also used drgs that block MA kinase pathways and assessed their ability to inibit nNOS induction. Even though U0126 and PD98059 are both MEK inhibitors, we fmd that U0126, but not PD98059 blocks nNOS induction and NOS activity in NGF-treated PC12 cells. Also, the p38 kinase inibitor, SB 203580, does not block nNOS induction in our clone ofPC12 cells. Since the JNK pathway is not activated in NGF-treated PC12 cells, we deterne that the Ras-ERK pathway and not the p38 or JN pathway is required for nNOS induction in NGF-treated PC12 cells. We find that U0126 is much more effective than PD98059 in blocking the RaERK pathway, thereby explaining the discrepancy in nNOS inhibition. We conclude that the RasERK pathway is required for nNOS induction. The activation of soluble guanylate cyclase and the production of cyclic GMP is one of the best characterized modes of NO action. Havig shown that inhibition of NOS blocks PC12 cell differentiation we tested whether nitric oxide acts through soluble guanylate cyclase to lead to cell cycle arest and neuronal differentiation. Unlike NOS inibition, the inibition of soluble guanylate cy1case does not block the induction of neuronal markers. Moreover, treatent ofNGF-treated, NOS-inhibited PC12 cells with a soluble analog of cyclic GMP was unable to restore differentiation of those cells. Hence cGMP is not a component of this pathway and we had to consider other mechanisms of NO acion. It has become increasingly evident that another manner by which NO may exert its effects is by S-nitrosylation of cysteine residues. We tested, in vitro whether nitric oxide may control p53 by S-nitrosylation and inactivation of the p53 negative regulator Hdm. Treatment of Hdm with a nitric oxide donor inhibits Hdm2-p53 binding, the first step in Hdm regulation of p53. The presence of cysteine or DTT blocks this inhibition of binding. Moreover, nitric oxide inibition ofHdm-p53 binding was found to be reversible. Sulfhydrl-sensitivity and reversibility are consistent with nitrosylation. Finally, we have identified a crtical cysteine residue that nitrc oxide modifies in order disrupt Hdm-p53 binding. Mutation of this residue from a cysteine to an alanine does not interfere with binding but rather eliminates the sensitivity ofHdm to nitric oxide inactivation.