3-Morpholinosydnonimine hydrochloride induces p53-dependent apoptosis in murine primary neural cells: a critical role for p21(ras)-MAPK-p19(ARF) pathway.

In some neurological disorders, excessive nitric oxide (NO, nitrogen monoxide) produced by inducible and/or neuronal nitric oxide synthases (iNOS and nNOS) is able to combine with superoxide (O(minus sign)(2)) to form peroxynitrite (ONOO(minus sign)), which can then induce p53-dependent neural apoptosis. In the present study, experiments using p53 knock-out mice primary neural cells revealed that 3-morpholinosydnonimine hydrochloride (SIN-1), a peroxynitrite donor, triggered apoptosis, while p53-transcriptional activity was effectively suppressed in the absence of p53 molecules. This shows that SIN-1 was able to induce p53-dependent apoptosis in murine primary neural cells. The mechanism responsible for the SIN-1-induced accumulation of p53 molecules was then analyzed. Western blot analysis indicated that p53 accumulation caused by SIN-1 did not require p53 phosphorylation, whereas SIN-1 treatment triggered MAP kinase (MAPK) phosphorylation and pretreatment with the MAP kinase kinase (MEK) inhibitor U0126 inhibited p53 accumulation. Pretreatment of the neural cells with lovastatin, an inhibitor of p21(ras) signaling, greatly inhibited the accumulation of p53 induced by SIN-1. Northern blot and immunofluorescence analyses revealed that primary neural cells treated with SIN-1 had increased levels of p19 alternate reading frame (p19(ARF)) mRNA and protein, which is induced by MAPK and stabilizes the p53 protein. Our findings clearly show that the p21(ras)-MAPK-p19(ARF) pathway has an essential role in p53-dependent apoptosis triggered by peroxynitrite in neural cells.