Neuronal nitric oxide synthase, as a downstream signaling molecule of c-jun, regulates the survival of differentiated PC12 cells

Abstract

The high expression of c-jun and neuronal nitric oxide synthase (nNOS) generally occurs in neurons following the generation of various animal models of central neuronal diseases. However, the mechanism between them in neuronal disease remains to be elucidated. Our previous studies demonstrated that the expression of c-jun always occurs prior to expression of nNOS in motoneuron injuries and suppression of c-jun expression by c-jun siRNA decreased nNOS expression in differentiated PC12 cells. The present study aimed to examine whether there was an association of up and downstream regulation or crosstalk between c-jun and nNOS in neurons. Using a culture of differentiated PC12 cells in vitro, the expression of nNOS and c-jun in cells was investigated by immunofluorescence. The nNOS inhibitor 7-nitroindazole (7-NI) was used in differentiated PC12 cells to downregulate the expression of nNOS. The optimal concentration of 7-NI on the viability and survival of cultured differentiated PC12 cells was selected using a 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium assay and the effects of 7-NI on the activity of constitutive nitric oxide synthase (cNOS) in differentiated PC12 cells were determined using a NOS Activity Detection kit. The effects of 7-NI on the gene expression of nNOS and c-jun were detected by western blot analysis. The results from the immunofluorescence demonstrated that the c-jun and nNOS protein were constantly expressed in PC12 cells. The cell viability of differentiated PC12 cells were significantly inhibited by treatment with 200 and 400 μmol/l 7-NI, and the expression levels of the nNOS protein were significantly inhibited by treatment with 200 μmol/l 7-NI. However, 7-NI had no significant effect on the protein expression level of c-jun and the total activities of cNOS. Based on our previous studies, which revealed that the nNOS gene was a downstream signaling molecule of the JNK/c-jun signaling pathway in cultured neurons, the expression of nNOS downstream was able to be regulated by c-jun which was the upstream molecule. Therefore, these results indicated that the association between them involved up and downregulation instead of crosstalk.