AP-1 mediates β-amyloid-induced iNOS expression in PC12 cells via the ERK2 and p38 MAPK signaling pathways

https://doi.org/10.1016/j.bbrc.2005.04.057Get rights and content

Abstract

Nitrosative stress with subsequent inflammatory cell death has been implicated in some neurodegenerative disorders such as Alzheimer’s disease (AD). Expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO) have been frequently elevated in AD. In this study, we have investigated the molecular mechanisms underlying nitrosative stress induced by β-amyloid (Aβ), a neurotoxic peptide associated with senile plaques formed in the brains of patients with AD. Exposure of rat pheochromocytoma (PC12) cells to the Aβ resulted in increased mRNA and protein expression of iNOS and generation of NO. NO can rapidly interact with superoxide anion, forming more reactive peroxynitrite. Treatment of PC12 cells with Aβ led to increased peroxynitrite production and nitrotyrosine formation. Aβ induced activation of redox sensitive transcription factor activator protein-1 (AP-1), and AP-1 antisense oligonucleotide abolished the Aβ-induced iNOS expression. Moreover, Aβ transiently activated extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) via phosphorylation. Pharmacologic inhibition of both enzymes or dominant-negative mutation of ERK2 or p38 MAPK effectively down-regulated DNA binding as well as transcriptional activity of AP-1 and subsequent iNOS expression and NO production. The above findings suggest that Aβ induces iNOS expression in PC12 cells through activation of AP-1 which is regulated by upstream kinases, such as ERK and p38 MAPK.

Section snippets

Experimental procedures

Chemical and biochemical reagents. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, horse serum, F-12, and N-2 supplement were provided from Gibco (Grand Island, NY, USA). Aβ25–35 was supplied from Bachem (Torrance, CA, USA). Aβ25–35 was dissolved in deionized distilled water at a concentration of 1 mM and stored at −20 °C. The stock solution was diluted to desired concentrations immediately before use and added to culture medium without the aging procedure. We note that both fresh

25–35-mediated iNOS expression and NO production in PC12 cells

We have previously reported that Aβ25–35 induces apoptosis in PC12 cells by provoking oxidative stress [25], [26]. To explore the possible involvement of nitrosative stress as an alternative mechanism responsible for Aβ25–35-induced PC12 cell death, expression of iNOS was measured at protein and mRNA levels by Western blot analysis and RT-PCR, respectively. Significantly elevated iNOS protein expression was detected at 12 h after the Aβ25–35 treatment, which steadily increased up to 36 h after Aβ

Discussion

Aβ is the major component of senile plaques, which has been considered to play a causal role in the development and progress of AD. The molecular mechanisms underlying Aβ-mediated neurotoxicity still remain to be elucidated, but there is convincing evidence for the involvement of nitrosative stress caused by increased accumulation of reactive nitrogen species (RNS), such as NO and peroxynitrite [28], [29]. In this study, exposure of PC12 cells to Aβ25–35 resulted in elevated iNOS protein and

Acknowledgment

This work was supported by the Korea Research Foundation Grant ES0022.

References (42)

Cited by (0)

View full text