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Induction of CHOP and apoptosis by nitric oxide in p53-deficient microglial cells
K Kawahara1, S Oyadomari, T Gotoh
1Department of Molecular Genetics, Kumamoto University School of Medicine, Honjo, Japan.
Abstract:
Excessive nitric oxide (NO) has been implicated in neurotoxicity after stresses such as ischemia. NO toxicity is generally thought to be mediated by the DNA damage-p53 pathway or mitochondrial dysfunction. We investigated the mechanism of NO toxicity by using murine microglial MG5 cells established from p53-deficient mice. When MG5 cells were exposed to bacterial lipopolysaccharide plus interferon-gamma, mRNA and protein for inducible NO synthase (iNOS) were markedly induced, and apoptosis occurred. Under these conditions, we found that mRNA and protein for CHOP/GADD153, a C/EBP family transcription factor which is involved in endoplasmic reticulum (ER) stress-induced apoptosis, are induced. iNOS mRNA was induced 2 h after treatment, whereas CHOP mRNA began to increase at 6 h with a time lag. CHOP mRNA was also induced by NO donors S-nitroso-N-acetyl-DL-penicillamine (SNAP) or NOC18, or a peroxynitrite generator 3-(4-morpholinyl)-sydnonimine hydrochloride (SIN-1). Bip/GRP78, an ER chaperone which is known to be induced by ER stress, was also induced by SNAP or SIN-1, indicating that NO causes ER stress. These results suggest that NO-induced apoptosis in MG5 cells occurs through the ER stress pathway involving CHOP, but is independent of p53.
Insights
Excessive nitric oxide (NO) causes neurotoxicity via endoplasmic reticulum (ER) stress and CHOP induction, independent of the p53 pathway. This finding reveals a novel mechanism in NO-induced cell death.
Area of Science:
- Neuroscience
- Cell Biology
- Molecular Biology
Background:
- Excessive nitric oxide (NO) is linked to neurotoxicity following ischemic events.
- Current understanding suggests NO toxicity involves DNA damage-p53 or mitochondrial pathways.
- The precise molecular mechanisms underlying NO-induced neurotoxicity require further elucidation.
Purpose of the Study:
- To investigate the specific mechanism of nitric oxide (NO) toxicity in neuronal cells.
- To determine the role of the p53 pathway and endoplasmic reticulum (ER) stress in NO-induced apoptosis.
- To utilize p53-deficient murine microglial cells for mechanistic studies.
Main Methods:
- Utilized p53-deficient murine microglial MG5 cells.
- Stimulated cells with bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-γ).
- Assessed mRNA and protein expression of inducible NO synthase (iNOS), CHOP/GADD153, and ER chaperone Bip/GRP78.
- Administered NO donors (SNAP, NOC18) and a peroxynitrite generator (SIN-1) to evaluate NO-specific effects.
Main Results:
- LPS plus IFN-γ induced iNOS and apoptosis in MG5 cells.
- CHOP/GADD153, an ER stress-related transcription factor, was upregulated following NO induction.
- NO donors and peroxynitrite generators induced both CHOP/GADD153 and the ER chaperone Bip/GRP78.
- CHOP mRNA induction exhibited a time lag relative to iNOS mRNA induction.
Conclusions:
- Nitric oxide (NO) induces apoptosis in microglial cells through the endoplasmic reticulum (ER) stress pathway involving CHOP/GADD153.
- This NO-induced apoptotic pathway is independent of the p53 tumor suppressor protein.
- The findings highlight ER stress as a critical mediator of NO-induced neurotoxicity.