Selective activation of the c-Jun N-terminal protein kinase pathway during 4-hydroxynonenal-induced apoptosis of PC12
1Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, Rockville, Maryland 20852, USA.
Abstract:
The by-product of lipid peroxidation, 4-hydroxynonenal (HNE), was shown to cause apoptosis in PC12 cells. In this study, we investigated the molecular mechanism of HNE-induced apoptosis in these cells. Specifically, we determined the effect of HNE on the activities of mitogen-activated protein (MAP) kinases involved in early signal transduction. Within 15 to 30 min after HNE treatment, c-Jun N-terminal protein kinase (JNK) was maximally activated, before it returned to control level at 1 h post-treatment. In contrast, activities of extracellular signal-regulated kinase and p38 MAP kinase remained unchanged from their baseline levels. Stress-activated protein kinase kinase (SEK1), an upstream kinase of JNK, was also activated within 5 min after HNE treatment and remained activated for up to 60 min. Marked activation of the JNK pathway through SEK1 and apoptosis signal-regulating kinase 1 (ASK1), an upstream kinase of SEK1, was demonstrated by the transient transfection of cDNA for wild-type SEK1 or ASK1 together with JNK into COS-7 cells. Furthermore, significant reductions in JNK activation and HNE-induced cell death were observed when either of the dominant negative mutant of SEK1 or ASK1 was cotransfected with JNK. Pretreatment of PC12 cells with a survival-promoting agent, 8-(4-chlorophenylthio)-cAMP, prevented both the HNE-induced JNK activation and apoptosis. Nonaldehyde, a nontoxic aldehyde, neither caused apoptosis nor JNK activation. Pretreatment of PC12 cells with SB203580, a specific inhibitor of p38 MAP kinase, had no effect on HNE-induced apoptosis. All these data suggest that the selective JNK activation by HNE is critical for the apoptosis of PC12 cells and that the HNE-mediated apoptosis is likely to be mediated through the activation of the ASK1-SEK1-JNK pathway without activation of extracellular signal-regulated kinase or p38 MAP kinase.
Insights
4-hydroxynonenal (HNE), a lipid peroxidation by-product, triggers apoptosis in PC12 cells by selectively activating the c-Jun N-terminal protein kinase (JNK) pathway. This HNE-induced cell death is mediated via the ASK1-SEK1-JNK signaling cascade.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Lipid peroxidation by-product 4-hydroxynonenal (HNE) is known to induce apoptosis in PC12 cells.
- The precise molecular mechanisms underlying HNE-induced apoptosis, particularly the involvement of mitogen-activated protein (MAP) kinases, require further elucidation.
Purpose of the Study:
- To investigate the molecular mechanisms of HNE-induced apoptosis in PC12 cells.
- To determine the specific role of MAP kinases, including c-Jun N-terminal protein kinase (JNK), in early signal transduction pathways following HNE exposure.
Main Methods:
- PC12 cells were treated with HNE, and the activation of MAP kinases (JNK, ERK, p38) and their upstream kinases (SEK1, ASK1) was assessed over time.
- Transient transfection assays using wild-type and dominant-negative mutants of SEK1 and ASK1, along with JNK, were performed in COS-7 cells to delineate pathway activation.
- The effects of a survival-promoting agent (8-(4-chlorophenylthio)-cAMP) and a p38 inhibitor (SB203580) on HNE-induced apoptosis and JNK activation were evaluated.
Main Results:
- HNE treatment led to rapid and transient activation of JNK and its upstream kinase SEK1, with maximal JNK activation observed between 15-30 minutes.
- Apoptosis signal-regulating kinase 1 (ASK1) and SEK1 were identified as key upstream activators of JNK in response to HNE, as demonstrated by transfection studies.
- Inhibition of the ASK1-SEK1-JNK pathway using dominant-negative mutants significantly reduced both JNK activation and HNE-induced cell death, while ERK and p38 MAP kinase activities remained unaffected.
Conclusions:
- Selective activation of the JNK pathway, mediated by the ASK1-SEK1 cascade, is critical for HNE-induced apoptosis in PC12 cells.
- The findings highlight a specific signaling pathway involved in oxidative stress-induced cell death, distinct from the activation of ERK or p38 MAP kinases.
- Targeting the ASK1-SEK1-JNK pathway may offer a therapeutic strategy for conditions involving HNE-mediated cellular damage.
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