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Activation of transcription factor AP-1 by extracellular ATP in PC12 cells
Abstract:
We have previously shown that extracellular ATP caused cell death in PC12 cells through activation of its receptors. Oxidative stress has been implicated as a mechanism of cell death caused by extracellular ATP. In the present study we examined the possible signal transduction cascades leading to cell death by extracellular ATP. We found, using the electrophoretic mobility shift assay, that transcription factor AP-1 DNA binding activity was stimulated by extracellular ATP. Northern blot analysis showed that mRNA levels of c-fos, c-jun were elevated after treatment with ATP. The stimulation was receptor mediated, since it was blocked by the ATP receptor antagonist, suramin. The stimulated AP-1 binding was also blocked by the antioxidant N-acetyl-L-cysteine, indicating that reactive oxygen species generated following ATP stimulation were involved in the induction of AP-1 activity. It appears that both translational and posttranslational events contributed to the increased AP-1 DNA binding since cyclohexamide (a protein synthesis inhibitor), genistein (tyrosine kinase inhibitor) and staurosporine (PKC inhibitor) each partially blocked the AP-1 activation. Changes in AP-1 DNA binding activity may modulate expression of target genes involved in cell death pathways.
Insights
Extracellular ATP triggers cell death in PC12 cells by activating transcription factor AP-1. This process involves oxidative stress and receptor signaling, influencing cell death pathways.
Area of Science:
- Cell Biology
- Neuroscience
- Biochemistry
Background:
- Extracellular ATP is known to induce cell death in PC12 cells via receptor activation.
- Oxidative stress is a proposed mechanism underlying ATP-induced cell death.
Purpose of the Study:
- To investigate the signal transduction pathways involved in extracellular ATP-induced cell death.
- To elucidate the role of transcription factor AP-1 in this process.
Main Methods:
- Electrophoretic mobility shift assay (EMSA) to assess AP-1 DNA binding activity.
- Northern blot analysis to measure c-fos and c-jun mRNA levels.
- Utilized receptor antagonist (suramin), antioxidant (N-acetyl-L-cysteine), and inhibitors (cyclohexamide, genistein, staurosporine) to probe signaling pathways.
Main Results:
- Extracellular ATP significantly stimulated AP-1 DNA binding activity and increased c-fos and c-jun mRNA levels in PC12 cells.
- ATP-induced AP-1 activation was receptor-mediated, as confirmed by blockade with suramin.
- The antioxidant N-acetyl-L-cysteine blocked AP-1 activation, implicating reactive oxygen species.
- Inhibitors of protein synthesis, tyrosine kinase, and PKC partially blocked AP-1 activation, suggesting both translational and posttranslational regulation.
Conclusions:
- Extracellular ATP activates transcription factor AP-1 in PC12 cells through a receptor-dependent mechanism involving oxidative stress.
- Both translational and posttranslational modifications contribute to the enhanced AP-1 DNA binding activity.
- Modulation of AP-1 activity by extracellular ATP may play a critical role in regulating target genes within cell death pathways.