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Cyclooxygenase-2 is a neuronal target gene of NF-kappaB
Barbara Kaltschmidt1, Ralf A Linker, Jinbo Deng
1Institute of Neurobiochemistry University of Witten/Herdecke, Stockumer Str, 10, D-58448 Witten, Germany. c.kaltschmidt@uni-wh.de
Background:
NF-kappaB is implicated in gene regulation involved in neuronal survival, inflammmatory response and cancer. There are relatively few neuronal target genes of NF-kappaB characterized.
Results:
We have identified the neuronal cyclooxygenase-2 (COX-2) as a NF-kappaB target gene. In organotypic hippocampal slice cultures constitutive NF-kappaB activity was detected, which was correlated with high anti-COX-2 immunoreactivity. Aspirin a frequently used painkiller inhibits neuronal NF-kappaB activity in organotypic cultures resulting in a strong inhibition of the NF-kappaB target gene COX-2. Based on these findings, the transcriptional regulation of COX-2 by NF-kappaB was investigated. Transient transfections showed a significant increase of COX-2 promoter activity upon stimulation with PMA, an effect which could be obtained also by cotransfection of the NF-kappaB subunits p65 and p50. In the murine neuroblastoma cell line NB-4, which is characterized by constitutive NF-kappaB activity, COX-2 promoter activity could not be further increased with PMA or TNF. Constitutive promoter activity could be repressed upon cotransfection of the inhibitory subunit IkappaB-alpha. EMSA and mutational analysis conferred the regulatory NF-kappaB activity to the promoter distal kappaB-site in the human COX-2 promoter.
Conclusions:
NF-kappaB regulates neuronal COX-2 gene expression, and acts as an upstream target of Aspirin. This extends Aspirin's mode of action from a covalent modification of COX-2 to the upstream regulation of COX-2 gene expression in neurons.
Insights
Nuclear factor-kappa B (NF-kappaB) regulates neuronal cyclooxygenase-2 (COX-2) gene expression. Aspirin inhibits NF-kappaB activity, impacting COX-2 expression, extending its known anti-inflammatory mechanism.
Area of Science:
- Neuroscience
- Molecular Biology
- Pharmacology
Background:
- Nuclear factor-kappa B (NF-kappaB) plays a role in neuronal survival, inflammation, and cancer.
- Few neuronal target genes of NF-kappaB have been identified.
Purpose of the Study:
- To identify neuronal target genes of NF-kappaB.
- To investigate the role of NF-kappaB in regulating cyclooxygenase-2 (COX-2) gene expression in neurons.
- To explore Aspirin's effect on NF-kappaB and COX-2 regulation.
Main Methods:
- Organotypic hippocampal slice cultures to assess NF-kappaB activity and COX-2 immunoreactivity.
- Transient transfection assays to analyze COX-2 promoter activity.
- Electrophoretic mobility shift assay (EMSA) and mutational analysis to identify regulatory elements.
Main Results:
- Neuronal cyclooxygenase-2 (COX-2) was identified as an NF-kappaB target gene.
- Constitutive NF-kappaB activity correlated with high COX-2 expression in hippocampal slices.
- Aspirin inhibited neuronal NF-kappaB activity and subsequent COX-2 expression.
- NF-kappaB subunits p65 and p50 increased COX-2 promoter activity.
- The distal kappaB-site in the human COX-2 promoter was identified as the regulatory element.
Conclusions:
- NF-kappaB directly regulates neuronal COX-2 gene expression.
- Aspirin acts upstream of COX-2 by inhibiting NF-kappaB activity.
- This study expands the understanding of Aspirin's mechanism of action beyond direct COX-2 inhibition to include upstream gene regulation.