Suppression of ischemia-induced fos expression and AP-1 activity by an antisense oligodeoxynucleotide to c-fos mRNA
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1Division of Restorative Neurology and Human Neurobiology, Baylor College of Medicine, Houston, TX 77030.
Abstract:
Activation of c-fos, an immediate early gene, and the subsequent expression of the Fos protein have been noted following focal cerebral ischemia. Fos and Jun form a heterodimer as activator protein 1 (AP-1), which transregulates the expression of several genes. To study the postischemic events related to c-fos expression, we suppressed the expression of c-fos by intraventricular infusion of an antisense oligodeoxynucleotide (anti-rncfosr115) of c-fos mRNA. The effectiveness of anti-rncfosr115 was confirmed first by its capability to block in vitro c-fos mRNA translation. In vivo, after intraventricular infusion of 32P-labeled anti-rncfosr115, the oligodeoxynucleotide was internalized within 6 hours and detectable also in the nucleic acids fraction up to 41 hours. Treatment of the recovered nucleic acids with RNase H separated the labeled oligodeoxynucleotide from the nucleic acid fraction, indicating an association of the antisense oligodeoxynucleotide and cellular RNA after uptake. When focal cerebral ischemia was induced 16 hours after the infusion of anti-rncfosr115, the postischemic increase in Fos expression and AP-1 binding activity were suppressed. Specificity of the effect of anti-rncfosr115 was suggested by its failure to suppress the DNA binding activity of nuclear cyclic AMP response elements. These results support the hypothesis that increased AP-1 binding activity following focal cerebral ischemia is dependent on Fos expression and can be inhibited in vivo by antisense c-fos oligodeoxynucleotides.
Insights
Researchers inhibited c-fos expression in focal cerebral ischemia using antisense oligodeoxynucleotides. This suppression reduced Fos protein and activator protein 1 (AP-1) activity, demonstrating a targeted approach to post-ischemic gene regulation.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- Focal cerebral ischemia triggers c-fos immediate early gene activation and Fos protein expression.
- Fos and Jun proteins form the activator protein 1 (AP-1) complex, regulating gene expression.
- Understanding post-ischemic c-fos expression is crucial for therapeutic interventions.
Purpose of the Study:
- To investigate the role of c-fos expression in post-ischemic events.
- To suppress c-fos expression in vivo using antisense oligodeoxynucleotides.
- To evaluate the effect of c-fos suppression on AP-1 activity following focal cerebral ischemia.
Main Methods:
- Intraventricular infusion of an antisense oligodeoxynucleotide (anti-rncfosr115) targeting c-fos mRNA.
- In vitro confirmation of anti-rncfosr115 efficacy in blocking c-fos mRNA translation.
- In vivo assessment of oligodeoxynucleotide uptake, stability, and association with cellular RNA using labeled compounds and RNase H.
- Induction of focal cerebral ischemia and measurement of Fos expression and AP-1 binding activity.
Main Results:
- The antisense oligodeoxynucleotide (anti-rncfosr115) was effectively internalized and remained detectable for up to 41 hours, associating with cellular RNA.
- Infusion of anti-rncfosr115 significantly suppressed the post-ischemic increase in Fos expression and AP-1 binding activity.
- The suppression was specific, as the DNA binding activity of nuclear cyclic AMP response elements remained unaffected.
Conclusions:
- Increased AP-1 binding activity following focal cerebral ischemia is dependent on Fos expression.
- Antisense c-fos oligodeoxynucleotides can effectively inhibit Fos expression and subsequent AP-1 activity in vivo.
- This study provides a foundation for using antisense technology to modulate gene expression in ischemic stroke.