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Cycloheximide induces nitric oxide synthase mRNA in vascular smooth muscle cells by prolonging mRNA lifetime
1Department of Endcrinology, Dokkyo University School of Medicine, Tochigi, Japan.
Abstract:
Bacterial lipopolysaccharide (LPS) and other immunostimulants induce an isoform of NO synthase (iNOS) in vascular smooth muscle (VSM) which produces large quantities of NO and profound vasodilation; this process has been implicated as the cause of gram-negative septic shock. Although regulation of iNOS has been considered to occur at the level of transcription, it is unclear whether post-transcriptional events also contribute to changes in iNOS mRNA expression. We show that cycloheximide (CH), an inhibitor of protein synthesis, induces iNOS mRNA in VSM and potentiates the induction of iNOS mRNA caused by LPS. For many early-response genes, protein-synthesis inhibitors enhance mRNA levels by increasing mRNA stability. Since iNOS mRNA contains multiple copies of a consensus sequence (AUUUA) in common with these eary-response genes and responsible for mRNA destabilization, we tested whether CH induces iNOS mRNA by prolonging mRNA lifetime. In the absence of CH, iNOS mRNA decayed with biphagic kinetics and a half-life of 2 h. In the presence of CH, half-life was prolonged to approximately 7 h. Our observations indicate that in VSM the stability of iNOS mRNA may be under the control of a labile protein factor which awaits identification and characterization. Regulation of mRNA lifetime represents a novel site for control of iNOS gene expression.
Insights
Bacterial lipopolysaccharide (LPS) induces inducible nitric oxide synthase (iNOS) mRNA in vascular smooth muscle (VSM). Protein synthesis inhibition prolongs iNOS mRNA stability, suggesting post-transcriptional regulation is key in septic shock.
Area of Science:
- Molecular Biology
- Physiology
- Immunology
Background:
- Bacterial lipopolysaccharide (LPS) and immunostimulants induce inducible nitric oxide synthase (iNOS) in vascular smooth muscle (VSM).
- This iNOS induction leads to nitric oxide (NO) production, causing vasodilation implicated in gram-negative septic shock.
- Regulation of iNOS is primarily considered transcriptional, but post-transcriptional control remains unclear.
Purpose of the Study:
- To investigate the role of post-transcriptional regulation in iNOS mRNA expression in VSM.
- To determine if protein synthesis inhibition affects iNOS mRNA stability.
- To elucidate novel regulatory mechanisms of iNOS gene expression.
Main Methods:
- Treatment of VSM cells with cycloheximide (CH), a protein synthesis inhibitor.
- Treatment of VSM cells with LPS.
- Measurement of iNOS mRNA levels and decay kinetics in the presence and absence of CH.
Main Results:
- Cycloheximide (CH) alone induced iNOS mRNA in VSM.
- CH potentiated LPS-induced iNOS mRNA levels.
- In the absence of CH, iNOS mRNA had a half-life of 2 hours; in the presence of CH, the half-life extended to approximately 7 hours.
- iNOS mRNA contains AUUUA sequences common to destabilized early-response genes.
Conclusions:
- iNOS mRNA stability is regulated by a labile protein factor in VSM.
- Post-transcriptional regulation, specifically mRNA stability, is a significant mechanism controlling iNOS gene expression.
- This finding offers a novel target for modulating iNOS expression in conditions like septic shock.