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Tumor necrosis factor downregulates an endothelial nitric oxide synthase mRNA by shortening its half-life
M Yoshizumi1, M A Perrella, J C Burnett
1Cardiovascular Biology Laboratory, Harvard School of Public Health, Boston, MA 02115.
Abstract:
Nitric oxide (NO), which accounts for the biological properties of endothelium-derived relaxing factor, is generated by NO synthase (NOS). The vascular endothelium contains two types of NOS: one is constitutively expressed (cNOS), and the other is inducible. Endothelium-mediated vasorelaxation is impaired in atherosclerotic vessels. To determine whether tumor necrosis factor (TNF)-alpha, which is commonly found in atherosclerotic lesions, has an effect on NOS message, we measured cNOS mRNA levels in TNF-treated human umbilical vein endothelial cells (HUVECs) by RNA blot analysis with a cNOS cDNA probe. TNF-alpha markedly reduced cNOS mRNA levels in HUVECs in a dose- and time-dependent manner. In response to 3 ng/mL TNF-alpha, cNOS mRNA levels began to decrease at 4 hours and diminished to only 5% of control levels at 24 hours. As little as 0.1 ng/mL TNF-alpha reduced cNOS mRNA levels by 50%. This reduction in cNOS message in response to TNF-alpha depended on protein synthesis as it was blocked by cycloheximide. In nuclear runoff experiments, TNF-alpha did not change the rate of cNOS gene transcription. cNOS mRNA is very stable under basal conditions, with a half-life of 48 hours; however, treatment with TNF-alpha shortened this half-life to 3 hours. TNF-alpha thus appears to decrease cNOS mRNA levels by increasing the rate of mRNA degradation. TNF-induced reductions in cNOS mRNA levels may have an important effect on impaired endothelium-mediated vasorelaxation in atherosclerosis.
Insights
Tumor necrosis factor-alpha (TNF-alpha) significantly decreases nitric oxide synthase (NOS) message in endothelial cells. This reduction in NOS mRNA, caused by increased degradation, may impair blood vessel relaxation in atherosclerosis.
Area of Science:
- Molecular Biology
- Cardiovascular Research
- Endothelial Cell Biology
Background:
- Nitric oxide (NO) is crucial for endothelium-derived relaxing factor activity, generated by NO synthase (NOS).
- Endothelial NOS (cNOS) is constitutively expressed in vascular endothelium.
- Impaired endothelium-mediated vasorelaxation is characteristic of atherosclerotic vessels.
Purpose of the Study:
- To investigate the effect of tumor necrosis factor-alpha (TNF-alpha) on cNOS mRNA levels in human umbilical vein endothelial cells (HUVECs).
- To elucidate the mechanism by which TNF-alpha influences cNOS mRNA expression.
Main Methods:
- RNA blot analysis was used to quantify cNOS mRNA levels in HUVECs treated with varying concentrations and durations of TNF-alpha.
- Nuclear runoff assays were performed to assess cNOS gene transcription rates.
- The role of protein synthesis in TNF-alpha's effect was evaluated using cycloheximide.
- mRNA half-life was determined under basal and TNF-alpha-treated conditions.
Main Results:
- TNF-alpha markedly reduced cNOS mRNA levels in HUVECs in a dose- and time-dependent manner.
- A significant decrease in cNOS mRNA was observed as early as 4 hours and diminished to 5% of control levels at 24 hours post-treatment with 3 ng/mL TNF-alpha.
- TNF-alpha treatment shortened the half-life of cNOS mRNA from 48 hours to 3 hours, indicating increased degradation.
- TNF-alpha did not alter the rate of cNOS gene transcription.
Conclusions:
- TNF-alpha decreases cNOS mRNA levels in endothelial cells primarily by accelerating mRNA degradation, not by affecting gene transcription.
- These findings suggest that TNF-alpha-induced downregulation of cNOS mRNA may contribute to the impaired endothelium-mediated vasorelaxation observed in atherosclerosis.