Dual effects of histone deacetylase inhibition by trichostatin A on endothelial nitric oxide synthase expression in endothelial cells

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Abstract

Inhibition of histone deacetylases by trichostatin A (TSA) has pleiotropic effects on gene expression. We demonstrated that at low dose (0.1 μg) TSA increased the eNOS mRNA levels, which was followed by a time- and dose-dependent down-regulation. Cycloheximide, a protein synthesis inhibitor, completely abolished TSA-induced decrease in eNOS expression, indicating that new protein synthesis is required for the inhibiting effect. Mevastatin—an inhibitor HMG-CoA reductase and geranylgeranylation reaction dose-dependently antagonized TSA-induced reduction. This mevastatin-mediated antagonism was completely abolished by geranylgeranylpyrophosphate, suggesting that geranylgeranyl modification is needed to activate the eNOS mRNA destabilizing factor—a mechanism responsible for statin-mediated eNOS upregulation.

Section snippets

Materials and methods

Reagents. TSA, cycloheximide (CHX), mevastatin, farnesylpyrophosphate (FPP), and geranylgeranylpyrophosphate (GGPP) were purchased from Sigma (St. Louis, MO). TSA, mevastatin, and CHX were dissolved in dimethyl sulfoxide (Me2SO) before use. The Clostridium botulinum C3 transferase was purchased from List Biological Laboratories (Campbell, CA) and dissolved in PBS. Mevastatin was chemically activated by alkaline hydrolysis prior to use. Briefly, after 5 mg mevastatin was dissolved in 200 μl Me2SO,

Dual effects of TSA on eNOS mRNA expression

TSA has been demonstrated to down-regulate eNOS expression in endothelial cells after 24 h treatment [6]. While our experiments confirmed this TSA-induced eNOS down-regulation, the true effects of the TSA on eNOS expression appeared to be time- and dose-dependent. Contrary to late inhibition (by 24 h), we observed an induction of eNOS in HUVECs (>2-fold increase) during early stage (as early as 15 min) of the TSA treatment (Fig. 1A). For the dose-dependent effects, we observed that at the low dose

Discussion

In the present study, we demonstrated that inhibition of HDACs by TSA had a dual effect on the eNOS expression in a time- and dose-dependent fashion. Although previous studies demonstrated that TSA down-regulated eNOS by 24 h [6], our study showed that TSA could activate eNOS at the early stage of TSA treatment (by 15 min), and at the low doses (0.1 μg/ml). This up-regulating effect is followed by a time- and dose-dependent repression in the eNOS mRNA expression. We suggest that HDAC inhibition

Acknowledgment

This study was supported by NIH Grant R01-066053. Dr. XL Wang is an AHA Established Investigator.

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