Abstract
The mechanism by which 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors increase endothelial nitric oxide synthase (eNOS) expression is unknown. To determine whether changes in isoprenoid synthesis affects eNOS expression, human endothelial cells were treated with the HMG-CoA reductase inhibitor, mevastatin (1-10 microM), in the presence of L-mevalonate (200 microM), geranylgeranylpyrophosphate (GGPP, 1-10 microM), farnesylpyrophosphate (FPP, 5-10 microM), or low density lipoprotein (LDL, 1 mg/ml). Mevastatin increased eNOS mRNA and protein levels by 305 +/- 15% and 180 +/- 11%, respectively. Co-treatment with L-mevalonate or GGPP, but not FPP or LDL, reversed mevastatin's effects. Because Rho GTPases undergo geranylgeranyl modification, we investigated whether Rho regulates eNOS expression. Immunoblot analyses and [35S]GTPgammaS-binding assays revealed that mevastatin inhibited Rho membrane translocation and GTP binding activity by 60 +/- 5% and 78 +/- 6%, both of which were reversed by co-treatment with GGPP but not FPP. Furthermore, inhibition of Rho by Clostridium botulinum C3 transferase (50 microg/ml) or by overexpression of a dominant-negative N19RhoA mutant increased eNOS expression. In contrast, activation of Rho by Escherichia coli cytotoxic necrotizing factor-1 (200 ng/ml) decreased eNOS expression. These findings indicate that Rho negatively regulates eNOS expression and that HMG-CoA reductase inhibitors up-regulate eNOS expression by blocking Rho geranylgeranylation, which is necessary for its membrane-associated activity.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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ADP Ribose Transferases / metabolism
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ADP Ribose Transferases / pharmacology
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Bacterial Toxins / pharmacology
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Botulinum Toxins*
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Cells, Cultured
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Cytosol / metabolism
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Cytotoxins / pharmacology
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Endothelium, Vascular / enzymology*
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Escherichia coli Proteins*
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GTP Phosphohydrolases / genetics
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GTP Phosphohydrolases / metabolism*
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GTP-Binding Proteins / genetics
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GTP-Binding Proteins / metabolism
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Gene Expression Regulation, Enzymologic / drug effects
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Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
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Guanosine Triphosphate / metabolism
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Humans
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Hydroxymethylglutaryl-CoA Reductase Inhibitors / pharmacology
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Kinetics
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Lipoproteins, LDL / pharmacology
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Lipoproteins, LDL / physiology
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Lovastatin / analogs & derivatives
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Lovastatin / pharmacology
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Membrane Proteins / genetics
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Membrane Proteins / metabolism
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Mevalonic Acid / pharmacology
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Nitric Oxide Synthase / genetics*
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Nitric Oxide Synthase Type III
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Polyisoprenyl Phosphates / pharmacology
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RNA Processing, Post-Transcriptional* / drug effects
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RNA, Messenger / genetics
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RNA, Messenger / metabolism*
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Sesquiterpenes
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ras Proteins / metabolism
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rhoA GTP-Binding Protein
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rhoB GTP-Binding Protein
Substances
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Bacterial Toxins
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Cytotoxins
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Escherichia coli Proteins
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Hydroxymethylglutaryl-CoA Reductase Inhibitors
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Lipoproteins, LDL
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Membrane Proteins
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Polyisoprenyl Phosphates
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RNA, Messenger
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Sesquiterpenes
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cytotoxic necrotizing factor type 1
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mevastatin
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Guanosine 5'-O-(3-Thiotriphosphate)
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farnesyl pyrophosphate
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Guanosine Triphosphate
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Lovastatin
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NOS3 protein, human
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Nitric Oxide Synthase
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Nitric Oxide Synthase Type III
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ADP Ribose Transferases
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exoenzyme C3, Clostridium botulinum
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Botulinum Toxins
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GTP Phosphohydrolases
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GTP-Binding Proteins
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ras Proteins
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rhoA GTP-Binding Protein
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rhoB GTP-Binding Protein
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geranylgeranyl pyrophosphate
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Mevalonic Acid