Post-transcriptional Regulation of Endothelial Nitric Oxide Synthase mRNA Stability by Rho GTPase*
- From the Cardiovascular Division, Brigham & Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115
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 μm), in the presence ofl-mevalonate (200 μm), geranylgeranylpyrophosphate (GGPP, 1–10 μm), farnesylpyrophosphate (FPP, 5–10 μm), 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]GTPγS-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 μg/ml) or by overexpression of a dominant-negative N19RhoA mutant increased eNOS expression. In contrast, activation of Rho byEscherichia 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.
Footnotes
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↵* This work was supported by National Institutes of Health Grant HL-52233 (to J. K. L.) and the Deutsche Forschungsgemeinschaft (to U. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Established Investigator of the American Heart Association. To whom correspondence should be addressed: Cardiovascular Division, Dept. of Medicine, 221 Longwood Ave., LMRC-322, Boston, MA 02115. Tel.: 617-732-6538; Fax: 617-264-6336; E-mail:jliao{at}rics.bwh.harvard.edu.
- Abbreviations:
- HMG-CoA
-
3-hydroxy-3-methylglutaryl coenzyme A
- NO
-
nitric oxide
- eNOS
-
endothelial (Type III) nitric oxide synthase
- LDL
-
low density lipoprotein
- FPP
-
farnesylpyrophosphate
- GGPP
-
geranylgeranylpyrophosphate
- GTPγS
-
guanosine 5′-O-(3-thiotriphosphate)
- C3 transferase
-
C. botulinum C3 transferase or exoenzyme
- CNF-1
-
E. colicytotoxic necrotizing factor-1
- LNMA
-
l-N ω-monomethylarginine
- β-Gal
-
β-galactosidase
- DRB
-
5,6-dichlorobenzimidazole riboside
- kb
-
kilobase(s)
- PAGE
-
polyacrylamide gel electrophoresis.
-
- Received June 5, 1998.
- Revision received July 7, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
