POSTTRANSCRIPTIONAL REGULATION OF ENDOTHELIAL NITRIC-OXIDE SYNTHASE MESSENGER-RNA STABILITY BY RHO-GTPASE

The mechanism by which 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors increase endothelial nitric oxide synthase (eNOS) expressio n is unknown. To determine whether changes in isoprenoid synthesis aff ects eNOS expression, human endothelial cells were treated with the HM G-CoA reductase inhibitor, mevastatin (1-10 mu M), in the presence of L-mevalonate (200 mu M), geranylgeranylpyrophosphate (GGPP, 1-10 mu M) , farnesylpyrophosphate (FPP, 5-10 mu M), or low density lipoprotein ( LDL, 1 mg/ml). Mevastatin increased eNOS mRNA and protein levels by 30 5 +/- 15% and 180 +/- 11%, respectively, Go-treatment with L-mevalonat e or GGPP, but not FPP or LDL, reversed mevastatin's effects. Because Rho GTPases undergo geranylgeranyl modification, we investigated wheth er Rho regulates eNOS expression. Immunoblot analyses and [S-35]GTP ga mma S-binding assays revealed that mevastatin inhibited Rho membrane t ranslocation and GTP binding activity by 60 +/- 5% and 78 +/- 6%, both of which were reversed by co-treatment with GGPP but not FPP. Further more, inhibition of Rho by Clostridium botulinum. C3 transferase (50 m u g/ml) or by overexpression of a dominant-negative N19RhoA mutant inc reased eNOS expression. In contrast, activation of Rho by Escherichia coli cytotoxic necrotizing factor-1 (200 ng/ml) decreased eNOS express ion. These findings indicate that Rho negatively regulates eNOS expres sion and that HMG-CoA reductase inhibitors up-regulate eNOS expression by blocking Rho geranylgeranylation, which is necessary for its membr ane-associated activity.