Monitoring the Cellular Effects of HMG-CoA Reductase Inhibitors In Vitro and Ex Vivo

Abstract

Objective-Inhibition of 3hydroxy3methylglutaryl-coenzyme A (HMG-CoA) reductase by statins and the subsequent reduction in Rho protein isoprenylation inactivates these important signaling molecules. The purpose of this study was to directly monitor statin effects on Rho proteins. Methods and Results-We used biphasic Triton X-114 system, 1-dimensional isoelectric focusing, and 2D-electrophoresis for the separation of modified and nonmodified Rho proteins. These methods were evaluated in human fibroblasts treated with simvastatin. 2D-electrophoresis, which proved to be the most sensitive method, revealed 2 major spots of identical molecular weight but different isoelectric points, with the more basic spot representing the carboxymethylated form of RhoA. In control cells, 90% of RhoA was fully modified (carboxymethylated). After treatment with simvastatin, a significant shift toward the unmethylated form was observed, representing inhibition of isoprenylation, which is a prerequisite to further modification. Similar shifts were observed for Rac1 and Cdc42. In freshly isolated peripheral blood mononuclear cells, a shift toward nonmodified RhoA was observed after treatment with atorvastatin in vitro and in vivo. There was a significant increase in unmethylated RhoA in statin-treated individuals versus control individuals. Key Words: RhoA Ⅲ isoprenylation Ⅲ carboxymethylation Ⅲ HMG-CoA reductase Ⅲ statins T herapy with 3hydroxy3methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) is associated with lipid-lowering and plaque-stabilizing effects, resulting from their interference with cholesterol biosynthesis. However, recent experimental and clinical evidence indicate that the beneficial effects of statins extend beyond cholesterol reduction, involving improved plaque composition, decreased inflammation, and ameliorated endothelial function. 1,2 These pleiotropic effects are mediated by the ability of statins to interfere directly with the posttranslational modification of signaling proteins, particularly small GTP-binding proteins of the Ras and Rho families. Because the membrane localization and function of Ras and Rho depend on the attachment of isoprenoid intermediates to these proteins, by inhibiting protein isoprenylation, statin treatment leads to accumulation of the inactive forms of Ras and Rho in the cytoplasm. Although farnesylation of Ras proteins and geranylgeranylation of Rho proteins are equally affected by statins, many of the beneficial cardiovascular effects of statins are thought to be a result of their interference with Rho protein signaling. 3 As interference with the geranylgeranyl modification of Rho proteins is one of the earliest steps related to the direct cellular actions of statins, it appears desirable to determine the relative amount of modified versus nonmodified protein. Some of the isoprenylated proteins, such as Ras proteins, can be separated from their nonmodified form by high-resolution SDS-polyacrylamide gel electrophoresis (SDS-PAGE). 4 However, for Rho family prot...

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