on September 23, 2004
Published online before print September 23, 2004, doi: 10.1161/01.ATV.0000145943.19099.a3
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Submitted on March 19, 2004
Accepted on September 2, 2004
Monitoring the Cellular Effects of HMG-CoA Reductase Inhibitors In Vitro and Ex Vivo
Iwona Cicha ;
From the Medical Clinic IV (I.C., N.S.-M., M.G.-S.) and Medical Clinic II (A.Y., C.D.G.), University of Erlangen-Nuremberg, Erlangen, Germany. Present address for N.S.-M.: Natural and Medical Sciences Institute (NMI), University of Tuebingen, Department of Biochemistry, Reutlingen, Germany.
* To whom correspondence should be addressed. E-mail: Goppelt-Struebe{at}rzmail.uni-erlangen.de.
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.
Conclusion--2D-electrophoresis is a sensitive method for detecting changes in the amount of nonisoprenylated Rho proteins, allowing monitoring the direct cellular effects of statins.
Key words: RhoA • isoprenylation • carboxymethylation • HMG-CoA reductase • statins
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