nb-598 and squalestatin-1

The role of mevalonic acid (MVA) and its products (isoprenoids) in cell proliferation prompted us to investigate the effect of drugs affecting diverse enzymatic steps of the MVA pathway on rat aorta smooth muscle cell (SMC) proliferation. Competitive inhibitors of HMG-CoA reductase (statins) decreased SMC proliferation in a dose-dependent manner. The inhibitory effect induced by simvastatin 3.5 microM (70% +/- 3.8 decrease) was prevented by addition of 100 microM MVA, (100% +/- 2.3), 10 microM farnesol (F-OH) (85% +/- 1.2) and 5 microM of all-trans geranylgeraniol (GG-OH) (precursor of prenylated proteins) (81% +/- 1.1), but not by 2-cis GG-OH (precursor of dolichols), squalene and ubiquinone. The same inhibitory effect was obtained with 6-fluoromevalonate (1-50 microM), an inhibitor of MVA-PP decarboxylase. Squalestatin 1 (1-25 microM) and NB-598 (1-10 microM), potent squalene synthase and epoxidase inhibitors, respectively, caused a complete inhibition of cholesterol synthesis without affecting SMC proliferation. Finally, BZA-5B (10-50 microM) a specific inhibitor of protein farnesyl tranferase (PFTase), inhibited SMC proliferation in a dose- (10-50 microM) and time-dependent manner, reaching 52% +/- 6.3 inhibition after 9 days, in the presence of 50 microM BZA-5B, without affecting cholesterol synthesis. This effect was partially prevented by mevalonate (76% +/- 3.2) and GG-OH (87% +/- 7.3) but not by F-OH. On the other hand, SMC proliferation was not affected by the closely related compound BZA-7B (93% +/- 4), which does not inhibit PFTase. Taken together, these findings support the involvement of specific isoprenoid metabolites, probably through farnesylated and geranylgeranylated proteins in cell proliferation.

Topics: Animals; Aorta; Benzodiazepines; Benzylamines; Bridged Bicyclo Compounds, Heterocyclic; Cell Division; Cholesterol; Dose-Response Relationship, Drug; Enzyme Inhibitors; Farnesyl-Diphosphate Farnesyltransferase; Male; Mevalonic Acid; Muscle, Smooth, Vascular; Oligopeptides; Rats; Rats, Sprague-Dawley; Thiophenes; Tricarboxylic Acids

The microsomal enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is subject to rapid degradation when cells are incubated with sterols or mevalonic acid (MVA). It has been shown that this rapid degradation is dependent upon both a sterol and another MVA-derived metabolite (Nakanishi, M., Goldstein, J. L., and Brown, M. S. (1988) J. Biol. Chem. 258, 8929-8937). In the current study, inhibitors of the isoprene biosynthetic pathway were used to define further this mevalonic acid derivative involved in the accelerated degradation of HMG-CoA reductase. The accelerated degradation of HMG-CoA reductase in met-18b-2 cells, which is induced by the addition of MVA, was inhibited by the presence of the squalene synthase inhibitor, zaragozic acid/squalestatin, or the squalene epoxidase inhibitor, NB-598. Accelerated degradation of HMG-CoA reductase was observed when NB-598-treated cells were incubated with both MVA and sterols. In contrast, the addition of MVA and sterols to zaragozic acid/squalestatin-treated cells did not result in rapid enzyme degradation. This MVA- and sterol-dependent degradation of HMG-CoA reductase persisted in cells permeabilized with reduced streptolysin O. Finally, the selective degradation of HMG-CoA reductase was also observed in rat hepatic microsomes incubated in vitro in the absence of ATP and cytosol. We conclude that the MVA-derived component that is required for the accelerated degradation of HMG-CoA reductase is derived from farnesyl disphosphate and/or squalene in the isoprenoid biosynthetic pathway. We propose that this component has a permissive effect and does not, by itself, induce the degradation of HMG-CoA reductase. We also conclude that the degradation of HMG-CoA occurs in the endoplasmic reticulum, and, once the degradation of HMG-CoA reductase has been initiated by MVA and sterols, all necessary components for the continued degradation of HMG-CoA reductase reside in the endoplasmic reticulum.

Topics: Animals; Benzylamines; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cells, Cultured; Enzyme Inhibitors; Hydrolysis; Hydroxymethylglutaryl CoA Reductases; Male; Mevalonic Acid; Microsomes, Liver; Rats; Rats, Sprague-Dawley; Sterols; Thiophenes; Tricarboxylic Acids