squalestatin-1 and mevastatin

Farnesyl pyrophosphate (FPP), a key intermediate in the mevalonate pathway and protein farnesylation, can act as an agonist for several nuclear hormone receptors. Here we show a novel mechanism by which FPP inhibits wound healing acting as an agonist for glucocorticoid receptor (GR). Elevation of endogenous FPP by the squalene synthetase inhibitor zaragozic acid A (ZGA) or addition of FPP to the cell culture medium results in activation and nuclear translocation of the GR, a known wound healing inhibitor. We used functional studies to evaluate the effects of FPP on wound healing. Both FPP and ZGA inhibited keratinocyte migration and epithelialization in vitro and ex vivo. These effects were independent of farnesylation and indicate that modulation of FPP levels in skin may be beneficial for wound healing. FPP inhibition of keratinocyte migration and wound healing proceeds, in part, by repression of the keratin 6 gene. Furthermore, we show that the 3-hydroxy-3-methylglutaryl-CoA-reductase inhibitor mevastatin, which blocks FPP formation, not only promotes epithelialization in acute wounds but also reverses the effect of ZGA on activation of the GR and inhibition of epithelialization. We conclude that FPP inhibits wound healing by acting as a GR agonist. Of special interest is that FPP is naturally present in cells prior to glucocorticoid synthesis and that FPP levels can be further altered by the statins. Therefore, our findings may provide a better understanding of the pleiotropic effects of statins as well as molecular mechanisms by which they may accelerate wound healing.

Topics: Animals; Bridged Bicyclo Compounds, Heterocyclic; Cattle; Cell Line; Cell Movement; Enzyme Inhibitors; Epithelial Cells; Farnesyl-Diphosphate Farnesyltransferase; Gene Expression Regulation; Glucocorticoids; Humans; Keratin-6; Keratinocytes; Ligands; Lovastatin; Polyisoprenyl Phosphates; Promoter Regions, Genetic; Receptors, Glucocorticoid; Sesquiterpenes; Signal Transduction; Tricarboxylic Acids; Wound Healing

Cholesterol and sphingolipids are abundant in neuronal membranes, where they help the organisation of the membrane microdomains involved in major roles such as axonal and dendritic growth, and synapse and spine stability. The aim of this study was to analyse their roles in presynaptic physiology. We first confirmed the presence of proteins of the exocytic machinery (SNARES and Ca(v)2.1 channels) in the lipid microdomains of cultured neurons, and then incubated the neurons with fumonisin B (an inhibitor of sphingolipid synthesis), or with mevastatin or zaragozic acid (two compounds that affect the synthesis of cholesterol by inhibiting HMG-CoA reductase or squalene synthase). The results demonstrate that fumonisin B and zaragozic acid efficiently decrease sphingolipid and cholesterol levels without greatly affecting the viability of neurons or the expression of synaptic proteins. Electron microscopy showed that the morphology and number of synaptic vesicles in the presynaptic boutons of cholesterol-depleted neurons were similar to those observed in control neurons. Zaragozic acid (but not fumonisin B) treatment impaired synaptic vesicle uptake of the lipophilic dye FM1-43 and an antibody directed against the luminal epitope of synaptotagmin-1, effects that depended on the reduction in cholesterol because they were reversed by cholesterol reloading. The time-lapse confocal imaging of neurons transfected with ecliptic SynaptopHluorin showed that cholesterol depletion affects the post-depolarisation increase in fluorescence intensity. Taken together, these findings show that reduced cholesterol levels impair synaptic vesicle exocytosis in cultured neurons.

Topics: Animals; Anticholesteremic Agents; Bridged Bicyclo Compounds, Heterocyclic; Calcium Channels, N-Type; Cells, Cultured; Cholesterol; Exocytosis; Fumonisins; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Immunoglobulin G; Lovastatin; Membrane Microdomains; Microscopy, Electron, Transmission; Models, Neurological; Neurons; Presynaptic Terminals; Rats; SNARE Proteins; Sphingolipids; Synaptic Vesicles; Synaptotagmin I; Tricarboxylic Acids

The mevalonate pathway produces many critical substances in cells, including sterols essential for membrane structure and isoprenoids vital to the function of many membrane proteins. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is a rate-limiting enzyme in the mevalonate pathway. Because cholesterol is a product of this pathway, HMG-CoA reductase inhibitors (statins) are used to treat hypercholesterolemia. Statins are also toxic to several malignancies, including acute myeloid leukemia (AML). Although this toxicity has been attributed to the inhibition of Ras/Rho isoprenylation, we have previously shown that statin toxicity in primary AML cells (AMLs) does not correlate with Ras isoprenylation or with activating Ras mutations. In other studies, we have shown that hypoxic and oxidant injuries induce cholesterol increments in renal tubule cells and that statins sensitize these cells to injury by blocking protective cholesterol responses. We now demonstrate that exposing particular AMLs to radiochemotherapy induces much greater cellular cholesterol increments than those seen in similarly treated normal bone marrow. Treatment of these AMLs with mevastatin or zaragozic acid (which inhibits cholesterol synthesis but not isoprenoid synthesis) attenuates the cholesterol increments and sensitizes cells to radiochemotherapy. The extent of toxicity is affected by the availability of extracellular lipoproteins, further suggesting that cellular cholesterol is critical to cell survival in particular AMLs. Because zaragozic acid does not inhibit isoprenoid synthesis, these data suggest that cholesterol modulation is an important mechanism whereby statins exert toxic effects on some AMLs and that cholesterol modulators may improve therapeutic ratios in AML by impacting cholesterol-dependent cytoresistance.

Topics: Acute Disease; Antineoplastic Agents; Bone Marrow Cells; Bridged Bicyclo Compounds, Heterocyclic; Cholesterol; Cytarabine; Daunorubicin; Drug Synergism; HL-60 Cells; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Leukemia, Myeloid; Lipoproteins, LDL; Lovastatin; Membrane Lipids; Protein Prenylation; Protein Processing, Post-Translational; Radiation-Sensitizing Agents; Tricarboxylic Acids; Tumor Cells, Cultured

Deficiency of nonsterol isoprenoids, intermediate metabolites of the cholesterol biosynthetic pathway, has been known to cause an inhibition of DNA synthesis and cell growth, and to induce apoptosis in nonneuronal cells. To investigate whether this is also the case in neurons, we examined the effect of a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor on the viability of neuronal cultures prepared from fetal rat brains. Treatment with compactin, a competitive inhibitor of HMG-CoA reductase, induced neuronal death in a dose-dependent manner. Concurrent treatment with cholesterol, beta-migrating very low density lipoprotein, mevalonate, or squalene substantially inhibited the induction of neuronal death by compactin. Cell death was also induced by treatment with squalestatin, which specifically inhibits cholesterol biosynthesis at a site downstream from the generation of nonsterol metabolites. Furthermore, squalestatin-induced neuronal death was inhibited by concurrent incubation with squalene but not mevalonate. In contrast, cell growth of proliferating cells such as NIH 3T3 and PC12 cells was exclusively dependent on the level of nonsterol isoprenoid products and not that of cholesterol. The results of this study clearly indicate that the viability of neurons, different from that of nonneuronal cells, depends on the intracellular cholesterol content and not on the intermediate nonsterol isoprenoid products.

Topics: 3T3 Cells; Acetates; Animals; Apoptosis; Bridged Bicyclo Compounds, Heterocyclic; Cell Survival; Cerebral Cortex; Cholesterol; Embryo, Mammalian; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Kinetics; Lipoproteins, VLDL; Lovastatin; Mevalonic Acid; Mice; Neurons; PC12 Cells; Rabbits; Rats; Rats, Sprague-Dawley; Squalene; Tricarboxylic Acids

Apolipoprotein E (apoE) allele epsilon4 is a major risk factor for Alzheimer's disease (AD); however, the molecular mechanism underlying the acceleration of the development of AD in patients possessing epsilon4 remains to be determined. To investigate the isoform-specific effects of apoE on neurons, primary neuron cultures were prepared from fetal rat cerebral cortices. Inhibition of de novo cholesterol synthesis by compactin, a 3-hydroxyl-3-methylglutaryl CoA reductase inhibitor, induced neuronal cell death in a dose dependent manner. In the presence of a sublethal dose of compactin, apoE4 with beta-migrating very low density lipoproteins (beta-VLDL) caused apoptotic cell death in neuronal cultures. The same results were obtained with inhibition of de novo cholesterol synthesis by sublethal doses of squalestatin, an inhibitor of squalene synthase. The de novo cholesterol synthesis was suppressed to a higher degree by apoE4 than by apoE3, administered with beta-VLDL in the presence or absence of compactin. Mevalonate and squalene, which are metabolites of the cholesterol synthesis pathway, protected neuronal cells from apoE4-induced cell death. These results may suggest that apoE4 may exhibit neurotoxic action when de novo cholesterol synthesis is suppressed to a certain level, and that apoE4 induces neuronal cell death through the suppression of de novo cholesterol synthesis via an undetermined isoform-specific mechanism.

Topics: Animals; Apolipoprotein E3; Apolipoprotein E4; Apolipoproteins E; Bridged Bicyclo Compounds, Heterocyclic; Cell Survival; Cells, Cultured; Cholesterol; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lipoproteins, VLDL; Lovastatin; Male; Neurons; Protein Isoforms; Rabbits; Rats; Rats, Sprague-Dawley; Tricarboxylic Acids