Inhibition of squalene synthase and squalene epoxidase in tobacco cells triggers an up-regulation of 3-hydroxy-3-methylglutaryl coenzyme a reductase.
To get some insight into the regulatory mechanisms controlling the sterol branch of the mevalonate pathway, tobacco (Nicotiana tabacum cv Bright Yellow-2) cell suspensions were treated with squalestatin-1 and terbinafine, two specific inhibitors of squalene synthase (SQS) and squalene epoxidase, respectively. These two enzymes catalyze the first two steps involved in sterol biosynthesis. In highly dividing cells, SQS was actively expressed concomitantly with 3-hydroxy-3-methylglutaryl coenzyme A reductase and both sterol methyltransferases. At nanomolar concentrations, squalestatin was found to inhibit efficiently sterol biosynthesis as attested by the rapid decrease in SQS activity and [(14)C]radioactivity from acetate incorporated into sterols. A parallel dose-dependent accumulation of farnesol, the dephosphorylated form of the SQS substrate, was observed without affecting farnesyl diphosphate synthase steady-state mRNA levels. Treatment of tobacco cells with terbinafine is also shown to inhibit sterol synthesis. In addition, this inhibitor induced an impressive accumulation of squalene and a dose-dependent stimulation of the triacylglycerol content and synthesis, suggesting the occurrence of regulatory relationships between sterol and triacylglycerol biosynthetic pathways. We demonstrate that squalene was stored in cytosolic lipid particles, but could be redirected toward sterol synthesis if required. Inhibition of either SQS or squalene epoxidase was found to trigger a severalfold increase in enzyme activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, giving first evidence for a positive feedback regulation of this key enzyme in response to a selective depletion of endogenous sterols. At the same time, no compensatory responses mediated by SQS were observed, in sharp contrast to the situation in mammalian cells.
Wentzinger
Laurent F
LF
Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique Unité Propre de Recherche 2357, 28 rue Goethe, 67083 Strasbourg, France.
Bach
Thomas J
TJ
Hartmann
Marie-Andrée
MA
D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002250:Carbon Radioisotopes; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics / Q000378:metabolism*; D008780:Methyltransferases / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010840:Phytosterols / Q000096:biosynthesis; D011106:Polyisoprenyl Phosphates / Q000096:biosynthesis; D012717:Sesquiterpenes; D013185:Squalene / Q000378:metabolism; D050603:Squalene Monooxygenase; D000077291:Terbinafine; D014026:Nicotiana / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D014233:Tricarboxylic Acids / Q000494:pharmacology; D014280:Triglycerides / Q000378:metabolism; D014315:Triterpenes; D015854:Up-Regulation / Q000187:drug effects
D019086:Bridged Bicyclo Compounds, Heterocyclic; D002250:Carbon Radioisotopes; D004791:Enzyme Inhibitors; D009281:Naphthalenes; D010840:Phytosterols; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D014233:Tricarboxylic Acids; D014280:Triglycerides; D014315:Triterpenes; C075117:squalestatin 1; C004808:farnesyl pyrophosphate; D013185:Squalene; D006903:Hydroxymethylglutaryl CoA Reductases; D010105:Oxygenases; D050603:Squalene Monooxygenase; C409822:24-methylene-lophenol-C24-methyltransferase; D008780:Methyltransferases; C019609:S-adenosyl-L-methionine-cycloartenol methyltransferase; D013186:Farnesyl-Diphosphate Farnesyltransferase; D010744:Phosphoric Monoester Hydrolases; C438007:prenyl-pyrophosphatase; D000077291:Terbinafine; C100089:cycloartenol