Abstract
A variety of amphiphiles inhibit plasma membrane cholesterol esterification and induce 3-hydroxy-3-methylglutaryl-coenzyme A reductase accumulation in cultured cells; among these are steroids, hydrophobic amines, phenothiazines, ionophores, colchicine, and lysophosphatides. It has been proposed that these amphiphiles signal a sterol deficiency to regulatory sites by blocking the movement of plasma membrane cholesterol into the cell (Lange, Y., and Steck, T. L. 1994. J. Biol. Chem. 269: 29371-29374). If this were the case, these agents also should enhance transcription of sterol responsive genes and stabilize 3-hydroxy-3-methylglutaryl-coenzyme A reductase. As a test of this hypothesis, the effect of the amphiphiles on such transcriptional and post-transcriptional events was assessed. A mouse embryo cell line was transfected with a construct containing the promoter for the human low density lipoprotein receptor upstream of the DNA sequence coding for chloramphenicol acyltransferase (CAT). Incubation of these cells for 7-18 h with the aforementioned agents caused the level of expression of the promoter/CAT construct to increase 2- to 9-fold. We showed further that the amphiphiles stimulated 3-hydroxy-3 methylglutaryl-coenzyme A reductase activity by increasing gene transcription as well as by decreasing degradation of the enzyme. These are the predicted homeostatic responses to cell cholesterol deficiency. These findings support the hypothesis that certain amphiphiles falsely signal a cholesterol deficiency to the intracellular sites regulating cholesterol homeostasis.
Highlights
A variety of amphiphiles inhibit plasma membrane cholesterol esterification and induce 3-hydroxy-3methylglutaryl-coenzyme A reductase accumulation in cultured cells; among these are steroids, hydrophobic amines, phenothiazines, ionophores, colchicine, and lysophosphatides
The biosynthesis of HMG-CoA synthase, HMG-CoA reductase, and LDL receptors all increase as cell cholesterol declines in various cell types (1).Expression of the genes for HMG-CoA synthase and the LDL receptor are under the control of a 10 bp sequence in its 5' flanking region, termed the sterol regulatory element-1 (2)
As most of the cholesterol in the cell is in the plasma membrane (4),how is its abundance sensed by these internal regulatory sites? A clue to this unknown mechanism comes from the recent finding that a broad class of amphiphiles inhibited esterification of cholesterol and stimulated HMG-CoA reductase activity and cholesterol biosynthesis (5).These agents included progesterone, monensin, chloroquine, imipramine, trifluoperazine, nigericin, colchicine, and lysolecithin
Summary
The medium in replicate dishes of cells transfected with the LDLR-CATgene construct was replaced with medium containing 1% human plasma-derivedserum. After 48 h incubation, the agents were added in ethanol or M e 6 0 (< 1%final),and the cells were incubated for a further 7 or 18 h before extraction and assay of CAT activity as described under Experimental Procedures. Triplicate flasks were used for each condition. Values are expressed relative to that of the control (mean f SD)
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