Sep 30, 1994 - Yvonne Lange*$ and Theodore L. Steckl. From the fDepartrnents ..... Murphy, B. D., and Silavin, S. L. (1989) Oxford Rev. Reprod. Biol. 11,179- ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 269, No. 47, Issue of November 25, pp. 29371-29374, 1994
Communication
0 1994 by T h e American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
we shall refer to as class 1 agents. In particular, 25-HC’ stimulates cholesterol esterification (6, 13-15) and conversion to steroids (13, 16); it also stimulates the secretion of cholesterol MODULATION BY AMPHIPHILES* from liver cells as LDL (14) and down-regulates HMG-CoA (Received for publication, August 25, 1994, and in revised form, reductase activity (17). Like cholesterol, oxysterols inhibit the September 30, 1994) expression of genes coding for HMG-CoA reductase and LDL receptors (1,2, 15). Yvonne Lange*$ and Theodore L. Steckl A second set of compounds, which we shall call class 2, has From the fDepartrnents of Pathology and Biochemistry, effects opposite t o those of excess cholesterol; these agents inRush-Presbyterian-St. Luke’s Medical Center, Chicago, Illinois 60612 and the Wepartment of Biochemistry and hibit cholesterol esterification, stimulate cholesterol biosyntheMolecular Biology, University of Chicago, sis, and counter the action of oxysterols. The first members of Chicago, Illinois 60637 this group t o be described were steroids such as progesterone (14, 18-21, 44) and hydrophobic amines like imipramine and Diverse amphiphiles act on cellular cholesterol metabolism as if signaling regulatory sites. One class (ox- the steroid U18666A (15, 22). We now show that class 2 conysterols) mimics the homeostatic effects of excess cell tains a wide variety of other amphiphiles, most of which interact with P-glycoproteins. Furthermore, the reciprocal antagocholesterol,inhibitingcholesterolbiosynthesisand nism of class 1 and class 2 agents supports an hypothesis stimulatingplasmamembranecholesterolesterificaintracellular cholesterol tion. A second classof amphiphiles has effects precisely for the mechanism underlying homeostasis. opposite to the oxysterols,i.e. they immediately inhibit plasmamembrane cholesterol esterification and proRESULTS gressively induce3-hydroxy-3-methylglutaryl-coenzyme Fig. 1 shows that the esterification of cholesterol in intact A reductase activity and cholesterol biosynthesis. This cells is inhibited by a variety of amphiphilic agents. Similar second class of agents includes steroids, hydrophobic amines, phenothiazines, ionophores, colchicine, cy- effects were also obtained with the agentsemployed in Table I tochalasins, and lysophosphatides, most of which inter- as well as with lysophosphatidylserine and lysophosphatiact with P-glycoproteins. dylethanolamine (not shown). This inhibition was immediate in These data support a general hypothesis describing all cases and was reversed by washing when tested with pro(a) Proteins regulating gesterone, monensin, imipramine, andchlorpromazine. Blockcellular cholesterol homeostasis. sterol metabolism are embedded in intracellular mem- ing protein synthesis with25 PM cycloheximide did not prevent by the local the inhibition of esterification by any agent tested (nigericin, branes where their activities are governed level of cholesterol. ( b ) Excess plasma membrane and lysophosphatidylcholine, monensin, progesterone, chloroquine, lysosomal cholesterol circulates through those intracel-and trifluoperazine). The inhibition was antagonized by 25-HC lularmembranesand sets thehomeostaticactivities (Fig. 2) as well as 7-ketocholesterol (not shown). therein. ( c ) The two classes of agents mentioned above As illustrated inTable I, the inhibitorsof cholesterol esteriaffect cholesterol homeostasis by increasing or decreasfication allstimulatedsterol biosynthesis and HMG-CoA of cholesterol at the ing, respectively, the ambient level reductase activity. Where tested (progesterone, monensin, and sites of regulation. lysophosphatidylcholine), the stimulation accelerated characteristically over a period of up to 20 h. Stimulation by progesterone was blocked by cycloheximide. None of several agents Cholesterol regulates its own abundanceinanimal cells tested (progesterone,monensin, lysophosphatidylcholine, nithrough feedback control of several homeostaticactivities. gericin, and trifluoperazine) had anappreciable effect on HMGThese include accretion by ingestion, biosynthesis, and choles- CoA reductase activity when addedt o homogenates. The effect terol esterhydrolysis; and consumption by cholesterol esterifi- of these agents on cholesterol biosynthesis in intact cells was cation, secretion, and transformation into steroids, bile acids, opposed by 25-HC and 7-ketocholesterol (not shown). These are and lipoproteins (1, 2). Although the sites that mediate these the attributes of class 2 agents. activities reside incytoplasmic organelles, almost all unesteriIt has been shown previously that the utilization of plasma fied cell cholesterol is confined to the plasma membrane(3-5). membrane cholesterol for esterification and steroidogenesis is An unanswered question is how the intracellular sites gauge inhibited by certain compounds which perturb calmodulin and regulate thepool in the plasma membrane. (namely trifluoperazine), acidic vacuoles (chloroquine and niRecent evidence suggests the existence of a sensor mechagericin),microtubules (colchicine), and microfilaments (cynism by which plasmamembrane cholesterol exceeding a class 2 agents isnow tochalasins) (7). That these inhibitors are threshold is transported to intracellular sites for conversion t o suggested by the fact that they also stimulate cholesterol bioesters and steroids (6-12). In thisregard, excess cell cholesterol synthesis and HMG-CoA reductase activity (Table I). Furthercan be mimicked by certain exogenous oxysterols (1,2), which more, the inhibitionof cholesterol esterification by these agents opposed by 25-HC (Fig. 2) and 7-ketocholesterol (not * This work was supported by National Institutes of Health Grants was shown); this should not be the case if their mode of action was HL28448 (toY. L.) and GM47282 (to T. L. S.). The costs of publication of this article were defrayedin part by the payment of page charges. This on the subcellular structures originally proposed (7). article must therefore be hereby marked‘‘aduertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviations and trivial names used are: 25-HC, 25-hydroxy8 To whom correspondence should be addressed: Dept. of Pathology, cholesterol; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; LDL, Rush-Presbyterian-St.Luke’s Medical Center, 1653W. Congress Pkwy,, low density lipoprotein; ER, endoplasmic reticulum; U18666A, 3-p-[2Chicago, IL 60612. (diethylamino)ethoxylandrost-5-en-l7-one.
Cholesterol Homeostasis
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Regulation of Cholesterol Homeostasis r
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0.0 0.5 1 .o FRACTIONAL CONCENTRATION FIG.1. Class 2 agents inhibit cholesterol esterification. Confluent monolayers of cultured rat hepatoma cells, line FU5AH (Il),were
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FIG. 2.Antagonistic effectsof 25-hydroxycholesterol andclass 2 agents on cholesterol esterification. FUBAH cells cultured in medium containing 10% fetal bovine serum (11) were suspended and assayed for cholesteryl ester synthesis as in Fig. 1. Bar 1, solvent control; bar 2,25-HC (50-80 p ~ ) bar ; 3, the indicated class 2 agent;bar 4, both the indicated agent plus25-HC. PG, 3 p~ progesterone; CYT, 5 p~ cytochalasin A; U, 2 pg/ml U18666A; MON, 3 PM monensin; NIG, 5 p~ nigericin; CLQ, 50 p~ chloroquine. Values, expressed as percent of control, were averages of duplicate samples or replicate experiments. Cytochalasins B and D had a lower potency than cytochalasin A (not shown).
TABLEI1 Effect of agents on esterification of plasmamembrane P'Clcholesterol Cultured hepatoma cells were labeled in suspension l'4Clcholeswith terol(11). Aliquots were prepared and incubated with agents as in Fig. washed and suspended in5 ml of 150 mM NaCI, 5 mM Nap, (pH7.51, 5 1. The incorporation of label into cholesteryl esters was determined m~ glucose. A total of 6 1 0 aliquots were dispensed from each flask following a 40-min incubationat 37 "C (11).In ExperimentA, cells were (containing -60 pg of cell cholesterol) and preincubated for 5 min at 37 "C with class 2 compounds in Me,SO or ethanol (
