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THE JOURNAL OF BIOLOGICAL CHEMISTRY(0 1990 by The American Society for Biochemistry and Molecular Biolog y, Inc. Vol. 265, No. 36, Issue of December 25, pp. 22474-22479, 199OPrinted in U.S.A.

Differential Regulation of Hepatic Triglyceride Lipase and3-Hydroxy-3-methylglutaryl-CoA Reductase Gene Expressionin a Human Hepatoma Cell Line, HepGZ*(Received for publication, March 12, 1990)

Steven J. Busch, Roger L. Barnhart , Gary A. Mart in, Margaret A. Flanagan, andRichard L. Jackson *From the Merrell Dow Research Institute, Cincinnati, Ohio 45215

The level of hepat ic tr iglyceride l ipase (H-TGL) syn-thesis and secret ion was examined in response tochanges in cholesterol biosynthesis in the human hep-atoma cell l ine HepG Z. Cells were f irst fed a l ipopro-tein-def icient serum-supplemented medium to el imi-nate exogenous cholesterol. Mevinolin, a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA ) reductase inhibitor,was then added at a concentrat ion (37 KM ) which in-hibited cholesterol biosynthesis by >85% and de-creased total cel l cholesterol f rom 36.1 to 27.4 pg/mlof cell protein. Mevinolin treatment caused a 4.9 f 0.8-fold increase in the amount of H-TGL act iv ity secretedinto the medium, a 1.8 ? 0.4-fold r ise in H-TGL-spe-cif ic mR NA, and a concurrent 14-fold increase inHMG -CoA reductase mRN A. Addit ion of 1 m M meva-ionic acid to normal or mevino lin-treated cells raisedthe cellular cholesterol content and decreased theamount of secreted H-TGL act iv ity to levels belowcontrol values. Mevalonic acid also prevented mevi-nolin-induction of H-TGL and HMG -CoA reductasemRN A, suggest ing a comm on regulatory step for H-TGL and HMG -CoA reductase. Exposure of cel ls tomevinolin and 25-hydroxycholesterol together re-sulted in a marked repression of HMG -CoA reductasemRN A levels, whereas these condit ions further en-hanced the secret ion of H-TGL act iv ity and the expres-sion of H-TGL mRN A. These results demonstrate adifferential role for 25-hyd roxycho lesterol in the reg-ulat ion of H-TGL and HMG -CoA reductase expression.

The l iver is largely responsible for the metabolism of cho-lesterol and under norm al condition s it is finely tuned torespond to the cholesterol requirements of the body (l-3). Denouo synthesis of cholesterol is regulated by the cellularcontent of sterol and intermediates from the cholesterol bio-synthet ic pathway at the level of the rate l imiting enzyme , 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. Al-though capable of de nouo synthesis, mos t t issues derivecholesterol through receptor-mediated uptake of circulat ing

* The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be herebymarked aduertisement in accordance with 18 U.S.C. Section 1734solely to indicate this fact.$ To whom reprint requests should be addressed: Merrell DowResearch Inst., 2110 E. Galbraith Rd., Cincinnati, OH 45215.

The abbreviations used are: HMG-CoA, 3-hydroxy-3-methylglu-taryl-CoA; H-TGL, hepatic triglyceride lipase; LDL, low densitylipoproteins; HDL, high density lipoproteins; LPDS , lipoprotein-deficient serum; MEM, Eagles minimal essential medium; PBS,phosphate-buffered saline; PIPE S, l-4-piperazinediethanesulfonicacid.

low density l ipoproteins (LDL). An increase in the expressionof the LDL receptor results in lower plasma cholesterol levelsdue to an increase in LDL catabolism (4). The receptor-mediated uptake of LDL is regulated by the cellular contentof cholesterol and, l ike HMG -CoA reductase, LDL receptorgene transcription is repressed as cellular cholestero l in-creases (5). Inhibit ion of cholesterol biosynthesis with mevin-Olin, a potent inhibitor of HMG-C oA reductase (6), results ina marked increase in the expression of the genes for bothHMG-C oA reductase and the LDL receptor.

In addit ion to the receptor-mediated pathway, delivery ofcholesterol to some extrahepat ic t issues has been postulatedto involve a mechanism independent of whole part icle l ipo-protein catabolism. Reaven et al. (7) showed that rat lutealand granulosa cells ut i l ize LDL cholesterol for steroidogenesiswithou t uptake and degradation of the lipoprotein particle.Steroidogen ic glands also utilize high den sity lipoprotein(HDL) cholesterol for steroidogenesis by a mechan ism that isindependent of HDL degradation (8). Several lines of evidencesuggest that hepat ic triglyceride l ipase (H-TGL) plays animportant role in the delivery of HDL cholesterol to the l iverand endocrine organs (9). Jansen and Hii lsmann (10) pro-posed that the H-TGL-catalyzed hydrolysis of HDL phospho-l ipids is associated with an increase in the free cholesterol-to-phospholipid ratio in the lipoprotein particle. As a resu lt, theequil ibrium of cholesterol movem ent into and out of thelipoprotein particle is shifted such that there is a net effluxof the sterol f rom the part ic le and subsequent uptake by thecell. The fact that H-TGL can contribute to cellular choles-terol content may suggest that i t is regulated similarly toHMG-C oA reductase and the LDL receptor.

Recen t ly, we (11) and others (12-14) have reported thecomplete cDN A sequence for rat and human H-TGL . Inhumans, the cDNA codes for a protein of 477 amino acids. Inthe present study, using a human hepatoma cell l ine, HepG2,this cDN A was ut i l ized to measure changes in H-TGL mRN Alevels. Under conditions which perturb cellular cholesterolhomeo stasis, we demonstrate that both H-TGL mR NA levelsand secreted enzyma tic act iv it ies are affected.

EXPERIMENTAL PROCEDURESMuterials-[2-4C]Acetate, sodium salt (53 mCi/mm ol), tri-[l-l%]

oleoyl-glycerol (54.3 mCi/mm ol), [w~P]~AT P (400 Ci/mmol), [a-P]UTP (800 Ci/mmol), and [a-3P]dCTP (400 Ci/mmo l) were pur-chased from Amersham Corp. GeneScreen hybridization transfermembranes were obtained from Du Pont-New Englan d Nuclear.Mevinol in was obtained from Merck. Guanidine thiocyanate waspurchased from Fluka AG (Federal Republic of Germany). Nucleicacid-modifying enzymes, restriction endonucleases, and RNase-freeSephadex G-50 columns were obtained from Boehringer Mannh eim.RNase T1 and all other molecular biology grade reagents were pur-chased from Bethesda Research Laboratories. Elutip-r minicolumn s

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Regulation of Hepatic Lipase 22475were from Schleicher & Schuell. Yeast RNA was from 5-3 (Paoli,PA). Eagles minimal essential medium (MEM), glutamine, and fetalbovine serum were purchased from GIBCO. Silica ge l IB2 Baker-flexthin layer chromatography sheets were from J. T. Baker C hemicalCo. RNase A (5 x crystallized) and all other reagents were obtainedfrom Sigma. LDL and HDL were isolated from normal human plasmaby ultracentrifugation in potassium bromide (KBr) between the den-sities 1.019-1.063 and 1.063-1.210 g/ml, respectively.Hepatom a Cells-HepG2 cells were obtained from American TypeCulture Collection (Rockville, MD) and mainta ined in Eagles MEMsupplemen ted with 10% fetal bovine serum and 2 m M glutamine(maintenance medium ) at 37 C in a humid ified air atmosphere of5% CO,. For passage o f cells, confluent cultures were trvpsinized with0.02% trypsin in 137 m M NaCl, 5 mM KCI, 4 m M NaHCOR, pH 7.4,containing 5 m M glucose and 0.05 mM EDTA. and renlated in 160-cm2 flasks (GIBCO) with maintenance medium . Cell viability wasdetermined by counting the cells in 0.4% trypan blue. Lipoprotein-deficient serum (LPDS) was prepared from fetal bovine serum byultracentrifugation in KBr at a density of 1.21 g/ml. A fter centrifu-gation for 48 h at 214,000 X g, the lipoprotein layer was removed byaspiration and the bottom fraction was dialyzed against phosphate-buffered saline (PBS, 10 mM potassium phosphate, pH 7.4, containing12 0 m M NaCl and 2.7 m M KCI) and sterile-filtered through a Corning0.22-pm cellulose acetate membran e (Corning Glass Works, C orning,NY) prior to use.Measurement of Cellular Cholesterol Biosynthesis-Cholesterol bio-synthesis was determined in cells by measuring the incorporation of[CJacetate into total cholesterol. Confluent monolayers of HepG2cells were established in 36-mm well plates (Falcon) and were fedwith maintenance media. Prior to each experiment, cells were refedwith 10% LPDS-MEM medium for 4 h. Then, mevinolin was addedto the cell cultures for either 2 or 24 h. Cells were pulsed with 1 &iof (C]acetate/well for 4 h. The mediu m was removed, and cellmonolayers were washed 2 times with PBS . Lipids were extracted byadding 2 ml of hexane:isopropanol (3:2, v/v) to each well and allowin gthe extraction to proceed for 2 h at room temperature taking care toavoid evaporation of the solvent. After removing the solvent, eachwell was washed 2 times with 1 ml of the same solvent. The lipidextracts were combined and dried under a stream of nitrogen, andeach pellet was then resolub ilized in 500 ~1 of the same solvent. Ten~1, containing 10 rg of unlabe led cholesterol and 5 rg of unlabe ledcholesterol oleate, added as carriers, were spotted on silica gel TLCstrips and developed in heptane:dieth yl ether:acetic acid (85:14:1, v/v/v). TLC strips were air-dried and stained with iodine vapor tolocate cholesterol and cholesterol oleate; spots corresponding to thesestandards were cut out, and radioactivity was determined . Followin glipid extraction, cell proteins were solubilized in 1 ml of 1 N NaOHand quantitated by the method of Lowry et al. (15) using bovineserum album in as a standard.

Assay for H-TGL Actiuity-H-TGL activity in the HepGP cellculture mediu m was quantitated by measuring the lipolytic activitytowards a substrate of tri-[1-C]oleoylglycerol emulsifie d with TritonN-101 (11). After 24 or 48 h of treatment, the culture mediu m (15ml) was removed and adjusted (to final concentrations) with aprotinin(SO kallikrein units/ml), glycerol (lo%), potassium phosphate (10mM), pH 6.8. Heparin-Sepharose (0.6 ml packed volume) was added,and the sample was incubated overnight at 4 C with g entle mixing.The heparin-Sepharose was pellete d by centrifugation at 2000 x g for15 min (Beckman GPKR centrifuge), and the supernatant fractionwas removed. The pellet was then resuspended in 1 ml of PB S andtransferred to a 6-ml polystyrene column (Kew Scientific, Columbus,OH). The centrifuge tube was rinsed twice with 1 ml of PBS , andthese washes were added to the column. The heparin-Sepharosecolumn was then washed with 4 ml of PBS . and bound H-TGL waseluted with 1.75 ml of 10 mM Tris-HCI, pH 6.8, 1 m M EDTA, 10%glycerol, 0.01% sodium azide, 1.4 M sodium chloride. H-TGL activitywas determined in triplicate as described previously (11).

Cell Total Cholesterol Determination-HepG 2 cells (approximately2 x 10) were trypsinized and transferred to conical 50.ml centrifugetubes. After centrifugation for 10 min at 2000 rpm (Beckman GPKRcentrifuge), the supernatant fraction was removed, and discarded.Cells were washed with two sequential washes of 15 ml of Hanksbuffer (Hanks balanced salt solution, GIBCO). Cells were extractedwith 5 ml of hexane:isopropanol (3:2, v/v) overnight at 4 C. Aftercentrifugation, the supernatant fractions were removed and saved.Cells were then reextracted with 2 ml of solvent for 30 min at roomtemperature. After centrifugation, the two fractions were combined,and the solvent was removed under a stream of nitrogen. Total

cholesterol was determined by gas chromatography after saponifica-tion and extraction with hexane; free cholesterol was determined byomittin g the saponification step. Cholestane was used as the internalstandard. A bonded-phase fused silica capillary column (10 m x 0.32mm inner diameter) coated with a SE-30-type coating (O.l-pm DB-1column; J&W Scientific, Rancho, Cordova, CA) was programmedfrom 80 to 290 C in a mode l 5790A gas chromatograph (Hewlett-Packard, Avondale, PA) equippe d with dual flame ionization detec-tors. The injector port and detector were mainta ined at 300 C, andthe helium linear velocity was 75 cm/s.Synthesis and Isolation of cRNA Probes-Clone pST668, used forsolution hybridization to quantitate H-TGL mRNA transcripts, is a668-bp S&I-SstI insert (from nucleotides 115 to 783) of H-TGLpHL220 (11) ligated into the polylinker Sat1 site of the vector, pSPG/T7-19 (Bethesda Research Laboratories). Antisense mRNA (cRNA)probes were synthesized on an isolated linear PuuII-EcoRI fragmentfrom the pST668 plasmid, containing the entire 668-bp internal H-TGL cDNA insert and the T7 polymerase bindin g site. These labe ledtranscripts were synthesized according to the recommendations ofthe manufacturer (Boehringer Mannhe im) with the followin g modi-fications. The synthesis was carried out in 50 pl containing 0.25 pgof DNA template and a final molar ratio of [(Y-P]UTP (800 Ci/mmol) to cold UTP of 7:12. The DNA template was removed byRNase-free DNase I digest at 37 C for 60 min. The cRNA probe wasthen precipitated overnight at -20 C in 0.3 M sodium acetate and2.5 volumes of ethanol. The cRNA probe was further purified (16) byloading onto a Schleicher & Schuell Elutip-r minicolumn in 1 ml ofloading buffer (0.5 M NaCl, 10 m M Tris acetate, pH 7.5, 1 m M EDTA)with 25 m M dithiothreito l. The column was washed 3 times with 0.5ml of loading buffer, and the RNA probe was eluted with 400 pl of 1M NaCl. The isolated probe was concentrated by adding 2 volumes ofethanol to the eluate and precipitated on dry ice. The H-TGL [,P]cRNA probe was resuspended in 80% deionized formamide (specificactivity = 2.02 X 10 cpm/pg).

Measurement of H-TGL mRNA Transcript Levels b.y Solut ion Hv-bridization-Track excess solution hybridization was carried omessentiallv as described bv Lee et al. (17). Total cellular RNA wasisolated from HepG2 cells by the guani dinium isothiocyanate proce-dure followe d by centrifugation over cesium chloride (18). The totalcellular RNA pellet was resolubilized in water and passed through aRNase-free Sephadex G-50 column prior to determin ing the nucleicacid concentration by absorbance at 260 nm.

The hybridization assay contained increasing amounts of HepG2cellular RNA (O-100 fig) and 200 pg of ,P-labeled cRNA dried downin a 1.5ml siliconized and diethyl pyrocarbonate-treated Microcen-trifuge tube. The reactions for standard curves includ ed increasingamo&ts of single-stranded M13m p18 DNA containing the full-lengthH-TGL cDNA insert from nHL 220 in mRNA sense orientation. 200pg of P-labeled cRNA, and total yeast RNA giving a final nucleicacid concentration of 100 rg/tube. Each RNA mixture and standardcurve reaction tube content was resolubilized in 10 kl of deionizedformamide, 6 ~1 of water, and 4 ~1 of a 5 x reaction buffer (2 M NaCl,5 m M EDTA, and 125 m M PIPE S, pH 6.8), and then covered with 50~1 of light mineral oil, heated for 5 min at 85 C, and incubated for20 h at 50 C. After incubation, the mineral oil layer was removed,and 300 ~1 of 2.5 X SET (375 m M NaCl. 75 m M Tris-HCl. DH 8. 5m M EDTA) containing 20 pg of RNase A and 700 units of RNase T,were ad ded to each tub e and incubated for 60 min at 37 C. TheRNase-resistant hybrids were precipitated by adding 300 fig of totalyeast RNA and 370 ~1 of cold 10% trichloroacetic acid and incubatedon ice for 15 min. The mixture was then collected on Whatman GF/C filters, washed with 20 ml of cold 5% trichloroacetic acid, dried,and radioactivity was determined in Beckman Ready-solvTM. Thecounts/min of the RNase-resistant 32P-labe led cRNA-mRNA hvbridwas plotted uersas tota l HepG2 RNA (m). The slope of the line wasused to calculate the number of H-TGL transcriptsjHepG2 cell basedon the mass of total RNA/HenG2 cell of 6.9 PE (19) and the nrobespecific activity (202 cpm/pg) (determined by the standard curve, seeinset Fig. 2).

Measurement of H-TGL and HMG-CoA reductase mRNA by North-ern Blot Analysis-Northern blots of HepG2 R NA were preparedusing 1.1% agarose-formaldehyde gels (18) to size-fractionate 50 figof total RN A/lane. The same quantity of RNA was loaded on eachlane as estimated by A&A? Ro ratio of 1.8 or greater. In addition , afterelectrophoresis a visual inspection of the 28 S and 18 S ribosomalRNA bands were made to assess the quantity of total RNA/lane .RNA was transferred to GeneScreen membranes according to theprocedures of the manufacturer. The 1.6-kilobase total H-TGL cDNA

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22476 Regulation of Hepatic Lipaseinsert pHL220 (11) was labeled by nick translat ion to a specif icact iv ity of 3 X lO* cpm/pg. The measurement o f H-TGL mRNA byNorthern blot analysis was performed as described previously (11)using hybridizat ion condit ions of Busch et al. (20). A probe for HMG-CoA reductase mR NA was isolated by cutt ing a 2.5-ki lobase BglI If ragment containing the majority of the human HMG -CoA reductasecDNA (21) from pHRed-102 (ATC C 57042) and purify ing the frag-ment by 2 cycles o f preparative gel electrophoresis. This fragmentwas then labele d by nick translation with [a-P]ATP and [,I-PICTP to a specif ic act iv ity of l-2 X lo9 cpm/F g. The Northern blotsprepared for measurem ent of H-TGL mR NA were used for thequant itation of HMG -CoA reductase mRN A under the fol lowingcondit ions: prehybridizat ion and hybridization were performed at42 C in 0.2% bovine serum albumin, 0.2% polyvinylchloride, 0.2%Ficoll , 50 m M Tris-HCl, pH 7.5, 0.1% sodium pyrophosphate, 1.0%sodium dodecyl sulfate, and 50% formamide; prehydriziat ion wasperformed for 4 h and hybridization for 48-72 h . The f inal wash ofthe blots was at 65 C for 45 min in 0.1 X SSC (1 x SSC = 0.15 MNaCl, 0.015 M sodium citrate, p H 7 .0), 1% sodium do decyl sulfate.

RESULTSThe incorporat ion of [14C]acetate into de rzouo-synthesizedcholesterol in HepG2 cells increased by 113% (from 16,654 to

35,553 cpm /mg of cell protein) in cells fed with LPDS-sup-plemented medium in place of fetal bovine serum supple-mented medium demonstrat ing that l ipoprotein deprivat ioninduces cholesterol biosynthesis in these cells. As expected,treatment of these l ipid-deprived HepG2 cells with the HMG -CoA reductase inhibitor, mevinolin, caused a decrease incholesterol biosynthesis (Fig. 1). The concentrat ion of mevi-nolin giving max imal inhibition (~35%) o f cholestero l biosyn-thesis after 6 h of exposure was 37 pM . In a separate experi-ment, cel ls were treated with 37 pM mevinolin for 24 h; thiscaused an inhibit ion of cholesterol biosynthesis by 85.1 f2.1% (mean f S.E., n = 3). This concentrat ion of mevinolinwas suff ic ient to block cholesterol biosynthesis for at least 48h without signif icant loss of viabil ity provided the mediumwas changed at 24-h intervals. Cell v iabil i ty with m evinolinwas 99.2 f 0.8 and 97.3 t- 0.6% at 24 and 48 h, respect ively(n = 3). Next, w e examined the cellular product ion of H-TGLwith the experimental condit ions in which cells were deprivedof exogenous l ipid and cholesterol biosynthesis was inhibited.Mevinolin addit ion to the cells was invariably associated withan increase in secreted H -TGL act iv ity. In nine separateexperiments (Fig. 2), H-TGL secreted act iv ity increased 4.9-C O.&fold (mean f S.E.). The increase in secreted H-TGLact ivity was accompanied by an increase in H-TGL mR NA

2 oL-7--r r -71-log [Mevinolin]

FIG. 1. Inhibiti on of cholesterol biosynthesis in HepG2 cellsby mevino lin. Confluent HepG2 cells were grown in maintenan cemedium. Medium was then replaced with glutamine-supplementedMEM containing 10% LPDS prepared as described under Experi-mental Procedures, and cells were incubated. After 4 h, mevino linwas added a nd the incubation continued for 2 h. After 2 h, [Clacetate was added, and following an additional 4 h of incubation theincorporation of radioactivity into cholesterol was determined asdescribed under Experimental Procedures. The results representthe mean of three separate analyses at each concentration of mevi-nolin.

0L784 bb,02y P

02

FIG. 2. Effects of mevin olin on H-TGL expression. HepG2cells were grown in glutamine-supplemented MEM containing LPDSfor 48 h and then treated with me dium alone (0) or medi um plusmevinolin (37 pM , 0) for 48 h. Left, example of solution hybridizationof total HepG2 RNAs for determin ing the number of H-TGL mRNAmolecules/HepG2 cell. Actual transcript number was determine dfrom the slope of the line and specific activity of cRNA probe usedas described under Experimental Procedures. Bar graph (right)represents mevinolin-ind uced H-TGL mRN A and secreted enzymeactivity over control for nine experiments (mean + S.E.). Controlactivities were 28 + 8.0 nmo l of oleic acid/h/culture. ss, single-stranded.

levels. The mR NA level, expressed as copy number/cell , wasdetermined by solut ion hybridization techniques as describedunder Experimental Procedures. Fig. 2 ( inset) shows thestandard curve used to quant itate the specif ic act iv ity of theprobe. For this representat ive experiment, mevinolin inducesa 4.8-fold increase (from 2.5 to 12.1 transcripts/cell) in H-TGL-specif ic mR NA. The copy number for mevinolin-treatedcells was averaged for the same nine experiments that wereused to quant itate H-TGL secreted a ct iv ity and is expressedrelat ive to control levels in Fig. 2. On average, a 1.8 + 0.4-fold increase in H-TGL specif ic mR NA was observed after 48h of mevinolin treatment.

Induct ion of H-TGL mRN A and secreted lipolyt ic act iv itywith cholesterol deprivation also correlated with an increasein apparent secreted mas s of H-TGL as observed by Westernblot analysis (Fig. 3). In this experiment, cel ls were fed LPDSfor 48 h (with refeeding every 24 h). Then, the medium wasremoved, and the cells were refed LPDS with or withoutmevinolin (37 gM). After 24 h, the medium from six f lasks ofcontrol cel ls (LPDS only) or six f lasks of mevinolin-treatedcells were extracted with heparin-Sepharose, H-TGL waseluted, and enzyme act iv ity determined. The Western blotwas developed using the H-TGL carboxyl-terminal-specificant ibody, P3, as described by Busch ef al. (22). As is shownin Fig. 3, the elut ion prof i les as measured by H-TGL act iv it iesindicate a 2.2-fold elevat ion in secreted act iv ity in mevinolin-treated cells. H-TGL immunoreact ive protein wa s greater inthe mevinolin-treated cells than in the control cel ls.

Next, we determined whether addit ion of mevalonic acid,the product of the HMG -CoA reductase-catalyzed react ion,could reverse the mevinolin-induced increase in H-TGL se-cret ion. When me valonic acid (1 m M) was added alone or incombinat ion with mevinolin (37 PM), there was a 53 and 65%decrease, respect ively, in secreted H-TG L act iv ity after 48 h(Table I) when compared to LPDS-fed control cel ls. Theseresults demonstrate that in bypassing the inhibited step andsupplying substrate for cholesterol b iosynthesis, H-TGL ac-t iv ity was repressed to below control levels. There was nodetectable change in H-TGL mR NA levels after treatmentwith mevalonic acid (data not shown) indicating that a post-transcript ional event may have been affected. In these exper-iments, the cholesterol content of HepG2 cells was measured

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Regulation of Hepatic Lipase2 12345676

?1 2 3 4 5 6 7 a 9Fract ion Number

FIG. 3. Mevino lin (Mev)-induced expression of H-TGLlipolytic activity correlates with increased p rotein mass.Twelve flasks of -60% confluent HepGP cultures were fed 15 ml ofLPDS supplemented medium for 48 h with changes in media every24 h. Immediately following the final refeeding, 6 of the 12 cultureswere adjusted to 37 pM mevin olin and a mixture of protease inhibitorswas added as previously described (22). Media for the six controlflasks also included the protease inhibitor mixture. After 24 h, medi afrom the control flasks or the six mevinolin-trea ted flasks were pooled(90 ml, final volum e) and extracted with 5 ml of heparin-Sepharos eas described under Experimenta l Procedures. Individu al l.O-mlfractions were eluted in 1.4 M NaCl elution buffer, and 175-J aliquotswere assayed for lipolytic activity in the medi um from control cells(0) or mevinolin-treate d cells (0). A Western blot analysis (inset) ofeach fraction was performed as described previously (22) on theremaining 0.82 ml.

TABLE IEffect of meuinolin and meualonic acid on HepG2 cell cholesterol

content and H-TGL secreted activityConflue nt cells were cultured for 2 days in MEM containing 10%

LPDS. Then, the medium was changed and mevalonic acid (1 mM )or mevinolin (37 pM ) or a combina tion, as indicated, was added. After24 h, the medium was removed and fresh m edium containing theappropriate additions was added. After another 24 h, both the mediumand the cells were collected and H-TGL activity and cell total cho-lesterol content were determine d as described under ExperimentalProcedures. The numbers in parentheses under Cell cholesterolare percentages of the untreated control. H-TGL activity is expressedas nanom oles of oleic acid released per h/15 m l of culture med ium(mean + S.D., n = 3 determinationsiculture).

Addi t ion Cel l protein Ce ll choles terol H-TGL ac tivityQ? g/mg cell pro.tein (%I

Untreated 10.8 36.1 (100) 42.4 + 1.8Mevalonic acid 13.2 45.3 (126) 20.2 f 6.0Mevinolin 10.2 27.4 (76) 169.0 + 29.0Mevinolin + mevalonic 17.2 51.4 (142) 15.3 + 1.1

acid

in order to determine the relationship between cellular cho-lesterol and H-TGL secretion (Table I). Mevalonic acid, whenadded either alone or with mevinolin, increased cell choles-terol content by 26 and 42%, respectively, suggesting thatcellular cholesterol content correlates inversely with H-TG Lsynthesis. In the absence of exogenous cholesterol, treatmentof cells with mevinolin reduced cellular cholesterol contentby 24% from 36.1 to 27.4 pg of total cholesterol/mg of cellprotein and increased H-TG L secretion by 4-fold (from 42 to169 nmol of oleic acid released per h/15 m l). Taken together,these results suggest that H-TGL expression is inverselycoupled to cellular cholesterol or to som e intermediate in thecholesterol biosynthetic pathway.

In the next experim ent, the time required for mevinolininduction of H-TGL was determined. Mevinolin was addedto HepG2 cells, and the amount of secreted H-TGL activity

and relative abundance in H-TGL mR NA were determinedby solution hybridization as a function of time (F ig. 4). Amaximal increase in secreted H-TGL activity and mR NAoccurred after 24 h of mevinolin treatm ent. By 32 h, the levelof secreted lipolytic activ ity had decreased in both controland mevinolin-treated cells while the amou nt of H-TG LmR NA had leveled off. These results demonstrate that amaximum in H-TGL induction due to mevinolin treatmentalone is observed by 24 h. Comparison of these results withprevious experiments indicated that the LPDS medium mus tbe changed every 24 h for max imal cell viability for bothcontrol and treated cells, and the fall in secreted a ctivities istherefore related to the LPDS medium and not to the mevi-nolin treatment.I t is well established that the level of HMG -CoA reductasemR NA is markedly induced by mevinolin treatment (23, 24).The addit ion of LDL or oxysterols to cells results in a subse-quent rise in cellular oxys terols and a strong repression ofHMG-C oA reductase mR NA levels (1, 2). We therefore ex-amined the effec t of lipoprotein feeding on H-TG L expressionin the absence or presence of mevino lin. The addition of LDL(30 pg of protein/ml) or HD L (100 pg of protein/ml) alonehad no consistent signif icant effect on the expression of H-TGL in HepG2 cells grown on LPDS (data not shown). Incontra st, the combination of both LDL and mevinolin inducedH-TGL secretion above that observed for mevinolin alone.Fig. 5 show s that in this experiment the addition of mevinolinto HepGP cells increased H-TGL secreted activity 2.4-fold.Incubation of mevinolin-treated HepG2 cells with LDL (30pg of protein/ml) further increased the amoun t of secreted H-TGL into the medium by 43% over mevinolin alone withouta signif icant increase in mR NA levels (Fig. 5). Mevinolinalone caused a 14-fold increas e in HM G-Co A reductasemR NA, and LDL addit ion with mevinolin reduced this levelby approxim ately 50% in 48 h (6.8-fold over control levels,Fig. 5, bottom). The synergist ic response to mevinolin andLDL required more than 24 h to occur, which suggests that it

" 50'; I12 24Time (hr)

FIG. 4. Time course for the effect of mevin olin treatmenton H-TGL expression. HepG2 cells were grown in maintena ncemedium. At time 0, the medium was removed and replaced with MEMcontaining 10% LPDS and 2 mM glutamine in the presence andabsence of mevin olin (37 PM). Flasks were harvested at the indicatedtimes, and secreted H-TGL activity (top panel) was determin ed forcontrol cells (0) and mevinolin-trea ted cells (O), as described underExperimen tal Procedures. H-TGL m RNA levels (bottom panel) foruntreated control cells (0) and mevinolin-treate d cells (0) weremeasured by solution hybridization.

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22478-Lc

Regulation of Hebatic Lipase

Control Mevinolin !+ievinolinii,.

*~~HMGCoAReductase

FIG. 5. Effect of mevino lin and LDL on H-TGL expression.Preconfluent HepG2 cells (-60%) were grown in MEM containing10% LPDS and 2 mM glutamine. Cells were refed every 24 h. After48 h, medi um was removed and replaced with fresh med ium (Control),medium containing mevinolin (37 pM) or medium containing mevi-nolin (37 pM) plus LDL (30 pg of protein/ml). H-TGL activity wasdetermined after a 48-h incubation in which medium was changedevery 24 h (n = 3 flasks/treatment). Relative mRNA levels of H-TGL and HMG-CoA reductase were determine d by Northern blotanalysis as described under Experimental Procedures. Bottom, theNorthern blot was first hvbridized with the H-TGL cDNA nrob e.Then, the probe was removed and the blot was rehybridized with theHMG-CoA reductase probe. Relative differences in mRNA levelswere determined by scanning densitometry as described previously(20).is not cholesterol itself but possibly oxysterols which areaffecting H-TGL expression. Moreover, since 24 h was re-quired to observe this effect suggests that LDL were notsimply stabilizing secreted H-TGL resulting in improved ac-tivity recoveries.To examine the effects of oxysterols themselves on H-TGLexpression, 25hydroxycholesterol was added to the cells inculture, and H-TGL enzymatic activity and mRNA levelswere determined. The addition of 25-hydroxycholesterol re-duced the amount of HMG-CoA reductase mRNA to unde-tectable levels even in the presence of mevinolin (Fig. 6,bottom). 25-Hydroxycholesterol alone produced a small butreproducible induction of secreted lipolytic activity but with-out an apparent increase in hybridizable H-TGL mRNA inthe same time frame (data not shown). In contrast, when 25-hydroxycholestero l and mevinolin were added simultaneously,H-TGL lipolytic activity and H-TGL mRNA levels werefurther induced beyond the levels observed for mevinolinalone (Fig. 6). These results suggest that repression of H-TGL expression is sensitive to the level of HMG-CoA reduc-tase or the concentration of intermediates of the cholesterolbiosynthe tic pathway at or beyond the level of mevalonic acid.

DISCUSSIONThe level of hepatic cellular cholesterol is controlled by thecoordinate regulation of biosynthesis, uptake from exogenoussources supplied through the LDL receptor-mediated path-way, and the nonreceptor-mediated uptake of LDL and HDLcholesterol. These results using a hepatoma cel l line showthat H-TGL expression is regulated as an integral part ofthese same regulatory pathways. Exposure of HepG2 cells tomevinolin, a potent inhibitor of the rate-limiting enzyme ofthe cholestero l biosynthetic pathway, caused an increase inthe level of secreted H-TGL lipolytic enzyme with a concom-itant increase in the level of H-TGL mRNA. A similar in-

H-XL -

HMGCoAReductose s

FIG. 6. Effect of mevino lin and 25-hydroxycholesterol onH-TGL expression. Preconfluent HepG2 cells (-60%) were grownin MEM supplemented with 10% LPDS and 2 mM glutamine. Cellswere refed every 24 h. After 48 h, cells were refed with fresh mediu m(Control) or mediu m containing mevinohn (37 PM) or mevinohn (37pM) plus 25-hydroxycholesterol (25-HC, 50 pg/ml). After 24 h, me-dium was removed and assayed for H-TGL activity (bar graph) (n =3, flasks/treatment). Northern blots of total HepG2 R NA were per-formed (bottom) as described in Fig. 5.

crease was observed for HMG-CoA reductase mRNA levelsin cells treated with mevinolin and is consistent with p reviousreports (23, 24). The increase in H-TGL mRNA, secretedprotein, and activity in the presence of mevinolin suggeststhat H-TGL may respond to regulatory controls similar tothat for HMG-CoA synthase, HMG-CoA reductase, and theLDL receptor (24).We have demonstrated in these studies that mevalon ic acid,in e ither the presence or absence of mev inolin, causes a 65 or53% decrease, respectively, of secreted H-TGL activity withno effect on H-TGL mRNA levels . Although its mechanismis unknown, the data are consistent with the suppression ofsecretion being mediated by a metabolite of melavonic acid.One possibility is the activation of a suppressor activity byisoprenylation. Several recent studies have demonstrated thesens itivity of protein isoprenylation and their subsequentfunction to inhibition of mevalonate biosynthesis by mevi-nolin (25-28). For example, the maturation and processing ofnuclear lamin A is sens itive to mevinolin treatment; additionof mevalonic acid to mevinolin-treated cells leads to lamin Aisoprenylation and maturation (25). The activation of c-H-r a SVd .1 2 in oocytes is blocked by the addition of mevinolin andagain restored by the addition of mevalonic acid (26). Inde-pendently, it has been shown that the ras gene productundergoes farnesylation as an integral step in its insertioninto the cell membrane (27). Lastly, there is evidence forposttranscriptional regulation of HMG-CoA reductase bynon-sterols derived from mevalonic acid (24, 29). It has beenproposed that the sens itivity of HMG-CoA reductase activ ityto mevalonic acid is mediated through the interaction of afarnesylated protein with the membrane-inserted reductasemolecule (24) or through modification of the reductase itselfat the consensus farnesylation site CAAX, where C is cysteine,A is any aliphatic residue, and X is any amino acid at thecarboxyl terminus (26). Farnesylation of a regulatory proteinin a simi lar manner may be required for H-TGL suppression.It is unlikely that H-TGL itself is farnesylated since it lacksthis consensus sequence at its carboxyl terminus. However, if

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Regulation of Hepatic Lipase 22479the posttranslat ional regulation of H-TGL secret ion is underthe control of a metabolite of mevalonic acid, then it mightbe regulated by isoprenylation of a regulatory protein.

Finally, we have shown in HepG2 cells that H-TGL mRN Alevels and secret ion are further enhanced by the addit ion of25-hydroxycholesterol or LDL to mevinolin-treated cells. 25-Hydroxycholesterol and LDL act through a comm on mecha-nism to down-regulate the transcription of the gene for HMG -CoA reductase (24). H-TGL expression, as measured bysteady-state mR NA levels, is insensit ive to the oxysterol andLDL by themselves . In contrast, in combinat ion with mevi-nolin, 25-hydroxycholesterol and LDL act synergist ical ly toincrease H-TGL expression and secret ion. Th ese data arguefor at least two points of regulation of H-TGL expression.One appears to be responsive to a metabolite of mevalonicacid, perhaps also mediated by isoprenylation. The other canonly be discerned in the absence of mevalonic acid synthesisand is responsive to the level of sterols, such as LDL-derivedcholesterol and oxysterols, or 25-hydroxycholesterol. I t haspreviously been shown that the regulation of the HMG -CoAreductase gene is sensit ive to both a mevalonate-derived com-ponent and a sterol component (24) and that both are neces-sary for the complete repression of the HMG-C oA reductasegene. The fact that the effect of sterols on H-TGL expressioncan only be seen in the absence of mevalonic acid synthesissuggests that the mevalonate-derived component is a repres-sor of H-TGL synthesis, and the sterol component is anact ivator only in the absence of mevalonate. Thus, we con-clude that the expression of the H-TGL and HMG -CoAreductase genes is similarly regulated by the concentrat ion ofmevalonate-derived metabolites, but dif ferent ial ly regulatedby the concentrat ion of sterols such as 25-hydroxycholesterol.

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