gene expression in HepC2 and Caco2 cells by palmitate, oleate, and 25-hydroxycholestero11

Transcription

1 Regultion of low density lipoprotein reeptor gene expression in HepG nd Co ells by plmitte, olete, nd 5-hydroxyholestero11 Ri Ajit K. Srivstv,' Hiroo I ~O,~ Mtthis Hess,4 Neelm Srivstv, nd Gustv Shonfeld Division of Atheroslerosis, Nutrition nd Lipid Reserh, Deprtment of Internl Mediine, Wshington University Shool of Mediine, 66 S. Eulid Avenue, Box 846, St. Louis, MO Abstrt Our in vivo studies in mie hve shown tht LDLreeptor gene expression is regulted differently in both liver nd intestine by dietry holesterol nd dietry sturted ft. While dietry holesterol serves to regulte t trnsriptionl levels, dietry ftty ids do not. To study the mehnism of regultion of LDL-reeptor by sturted ft nd holesterol t the ellulr level, where ny seondry effets of long-term feeding in vivo re minimized, we used the ultured heptom nd olon rinom ells, He G nd Co. LDL-reeptor tivity ws determined by 1y51-lbeled LDL binding nd uptke, LDLreeptor protein by Western blotting, LDLreeptor mrna by RNse protetion ssy, nd reltive rtes of LDL-reeptor mrna trnsription by nuler 'run-off ssy. Inubtion of ells in lipoproteindefiient serum (LPDS) for 48 h progressively indued LDLreeptor tivity nd LDL-reeptor protein by 5- to 6-fold in HepG ells nd - to 3-fold in Co ells. Absolute levels of LDL-reeptor mrna nd reltive rtes of LDL-reeptor mrna trnsription lso inresed in prllel to the LDLreeptor tivity nd protein levels in both ell lines. These dt suggest tht LPDS indued the LDLreeptor gene by trnsriptionl mehnism. The suppressive effet of 5-hydroxyholesterol on LDL-reeptor regultion ws studied by inubting HepG nd Co ells grown either in 1% FCS or 1% LPDS for 4 h nd then for -4 h with vrious doses of 5-hydroxyholesterol. In HepC ells, LDL-reeptor tivity nd protein mss progressively deresed to 5% of zero time ontrols over 4 h. LDLreeptor mrna levels nd reltive rtes of trnsription deresed in prllel. In Co ells, 5-hydroxyholesterol lowered LDL-reeptor tivity, mrna, nd trnsription by -35%. To exmine the effets of plmitte on LDL-reeptor regultion, plmitte ws omplexed with lbumin. Plmitte deresed LDL-reeptor tivity by 5% in HepC ells without ltering LDL-reeptor mss, mrna levels, or rtes of mrna trnsription. Similrly, in Co ells, plmitte deresed LDL-reeptor tivity nd protein mss 3% of ontrols, but did not hnge LDL-reeptor mrna levels nd/or rtes of trnsription. The ombintion of plmitte (.8 mm) nd 5-hydroxyholestero1 (.5-5 pg,"l) suppressed LDL-reeptor tivity by 65% in HepG ells nd by 5% in Co ells. However, LDL-reeptor mrna deresed by -5% in HepC ells nd 3-4% in Co ells. Thus, there were further dereses in LDL-reeptor tivity nd mrna levels when plmitte nd 5-hydroxyholesterol were present together in the medi s ompred to 5-hydroxyholesterol lone. Olete did not ffet LDL-reeptor tivity. 811 Thus, ) exogenously dded 5-hydroxyholesterol regultes LDL-reeptor gene expression by trnsriptionl mehnism, the mplitude of regultion being greter in HepC thn in Co ells; b) exogenously dded plmitte regultes LDL-reeptor gene expression by posttrnsriptionl mehnism, possibly t the posttrnsltionl level; nd ) ombintion of holesterol nd plmitte hve dditive effets on the regultion of the LDL-reeptor gene in prt vi posttrnsriptionl nd in prt vi trnsriptionl mehnismssrivstv, R. A. K., H. Ito, M. Hess, N. Srivstv, nd G. Shonfeld. Regultion of low density lipoprotein reeptor gene expression in HepC nd Co ells by plmitte, olete, nd 5-hydroxyholestero1.J Lipid Res : Supplementry key words LDLreeptor - plmitte - 5-hydroxyholesterol HepC Co Elevtion of low density lipoproteins (LDL) levels in irultion is one of the mjor risk ftors for oronry rtery disese (1). LDL levels re determined by blne between rtes of prodution nd rtes of lerne of LDLs from irultion. The mjority of plsm LDL lerne ours vi LDL-reeptor-medited uptke by Abbrevitions: LDL, low density lipoprotein; FCS, fetl lf serum; LPDS, lipoproteindefiient serum; LDL-R, low density lipoprotein reeptor; PBS, phosphte-buffered sline; BSA, bovine serum lbumin; DMEM, Dulbeo's modified Egle's medium; DTT, dithiothreitol; TCA, trihloroeti id; EDTA, ethylenedimine tetr eti id; EGTA, ethylene glyol tetr eti id. 'Presented in prt t the 66th Sientifi Session of the Amerin Hert Assoition, November 1993, New Orlens.?To whom orrespondene should be ddressed. :'Present ddress: 1st Deprtment of Internl Mediine, Teiko University Shool of Mediine, , Kgd, Itbshi-ku, Tokyo 173, Jpn. 4Present ddress: /o Prof. Dr. Autr Wlli, Institut fur Klinishe Chemie, Klinium Grosshdern, Mrhioninistr. 15,81377 Munhen, Germny. Downloded from by guest, on June 19, Journl of Lipid Reserh Volume 36, 1995

2 heptoytes. The onentrtions of these lipoprotein prtiles re influened by nutritionl nd hormonl stimuli (-5). For exmple, dietry holesterol nd sturted ft n seprtely nd together elevte LDL holesterol in humns, nd ltertion of hepti LDL-reeptor tivities is one wy tht dietry lipids n hnge plsm LDL onentrtions. Dietry lipids gin ess to heptoytes vi hylomiron remnnts. Dietry holesterol is thought to derese hepti LDL-reeptor protein nd tivity by trnsriptionl mehnisms in vivo (, 5). Similrly, in ultured ell lines exogenously dded holesterol or oxidized holesterol derivtives effiiently regulte LDL-reeptor tivity (6-8). This regultion is medited by the intertions of steroid reeptor element in the promoter region of the LDL-reeptor gene with steroid reeptor element binding protein (9). Diets high in sturted fts in the bsene of dietry holesterol lso inrese plsm LDL-holesterol levels (, lo), while onomitntly, LDLreeptor tivities deline in livers. The regultion by ftty ids ppers to be posttrnsriptionl ( 1). Combintions of dietry sturted ftty ids nd dietry holesterol hve dditive effets on plsm LDL levels (11, 1). It hs been suggested tht the dietry sturted ftty id-holesterol ombintion my regulte LDL-reeptors by ltering sizes of puttive intrellulr regultory pools of holesterol nd hene presumbly LDL-reeptor trnsription (1). Our hypothesis ws tht holesterol nd ftty ids, when dministered seprtely, operted t different loi to regulte LDL-reeptor gene expression. But when dministered together, holesterol nd sturted ftty ids ould operte through similr moleulr mehnisms, e.g., regultion of mrna levels. We used ultured ells in order to minimize ny seondry effets due to dpttions tht my our in whole orgnisms in vivo. Both HepG nd Co ells were used beuse they re dequte models of humn heptoytes nd enteroytes, respetively, i.e., they serete lipoproteins nd polipoproteins, nd their LDL-reeptors re regulted by physiologi perturbtions (8, 13, 14), nd more importntly, beuse LDL-reeptors of liver nd smll intestine ppered to respond dissimilrly to dietry or hormonl perturbtions (,4, 5). (FCS, J. R. Sientifi, Woodlnd, CA),.1 mm nonessentil mino ids, mm L-glutmine, 1 U/ml peniillin, nd 1 pg/ml streptomyin. Identil medi were used for both HepG nd Co ells in order to filitte omprisons between the two ell lines. Cells were fed fresh medium every dys nd mintined in humidified inubtor equilibrted with 5% COd95% ir. Cells were plted t density of.5-3 x 15/1 m in m tissue ulture flsks (Costr, Cmbridge, MA). When the ell lyers rehed bout 8% onfluene, the ells were dissoited by inubtion with.5% trypsin/.% EDTA for 5 min t 37 C nd pssged t split rtio of pproximtely 1:lO. Cells were plted on 3.5" &well plsti dishes (Costr, Cmbridge, MA) for 15I-lbeled LDL uptke nd binding studies, on 1-mm tissue ulture dishes (Corning Glss Works, Corning, NY) for ell membrne preprtions for LDL-reeptor immunoblotting, or in -mm Costr flsks for mrna quntittion. When the ells were 8% onfluent, the mintenne medium ws removed, monolyers were wshed twie with phosphte-buffered sline (PBS), nd DMEM ontining either 1% FCS or 1% lipoproteindefiient serum (LPDS) ws dded to eh dish. After inubtion for 6-48 h ellulr LDL-reeptor protein nd tivities, mrna levels, nd trnsription rtes were determined. To test the effets of 5-hydroxyholesterol nd ftty ids, ells were preinubted either in 1% FCS or 1% LPDS for 4 h, then ells were wshed twie with PBS nd inubted for the indited times in serum-free DMEM supplemented with one of the following dditions to give the finl desired onentrtions of 5-hydroxyholesterol in.5% ethnol with or without plmitte or olete omplexed to BSA; or.16 mm BSA lone. Plmitte or olete (Sigm Chemil Co., Sint Louis, MO) were omplexed with ftty id-free BSA (Sigm Chemil Co., MO) s previously published (15). Most frequently BSA present t.16 mm, but onentrtions were vried to obtin desired ftty id/bsa molr rtios. Complexes were still-filtered before use. As 5- hydroxyholesterol ws dissolved in ethnol, the sme mount of ethnol (.5% finl onentrtion) ws dded to the ontrol ells. Ethnol did not ffet ellulr protein ontents per dish. Downloded from by guest, on June 19, 18 Cell ulture MATERIALS AND METHODS HepG ells were obtined from the Amerin Type Culture Colletion t pssge 77 (ATCC, Rokville, MD). Co ells (pssge 41) were generous gift from Dr. Jeffrey Field, University of Iow. Stoks of both ell lines were grown in Dulbeo's modified Egle's medium (DMEM) supplemented with % fetl lf serum 151-lbeled LDL uptke by ultured ells LDL nd LPDS were prepred by ultrentrifugtion of serum obtined from helthy humn subjets using the method of Hvel, Eder, nd Brgdon (16). LDL ws iodinted with NlZ5I (17). More thn 95% of the rdiotivity ws preipitble with 1% trihloroeti id (TCA). Speifi tivities of '5I-lbeled LDL vried between 3 to 5 pm per ng of protein. Sriustu et l. LDL reeptor gene expression in HepG nd Co ells 1435

3 15I-lbeled LDL binding nd uptke by the ells were determined in ells grown in 35-mm dishes (18). Cell medi were s indited for individul experiments. Then, 1 ml of DMEM ontining mg/ml of BSA nd 5 pg/ml of lz5i-lbeled LDL in the presene or bsene of 5 pg/ml of ntive LDL were dded. Inubtions t 4 C or t 37 C lsted for 3 h. The medi were then removed nd ll subsequent opertions were rried out t 4 C. Eh ell monolyer ws wshed three times with ml of buffer A ontining 5 mm Tris-HC1 (ph 7.4) buffer,.15 M NCl, nd mum1 BSA, fter whih further ml of buffer A ws dded nd the monolyer ws wshed finlly with ml of buffer ontining 5 mm Tris-HC1 (ph 7.4) nd.15 M NCl. The ells were then dissolved in 1 ml of.1 N NOH nd olleted for lz5i ounting in gmm ounter nd for the mesurement of ell protein onentrtions by the method of Lowry et l. (19). Cell membrne preprtion For LDL-reeptor immunoblotting, ells were grown on 1-mm ulture dishes. After inubtion with the test substrtes for the indited times, ells were pled on ie, wshed three times with.15 M NCl t 4"C, srped from the inubtion dishes with rubber poliemn, nd briefly entrifuged. The ell pellet ws suspended in 1-fold (ompred with pellet volume) exess of reeptor solubilizing buffer ontining 1.6% Triton X- 1,.3 mm leupeptin, 5 M ure, nd 1.5 mm phenylmethylsulfonyl fluoride (PMSF). After homogeniztion of the ells on ie in 1.5 ml tissue homogenizer nd inubtion on ie for 3 min, the resulting suspension ws entrifuged in Bekmn TL-1 Ultrentrifuge t 1, g for 1 min. The superntnt ws removed nd stored in liquid nitrogen (). LDCreeptor protein quntifition Six perent polyrylmide seprtion gels nd 3% polyrylmide stking gels, eh ontining.1% SDS, were prepred s desribed (4). Solubilized ellulr membrnes were mixed with solution of.5% SDS, 5% glyerol, nd.5% bromophenol blue in rtio of 4: 1. One hundred mirogrm of totl HepG ell membrne or pg of Co ell membrne protein were eletrophoresed without preheting nd in the bsene of dithiothreitol (DIT) for 3 h t 4 C in buffer ontining 5 mm Tris-glyine (ph 8.6) nd.1% SDS. Eletrotrnsfer of proteins seprted on polyrylmide gels ws performed essentilly s desribed by Towbin, Stehelin, nd Gordon (1) with the exeption tht for nondenturing gels methnol ws omitted from the trnsfer buffer. Immobilin-P membrne (Millipore Corp., Bedford, MA) ws presoked in methnol, wshed with wter, nd then soked in trnsfer buffer (5 mm Tris, 19 mm glyine) for 15 min. The membrne ws pled on the node side of the gel between two sheets of porous filter pper nd eletrotrnsferred t 4 C t 4 V/m. After the trnsfer, the membrne ws immersed in bovine Lto Trnsfer Tehnique Optimizer (BLO?TO) bloking buffer onsisting of 5% (w/v) nonft powdered dry milk in PBS with.1% ntifom A emulsion (Sigm) nd.1% merthiolte (Sigm) for 1 h. Next, the membrne ws inubted overnight t room temperture in BLOTTO ontining 1 pg/ml of mouse monolonl nti-humn LDL-reeptor ntibody (IgG-C7),(generously provided by Drs. Y. K. Ho, Joseph L. Goldstein, nd Mihel S. Brown, Deprtment of Moleulr Genetis, University of Texs Southwestern Medil Center, Dlls). The membrne ws then wshed with 5 ml of.1% Tween-8 nd.1% Triton X-1 in PBS. The membrne ws inubted for 4 h t room temperture in BLOTTO ontining 1*51-lbeled got nti-mouse IgG ntibody (pproximtely 7.5 x lo5 pm/ml), nd then wshed gin with 5 ml of.1% Tween8 nd.1% Triton X-1 in PBS. Bnds were visulized on rdiogrphi film using utordiogrphy, nd bnd densities were determined using n imge nlysis system (JAVA, Jndel Sientifi, Corte Meder, CA). Quntifition of LDCreeptor mrna Cells were grown in -m ulture flsks. Immeditely fter removl of the ulture medium, ells were wshed with serum-free DMEM, srped off by rubber poliemn, nd totl RNA ws isolted by single-step RNA isoltion method (). Conentrtions of LDL-reeptor mrna were determined by n RNse protetionsolution hybridiztion ssy s desribed (3). For the preprtion of riboprobes nd RNA stndrd, bp DNA frgment of lone pldlr3 (ATCC, Rokville, MD) ws subloned into BmHI nd EoRI sites of pgemszf(+) vetor (Promeg Corp., Mdison, WI). After onfirming the orienttion of the insert by sequening, 3P-lbeled RNA probe nd RNA stndrd were synthesized by in vitro trnsription using T7 or SP6 RNA polymerses. The totl ellulr RNA nd different onentrtions of RNA were hybridized with 3P-lbeled RNA probe (5, pm). The hybridized probe ws olleted on glss fiber filters fter TCA preipittion nd ounted in sintilltion ounter. A stndrd urve ws generted by hybridizing the probe with sense RNA stndrd nd bsolute levels of LDLreeptor mrna were lulted. As n internl ontrol, the mrna for y-tin ws lso quntified using 67 bp 'ftin DNA subloned t HindIII-XbI sites of pgemszf(+) vetor. To prepre riboprobe the reombinnt plsmid ws linerized with Hind111 nd T7 RNA polymerse ws used. RNse protetion ssy ws performed in similr Downloded from by guest, on June 19, Journl of Lipid Reserh Volume 36, 1995

4 o I I Riboprobe Proteted Frgment 1 Mesurements of reltive rtes of LDLreeptor mrna trnsription Fig. 1. Autordiogrm of RNse protetion ssy. Totl RNA from HepC ells (5 pg) nd from Co ells (5 pg) were hybridized with LDLreeptor riboprobe (1,pm), treted with RNse to digest unhybridized RNA nd riboprobes, preipitted with isopropnol, dissolved in loding buffer nd seprted in sequening gel. Lne 1, RNA moleulr size mrkers synthesized in vitro in the sme wy s the riboprobes using mixture of linerized plsmids obtined from Ambion; lne, LDLreeptor riboprobe; lne 3, riboprobe hybridized with yest trna; FCS, ells inubted in 1% fetl lf serum: LPDS, ells inubted with 1% lipoproteindefiient serum; 5-OH, ells inubted with 5 p d m l 5-hydroxyholestero1ontining 1% FCS medi. The positions of riboprobe nd the proteted frgments re indited. wy s desribed for the LDL-reeptor. Figure 1 shows the proteted frgment of the LDL-reeptor mrna in n RNse protetion ssy. Flututions in LDL-reep tor mrna by LPDS nd 5-hydroxyholesterol re lso evident in HepG nd Co ells. Preprtion of nulei from ultured ells Cells were grown in m ulture flsks, nd were wshed nd srped off by rubber poliemn s bove. Nulei were prepred s previously desribed (,4) with some modifitions for the ultured ells. Cells were wshed with.5 ml of ieold wsh solution ( mm. Tris-HCI, ph 7.5, 15 mm NCl, 1.1 mm surose) nd pelleted by entrifugtion t 3 g t 4 C for 1 min. The ells were resuspended in.5 ml of ieold hypo- Srivstu et l. Detils of nuler 'run-off ssy hve been desribed erlier (, 4). The trnsription ssy ws performed in p1 of buffer ontining 1 mm Tris-HC1,pH 7.9,5 mm NCl,.4 mm EDTA,.1 mm PMSF, 1. mm DTT, 1 mg/ml heprin sulfte, mm MnC1, 4 mm MgC1, 1 mm retine phosphte, 1 mm eh of ATP, CTP, nd GTP, 4 U humn plentl ribonulese inhibitor, 3% glyerol, 1 million nulei nd 1 pci [-3P]UTP. Trnsription ws llowed to our t 6 C for min in the presene or bsene of pg/ml -mnitin nd the retion ws terminted by the ddition of 3 U of DNse I (RNse-free).Nuler RNA ws then isolted using RNAzolTM B. The isolted RNA ws wshed with 7% ethnol nd dissolved in hybridiztion buffer ( mm PIPES, ph 6.7, 5% deionized formmide, mm EDTA,.8 M NCl,.% SDS,.% fioll,.% polyvinylpyrrolidone,.% BSA, 5 p d m l dentured slmon sperm DNA). An liquot ws ounted to determine the inorportion of ["P]UTP in RNA. The extrted nuler RNA ws hybridized to membrne bound LDLreeptor DNA (1.9 kb) t 4 C for 5 h. A 1.9 kb LDL-reeptor DNA of lone pldlr3 ws subloned into BumHI site of pgem3zf(+) vetor. As n internl ontrol 67 bp humn y-tin DNA frgment subloned into Hind11 nd XbuI site ws used. This lone ws kindly provided by Dr. Cly Semenkovih, Deprtment of Internl Mediine, Wshington University, Sint Louis. For bkground ounts nonreombinnt pgem vetor ws linerized nd hybridized the LDL reeptor gene expression in HepG nd Co ells 1437 Downloded from by guest, on June 19, 18 toni solution ( mm Tris-HC1, ph 8., 4 mm MgC1, 6 mm CC1,.5 mm DTT) nd llowed to remin on ie for 5 min. After the ddition of.5 ml of lysis buffer (.6 M surose,.% Nonidet P-4,.5 mm DTT), ells were homogenized with the tight-fitting pestle of homogenizer. The homogente ws entrifuged t 1,5gt 4 C for 1 min nd the pellet ws resuspended in.5 ml of.3 M surose solution in buffer B (6 mm KCl, 15 mm NCl,.15 mm spermine,.5 M spermidine, 14 mm P-merptoethnol,.5 mm EGTA, mm EDTA, 15 mm HEPES buffer, ph 7.5). The rude nuler suspension ws lyered over.5 ml ushion of 1.5 M surose in buffer C (sme s buffer B but EGTA nd EDTA onentrtions redued to.1 mm eh) nd entrifuged for 1h t 4 C t 45, rpm in Bekmn TLlOO Ultrentrifuge. The len nulei were trnsferred to mirofuge tube nd suspended in old nulei storge buffer ( mm Tris-HC1, ph 7.9, mm NCl,.5 mm EDTA,.85 mm DTT,.15 mm PMSF, 5% glyerol). Prepred nulei were either used immeditely for in vitro nuler run-off ssy or stored t -7 C for up to 4 weeks without loss in tivity. HepG Co

5 loo. - A 9 C 8' 6ol 7 p 5 E Y ' o C =I 3 41 /'+ I loo )B I 1 n -l i - *OF *, ' D Fig.. Effets of LPDS on LDLreeptor gene expression in HepC ells. HepC ells were grown to 8% onflueny in % FCS, wshed, nd then inubted with 1% LPDS or FCS for the times indited. A Shows binding of iodinted LDL isolted from humn blood. This ssy ws performed t 4 C s desribed in the text. Eh ssy point indites men vlues of duplite dishes. Cirles represent vlues obtined with 1% FCS nd tringles indite vlues obtined with 1% LPDS medi. B: Shows binding nd uptke of iodinted LDL performed t 37 C. This ssy ws lso performed in duplite nd the men vlues re plotted. C: Shows bsolute levels of LDL-reeptor mrna quntified by RNse protetion ssy. Eh ssy ws performed in triplite using totl RNA from three dishes, nd 5 pg totl RNA ws tken for eh ssy. Detils of LDLreeptor mrna quntifition re desribed in the text; *, signifintly different ompred to initil vlues nd vlues obtined with FCS. Eh vlue represents men f SEM. As n internl ontrol bsolute levels of y-tin were lso detrmined. These did not hnge in the LPDS medi. D: Shows results of reltive rtes of LDL-reeptor mrna trnsription. Nuler 'run-off ssy ws performed s desribed in the text. Eh ssy ws performed in triplite nd the vlues represent men f SEM. As n internl ontrol the reltive rtes of y-tin mrna trnsription were lso performed. The mximum hnge ws 1% inrese in the rtes of y-tin mrna trnsription t the 4-h time point. Experiments shown here were repeted nd similr results were obtined. Downloded from by guest, on June 19, 18 sme wy. After the hybridiztions, the membrne ws wshed s desribed (), nd ounted. Trnsription rtes were expressed s perent in reltion to ontrol group tht ws ssigned vlue of 1%. RESULTS Effets of 1% LPDS or 1% FCS on LDLreeptor gene expression Medi of 8% onfluent ells were hnged from % FCS to 1% LPDS (or 1% FCS s indited) for vrying lengths of time to up-regulte LDL-reeptors. As expeted, LDL-reeptor mss nd tivity inresed progressively with inresing inubtion time nd rehed mximum of 5- to 6-fold inrese fter 4 h of inubtion in 1% LPDS (Fig. A nd B). Inubtion in 1% FCS produed muh lower degrees of LDL-reeptor up-regultion. Prllel inreses re noted in LDL-reeptor mrna levels nd reltive rtes of trnsription (Figs. C nd D). The mrna for y-tin ws lso quntified. No signifint hnges were seen. LDL-reeptor tivity nd mss lso inresed in prllel with LDL-reeptor mrna levels nd rtes of trnsription in Co ells 1438 Joumd of Lipid Reserh Volume 36, 1995

6 4 h 35 Q, te v Q, Y 15 m J 5 5 B '" oo 1 D P). m 4- -, - I - -7 I Fig. 3. Effets of LPDS medi on LDLreeptor gene expression in Co ells. The experimentl detils re provided in the legend to Fig.. A Iodinted LDL binding t 4 C; B: iodinted LDL uptke nd degrdtion t 37 C; C: LDLreeptor mrna; D LDLreeptor mrna trnsription. Filled irle represents vlues obtined with 1% FCS medi nd tringle represents vlues obtined with 1% LPDS medi. Downloded from by guest, on June 19, 18 (Fig. 3). However, LDL-reeptors were indued less in Co ells thn in HepG ells. Effets of 5-hydroxyholesterol on LDLreeptor expression In HepG ells grown in 1% FCS or 1% LPDS for 48 h (down from the % FCS used for routine mintenne) showed 5-hydroxyholestero1 dose-dependent (Fig. 4) nd time-dependent (Fig. 5) dereses in the LDL-reeptor tivity. Thus,.1 nd 5 pg of 5-hydroxyholesterol deresed LDL-reeptor tivity by % nd 5%, respetively (Fig. 4). In the presene of.5 pg/ml of 5-hydroxyholestero1, LDL-reeptor tivity deresed to -5% of bsl over 4 h (Figs. 5A nd 5C). LDL-reeptor binding ws higher in ells whose reeptors hd been up-regulted in LPDS, nd the extent of 5-hydroxyholesterol-indued derese ws lso greter. Nevertheless, the 5-hydroxyholesterol-indued frtionl dereses in 1% FCS inubted ells suggested tht 1% FCS lone did not produe omplete down-regultion of reeptors. LDL-reeptor protein mss deresed in prllel with LDL-reeptor tivity (Figs. 5A nd 5C). LDL-reeptor mrna levels nd reltive rtes of trnsription lso deresed fter s little s 6 h of inubtion of HepG ells in 1% FCS (Fig. 5B) or with 1% LPDS (Fig. 5D). Results in Co ells resembled those in HepG ells, Le., 5-hydroxyholesterol (5 pg/ml) deresed LDL-reeptor tivity nd protein mss to 65% nd 6% of initil vlues, respetively (Fig. 6A), while onomitntly, LDL-reeptor mrna onentrtions nd reltive rtes of trnsription both deresed to bout 6% (Fig. 6B). Sn'ustv et l. LDL reeptor gene expression in HepG nd Co ells 1439

7 -I n 15 i J 5-. for ytin remined unhnged (Fig. 9). Inubtion of HepC ells in 5 pg/ml 5-hydroxyholesterol nd.8 mm plmitte (plmitte/bsa rtio of 5) lowered LDLreeptor mrna to greter extent thn did plmitte or 5-hydroxyholesterol lone (Fig. 1). Control inubtion of ells in the sme mounts of BSA ( mm) used to produe the vrious ftty id/bsa rtios, in the bsene of either ftty ids or 5-hydroxyholesterol, hd no effet on ny of the prmeters mesured (not shown). DISCUSSION Hydroxyholesterol (ug/ml) Fig. 4. Effets of 5-hydroxyholesterol onentrtions on LDL-reeptor tivity in HepG ells. HepG ells were grown to 8% onflueny, wshed with PBS, inubted in 1% LPDS for 4 h, nd then further inubted with DMEM ontining vrying mounts of 5-hydroxyholesterol for nother 4 h. The vlues represent mens of duplite dishes. Effets of olete, plmitte, nd 5-hydroxyholesterol ombintions on LDCreeptor expression Three onentrtions of olete t three different olete/bsa molr rtios did not ffet LDL-reeptor tivity (Fig. 7A). By ontrst, mximum -3% derese in LDL-reeptor tivity ws noted fter 4 h inubtion in.5 mm plmitte omplexed with BSA in rtio of 9:l (Fig. 7B). Lesser degrees of lowering were obtined t lower onentrtions nd molr rtios. In different experiment performed with.8 mm plmitte/.16 mm BSA, LDL-reeptor tivities fell by -3%, but LDL-reeptor protein, mrna levels nd rtes of trnsription were not ltered (Tble 1). Addition of 1 ~g,"15-hydroxyholesterol to plmitte produed lowering of LDL-reeptor tivities t every onentrtions of plmitte nd molr rtio of plmitte/bsa (Fig. 7B). Conversely, dding fixed mount of plmitte (.8 mm) to vrying doses of 5-hydroxyholesterol lso produed dditive effets on LDLreeptor tivities in both HepC nd Co ells (Fig. 8). Comptible results were obtined whether preinubtions were in 1% LPDS or 1% FCS. In onfirmtion of the experiment reported in Tble 1,.5 mm plmitte in the bsene of 5-hydroxyholesterol did not lter LDL-reeptor mrna levels, while ddition of vrying onentrtions of 5-hydroxyholesterol to medi ontining the.5 mm plmitte deresed LDL-reeptor mrna in 5-hydroxyholesterol dose-dependent mnner while the mrna Mny investigtors hve used HepG ells s model system to study vrious spets of the regultion of holesterol biosynthesis nd metbolism nd LDL-reeptor regultion (7,8,4-9). The im of the present investigtion ws to sertin how sturted ftty ids in the bsene or presene of 5-hydroxyholesterol deresed LDL-reeptor tivity. It hd been reported previously tht holesterol or its derivtives, prtiulrly 5-hydroxyholestero1, effiiently redued LDL-reeptor tivity, protein nd mrna levels in vivo (,4), nd in ultured ells (644). Our dt onfirm nd extend these findings in Co nd HepC ells. We show tht removl of extrellulr soures of holesterol {ie., ulturing in 1% LPDS) used inreses in LDL-reeptor tivity, protein, mrna, nd rtes of trnsription, while supplying externl soures of 5-hydroxyholesterol produed time- nd dose-dependent dereses tht were in the sme diretion nd similr in mgnitude. The findings were like for both ell types, but the mplitudes of the vritions were greter for HepG ells. Swithing either HepG or Co ells from % to 1% FCS produed only smll degrees of up-regultion of reeptors, while ddition of 5-hydroxyholesterol to 1% FCS ells produed reeptor inhibition, suggesting tht 1% FCS did not ompletely suppress reeptor tivity. In humn fibroblsts, stimultion of LDL-reeptor tivity nd mrna levels by LPDS ws not similr in mgnitude. Therefore, dditionl regultion of LDL-reeptor gene t the trnsltionl level ws suggested (4). However, unlike in fibroblsts, omplete down-regultion of LDL-reeptor gene ws not observed in HepGP ells when they were inubted with 5-hydroxyholesterol. We found bout 4-57 inhibition in reeptor tivity nd mrna levels of LDL-reeptor. Similr results hve been obtined in other studies where LDL-reeptor mrna ws quntified by slot-blot tehnique (7, 6, 9). More effetive suppression of LDL-reeptors by 5-hydroxyholesterol thn by LDL hs been reported (7,s). However, EVLDL nd 5-hydroxyholesterol down-regulted LDLreeptor tivity to lmost the sme extent (8). Bsed on our Downloded from by guest, on June 19, Journl of Lipid Reserh Volume 36,1995

8 5 A L t Q ln I- O z U E A s 1'1 B C n Q * W s C E L Q t 15 1 ln I- O C lo 5 Z U E 5 -J D L?.+ * * * Fig. 5. Effets of 5-hydroxyholesterol on LDL-reeptor gene expression in HepG ells. HepC ells were grown to 8% onflueny in % FCS, wshed with old PBS, nd inubted in medi ontining 1% FCS or 1% LPDS. After 4 h inubtion, the ells were wshed with PBS nd inubted with DMEM,.5% (vol/vol) ethnol, nd.5 pg."l of 5-hydroxyholestero1 for the indited times. Pnels And B show results obtined with ells preinubted with 1% FCS, nd pnels C nd D show vlues obtined with ells preinubted in 1% LPDS. Cells were lso inubted with equivlent mounts (.5% vol/vol) of ethnol but without 5-hydroxyholesterol. After eh inubtion period, LDLreeptor tivity, protein mss, mrna, nd reltive rtes of trnsription were mesured. Pnels A nd C show LDLreeptor tivity (irles) nd protein (tringles). Pnels B nd D show LDLreeptor mrna (irles) nd trnsription (tringles). Results re mens of triplite dishes f SEM. *Signifintly different ompred to zero time vlues. The bsolute vlues t zero time point for HepC ells preinubted with FCS were: LDL-reeptor tivity, f ng 151-lbeled LDL/mg ell protein, LDLreeptor mrna, 43.1 pg/pg RNA. The densitometri snning of LDLreeptor protein bnd nd ounts of LDL-reeptor mrna trnsription were ssigned vlue of 1. The bsolute vlues t zero time point for HepG ells preinubted in LPDS were: LDL-reeptor tivity, 59.6 i 5.5 ng '51-lbeled LDL/mg ell protein, LDLreeptor mrna, f 6 pg/fg RNA. Downloded from by guest, on June 19, 18 dt, we suggest tht P-VLDL my lso down-regulte LDL-reeptor gene by trnsriptionl mehnism s seen with 5-hydroxyholestero1. The differenes in the extent of down-regultion of LDL-reeptor gene by LDL nd BVLDL (8) my reflet the bilities of these prtiles to deliver holesterol to intrellulr pools nd regultory sites for the LDL-reeptor (9). In order to understnd how sturted ftty ids regulte the LDL-reeptor gene, HepG nd Co ells were inubted with vrying olete nd plmitte onentrtions nd plmitte/bsa molr rtios. Olete produed no effets, while plmitte produed modest dereses in LDL-reeptor tivity in both ell lines. In ontrst, with 5-hydroxyholesterol, plmitte evoked no responses in LDL-reeptor protein or mrna levels, or in rtes of mrna trnsription. These findings re in greement with the in vivo studies where sturted ft did not lter LDL-reeptor mrna (, 5), yet reep- Srivstuvu et ul. LDL reeptor gene expression in HepG nd Co ells 1441

9 15 1 A T T 15 1 B U C m z n E Fig. 6. Effets of 5-hydroxyholesterol on LDL-reeptor in Co ells. Experimentl design ws sme s shown in Fig. 5, but the results obtined with ells preinubted further with 1% FCS nd inubted for nother 4 h with DMEM nd 5 pg,"l5-hydroxyholesteroi in.5% ethnol re only shown. LDL-reeptor tivity, mss, mrna, nd trnsription were determined. A: Shows LDL-reeptor tivity (filled br) nd LDL-reeptor protein mss (digonl br); B: Shows LDL-reeptor mrna (filled br) nd mrna trnsription (digonl br). *, Signifintly different when ompred to ontrol nd Lero hour vlues. Similr results were obtined when Co ells were preinubted with 1% LPDS for 4 h, wshed, nd inubted with DMEM ontining equivlent mounts of 5-hydroxyholesterol. The bsolute vlues t zero time point preinubted in FCS were: LDL-reeptor tivity, 3..3 ng "SI-lbeled LDL/mg protein, LDL-reeptor mrna, 4.94 k 1. pg/fg RNA. The zero time point vlues preinubted with LPDS were: LDL-reeptor tivity 14.4 ng "'I-lbeled LDL/mg protein, LDL-reeptor mrna, 8.86 Pg/M RNA _ > I Q, 5 I: 1 n 5 Olete Plmitte Downloded from by guest, on June 19, C C BSA-Olei id rtios BSA-Plmitte Rtios Fig. 7. Effets of olete nd plmitte on LDLreeptor tivity in HepG ells. Three different onentrtions of olete (.5 mm,.5 mu, 1. mm) were omplexed with BSA in vrying olete/bsa rtios s indited. Two onentrtions (.5 mm nd.5 mm) of plmitte t three different molr rtios were used. HepC ells were grown to 8% onflueny in % FCS, wshed with old PBS, nd inubted for 4 h in 1% FCS, wshed gin, nd then inubted with the indited ftty id-bsa omplex in DMEM. At the end of 4 h inubtion, LDL-reeptor tivity ws mesured. Left pnel, tringles,.5 mm; squres,.5 mm; irles, 1 mbi olete. Right pnel, tringles,.5 mu; irles,.5 mu; nd dotted lines with squres,.5 mm plmitte plus 1 pg/ml 5-hydroxyholesterol. The vlues presented re mens of duplite dishes nd given s perents of zero hour vlues. Similr results were obtined when LDL-reeptors were up-regulted by preinubtion with 1% LPDS nd then trnsferred to DMEM ontining indited mounts of ftty id-bsa omplex (not shown). 144 Journl of Lipid Reserh Volume 36, 1995

10 / HepG A > L l $ 5 [r hydroxyholesterol (uglml) hydroxyholesteroI (uglml) 15 HepG 16 1 W b. Q 5 -I Hydroxyholesterol (Pglml) 5 1 > L n l 5 -I HydroxyholesteroI (pglml) Fig. 8. Effets of ombintion of 5hydroxyholesterol nd plmitte (.8 mm) on LDL-reeptor tivity in HepC nd Co ells. Plmitte ws omplexed with bovine serum lbumin. Cells were grown to 8% onflueny in % FCS, wshed with PBS inubted for 4 h in 1% FCS (pnels A nd B) nd then inubted in DMEM ontining.8 mm plmitte,.16 mm BSA, nd the indited mounts of 5-hydroxyholestero1. Initil (zero point) vlues represent vlues obtined in the bsene of plmitte nd 5-hydroxyholesterol, nd rest of the vlues represent perents of the initil vlues. Cirles represent vlues obtined in the presene of 5-hydroxyholesterol lone; tringles represent vlues obtined in the presene of.16 mm bovine serum lbumin nd vrying onentrtions of 5-hydroxyholestero1; squres represent vlues obtined in the presene of.8 mm plmitte plus vrying onentrtions of 5-hydroxyholesterol. Eh ssy ws performed in triplite nd the experiment ws repeted. *, Signifintly different ompred to initil vlues. The bsolute vlues of LDLreeptor tivity t zero time point without 5-hydroxyholesterol for HepG ells were: DMEM, 37.3 f 3.6, DMEM + BSA (.16 mm), 33 f 3, DMEM + plmitte/bsa (.8 mm nd.16 mm, respetively), 36.3 ng 151-lbeled LDL/mg proteins. For Co ells, the vlues were: DMEM + BSA, 1 f 1.15, DMEM + plmitte/bsa, 1.9 f.9. C nd D show effets of ombintion of plmitte (.8 mm) nd 5-hydroxyholesterol on LDLreeptor tivity in HepG nd Co ells preinubted with 1% LPDS for 4 h, wshed with old PBS, nd inubted with DMEM nd indited mounts of plmitte/bsa lone or in ombintion with 5-hydroxyholesterol. The zero time point vlues of LDLreeptor tivity for HepG ells were: DMEM + BSA, 77.6 f 7, DMEM + plmitte/bsa, 76.1, Co ells, DMEM + BSA 5.1 f.16, DMEM + plmitte/bsa, 3.9 f 1.9 ng 151-lbeled LDL/mg protein. Downloded from by guest, on June 19, 18 tor-medited lerne of LDL from plsm deresed (3, 31), nd plsm LDL levels inresed s onsequene. Thus, it is ler tht the suppressive effet of ftty ids is most likely to be posttrnsltionl. The deresed LDL-reeptor tivity is not due to the presene of ftty ids in the medi during the binding ssy. Bihin nd ssoites (3) hve reported tht ftty ids diretly ffet LDL binding to fibroblst LDL-reeptors. However, in our experiments the ftty id-bsa om- plexes were removed, ells were wshed with DMEM, nd the ssy ws then performed in the bsene of dded ftty ids. Plmiti id produed the effet on LDL-reeptor binding nd olete did not, demonstrting the speifiity of the sturted ftty id effet nd onfirming the bsene of ny effets of medi ftty ids on the binding ssys. The tion of plmitte is not unique. Posttrnsriptionl regultion of the LDLreeptor hs been reported for growth hormones (33). Srivstuu et l. LDL reeptor gene expressin in HepG nd Cof ells 1443

11 TABLE 1. Effets of plmitte/bsa (.8 m~/.16 mm) on LDL-reeptor gene expression in HepG nd Co ells Inubtion Cell Types Medi Time LDL-Reeptor Ativity" LDL-Reeptor Proteinb LDLReeptor mrna LDL-Reeptor mrna Trnsriptiond h HepG BSA 4 HepG BSA/plmitte 4 Co BSA 4 Co BSA/plmitte It f f f f f f f.8 1 f f f f * f f f f f f f f f f f f f * f f k f 7.8 'LDL-reeptor tivity shown s ng of '51-lbeled LDL per mg protein. bldl-reeptor protein ws determined by Western blotting nd vlues represent densitometri snning of LDL-reeptor protein bnd. Vlues t zero time point hve been ssigned 1. CLDL-reeptor mrna ws determined by RNse protetion ssy using 5 pg totl RNA for eh ssy. Results re in pg nirna/pg totl RNA. dldl-reeptor mrna trnsriptions were mesured by "nuler run-off' ssy on isolted nulei. Vlues obtined t zero time point hve been ssigned 1. Cells were grown to 8% onflueny in % FCS, wshed with old PBS nd inubted in 1% LPDS for 4 h to upregulte LDL-reeptor, gin wshed with old PBS, nd then inubted for indited time in DMEM ontining either BSA lone or with plmitte. h r Q) N Y- O s v Z E Downloded from by guest, on June 19, 18 C P+O P+.5 P+1. P+.5 5-hydroxyholesteroI (pg) Fig. 9. Effets of plmitte on LDL-reeptor nd y-tin mrna in HepG ells. Cells were grown to 8%onflueny in % FCS, wshed, inubted either in 1% FCS (irles) or in 1% LPDS (tringles) for 4 h, nd then further inubted in DMEM ontining vrious onentrtions nd molr rtios of plmitte/bsa. At the end of 4 h, totl RNA were prepred nd LDLreeptor (filled irles nd tringles) nd y-tin (empty irles nd tringles) mrna were quntified by RNse protetion ssy shown in Fig. 1. C indites ontrol levels fter 4 h in 1% LPDS or 1% FCS. P indites levels fter 4 h in plmitte (.5 mm) but in the bsene of 5-hydroxyholesterol. P +.5, P + 1., nd P +.5 indite mrna levels fter 4 h in.5 mm plmitte to whih the indited inresing onentrtions of 5-hydroxyholesterol (in pg/ml) were dded. Vlues represent mens of duplite dishes. Clerly, plmitte lone did not ffet LDL-reeptor mrna levels (ompre C vs. P + ); dding 5-hydroxyholesterol produed lower LDL-reeptor mrna levels (P + vs. P +.5, et.). 4 Fig. 1. Effets of ombintion of 5-hydroxyholesterol nd plmitte on LDL-reeptor mrna in HepG ells. Cells were grown to 8% onflueny in % FCS, wshed with PBS, inubted for 4 h in 1% FCS, nd then further inubted for 4 h with DMEM nd plmitte- BSA (.8 mm nd.16 mm, respetively) or 5 pg/ml of 5-hydroxyholesterol. Filled br, DMEM plus ethnol; digonl br, DMEM plus ethnol plus BSA; dotted br, DMEM nd ethnol plus 5-hydroxyholesterol plus BSA; empty br, DMEM plus ethnol, plus 5-hydroxyholesterol plus BSA plus plmitte. Vlues of LDL-reeptor mrna re in % of zero hour vlues nd represent mens of duplite dishes Journl of Lipid Reserh Volume 36, 1995

12 The posttrnsltionl mehnism( s) by mens of whih sturted ftty ids redue LDL-reeptor tivity is(re) still not ler, but some possibilities re suggested by reent study on the effets of plmiti nd linolei ids on triglyeride synthesis nd lipoprotein seretion in Co ells (34). Plmiti id inresed intrellulr sturted phospholipid ell membrne ontents of ells by muh more thn did linolei id, while linolei id inresed the triglyeride ontents of ells nd the seretion of triglyerides from ells more thn did plmiti id. The umultion of the newly synthesized sturted phospholipid in ells inubted with plmiti id my lter the yling of LDL-reeptors between the inside nd the outside of ells or it my lter the physil hrteristis of membrnes nd ffet the tivity of LDL-reeptors by this mehnism. Combining plmitte with 5-hydroxyholestero1 produed dditive effets on LDL binding in both ell lines t eh onentrtion of plmitte nd plmitte/bsa molr rtio tested. Adding inresing mounts of 5-hydroxyholesterol to fixed mounts of plmitte lso produed dditive effets. In hmsters, dietry sturted ft ugmented the effets of dietry holesterol in suppressing hepti LDL-reeptor tivity ( 1). With respet to mrna levels, while 5-hydroxyholesterol lone down-regulted LDL-reeptor mrna levels to mximum of -5% of ontrol vlues nd plmitte lone did not signifintly redue LDL-reeptor mrna levels t ll, ombintions of 5-hydroxyholesterol nd plmitte hd dditive effets on LDL-reeptor mrna levels (Fig. 1). These results support the postulte by Dumerie, Woollett, nd Dietshy (1) tht the ombintion of dietry sturted ftty ids nd holesterol my be regulting LDL-reeptor tivity by ltering the sizes of regultory pools of holesterol. The implition of their postulte is tht the ombintion my be enhning trnsriptionl regultion. Thus, sturted ftty ids my be suppressing LDL-reeptors by two different mehnisms, one ffeting membrne fluidity nd/or reeptor reyling nd the other in onjuntion with holesterol, ffeting rtes of reeptor mrna trnsription. In onlusion, our dt demonstrte tht the LDL-reeptor gene in humn heptom ell line nd humn olon rinom ell line is subjet to up-regultion by LPDS, nd down-regultion by 5-hydroxyholestero1 t the trnsriptionl level. Sturted ft regulted LDL-reeptor in both ell lines by posttrnsltionl mehnism. We lso showed tht the effet of ombintion of 5-hydroxyholesterol nd sturted ft on LDL-reeptor tivity nd mrna levels ws dditive. Thus, holesterol nd sturted ft when dministered seprtely ffet the LDL-reeptor gene t different loi of regultion. When dministered together, sturted ftty ids my enhne the regultory effets of holesterol t the trnsriptionl level, perhps by ffeting regultory pools of holesterol (1, 1, 3, 31). I This work ws supported by NIH grnt R1 HL4466 nd DRTC grnt NIH5 P6 DK 579. Mnusript reeived 7 Jul~ 1994 nd in revised form I3 Fetnuly REFERENCES 1. Gofmn, J. W., W. Young, nd R. Tndy Ishemi hert disese, theroslerosis nd longevity. Cirultion. 34: Srivstv, R. A. K., S. Jio, J. Tng, B. Pfleger, R. T. Kithens, nd G. Shonfeld In vivo regultion of low density lipoprotein reeptor nd polipoprotein B gene expressions by dietry ft nd holesterol in inbred strins of mie. Biohim. Biophys. At Srivstv, R. A. K., J. Tng, E. S. Krul, B. Pfleger, R. T. Kithens, nd G. Shonfeld Dietry ftty ids nd dietry holesterol differ in their effet on the in vivo regultion of polipoprotein A-I nd polipoprotein A-I1 gene expression in inbred strins of mie. Biohim. Biophys. At Srivstv, R. A. K., D. Bumnn, nd G. Shonfeld In vivo regultion of low density lipoprotein reeptors by estrogen differs t the post-trnsriptionl level in rt nd mouse. Eur. J. Biohem. 16: Sori-Thoms, M., M. D. Wilson, F. L. Johnson, D. L. Willims, nd L. L. 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