Subcellular localization of squalene synthase in human hepatoma cell line Hep G2

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Biochimica et Biophysica Acta, 1t26(1~)2) 114-tl8 © 1992El~vier SciencePublishersB,V. All righls reserved1XX15-2760/92/$05,110

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Subcellular localization of squalene synthase in human hepatoma cell line Hep G2 Louis H. Cohen ", Marieke Griffioen a, Carlo W.T. van Roermund b and Ronald J.A. Wanders b TNO Inmtute for Ageing and Va~cu!arResearch. Gaubius Laboratory. Leiden (Netherlands~ and ~ Department of Pediatrics, Uhi~'er~io"Hospital Amsterdam, Amsterdam ~Netherlmtds)

(Received24 October I~)l ) (Revisedmanuscriptreceived12 February 1992)

Key ~ords: Clmlestero!biosynthesis:Squalen¢synthas¢:HMG-CoArcductasc; Pcroxisome;U 18666A:(Human) Us&~ the Hep G2 cell line as a model for the human hepalocytc the question was studied whether Hcp G2-peroxisomes could b¢ abk: ,o synthesize cholesterol. Hop G2 cell homogenates werc applied to dcnsity gradient cc,itrifugation on Nycodeuz, rcsuhing in good separation between the organelle~. The different organeile fractions were characterized by assaying the [oelo~ing marker enzymes: catala~ for pcroxisomcs, glutamate, dehydrogcnasc for mitochondria and esterasc for endoplasmic rei£culum. Squalcnc .synthasc actkity was not dctectable in the peroxisoma! fraction, incubatim. ~f Hop G2 cells with UI8666A, an mh/bitor o[ the cholesterol s~mhesis at the site of oxidosqualene cyclase, together with heavy high density lipoprotein, which stimulates the cffiux of cholesterol, led to a marked increase in thc activity of squalene ~nma-,,e as well as HMG-CoA reductase, whc~as no signii'~nt clt~.c~ on the marker enzymes was observed. Neither enzyme activity was detectable in the peroxisomal dcl~si~ gradient fr~'tion, suggesting that in Hop G2-pcroxisomcs cholcstcrol synthesis frola the water-soluble early intermediates c~ the paeh~ay canrmt rake place. Both stimulated and aon-~timulated cells gave rise to preparations where squaiene synthase a~-ti~iw ~as comigrafing ~ith the rcductasc activity at thc lower density side of the microsomal fraction; however, it was also p:cseni at the high density side of the microsomal peak, where rcdpcta~ activity was not detected.

ialrodnctioa Despite intensive investigations on the pathway of cholesterol biosynthesis there is still unccrtainty with regard to *.he subce|lular distribuiion of the eazyme~ involved in this pathway. Until recently it was generally bclic,'cd that synthesis of cholesterol from farnesyl pt, roph~osphate, the last of the early non-lipid intermediates, occarred [n the endoplasmic reticulum only [!]. ~er, rat liver pcroxisomes were aLqo found to possess cholesterol synti'.esizing capacity [2]. Indeed, two enzymes of this pathway were reported to be present to some extent in rat pcroxisomal preparations: HMG-CoA reductase (EC I.I.I.:M,), tlle major"

Abbreviations: HMG-CoA. 3-hydro~'-3-methk'lglutaryl-coenzx~eA; hHDL he~W high density tilmprotein: SElL smoot~ endoplasmic

:¢ticalam: REIL nsugh endoplasmicreticulum:DMEM, Dulbccco's madifmd E~ghf~~s',cnlial reed;am. Co~r~s,,~der,ce: L.H. C ~ n . Gaubius LaboratoryIWO-TNO. P.O. Boa 430. L~]0 AK Leiden, Nelherlands.

rate limiting enzyme of cholesterol synthesis [3,4], and an acetoacetyl-CoA thiolase [5]. Others (see, e.g., Ref. 6), however, localized the reducta~ solely in the smooth endoplasmic reticulnm (SER) of rat liver cells. The p r e ~ n c e of these enzymes does not provide unambiguous proof that they play a role in peroxisomal cholesterol synthcsis, because the products of the reactions catalyzed by these enzymes are water soluble and may be processed to cholesterol or other isoprene-related compounds elsewhere in the cell. Because squalene is the first lipid intermediate in the pathway and is committ.ed to be ultimately convcrted into cholesterol, we studied the possible peroxisomai localization of the enzyme squalene synthase (EC 2.5.1.21). For se~,eral years we have been using the human hepatoma cell line Hop (52 as a model for the study of chc,lesterol synthesis in the human hepatocyte [7-10] and we have found that the activity of this enzyme is high in these cells [1 !]. We observed further, that in Hep G2 cells ~ u a l e n e synthase is regulated by sterols [i 1], probably in the same way as has been reported for other enzymes of the cholesterol synthetic pathway [10,12] at the level of gene expression.

!15 In this paper we demonstrate that squalene sy~lLhase activity was solely observed in microsomal preparations from Hep G2 cells, cultured under different conditions. Materials and Methods

Recovery of HMG-CoA reductase and squalene synthase activities in the gradient fractions were 6782% and 70-91%, respectively. Recoveries of the marker enzymes varied between 80-120%. Protein concentrations were measured as described by Bradford [19].

Materials

Results

U 18666A (3~-[2-(diethylamino)ethoxy]androst-5-en17-one) wa:~a girl from Upjohn, Kalamazoo, MI, USA. [3-14C]HMG-CoA and R-[2-14C]mevalanolactone were purchased from Amersham; all other radiolabeled chemicals were from New England Nuclear. [I,5,9~4C]Farnesyl pyrophosphate was prepared enzymically from R-[2-14C]mevalanolactone as described previously [11]. Nycodenz was obtained from Nycomed AS, Oslo, Norway. Human hHDL (density 1.16-1.20 g/ml) was isolated from freshly collected 13ooled blood [13]. All other chemicals were of the highest purity available.

Human hepatoma cell line Hep G2 The cells were cultured in 75 c m 2 flasks in DMEM supplemented with 10% (w/v) fetal calf serum [14]. In order to increase the activity of the enzymes studie0 the cells were incubated with DMEM supplemented with 1% palmitate-loaded human serum albumin [10], 20/zM of UI8666A and 20% human hHDL for 20 h befole harvesting.

Organelle separation by Nycodenz equilibrium density gradient centrifugation Hep G2 cells (28-38 mg of cellular protein) were taken up in 6 ml of cold homogenization buffer (250 mM sucrose, 2 mM EDTA, 0.1% (v/v) ethanol, 2 mM MOPS-NaOH (pH 7.4)) and a postnuclear supernatant was subsequently prepared by 50 up-and-down strokes in a glass, tight-fitting, motor-driven (1000 rpm) Potter.Eivehjem homogenizer followed by centrifugation for 10 rain at 600×g. 4 ml of the. resulting postnuclear supernatant was loaded onto a 35 ml preformed Nycodenz-gradient, centrifuged and fractionated (1.7 mI fractions), as described before for rat liver homogenate [15]. All handlings were performed at 4°C.

Enzyme assays Several enzyme activiLies were determined in the gradient fractions. We described previously the determination of squalene synthase activity [10]. of HMGCoA reductase activity (Ref. 7; [!4C]HMG.CoA concentration was 0.5 raM), of glutamate dehydrogenase (EC 1.4.1.2) activity [16], of catalase (EC 1.11.1.6) activity [17] and of esterase (EC 3.1.1.2) activity [18], The data shown in 1he figures are typical examples from two separately performed experiments,

Hep G2 cells, cultured in normal growth medium (DMEM-10% foetal calf serum), were homogenized and after a low-speed centrifugation in order to discard nondisrupted cells and nuclei, the organelles were separated by Nycodenz-density gradient centrifugation. Marker enzyme activities (catalas¢ for pcroxisomes, glutamate dehydrogenase for mitochondria and esterase for the microsomai fraction), measured in the gradient fractions, revealed a good separation of the peroxisomes and the endoplasmic reticulum membranes (Fig. IA). In Fig. 1B the results of the determinations of squalene synthase and HMG-CoA reductase activity in the gradient fractions are depicted. Squalene synthase activity comigrated with the microsomal marker enzyme and was not detectable in the peroxisomal fractions. Also no comigration of the reducta~¢ activity with the peroxisomai marker was observed, although a small percentage of this activity was found in fractions different from the microsomes at higher densities (fractions 5-10 in Fig. IB). However, the all-over reductase activity was rather low, as expected for the values in Hep G2 cells cultured under these conditions (activity in the cell homogenate: 64 pmol/min per mg protein), in order to exclude a possible inhibitory effect of Nycodenz on the two enzyme activities we performed the assay in Hep G2 cell homogenates in the presence of different quantities of Nycodenz. At 18% (w/v) of Nycodenz, the maximal concentration in the assay when the peroxisomal frac. :ions of *.he density gradient were used, squalene syn~ thase and HMG.CoA reductase activity were found to be inhibited by 25% and 14%, respectively. Performing the assay in a mixture of the peak fractions of both peroxisomal and microsomal fraction showed, apart from the dilution factor, no additional decrease of the microsomal activity, excluding the presence of an inhibitory agent in the peroxisomal fraction (results not shown). Addition of a detergent prior to the determination of squalene synthase activity did also not reveal significant activity in the peroxisomal fraction. In order to investigate the possibility that peroxisoreal squalene synthase and HMG-CoA reductase may be detectable after induction of these activities by lowering of the regulatory steroi ,,x~ol, Hep G2 cells were incubated for 20 h in the presence of either 20 /zM of UI8666A together with 20% of hHDL or 10%

116 foetal calf serum as control. We showed previous' that in He;, G2 cells ~ / . t M of the oxidosqualene cyclase inlfibitor U18666A totally blocked cholesterol s~ynthesis and increased HMG-CoA reductase activity [9] as well as squalene synthase activity [11]. Incubation with h H D L which [s thought to increase the sterol efflux from the cells, also increases both activities in Hep G2 cells [8,11,20]. The marker enzyme activity distribution in the Nycodertz-density gradient from the h H D L / U18666A-incubated cells ~Fig. 2) showed that microspinal contamination of the peroxisomal fractions was hardly present. As depicted in Fig. 3A and B both

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and Methods. The activities of the marker enzymes, squalene sy~+thase and HMG-CoA reduclase were measured in the gradient fractions and the relative distribulion calculation. See Fig. 3 for absolute values. Symbolsare the same as used in the legend of Fig. 1.

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traction Fig. I. Separation of organdies from Hop G2 ceils by Nycodenz equilibrium density centrifugation and distribulion of squalene synthese and HMG-CoA reductase activity. Hep G2 cells were homogenized and the postnuclear fraction, containing 13 mg of protein, was centrifuged on a Nycodenz pre[ormed density gradient as described in Materials and Methods. Fractions were. collected from bottom to top and I.A) marker enzyme activities w~:re determined: tot pcrnxisprees catalase (.). for mitochondri,+glutamate dehydrogenase (A) and for microsomes esterase (v). (B) Distribution of squalene synIhase (Ill) and HMG-CoA reductase (1:3) activity over the gradient fractions. The activities of lhe fractitms are e:.pre~ed as percentage of the total activitymeasured in the gradient which was for squa!cnc _~ymhaseavd HMG-CoA reductasc 67 and 82%, respectively',of the activities measured in the Hep G2 cell Imstnuclear fraction (respective activities: 1.33 nmol/min per mg and il ! pmol/min per mg of protein).

squalene synthase and HMG-CoA reductase activity were markedly increased, whereas the marker enzyme activities were net significantly influenced by the treatmerit (Fig. 3C-E). The protein distribution was about the sam:~ iv both gradients (Fig. 3F). Notwithstanding the large increase in total activity, squalene synthase is still not detectable in the peroxisomal fractions. The activity of HMG-CoA reductase in the peroxisomes was also extremely low, if present at all. Control experiments to exclude the presence of an ial',ibito~ agent in the peroxisomal fraction, as mentioned above, were performed and did not affect these results. Again some activity was observed in fractions which had a higher density than the bulk of the ER membranes (fraction 7-10 in Fig. 3A and B), however, it is not clear from these experiments whether this activity is attached to particles different from microsomes. Close inspection of the data such as shown in Figs. I B and 2 revealed that in all cases squalene synthase ac*ivity [tad the same density distribution as that of the microsomal marker esterase, whiie the HMG-CoA reductase activity peak is always found more to the lower density side of the microsomai fraction. To rdle out the possibility that e n ~ m e s present in the higher density fractions, such as the mitochondrial HMG-CoA lyase, may disturb the reductase assay, microsomal fractions of lower density were mixed with the higher density ER fraction and reductase activity determined. No decreasing effect was observed (not shown). These eesults suggest that squalene synthas¢ is present both in the same microsomal membranes to which reductase is linked, and also in another, higher density ER fraction of Hep G2 cells.

117 Discussion

From the experiments described in this paper we conclude that in H e p G2 cells squalene synthase activity is not detectable in the pero×isomal fraction, even after a strong e n h a n c e m e n t of this activity by U 1 8 6 6 6 A / h H D L - t r e a t m e n t of the cells. This seems also to be the case for the H M G - C o A reductase activity in the peroxisomal fractioa from the U 1 8 6 6 6 A / h H D L - t r e a t e d cells, One can deduce from our data that it seems unlikely that in H e p G2-peroxisomes cholesterol is synthesized from early intermeliates in the pathway. O u r findings deviate from the

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observation that rat liver peroxisomes contain s+me H M G - C o A reductase [4] and were found to be able to synthesize cholesterol from m+valonat~ [2]. Although Singer et al, [6J could not confixm the pero×isomal localization of the reductase in rat liver as observed by Keller et al, [4], it may be possible that our observation of" the absence of both enzymes in peroxisomes is characteristic for H e p G2 cells, it was reported previously that Hep G2 cells are deficient in the peroxisomal processing of 3m,7o~,12~-trihydroxy-5#-cholestan26-oic acid (THCA) [21], However, others [22] found no detectable defects in the side chain oxidation of C27 sterols in these cells and moreover+ no accumulation of

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Fig. 3. Absolute values of squalene synihase, HMG-CoA reductase and marker exizymeactivilies after separation of organelles from c~qtrol and hHDL/U 18666A-treated I lep G2 cells on Nycodenzdensily gradienls. Postnuclear superna|ant was isol~.ted f~'omcontrol Hep G2 cells Co) and hHDL/U 186f~A-incubated cells (o) and 2! and 20 mg of protein, respectively,were load¢d on Nycodenz gradients. After ¢catrif~gation tile different enzyme activitieswere measured in the fraclions (i.7 ml) of both gradients, (A) Squalere s>nthaseactivity: the recoverywar,70 and 86¢;. of the activiiies measured in ihe posinuctear fraclions from the control (|.64 nmol/min per mR of prGtein) and hHDL/U186f~A-treated (5.20 nmol/min per mg of protein} cells, respectively:(B) HMG-CoA reductase activity: the recoverywas 68 and 71% of the postnuclear activities(26 and 412 pmol/min per mg of protein for control and lnHDI./UI8666A-treated ceils, respectively): (C) cata!as¢ aclivily: (D) glutamate dehydrogenase aclivity: and (E) esterasc activity. The recoveries of the marker en~me activities varied between 80-12P,%: (F) protein concentratien of the gradient fractions.

118

THCA was observed it; the Hep G2 cell line, which was used in the present study ~Dr. H.M.G. Princcn, personal communication). Besides that, we ~;~owed recently [23] that several other peroxisomal enzyrr.es are still present in these Hep G2 cells. Whether squalene synthase activity is also not present in peroxisomes from human hepatocytes is not yet clear. From our data we also conclude that squalene synthase resides in ER membranes which are in part different from those containing HMG-CoA reductase, it was pr~:viously reported [24] that different parts of the cholesterol biosynthetic pathway were performed in different membranes of human fibroblasts and rat liver. Some authors localized HMG-CoA reduetase in the SER [25], however, others (e.g., Ref. 26) observed additional activity in the RER. The nature of the high density ER membranes containing the squalene synthase activity has to be investigated further. Acknowledgments Mrs. L. de Pagter is gratefully acknowledged for her skilful assistance in culturing and maintaining the Hep G2 cell line. The Upjohn company is thanked for providing us with UI8666A, and the authors thank Mrs. M. Horsting for excellent handling of the manuscript. References ! Che~terton, C.J. (t96811. Biol. Chem. 243, 1147-1151, 2 Thompmn, S.L, Burrows, R., Laub, R.J. and Krisans. S,K. ( 19871 J. Biol. Chem. 262, 17420-17425. 3 Keller. G.A.. Barton, M.C.. Shapiro. D. and Singer. S.J. rl985) Proc. Natl. Acad. Sci. USA 82, 770-774. 4 Keller, G.A.. Pazirandeh. M. anti Krisans, S.K. (1086) I. Celt Biol. 123, 875-88.6. 5 Thompson, S.L. and Krisa,,s. S.K. (199{)) J. Biol. Chem. 265. 5731-5%5. 6 Singer, 1.1., Sc,~tt, S., Kazazis, D.M. and lluff. J.W. ~195~"' Proc. Natl. Acad. Sci. USA ~5, 52lvl-5268.

7 C~hen. LH., Griffitmn, M., Havekes, L., Schouten, D., Van Hinsbergh. V, and Kempen. H,J. (1984) Bk~ehem. J. 222, 35-39. 8 Cohen, LH,. Griffioen, M. and 8oogaard, A, (1985) in Cholesterol Metabolism in Health and Disease: Studies in the Netherlands (Beynen, A.C., et al., eds.L pp. 53-60. Ponsen en Ltmljen, Wageningen. q Bongaard, A,, Griffioen, M. and Cohen. L,H, (1987} Biochem. J, 241. 345-351. t0 Cohen, LH. and Griffioen, M. (19881 Bioehem. J. 255, 61-69. i I Cohen, LH., Van Micrl, E. and Griffioen. M. (1989) Biochim. Biophys, Acta ItX)2, 69-73. 12 Rosscr, D.S.E.. Ashby. M,N.. Ellis, J.L. and Edwards, P.A. (1989) J. Biol. Chem. 264, 12653-12t~56. 13 Redgrav¢, T.G.. Roberts. D,C.K. and West, C.E. (19751 Anal. Biochem, 65, 42-49. 14 Havekes, LM.. Van Hiasbergh. V.W,M., Kempen, H.L and Emeis. J.J. (19831 Bitx:hem. J. 214. 951.95C 15 Wanders, R.J.A., Van Roermund. C.N.'[.. Van Wijland, M.J.A., S~hutgens, R.B.H., l-lcikoop. J.. Van den Bosch, H., Schram, A.W. and Tager, J.M. (t9871 J. Clin. Invest, 81h 1778-1783. 16 Cohen. LH.. Griffioen, M.. Wanders. R.J.A,, Van Roermund, C.W.T., Huysmans, C.M.G. :rod Princen, H.M.G. ( 191q6}Biochem. Biophys, Res. Cummun. 138, 335-341. 17 Wanders, R.J.A., Kos, M., Roest. B., Meyer, A.J.. Schrakamp, G., Heymans. H.S.A., Tcgelaers, W.H,H., Van den l~.~sch, H., Schutgens, R.B.tl. and Ta3er, J.M. (1984) Biochem. Biophys. Res. Commuo. 123. i~154-1061. 18 Wanders, R J.A., Romeijn, G,J., Sehutgens, R.B.H. and "lager. J.M. (1989) Piachem. Biophy,, Res. Commun. 164, 55(I-555. 19 Bradford, M.M (1976) Anal. Biochem. 72, 248-254, 211 Havekes, L,M,. Schouten. D., De Wit, E,C.M., Cohen. L.H.. GrifEoen. M., Van Hinsbergh, V,W.M and Princen, H.M.G. (lqK6) Biochim. BiGphys. Acta 875, 236.-246. 21 Everson. G.T. anti Polokeff, M.A. (itJ86} L Biol. Chem. 761, 2197-220I. 22 Javilt, M.B., PfelTer. R.. Kok. E.. Burnslein, S.. C-:;hen, B.l, and I~udai. K, (lq89)J. Biol. Chem. 264, !t1384-103~7. 23 Wanticrs R.J.A. VarT Roermund, C.W.T., Griffioen, M. and Cohen, L.H.J.lq91i Biochim. liiophys. Acla 1115, 54--iq~. ")4 Lange. Y. and Muraski, M.F. (t987) J. Biol. Chem. 262. 44334436. 25 Orci. L.. Bro~n, M.S.. Goldslein. J.S,. Garckt-Sequra, L.M. and Anoers:m. R.G.W. (!'4841 Celt 36, 835-845. 26 Reinhart, M r , Biltheimer, J.T., Faust, J.R. and Oaylor. J.L. (1987) I. Bit,i. Chem. 262. %4q)-9655.