Activation of Liver X Receptors Prevents Statin-inducedDeath of 3T3-L1 Preadipocytes*□S

Received for publication, January 28, 2008, and in revised form, May 16, 2008 Published, JBC Papers in Press, May 16, 2008, DOI 10.1074/jbc.M800720200

Lise Madsen‡§1,2, Rasmus K. Petersen‡¶1, Knut R. Steffensen�, Lone M. Pedersen‡, Philip Hallenborg‡, Tao Ma‡,Livar Frøyland§, Stein Ove Døskeland**, Jan-Åke Gustafsson�, and Karsten Kristiansen‡3

From the ‡Department of Biochemistry and Molecular Biology, Campusvej 55, University of Southern Denmark, 5230 Odense M, Denmark,the §National Institute of Nutrition and Seafood Research, Postboks 2029, 5817 Bergen, Norway, ¶BioLigands, InternationalScience Park, 5230 Odense, Denmark, the �Department of Biosciences and Nutrition, Karolinska Institutet, NOVUM,14157 Huddinge, Sweden, and the **Department of Biomedicine, Medical Faculty, University of Bergen, 5097 Bergen, Norway

The biological functions of liver X receptors (LXRs) � and �have primarily been linked to pathways involved in fatty acidand cholesterol homeostasis. Here we report a novel role of LXRactivation in protecting cells from statin-induced death. When3T3-L1 preadipocytes were induced to differentiate by standardisobutylmethylxanthine/dexamethasone/insulin treatment inthe presence of statins, they failed to differentiate and under-went massive apoptosis. The simultaneous addition of selectiveLXR agonists prevented the statin-induced apoptosis. By usingmouse embryo fibroblasts from wild-type (LXR��/�/LXR��/�),LXR� knock-out mice (LXR��/�/LXR��/�), LXR� knock-outmice (LXR��/�/LXR��/�), and LXR double knock-out mice(LXR��/�/LXR��/�) as well as 3T3-L1 cells transduced withretroviruses expressing either wild-type LXR� or a dominantnegative version of LXR�, we demonstrate that the response toLXR agonists is LXR-dependent. Interestingly, LXR-mediatedrescue of statin-induced apoptosis was not related to up-regula-tion of genes previously shown to be involved in the antiapopto-tic action of LXR. Furthermore, forced expression of Bcl-2 didnot prevent statin-induced apoptosis; nor did LXR actiondepend on protein kinase B, whose activation by insulin wasimpaired in statin-treated cells. Rather, LXR-dependent rescueof statin-induced apoptosis in 3T3-L1 preadipocytes requiredNF-�B activity, since expression of a dominant negative versionof I�B� prevented LXR agonist-dependent rescue of statin-in-duced apoptosis. Thus, the results presented in this paper pro-vide novel insight into the action of statins on and LXR-depend-ent inhibition of apoptosis.

Inhibitors of the rate-limiting enzyme of the mevalonatepathway, 3-hydroxy-3-methylglutaryl-CoA reductase, collec-tively known as statins, are well established effective agents

used in the treatment of hypercholesterolemia (1, 2). Some ben-eficial effects of statins cannot solely be ascribed to the loweringof low density lipoprotein cholesterol, and these effects are col-lectively termed pleiotropic effects (3). Statins induce cell deathin a number of different cell lines (4), and it appears that espe-cially malignant cells are dependent on isoprenoids for survival(5–8). Although tumor cells derived from acute myelogenousleukemia undergo apoptosis when treated with statins, myeloidprogenitor cells from normal bone marrow or cord blood donot (9, 10). Statins are well tolerated by humans and consideredsafe (11, 12); hence, the concept of using statins for cancer treat-ment is receiving considerable attention (13, 14).The precise mechanism by which statins induce apoptosis is

not yet elucidated, and it remains unclear which proteins andsignal transduction cascades are involved. Inhibition of 3-hy-droxy-3-methylglutaryl-CoA reductase results in decreasedfarnesylation and geranylgeranylation of several proteins essen-tial for cellular proliferation and survival, such as members ofthe Ras and Rho families, but mechanistic studies also showthat the expression of the proto-oncogene, Bcl-2, is down-reg-ulated in transformed cells undergoing apoptosis in response tostatin exposure (8). Bcl-2 is a key regulator prolonging cell sur-vival by blocking apoptosis (15, 16). Statins down-regulateBcl-2expression levels in colon cancer cells (17), neuroblasts (18),glioma cells (19), humanhepatocytes (20), human breast cancercells (21), and acute myelogenous leukemia cell lines (22).Forced expression of Bcl-2 is able to inhibit statin-induced apo-ptosis in colon cancer cells (17) and in NIH-3T3 fibroblasts(23), and the Bcl-2-related Bcl-xL protects murine tubular cellsfrom statin-induced apoptosis (24). Both Bcl-2 (25–28) andBcl-xL (29) are up-regulated by activation of NF-�B4 in certaincell lines. NF-�B activation has been shown to protect againstapoptosis in different cell lines (30–32), and forced expressionof NF-�B has been reported to increase cell viability and sup-press apoptosis (30–32). Interestingly, the reported effects ofstatins on different cell types comprise repression of NF-�Bactivation (33, 34) as well as activation of NF-�B (35), underlin-

* This work was supported by the Danish Natural Science Research Council,the Norwegian Research Council, and the NOVO Foundation. The costs ofpublication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement” inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. 1.

1 Both authors contributed equally to this work.2 To whom correspondence may be addressed. Fax: 47-55-90-52-99; E-mail:

lise.madsen@bmb.sdu.dk.3 To whom correspondence may be addressed. Fax: 45-6550-2467; E-mail:

kak@bmb.sdu.dk.

4 The abbreviations used are: NF-�B, nuclear factor-�B; DMEM, Dulbecco’smodified Eagle’s medium; ERK, extracellular signal-regulated kinase; I�B,inhibitory factor NF-�B; IKK, I�B kinase; LXR, liver X receptor; LXR-DN, dom-inant negative LXR; MEF, mouse embryo fibroblast; PKB, protein kinase B;RT, reverse transcription; qPCR, quantitative PCR; FBS, fetal bovine serum;PPAR�, peroxisome proliferator-activated receptor �; IGF, insulin-likegrowth factor.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 33, pp. 22723–22736, August 15, 2008© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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ing the cell type-dependent complexity of statin action. Otherprosurvival pathways down-regulated by statins include PKBactivation and nuclear translocation via a P2X7 purinergicreceptor and mTOR-dependent signaling pathway (36, 37).The nuclear receptor liver X receptor (LXR) exists in two

isoforms, LXR� and LXR�. Whereas LXR� is ubiquitouslyexpressed, LXR� is preferentially expressed in liver, adiposetissue, small intestine, and macrophages (38, 39). Both recep-tors are activated by oxysterols (40) and have primarily beenlinked to pathways involved in fatty acid and cholesterol home-ostasis (41, 42). Activation of LXR induces expression of severalATP-binding cassette transporters, which mediate cholesterolefflux from cells (43).Whereas LXR� is expressed in both prea-dipocytes and adipocytes, the expression of LXR� is stronglyinduced during adipocyte differentiation (44, 45). Recently,LXR has also been implicated in cell survival and apoptosis.Gene array analysis revealed that expression of a subset of bothpro- and antiapoptotic genes is regulated in an LXR-dependentmanner (46, 47). LXR-null macrophages undergo acceleratedbacterially induced apoptosis (48), and activation of LXR pre-vents bacterially inducedmacrophage apoptosis (49). Finally, itwas reported that inactivation of LXR� leads to neuronaldegeneration (50).Although the beneficial effects of statins on lipid homeostasis

are well documented, the effects of statins on glucose homeo-stasis and adipocyte function are less well understood, and con-flicting results have been reported (51–55). Here we report thatthe lipophilic statin, simvastatin, induced apoptosis in differen-tiating 3T3-L1 preadipocytes and mouse embryo fibroblasts(MEFs) when administered during the first 4 days of the differ-entiation program. The addition ofmevalonate, but not choles-terol or farnesol, rescued survival and differentiation, and theaddition of geranylgeraniol partly rescued differentiation.Insulin-dependent activation of PKB was impaired in statin-treated 3T3-L1 preadipocytes.We show that the addition of LXRagonists rescuedsurvival anddifferentiation. InhibitionofPKBdidnot prevent LXR agonist-mediated rescue of cell survival but, asexpected, abolished adipocyte differentiation. By using MEFsdeficient for LXR� or LXR� or both and by using retroviralexpression of a dominant negative form of LXR, we demon-strated that LXR agonist-dependent rescuewas indeed depend-ent on LXR activity. Interestingly, LXR-mediated rescue of sta-tin-induced apoptosis was not related to up-regulation of thegenes, AIM/SP�/Api6 and Bcl-xL, previously shown to beinvolved in the antiapoptotic action of LXR in macrophages(48, 49). Furthermore, forced expression of Bcl-2 did not pre-vent statin-induced apoptosis. Rather, activation of LXR pre-vented statin-induced apoptosis in 3T3-L1 preadipocytes in amanner dependent on NF-�B activity, possibly via an up-regu-lation ofCARD14, known to activate I�Bkinase to induce phos-phorylation and degradation of I�B� (56). Thus, the resultspresented in this paper add novel facets to the action of statinson adipocyte differentiation and LXR-dependent inhibition ofapoptosis.

EXPERIMENTAL PROCEDURES

Cell Culture andDifferentiation—3T3-L1 cells were culturedto confluence in DMEM supplemented with 10% calf serum.

Two days postconfluent (designated day 0) cells were inducedto differentiate with Dulbecco’s modified Eagle’s medium(DMEM) supplemented with 10% fetal bovine serum (FBS), 1�M dexamethasone (Sigma), 0.5 mM isobutylmethylxanthine(Sigma), and 1 �g/ml insulin (Roche Applied Science). Inhibi-tors and ligands were dissolved in Me2SO and added when dif-ferentiation was induced. Cells not treated with ligands orinhibitors received similar volumes of Me2SO. After 48 h, themedia were replaced with DMEM supplemented with 10% FBSand 1 �g/ml insulin. The cells were subsequently refed every48 h with DMEM supplemented with 10% fetal bovine serum.When present, 5�M simvastatin (Calbiochem)was added every48 h. Simvastatinwas converted to the active formby dissolvingit in absolute ethanol, followed by the addition of 1 M NaOH toa final concentration of 100 mM. The simvastatin solution wasneutralized with 1 MHCl and diluted in vehicle (Me2SO) beforeuse.MEFs—MEFs were isolated from 13.5-day embryos from

crosses between LXR��/�/LXR��/� (57) or p53�/� (58) het-erozygotes, respectively. The isolatedMEFs were genotyped byPCR. MEFs were grown in AmnioMax basal medium (Invitro-gen) supplemented with 7.5% FBS, 7.5% AmnioMax-C100 sup-plement, 2mM glutamine, 62.5�g/ml penicillin, and 100�g/mlstreptomycin (growth medium) in a humidified atmosphere of5% CO2 at 37 °C. Medium was changed every second day. Fordifferentiation, 2-day postconfluent cells (day 0) were treatedwith growthmedium containing 1�M dexamethasone (Sigma),0.5 mM isobutylmethylxanthine, and 5 �g/ml insulin for 2 days.From day 2, medium contained 5 �g/ml insulin and waschanged every second day.Oil-Red-O Staining—Staining of lipid by Oil-Red-Owas per-

formed as described previously (59).Microscopical Assessment of Apoptosis—Cells were grown on

collagen-coated coverslips in 12-well plates. Cells were fixed in4%paraformaldehyde in PBS for 15min, washed in PBS, stainedwith 2.5�g/�l Hoechst 33258 (Sigma B-2883) in PBS for 5min,and washed three times for 5 min each in PBS. The cells wereexamined by fluorescencemicroscopy to assess chromatin con-densation and by phase-contrast microscopy to assess apopto-tic cell budding and shrinkage.Whole Cell Extracts and Western Blot Analysis—Whole cell

extracts, electrophoresis, blotting, visualization, and strippingof membranes were performed as described (59). Primary anti-bodies used were rabbit anti-phospho-PKB (Ser-473), rabbitanti-PKB (Cell Signaling Technology), rabbit anti-phospho-IKK� (Ser-181), rabbit anti-IKK�/�, rabbit anti-I�B�, mouseanti-Bcl-2, mouse anti-PPAR�, and rabbit anti-TFIIB (SantaCruz Biotechnology, Inc., Santa Cruz, CA). Secondary antibod-ies were horseradish peroxidase-conjugated anti-mouse oranti-rabbit antibodies obtained from DAKO.De Novo Cholesterol Synthesis—Cells were incubated with

[1(2)-14C]acetic acid, sodium salt (0.2�Ci/mlmedium) (Amer-sham Biosciences) for 4 h, harvested in water, and frozen. Cel-lular lipids were extracted from the cell suspensions by themethod of Folch, Lees, and Sloane-Stanley (60), with minormodifications. The cell suspension was mixed with 20 volumesof chloroform/methanol (2:1, v/v) and 4 volumes of 0.9% NaCl,pH 2. The organic phase was evaporated under N2, and the

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lipids were dissolved in n-hexane and separated by TLC onsilica gel plates developed in hexane/diethyl ether/acetic acid(80:20:1, by volume). The bands were detected with iodinevapor, cut into pieces, and assayed for radioactivity by scintilla-tion counting.Cellular Levels of Triacylglycerols—Cells grown in 6-well

plateswere harvested in 1ml ofwater and frozen. The cellsweresonicated, and the cellular levels of triacylglycerols were meas-ured on an AXON Byer spectrophotometer using theTRINDER reaction kit from BioMerieux.Plasmids—The retroviral expression vector pBABE-PPAR�2

was constructed by inserting an SmaI fragment containing full-length mouse PPAR�2 cDNA into the SnaBI site of pBABE-puro. pBABE-LXR� and pBABE-LXR�DN were a kind gift ofDr. Peter Åkerblad. pWZL-Bcl-2 was kindly provided by Dr.Camilla Krakstad, and pLZRS-I�B�-mut (61) was kindly pro-vided by Dr. Paul Khavari. The pBABE-ADD1-DN was kindlyprovided by Dr. Bruce M. Spiegelman (62). The reporter con-struct containing three tandem copies of the �B site (p(�B)3-luc�; Stratagene, La Jolla, CA) has been described previously(63).Transient Transfections—50% confluent 3T3-L1 cells in

12-well dishes were transiently transfected by the calciumphosphate method. Each well received, 50 ng of p(�B)3-luc�reporter and 25 ng of CMV-�-galactosidase were used for nor-malization. 6 h after transfection, the medium was changed,and cells were incubated for 20 h with tumor necrosis factor-�or vehicle (Me2SO) as indicated.Retrovirus Production and Transduction—Phoenix cells

were transfected with viral DNA at 50% confluence. Two dayspost-transfection, the virus-containingmediawere collected bycentrifugation and immediately used to infect 30–40% conflu-ent 3T3-L1 cells by mixing viral supernatant 1:1 with DMEMsupplemented with 10% calf serum. Polybrene (Sigma) wasadded to a final concentration of 7 �g/ml. After 24 h, the trans-duced cells were split and subjected to selection (3 �g/ml puro-mycin (InvivoGen)) (I�B�-mut- and vector-transduced 3T3-L1cells were not subjected to selection). Approximately 3 days later,the selected clones were pooled and replated for differentiation.RT-qPCR—Total RNA was prepared from cells using the

RNeasy minikit (Qiagen) according to the manufacturer’sinstructions. One �g of total RNA was reverse transcribed intocDNA using SuperscriptII and random hexamer primers(Invitrogen). The concentration and quality of the purified totalRNA were determined spectrophotometrically at A260 and bythe A260/A280 ratio, respectively. mRNA expression levels werequantified using the ABI 7500 instrument and the SYBR greentechnology (Applied Biosystems, Foster City, CA). All primerswere designedwith the Primer Express� software, version 2.0, aprogram specifically provided for primer design using ABIqPCR instruments. 100 nM SYBR green assay primers wereused, and for each primer pair, a dissociation curve analysis wascarried out to ensure the specificity of the qPCR amplification.We calculated relative changes employing the comparative CTmethod using 18 S or TBP as the internal reference gene.The primers (upstream and downstream) were as follows:

TBP, 5�-ACCCTTCACCAATGACTCCTATG and 5�-ATGA-TGACTGCAGCAAATCGC; LXR�, 5�-CCGACAGAGCT-

TCGTCC and 5�-CCCACAGACACTGCACAG; LXR�, 5�-TACATCGTGGTCATCTTAGAG and 5�-GGCACAGCT-CATGGC; ABCA1, 5�-TGGTGTCTGTACCGCAAGC and5�-CCCCATTACATAACACATGCC; ABCG1, 5�-CTGTG-AGCCTCAGTTTCCCCand 5�-GGTAAGTCCAATTCGGA-CCC; Nip3, 5�-TTAATCAGGATCCAAAGCTGCTTA and5�-GGCAGGCTTTAAAACCATGCT; CIDE-A, 5�-CCGAG-TACTGGGCGATACAGA and 5�-GGTTACATGAACCAG-CCTTTGG; CIDE-B, 5�-GGATGTTGTCATACGGCCTAGGand 5�-AAGGTGATGCGGGCGA; Aatk, 5�-GCCCAGTT-GGAGTGCAGC and 5�-GTGGAACGGAGGTCAGCG;TIA1, 5�-GGATGGGACCCAATTACAGTGT and 5�-GCAG-GCTGACTAGGCAACATG; Bax, 5�-CACGGACTCCCC-CCGA and 5�-GAAGTTGCCATCAGCAAACATG; p53, 5�-TGCACAAGCGCCTCTCC and 5�-CGCGGATCTTGAG-GGTGA; Bcl2l2, 5�-ACCTTCTCTGACCTGGCCG and5�-GAAACCTGGGTGAAGCGTTG; Mcl-1 5�-GACGGCCT-TCCAGGGC and 5�-CGAGAAAAAGATTTAACATCGCC;Bad, 5�-CTCCGAAGGATGAGCGATGA and 5�-GCGAGG-AAGTCCCTTGAAGG; Bcl-2, 5�-CTGGGATGCCTTTGT-GGAAC and 5�-GAGACAGCCAGGAGAAATCAAAC;CARD14, 5�-TGGACAAAGCCGCTGTCAG and 5�-AGCAC-ACGCTAGGACCACCT; Bcl-xL, 5�-GCGGCTGGGACACT-TTTGT and 5�-TCTCGGCTGCTGCATTGTT; AIM/SP�,5�-CCAGACAGTGACCTCCTCTTCAT and 5�-ACCCTG-GCAGTGCCCC; CARD9, 5�-GCATCTTTCACTGACCC-ATGTAAG and 5�-CCAGGGCTCTTGGGAACC; SREBP1c,5�-GGAGCCATGGATTGCACATT and GCTTCCAGAGA-GGAGGCCAG; SREBP2, 5�-GCGTTCTGGAGACCATGGand 5�-ACAAAGTTGCTCTGAAAACAAATC.

RESULTS

LXR Agonists Rescue Statin-induced Cell Death—When 2days postconfluent 3T3-L1 preadipocytes are treated withisobutylmethylxanthine, dexamethasone, and insulin, they syn-chronously reenter the cell cycle and undergo two sequentialrounds of mitosis in a process known as mitotic clonal expan-sion and subsequently express genes that control the adipocytephenotype (64). During this phase, statin treatment inducedsevere cell death (Fig. 1A). By contrast, statin was well toleratedif added 4 days after induction (Fig. 1A), when the cells aregrowth-arrested (64). The statin-induced cell death was pre-vented by the LXR agonist T0901317 but only when it was pres-ent fromday 0 (Fig. 1B). The protective effect was not restrictedto the Tularik compound T0901317, since the LXR agonistGW3965 also prevented statin-induced cell death (Fig. 1C).Hoechst staining of nuclei revealed that statin-induced celldeath was associated with pronounced hypercondensation ofnuclear chromatin (Fig. 1C). Furthermore, apoptotic cell bud-ding and shrinkage was observed upon treatment with simvas-tatin (results not shown). This indicates that simvastatininduced an apoptotic type cell death in 3T3-L1 preadipocytes.RT-qPCR analysis confirmed that T0901317 treatment

induced known LXR target genes, including the ones involvedin cholesterol transport, and demonstrated that they were up-regulated also in the presence of simvastatin (Fig. 1E). The acti-vation of LXR can stimulate the expression also of other cellulartransport systems (65). To exclude the possibility that activa-

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tion of LXR simply led to transport of simvastatin out of thecells, the effect of simvastatin on cholesterol synthesis wasdetermined in the absence and presence of the LXR agonistT0901317. Although T0901317 tended to increase cholesterolsynthesis in the absence of simvastatin, it failed to rescue cho-lesterol synthesis in the presence of simvastatin (Fig. 1D).We considered next if treatment with LXR agonists worked

by normalizing the expression ofmembers of the SREBP (sterolregulatory element-binding protein) family, known to beaffected by statin-induced cholesterol depletion in preadipo-cytes (66). Furthermore, expression of SREBP-1c also known as

adipocyte determination and differentiation-dependent factor1 (ADD1) is induced by LXR (67). Treatment with simvastatinmarkedly reduced the expression of SREBP-1c, and althoughthe addition of T0901317 induced expression of SREBP-1c, itfailed to restore the expression level to that of cells not treatedwith simvastatin. As expected, simvastatin treatment increasedexpression of SREBP-2, and the addition of T0901317 furtherincreased the expression in the absence or presence of simvas-tatin (supplemental Fig. 1A). The expression data, therefore,did not support that T0901317-dependent survival was linkedto a restoration of SREBP-1c or SREBP-2 expression. The

FIGURE 1. LXR agonists prevent statin-induced cell death in 3T3-L1 cells. Two-day postconfluent 3T3-L1 cells were induced to differentiate by treatmentwith isobutylmethylxanthine, dexamethasone, and insulin, as described under “Experimental Procedures.” From day 4, medium consisted of DMEM with 10%FBS and was changed every second day. A, 5 �M simvastatin dissolved in Me2SO or Me2SO alone was added when differentiation was induced on day 0 or onday 2 or 4 and was present throughout the remaining part of the differentiation period. On day 8, the cells were stained with Oil-Red-O and photographed(�100 magnification). B, 5 �M simvastatin was included from the time of induction at day 0 and was present throughout the differentiation period. 1 �M

T0901317 was included on day 0, 2, or 4 and was present throughout the remaining part of the differentiation period. On day 8, the cells were stained withOil-Red-O and photographed (�100 magnification). C, 5 �M simvastatin dissolved in Me2SO or Me2SO alone was added from the time of induction at day 0 inthe absence or presence of 1 �M T0901317 or 1 �M GW3965. All chemicals were present throughout the differentiation period. The cells were stained withOil-Red-O or Hoechst on day 4. D, 5 �M simvastatin dissolved in Me2SO or Me2SO alone was added from the time of induction at day 0 in the absence or presenceof 1 �M T090131. The media contained U-14C-labeled acetic acid. After 4 h, the cells were harvested, and labeled cholesterol was extracted, isolated on TLC, andquantitated by scintillation counting. E, 5 �M simvastatin dissolved in Me2SO or Me2SO alone was added from the time of induction at day 0 in the absence orpresence of 1 �M T0901317. On day 4, RNA was harvested, and the expression levels of LXR�, LXR�, ABCG1 (ATP-binding cassette, subfamily G (WHITE), member1) and ABCA1 (ATP-binding cassette, subfamily A (ABC1), member 1) were analyzed by real time RT-PCR. One representative experiment out of 3–5 independ-ent experiments performed in duplicates or triplicates is shown (A–C). The bars represent mean � S.D. of four experiments (D–E).

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notion that SREBP-1 activity was not required for rescue ofstatin-induced cell death received further support from exper-iments where preadipocytes were transduced with a retrovirusexpressing a dominant negative version of SREBP-1,ADD1-DN (62). This blunted the expression of the SREBP-1-responsive genes ACC1 and FAS in response to treatment withT0901317 (supplemental Fig. 1, B and C). However, adminis-tration of T0901317 still rescued the cells from statin-induced

cell death (supplemental Fig. 1D).As reported previously (62), expres-sion of ADD1-DN inhibited adipo-cyte differentiation, as determinedby Oil-Red-O staining. Of note,treatment with T0901317 also par-tially restored lipid accumulation incells expressing the dominant nega-tive form of SREBP-1 (supplementalFig. 1D).The fact that statin treatment did

not induce apoptosis if adminis-tered after day 4 of the differentia-tion program, when PPAR�2expression is induced and the cellsbegin to accumulate triacylglycerollipid droplets, might imply thatstatin-dependent repression ofPPAR�2 expression was involved inthe induction of apoptotis (54). Toinvestigate this possibility, we trans-duced 3T3-L1 cells with an emptyretrovirus or a retrovirus expressingPPAR�2. Fig. 2A illustrates theoverexpression of PPAR�2 in thetransduced 3T3-L1 cells. The cellswere subjected to the standard pro-tocol for induction of adipocyte dif-ferentiation and treated with differ-ent combinations of the PPAR�agonist rosiglitazone, the LXR ago-nist T0901317, and simvastatin, asshown in Fig. 2B. Forced expressionof PPAR�2 in 3T3-L1 cells led to theexpected enhancement of adipocytedifferentiation as determined byaccumulation of triacylglycerol (Fig.2C). However, in relation to statin-induced apoptosis, no differencesbetween cells transduced with theempty retroviral vector or the retro-viral vector expressing PPAR�2were observed. In both cases, statinwas able to induce cell death whenadministered from day 0, irrespec-tive of whether rosiglitazone wasadded or not, and the simultaneousaddition of T0901317 protected thecells from apoptosis and partly res-cued differentiation (Fig. 2, B and

C). Thus, the statin-induced apoptosis was independent ofPPAR�2 expression.Statin-induced Cell Death Is Prevented by Mevalonate and

Geranylgeraniol—Statins are competitive inhibitors of the3-hydroxy-3-methylsglutaryl-CoA reductase, the rate-limitingstep in the mevalonate biosynthetic pathway. This pathwaygives rise to not only cholesterol but also ubiquinone, dolichol,and other isoprenoids, farnesol and geranylgeranol, which are

FIGURE 2. Expression of PPAR�2 is insufficient to prevent statin-induced cell death in 3T3-L1 cells. 3T3-L1cells were retrovirally transduced by a PPAR�2-expressiong vector or an empty vector and grown to conflu-ence after selection. A, overexpression of PPAR�2 was verified by Western blotting. B and C, transduced 3T3-L1cells were grown to 2 days postconfluence and induced to differentiate as described under “ExperimentalProcedures.” Cells were treated with 5 �M simvastatin in the absence or presence of 1 �M T0901317 or 0.5 �M

rosiglitazone (Rosi). On day 8, the cells were stained with Oil-Red-O and photographed (B), or the cells wereharvested and the cellular content of triacylglycerol was measured (C). One representative experiment of threeindependent experiments performed in duplicates or triplicates is shown (A and B). The bars represent mean �S.D. of three experiments performed in triplicates (C).

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involved in regulation of cellular proliferation. As anticipated,mevalonate rescued statin-induced cell death and restored dif-ferentiation of statin-treated cells (Fig. 3A). By contrast, theaddition of cholesterol or farnesol did not rescue statin-treatedcells from cell death (Fig. 3, B and C), whereas the addition ofgeranylgeraniol rescued the cells from statin-induced cell deathbut only partly restored adipocyte differentiation (Fig. 3D).Insulin/IGF-1 stimulation plays a pivotal role in the survival

of preadipocytes and induction of adipocyte differentiation.These processes have been shown to be intimately associatedwith prenylation of members of the Ras and RhoA families.Insulin stimulates the activity of farnesyl and geranylgeranyl

transferases (68), and pharmacolog-ical or genetic inhibition of thesetransferases impairs insulin signal-ing and prevents adipocyte differen-tiation (69–71). Impaired prenyla-tion also interferes with activationof ERK in 3T3-L1 preadipocytes andadipocytes (68, 69). In line with aprevious study showing that simvas-tatin impaired insulin/IGF-1 signal-ing (72), we found that simvastatinalmost completely prevented theincrease in IGF-1-mediated PKBphosphorylation in 3T3-L1 cells(Fig. 4A). To know if ERK- and PKB-dependent signaling had a majorrole in LXR agonist-dependent res-cue of statin-induced cell death, wetreated 3T3-L1 preadipocytes withstandard adipogenic inducers anddifferent combinations of simvasta-tin and inhibitors of the ERKand thePKB signaling pathways. Inhibitionof either the ERK or PKB activationdid not interfere with the ability ofT0901317 to protect the cells fromapoptosis, but as expected, inhibi-tion of PKB activation preventedadipocyte differentiation with accu-mulation of lipid (Fig. 4B). We con-clude that rescue of statin-treatedcells from death by LXR activationproceeds in the absence of activesignaling via ERKs or PKB and thatprotection of preadipocytes againstdeath can be dissociated fromdifferentiation.The Antiapoptotic Response to

LXR Agonists is LXR-dependent,and Overexpressed LXR Can ProtectEven in the Absence of AddedAgonists—LXR� is constitutivelyexpressed in both preadipocytesand mature adipocytes (44),whereas LXR� is induced duringadipogenesis and has much higher

expression in adipocytes than in preadipocytes (45). Growth-ar-rested 3T3-L1 cells on day 4 expressed relatively high levels ofLXR�anddidnotundergoextensive simvastatin-inducedapopto-sis even in the absence of added LXR agonists, suggesting that theendogenous level and/or activity of LXR was sufficient to protectthem against statin-induced death (Fig. 1A). If this is true, weexpected even the growth-arrested 3T3-L1 cells (day 4) to becomesensitive to simvastatin if LXRexpression/activitywas experimen-tally attenuated. Conversely, 3T3-L1 cells undergoing mitoticclonal expansion shouldbecome resistant to statins if LXRexpres-sion was ectopically increased. To test this notion, 3T3-L1 cellswere transduced with retroviruses expressing wild-type LXR�, a

FIGURE 3. Mevalonate and geranylgeraniol, but not cholesterol and farnesol, rescue 3T3-L1 cells fromstatin-induced cell death. 3T3-L1 cells were induced to differentiate as described under “Experimental Pro-cedures.” Cells were treated with 5 �M simvastatin in the absence or presence of 250 �M mevalonate (A); 1, 5, or10 �g/ml cholesterol (B); 1, 10, or 50 �M farnesyl pyrophosphate triammonium (C); or 1, 10, or 50 �M gera-nylgeranyl pyrophosphate triammonium salt (D). On day 8, the cells were stained with Oil-Red-O and photo-graphed, or the cells were harvested, and the content of triacylglycerol was measured. One representativeexperiment of three or four independent experiments performed in duplicates or triplicates is shown. The barsrepresent mean � S.D. of three or four experiments performed in triplicates.

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dominant-negative LXR� (LXR-DN), or an empty vector, and thetransduced cells were then treated with simvastatin and/or LXRagonists from day 0 or day 4.Judging by the expression of known LXR-responsive gene

ABCA1 (ATP-binding cassette, subfamily A,member 1), forcedexpression of LXR� sensitized the cells to agonist treatment,whereas LXR-DN abolished the effects of LXR agonist treat-ment (Fig. 5A). Furthermore, forced expression of LXR� res-cued the cells from simvastatin-induced cell death, also in theabsence of exogenous LXR ligands. As previously reported (45),the addition of LXR agonists to 3T3-L1 preadipocytes withforced expression of LXR prevented adipocyte differentiation,further demonstrating that LXR-dependent rescue of statin-induced apoptosis was independent of cellular differentiation.On the other hand, ablation of LXR activity by expression ofLXR-DN sensitized the cells to simvastatin-induced cell death,which was not prevented by T0901317 or GW3965 (Fig. 5B).Similarly, growth-arrested 3T3-L1 cells became sensitive tosimvastatin when LXR-DN was expressed, further corroborat-ing the notion that cell survival was LXR-dependent (Fig. 5B).

In order to decipher whether cell survival was LXR subtype-selective, we used primary MEFs from LXR��/� and LXR��/�

as well as LXR��/�/LXR��/� and wild-type embryos. TheMEFs were treated with adipogenic inducers as describedunder “Experimental Procedures” and treated with simvastatinand/or the LXR agonist T0901317 from day 0, as shown in Fig.6. As previously described, LXR�was expressed at a low level inwild-type undifferentiated MEFs (73), but similar to 3T3-L1cells, expression of LXR� was induced upon induction of adi-pocyte differentiation (Fig. 6A). Expression of LXR� tended tobe slightly higher in LXR��/�/LXR��/� MEFs than wild-typeMEFs (Fig. 6A). To examine the ability of LXR agonists toinduce expression of a canonical LXR-responsive gene, day 0MEFs were treated for 24 h with T0901317 or GW3965, andexpression of ABCA1 was determined by RT-qPCR analysis.Fig. 6B shows that both LXR agonists induced ABCA1 expres-sion more strongly in wild-type than in LXR��/�/LXR��/�

MEFs, suggesting that even the loss of the low level of LXR� inwild-type cells could impact ABCA1 expression. No inductionof ABCA1 was observed in LXR��/�/LXR��/� or LXR��/�/

FIGURE 4. Rescue of statin-treated cells by LXR activation proceeds in the absence of active signaling via ERKs or PKB. A, 3T3-L1 cells were grown to 2days postconfluence and then treated with dexamethasone and increasing concentrations of IGF-1 in the absence or presence of 5 �M simvastatin, as indicatedin the figure. After 15 min, whole cell extracts were prepared, and the levels of phosphorylated PKB and total PKB were determined by Western blotting.Autoradiographs were analyzed by densitometric scanning, and the levels of phosphorylated PKB relative to total PKB were determined. The error barsrepresent S.D. (n � 3). B, 3T3-L1 cells were grown to 2 days postconfluence and induced to differentiate as described under “Experimental Procedures.” 5 �M

simvastatin and 1 �M T0901317 were dissolved in Me2SO and added when differentiation was induced on day 0 in the absence or presence of 1 �M T0901317and 10 �M U0126, 100 nM wortmannin, or 20 �M Ly294002. On day 2 and day 4, the cells were stained with Oil-Red-O and photographed (�100 magnification).One representative experiment of three independent experiments is shown.

FIGURE 5. The response to LXR agonists is LXR-dependent in 3T3-L1 cells. 3T3-L1 cells were retrovirally transduced by vectors expressing LXR� or LXR-DNor an empty vector and grown to confluence after selection. A, transduced 3T3-L1 cells were grown to 2 days postconfluence and treated with the LXR agonist1 �M T0901317, 1 �M GW3965, or Me2SO for 24 h. RNA was harvested, and the expression level of ABCA1 (ATP-binding cassette, subfamily A (ABC1), member1) was measured by RT-qPCR. The bars represent mean � S.D. of four experiments. B, transduced 3T3-L1 cells were grown to 2 days postconfluence andinduced to differentiate. 5 �M simvastatin dissolved in Me2SO or Me2SO alone was added when differentiation was induced on day 0 or on day 4 and waspresent throughout the remaining part of the differentiation period in the absence or presence of 1 �M T0901317 or 1 �M GW39655. On day 8, the cells werestained with Oil-Red-O and photographed (�100 magnification). One representative experiment of three independent experiments performed in duplicatesor triplicates is shown.

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LXR��/� MEFs (Fig. 6B). Simvastatin induced cell death morestrongly in LXR��/�/LXR��/� than in wild-type, LXR��/�/LXR��/�, or LXR��/�/LXR��/� MEFs (Fig. 6C), suggestingthat the complete loss of LXR expression sensitized the cells tostatin-dependent cell death.The LXR agonist T0901317 rescued statin-treated cells from

apoptosis in LXR��/�/LXR��/� MEFs as well as in wild-typeMEFs (Fig. 6C). The rescue was abolished, however, in LXR��/�/LXR��/� and LXR��/�/LXR��/� MEFs (Fig. 6C). This resultsuggested that LXR� was responsible for the antiapoptotic

effect of LXR agonists in nondiffer-entiated cells. The very low LXR�expression in undifferentiatedMEFs, although apparently able toprovide some protection againstsimvastatin in the absence of addedLXR agonist (see above), was prob-ably insufficient tomediate the anti-apoptotic effect of T0901317. Sinceforced expression of LXR� rescuedundifferentiated cells from statin-induced cells death (Fig. 5), ourresults collectively indicate thatboth LXR� and LXR� are able topartially protect against statin-in-duced death in the absence of addedLXR agonist and are required tomediate the pronounced protectionafforded by added LXR agonist.Regulation of Expression of Genes

Involved in Apoptosis and Survivalby Simvastatin and the LXR AgonistT0901317—Since LXR is a wellestablished transcription factor, anattractive possibility would be thatactivation of LXR reversed a statin-dependent up-regulation of proapo-ptotic genes or down-regulation ofantiapoptotic genes. In fact, theexpression of several genes involvedin apoptosis or survival has beenreported to be regulated in responseto administration of LXR agonists(47). Based on these reports and anAffymetrixTM-based expressionanalysis of 3T3-L1 preadipocytestreated with simvastatin and LXRagonists (not shown), we selected asubset of genes for further analysisby RT-qPCR. Treatment of cellswith T0901317 reduced the expres-sion of CIDE-A, CIDE-B, and Nip3,as well as Aatk and TIA1, but to asimilar degree in the absence andpresence of simvastatin (Fig. 7, row1). Certain other genes involved inapoptosis, such asp53 andBax, havebeen reported to be up-regulated by

statins in different cell lines, and expression of p53 and Baxwasalso up-regulated by simvastatin in 3T3-L1 cells (Fig. 7, row 2).Concomitant treatment with the LXR agonist T0901317 sup-pressed the up-regulation of p53 but notBax (Fig. 7, row 2). Theeffects were selective, since expression of several additionalgenes involved in apoptosis or survival, such as Bcl2l2, Mcl-1,and Bad, was unaffected by either simvastatin or T0901317treatment (Fig. 7, row 2).LXR-dependent gene expression was recently shown to be

important for macrophage survival (48, 49). AIM (apoptosis

FIGURE 6. The response to LXR agonists is LXR-dependent in MEFs. MEFs were isolated from LXR��/�,LXR��/�, LXR��/�/LXR��/�, and wild-type embryos and grown to confluence in AmnioMax basal mediumsupplemented with 7.5% FBS, 7.5% AmnioMax-C100 supplement, and 2 mM L-glutamine. A and C, MEFs weregrown to 2 days postconfluence and induced to differentiate. 5 �M simvastatin dissolved in Me2SO or Me2SOalone was added when differentiation was induced on day 0 in the absence or presence of 1 �M T0901317 andwas present throughout the remaining part of the differentiation period. RNA was harvested at the indicatedtime points, and the expression levels of LXR� and LXR� were measured by RT-qPCR (A). On day 8, the cellswere stained with Oil-Red-O and photographed (�100 magnification) (C). B, MEFs were grown to 2 dayspostconfluence and treated with the LXR agonist 1 �M T0901317, 1 �M GW3965, or Me2SO for 24 h. RNAwas harvested, and the expression level of ABCA1 (ATP-binding cassette, subfamily A (ABC1), member 1)was measured by RT-qPCR. The bars represent mean � S.D. of three experiments performed in triplicates(A–B). One representative experiment of three independent experiments performed in duplicates ortriplicates is shown (C).

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inhibitor of macrophages), also known as SP� or Api6, is regu-lated in an LXR-dependent manner and protects macrophagesfrom bacteria-induced apoptosis (48, 49). In 3T3-L1 cells, sur-prisingly, AIM/SP�/Api6 was up-regulated by simvastatintreatment in the absence but not in the presence of T0901317(Fig. 7, row 3). Thus, expression of AIM/SP�/Api6 was not up-regulated by LXR activation in 3T3-L1 cells. The expression ofBcl-xL, an antiapoptotic form ofBcl-x, also known to be up-reg-ulated by LXR agonist inmacrophages, was not up-regulated byT0901317 in 3T3-L1 cells (Fig. 7, row 3). Collectively, these

results illustrate the cell-specific regulation of LXR-responsivegenes and clearly demonstrate that the mechanism for LXR-mediated prevention of bacteria-induced apoptosis in macro-phages is not operative in the context of LXR-dependent rescueof statin-mediated apoptosis in 3T3-L1 preadipocytes.The RT-qPCR analysis further revealed that expression of a

major gene involved in the prevention of apoptotic cell death,Bcl-2, was up-regulated by T0901317 and further enhanced inthe presence of simvastatin (Fig. 7, row 3). In addition, expres-sion of CARD14 (caspase recruitment domain-containing 14)

FIGURE 7. LXR agonists down-regulate the expression of proapototic genes. 3T3-L1 cells were grown to 2 days postconfluence and induced to differen-tiate. 5 �M simvastatin dissolved in Me2SO or Me2SO alone was added when differentiation was induced on day 0 in the absence or presence of 1 �M T0901317.RNA was harvested on day 4, and expression levels of NIP3 (BCL2/adenovirus E1B-interacting protein 1), CIDE-A (cell death-inducing DNA fragmentation factor,� subunit-like effector A), CIDE-B (cell death-inducing DNA fragmentation factor, � subunit-like effector B), Aatk (apoptosis-associated tyrosine kinase), TIA1(cytotoxic granule-associated RNA-binding protein 1), p53, Bax (Bcl2-associated X protein), Bad (Bcl-associated death promoter), Mcl-1 (myeloid cell leukemiasequence 1), Bcl2l2 (Bcl2-like 2), AIM (apoptosis inhibitor expressed by macrophages), also known as SP�/CT-2/Api6, Bcl-xL (Bcl2-like 1), CARD9 (caspaserecruitment domain family, member 9), Bcl-2 (B-cell leukemia/lymphoma 2), and CARD14 (caspase recruitment domain-containing protein 14) were measuredby RT-qPCR. The bars represent the mean � S.D. of four experiments.

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was up-regulated by T0901317, both in the absence and pres-ence of simvastatin (Fig. 7, row 3).Statin-induced Cell Death Is Independent of p53 and Cannot

Be Rescued by Bcl-2 Overexpression—Based on the aboveresults (Fig. 7), we studied whether the prevention of simvasta-tin-enhanced p53 expression or the enhancement of Bcl-2induction by LXR agonist was relevant for cell survival. p53 is akey regulator of cell death, and p53 deficiency or p53 inhibitionprotects certain cell types from a wide variety of acute apopto-sis-inducing agents (74). The finding that T0901317 treatmentprevented both simvastatin-induced cell death and p53 induc-tion indicated that simvastatin-induced cell death might beconnected with induction of p53. To examine this possibility,we investigated whether simvastatin was able to induce celldeath in p53-deficientMEFs induced to undergo adipocyte dif-ferentiation. MEFs isolated from p53�/� embryos were treatedwith simvastatin in the absence and presence of T0901317. Thelack of p53 did not affect simvastatin-induced cell death or theability of T0901317 to counteract the effects of simvastatin (Fig.8A). Therefore, although p53 expression is regulated inresponse to simvastatin and T0901317 administration, p53 isdispensable for statin-induced cell death.Since forced expression ofBcl-2 has been reported to prevent

statin-induced apoptosis in colon cancer cells (17) and in NIH-3T3 fibroblasts (23), the significant up-regulation of Bcl-2expression by T0901317 in the presence of simvastatin wasintriguing and suggested that statin-induced cell death mightbe counteracted by up-regulation of Bcl-2 expression. To testthis, we stably transduced 3T3-L1 preadipocytes with an emptyvector or a vector expressing Bcl-2. Western blotting verified astrong overexpression of Bcl-2 that by far exceeded the levelsobserved in cells transduced with the empty vector and wassimilar in cells treated with vehicle, simvastatin, and/orT0901317 (Fig. 8B). Surprisingly, forced expression did notdiminish statin-induced cell death, indicating that up-regula-tion of Bcl-2 expression was insignificant for T0901317-medi-ated rescue of cell death (Fig. 8C).LXR-mediated Protection against Simvastatin-induced

Apoptosis Requires NF-�B Activity—Although NF-�B has beenimplicated in activation of apoptosis in several cell lines (75–77), activation of NF-�B also plays a key role in the protectionagainst apoptosis in many cell types and cell lines (30–32),including 3T3-L1 cells (78). In this context, it was of interestthat the expression of CARD14 was strongly enhanced in cellstreated with simvastatin and T0901317 (Fig. 7, row 3). CARD14has been shown to associate with the CARD domain of BCL10,forming a signaling protein complex activating NF-�B throughthe IKK complex (56). Hence, we hypothesized that NF-�Bactivitymight be involved in the LXR agonist-dependent rescueof statin-induced cell death. This hypothesis was further sup-ported by the finding that T0901317 augmented the phospho-rylation of IKK� in 3T3-L1 cells in the absence or presence ofsimvastatin (Fig. 9A). To investigate the importance of NF-�B-dependent signaling, we stably transduced 3T3-L1 cells with avector expressing a dominant negative (nonphosphorylatable)form of I�B�. Overexpression of themutated I�B�was verifiedby Western blotting (Fig. 9B). The inability of the stably trans-duced cells to activate NF-�B-dependent transactivation was

confirmed in transient transfection experiments, demonstrat-ing that tumor necrosis factor-� was unable to induce a �B-re-sponsive reporter gene in cells overexpressing themutant formof I�B� (Fig. 9C).The vector-transduced cells and cells with forced expression

of the mutant I�B� were subsequently induced to differentiatein the presence or absence of simvastatin, and the effect on cell

FIGURE 8. Statin-induced cell death is independent of p53 and cannot berescued by Bcl-2 overexpression. A, MEFs were isolated from p53�/�

embryos; grown to 2 days postconfluence in AmnioMax basal medium sup-plemented with 7.5% FBS, 7.5% AmnioMax-C100 supplement, and 2 mM

L-glutamine; and then induced to differentiate. 5 �M simvastatin dissolved inMe2SO or Me2SO alone was added when differentiation was induced on day0 and was present throughout the remaining part of the differentiationperiod in the absence or presence of 1 �M T0901317. On day 2 and day 4, thecells were stained with Oil-Red-O and photographed (�100 magnification). Band C, 3T3-L1 cells were retrovirally transduced with a vector expressing Bcl-2or an empty vector and grown to confluence. B, whole cell extracts wereprepared and analyzed for expression of Bcl-2 by Western blotting. An anti-body recognizing TFIIB (transcription factor IIB) was used as control for equalloading. C, 3T3-L1 transduced with Bcl-2 or an empty vector were grown to 2days postconfluence and induced to differentiate. 5 �M simvastatin dissolvedin Me2SO, or Me2SO alone, were added when differentiation was induced onday 0 in the absence or presence of 1 �M T0901317, and was present through-out the remaining part of the differentiation period. On days 2 and 4, the cellswere stained with Oil-Red-O and photographed (�100 magnification). Onerepresentative experiment of two independent experiments performed induplicates or triplicates is shown.

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death of added T0901317 was determined. Regardless of theexpression of I�B�, simvastatin strongly induced cell death. Invector-transduced cells, administration of T0901317 rescuedthe cells, but in cells overexpressing the mutated I�B�,T0901317 was unable to prevent apoptosis (Fig. 9D). Takentogether, these results indicate, surprisingly, that NF-�B activ-ity was required for LXR-dependent rescue of statin-inducedcell death.

DISCUSSION

The present report provides novel information on the effectsof a commonly prescribed lipophilic statin, simvastatin, on

fibroblastic cells capable of under-going adipocyte differentiation,namely the preadipocyte cell line3T3-L1 and mouse embryo fibro-blasts. Adipocytes are importantplayers in the control of whole bodylipid and glucose homeostasis, andthe effects of statins prescribed forthe treatment of hypercholesterol-emia, often associated with obesity,are therefore not only of interestfrom a basic scientific point of viewbut also in a more clinical perspec-tive. It should, however, be pointedout that the concentration of simv-astatin used in this article (5 �M)and the concentrations of statinsused in most in vitro studies arehigher than the 1 �M plasma con-centration achieved in vivo (79, 80).It is generally observed that statinsaffect negatively the differentiationof adipocytes (51–55). Variousmechanisms have been suggested,but upon close inspection of manyreports, it appears that the statinsmost potently affected adipogenesisduring the first 4 days of the differ-entiation program, as shown explic-itly in the present study. Regardingthe molecular mechanism, it hasbeen reported that pitavastatin,another lipophilic statin, preventedadipocyte differentiation of 3T3-L1preadipocytes by inhibiting PPAR�expression but not expression ofC/EBP�, and interestingly pitavas-tatin also prevented the normaldown-regulation of Pref-1 expres-sion (54). Here we show that forcedexpression of PPAR�2 did not res-cue adipocyte differentiation in thepresence of simvastatin. Since ele-vated and sustained expression ofPref-1 is associated with an inhibi-tion of IGF-1/insulin signaling (81),

it is possible that the statin-dependent down-regulation of IGF-1/insulin signaling (72) (this work) combined with a sustainedexpression of Pref-1 prevents adipogenesis even in 3T3-L1 cellswith forced expression of PPAR�2. Impaired differentiation ofstatin-treated preadipocytes also clearly relates to impairedIGF-1/insulin signaling due to statin-induced inhibition of thesynthesis of farnesyl and geranylgeranyl precursors for modifi-cation of members of the Ras and Rho families of small G-pro-teins (51, 68–71).Only a few reports have investigated statin-dependent apo-

ptosis in preadipocytes or adipocytes (52, 82). We show thatstatins when administered during the first days of the differen-

FIGURE 9. LXR-mediated protection against simvastatin-induced apoptosis requires NF-�B activation.A, 3T3-L1 cells were grown to 2 days postconfluence and induced to differentiate. 5 �M simvastatin dissolvedin Me2SO or Me2SO alone was added when differentiation was induced on day 0 in the absence or presence of1 �M T0901317. Whole cell extracts were prepared on day 4 after induction and analyzed for the presence ofphosphorylated IKK� and total IKK�/� by Western blotting. Autoradiographs were analyzed by densitometricscanning, and the levels of phosphorylated IKK� relative to total IKK�/� were determined. The error barsrepresent S.D. (n � 3). B–D, 3T3-L1 cells were retrovirally transduced by a nondegradable I�B mutant or anempty vector and grown to confluence. B, whole cell extracts were prepared and analyzed for the presence ofI�B by Western blotting. An antibody recognizing TFIIB (transcription factor IIB) was used as control for equalloading. C, transduced 3T3-L1 cells were grown to 2 days postconfluence and transfected with a p(�B)3-lucreporter. Medium was changed after 6 h, and the cells were subsequently treated with tumor necrosis factor-�or water for 20 h prior to harvest. In all transfections, empty expression vector was added to ensure equalpromoter load, and an SV40-�-galactosidase construct was used for normalization. Reporter activity was nor-malized to �-galactosidase values, and -fold induction is presented as the mean � S.D. Transfections wereperformed in triplicate and measured in duplicates. D, transduced 3T3-L1 cells were grown to 2 days postcon-fluence and induced to differentiate. 5 �M simvastatin dissolved in Me2SO or Me2SO alone was added whendifferentiation was induced on day 0 and was present throughout the remaining part of the differentiationperiod in the absence or presence of 1 �M T0901317. On days 2 and 4, the cells were stained with Oil-Red-O andphotographed (�100 magnification). One representative experiment of three independent experiments per-formed in duplicates or triplicates is shown.

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tiation process elicited pronounced 3T3-L1 cell death withnuclear morphology of apoptosis. Statin-induced cell deathwas, as expected, prevented by the addition of mevalonate butnot by the addition of cholesterol or farnesol. The addition ofgeranylgeraniol rescued the cells from statin-induced death butonly partly restored differentiation and lipid accumulation. Therescue of cell death and partial rescue of differentiation bygeranylgeraniol point to the involvement of impaired geranylgera-nylationofmembers of theRho family ofG-proteins. This conclu-sion is in linewith the finding that geranylgeranyl-pyrophosphate/geranylgeraniol, but not cholesterol or farnesyl-pyrophosphate/farnesol, was able to rescue stain-induced cell death of corticalneurons (83) andmyotubes (84).Members of the Rho family of G-proteins are crucially

involved in IGF-1/insulin signaling, survival, and adipocyte dif-ferentiation (85–88); hence, it is not surprising that perturba-tion of geranylgeranylation impacts seriously on preadipocytesinduced to undergo adipocyte differentiation. It is possible thattreatment with differentiation inducers per se causes a condi-tion of stress in the cells and that this aggravates the effects ofstatins. Along this line, it was reported that simvastatin did notaffect 3T3-L1 fibroblasts, which were not induced to undergodifferentiation (55).Surprisingly, we discovered that statin-induced cell death

was prevented by the simultaneous addition of LXR agonists.The effect was not just observed for the classical T0901317 LXRagonist but could also be reproduced using the more selectiveLXR agonist GW3965. Rescue from statin-induced cell deathwas not dependent on the activity of SREBP-1. By using a com-bination of forced expression of wild-type and dominant nega-tive LXR in 3T3-L1 cells with the use of primary MEFs isolatedfrom LXR��/� and LXR��/� as well as LXR��/�/LXR��/�

andwild-type embryos, we provided evidence that the rescue ofstatin-induced cell death was dependent on the expressionand/or activity of either LXR� or LXR�. Thus, our data did notindicate subtype-specific effects of LXR in relation to the rescueof statin-induced cell death. This finding distinguishes LXR-de-pendent rescue of statin-induced cell death in 3T3-L1 cellsfrom the LXR-dependent rescue of bacterially induced apopto-sis of macrophages, which is orchestrated via an LXR�-selec-tive up-regulation of AIM/SP�/Api6 (48). Furthermore, in thecase of LXR-dependent rescue of statin-induced cell death in3T3-L1 cells, we did not observe any up-regulation of AIM/SP�/Api6 expression by administration of an LXR agonist; incontrast, we observed a marked up-regulation of AIM/SP�/Api6 expression by simvastatin.Since LXR signaling is induced by insulin (89), we speculated

whether LXR activation could override the statin-inducedabrogation of IGF-1/insulin survival signals impinging on alter-native pathways for PKB and/or ERK-dependent signaling.However, LXR-mediated rescue of statin-induced cell deathproceeded unabated in the presence of inhibitors of PI3-K/PKBand ERK activation, suggesting that LXR-dependent rescue didnot require signaling along these pathways. The expression ofp53was up-regulated by simvastatin, but this up-regulationwasprevented by the addition of T0901317, suggesting that p53-mediated processes might be involved in statin-induced celldeath. However, the addition of simvastatin to p53-deficient

MEFs induced to undergo adipocyte differentiation elicited thesame degree of cell death as was observed in wild-type MEFs.Statins are reported to down-regulate expression of Bcl-2

and/or Bcl-xL in several different cell lines (17–20, 22, 24).However, in 3T3-L1 cells, simvastatin did not affect expressionof either Bcl-2 or Bcl-xL. Expression of Bcl-2 was, however,increased by T0901317, especially in the presence of simvasta-tin. Forced expression of Bcl-2 inhibits statin-induced apopto-sis in colon cancer cells (17), whereas Bcl-xL overexpressionprotects from apoptosis induced by statins in murine tubularcells (24). Thus, up-regulation of Bcl-2might contribute to theLXR-mediated rescue of simvastatin-induced cell death also in3T3-L1 cells. Unexpectedly, forced overexpression of Bcl-2 didnot prevent statin-induced cell death.A surprising clue to a possible mechanism involved in LXR-

mediated rescue came from the observation that T0901317increased the expression of CARD14, a protein reported to beinvolved in IKK activation, leading to increased NF-�B activity(56). In keeping with this, we observed that T0901317 admin-istration increased IKK� phosphorylation and, furthermore,that inhibition of NF-�B activity by overexpression of a non-degradable form of I�B� completely prevented LXR-depend-ent rescue of statin-induced cell death. The finding that LXR/LXR agonist-dependent rescue relied on NF-�B activity wasunexpected, since it is well established that LXR/LXR agonistsnormally down-regulate NF-�B activity or prevent NF-�B acti-vation (90). However, it is conceivable that the perturbation ofsignaling bymembers of the Rho family ofG-proteins caused byimpaired prenylation changes the normal repressive mode ofLXR in a manner analogous to that observed for the statin-induced switch between an activating and a repressive state ofPPAR� (91). In conclusion, in this paper, we describe a novelmechanism for LXR-dependent rescue of cell death that doesnot depend on regulation of the previously described mediatorof LXR-dependent rescue of apoptosis or regulation of the nor-mally involved pro- or antiapoptotic genes. Rather, the mecha-nism involves a novel and puzzling positive collaborationbetween LXR- and NF-�B-dependent pathways. Consideringthe common use of statins for treatment of hypercholesterol-emia, the emerging use of statins combined with nuclear recep-tor agonists for prevention of coronary heart diseases and thepossibilities to employ statins for cancer treatment, furtherstudies of the interplay between statins and LXR agonistsappear warranted.

Acknowledgments—We thank Dr. Stephen N. Jones for p53�/� MEFsand Dr. Camilla Krakstad, Dr. Paul Khavari, Dr. Bruce M.Spiegelman, and Dr. Peter Åkerblad for valuable plasmids.

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LXR Activity Prevents Statin-induced Death of Preadipocytes

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Karsten KristiansenHallenborg, Tao Ma, Livar Frøyland, Stein Ove Døskeland, Jan-Åke Gustafsson and

Lise Madsen, Rasmus K. Petersen, Knut R. Steffensen, Lone M. Pedersen, PhilipPreadipocytes

Activation of Liver X Receptors Prevents Statin-induced Death of 3T3-L1

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