Role for sterol regulatory element binding protein in the regulation of farnesyl diphosphate synthase and in the control of cellular levels of cholesterol and triglyceride: evidence from sterol regulation-defective cells

Simon M. Jackson,*$t Johan Ericsson,*,t James E. Metherall,** and Peter A. Edwards11**t9§ Departments of Biological Chemistry* and Medicinet and the Molecular Biology Institute,§ University of California Los Angeles, Los Angeles, CA 90095, and Department of Human Genetics,** Program in Human Molecular Biology and Genetics, University of Utah, Salt Lake City, UT 84112

Abstract In order to define the factors involved in the regu- lation of farnesyl diphosphate (FPP) synthase, we used sterol regulation-defective (SRD) cell lines that constitutively ex- press either high (SRD-2) or low (SRDB) levels of transcrip- tionally active sterol regulatory element binding protein (SREBP). FPP synthase mRNA levels were high in SRD-2 cells and low in SRD-6 cells and were unaffected by the addition or removal of sterols from the media. In contrast, the mRNA levels in parental C H 0 7 cells were regulated by sterols. SRD- 2, SRD-6, and CHO-7 cells were also transiently transfected with plasmids containing FPP synthase promoter-reporter genes. Reporter gene activity was significantly higher in SRD- 2 cells than in either SRDG or CHO-7 cells, consistent with a higher rate of transcription of the reporter gene in SRD-2 cells. The high expression of the reporter gene in SRD-2 cells was not observed when the FPP synthase promoter contained a three base pair mutation within an SREBP binding site, termed sterol regulatory element-3 (SRE-3). These ob- servations are consistent with the hypothesis that high levels of transcription of the FPP synthase gene are de- pendent on the availability of transcriptionally active SREBP.m We also demonstrate that the incorporation of radioactive acetate into both cholesterol and fatty acids was enhanced in SRD-2 cells as compared to CHO-7 or SRDG cells. Finally, we demonstrate that the concentrations of cho- lesterol, cholesteryl ester, and triglyceride were all signifi- cantly elevated in SRD-2 cells. We conclude that SREBP is involved not only in the regulation of FPP synthase and cholesterogenesis but also in fatty acid and triglyceride syn- thesis.-Jackson, S. M., J. ETicsson, J. E. Metherall, and P. A. Edwards. Role for sterol regulatory element binding protein in the regulation of farnesyl diphosphate synthase and in the control of cellular levels of cholesterol and triglyceride: evi- dence from sterol regulation-defective cells.]. Lipid Res. 1996. 37: 1712-1721.

Cellular cholesterol homeostasis is a critical compo- nent of normal cell function (1). Extensive studies over the last decade have demonstrated that a number of enzymes in the cholesterol biosynthetic pathway, includ- ing HMGCoA synthase, HMG-CoA reductase, FPP syn- thase, and squalene synthase, are regulated at the level of transcription, in response to changing levels of cellu- lar sterols (1-3). The transcription of these genes, to- gether with the LDL receptor gene, is low under condi- tions where cells accumulate cholesterol and is high in cells that are deprived of sterols (1-3). These changes result in altered rates of both cholesterol synthesis and endocytosis of LDL such that cellular sterol homeostasis is maintained.

Recently, the molecular basis for cholesterol-regu- lated transcription of the LDL receptor and HMG-CoA synthase genes was elucidated in a series of elegant studies. The promoters of these two genes were shown to contain a 10 bp sterol regulatory element-1 (SRE-1) sequence that was required for high levels of transcrip- tion (1). Subsequent studies identified an SRE-1 binding protein (SREBP) that bound to the SRE-1 identified in

Abbreviations: SRE, sterol regulatory element; SREBP, sterol regulatory element-binding protein; SRD, sterol regulationdefective; LPDS, lipoprotein-deficient fetal calf sera; FBS, fetal bovine sera; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; CHO, Chinese hamster ovary; ADDl , adipocyte differentiation and determination-dependent factor-1; IPP, isopentenyl diphosphate; FPP, farnesyl diphosphate; CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; LDL, low density lipoprotein; bp, base pairs.

'To whom correspondence should be addressed. Supplementary key words SREBP SRD-2 SRD-6

1712 Journal of Lipid Research Volume 37,1996

by guest, on June 13, 2018w

ww

.jlr.orgD

ownloaded from

the promoters of the LDL receptor and HMG-CoA synthase genes (4-6). SREBP is synthesized as a 125 kDa precursor that is localized to the endoplasmic reticulum (4). Low intracellular sterol levels result in increased proteolytic cleavage of the precursor form of SREBP and the release of the amino terminal, transcriptionally active 68 kDa mature SREBP fragment (4). The mature form of SREBP enters the nucleus and binds to specific DNA sequences in the promoters of target genes (4). High intracellular sterol levels prevent the proteolytic cleavage of the precursor SREBP and thus prevent the SREBP-dependent increase in transcription of target genes. There are two distinct SREBP genes, SREBP-1 and SREBP-2, that produce, as a result of differential splicing, five proteins (6, 7). The functional importance of these different SREBPs is not known.

The rat homologue of SREBP (ADD-1) was cloned independently based on the ability of the protein to bind in vitro to an E-box motif present in the promoter of the fatty acid synthase gene (8,9). Surprisingly, there is only a 5/ 10 identity between the nucleotide sequence sur- rounding and including the E-box motif of the fatty acid synthase gene and the SRE-1 of the LDL receptor gene. Recent studies demonstrate that the transcription of both the fatty acid synthase and HMG-CoA synthase genes are regulated in parallel in response to either changes in sterol availability to the cells or overexpres- sion of SREBP, consistent with the binding of SREBP to either an SRE-1 or E-box motif (10).

FPP synthase is a soluble enzyme that is localized to peroxisomes (1 1). It catalyzes the formation of FPP from isopentenyl diphosphate and dimethylallyl diphosphate (12). FPP is a precursor of many essential molecules including cholesterol, ubiquinone, dolichol, geranylger- any1 diphosphate, heme a, steroid hormones, and bile acids and is also required for the post translational prenylation of certain proteins (12, 13). Moreover, cat- abolites of FPP have recently been proposed to function as signaling molecules involved in such diverse areas as protein degradation (14-16) and nuclear receptor de- pendent gene regulation (17). In addition, isopentenyl diphosphate and dimethylallyl diphosphate, the two substrates for the reaction catalyzed by FPP synthase, and related prenyl diphosphates (geranyl, farnesyl, or geranylgeranyl diphosphate) are all recognized by yS T-cell receptors and result in T-cell proliferation (18).

We have previously reported that transcription of the FPP synthase gene is regulated by sterols (2, 19-21). At least four SRE-l-like sequences are present in the 319 bp proximal promoter of the FPP synthase gene (2). However, deletion or mutagenesis of these SRE-l-like sequences in a number of FPP synthase promoter-re- porter constructs did not block sterol-regulated expres- sion (2, 19). Subsequent studies demonstrated that the

high level of expression of FPP synthase promoter-re- porter gene constructs that occurs when cells are incu- bated in steroldepleted media requires two transcrip- tion factors, namely NF-Y and SREBP (20, 21). NF-Y binds to an inverted CCAAT box and SREBP binds to a novel DNA sequence termed S a 3 (20,21). SRE-3 has 6/10 identity with the SRE-1 identified in the promoter of the LDL receptor gene and 5/10 identity with the E-box motif of the Si4 gene (21). A chloramphenicol acetyltransferase (CAT) reporter gene, driven by a 61 bp fragment of the FPP synthase promoter that con- tained the inverted CCAAT box and the SRE-3 se- quence, was regulated by exogenously added sterols or by co-transfection of a plasmid encoding mature SREBP- 1 (21). In contrast, induction of the reporter gene that resulted from co-expression of mature SREBP-1 was prevented when the promoter contained a 3 bp muta- tion within SRE-3 (21). We interpreted these results to indicate that SREBP-1 bound to SRE-3 and, in the presence of bound NF-Y, activated transcription of the FPP synthase promoter-reporter gene.

In the current report, we used mutant Chinese ham- ster ovary (CHO) cell lines to more clearly define the SREBP involvement in the transcriptional regulation of the endogenous FPP synthase gene. The mutant sterol regulation-defective (SRD) cell lines were generated by exposure of CHO-7 cells, a subline of CHO-K1 cells, to either gamma-irradiation (22) or methanesulfonic acid ethyl ester (23). The "agenized cells were selected for growth on either lipoproteindeficient medium conuin- ing 25-hydroxycholesterol (SRD-2) (22) or medium con- taining amphotericin B (SRD-6) (23). These selection procedures resulted in the death of parental CHO-7 cells.

The SRD-2 cell line was shown to contain a hybrid gene encoding the transcriptionally active component of SREBP-2 fused to 378 amino acids of an unidentified gene (24). Constitutive expression of the mutant SREBP- 2-hybrid gene in SRD-2 cells circumvents the sterol-regu- lated proteolytic release of the mature 68 kDa SREBP-2. Consequently, the LDL receptor, HMG-CoA synthase, and HMG-CoA reductase genes are highly expressed in a constitutive fashion (22, 25). The mutation in the SRD-6 cell line has not yet been elucidated. However, the low mRNA levels of the LDL receptor, HMG-CoA synthase, and HMG-CoA reductase in SRD-6 cells incu- bated either in the absence or presence of sterols sug- gests that there is little or no mature SREBP (23).

We postulated that if the endogenous FPP synthase gene is regulated by an SREBP/SRE4 interaction, then the levels of the endogenous FPP synthase mRNA would display the same characteristics as the mRNAs for the LDL receptor, HMG-CoA synthase and HMGCoA re- ductase in the SRD cell lines. We now show this to be

Jackson et al. Regulation of FPP synthase by SREBP 1713

by guest, on June 13, 2018w

ww

.jlr.orgD

ownloaded from

the case. We also demonstrate that the induction of an FPP synthase promoter-reporter construct by co-trans- fected SREBP-2 is greater in SRD-6 YCHO-7 YSRD-2 cells, consistent with a lack of functionally mature SREBP in the former cells. In addition, the current studies demonstrate that constitutive expression of SREBP-2 in SRD-2 cells is associated with elevated levels of cholesterol, cholesteryl ester, and triglyceride. Thus, SREBP-2 appears to play an important role in more than one lipid biosynthetic pathway.

MATERIALS AND METHODS

Materials

Cell culture supplies were purchased from Gibco BRL and lipoprotein-deficient serum (LPDS) was obtained from PerImmune (Rockville, MD). The HMG-CoA syn- thase-luciferase promoter-reporter plasmid construct (pSynSRE) containing nucleotides -324 to -225 of the proximal promoter (26), pTKCIII-CAT reporter plas- mid, and the CMV-CS2 plasmid (SREBP-2 expression vector encoding the transcriptionally active mature form, amino acids 1-485) were kindly provided by Dr. Timothy Osborne (Department of Molecular Biology and Biochemistry, University of California, Imine). The pTKCIII-CAT plasmid was used to generate either pTKCIII-0.061 containing 61 bp (-293 to -233) of the FPP synthase proximal promoter containing the inverted CCAAT box and SRE-3 (20, 21) or pTKCIII-O.O6lq, in which the CAC nucleotides within the SRE-3 (-259 to -257) were mutated to ACA (21). The wild-type 61 bp promoter was also ligated into pG.LUC (kindly provided by Dr. Jonathon Tugwood, Zeneca Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, Eng- land) which contains the rabbit P-globin promoter (-109 to +lo) to produce pGL-0.061. [14C]sodium acetate (58 Ci/mol) and [@‘P]dCTP (3000 Ci/mmol) were pur- chased from NEN. [3H]IPP (15 Ci/mmol) was prepared as described previously (27). [“%]chloramphenicol (56 Ci/mol) was obtained from Amersham. Reagents for lipid analysis were supplied by Sigma. 3,4Epoxy-3- methyl-l-butyl diphosphate was a generous gift from Dr. Dale Poulter (Department of Chemistry, University of Utah, Utah).

Cell culture

CHO cells were cultured in Hams F12 supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% Con. CHO-7 cells were grown in DMEM/Hams F12 (1:l) supplemented with 5% LPDS. SRD-2 and SRD-6 cells were cultured in the same media supplemented with

either 25-hydroxycholestero1(0.5 pglml) or cholesterol (5 pg/ml) and mevalonic acid (200 pM), respectively (22, 23).

Cell transfections

CHO-7, SRD-2, and SRD-6 cells were cultured in 60-mm dishes until they were approximately 40% con- fluent. The cells were then transiently transfected with 12.5 pg plasmid DNA (reporter plasmid plus CMV-b galactosidase and carrier plasmid) as described (19) using the Stratagene MBS kit. Where indicated, cells were also co-transfected with the indicated amount of CMV-CS2, a plasmid encoding the mature SREBP-2. After transfection, cells were incubated for approxi- mately 24 h in Hams F12 supplemented with either 10% FBS, 10% LPDS, or 10% LPDS plus sterols (10 pg/ml cholesterol and 1 pg/ml25-hydroxycholestero1) as indi- cated in the legends. Cells were lysed in cell culture lysis buffers appropriate for either luciferase reporter gene constructs or CAT reporter gene constructs, according to the supplier (Promega Corporation). Luciferase, CAT, and P-galactosidase activities were measured in the cell extracts as described (2, 19). The bgalactosidase activity was used to normalize luciferase and CAT activi- ties and thus correct for differences in transfection efficiencies (21).

Lipid synthesis

CHO-7, SRD-2, and SRD-6 cells were cultured in 100-mm culture dishes until they reached 30-50% con- fluency. The cells were then incubated for 24 h in Hams F12 supplemented with 10% FBS. [l*C]sodium acetate ( 1.67 pCi/ml) was added to 5 ml new media and the cells incubated for 2 h. Cells were washed extensively with phosphate-buffered saline and then treated with 1 ml of ethanolic potassium hydroxide solution. The solubilized cells were incubated for 18 h at 60°C in sealed glass vials. Nonsaponifiable lipids were extracted into hexane and the radioactivity in the organic phase was determined by scintillation counting. The aqueous phase was acidi- fied with 5 N HCl, incubated at 37°C for 2 h, and the saponifiable lipids were extracted into hexane.

Lipid mass

Cells were cultured in 100-mm culture dishes until they reached 20-30% confluency. They were washed with phosphate-buffered saline and then incubated for 48 h in Hams F12 supplemented with either 10% FBS or 10% LPDS, washed extensively with phosphate-buff- ered saline, lysed in 0.25 ml of cell reporter lysis reagent (Promega Corporation), and sonicated for 2 x 10 sec.

1714 Journal of Lipid Research Volume 37, 1996

by guest, on June 13, 2018w

ww

.jlr.orgD

ownloaded from

Cholesteryl ester, unesterified cholesterol, and triglyceride mass levels were determined within these cell preparations by enzymatic procedures (28). Assays were performed in 96-well microtiter plates (Costar 3598) using a Biomek 1000 automated laboratory work- station (Beckman). Lipid control samples of known concentrations were analyzed in each plate to assure accuracy. Lipid values were normalized to protein level as determined by the Bradford protein assay (29).

~ ~

CHO-7 - - -

(31) was generated by random priming (Pharmacia kit). Blots were hybridized in Rapid-hyb (Amersham) and subsequently washed as specified by the supplier. Air- dried blots were exposed to X-ray film and the band density was quantified using a Molecular Dynamics den- sitometry system. The filters were subsequently hybrid- ized with a radiolabeled probe to 18s RNA (pN2911, ATCC). The relative amount of 18s RNA per lane was determined by densitometry and used to normalize the values obtained with the FPP synthase probe.

SRD-2 + + + - - - + + +

Northern blot hybridization

CHO-7 + + + - - - - - -

Enzyme assay

SRD-6 + + +

Total RNA was isolated from cells incubated for 24 h in Hams F12 supplemented with 10% LPDS in the absence or presence of sterols (10 pg/ml cholesterol and 1 pg/ml 25-hydroxycholesterol) using Trizol reagent (Gibco BRL). Ten-pg aliquots of total RNA were frac- tionated by electrophoresis on 1% agarose/formalde- hyde gels (30). The RNA was capillary transferred to a nylon membrane and cross-linked with U V light. An [a3*P]dCTP radiolabeled cDNA probe to FPP synthase

Cells were homogenized in order to release FPP syn- thase from peroxisomes. FPP synthase enzyme activity was determined in cell lysates as previously described, but with minor modification (32). Briefly, cell lysates (10 pg) were incubated with [3H]IPP (56 Ci/mol) and GPP (25 pM) in a buffer (10 mM KF, 1 mM DTT, 5 mM MgCIz, 25 mM HEPES, pH 6.8, final volume 50 p1) at 37°C. The reaction was stopped after 15 min and the samples were

Sterols A

FPPS

Relative mRNA concentration

Sterols

FPPS Relative mRNA concentration

4 2 3 4 5 6 7 8 9 1 0 1 1 1 2

i

. -*- Y

m - m 100i5.3 I 32 k 1.2 I 315234.9 I 330~38.1 I

10025.0 I 46.353.8 I 41.727.1 I 51.3k5.5 1

B ~ - ~ u w F d y l l o a 3 c -- 10s - J : ?*V

1 2 3 4 5 6 7 8 9 I O - f l 12 13 14 15 16 17 18 19 20 21 22 23 24

Fig. 1. FPP synthase mRNA levels in parental and mutant cell lines. Cells were incubated for 24 h in Hams F12 supplemented with 10% LPDS in the absence or presence of sterols (10 pg/ml cholesterol and 1 pg/ml 25-hydroxy- cholesterol) as indicated. RNA was isolated from 24 individual dishes and analyzed as described under Materials and Methods. (A) The filter was hybridized with a probe to FPP synthase and exposed to film. The normalized levels of FPP synthase mRNAs f SD are indicated and are given relative to the level obtained in CHO-7 cells incubated in the absence of sterols. (B) The filter was rehybridized with an 18s RNA probe and exposed to film.

Jackson et al. Regulation of FPP synthase by SREBP 1715

by guest, on June 13, 2018w

ww

.jlr.orgD

ownloaded from

hydrolyzed at 37°C for 30 min (32). The radiolabeled product was extracted into hexane and the radioactivity was determined by scintillation counting.

Western blot hybridization

Sonicated cell lysates (25 pg) were subjected to SDS polyacrylamide gel electrophoresis on 10% acrylamide gels under reducing conditions. Proteins were electro- transferred to nitrocellulose membrane (Amersham) and probed with affnity-purified anti-FPP synthase an- tibody (33) using the ECL detection system (Amer- sham). The antibody recognizes both FPP synthase at 39 kDa and a second unidentified protein at 42 kDa.

RESULTS

Expression and regulation of FPP synthase in SRD-2, SRD-6, and parental CHO-7 cells

FPP synthase mRNA levels in parental CHO-7 cells were regulated 2- to 3-fold in response to the sterol content of the incubation media; the levels were high when the cells were incubated for 24 h in media supple- mented with 10% LPDS and low when the cells were incubated in LPDS supplemented with cholesterol and 25-hydroxycholesterol (Fig. lA, lanes 1-3 vs. 4-6). In contrast, FPP synthase mRNA levels in SRD-2 cells were unaffected by the sterol status of the media (Fig. lA, lanes 7-12); the levels were 3- and 10-fold higher than the corresponding levels observed in CHO-7 cells incu- bated under identical conditions (Fig. lA, lanes 7-12 vs. 1-6). FPP synthase mRNA levels in SRD-6 cells were unregulated by sterols (Fig. lA, lanes 19-24) and a p proximated the levels observed in parental cells incu- bated under repressing conditions (Fig. 1 A, lanes 16- 18).

Differential expression of FPP synthase protein and enzyme activity in mutant SRD-2 and SRD-6 cells

FPP synthase enzyme mass and activity were esti- mated in the wild-type, CHO-7, and mutant cell lines after incubation of the cells for 48 h in the presence of normal growth media (Hams F12 supplemented with 10% fetal bovine serum). These growth conditions are known to repress, nearly maximally, the expression of FPP synthase mRNA, as a result of the lipoproteins present in the bovine serum. Figure 2A indicates that the levels of the 39 kDa FPP synthase protein were significantly higher in mutant SRD-2 cells than in wild- type CHO, parental CHO-7, or mutant SRD-6 cells. The identity of a 42 kDa protein that is recognized by the

antibody to FPP synthase (Fig. 2A) has not been estab- lished.

The FPP synthase enzyme activity was 6- and 9-fold higher in the SRD-2 cell cytosolic fraction than in the corresponding fraction obtained from CHO-7 cells or SRD-6 cells, respectively (Fig. 2B). These differences were unaffected by the presence of 200 pM 3,4-epoxy-3- methyl-l-butyl diphosphate, an inhibitor of isopentenyl diphosphate isomerase (data not shown).

Taken together, these results indicate that FPP syn- thase mRNA, protein levels, and enzyme activity levels are enhanced in SRD-2 cells, as compared to the parental CHO-7 or mutant SRD-6 cells. As expected, these same parameters are not significantly different between CHO-7 and CHO cells (Fig. 2A, data not shown).

A I CHO I CHO-7 1 S R D - E I SRD-6 I - - 46kDt1--~- .I

30 kDo - FPPS -C

Y Q) 8o01 - m c n

c -- a 600- O E n.2

> .-

CHO-7 SRD-2 SRD-6

Fig. 2. FPP synthase enzyme mass and activity in different cell lines. Cells in 100-mm culture dishes were preincubated for 48 h in Hams F12 supplemented with 10% FBS and then washed extensively with phosphate-buffered saline. (A) Cell lysates were prepared, sonicated, and aliquots were subjected to Western blot analysis with affnity-pu- rified anti-FPP synthase antibody as described in Materials and Meth- ods. The migration of the 39 kDa FPP synthase (FPPS) protein is indicated. A second, unidentified, protein of approximately 42 kDa is also recognized by the antibody. The positions of the 30 and 46 kDa molecular weight standards are shown. (B) The FPP synthase activities in the lysates obtained from the indicated cell types are shown. Data are presented as mean f SD (n = 4 individual plates) and expressed as a percentage of the value obtained for the CHO-7 cells. The 100% value was equivalent to 2.2 nmol IPP incorporated/mg protein/h.

1716 Journal of Lipid Research Volume 37,1996

by guest, on June 13, 2018w

ww

.jlr.orgD

ownloaded from

400 1 ul

P -0 2 300

CHO-7

T

SRD-2 SRDS Fig. 3. Incorporation of radiolabeled acetate into lipids in different cell lines. Cells were incubated for 24 h in Hams F12 supplemented with 10% FBS and then pulsed for 2 h with [ ''C]acetate (1.67 KCi/ml). Cells were lysed and the radioactivity present in the nonsaponifiable (open bars) or saponifiable (solid bars) lipid fractions was determined as described in Materials and Methods. Data are presented as the mean f range (n = 2) and expressed as a percentage of the values obtained for the CHO-7 cells. Absolute amounts of radioactivity recovered in the nonsaponifiable and saponifiable lipid fractions of the CHO-7 cells (100%) were 16,290 and 33,800 dpm/pg cell pro- tein/h, respectively. The results are representative of two different experiments.

Lipid synthesis and lipid mass in SRD-2, SkD-6, and parental cells

The differences in gene expression in'the mutant and CHO-7 cells might be expected to affect both the relative rates of sterol synthesis and the endogenous cholesterol levels within the cell. Figure 3 (open bars) shows that the incorporation of [ 14C]acetate into nonsaponifiable lipids was 2.8- to 4.6-fold greater in SRD-2 cells than in CHO-7 or SRD-6 cells. Analysis of the lipid by thin-layer chromatography indicated that most of the radiolabel co-migrated with squalene or cholesterol standards (data not shown).

The incorporation of [ 14C]acetate into saponifiable lipids was 3.3- to 7-fold greater in SRD-2 cells than in CHO-7 or SRD-6 cells (Fig. 3, solid bars). Analysis of the saponifiable lipid fractions on thin-layer chromatogra- phy indicated that essentially all of the radiolabel co-mi- grated with a free fatty acid standard (data not shown). These changes in the relative rates of incorporation of radiolabeled acetate into cholesterol and fatty acids could result from increased absolute rates of synthesis of these lipids in SRD-2 cells or to differences in the acetate pool size in the different cell lines. Consequently, we determined the mass of specific lipids in the different cell lines in order to determine whether there were physiological consequences to the altered gene expres- sion and apparent increase in lipid synthesis.

Analyses of the cells indicated that when they were cultured in media supplemented with either 10% FBS (Table 1) or 10% LPDS (Table 2), SRD-2 cells had increased steady-state concentrations of cholesteryl es- ter, unesterified cholesterol, and triglyceride as com- pared to the other cell types. In contrast, the levels of these same lipids were not significantly different be- tween SRD-6 and the parental CHO-7 cells cultured in the presence of FBS (Table 1). SRD-6 cells do not survive when cultured in the presence of LPDS (and in the absence of added sterols and mevalonic acid) and thus preclude analysis under these latter conditions.

Expression of FPP synthase promoter-reporter gene constructs in mutant and parental cells

All of the cis-elements required for sterol-regulated transcription of the FPP synthase promoter-reporter gene are contained within a 61 bp sequence of the proximal promoter (21). This 61 bp fragment was placed upstream of a minimal thymidine kinase promoter-CAT gene to generate a promoter-reporter plasmid (pTKCIII-0.061; 61wt) (21). This plasmid, together with a plasmid encoding &galactosidase under the control of a CMV promoter, was transiently transfected into the different cell lines. The cells were then incubated for 20

TABLE 1. Lipid mass levels in SRD, CHO-7, and wild-type cells incubated under normal conditions

Lipid Lipid Mass

CHO CHO-7 SRDZ SRDG pg/mg cell protein

Free cholesterol 16.4 f 0.6 17.2 f 4.2 39.1 f 2.Ba 14.0 f 1.9

Cholesteryl ester 3.3 f 0.3 5.3 f 1.8 20.9 f 1.3= 3.2 k 1.0

Triglyceride 3.6 f 4.2 5.5 f 3.1 37.4 f 3.3a 5.6 f 1.8 ~

Cells were incubated for 48 h in Hams F12 supplemented with 10% FBS, washed extensively with phosphate-buffered saline, lysed, and sonicated as described in Materials and Methods. Lipid levels were determined by enzymatic procedures and the data, normalized to protein, are presented as mean f SD (n = 4).

"P < 0.001 versus parental CHO-7.

Jackson et al. Regulation of FPP synthase by SREBP 1717

by guest, on June 13, 2018w

ww

.jlr.orgD

ownloaded from

TABLE 2. Lipid mass levels of SRD-2, CHO-7, and wild-type cells incubated in 10% LPDS

Lipid Mass Lipid CHO CH07 SRD-2

pg/mg cell protein

Free cholesterol 11.1 f 0.5 10.3 f 0.7 16.5 f 1.5a

Cholesteryl ester 0.3 f 0.3 0.4 f 0.3 4.5 f O.ga

Triglyceride 5.7 f 1.1 6.2 f 0.8 9.3 f 2.1b

The cells were incubated for 48 h in the presence of Hams F12 supplemented with 10% LPDS. Lipid analyses were as described in the legend to Table 1.

'P < 0.001 versus parental CHO-7. bP < 0.05 versus parental CHO-7.

h in media supplemented with 10% fetal bovine serum. Figure 4 (open bars) demonstrates that CAT activity was 7.7-fold higher in SRD-2 than in CHO-7 cells. In con- trast, CAT activity in SRD-6 cells was 84% of that ex- pressed in CHO-7 cells (Fig. 4, open bars).

In agreement with earlier studies (22), transient trans- fection of these cells with an HMGCoA synthase-pro- moter-luciferase reporter construct resulted in a signifi- cantly greater luciferase expression in SRD-2 cells than in CHO-7 cells (Fig. 4, solid bars). The luciferase activity in SRD-6 cells was 65% of that expressed in the parental CHO-7 cells (Fig. 4, solid bars). Thus, the relative changes in activity of the FPP synthase and HMGCoA synthase promoter-reporter constructs, after transfec- tion into the different cell types, were similar (Fig. 4).

We have previously reported that a 3 bp mutation in SRE-3 (mutation q in reference 21) prevents sterolde- pendent regulation of an FPP synthase promoter-re- porter construct (21). This mutation was shown to pre- vent the binding of purified recombinant SREBP-1 to a DNA fragment containing the SRE-3 sequence (21). Figure 5 shows the results obtained when cells were transiently transfected with a plasmid containing the CAT reporter gene under the control of 61 bp of either wild-type FPP synthase promoter (pTKCIII-0.061; 61wt) or the same 61 bp containing the q mutation (pTKCIII- 0.061q; 61q). The cells were then incubated in the presence of sterols in order to suppress the release of the endogenous, transcriptionally active SREBP (4). The CAT activity in SRD-2 cells was 7-fold higher than that in CHO-7 cells after transfection with the wild-type promoter construct (Fig. 5). This high level of CAT activity was not observed when the FPP synthase pro- moter contained the q mutation (Fig. 5). Thus, the high level of CAT expression in SRD-2 cells requires that the transcriptionally active SREBP-2, produced constitu- tively in SRD-2 cells (24), binds to SRE-3. As expected, the CAT activities obtained after transient transfection of CHO-7 cells with either the wild-type or mutant reporter genes were similar (Fig. 5) , consistent with the

presence of little or no transcriptionally active SREBP in the nuclei of CHO-7 cells incubated in the presence of excess sterols.

Figure 6 shows the results of experiments in which CHO-7, SRD-2, or SRD-6 cells were transfected with a luciferase reporter gene under the control of 61 bp of the FPP synthase promoter (pCLo.061) and increasing amounts of the plasmid encoding the 68 kDa mature SREBP-2. The cells were then incubated for 24 h under inducing conditions (10% LPDS). These latter incuba- tion conditions promoted the near maximal release of the mature endogenous SREBP from the endoplasmic reticulum of the CHO-7 cells. The results indicate that the luciferase activity was induced in all cell types in an SREBP-2 dosedependent manner (Fig. 6). The maximal luciferase activities (relative light units/fbgalactosidase activity/min), obtained in the presence of 100 ng of the SREBP-2 plasmid, were similar for all three cell types; the values were 1.54 x lo6, 1.41 x lo6, and 1.25 x lo6 for CHO-7, SRD-6. and SRD-2 cells, respectively (Fig. 6). However, the maximum fold increase in luciferase activ- ity, as a result of SREBP-2 co-expression, was greater in SRD-6 cells (9-fold) than in CHO-7 cells (&fold) or SRD-2

1000

.- 0

2 t

> .- - 750

E 500 a U Q) >

a [r

.- 3 250 -

0 CHO-7 SRD-2 SRD-6

Fig. 4. Transient transfection of FPP synthase- and HMG-CoA s p thase-promoter-reporter constructs into mutant or parental CHO-7 cells. The indicated cell types were transiently transfected with CMV- Bgaactosidase and either pTKCIII-O.061 (FPP synthase) (open bars) or pSynSRE (HMG-CoA synthase) (solid bars) promoter-reporter gene constructs, and the cells were incubated for 20 h in Hams F12 supple mented with 10% FBS. Enzyme activities were determined in cell lysates isolated from four separate plates for each cell type and each reporter plasmid, as described in Materials and Methods. In order to normalize for differences in transfection efficiencies, aliquots of the cell lysates, which contained equal amounts of Bgalactosidase activity, were used to assay for the activity of CAT or luciferase as described in Materials and Methods. The data are presented as mean f SD (n = 4) and expressed as a percentage of the value obtained for the C H 0 7 cells. The 100% value (CH07 cells) for the CAT assay was equivalent to conversion of 9.5% of the radiolabeled chloramphenicol to the acetylated product. The 100% value for the luciferase activity corre- sponded to 202,500 relative light units.

1718 Journal of Lipid Research Volume 37,1996

by guest, on June 13, 2018w

ww

.jlr.orgD

ownloaded from

!Cell type1 CHO-7 SRD-2 I 1 Fig. 5. Enhanced activity of FPP synthase promoter-reporter con- structs in SRD-2 cells requires a functional SREBP binding site. Wild- type (pTKCIII-0.061; 61wt) and mutant (pTKCII1-O.06lq; 61q) con- structs were transiently transfected together with the CMV-bgalactosidase construct into CHO-7 and SRD-2 cells. After transfection, cells were incubated for 20 h in Hams F12 supplemented with 10% LPDS and sterols (10 pg/ml cholesterol and 1 pg/ml 25-hydroxycholesterol). The CAT activities, corrected for minor vari- ations in transfection efficiency, were determined as described in Materials and Methods. Data are presented as the mean values ob- tained from duplicate dishes and expressed as a percentage of the value obtained for CHO-7 cells transfected with the wild-type pro- moter-reporter construct. Duplicate values varied by less than 10%. The 100% value (61wt in CHO-7 cells) was equivalent to conversion of 1% of the radiolabeled substrate to product. The data are repre- sentative of two experiments.

cells (1.6-fold). Thus, SRD-2 cells that constitutively ex- press a transcriptionally active form of SREBP-2 are the least responsive to the SREBP-2 derived from the co-ex- pressed plasmid (Fig. 6). The latter results suggest that SRD-2 cells normally contain levels of transcriptionally active SREBP that are nearly sufficient to maximally stimulate the transfected promoter-reporter construct. The greater responsiveness of SRDB cells to co-ex- pressed SREBP-2 would be consistent with the proposal that these cells produce very little or no endogenous mature SREBP.

DISCUSSION

We recently reported that the promoter of the rat FPP synthase gene contains a novel sequence, termed SRE-3, to which recombinant SREBP-1 can bind in vitro (21). Co-transfection of cells with FPP synthase promoter-re- porter constructs, together with a plasmid that constitu- tively expressed the 68 kDa mature SREBP-1, resulted in enhanced activity of the reporter gene (21). This SREBP-stimulated activity of the reporter gene was abol-

ished by mutation of a core trinucleotide sequence within the FPP synthase promoter SRE-3 (21). In the current studies we have used sterol regulation-defective (SRD) cells (22-25) to determine whether the transcrip tion of the endogenous FPP synthase gene is regulated by SREBP in a manner consistent with the interaction of SREBP-2 with the SRE-3 in the FPP synthase pro- moter.

The results in the present report indicate that the endogenous FPP synthase mRNA levels are regulated normally in parental CHO-7 cells in response to the addition or removal of sterols from the media (Fig. 1). In contrast, FPP synthase mRNA levels are expressed at high, unregulated levels in SRD-2 cells and at low, un- regulated levels in SRD-6 cells (Fig. 1). The relative changes in FPP synthase mRNA levels in the different cell types under these different incubation conditions are similar to those reported for the mRNAs for HMG- CoA synthase and the LDL receptor (22,23). The sterol- regulated transcription of these latter two genes is known to be dependent, in part, on the binding of SREBP to the 10 bp SRE-1 (4-6). Based on these results, we conclude that SREBP has a physiological role in the transcriptional regulation of the endogenous FPP syn- thase gene.

The apparent increase in the rate of synthesis of nonsaponifiable lipids in SRD-2 cells, as compared to SRD-6 or parental CHO-7 cells (Fig. 3), is consistent with the increased endogenous protein and/or mRNA levels of FPP synthase (Figs. 1 and 2), HMG-CoA synthase and HMG-CoA reductase (22, 25). However, the enhanced sterol mass in the SRD-2 cells cultured in the presence of 10% FBS (Table 1) could result from both an increase in sterol synthesis and an increase in endocytosis of LDL that is likely to occur as the result of the enhanced expression of the LDL receptor mRNA in SRD-2 cells (22, 25). However, when cells were cultured in 10% LPDS, in the absence of LDL, SRD-2 cells still exhibited increased levels of cholesterol, cholesteryl ester, and triglyceride when compared to the other cell types (Ta- ble 2). The increased mass of these lipids in SRD-2 cells is likely to result, in part, from increased lipid (choles- terol, fatty acid and triglyceride) synthesis.

The recent observation that co-expression of SREBP with a fatty acid synthase promoter-reporter gene re- sulted in stimulation of this reporter gene is consistent with SREBP having a role both in the transcriptional regulation of fatty acid synthase (10) and in the in- creased biosynthesis of fatty acids noted in the current studies. Further experiments will be required to deter- mine whether SREBP also regulates other genes in- volved in the synthesis of triglycerides.

The current study provides strong evidence that the sterol-regulated transcription of the rat FPP synthase

Jackson et al. Regulation of FPP synthase by SREBP 1719

by guest, on June 13, 2018w

ww

.jlr.orgD

ownloaded from

gene in vivo is dependent, in part, on the binding of SREBP to SRE-3 in the FPP synthase promoter. The finding that SREBP functionally binds to at least three distinct sequences, SRE-1, SRE-3, and the E-box (4, 8, 21), may allow the identification of new target genes that are regulated by this unique transcription factor. In this context, a recent study demonstrated that ADD- l/SREBP bound to two sequences in the proximal promoter of the rat squalene synthase gene (3). One binding site corresponded to an SRE-1 sequence while the other had 11/12 identity with the nucleotides sur- rounding the SRE-3 sequence identified in the FPP synthase promoter. It will be of interest to determine whether both cis elements are important for the sterol- dependent regulation of squalene synthase.

Based on the observation that the mRNA levels for HMG-CoA reductase, HMGCoA synthase, and LDL receptor genes were low in SRD-6 cells, Evans and Metherall (23) proposed that these cells may lack func- tional SREBP. The current finding that the fold induc-

7.5-

5.0-

2.5-

1- 0 25 50 75 100

SREBP (ng)

Fig. 6. SREBP increases the activity of an FPP synthase promoter- reporter construct to different degrees in CHO-7, SRD-6, and SRD-2 cell lines. Cells were transiently transfected with the FPP synthase promoter-luciferase reporter construct (pGLO.061) together with the CMV-bgalactosidase construct in the absence or presence of the indicated amount of the plasmid encoding the mature form of SREBP-2 (CMV-CS2). After transfection, the CHO-7 (A), SRD-6 (O), and SRDP (13) cells were incubated in Hams F12 supplemented with 10% LPDS for 20 h. Each value is the average from two individual plates of cells and the variation was less than 10%. The fold induction was determined by comparison of the luciferase activities, normalized for transfection efficiencies, obtained from cells incubated in the presence versus absence of CMV-CS2 (SREBP). The luciferase activi- ties, obtained from cells that were not transfected with CMV-CS2, were 257,608, 154,953, and 777,402 relative light units for CHO-7, SRD-6, and SRD-2 cells, respectively.

tion of the FPP synthase promoter-reporter construct in response to co-transfected mature SREBP-2 was greater in SRD-6 cells, as compared to either SRD-2 or CHO-7 cells, provides strong indirect evidence that SRD-6 cells lack transcriptionally functional SREBP. However, there are two distinct genes, one that encodes SREBP-1 and another that encodes SREBP-2 (6, 7). As in vitro studies demonstrate that SREBP-1 and SREBP-2 are inter- changeable with respect to target gene induction (6), one protein would be expected to rescue the deficit of the other. Thus, it seems likely that the mutation in SRD-6 cells results in a defect that affects the expression and/or processing of both SREBP-1 and SREBP-2. Fur- ther studies will be required to determine whether SRD-6 cells are defective in the activity of the sterol-de- pendent protease that releases mature SREBP from the endoplasmic reticulum (4).1

We thank Bernard Lee, Aimie Goto, and Sharda Charugundla for excellent technical assistance, and Dr. Timothy Osborne for kindly providing the pTKCIII-CAT and CMV-CS2 plas- mids. This work was supported by Grant HL 30568 (to P.A.E.) from the National Institutes of Health and a grant from the Laubisch Fund (to P.A.E.). J.E. is the recipient of an American Heart Association, Greater Los Angeles Affiliate, Postdoc- toral Fellowship. J.E.M. is an Established Investigator of the American Heart Association. Manuscript received 12 February 1996 and in reuisedfonn 10 May 1996.

REFERENCES

1. Goldstein, J. L., and M. S. Brown. 1990. Regulation of the mevalonate pathway. Nature. 343: 425-430.

2. Spear, D. H., S. Y. Kutsunai, C. C. Correll, and P. A. Edwards. 1992. Molecular cloning and promoter analysis of the rat liver farnesyl diphosphate synthase gene.]. Biol. Chem. 267: 14462-14469.

3. Guan, G., G. Jiang, R. L. Koch, and I. Shechter. 1995. Molecular cloning and functional analysis of the pro- moter of the human squalene synthase gene. J. Biol. Chem.

.4. Wang, X., R. Sato, M. S. Brown, X. Hua, and J. L. Gold- stein. 1994. SREBP-1, a membrane bound transcription factor released by sterol-regulated proteolysis. Cell. 77:

5. Wang, X., M. R. Briggs, X. Hua, C. Yokoyama, J. L. Goldstein, and M. S. Brown. 1993. Nuclear protein that binds to sterol regulatory element of low density lipopro- tein receptor promoter. 11. Purification and charac- terizati0n.J. Biol. Chem. 268 14497-14504.

6. Hua, X., C. Yokoyama, J. Wu, M. R. Briggs, M. S. Brown, and J. L. Goldstein. 1993. SREBP-2, a second basic-helix- loop-helix-leucine zipper protein that stimulates tran- scription by binding to a sterol regulatory element. PTOC. Natl. Acad. Sci. USA. 90: 11603-1 1607.

7. Yokoyama, C., X. Wang, M. R. Briggs, A. Adman, J. Wu, X. Hua, J. L. Goldstein, and M. S. Brown. 1993. SREBP-1, a basic-helix-loopheli-leucine zipper protein that con-

270: 21958-21965.

53-62.

1720 Journal of Lipid Research Volume 37,1996

by guest, on June 13, 2018w

ww

.jlr.orgD

ownloaded from

trols transcription of the low density lipoprotein receptor gene. Cell. 75: 187-197.

8. Tontonoz, P., J. B. Kim, R. A. Graves, and B. M. Spiegel- man. 1993. ADDl: a novel helix-loophelix transcription factor associated with adipocyte determination and differ- entiation. Mol. Cell. Biol. 13: 4753-4759.

9. Kim, J. B., G. D. Spotts, Y. Halvorsen, H. Shih, T. Ellen- berger, H. C. Towle, and B. M. Spiegelman. 1995. Dual DNA binding specificity of ADDl/SREBP controlled by a single amino acid in the basic heli-loophelix domain. Mol. Cell. Biol. 15: 2582-2588.

10. Bennett, M. K., J. M. Lopez, H. B. Sanchez, and T. F. Osborne. 1995. Sterol regulation of fatty acid synthase prom0ter.J. Biol. Chem. 270 25578-25583.

11. Krisans, S. K., J. Ericsson, P. A. Edwards, and G-A. Keller. 1994. Farnesyl diphosphate synthase is localized in perox- isomes. J. Biol. Chem. 2 6 9 14165-14169.

12. Poulter, C. D., andH. C. Rilling. 1991. Prenyl transferases and isomerases. In Biosynthesis of Isoprenoid Com- pounds. J. W. Porter and S. L. Spurgeon, editors. J. Wiley and Sons, New York. Vol. 1. 162-224.

13. Maltese, W. A. 1990. Posttranslational modification of proteins by isoprenoids in mammalian cells. FASEB J. 4:

14. Correll, C. C., L. Ng, and P. A. Edwards. 1994. Identifica- tion of farnesol as the non-sterol derivative of mevalonic acid required for the accelerated degradation of 3-hy- droxy-3-methylglutaryl coenzyme A reductase. J. Biol.

15. Bradfute, D. L., and R. D. Simoni. 1994. Non-sterol com- pounds that regulate cho1esterogenesis.J. Biol. Chem. 269:

16. Giron, M. D., C. M. Havel, and J. A. Watson. 1994. Mevalonate-mediated suppression of 3-hydroxy-3-methyl- glutaryl coenzyme A reductase function in a-toxin-perfo- rated cells. h o c . Natl. Acad. Sn'. USA. 91: 6398-6402.

17. Forman, B. M., E. Goode, J. Chen, A. E. Oro, D. J. Bradley, T. Perlmann, D. J. Noonan, L. T. Burka, T. McMorris, W. W. Lamph, R. M. Evans, and C. Weinberger. 1995. Iden- tification of a nuclear receptor that is activated by farnesol metabolites. Cell. 81: 687-693.

18. Tanaka, Y., C. T. Morita, Y. Tanaka, E. Nieves, M. B. Brenner, and B. R. Bloom. 1995. Natural and synthetic non-peptide antigens recognized by human y,S T cells. Nature. 375: 155-158.

19. Spear, D. H., J. Ericsson, S. M. Jackson, and P. A. Edwards. 1994. Identification of a 6-base pair element involved in the sterol-mediated transcriptional regulation of farnesyl diphosphate synthase. J. Biol. Chem. 269: 25212-25218.

20. Jackson, S. M., J. Ericsson, T. F. Osborne, and P. A. Edwards. 1995. NF-Y has a novel role in sterol-dependent transcription of two cholesterogenic genes. J. Biol. Chem.

3319-3328.

C h m . 269: 17390-17393.

6645-6650.

270 21445-21448.

21. Ericsson, J., S. M. Jackson, B. Lee, and P. A. Edwards. 1996. Sterol regulatory element binding protein binds to a cis element in the promoter of the farnesyl diphosphate synthase gene. Proc. Natl. Acad. Sci. USA. 93: 945-950.

22. Metherall, J. E., J. L. Goldstein, K. L. Juskey, and M. S. Brown. 1989. Loss of transcriptional repression of three sterol-regulated genes in mutant hamster cells. J. Biol.

23. Evans, M. J., and J. E. Metherall. 1993. Loss of transcrip tional activation of three sterol-regulated genes in mutant hamster cells. Mol. Cell. Biol. 13: 5175-5185.

24. Yang, J., M. S. Brown, Y. K. Ho, and J. L. Goldstein. 1995. Three different rearrangements in a single intron trun- cate sterol regulatory element binding protein-:! and pro- duce sterol-resistant phenotype in three cell lines: role of introns in protein evolution. J. Biol. Chem. 270

25. Dawson P. A., J. E. Metherall, N. D. Ridgeway, M. S. Brown, and J. L. Goldstein. 1991. Genetic distinction between sterol-mediated transcriptional and posttran- scriptional control of 3-hydroxy-3-methylglutaryl-coen- zyme A reductase.J. Bwl. Chem. 266 9128-9134.

26. Vallett, S. M., and T. F. Osborne. 1994. Two separate sites contribute to Ap-1 activation of the promoter for 3-hy- droxy-3-methylglutaryl coenzyme A synthase. Nwkic. Ac-

27. Ericsson, J., A. Thelin, T. Chojnacki, and G. Dallner. 1991. Characterization and distribution of cisprenyl transferase participating in liver microsomal polyisoprenoid biosyn- thesis. Eur. J. Biochem. 202: 789-796.

28. Warnick, G. R. 1986. Enzymatic methods for quantifica- tion of lipoprotein lipids. Methods Enzymol. 129: 101-123.

29. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein util- izing the principle of proteindye binding. Anal. Bwchem.

30. Maniatis, T., E. F. Fritsch, andJ. Sambrook. 1989. Molecu- lar Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press, Plainview, NY. 7.37-7.52.

31. Clarke, C. F., R. Tanaka, K. Svenson, M. Walmsley, A. M. Fogelman, and P. A. Edwards. 1987. Molecular cloning and sequence of a cholesterol-repressible enzyme related to prenyltransferase in the isoprene biosynthetic pathway. Mol. Cell. Biol. 7: 3138-3146.

32. Runquist, M., J. Ericsson, A. Thelin, T. Chojnacki, and G. Dallner. 1992. Biosynthesis of trans,trans,trans~eranylger- any1 diphosphate by the cytosolic fraction from rat tissues. Biochem. Biophys. Res. Commun. 186 157-165.

33. Joly, A., and P. A. Edwards. 1993. Effect of sitedirected mutagenesis of conserved aspartate and arginine residues upon farnesyl diphosphate synthae activity.J. Biol. Chem.

C h m . 264 15634-15641.

12152-12161.

idF Res. 22: 5184-5189.

72: 248-254.

268 26983-26989.

Jackson et al. Regulation of FPP synthase by SREBP 1721

by guest, on June 13, 2018w

ww

.jlr.orgD

ownloaded from