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(Circulation Research. 2001;88:268.)
© 2001 American Heart Association, Inc.

Molecular Medicine

LDL Downregulates CYP51 in Porcine Vascular Endothelial Cells and in the Arterial Wall Through a Sterol Regulatory Element Binding Protein-2–Dependent Mechanism

Cristina Rodríguez, José Martínez-González, Sonia Sánchez-Gómez, Lina Badimon

From the Cardiovascular Research Center, Instituto de Investigaciones Biomédicas de Barcelona/Consejo Superior de Investigaciones Cientificas–Institut de Recerca Hospital de la Santa Creu i Sant Pau–UAB, Barcelona, Spain.

Correspondence to Dr Lina Badimon, IIBB-CSIC, C/Jordi Girona, 18-26, 08034 Barcelona, Spain. E-mail lbmucv{at}cid.csic.es

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—Hypercholesterolemia is associated with endothelial dysfunction and atherosclerotic lesion formation. By mRNA–differential display analysis, we have identified lanosterol 14-demethylase (CYP51) as a gene highly regulated by native LDLs (nLDLs) in endothelial cells. CYP51 is a cytochrome P-450 enzyme involved in the postsqualene phases of cholesterol biosynthesis. CYP51 mRNA levels decrease in nLDL-treated cells in a dose- and time-dependent manner (9-fold after 24 hours with 180 mg of LDL cholesterol per deciliter), an effect that is blocked by cycloheximide. In parallel, sterol regulatory element (SRE) binding protein-2 (SREBP-2) expression falls (10-fold), without alteration in SREBP-1 level. N-Acetyl-leucyl-leucyl-norleucinal, which inhibits catabolism of the active form of SREBPs, abolished the effect of high concentrations of nLDL on CYP51 expression. Gel-shift assays performed with the SRE of the cyp51 gene (cyp51-SRE) revealed a diminished SREBP-SRE interaction in LDL-treated cells. Moreover, nLDLs downregulate CYP51 promoter activity in transfection assays. Thus, atherogenic levels of nLDL downregulate endothelial CYP51 mRNA levels through a reduction in SRE–SREBP-2 interaction. Additionally, SREBP-2 and CYP51 mRNA levels are decreased in the arterial wall of hypercholesterolemic pigs. In summary, we have described for the first time, both in in vivo and in vitro systems, that CYP51 is expressed in the vascular wall and that it is downregulated together with SREBP-2 by high levels of nLDL. Because this transcription factor controls multiple cell lipid metabolism pathways, its regulation by nLDL could play a key role in lipid-mediated endothelial dysfunction.

Key Words: SREBP-2 • endothelium • CYP51 • LDL • hypercholesterolemia

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
High LDL levels are a well-known risk factor for atherosclerosis. Hypercholesterolemia produces endothelial dysfunction,^{1 2} an increase of leukocyte recruitment by an increased expression of adhesion molecules,³ a decrease in endothelium-dependent vasodilation,⁴ and alterations in the thrombosis/fibrinolysis balance.⁵

Recently, it has been reported that sterol regulation of a great number of genes involved in lipid metabolism is performed through sterol regulatory element (SRE) binding proteins (SREBPs).⁶ SREBPs are transcription factors localized in the endoplasmic reticulum that are synthesized as precursors. When cellular sterol levels fall, a proteolytic cascade cleaves the precursor, releasing the mature form into the cytosol.^{7 8} Mature SREBPs translocate to the nucleus and modulate transcription of target genes by binding to the SRE, a nonpalindromic 10-bp motif. In sterol-loaded cells, the proteolytic activity is inhibited, nuclear levels of SREBPs fall quickly, and in consequence the transcription rate of genes regulated by this pathway decreases.^{9 10} Three isoforms of SREBPs (1a, 1c, and 2) have been described that seem to be independently regulated.^{9 11 12 13} SREBP-2 is involved preferentially in cholesterol homeostasis, whereas SREBP-1 controls fatty acid metabolism.¹⁰

The aim of this study was to analyze the effect of high levels of native LDL (nLDL) on endothelial gene expression. For this purpose, we used a highly sensitive mRNA–differential display (DD) technique that led us to identify the endothelial lanosterol 14-demethylase (CYP51) as an enzyme highly sensitive to downregulation by nLDL. CYP51 is a cytochrome P450 involved in postsqualene phases of cholesterol biosynthesis.¹⁴ We have observed that CYP51 and SREBP-2 mRNA levels decreased in parallel by nLDL treatment, with a low SREBP–cyp51-SRE binding. These data suggest that the SREBP pathway may be involved in nLDL regulation of CYP51 expression in endothelial cells. Interestingly, we have detected a decrease in CYP51 and SREBP-2 mRNA levels in the arterial wall of hypercholesterolemic pigs. Thus, SREBP-2 modulation by nLDL could play a crucial role in the regulation of genes involved in lipid-mediated endothelial dysfunction and atherosclerotic lesion formation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Endothelial Cell Culture
Porcine aortic endothelial cells (PAECs) were obtained from adult normolipemic animals as described.¹⁵ Cells were grown in M199 (Gibco) supplemented with 10% FCS and antibiotics (0.1 mg/mL streptomycin, 100 U/mL penicillin). Forty-eight hours after plating, cells were placed in 1% porcine lipoprotein–deficient serum for 24 hours. Then, serum-depleted medium and nLDL (140 and 180 mg cholesterol per deciliter) were added for another 24 hours. In some experiments, cells were maintained in 10% FCS, and 48 hours after seeding, fresh medium (10% FCS) and LDL were added for another 24 hours. Levels of thiobarbituric acid reactive substances (TBARS) in supernatants showed no differences between control and LDL-treated cells. Cell viability was determined by trypan blue exclusion. Porcine lipoprotein–deficient serum was obtained by density gradient ultracentrifugation.

Animals
Female pigs (body weight at initiation, 32±4 kg) were divided into 2 groups, as follows: normolipemic animals (n=8), which were fed with normal chow, and hyperlipemic animals (n=5), which were fed with a cholesterol-rich diet (2% cholesterol, 1% cholic acid, and 20% beef tallow) for 100 days.^{16 17} Plasma cholesterol levels and hematologic parameters were measured at baseline and at euthanization. At the end of the 100 days, the animals were euthanized with a thiopental overdose. Because the porcine model of atherosclerosis develops lesions initially in the abdominal aorta, rings of abdominal aorta were collected and frozen in liquid N₂ to measure gene expression. All procedures were in accordance with institutional guidelines and followed the American Physiological Society guidelines for animal research.

Plasma Biochemistry
Plasma total cholesterol was determined with an automatic analyzer (Kodak Ektachem DT System). Plasma lipoproteins (HDL, LDL, and VLDL cholesterol) were fractionated using the validated methods of the Lipid Research Clinic Program¹⁸ and quantified spectrophotometrically (Kontron Instruments).

LDL Isolation
Porcine LDL were obtained from fresh nonfrozen plasma by sequential ultracentrifugation (density=1.019 to 1.063 g/mL). LDLs used in the experiments were <72 hours old. The purity of LDL was assessed by agarose gel electrophoresis (Paragon System, Beckman). LDL samples had no detectable levels of endotoxin (Limulus Amebocyte Lysate test, BioWhittaker), and thiobarbituric acid reactive substances values were <1.5 nmol malonaldehyde per milligram protein.

mRNA-DD Analysis
Total RNA was isolated using Quick-Prep total RNA kit (Pharmacia) or Ultraspec (Biotecx) according to the manufacturer. mRNA-DD analysis was performed with the Delta RNA fingerprinting kit (Clontech) according to the manufacturer. Bands upregulated or downregulated by LDL treatment were cut out, and DNAs were eluted and reamplified with the same primers used in reverse transcriptase–polymerase chain reaction (RT-PCR)–DD. Reamplified products were cloned into the pGEM-T easy vector (Promega) and sequenced with the ABI Prism dRhodamine Terminator cycle sequencing kit (Perkin Elmer) and the T7 promoter sequencing primer (Promega). Comparison of DNA homology with databases (GenBank) was performed using BLAST program (available at http://www.ncbi.nlm.nih.gov).

Semiquantitative RT-PCR
Total RNA was obtained as described above, and CYP51 and SREBP-2 mRNA levels were analyzed by semiquantitative RT-PCR with specific oligonucleotides and the PCR digoxigenin (DIG) labeling mix (Roche Molecular Biochemicals). The specific oligonucleotides selected were as follows: CYP51 upper primer, 5'-TTC AGA CGC AGG GAC AGA GC-3', and CYP51 lower primer, 5'-CAA CGA TGA CGC CCA GCT CC-3'. SREBP-2 mRNA levels were analyzed with the SREBP-2 upper primer, 5'-TGG GAC CAT TCT GAC CAC AA-3', and the SREBP-2 lower primer, 5'-GCC ACA GGA GGA GAG TCT GG-3'. Levels of GAPDH were used to normalize results using the following primers: GAPDH upper primer, 5'-TTC ACC ACC ATG GAG AAG GC-3', and GAPDH lower primer, 5'-GCA GGG ATG ATG TTC TGG GC-3'. PCR products (10 µL) were resolved by electrophoresis in 1.5% to 2% agarose gels and transferred onto nylon membranes (Nytran-Plus; Schleicher and Schuell) by a standard capillary technique. Blots were UV cross-linked. Detection of DIG-labeled nucleic acids was performed with an anti-DIG antibody linked to alkaline phosphatase, and disodium3-(4-methoxyspiro-[1,2-dioxetane-3-2'(5'chloro)-tricyclo[3.3.1.13.7]decane]-4-yl)phenyl phosphate (CSPD) was used as substrate.

Western Blot Analysis
Whole-cell extracts (100 µg) were separated by SDS-PAGE (7.5%) and transferred to a nitrocellulose filter (Bio-Rad). Membranes were incubated with a rabbit polyclonal antibody against SREBP-1 (Santa Cruz Biotechnology, Inc) used at a dilution of 1:1000. Equal loading of protein in each lane was verified by Pounceau staining.

Electrophoretic Mobility Shift Assay (EMSA)
Nuclear extracts from PAECs were obtained by the method of Dignam et al.¹⁹ The doubled-stranded probe corresponding to the cyp51-SRE was obtained as described previously.²⁰ cyp51-SRE was end-labeled with [-³²P]ATP and T4 polynucleotide kinase. Nuclear extracts (10 µg) were incubated for 15 minutes on ice in a final volume of 20 µL with 1 µg of poly(d[I-C]) in (in mmol/L) Tris-HCl (pH 8) 25, MgCl₂ 4, DTT 0.5, EDTA 0.5, and KCl 60, and 5% glycerol. Then, 30 000 cpm of ³²P–end-labeled SRE was added, and incubation proceeded for an additional 30 minutes. DNA-protein complexes were resolved on a 5% polyacrylamide gel at 4°C in 0.5x Tris-buffered EDTA. Free probe and shifted bands were detected by autoradiography.²¹

Transient Transfection and Measurement of Luciferase Activity
The CYP51 promoter containing the SRE element was subcloned from the CYP51-CAT construct, kindly provided by Dr D. Rozman (Institute of Biochemistry, Medical Center for Molecular Biology, University of Ljubljana, Slovenia), into the pGL3 basic vector (Promega) by digestion with KpnI and BglII (pGL3-CYP51). Transient transfection assays of semiconfluent (60% to 70%) PAECs (passages 2 to 3) were performed with 1 µg/well of pGL3-CYP51, 0.03 µg/well of pSVß-gal (Promega) as internal control vector, and 3 µL of lipofectin (Life Technologies) according to the manufacturer’s protocol. After 5 hours of exposure to the DNA-liposome complexes, cells were washed and incubated with or without LDL (180 mg/dL) for 24 hours in M199 supplemented with 1% FCS. Luciferase and ß-galactosidase activities were measured in cell lysates using a luminometer (Anthos Lucy 1.0) according to the manufacturer (Promega). Results were normalized by ß-galactosidase activity.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CYP51 Is Expressed in Arterial Endothelial Cells and CYP51 mRNA Is Downregulated by Atherogenic Concentrations of LDL
PAECs maintained in serum-free medium (24 hours) were incubated with atherogenic levels of nLDL (140 and 180 mg/dL, 24 hours). mRNA-DD analyses were carried out by RT-PCR using different primer combinations. Only reproducible differentially displayed bands obtained with cDNAs synthesized from independent samples and with different cDNA concentrations were further analyzed. A total of 7 bands were differentially regulated by atherogenic nLDL concentrations. Figure 1A (a and b) shows the result corresponding to band 2. Band 2, amplified with primers P8 and T6, was cloned, sequenced, and further analyzed. Sequence analysis revealed that band 2, which is 352 bp of the porcine lanosterol 14-demethylase (CYP51), corresponds to positions between 1353 and 1652 bp (GenBank accession No. AB009988).

View larger version (20K): [in this window] [in a new window]   Figure 1. CYP51 is downregulated by atherogenic LDL concentrations in PAECs. A, PAECs were incubated with nLDL (140 and 180 mg/dL) in serum-depleted medium for 24 hours and mRNA-DD analysis was performed as described in Materials and Methods. Arrow shows a band downregulated by LDL in 2 independent RNA samples (a and b). This cDNA was cloned and sequenced showing identity with porcine CYP51 gene. B, Densitometric quantification and representative RT-PCR corresponding to CYP51 from PAECs incubated with nLDL (180 mg/dL, 24 hours) in serum-free medium. Data were normalized by GAPDH mRNA levels and are expressed as mean±SD; n=5. C, Representative RT-PCR assay corresponding to CYP51 from PAECs incubated with nLDL (180 mg/dL, 24 hours) in medium containing 10% FCS. CT indicates control.

The downregulation of porcine CYP51 by atherogenic levels of nLDL was confirmed by semiquantitative RT-PCR. nLDL treatment (180 mg/dL, 24 hours) significantly reduced CYP51 mRNA levels (8.8±1.73 [control] versus 1±0.35 [nLDL]; P<0.001) in PAECs maintained in the absence of FCS (Figure 1B). The effect of atherogenic nLDL levels on CYP51 mRNA levels was observed even in the presence of 10% FCS. In these conditions, basal CYP51 mRNA levels that were 13-fold lower than in the absence of serum (not shown) were significantly reduced by nLDL treatment (2.5-fold) (Figure 1C). The effect of nLDL was time- and dose-dependent. nLDL decreased CYP51 expression even at 50 mg/dL, the minimal concentration tested, and maximal downregulation of CYP51 mRNA was obtained at 180 mg/dL (Figure 2A). On the other hand, the time-course assays performed with the highest nLDL concentration assayed (180 mg/dL) showed that the reduction in CYP51 expression was already observed after 9 hours of incubation and was maximal after 24 hours (Figure 2B).

View larger version (16K): [in this window] [in a new window]   Figure 2. Effect of nLDL on CYP51 mRNA levels. A, Dose-response assay performed in absence of FCS with PAECs treated for 24 hours with nLDL. B, PAECs were incubated with nLDL (180 mg/dL) in serum-depleted medium during the times indicated. CYP51 mRNA levels were determined by RT-PCR using 1 µg of total RNA. Results were normalized by GAPDH mRNA level and are expressed as percentage of controls.

Effect of Cycloheximide and N-Acetyl-Leucyl-Leucyl–Norleucinal (ALLN) on nLDL CYP51 Downregulation
PAECs were exposed to cycloheximide (2 µg/mL) and nLDL for 9 hours. As shown in Figure 3A, cycloheximide blocked the reduction of CYP51 mRNA levels caused by nLDL, which suggests that new protein synthesis is necessary for the nLDL effect. A similar pattern was obtained by coincubation with ALLN, an inhibitor of neutral cysteine proteases (Figure 3B). ALLN (25 µmol/L) abolished CYP51 mRNA reduction caused by nLDL (180 mg/dL, 9 hours).

View larger version (12K): [in this window] [in a new window]   Figure 3. Effect of ALLN and cycloheximide on CYP51 expression. PAECs were incubated with nLDL (180 mg/dL) alone or in combination with cycloheximide (CHX, 2 µg/mL) (A) or 25 µmol/L ALLN (B) for 9 hours. CYP51 mRNA levels were determined by RT-PCR from 1 µg of total RNA. Results were normalized by GAPDH mRNA levels.

Effect of nLDL on SREBP Levels
Western blot analysis revealed no changes in SREBP-1 protein levels after 5 or 9 hours of nLDL stimulation (Figure 4A). SREBP-2 protein levels could not be analyzed because of the absence of a functional commercially available antibody. Because it has been reported that SREBP-2 mRNA expression is self-regulated through a SRE-1 element present in the SREBP-2 promoter,²² we determined SREBP-2 mRNA levels by RT-PCR. Figures 4B and 4C show the decline in SREBP-2 mRNA levels with nLDL treatment in a dose- and time-dependent manner. Maximal reduction was achieved after 24 hours of incubation with 180 mg/dL of nLDL. Therefore, there is a parallel nLDL downregulation on CYP51 and SREBP-2 expression. Because of the presence of a SRE in the CYP51 promoter, our results may again indicate a possible role of SREBP-2 in nLDL CYP51 regulation.

View larger version (13K): [in this window] [in a new window]   Figure 4. Effect of nLDL on SREBP levels. A, PAECs were incubated with nLDL (180 mg/dL) for 9 hours, and levels of mature and precursor forms of SREBP-1 were determined by Western blot (100 µg/lane). Shown is a representative autoradiography from 2 different assays. CT indicates control. B, Changes in SREBP-2 mRNA levels in PAECs treated with different concentrations of nLDL for 24 hours. Shown is an autoradiography of a representative RT-PCR analysis. C, Time-course assays performed with PAECs incubated for times indicated with 180 mg/dL nLDL. SREBP-2 mRNA levels were determined by RT-PCR and quantified by densitometric analysis. Results are expressed as percentage of control value.

Effect of nLDL on SREBP cyp51-SRE Interaction
To gain further insight into nLDL/SREBP-2–mediated CYP51 regulation, PAECs were incubated with nLDL (180 mg/dL) for 24 hours. Nuclear extracts obtained from these cells were used in gel-shift assays performed with a cyp51–SRE-1 probe that binds to SREBPs.²⁰ The intensity of shifted bands produced by nuclear extracts from PAECs treated with nLDL was significantly lower than that of control cells (Figure 5A). In contrast, the intensity of shifted bands obtained with the Oct-1 probe were not modified by nLDL treatment, confirming the specificity of these results. The SREBP/SRE binding was specific, because competition with unlabeled probe reduced the intensity of shifted bands. Thus, the decrease of CYP51 mRNA produced by nLDL could be mediated by a reduction in the SREBP-SRE binding at the level of the CYP51 promoter.

View larger version (21K): [in this window] [in a new window]   Figure 5. A, Effect of nLDL on cyp51-SRE–SREBP interaction. Shown is a representative EMSA performed with a specific 32P-labeled cyp51-SRE probe and nuclear extracts (+NE [10 µg]; -NE indicates without nuclear extracts) obtained from PAECs incubated with or without nLDL (180 mg/dL, 24 hours). Competition with an unlabeled cyp51-SRE probe is shown. Nuclear extracts were incubated in parallel with an unrelated Oct-1 probe. B, Reduction of CYP51 promoter activity by nLDL. PAECs were transfected with the pGL3 basic vector or the pGL3-CYP51 construct together with the control plasmid pSVß-gal. Cells were incubated without nLDL (CT) or with nLDL (LDL) (180 mg/dL) for 24 hours before lysis and determination of luciferase and ß-galactosidase activities. Results normalized by ß-galactosidase activity are expressed as a percentage of controls and are mean±SD of 2 independent assays performed in quintuplicate.

Effect of nLDL on CYP51 Promoter Activity
The EMSAs suggested that nLDL treatment of PAECs could produce a reduction in CYP51 promoter activity mediated by its SRE element. To confirm this hypothesis, a CYP51 promoter luciferase reporter construct containing 648 bp of the 5'-region of the human CYP51 gene, including the cyp51-SRE element,²⁰ was transfected into PAECs. As we expected, nLDL treatment (180 mg/dL, 24 hours) reduced CYP51 promoter activity 3 fold (Figure 5B). These results together with the EMSA data confirmed that nLDL downregulation of CYP51 is mediated by a reduction in CYP51 promoter activity through the SRE element.

CYP51 mRNA Levels After Forskolin Treatment
Recently, Rozman et al²⁰ described a CYP51 cAMP/CREM regulation mechanism in spermatids. To evaluate the role of cAMP in endothelial CYP51 regulation, PAECs were incubated with forskolin (25 µmol/L, 9 hours), an activator of adenylate cyclase, to increase intracellular levels of cAMP. As shown in Figure 6, forskolin was unable to increase basal CYP51 mRNA levels or to abolish nLDL downregulation of CYP51 mRNA.

View larger version (24K): [in this window] [in a new window]   Figure 6. Effect of forskolin on CYP51 mRNA levels. Shown is a representative RT-PCR assay performed with PAECs incubated with nLDL (180 mg/dL, 9 hours) with or without forskolin (25 µmol/L).

Effect of Hypercholesterolemia on SREBP-2 and CYP51 mRNA Levels in Porcine Abdominal Aorta
To determine whether the in vitro results were indeed showing an in vivo regulation of vascular SREBP-2 and CYP51 by LDL cholesterol, mRNA levels for both genes were analyzed in abdominal aorta samples from normolipemic pigs and from animals fed with a hypercholesterolemic diet. Animals fed with the hypercholesterolemic diet showed a modified plasma lipid profile in comparison with normolipemic animals. Plasma cholesterol levels were higher in hypercholesterolemic pigs as a result of an increase in LDL levels (Table). SREBP-2 and CYP51 mRNA levels were significantly reduced in abdominal aorta of hypercholesterolemic animals, which demonstrates that plasma LDL-cholesterol levels regulate vascular expression of these genes in vivo (Figure 7).

View this table: [in this window] [in a new window]   Table 1. Plasma Lipid Profile in Normolipemic and Hyperlipemic Pigs

View larger version (17K): [in this window] [in a new window]   Figure 7. Effect of hypercholesterolemia on vascular SREBP-2 and CYP51 mRNA levels. A, Densitometric analysis of SREBP-2 mRNA levels in abdominal aorta samples from normolipemic pigs (CONTROL) and hyperlipemic animals (HYPER). B, CYP51 mRNA levels in the vessel wall of control pigs and pigs fed with a hyperlipemic diet. C, Representative RT-PCR assay showing SREBP-2 and CYP51 mRNA levels from abdominal aorta samples of normolipemic animals (lanes 1 through 3) and hyperlipemic animals (lanes 4 through 6). Results were normalized by GAPDH mRNA levels.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The effect of LDL on endothelial cells is usually analyzed using oxidized LDL. Although the effect of atherogenic levels of nLDL on vascular cell adhesion molecules^{23 24 25} and endothelial NO synthase expression²⁶ has been reported, the effect of high LDL concentrations on endothelial cell gene expression is largely unknown. Using a highly sensitive method, we have identified CYP51 andSREBP-2 as 2 genes regulated by nLDL in arterial endothelial cells in culture and in vivo in the arterial wall of hypercholesterolemic animals.

CYP51 is a ubiquitous cholesterogenic gene involved in the postsqualene phases of cholesterol biosynthesis. The expression levels of CYP51 are high in testis, ovary, liver, adrenal gland, prostate, kidney, and lung and low in other tissues such as small intestine, spleen, and colon.¹⁴ However, endothelial CYP51 expression has not been previously reported. CYP51, which is significantly expressed in PAECs, is downregulated by atherogenic concentrations of nLDL in a dose- and time-dependent manner. Downregulation of CYP51 by oxysterols has also been observed in adrenocortical H295 cells and in hepatoma cells (HepG2) maintained in lipoprotein-free medium. Moreover, upregulation of CYP51 by serum deprivation has been observed in H295 cells but not in HepG2 cells, probably as a result of the dysregulation of cholesterol pathway observed in different carcinoma cell lines.²⁷ We have observed CYP51 downregulation by nLDL even in the presence of serum in endothelial cells. Moreover, this gene is significantly expressed in vivo in porcine aorta, in which it is downregulated by hypercholesterolemia. Thus, CYP51 is a gene closely regulated in vascular cells by changes in nLDL concentrations.

Promoter analysis of human cyp51 gene detected 2 potential SRE sites; thus, to analyze the molecular mechanisms involved in the regulation of CYP51 by LDL cholesterol, we have studied the role of SREBPs. ALLN, which inhibits the catabolism of the mature form of SREBPs, blocked nLDL downregulation of CYP51 mRNA, supporting the hypothesis that a decrease in SREBP levels would mediate CYP51 downregulation by nLDL. This effect of ALLN is in agreement with the effect produced by this compound on other enzymes involved in cholesterologenesis, such as 3-hydroxy-3-methylglutaryl (HMG)–coenzyme A (CoA) synthase, that are regulated by SREBPs.^{6 28} We also analyzed the effect of nLDL on SREBP-1 and SREBP-2 expression. We observed a differential regulation on SREBP-1 and SREBP-2 in endothelial cells. SREBP-2 mRNA levels decreased in a dose- and time-dependent manner, whereas SREBP-1 levels remained unchanged after nLDL treatment. This result is in agreement with the regulation of SREBP-2 by sterols observed in cell transfection assays²² and with the differential regulation of SREBP-1 and -2 produced by lipid-lowering drugs in the liver.¹³ The regulation of SREBP-2 and CYP51 by nLDL showed a similar pattern. Moreover, the depletion in SREBP-2 mRNA levels correlated with a decrease in SREBP–cyp51-SRE binding as observed by EMSAs. The cyp51-SRE probe used in these assays binds to SREBPs²⁰ and is identical to the pig cyp51-SRE described recently.²⁹ Because SREBP-1 protein levels were unaffected by nLDL, the differences in SREBP–cyp51-SRE observed in treated cells should be attributed to variations in nuclear mature SREBP-2. In addition, nLDL downregulated CYP51 promoter activity in transient transfection assays. The construct used in these assays contains the CYP51–SRE-1 element and controls sterol-dependent regulation of CYP51.²⁰ Taken together, these data suggest that CYP51 and other genes containing functional SRE elements could be regulated by nLDL through a decrease in both SREBP-2 mRNA and protein levels. In addition, the increase in activator protein (AP)–1 binding capacity observed in endothelial cells treated with nLDL³⁰ could contribute to the coordinate regulation by these 2 transcription factors of a wide set of genes in the endothelium exposed to hyperlipemic conditions. This hypothesis is supported by our results obtained in vivo in which SREBP-2 is downregulated by hypercholesterolemia in porcine aorta.

It has been described that transcription factors other than SREBPs, such nuclear factor (NF)-Y or Sp1, are necessary for the sterol-mediated transcriptional regulation of target genes.^{31 32} In contrast, other factors such as the Ying Yang 1 (YY1) protein could act as negative regulators disrupting SREBP-Sp1 interaction or displacing NF-Y.^{33 34} The CYP51 promoter contains multiple regulatory sequences recognizing cAMP response element binding protein (CREB), Sp1, AP-1, and AP-4 transcription factors. The cAMP-response element (CRE) motif present in the CYP51 promoter is involved in CYP51 induction during spermatogenesis. However, CYP51 expression in liver seems to be controlled only by SREBPs.²⁰ On the contrary, CREB is required for maximal sterol-mediated transcriptional activation of the HMG-CoA synthase promoter that contains a CRE overlapping with a SRE-1 motif.³⁵ On the basis of our results, a cAMP-dependent signaling pathway would not be involved in endothelial CYP51 regulation, given that we have observed that forskolin was unable to reverse nLDL CYP51 downregulation or increasing basal CYP51 mRNA levels. Other transcription factors could act as coregulators with SREBPs in the transcriptional control of CYP51 by nLDL, as suggested by the cycloheximide reversal of nLDL CYP51 downregulation. Moreover, it is possible that organ-specific relative levels between coactivators and corepressors might be involved in CYP51 endothelial sterol regulation.

Dietary cholesterol and fatty acids regulate in vivo SREBP expression.^{13 36} On the other hand, it has been reported that accelerated atherogenesis induced in mice by cyclosporin treatment could be due to an increase in hepatic SREBP-2 expression,³⁷ and SREBPs have also been involved in the regulation of LDL receptor promoter activity by insulin and platelet-derived growth factor linked to the mitogen-activated protein kinase signaling pathway.^{38 39} Therefore, SREBPs alone or with other transcription factors regulate transcriptional activity of multiple genes involved in lipid metabolism, including LDL receptor,¹¹ HDL receptor,⁴⁰ HMG-CoA synthase,³⁵ stearoyl-CoA desaturase,⁴¹ peroxisome proliferator-activated receptor ,⁴² acetyl-CoA carboxylase,³¹ and lipoprotein lipase.⁴³ In vivo studies on SREBP expression were primarily performed with hepatic samples. To our knowledge, this is the first report showing in vivo SREBP-2 expression and regulation by high levels of plasma cholesterol in the vessel wall. Thus, a plausible arterial downregulation of SREBP levels due to systemic hypercholesterolemia can alter vascular homeostasis and may play a key role in the cascade of cellular dysfunctions associated with the atherosclerotic process.

	Acknowledgments

 
This study has been made possible by funds provided by Merck Sharp & Dohme, Spain. Support for studies on atherosclerosis has been partially provided by a Fondo de Investigación Sanitaria Grant 98/715, and Catalana Occidente. S. Sánchez-Gómez is a predoctoral fellow of the Fundación de Investigación Cardiovascular. We thank Dr T. Royo (Cardiovascular Research Center, Barcelona, Spain) for her help with PAEC cultures. We thank Dr D. Rozman for providing the CYP51-CAT construct to perform the transfection experiments. We are indebted to Olga Bell, Margarita García, and Pablo Catalina for technical assistance.

	Footnotes

 
Original received September 5, 2000; revision received December 12, 2000; accepted December 12, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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