© 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
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 IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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).
|
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).
|
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).
|
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.
|
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.
|
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.
|
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).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 molecules23 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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Andrews HE, Bruckdorfer KR, Dunn RC, Jacobs M. Low density lipoproteins inhibit endothelium-dependent relaxation in rabbit aorta. Nature. 1987;327:237–239.[Medline] [Order article via Infotrieve]
2.
Zeiher AM,
Drexler H, Wollschlager H, Just H. Modulation of coronary vasomotor
tone in humans: progressive endothelial dysfunction with different
early stages of coronary atherosclerosis.
Circulation. 1991;83:391–401.
3.
Li H,
Cybulsky MI, Gimbrone MA, Libby P. An atherogenic diet rapidly induces
VCAM-1, a cytokine-regulatable mononuclear leukocyte adhesion molecule,
in rabbit aortic endothelium. Arterioscler
Thromb. 1993;13:197–204.
4.
Casino PR,
Crescence MK, Quyyumi AA, Hoeg JM, Panza JA. The role of nitric oxide
in endothelium-dependent vasodilation of hypercholesterolemic patients.
Circulation. 1993;88:2541–2547.
5. De Caterina R. Endothelial dysfunctions: common denominators in vascular disease. Curr Opin Lipidol. 2000;11:9–23.[Medline] [Order article via Infotrieve]
6. Wang X, Sato R, Brown MS, Hua X, Goldstein JL. SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis. Cell. 1994;77:53–62.[Medline] [Order article via Infotrieve]
7. Russell A, Boyd D, Brown MS, Li WP, Nohturfft A, Goldstein JL, Espenshade PJ. Transport-dependent proteolysis of SREBP: relocation of site-1 protease from Golgi to ER obviates the need for SREBP transport to Golgi. Cell. 1999;99:703–712.[Medline] [Order article via Infotrieve]
8.
Brown MS,
Goldstein JL. A proteolytic pathway that controls the cholesterol
content of membranes, cells and blood.
Proc Natl Acad Sci
U S A. 1999;96:11041–11048.
9. Brown MS, Goldstein JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell. 1997;89:331–340.[Medline] [Order article via Infotrieve]
10. Horton JD, Shimomura I. Sterol regulatory element-binding proteins: activators of cholesterol and fatty acid biosynthesis. Curr Opin Lipidol. 1999;10:143–150.[Medline] [Order article via Infotrieve]
11. Yokohama C, Wang X, Briggs MR, Admon A, Wu J, Hua X, Goldstein JL, Brown MS. SREBP-1, a basic helix-loop-helix leucine zipper protein that controls transcription of the LDL receptor gene. Cell. 1993;75:187–197.[Medline] [Order article via Infotrieve]
12.
Hua X,
Yokoyama C, Wu J, Briggs MR, Brown MS, Goldstein JL, Wang X. SREBP-2, a
second basic-helix-loop-helix-leucine zipper protein that stimulates
transcription by binding to a sterol regulatory element.
Proc Natl Acad Sci
U S A. 1993;90:11603–11607.
13. Sheng Z, Otani H, Brown MS, Goldstein JL. Independent regulation of sterol regulatory element binding proteins 1 and 2 in hamster liver. Proc Natl Acad Sci U S A. 1995;922:935–938.
14.
Strömstedt
M, Rozman D, Waterman MR. The ubiquitously expressed human CYP51
encodes lanosterol 14 -demethylase, a cytochrome p450 whose
expression is regulated by oxysterols.
Arch Biochem Biophys. 1996;329:73–81.[Medline]
[Order article via Infotrieve]
15. Colomé C, Martínez-González J, Vidal F, de Castellarnau C, Badimon L. Small oxidative changes in atherogenic LDL concentrations irreversibly regulate adhesiveness of human endothelial cells: effect of the lazaroid U74500A. Atherosclerosis. 2000;149:295–302.[Medline] [Order article via Infotrieve]
16. Pueyo Palazón C, Alfon J, Gaffney P, Berrozpe M, Royo T, Badimon L. Effects of reducing LDL and increasing HDL with gemfibrozil in experimental coronary lesion development and thrombotic risk. Atherosclerosis. 1998;136:333–345.[Medline] [Order article via Infotrieve]
17.
Alfon J,
Royo T, García-Moll X, Badimon L. Platelet deposition on eroded
vessel wall at stenotic shear rate is inhibited by lipid-lowering
treatment with atorvastatin. Arterioscler
Thromb Vasc Biol. 1999;19:1812–1817.
18. Lipid Research Clinic Program. Manual of Laboratory Operation. Washington DC: Department of Health, Education, and Welfare (DHHS) Publication No NIH75–628. Washington DC: U.S. Government Printing Office; 1974.
19.
Dignam JD,
Lebovitz RM, Roeder R. Accurate transcription initiation by RNA
polymerase II in a soluble extract from isolated mammalian nuclei.
Nucleic Acids Res. 1983;11:1475–1479.
20.
Rozman D,
Fink M, Firmia M, Sassone-Corsi P, Waterman MR. Cyclic adenosine
3',5'-monophosphate (cAMP)/cAMP-responsive element modulator
(CREM)-dependent regulation of cholesterogenic lanosterol 14
-demethylase (CYP51) in spermatids. Mol
Endocrinol. 1999;13:1951–1962.
21.
Rodríguez
C, Noé V, Cabrero A, Ciudad CJ, Laguna JC. Differences in the
formation of PPAR-RXR/acoPPRE complexes between responsive and
nonresponsive species on fibrate administration.
Mol Pharmacol. 2000;58:185–93.
22.
Sato R,
Inoue J, Kawabe Y, Kodama T, Takano T, Maeda M. Sterol-dependent
transcriptional regulation of sterol regulatory element-binding
protein-2. J Biol Chem. 1996;271:26461–26464.
23.
Smalley
DM, Lin JHC, Curtis ML, Kobari Y, Stemerman MB, Pritchard KA. Native
LDL increases endothelial cell adhesiveness by inducing intercellular
adhesion molecule-1. Arterioscler Thromb
Vasc Biol. 1996;16:585–590.
24. Lin JHC, Zhu Y, Liao HL, Kobari Y, Groszek L, Stemerman MB. Induction of vascular cell adhesion molecule-1 by low-density lipoprotein. Atherosclerosis. 1996;127:185–194.[Medline] [Order article via Infotrieve]
25. Allen S, Khan S, Al-Mohanna F, Battern P, Yacoub M. Native low density lipoprotein-induced calcium transients trigger VCAM-1 and E-selectin expression in cultured human vascular endothelial cells. J Clin Invest. 1998;101:1064–1075.[Medline] [Order article via Infotrieve]
26. Vidal F, Colomé C, Martínez-González J, Badimon L. Atherogenic concentrations of native low-density lipoproteins down-regulate nitric-oxide-synthase mRNA and protein levels in endothelial cells. Eur J Biochem. 1998;252:378–384.[Medline] [Order article via Infotrieve]
27.
Siperstein
MD, Gyde AM, Morris HP. Loss of feedback control of
hydroxymethylglutaryl coenzyme A reductase in hepatoma.
Proc Natl Acad Sci
U S A. 1971;68:315–317.
28. Mascaro C, Ortiz JA, Ramos MM, Haro D, Hegardt FG. Sterol regulatory element binding protein-mediated effect of fluvastatin on cytosolic 3-hydroxy-3-methylglutaryl-coenzyme A synthase transcription. Arch Biochem Biophys. 2000;374:286–292.[Medline] [Order article via Infotrieve]
29.
Kojima M,
Morozumi T, Onishi A, Mitsuhashi T. Structure of the pig sterol
14-demethylase (CYP51) gene and its expression in the testis and
other tissues. J Biochem. 2000;127:805–811.
30.
Zhu
Y, Lin JHC, Liao HL, Friedli O, Verna L, Marten NW, Straus DS,
Stemerman MB. LDL induces transcription factor activator protein-1 in
human endothelial cells. Arterioscler
Thromb Vasc Biol. 1998;18:473–480.
31.
López
JM, Bennett MK, Sánchez HB, Rosenfeld JM, Osborne TF. Sterol
regulation of acetyl coenzyme A carboxylase: a mechanism for coordinate
control of cellular lipid. Proc Natl Acad
Sci
U S A. 1996;93:1049–1053.
32.
Bennett MK,
López JM, Sánchez HB, Osborne TF. Sterol regulation of fatty acid
synthase promoter. J Biol
Chem. 1995;270:25578–25583.
33.
Ericsson J,
Usheva A, Edwards PA. YY1 is a negative regulator of transcription of
three sterol regulatory element-binding protein-responsive genes.
J Biol Chem. 1994;274:14508–14513.
34.
Bennett MK,
Ngo TT, Athanikar JN, Rosenfeld JM, Osborne TF. Co-stimulation of
promoter for low density lipoprotein receptor gene by sterol regulatory
element-binding protein and Sp1 is specifically disrupted by the yin
yang 1 protein. J Biol
Chem. 1999;274:13025–13032.
35.
Dooley KA,
Bennett MK, Osborne TF. A critical role for cAMP response
element-binding protein (CREB) as a coactivator in sterol-regulated
transcription of 3-hydroxy-3-methylglutaryl coenzyme A synthase
promoter. J Biol Chem. 1999;274:5285–5291.
36.
Manabu JX,
Nakamura T, Cho HP, Clarke SD. Sterol regulatory element binding
protein-1 expression is suppressed by dietary polyunsaturated fatty
acids. J Biol Chem. 1999;274:23577–23583.
37. Wu J, Zhu YH, Patel SB. Cyclosporin-induced dyslipoproteinemia is associated with selective activation of SREBP-2. Am J Physiol. 1999;277(6 pt 1):E1087–E1094.
38.
Streicher
R, Kotzka J, Müller-Wieland D, Siemeister G, Munck M, Avci H, Krone W.
SREBP-1 mediates activation of the low density lipoprotein receptor
promoter by insulin and insulin-like growth factor-1.
J Biol Chem. 1996;271:7128–7133.
39.
Kotzka J,
Müller-Wieland D, Roth G, Kremer L, Munck M, Schürmann S, Knebel B,
Krone CW. Sterol regulatory element binding proteins (SREBP)-1a and
SREBP-2 are linked to the MAP-kinase cascade.
J Lipid Res. 2000;41:99–108.
40.
López D,
McLean MP. Sterol regulatory element-binding protein-1a binds to
cis elements in the promoter of
the rat high density lipoprotein receptor SR-B1 gene.
Endocrinology. 1999;140:5669–5681.
41.
Tabor DE,
Kim JB, Spiegelman BM, Edwards PA. Identification of conserved
cis-elements and transcription
factors required for sterol-regulated transcription of stearoyl-CoA
desaturase 1 and 2. J Biol
Chem. 1999;274:20603–20610.
42.
Fajas L,
Schoonjans K, Gelman L, Kim JB, Najib J, Martin G, Fruchart JC, Briggs
M, Spiegelman BM, Auwerx J. Regulation of peroxisome
proliferator-activated receptor expression by adipocyte
differentiation and determination factor 1/sterol regulatory element
binding protein 1: implications for adipocyte differentiation and
metabolism. Mol Cell Biol. 1999;19:5495–5503.
43.
Yang WS,
Deeb SS. Sp1 and Sp3 transactivate the human lipoprotein lipase gene
promoter through to a CT element: synergy with the sterol regulatory
element binding protein and reduced transactivation of a naturally
occurring promoter variant. J Lipid
Res. 1998;39:2054–2063.
This article has been cited by other articles:
 |
|
 |
|
  C. Rodriguez, J. F. Alcudia, J. Martinez-Gonzalez, A. Guadall, B. Raposo, S. Sanchez-Gomez, and L. Badimon Statins normalize vascular lysyl oxidase down-regulation induced by proatherogenic risk factors Cardiovasc Res, May 22, 2009; (2009) cvp136v2. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Y. Tamboli, K. Prager, D. R. Thal, K. M. Thelen, I. Dewachter, C. U. Pietrzik, P. St. George-Hyslop, S. S. Sisodia, B. De Strooper, M. T. Heneka, et al. Loss of {gamma}-Secretase Function Impairs Endocytosis of Lipoprotein Particles and Membrane Cholesterol Homeostasis J. Neurosci., November 12, 2008; 28(46): 12097 - 12106. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Fink, J. Acimovic, T. Rezen, N. Tansek, and D. Rozman Cholesterogenic Lanosterol 14{alpha}-Demethylase (CYP51) Is an Immediate Early Response Gene Endocrinology, December 1, 2005; 146(12): 5321 - 5331. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Crespo, J. Martinez-Gonzalez, J. Rius, and L. Badimon Simvastatin inhibits NOR-1 expression induced by hyperlipemia by interfering with CREB activation Cardiovasc Res, August 1, 2005; 67(2): 333 - 341. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Llorente-Cortes and L. Badimon LDL Receptor-Related Protein and the Vascular Wall: Implications for Atherothrombosis Arterioscler. Thromb. Vasc. Biol., March 1, 2005; 25(3): 497 - 504. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Rius, J. Martinez-Gonzalez, J. Crespo, and L. Badimon Involvement of Neuron-Derived Orphan Receptor-1 (NOR-1) in LDL-Induced Mitogenic Stimulus in Vascular Smooth Muscle Cells: Role of CREB Arterioscler. Thromb. Vasc. Biol., April 1, 2004; 24(4): 697 - 702. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Rodriguez, B. Raposo, J. Martinez-Gonzalez, V. Llorente-Cortes, G. Vilahur, and L. Badimon Modulation of ERG25 expression by LDL in vascular cells Cardiovasc Res, April 1, 2003; 58(1): 178 - 185. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Martinez-Gonzalez, J. Rius, A. Castello, C. Cases-Langhoff, and L. Badimon Neuron-Derived Orphan Receptor-1 (NOR-1) Modulates Vascular Smooth Muscle Cell Proliferation Circ. Res., January 10, 2003; 92(1): 96 - 103. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Llorente-Cortes, M. Otero-Vinas, S. Sanchez, C. Rodriguez, and L. Badimon Low-Density Lipoprotein Upregulates Low-Density Lipoprotein Receptor-Related Protein Expression in Vascular Smooth Muscle Cells: Possible Involvement of Sterol Regulatory Element Binding Protein-2-Dependent Mechanism Circulation, December 10, 2002; 106(24): 3104 - 3110. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Bank and V. W.M. van Hinsbergh Lysyl Oxidase: New Looks on LOX Arterioscler. Thromb. Vasc. Biol., September 1, 2002; 22(9): 1365 - 1366. [Full Text] [PDF]
|
|
|
|
|
|
C. Rodriguez, B. Raposo, J. Martinez-Gonzalez, L. Casani, and L. Badimon Low Density Lipoproteins Downregulate Lysyl Oxidase in Vascular Endothelial Cells and the Arterial Wall Arterioscler. Thromb. Vasc. Biol., September 1, 2002; 22(9): 1409 - 1414. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Martinez-Gonzalez, B. Raposo, C. Rodriguez, and L. Badimon 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibition Prevents Endothelial NO Synthase Downregulation by Atherogenic Levels of Native LDLs : Balance Between Transcriptional and Posttranscriptional Regulation Arterioscler. Thromb. Vasc. Biol., May 1, 2001; 21(5): 804 - 809. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||