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(Received for publication, May 2, 1996, and in revised form, July 29, 1996)
From the Department of Biochemistry, Queen's University,
Kingston, Ontario K7L 3N6, Canada
Previously, we demonstrated that when
two human hepatoma cell lines, Hep3B and HepG2, were exposed to
gemfibrozil, a hypolipidemic drug, a 2-fold induction in apolipoprotein
A-I (apoA-I) mRNA levels resulted. To determine if mRNA
stabilization was responsible for the changes in apoA-I mRNA
levels, the half-lives for apoA-I mRNA were measured in the
presence of actinomycin D with and without gemfibrozil. These
experiments revealed no differences in stability. However, nuclear
run-off assays indicated that the transcription rate of the apoA-I gene
was increased 2-fold in gemfibrozil-treated cells. Transient
transfection experiments also indicated that the induction of apoA-I
mRNA level in response to gemfibrozil is mediated at the
transcriptional level. We have identified two copies of the
``drug-responsive element'' (DRE) in the apoA-I promoter region that
may be responsible for the increase in apoA-I transcriptional activity
by gemfibrozil. Using gel mobility shift assays with a synthetic DRE
oligonucleotide, we have demonstrated that exposure of Hep3B and HepG2
cells to gemfibrozil resulted in strong induction of a protein-DNA
complex. The formation of this complex is highly sequence-specific as
indicated by the DNA competition experiments. The drug-inducible
nuclear proteins bind to the DRE of the human apoA-I gene with an
apparent Kd of 4.1 nM. Methylation
interference experiments have localized the contact sites of nuclear
factors to the DRE region. Southwestern blot analyses have identified
two groups of drug-inducible nuclear proteins with molecular masses of
approximately 30 and 15 kDa. When a copy of synthetic DRE
oligonucleotide was inserted upstream of the thymidine kinase promoter
and luciferase reporter construct, a significant 2-fold induction in
luciferase activity was observed in the presence of gemfibrozil
following transient transfection of two human hepatoma cell lines,
HepG2 and Hep3B. However, a plasmid containing one copy of mutated
apoA-I-DRE oligomer did not confer responsiveness to gemfibrozil
treatment. Furthermore, pGL2 (apoA-I ApoA-I1 is the major constituent of
plasma high density lipoprotein (HDL) and participates in cholesterol
ester formation as a cofactor for lecithin-cholesterol acyltransferase
(1). It has been proposed that apoA-I, together with
lecithin-cholesterol acyltransferase, promotes cholesterol efflux from
peripheral tissues and carries the excess cholesterol to the liver for
catabolism (1, 2, 3). This process is known as ``reverse cholesterol
transport.'' Epidemiological and genetic studies have indicated that
levels of plasma HDL are inversely correlated with atherosclerotic risk
(4, 5, 6, 7, 8). Finally, evidence that apo AI expression is a major determinant
of atherosclerosis comes from the work of Rubin et al. (9),
which indicated that transgenic mice expressing high amounts of human
apoA-I were significantly protected from the development of fatty
streak lesions after exposure to high fat diets.
There are a number of drugs widely used for reducing the progression of
coronary heart diseases by altering lipoprotein metabolism. One of the
most extensively studied is gemfibrozil. The Helsinki Heart Study (10)
demonstrated that the elevation of HDL cholesterol resulting from use
of this drug had a more protective effect in reducing atherosclerotic
risk than the lowering of LDL cholesterol. Recently, we have
demonstrated that exposure of two human hepatoma cell lines, HepG2 and
Hep3B, to gemfibrozil resulted in a 2-fold induction of apoA-I mRNA
(11). The cis- and trans-acting elements involved in apoA-I gene
transcription have been extensively studied (12, 13, 14). Based on
transient transfection studies, it was concluded that the DNA region
located between nucleotides It is well known that the cytochrome P-450 system is responsible for
the metabolism of a variety of drugs (15). The induction of the
CYP1A1 gene is mediated by the interaction of the aryl
hydrocarbon receptor (AhR) together with its ligand to several DNA
recognition sites within a dioxin-responsive enhancer upstream of the
CYP1A1 gene (16, 17, 18). While examining the structure of the
human apoA-I gene promoter a sequence match with the 5 Complementary pairs of oligonucleotides were
synthesized on a Biosearch model 8600 DNA synthesizer at Queen's
University, Department of Biochemistry, Core Facility for Protein/DNA
Chemistry. Sequences of the synthetic oligomers are indicated in Table
I.
Nucleotide sequences of oligomers used for competition gel mobility
shift studies
Human hepatoma cell lines, Hep3B and HepG2, were obtained from the American Type Culture Collection (Rockville, Maryland). Cells were grown in T75 flasks containing 20 ml of Eagle's minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) as described previously (23). Freshly confluent monolayers were washed twice with MEM and then incubated with fresh medium for 24 h in the presence of gemfibrozil, dissolved in ethanol, to give a final concentration of 40 µg/ml. Control cells were incubated with an equal volume (20 µl) of ethanol. In some experiments, gemfibrozil and cycloheximide were added to the cells to give final concentrations of 40 and 10 µg/ml, respectively. Where noted, Hep3B or HepG2 cells were also treated with or without gemfibrozil and in the presence of actinomycin D, ranging from 0.625 to 10.0 µg/ml, for various time periods as described in the figure legends. Cell viability was routinely monitored by trypan blue exclusion and lactate dehydrogenase leakage as described previously (23). In all experiments the number of dead cells never exceeded 5% of the total number of cells. RNA Isolation and Northern Blot AnalysesTotal cellular RNA
was isolated by extraction with guanidine-HCl, as described (11). For
Northern blotting, 20 µg of total RNA were denatured by treatment
with glyoxal, subjected to electrophoresis on a 1.5% agarose gel, and
transferred to Zeta-probe GT membrane (Bio-Rad) as described previously
(11). Blots were prehybridized and hybridized with nick-translated
apoA-I and Nuclei were prepared according to the method of Bartalena et al. (24). Isolated nuclei were resuspended in storage buffer (10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 0.5 M D-sorbitol, 2.5% Ficoll, 50% glycerol, 10 mM spermidine, 20 mM DTT, 0.4 units/ml RNasin, 1 mM phenylmethylsulfonyl fluoride) at concentration of 2-3 × 108 nuclei/ml. An in vitro nuclear run-off transcription assay was carried out as described (24) with minor modifications (25). Specific gene transcripts were quantified by hybridization to filter-bound DNA. The recombinant pBS apoA-I used in this study was prepared by ligating the 0.6-kb human apoA-I insert from pkT apoA-I into the PstI site of the pBS vector. Preparation of Zeta-probe GT nylon filters was as outlined (24). Hybridization, washing, and elution of bound, labeled RNA were carried out as described previously (26). Nonspecific hybridization to each filter was determined by carrying out transcription with labeled pGEM 3Z control vector. The relative rates of transcription were calculated in terms of parts per million, after subtraction of control values. Total incorporation into RNA per assay ranged from 1 × 107 dpm to 5 × 107 dpm, all of which was used for each hybridization selection. Throughout the analysis, 3H sense strand RNAs produced by transcription with Sp6 polymerase were used to normalize for differences in hybridization efficiency. The efficiency ranged from 30 to 35%. Preparation of Nuclear ExtractsHep3B and HepG2 cells were
grown as described above, and 3-6 × 108 cells were
used to prepare nuclear extracts. Nuclear extracts were prepared by a
modification of the method of Dignam et al. (27). The
modification was as follows: cells were suspended in 2 volumes of
buffer (10 mM HEPES (pH 8.0), 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM DTT, and 1 mM phenylmethylsulfonyl fluoride), and incubated on ice for
15 min. The swelled cells were then passed through a 231/2-gauge
needle five times followed by a 261/2-gauge needle for another
five strokes instead of using a Dounce homogenizer. Nuclear extracts
were dialyzed against binding buffer (25 mM HEPES at pH
8.0, 12.5 mM MgCl2, 20% glycerol, and 50 mM KCl) and stored as aliquots at For mobility shift assays,
nuclear extracts were incubated with 100 µg of poly (dI-dC) in
binding buffer containing 5 mM DTT and 5 µM
ZnCl2 on ice for 30 min. Then 2 fmol (10,000 cpm)
5 Equal volumes of nuclear extract
and sample buffer (5% SDS, 5 mM Tris/HCl, pH 6.8, 200 mM DTT, 20% glycerol, and 0.5% pyronin Y) were mixed and
subjected to electrophoresis on a 10% SDS-polyacrylamide gel at room
temperature. Protein samples were electrotransferred to a Millipore
Immobilon P membrane in 25 mM Tris-HCl, 192 mM
glycine, 20% (v/v) methanol, and 0.002% SDS at room temperature for
4 h. Hybridization of 5 A 491-bp DNA fragment of the human
apoA-I promoter between nucleotides Plasmid pGL2 (apoA-I The vector designated pGL2 TK/luc (kindly provided by Dr. M. Petkovich,
Department of Biochemistry, Queen's University) was derived from the
pGL2-Basic vector in which the thymidine kinase promoter was inserted
upstream of the luciferase gene. Plasmid pGL2 (apoA-I-DRE)TK/luc was
constructed by cloning a synthetic oligo-DRE (between nucleotide
positions The human hepatoma cell line, Hep3B, was maintained as monolayers on 100-mm plates in MEM supplemented with 10% FBS. Transient DNA transfections were performed by the calcium phosphate precipitation procedure described by Gorman et al. (33). Ten µg of total DNA was used per plate, including 4 µg of reporter DNA and 6 µg of carrier pGL2 Basic Vector. After glycerol shock, the cells were washed twice with phosphate-buffered saline and once with MEM and cultured in MEM + 10% FBS in the absence or presence of gemfibrozil (40 µg/ml) for 24 h with changes of fresh medium. Transfected Hep3B cells were harvested by washing three times in
phosphate-buffered saline and assayed for luciferase activity as
described (34). In all transfections, 5 µg of an internal control
plasmid (pSG Single-stranded synthetic
oligonucleotides corresponding to the upper or lower strands of the DRE
from the human apoA-I gene were radiolabeled at their 5 Effect of Actinomycin D on the Induction of Apolipoprotein AI mRNA Levels by Gemfibrozil Previously, we demonstrated that exposure of HepG2 and Hep3B cells to gemfibrozil resulted in a 2-fold induction in apoA-I mRNA levels (11). To determine whether inhibition of RNA synthesis affects the induction of apoA-I mRNA levels, Hep3B cells were treated both in the absence and presence of increasing amounts of actinomycin D for 30 min prior to the addition of gemfibrozil. Actinomycin D at concentrations greater than or equal to 0.625 µg/ml were able to block the increase in apoA-I mRNA levels that was observed when the cells were treated with gemfibrozil alone. Under the same regimen of drug treatment, similar results were also obtained using HepG2 cells (data not shown). To examine if mRNA stabilization is responsible for the observed
induction of apoA-I mRNA levels by gemfibrozil, the half-lives for
apoA-I mRNA were determined in the presence of actinomycin D (1 µg/ml) with and without gemfibrozil. Hep3B cells were pretreated with
gemfibrozil (40 µg/ml) or ethanol (drug vehicle) for 24 h. Cells
were then cultured for additional periods ranging from 0 to 8 h in
the presence of actinomycin D. As shown in Fig. 1, the
level of apoA-I mRNA was approximately 2-fold higher in
gemfibrozil-treated cells compared with control cells. After the
addition of actinomycin D the levels of apoA-I mRNA decreased at
the same rate in the absence or presence of gemfibrozil, while levels
of actin mRNA remained relatively constant (Fig. 1, top
panel). The apparent half-lives obtained for apoA-I mRNAs
following the addition of actinomycin D in the absence or presence of
gemfibrozil were approximately 5.6 ± 0.5 h and 5.7 ± 0.4 h, respectively. These data were analyzed by multivariate
statistics and F test using the SAS (Statistical Analysis Systems)
computer program. The results indicated that there was no significant
difference (p > 0.05) between the two half-lives.
Fig. 1. Kinetic studies of apoA-I mRNA levels in the absence or presence of gemfibrozil. Newly confluent Hep3B cells were preincubated for 18 h in MEM and 10% FBS in the absence or presence of gemfibrozil (40 µg/ml). Cells were then incubated with actinomycin D (1 µg/ml). RNA was isolated 0, 1, 2, 3, 4, 6, and 8 h later, and the levels of -actin and apoA-I
mRNAs were determined by Northern analysis (top panel).
All results were normalized using densitometric scans of Northern blots
probed with radiolabeled -actin to correct for loading variations.
The normalized apoA-I mRNA levels in arbitrary units are presented
on a log scale to obtain a linear relationship. Open and solid circles
represent RNA samples isolated from cells cultured in the presence and
absence of gemfibrozil, respectively. Results are mean ± S.E. of
three experiments.
[View Larger Version of this Image (25K GIF file)]
Analysis of the Stimulation of Transcription by Gemfibrozil Treatment The transcription rate of the apoA-I gene was measured
using isolated nuclei from Hep3B cells cultured in the absence or
presence of gemfibrozil treatment. The effect of gemfibrozil on apoA-I
transcription rates at various times is shown in Fig. 2.
The rate of transcription of the apoA-I gene increased approximately
2-fold between 18 and 24 h after stimulation.
Fig. 2. Time course of transcription rates of apoA-I genes in Hep3B cells cultured in the absence or presence of gemfibrozil. Transcription rates were determined by nuclear run-off assays using nuclei isolated from Hep3B cells cultured in the absence or presence of gemfibrozil (40 µg/ml) as described under ``Experimental Procedures.'' Nuclei (2-3 × 107) were isolated at 0, 2, 4, 10, 18, and 24 h. Hybridization of the newly synthesized [32P]RNA to plasmids containing an apoA-I insert were carried out in triplicate. Nonspecific hybridization to each filter was determined by performing transcription with labeled pGEM3Z control vector. Filters were washed extensively, and bound radioactivity was measured by liquid scintillation counting. Relative transcription rates were calculated as parts per million (ppm)/filter = ((counts per min (cpm)/filter) cpm
background)/amount of [32P] RNA used in hybridization
(input count). These numbers were then normalized for hybridization
efficiency as measured by binding of known amounts of 3H
riboprobes added to the hybridization mixture. This was then corrected
for the size to the specific gene: ppm/gene = ((ppm/filter)/percentage of hybridization) × (gene size/cDNA
insert size). Sizes of the apoA-I gene and apoA-I cDNA fragments
were 2 and 0.6 kilobases, respectively. Results are mean ± S.E.
expressed in percentage of the O-h time point of three independent
experiments.
[View Larger Version of this Image (17K GIF file)]
Interactions of Drug-inducible Nuclear Factors Isolated from Hepatoma Cells with the DREs of the Human apoA-I Gene The
possibility that induction of apoA-I transcription by gemfibrozil
involved protein-DNA interactions at the DREs located between
nucleotides Fig. 3. Gel mobility shift analysis. Synthetic double-stranded oligonucleotides corresponding to the DNA sequence between nucleotides 79 and 44 upstream from the transcription start
site (+1) of the human apoA-I gene, designated as oligo-apoA-I-DRE,
were used as probes to study protein-DNA interactions. In addition,
synthetic double-stranded Sp1 and AP1 consensus sequences were also
utilized as probes to determine their activities before and after
gemfibrozil treatment. 32P-Labeled oligo-apoA-I-DRE,
oligo-Sp1, and oligo-AP1 (1 × 104 cpm) were incubated
with no protein (Blank) or with nuclear extracts from Hep3B
cells untreated (Control) or treated with gemfibrozil (40 µg/ml) as described under ``Experimental Procedures.'' Similar
results were also observed when nuclear extracts isolated from HepG2
cells were used.
[View Larger Version of this Image (54K GIF file)]
Competition Gel Mobility Shift Studies In order to determine
the specificity of binding of the drug-inducible nuclear proteins,
competition gel retardation assays were performed with several
different double-stranded oligomers (Table I). In
nuclear extract from gemfibrozil-treated Hep3B cells, binding to the
labeled apoA-I-DRE probe was efficiently blocked by competition with a
50-100-fold molar excess of unlabeled apoA-I-DRE (Fig.
4). The DNA fragments ((P-450(I) and P-450(II)) of the
CYP1A1 gene (19), which contain a DNA sequence very similar
to apoA-I-DRE, also competed efficiently with the probe. Furthermore,
the AP1 and AP2 consensus binding elements (37) were able to compete
efficiently with the probe for binding to the nuclear factor(s).
However, no competition was observed with a 200-fold molar excess of
the GC-rich, Sp1 consensus sequence (37) as well as the core
recognition motif for the AhR (16, 17). Mutation of 10 nucleotides
within the DRE motif of the human apoA-I gene also showed no
competition with the apoA-I-DRE probe (Fig. 4). Results of these
competition studies are summarized in Table I.
Fig. 4. Competitive gel mobility shift studies. The specificity of the drug-inducible nuclear proteins toward the oligo-apoA-I-DRE was determined by comparative competition experiments using unlabeled synthetic oligo apoA-I-DRE, oligo-P-450(I), oligo-P-450(II), oligo-AhR, oligo-AP1, oligo-AP2, oligo-Sp1, and oligo-M1. The sequences of these oligonucleotides are described in Table I. Gel mobility shift assays were performed by using constant amounts of nuclear proteins (5 µg) isolated from gemfibrozil-treated Hep3B cells and labeled oligo-apoA-I-DRE (25 ng) as well as various amounts of unlabeled oligonucleotides as indicated in the figure. The concentrations of the competitors are in molar excess, relative to the concentration of the labeled DNA. The bands representing specific protein-DNA complexes were identified by autoradiography, excised from the gel, and quantitated by Cerenkov counting. 32P-Labeled oligo-apoA-I-DRE was also incubated with with nuclear extracts from control Hep3B cells. Similar results were also observed when nuclear extracts isolated from HepG2 cells were used (data not shown). [View Larger Version of this Image (22K GIF file)]
Measurement of Relative Binding Affinity of the Drug-inducible Nuclear Proteins for Oligo-DRE The relative DNA binding affinity
of oligo-DRE was measured by saturation binding assays using gel
mobility shift analysis in which constant amounts of nuclear extracts
were titrated with increasing amounts of labeled oligo-DRE (Fig.
5). The data were plotted by the method of Scatchard
(38). The drug-inducible nuclear proteins bound to the DRE of the human
apoA-I gene with an apparent Kd of 4.1 nM.
Fig. 5. Affinity measurement of gemfibrozil-inducible nuclear proteins for the oligo-apo AI-DRE. The relative DNA binding affinity of oligo-DRE was measured by saturation binding assays using gel mobility shift analysis in which a constant amount (5 µg) of nuclear extract isolated from gemfibrozil-treated Hep3B cells was titrated with increasing amounts (0-500 fmol) of labeled oligo-apoA-I-DRE. The drug-inducible nuclear proteins bound to the oligo-DRE with an apparent Kd of 4.1 nM as determined by Scatchard analysis. Similar results were observed in three separate experiments. [View Larger Version of this Image (43K GIF file)]
Methylation Interference Analyses To more precisely localize
the contact sites of nuclear factors to the DRE region, methylation
interference studies were performed. Protein binding was impaired by
premethylation of guanines in any of seven positions: Fig. 6. Methylation interference footprinting analysis of the human apoA-I gene DRE. End-labeled, partially methylated synthetic oligo-DRE ( 79 to 44, coding or noncoding
strand) was incubated with nuclear extracts isolated from
gemfibrozil-treated Hep3B cells (Gemfibrozil) or untreated
cells (Control). Complexed and free fractions were separated
on a nondenaturing low ionic strength mobility shift assay gel. Bound
and free DNA fractions were eluted from the 5% acrylamide gel prior to
piperidine cleavage at methylated sites. Products were then separated
on a 20% sequencing gel. Methylated regions that interfered with
protein binding are indicated, and binding is summarized at the
bottom. Lane G + A indicates Maxam and Gilbert G + A reaction. *, guanines methylated at the N7 position
(major groove); , adenines methylated at the N3 position
(minor groove). Similar results were observed in three independent
experiments.
[View Larger Version of this Image (36K GIF file)]
Southwestern Blot Analyses To further characterize the
nuclear factors that bind to the oligo-DRE, Southwestern analyses were
carried out. As indicated by a solid horizontal bar beside
lane 2 in Fig. 7, the labeled oligo-DRE bound
to a protein band of approximately 28 kDa in size. This band appeared
to be inducible by gemfibrozil treatment, since no such band was
detectable in the nuclear extracts of control Hep3B cells. The
32P-labeled oligo-DRE also bound to several proteins that
were present in both gemfibrozil-treated and untreated samples. Two of
these proteins have molecular masses of approximately 68 and 42 kDa,
indicated by an open square and a star,
respectively (Fig. 7). These two protein bands gave similar signals on
the x-ray autoradiograph. Therefore, they could be used as internal
controls for protein loading. In addition, three other protein bands
with molecular masses of 30 kDa (open circle), 15 kDa
(open triangle), and 14.5 kDa (solid square) were
also induced by gemfibrozil treatment.
Fig. 7. Southwestern blot analyses. Nuclear proteins were isolated from Hep3B cells cultured in the absence (C) or presence of gemfibrozil (G), fractionated by 10% SDS-polyacrylamide gel electrophoresis, transferred onto an Immobilon P membrane, and probed with 32P-labeled oligo-DRE (lanes 1 and 2), oligo-P-450(I) (lanes 3 and 4), oligo-P-450(II) (lanes 5 and 6), and oligo-AhR (lanes 7 and 8), respectively. Molecular weight markers are indicated by solid lines beside lane 1. Similar results were observed in three separate studies. [View Larger Version of this Image (60K GIF file)]
To study the specificity of these drug-inducible protein bands toward the oligo-DRE, Southwestern blot analyses were performed using 32P-labeled oligo-P-450(I), oligo-P-450(II), and oligo-AhR separately. It was observed that two protein bands with masses of approximately 30 and 15 kDa could bind to oligo-P-450(I) and oligo-P-450(II) (Fig. 6, lanes 3-6). Furthermore, these two bands were highly inducible upon gemfibrozil treatment. It was also noteworthy that no nuclear proteins from control and gemfibrozil-treated cells interacted with oligo-AhR (Fig. 7, lanes 7 and 8). The data demonstrated that the drug-inducible bands bound specifically to the DRE consensus sequence. Functional Analysis of ApoA-I-DRETo analyze the role of
apoA-I-DRE in the regulation of human apoA-I gene expression in
response to gemfibrozil, we carried out transient transfection
experiments using a series of pGL2-derived luciferase reporter
plasmids. The construction of these plasmids is described in detail
under ``Experimental Procedures.'' Plasmids were transfected into
Hep3B cells in the absence or presence of gemfibrozil (40 µg/ml), and
luciferase activity was measured. As shown in Fig.
8A, both pGL2 (apoA-I Fig. 8. A schematic representation of pGL2-apoA-I and pGL2-apoA-I-DRE TK/luciferase reporter gene constructs and analysis of luciferase activity in transfected Hep3B cells. Diagrams at left represent the pGL2-apoA-I-luc chimeric construct series that contain sequences 491 to +1 and 250 to +1 of the human apoA-I
proximal promoter region (panel A). Panel A also
shows a schematic representation of the pGL2 (apoA-I 250 mutant DRE).
This construct contains an internal mutation in which the 10 nucleotides involved in possible contact with nuclear factors were
substituted (G T, C A, T G, and A C). The pGL2-TK-luc
series are constructed with synthetic DRE or mutated DRE (in which the
last eight nucleotides of each DRE consensus sequence were mutated: G
T, C A, T G, and A C) in front of TK promoter
(panel B). Construction of these plasmids is described under
``Experimental Procedures.'' Freshly seeded Hep3B cells were
transfected with the reporter plasmid and -galactosidase as internal
control to normalize for differences in transfection efficiency. Cells
were then cultured in the absence or presence of gemfibrozil (40 µg/ml) for 24 h prior to harvesting. The graph on the
right represents relative luciferase activity in transfected
cells cultured in the absence (control) or presence of
gemfibrozil. Luciferase activities of the constructs are expressed
relative to that of pGL2 promoter vector in panel A and pGL2
TK/luc in panel B, respectively. Results are mean ± S.E. for six independent experiments carried out in triplicate. **,
significantly different from control (p < 0.001, two-tail t test). The above noted plasmids were also
transfected into HepG2 cells. Similar relative luciferase activities of
the constructs were also observed in HepG2 cells compared with Hep3B
cells (data not shown).
[View Larger Version of this Image (33K GIF file)]
In order to further demonstrate that the DRE was able to confer responsiveness to gemfibrozil, transient transfection experiments were carried out using a series of pGL2 TK/luc constructs. The control vector pGL2TK/luc demonstrated no change in luciferase activity in response to gemfibrozil treatment (Fig. 8). To determine if binding of nuclear factors to the DRE is necessary for gemfibrozil-mediated induction of apoA-I gene expression, one copy of synthetic DRE oligonucleotide was inserted upstream of the thymidine kinase promoter, and the construct was tested for inducibility by gemfibrozil. After transient transfection of these constructs into Hep3B cells, pGL2 (apoA-I DRE)TK/luc showed luciferase activity approximately 4-fold higher than that observed by the control vector pGL2 TK/luc. This suggests that the DRE can act as an enhancer that may increase expression of the reporter gene. Furthermore, the luciferase activity of pGL2 (apoA-I-DRE)TK/luc was significantly increased by an additional 2-fold in the presence of gemfibrozil. However, a plasmid containing one copy of the mutated apoA-I-DRE oligomer, pGL2 (apoA-I-mutant DRE)TK/luc, lost the enhancer ability and responsiveness to gemfibrozil treatment (Fig. 8). Previously, we have demonstrated that exposure of two cultured human hepatoma cell lines, Hep3B and HepG2, to gemfibrozil resulted in a 2-fold induction in apoA-I mRNA level (11). Based on the results of the present report, the increase in apoA-I mRNA level upon gemfibrozil treatment is due primarily to increased rates of transcription of this gene. First, in actinomycin D-treated cells, the half-life of apoA-I mRNAs was not significantly affected by gemfibrozil treatment (Fig. 1). Second, both in vitro nuclear run-off transcription assays (Fig. 2) and transient transfection experiments (Fig. 8) indicated a significant 2-fold increase in apoA-I gene expression in the presence of gemfibrozil. In the presence of actinomycin D, the half-life of apoA-I mRNA was 5.6 ± 0.5 h in control Hep3B cells, in agreement with our previous studies, which measured apoA-I mRNA half-life by pulse-chase methods (25). By contrast, a recent report by Vandenbrouck and co-workers (39) has shown that the half-life of apoA-I mRNA is about 15 h in control HepG2 cells. Consequently, we also have performed kinetic studies in the presence of actinomycin D to provide a measurement of apoA-I mRNA half-life in HepG2 cells. Our results indicated that in HepG2 cells, apoA-I mRNA degraded with a half-life of 12.0 ± 1.5 h.2 Thus, the data suggest that the rate of degradation of apoA-I mRNA is at least 2-fold faster in control Hep3B cells compared with control HepG2 cells. The difference between the half-lives in these two cell lines might partially explain the 2-fold higher steady state level of apoA-I mRNA in control HepG2 cells compared with control Hep3B cells (11). Recently, many studies have been focussed on the use of fibric acid
derivatives to treat diet-resistant hyperlipidemic patients (40, 41, 42, 43, 44).
Fibrates are used widely to lower plasma cholesterol and triglyceride
levels as a treatment for coronary heart disease. However, these
compounds also cause peroxisome proliferation and, in some cases,
hepatocarcinogenesis in rodents (45, 46). It has been proposed that
fibrates and other peroxisome proliferators activate a member of the
steroid hormone receptor superfamily, the peroxisome
proliferator-activated receptor (PPAR) (47, 48, 49, 50, 51). The first PPAR was
isolated from a mouse liver cDNA library by Isseman and Green (47).
It appears that one of the physiological roles of PPAR is to regulate
fatty acid homeostasis. PPAR binds to a specific response element
(PPRE) located upstream of the target genes (48, 52, 53, 54, 55), which
consists of an almost perfect direct repeat of the sequence TGACCT
separated by one base pair. PPAR binds to these PPREs through
heterodimerization with the 9-cis-retinoic acid receptor, retinoid X
receptor Studies from both humans and rodents indicate that gemfibrozil causes
an increase in the levels of plasma HDL and/or apoA-I (10, 40, 66, 67, 68, 69).
Saku et al. (66) observed a 36% increase in HDL cholesterol
and a 29% rise in apoA-I levels when their patients with familial
hypertriglyceridemia were treated with gemfibrozil. This was
accompanied by an increase in the synthetic rate of apoA-I by 27% with
no change in the fractional clearance rate. The results from our
studies support, in part, Saku's findings and demonstrate that
gemfibrozil may act by selectively increasing expression of the apoA-I
gene in human liver. Elucidation of the mechanism by which gemfibrozil
modulates apoA-I gene expression requires the identification of
promoter elements and transcription factors responsible for mediating
the biological response to these agents. We have identified two copies
of the DRE in the apoA-I promoter that may be responsible for the
increase in apoA-I transcriptional activity by gemfibrozil. These two
decanucleotide elements, spaced 10 nucleotides apart in an inverted
repeat, are located between nucleotides The competition mobility shift experiments presented in Fig. 4 shows that the nuclear factor(s) that bind(s) apoA-I-DRE is highly sequence-specific. A 25-fold molar excess of a double-stranded oligonucleotide homologous to apoA-I-DRE competed strongly for binding with the 32P-labeled apoA-I-DRE probe, while an equivalent molar excess of mutated apoA-I-DRE resulted in virtually no competition with the apoA-I-DRE probe. Furthermore, the DRE consensus sequence of CYP1A1 (P-450(I) and P-450(II)) competed efficiently for factor binding to the apoA-I-DRE. However, no competition was observed with 200-fold molar excess of the Sp1 consensus sequence or the xenobiotic consensus sequence for the AhR. It is worth noting that both the AP1 and AP2 consensus sequences compete efficiently for protein binding to the apoA-I-DRE. One possible explanation for this observation is that nuclear factor(s) that recognize(s) the apoA-I-DRE can also bind to the AP1 and AP2 consensus sequences. Alternatively, it is possible that the protein-DNA complex observed in the mobility shift assay consisted of multiple proteins of which AP1 and AP2 binding proteins are two of the potential candidates. Results from the present studies suggest that the former may be the more likely possibility. First, our methylation interference experiments indicated that the contact sites of the nuclear factors with the DRE region did not resemble those of the AP1 (73) or AP2 (74, 75) DNA recognition sites. Second, Williams and Tjian (74) have demonstrated that HepG2 cells express neither AP2 mRNA nor protein. Third, the two groups of gemfibrozil-induced nuclear proteins detected by Southwestern blot analyses have molecular masses of 15 and 30 kDa, whereas AP1 and AP2 proteins have molecular masses between 40 and 75 kDa (75). Finally, we have performed supershift experiments to determine whether c-Jun and c-Fos protein interact with the DRE binding site. For supershift assays, the nuclear extracts isolated from gemfibrozil-treated cells were preincubated with affinity-purified polyclonal antibodies raised against c-Jun and c-Fos (Oncogene Science) at 4 °C for either 2 h or 18 h before the standard gel mobility shift studies. Both sets of experiments showed no effect on the binding activity of DRE oligonucleotide to the gemfibrozil-treated nuclear extract when either anti-c-Jun or anti-c-Fos antibodies were used (data not shown). The specific protein binding to oligo-DRE was observed with nuclear extracts from gemfibrozil-treated cells but not from cytosolic extracts (data not shown). Consequently, it was not clear whether inducible binding was due to a drug-induced translocation of a preexisting factor from the cytosol to the nucleus or was attributable to its de novo synthesis. The results of inhibiting protein synthesis with cycloheximide suggest the latter is the case. At the moment, we are uncertain whether this involves synthesis of the factors themselves or of ancillary proteins or enzymes required for binding activity. In order to establish a functional role for the DRE in apoA-I gene
transcription in response to gemfibrozil treatment, we have carried out
transient transfection experiments using pGL2-derived luciferase
reporter plasmids. Constructs that contain nucleotides In conclusion, we have identified a group of gemfibrozil-inducible nuclear proteins that bind specifically to the DRE region of the human apoA-I gene. This protein-DNA interaction may provide some insights into a novel drug-response mechanism by which drug-inducible trans-acting nuclear factors modulate apoA-I gene expression. Further experiments will be required to further understand the relationship between the structure and the function of these induced nuclear proteins in the regulation of human apoA-I transcription. * This work was supported by grants from the Medical Research Council of Canada and by an Ontario Heart and Stroke Foundation Career Investigator Award (to S.-P. T.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 613-545-2826;
Fax: 613-545-6830.
1 The abbreviations used are: apoA-I, apolipoprotein A-I; HDL, high density lipoprotein; P-450, cytochrome P-450; AhR, aryl hydrocarbon receptor; MEM, minimal essential medium; FBS, fetal bovine serum; DTT, dithiothreitol; DRE, drug-responsive element; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator response element; bp, base pair(s); PCR, polymerase chain reaction; AP1 and AP2, activator proteins 1 and 2, respectively. 2 C. Cuthbert, Z. Wang, and S.-P. Tam, unpublished results. 3 S.-P. Tam and S. Green, unpublished results. We thank Sandra Caine and Marilyn McCallum for excellent secretarial skills and express our appreciation to Dr. R. G. Deeley for critical comments.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc. This article has been cited by other articles:
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