| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 275, Issue 7, 5214-5221, February 18, 2000
§,,
From the Department of Veterans Affairs Palo Alto
Health Care System, Palo Alto, California 94304 and
¶ Monsanto, St. Louis, Missouri 63017
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
The cytokine oncostatin M (OM) activates human
low density lipoprotein receptor (LDLR) gene transcription through a
sterol-independent mechanism. Previous studies conducted in our
laboratory have narrowed the OM-responsive element to promoter region
Previous studies of the human low density lipoprotein receptor
(LDLR)1 promoter identified a
177-base pair fragment of the 5'-flanking DNA from In addition to cholesterol, LDLR transcription can also be regulated by
nonsterol mediators, including hormones (10), growth factors (11),
cytokines (12), and second messengers such as cyclic AMP (13). However,
the cis-acting elements that control LDLR transcription
through sterol-independent pathways have not been identified.
To elucidate the molecular mechanisms underlying sterol-independent
regulation, we previously dissected the promoter region that could be
responsible for cytokine oncostatin M (OM)-induced activation of LDLR
transcription (15), as OM has been shown to increase the expression of
LDLR protein and mRNA independent of intracellular cholesterol
(14). We have shown that mutations of the SRE-1 site or deletion of the
repeat 2 sequence to completely eliminate SRE-binding protein binding
has no effect on OM inducibility of LDLR promoter activity (16).
Deletion analysis further narrowed down the OM-responsive element in
the promoter region from In this study, we performed detailed mutagenic analysis in this
promoter region to identify the cis-regulatory element that mediates the OM activity of the LDLR promoter. We provide the first
evidence to demonstrate that OM activity is mediated through promoter
region Cells and Reagents--
The human hepatoma cell line HepG2 was
obtained from American Type Culture Collection (Manassas, VA) and was
cultured in Eagle's minimal essential medium supplemented with 10%
fetal bovine serum (Summit Biotechnology, Fort Collins, CO). Antibodies
specific to the following proteins were obtained from Santa Cruz
Biotechnology for use in supershift EMSAs: C/EBP Plasmid Vectors--
Construction of plasmids pLDLR234,
pLDLR-R3, and pLDLR-TATA has been described previously (17). To
construct the mutant vectors (pLDLR-R3-mu6 to pLDLR-R3-mu11),
double-stranded oligonucleotides containing the LDLR promoter sequence
from
The vector pLDLR-GAL4 was constructed by cloning a double-stranded
oligonucleotide containing the LDLR promoter sequence from
The plasmid pSG-GAL4-Sp1 encoding the yeast transcription factor GAL4
DNA-binding domain and the N-terminal glutamine-rich activation domain
of Sp1 and pSG-GAL4 DBD encoding the GAL4 DNA-binding domain only (18)
were kindly provided by Dr. Stephen Smale (UCLA). The plasmid pFA-GAL4
encoding the full-length GAL4 protein was generously provided by Dr.
ChaoFeng Zheng (Stratagene). The plasmid pEF-NFIL6 was a gift from Dr.
Shizuo Akira (Hyogo College of Medicine, Hyogo, Japan).
Site-directed Mutagenesis--
The plasmid pLDLR234 was used as
a template for making mutants utilizing the QuikChangeTM
site-directed mutagenesis kit (Stratagene). Correct clones were screened by restriction digestion and verified by dideoxy sequencing.
Preparation of Nuclear Extracts--
Nuclear extracts were
prepared by the method of Dignam et al. (19), except that
buffer A was supplemented with 1 mM
Na3VO4, 1 mM NaF, 1 mM
Na2MoO4, 1 mM Electrophoretic Mobility Shift Assays--
Oligonucleotide
probes were annealed and labeled by 3'-fill-in using Klenow fragment in
the presence of [
The sense sequences of EMSA probes were as follows: TATA1+2,
5'-CATTGAAATGCTGTAAATGACGTGGGCCCC-3'; repeat 3, 5'-TTCGAAACTCCTCCCCCTGCTAG-3'; and p53,
5'-AGCTAGGGCTTGCTTGAACAGGGTCT-3'. The Sp1- and p53-binding sites are underlined.
Transient Transfection Assays--
HepG2 cells were transiently
transfected with plasmid DNA by the calcium phosphate coprecipitation
method (16). To demonstrate sterol-independent regulation, cells
transfected with pLDLR234 or mutant vectors were cultured in medium
containing 10% lipoprotein-depleted serum (LPDS) or LPDS plus
cholesterol (10 µg/ml cholesterol + 1 µg/ml 25-hydroxycholesterol).
Twenty hours after transfection, OM (50 ng/ml) or dibutyryl cAMP at the
indicated concentrations was added. After 4 h of treatment, cells
were lysed, and luciferase and Statistical Analysis--
Comparisons of experimental data were
analyzed by a two-tailed Student's t test. A p
value < 0.05 was considered to indicate a statistically
significant difference.
Our previous study showed that the minimal LDLR promoter construct
pLDLR-R3 responds to OM stimulation to a degree similar to that of the
full promoter construct pLDLR234, suggesting that the OM-responsive
element is localized within promoter region 
52 to +13, which contains the repeat 3 and two TATA-like sequences.
We now identify LDLR promoter region 17 to 1 as a
sterol-independent regulatory element (SIRE) that is critically
involved in OM-, transcription factor CCAAT/enhancer-binding protein
(C/EBP)-, and second messenger cAMP-mediated activation of LDLR
transcription. The SIRE sequence overlaps the previously described
TATA-like element and consists of an active C/EBP-binding site (17 to
9) and a functional cAMP-responsive element (CRE) (8 to 1). We
demonstrate that (a) mutations within either the C/EBP or
CRE site have no impact on basal or cholesterol-mediated repression of
LDLR transcription, but they completely abolish OM-mediated activation
of LDLR transcription; (b) replacing the repeat 3 sequence that
contains the Sp1-binding site with a yeast transcription factor
GAL4-binding site in the LDLR promoter construct does not affect OM
inducibility, thereby demonstrating that OM induction is mediated
through the SIRE sequence in conjunction with a strong activator bound
to the repeat 3 sequence; (c) electrophoretic mobility
shift and supershift assays confirm the specific binding of
transcription factors C/EBP and cAMP-responsive element-binding protein
to the SIRE; (d) cotransfection of a human C/EBP
expression vector (pEF-NFIL6) with the LDLR promoter construct pLDLR234
increases LDLR promoter activity; and (e) OM and dibutyryl
cAMP synergistically activate LDLR transcription through this
regulatory element. This study identifies, for the first time, a
cis-acting regulatory element in the LDLR promoter that is
responsible for sterol-independent regulation of LDLR transcription.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
142 to +35,
relative to the major transcription start site, that is sufficient for
controlling basal transcription as well as negative feedback regulation
by cholesterol and its derivatives (1-3). The positive regulatory
elements within this region were identified as three GC-rich imperfect
16-base pair direct repeats and two TA-rich TATA-like sequences of 7 base pairs each. Repeats 1 and 3 contain transcription factor
Sp1-binding sites. Interference in Sp1 binding to either repeat
severely decreases basal transcriptional activity. Sterols regulate
LDLR transcription through a 10-base pair sequence within repeat 2 designated as sterol regulatory element-1 (SRE-1) (4, 5). When
intracellular cholesterol is low, SRE-binding protein-1 and -2 bind to
the SRE-1 sequence and interact with Sp1 in repeat 3, thereby
activating LDLR transcription (6-9). To date, no studies have been
conducted to investigate further how the TATA-like sequences affect
LDLR transcription, and the trans-acting factors that bind
to this promoter region have not been identified.
52 to +13 (17). This region contains the two
TATA-like sequences and repeat 3.
17 to 1 in conjunction with the strong activator Sp1 bound
to the repeat 3 sequence. We have designated this promoter region (17
to 1) as a sterol-independent regulatory element (SIRE). The SIRE
overlaps with the previously described TATA-like sequences and consists
of an active CCAAT/enhancer-binding protein (C/EBP)-binding site (17
to 9) and a functional cAMP-responsive element (CRE) (8 to
1).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, C/EBP,
C/EBP
, CREB-1, CREB-2, CREM-1, CREB/ATF-1 (recognizes all CREB/ATF
family proteins), ATF-1, ATF-2, c-Jun, JunB, c-Fos, XBP-1, CREB-binding
protein, and TATA-binding protein.
52 to +13 with single-base substitutions were synthesized with
SacI and HindIII sites at the 5'- and 3'-ends,
respectively, and inserted into pGL3-basic vector. The mutant sequences
are shown in Fig. 1.
36 to +13
and a GAL4-binding site with SacI and
HindIII sites (italic) at the 5'- and 3'-ends
(CtcggagtactgtcctccgatcgtagaaacctcacattgaaatgctgtaaatgacgtgggccccgagtgcaatA) into pGL3-basic vector. The GAL4 sequence is underlined. The
vector pLDLR-GAL4-TATA2MU was constructed as described above with the exception that the TATA2 site contains a 3-base substitution (TGTAAA 
TGcggA).
-glycerophosphate,
10 nM okadaic acid, 10 nM cypermethrin, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 µg/ml pepstatin A, and 2 mM dithiothreitol.
-32P]dCTP. Each binding reaction was
composed of 10 mM HEPES, pH 7.8, 2 mM
MgCl2, 2 mM dithiothreitol, 80 mM
NaCl, 10% glycerol, 1 µg of poly(dI-dC), 1 µg of bovine serum
albumin, and 6 µg of nuclear extract in a final volume of 20 µl.
Nuclear extracts were incubated with 0.4-0.5 ng of
32P-labeled double-stranded synthetic oligonucleotide probe
(40-80 × 103 cpm) for 10 min at room temperature.
The reaction mixtures were loaded onto a 5% polyacrylamide gel and run
in TGE buffer (50 mM Tris base, 400 mM glycine,
and 1.5 mM EDTA, pH 8.5) at 180 V for 3 h at 4 °C.
The gels were dried and visualized on a PhosphorImager. In competition
analysis, nuclear extracts were incubated with a 2-200-fold molar
excess of unlabeled competitor DNA for 5 min prior to the addition of
the labeled probe. For supershift assays, antibody was incubated with
nuclear extract for 30-60 min at room temperature prior to the
addition of the probe.
-galactosidase activities were
assayed. Absolute luciferase activity was normalized against
-galactosidase activity to correct for transfection efficiency.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
52 to +13, which contains
the repeat 3 and two TATA-like sequences (17). To firmly examine the
relationship between Sp1 binding to the repeat 3 sequence and OM
inducibility, several mutant vectors with single-base substitutions
(R3-mu6 to R3-mu11) within the core Sp1-binding site (CTCCTCCC) were
constructed by insertion of double-stranded oligonucleotides containing
LDLR promoter sequence 52 to +13 with designed mutations into the
pGL3-basic-luciferase vector. These reporters, along with the
-galactosidase expression vector BG2-gal, were transiently
transfected into HepG2 cells. Fig.
1A illustrates a
representative experiment that shows the normalized luciferase
activities of untreated and OM-treated HepG2 cells transfected with
each individual vector. These data show that pLDLR-TATA had very low
luciferase activity at a level similar to that of the promoterless
vector pGL3-basic. The effect of OM on this vector was insignificant,
generally <5% of the OM activity on the vector pLDLR-R3. Including a
single copy of repeat 3 sequence in the pLDLR-TATA vector increased
promoter activity ~4-5-fold, and vector pLDLR-R3 responded to OM
stimulation with a 3-fold increase in promoter activity. The effects of
repeat 3 mutation on basal and OM-induced promoter activities are
presented in Fig. 1 (B and C) using data derived
from five independent transfections. Fig. 1B compares the
basal promoter activities of all repeat 3 mutants. The response of each
mutant to OM stimulation is illustrated in Fig. 1C. With the
exception of R3-mu9, which did not change basal or OM-induced promoter
activity, all other mutants reduced basal promoter activity nearly to
the level of pLDLR-TATA. However, none of these mutations abolished OM
activity completely. These data suggest that Sp1 binding to the repeat
3 sequence is required for OM-induced activation of LDLR transcription,
but the repeat 3 sequence may not be the only cis-regulatory
element in this promoter region (52 to +13) to mediate OM
activity.
View larger version (25K):
[in a new window]
Fig. 1.
Effects of mutations of the repeat 3 sequence
on Sp1-mediated basal promoter activity and on OM-stimulated promoter
activity. HepG2 cells were transfected with wild-type pLDLR-R3
(R3-wt) and the mutant vectors pLDLR-R3-mu6 to pLDLR-R3-mu11
along with a -galactosidase expression vector, BG2-gal. After
transfection, cells were cultured in Eagle's minimal essential medium
containing 10% fetal bovine serum for 20 h prior to treating
cells with OM at a concentration of 50 ng/ml for 4 h. Assays of
luciferase and -galactosidase activities were conducted as described
(16). A, one representative experiment in which triplicate
wells were assayed; B and C, summarized results
of five independent transfections. In B, the normalized
basal luciferase activity of each vector is expressed as the percent of
the luciferase activity of the pLDLR-TATA vector. In C, the
response of each mutant vector to OM stimulation was compared with the
response of the wild-type vector (pLDLR-R3) to OM.
Gel shift assays using HepG2 nuclear extract and a
32P-labeled oligonucleotide containing the repeat 3 sequence were conducted to examine the Sp1 binding to the mutant repeat
3 sequences. As shown in Fig. 2, Sp1 and
Sp3 binding to the probe was competed by the double-stranded
oligonucleotides containing either the wild-type repeat 3 sequence or
the mutant sequence in R3-mu9, but it was not competed by the
oligonucleotides that carry the same mutations as those in the vectors
of R3-mu6, R3-mu7, R3-mu8, R3-mu10, and R3-mu11. These data clearly
demonstrate that loss of the basal promoter activities of these mutants
was due to loss of Sp1 binding.
|
To examine the functions of the DNA sequence 3' of repeat 3 in basal
and OM-stimulated transcription, site-directed mutagenesis was
conducted on the pLDLR234 vector to generate mutations within the two
TATA-like sequences, TATA1 (23 to 17) and TATA2 (14 to 8), and
a putative CRE (8 to 1) that overlaps with TATA2. Fig.
3 shows that none of the mutations
affected cholesterol regulation; promoter activities of all of the
constructs were repressed by sterol to a similar degree. However, OM
inducibility was completely abolished in the TATA2 mutant (TATA2a) and
the CRE mutant (CREMU1) and only partially reduced in the TATA1 mutant
(TATA1b).
|
Computer-aided sequence analysis suggested that the TATA-like sequences
contain a potential C/EBP-binding site (TGCTGTAAA, 17 to 9) that
includes the 3'-base pair of TATA1 and the TATA2 sequence except the
most 3'-base pair of TATA2, which is included in the putative CRE site
(Table I). To further demonstrate that OM
activity is mediated through the putative C/EBP site and the CRE site,
several reporter vectors with altered nucleotides in the 5'-region of
the C/EBP site (TATA1a) and the 3'-region of the CRE site (TATAMU3d and
TATAMU3e) were constructed, along with two additional mutants of the
C/EBP site (TATA2b) and the CRE site (TATAMU3c). The results are
summarized in Table I. The mutation in TATA1a with base substitutions
in the 5'-end of TATA1 but outside the C/EBP site lowered basal
activity to 59% of the wild-type level, but had no effect on OM
inducibility. In comparison, the mutation in TATA1b lowered basal
promoter activity to 69% of the wild-type level and also slightly
decreased OM activity from 4- to 2.9-fold. This is most likely due to
the A-to-G substitution immediately adjacent to the C/EBP-binding site.
These data suggest that the TATA1 site contributes to basal promoter
activity, but is dispensable for the OM response. In contrast, basal
promoter activities were unchanged in C/EBP mutants TATA2a and TATA2b
and in the CRE mutant CREMU1 and only slightly decreased in the CRE mutant TATAMU3c, but OM stimulation in these mutant vectors was completely abolished. Farther downstream mutations in the 3'-region of
the CRE site slightly decreased basal promoter activity (73-83% of
the wild-type level) without affecting OM-induced promoter activity.
These data clearly localized a novel SIRE in promoter region 17 to
1 that is critically involved in the OM-mediated activation of LDLR
transcription.
|
To determine whether Sp1 binding to repeat 3 is necessary for OM to
function through the SIRE, the repeat 3 sequence in pLDLR-R3 was
replaced by a GAL4-binding site to create the vector pLDLR-GAL4, and
the repeat 3 sequence in a TATA2 mutant of pLDLR-R3 was replaced by a
GAL4 site to generate pLDLR-GAL4-TATA2MU. These two reporter constructs
were cotransfected with expression vectors that express the GAL4
DNA-binding domain only (GAL4 DBD), the full-length GAL4 protein
(GAL4), or a fusion protein containing the GAL4 DNA-binding domain and
the Sp1 activation domain (GAL4-Sp1). The results in Fig.
4 show that the promoter activities of
both wild-type pLDLR-GAL4 and mutant pLDLR-GAL4-TATA2MU were increased
by cotransfection with transcription factor GAL4 or the GAL4-Sp1 fusion
protein, but were not affected by cotransfection with the GAL4 DBD,
which does not contain a transactivation domain. OM did not increase wild-type promoter activity with cotransfection of the GAL4 DBD. However, OM increased the pLDLR-GAL4 promoter activities to the same
extent (2-3-fold) with cotransfection of either GAL4 or GAL4-Sp1. This
OM activity was totally abolished in the promoter construct pLDLR-GAL4-TATAMU2, which carries a mutation in the SIRE site. These
results clearly demonstrate that the OM activity of LDLR transcription
is mediated through the SIRE site in conjunction with a strong
activator bound to the repeat 3 sequence.
|
To detect transcription factors that bind to the SIRE, gel shift assays
using a 32P-labeled oligonucleotide probe (TATA1+2)
containing LDLR promoter sequence 25 to +5 were performed with
nuclear extracts prepared from untreated and OM-treated HepG2 cells.
Three DNA-protein complexes (C1, C3, and C4) were detected in control
extracts, and 4 complexes (C1, C2, C3, and C4) were detected in
OM-treated extracts (Fig. 5A).
Formation of these complexes was inhibited by a 100-fold molar excess
of the unlabeled oligonucleotide TATA1+2, but was not inhibited by a
100-fold molar excess of oligonucleotides containing binding sites for
Sp1 (repeat 3) or p53, demonstrating the specificity of the binding. In
contrast to the TATA1+2 probe, the binding of Sp1 to the
32P-labeled repeat 3 probe was not altered by OM treatment
(Fig. 5B). Fig. 6 shows the
time-dependent induction of the C2 complex by OM. C2 was
detected in OM-treated cells after 30 min, and the intensity of C2
appeared to increase with longer OM treatment. Interestingly, the
OM-induced appearance of the C2 complex on the SIRE element was totally
inhibited by treating cells with U0126, an inhibitor of the
extracellular signal-regulated kinase (ERK) upstream kinase MEK (20).
These data suggest that the binding of C2 to SIRE site depends on the
ERK-induced phosphorylation of protein components of C2.
|
|
Supershift EMSA analyses with antibodies directed against several
members of the C/EBP and CREB families were conducted to characterize
the transcription factors present in these DNA-protein complexes. The
C3 complex in both control extracts (Fig.
7A) and OM-treated extracts
(Fig. 7B) was completely supershifted by anti-CREB-1 (lanes 2 and 3) and anti-CREM-1 (lane
8) antibodies. Antibodies against ATF-1 (lane 4) and
ATF-2 (lane 5), two other members of the CREB family,
supershifted the C1 complex and also slightly decreased the intensity
of C3. Anti-C/EBP antibody produced a supershift band and slightly
decreased the intensity of C3 in control extracts (lane 9).
In OM-treated extracts, the intensities of C3 and OM-induced C2 were
both slightly decreased by anti-C/EBP antibody. Anti-C/EBP
antibody produced a supershift band only in the OM-treated extracts
(Fig. 7B, lane 11), whereas anti-C/EBP antibody generated a faint supershift band in both control and OM-treated extracts (lane 10). In comparison with the
control extract, the intensities of supershifted bands produced by
antibodies against ATF-1 (Fig. 7B, lane 4) and
ATF-2 (lane 5) with OM-treated extract were decreased. In
contrast, antibodies against CREB-binding protein (lane 6),
CREB-2 (lane 7), and the TATA-binding protein and
TATA-binding protein-associated factors TAF130 and TAF100 (data not
shown) had no effect.
|
It is known that the consensus binding site (TGACT) for activator
protein-1 (AP-1), which is composed of a c-Jun homodimer or a c-Jun and
c-Fos heterodimer, overlaps with the CRE site (TGACGTCA) (21, 22).
Since OM has been shown to stimulate AP-1 activity (23), we were
interested to determine whether the OM-induced C2 complex contains
AP-1. Supershift assays with antibodies that recognize c-Jun, JunB,
c-Fos, and XBP-1 were performed using OM-treated nuclear extract. XBP-1
is another member of the leucine zipper family of transcription factors
that also binds to the CRE, and XBP-1 was shown to be regulated by
interleukin-6 (24). Fig. 7C shows that antibodies to c-Fos,
JunB, and XBP-1 had no effect on any of the four complexes. All three
antibodies to c-Jun supershifted the C1 complex, but none of these
anti-c-Jun antibodies affected the C2 complex. These data suggest that
the C1 complex may be a heterodimer of ATF and c-Jun; c-Jun is not
present in the C2 complex. Additional gel shift assays using control
extract detected a weaker supershift band with anti-c-Jun antibodies as
compared with that seen in OM-treated extract (data not shown),
suggesting that OM stimulation may increase the c-Jun DNA-binding
activity. Together, these supershift EMSA analyses suggest that 1) C3
is predominantly composed of homodimers and/or heterodimers of members of the CREB family (C3 may also contain a small amount of C/EBP); 2)
C1 may contain heterodimers between ATF and c-Jun; 3) C4 contains transcription factors that do not appear to belong to the CREB or C/EBP
family; and 4) the nature of the C2 complex is presently unknown. C2
may contain a novel factor, as none of the antibodies that recognized
most, if not all, transcription factors that could bind to the SIRE
sequence significantly affected this complex.
To determine the relationship between C/EBP binding and promoter
activity, we cotransfected HepG2 cells with pLDLR234 and pEF-NFIL6,
which encodes NFIL-6, a human homologue of rat C/EBP (25, 26). Fig.
8 shows that under cholesterol-replete
conditions, pEF-NFIL6 increased LDLR promoter activity in a
dose-dependent manner. At a low concentration of pEF-NFIL6,
the stimulatory effect of OM was diminished, whereas at a higher
concentration of pEF-NFIL6 that produced a >10-fold increase in basal
promoter activity, OM stimulation was abrogated. We speculate that the
loss of response to OM stimulation is through saturation of the binding
site (SIRE) on the LDLR promoter construct due to the overexpression of
NFIL-6. These results, together with the EMSA data presented in Fig. 7, suggest that the C/EBP site within this promoter region may be a
functional cis-acting element that positively regulates LDLR transcription.
|
Takagi and Strauss (13) have reported that 8-bromo-cAMP increases LDLR
mRNA without stimulating LDLR promoter activity. This led to the
conclusion that the upstream promoter of the human LDLR gene does not
contain a classical CRE. To examine the function of the identified CRE
site (8 to 1) in the regulation of LDLR transcription, HepG2 cells
were transfected with the full-length LDLR promoter construct pLDLR234
(143 to +35) or a reporter containing only the TATA-like element,
pLDLR-TATA (36 to +13) (17). Fig. 9A shows that dibutyryl cAMP
by itself is a weak stimulator of LDLR transcription, only increasing
the luciferase activity of pLDLR234 by 2-fold without a significant
effect on pLDLR-TATA at concentrations up to 5 mM. However,
in the presence of OM, luciferase activity was markedly increased up to
8-fold for the full-length promoter and up to 3-fold for the minimal
promoter construct in a dibutyryl cAMP dose-dependent
manner. This synergistic effect of cAMP and OM depends on the presence
of both intact C/EBP and CRE sites, as mutation in either site greatly
reduced the synergistic activation of the LDLR promoter (Fig.
9B). These data suggest that interaction of activated CREB
through protein kinase A-induced phosphorylation with an OM-activated
transcription factor(s) leads to the formation of a more robust
transcriptional activator complex.
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
A number of studies have demonstrated the modulated expression of LDLR by nonsterol regulators that are structurally and functionally distinct from cholesterol and its derivatives. In spite of the demonstration that most of these nonsterol mediators such as cytokines and growth factors regulate LDLR expression at the transcriptional level, the molecular mechanisms for sterol-independent regulation are totally unknown, as neither the cis-regulatory elements on the LDLR promoter nor the trans-acting factors that mediate these regulations have been identified. In this study, we identified, for the first time, a SIRE in the human LDLR promoter that is responsive to the cytokine oncostatin M.
The OM-responsive element was previously narrowed down to promoter
region 52 to +13. This region contains the Sp1-binding site and the
two TATA-like sequences. To distinguish Sp1-mediated constitutive
promoter activity from OM-stimulated promoter activity, we made
single-base mutations across the core Sp1-binding site within the
repeat 3 sequence. These mutations revealed that OM activity was
diminished when Sp1 binding was abrogated, but was not concurrently
lost (Fig. 1). One explanation would be that OM induced a DNA-binding
protein to bind this region, and mutations affected this factor
differently than Sp1 binding. However, failure of the detection of an
OM-induced DNA-binding activity in the repeat 3 sequence excluded this possibility.
In searching for additional regulatory elements 3' of repeat 3, a total
of eight mutants that encompass the two TATA-like sequences were
constructed and examined for their effects on basal and OM-induced
promoter activities. The two mutants TATA1a and TATA1b, which targeted
the TATA1 sequence, lowered basal promoter activity to 60-70% of the
wild-type level without affecting OM activity, thereby suggesting that
this region is not involved in the action of OM, but may contribute to
basal promoter activity. We have conducted gel shift assays using a
probe containing the TATA1 sequence (30 to 13) to detect nuclear
proteins from HepG2 cells that could bind to the TATA1 sequence.
However, no DNA-protein complex was detected (data not shown).
Therefore, at present, it is not clear how the TATA1 sequence regulates
LDLR transcription. In contrast to the TATA1 mutations, the OM
inducibility of LDLR promoter activity was completely abrogated by
mutations within TATA2 and its 3'-flanking sequence containing a
potential CRE site. Additional mutations downstream from the CRE site
did not affect OM activity. Taken together, these eight mutants defined promoter region 17 to 1 as the cis-regulatory element
that is responsible for OM-induced activation of LDLR transcription. As revealed by a computer-aided sequence analysis, this promoter region
consists of continuous binding sites for C/EBP (17 to 9) and CREB
(8 to 1). Because this promoter region not only mediated the effect
of OM, but also responded to cAMP stimulation (Fig. 9), we designated
this promoter region as a SIRE.
Next, we were interested to know the relationship between the SIRE and the Sp1-binding site in repeat 3. To determine whether Sp1 binding to repeat 3 is necessary for the SIRE to respond to OM, we replaced repeat 3 in pLDLR-R3 with a GAL4-binding site to create vector pLDLR-GAL4. The basal promoter activity of pLDLR-GAL4 in HepG2 cells that did not express GAL4 or GAL4-Sp1 was very low and did not respond to OM stimulation in a way similar to pLDLR-TATA. OM stimulation was restored equally when the promoter was activated by coexpression of either full-length GAL4 protein or the fusion protein containing the GAL4 DNA-binding domain and the Sp1 activation domain in a manner similar to pLDLR-R3. The mutation in the TATA2 site of pLDLR-GAL4 totally abolished OM stimulation. The same results were obtained with coexpression of GAL4-VP16 fusion protein (data not shown). These results strongly suggest that regulation of LDLR transcription by OM is mediated through the SIRE site. The repeat 3 sequence is dispensable, but a binding site for a strong transcriptional activator in a location proximal to the SIRE is necessary for the SIRE to function as an independent cis-acting element to activate LDLR transcription.
To understand the molecular basis of the SIRE, gel shift and supershift assays were performed to characterize the transactivators that interact with this element. In HepG2 nuclear extract without OM treatment, three DNA-protein complexes (C1, C3, and C4) were formed with the TATA1+2 probe, which contains the SIRE site. The C1 and C3 complexes were shown to contain homodimers or heterodimers of members of the CREB and C/EBP families (Fig. 7A). CREB or C/EBP binds to the SIRE independently, as mutations abolishing the CRE site did not affect C/EBP binding, and conversely, CREB binds the SIRE probe containing a mutation within the C/EBP site (data not shown).
In OM-treated HepG2 nuclear extract, in addition to C1, C3, and C4, an additional complex (C2) was formed. We found that binding of the C2 complex to the TATA1+2 probe required the intact C/EBP and CRE sites because mutation in either site eliminated C2 binding to the probe (data not shown). This is in contrast to the independent interaction of CREB and C/EBP with the SIRE sequence. Moreover, induction of C2 by OM depends on ERK activation, as treating cells with OM in the presence of U0126, an inhibitor to the ERK upstream kinase MEK, totally prevented C2 formation without an effect on the other three complexes. Previously, we have shown that U0126 abolished the OM induction of endogenous LDLR mRNA and decreased the OM-stimulated promoter activity of pLDLR-R3 (17). Thus, the inhibitory effect of U0126 on the C2 complex strongly suggests that the SIRE is a cis-acting element that mediates the extracellular signal transmitted through the mitogen-activated protein kinase pathway to regulate LDLR transcription. We speculate that the C2 complex contains protein components that are substrates of ERK. The nature of the C2 complex is presently unknown, as gel shift assays with antibodies directed against members of the CREB, C/EBP, and AP-1 families; TATA-binding protein; TAF130; and TAF100 failed to supershift or abolish the C2 complex. These experiments suggest that C2 may contain a novel transcription factor. The identity of the C2 is currently under further investigation.
The involvement of AP-1 in LDLR transcription deserves further investigation. In supershift assays, although c-Jun was detected in the C1 complex, pure recombinant c-Jun could not bind to the TATA1+2 probe under similar binding conditions. Interestingly, when recombinant c-Jun was mixed with nuclear extracts prepared from control or OM-treated HepG2 cells, the intensity of C1 was increased, and anti-c-Jun antibodies completely supershifted the C1 complex (data not shown). These data suggest that the c-Jun homodimer is not able to bind to the SIRE, but c-Jun may dimerize with ATF and consequently bind to the SIRE. The relationship between phosphorylation of c-Jun and formation of the OM-induced C2 complex is currently under investigation.
In summary, we have identified the SIRE as a novel sterol-independent
cis-acting regulatory element in region 17 to 1 of the
human LDLR promoter. This element overlaps the previously described
TATA-like sequences. It contains contiguous binding sites for the
transcription factors C/EBP and CREB. The SIRE is not involved in the
classical feedback regulation by cholesterol, but is critically
involved in regulations mediated by oncostatin M, cAMP, and C/EBP. We
hypothesize that the SIRE acts to integrate different signaling
pathways controlling the transcription of the LDLR gene independent of
cellular cholesterol. These novel findings provide a molecular basis
for the mechanism of sterol-independent regulation of LDLR and link
extracellular signaling events to promoter elements on the LDLR gene.
This work opens up a new avenue for developing alternative
pharmaceutical interventions to lower plasma low density lipoprotein
cholesterol via a mechanism of action distinct from the
classical inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Cong Li and Charlotte L. Scholtz for technical assistance and Linda M. Boxer for discussion.
| |
FOOTNOTES |
|---|
* This work was supported by a merit review from the Department of Veterans Affairs.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: 154P, VA Palo Alto Health Care System, 3801 Miranda Ave., Palo Alto, CA 94304. Tel.: 650-493-5000 (ext. 64411); Fax: 650-849-0251; E-mail: liu@icon. palo-alto.med.va.gov.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: LDLR, low density lipoprotein receptor; SRE, sterol regulatory element; OM, oncostatin M; SIRE, sterol-independent regulatory element; C/EBP, CCAAT/enhancer-binding protein; CRE, cAMP-responsive element; CREB, CRE-binding protein; CREM, cAMP-responsive element modulator; EMSA, electrophoretic mobility shift assay; ATF, activating transcription factor; XBP, X-box-binding protein; DBD, DNA-binding domain; LPDS, lipoprotein-depleted serum; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/ERK kinase; AP-1, activator protein-1.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. |
Sudhof, T. C.,
Van der Westhuyzen, D. R.,
Goldstein, J. L.,
Brown, M. S.,
and Russell, D. W.
(1987)
J. Biol. Chem.
262,
10773-10779 |
| 2. |
Dawson, P. A.,
Van der Westhuyzen, D. R.,
Sudhof, T. C.,
Brown, M. S.,
and Goldstein, J. L.
(1988)
J. Biol. Chem.
263,
3372-3379 |
| 3. | Sudhof, T. C., Russell, D. W., Brown, M. S., and Goldstein, J. L. (1987) Cell 48, 1061-1069[CrossRef][Medline] [Order article via Infotrieve] |
| 4. |
Smith, J. R.,
Osborne, T. F.,
Goldstein, J. L.,
and Brown, M. S.
(1990)
J. Biol. Chem.
265,
2306-2310 |
| 5. |
Briggs, M. R.,
Yokoyama, C.,
Wang, X.,
Brown, M. S.,
and Goldstein, J. L.
(1993)
J. Biol. Chem.
268,
14490-14496 |
| 6. |
Wang, X.,
Briggs, M. R.,
Yokoyama, C.,
Goldstein, J. L.,
and Brown, M. S.
(1998)
J. Biol. Chem.
268,
14497-14504 |
| 7. | Wang, X., Sato, R., Brown, M. S., Hua, X., and Goldstein, J. L. (1994) Cell 77, 53-62[CrossRef][Medline] [Order article via Infotrieve] |
| 8. | Yokoyama, C., Wang, X., Briggs, M. R., Admon, A., Wu, J., Hua, X., Goldstein, J. L., and Brown, M. S. (1993) Cell 75, 187-197[CrossRef][Medline] [Order article via Infotrieve] |
| 9. |
Sanchez, H. B.,
Yieh, L.,
and Osborne, T. F.
(1995)
J. Biol. Chem.
270,
1161-1169 |
| 10. |
Rudling, M.,
Norstedt, G.,
Olivecrona, H.,
Reihner, E.,
Gustafsson, J.,
and Angelin, B.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
6983-6987 |
| 11. | Pak, Y. K., Kanuck, M. P., Berrios, D., Briggs, M. R., Cooper, A. D., and Ellsworth, J. L. (1996) J. Lipid Res. 37, 985-998[Abstract] |
| 12. |
Stopeck, A. T.,
Nicholson, A. C.,
Mancini, F. P.,
and Haijar, D. P.
(1993)
J. Biol. Chem.
268,
17489-17494 |
| 13. | Takagi, T., and Strauss, J. F. (1988) Can. J. Physiol. Pharmacol. 67, 968-973 |
| 14. |
Grove, R. I.,
Mazzucco, C. E.,
Radka, S. F.,
Shoyab, M.,
and Kiener, P. A.
(1991)
J. Biol. Chem.
266,
18194-18199 |
| 15. | Liu, J., Grove, R. I., and Vestal, R. E. (1994) Cell Growth Differ. 5, 1333-1338[Abstract] |
| 16. | Liu, J., Streiff, R., Zhang, Y. L., Vestal, R. E., Spence, M. J., and Briggs, M. R. (1997) J. Lipid Res. 38, 2035-2048[Abstract] |
| 17. |
Li, C.,
Kraemer, F. B.,
Ahlborn, T. E.,
and Liu, J.
(1999)
J. Biol. Chem.
274,
6747-6753 |
| 18. | Emami, K. H., Navarre, W. W., and Smale, S. T. (1995) Mol. Cell. Biol. 15, 5906-5916[Abstract] |
| 19. |
Dignam, J. D.,
Lebovitz, R. M.,
and Roeder, R. C.
(1983)
Nucleic Acids Res.
11,
1475-1489 |
| 20. |
Favata, M. F.,
Horiuchi, K. Y.,
Manos, E. J.,
Daulerio, A. J.,
Stradley, D. A.,
Feeser, W. S.,
Van Dyk, D. E.,
Pitts, W. J.,
Earl, R. A.,
Hobbs, F.,
Copeland, R. A.,
Magolda, R. L.,
Scherle, P. A.,
and Trzaskos, J. M.
(1998)
J. Biol. Chem.
273,
18623-18632 |
| 21. |
Lalli, E.,
and Sassone-Corsi, P.
(1994)
J. Biol. Chem.
269,
17359-17362 |
| 22. | Lee, K. A. W., and Masson, N. (1993) Biochim. Biophys. Acta 1174, 221-233[Medline] [Order article via Infotrieve] |
| 23. |
Botelho, F. M.,
Edwards, D. R.,
and Richards, C. D.
(1998)
J. Biol. Chem.
273,
5211-5218 |
| 24. | Wen, X. Y., Stewart, A. K., Sooknanan, R. R., Henderson, G., Hawley, T. S., Reimold, A. M., Glimcher, L. H., Baumann, H., Malek, L. T., and Hawley, R. G. (1999) Int. J. Oncol. 15, 173-178[Medline] [Order article via Infotrieve] |
| 25. |
Isshiki, H.,
Akira, S.,
Tanabe, O.,
Nakajima, T.,
Shimamoto, T.,
Hirano, T.,
and Kishimoto, T.
(1990)
Mol. Cell. Biol.
10,
2757-2764 |
| 26. | Akira, S. (1997) Int. J. Biochem. Cell Biol. 29, 1401-1418[CrossRef][Medline] [Order article via Infotrieve] |
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  B. Morin, L. A. Nichols, K. M. Zalasky, J. W. Davis, J. A. Manthey, and L. J. Holland The Citrus Flavonoids Hesperetin and Nobiletin Differentially Regulate Low Density Lipoprotein Receptor Gene Transcription in HepG2 Liver Cells J. Nutr., July 1, 2008; 138(7): 1274 - 1281. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Abidi, W. Chen, F. B. Kraemer, H. Li, and J. Liu The medicinal plant goldenseal is a natural LDL-lowering agent with multiple bioactive components and new action mechanisms J. Lipid Res., October 1, 2006; 47(10): 2134 - 2147. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Ishihara, T. Yamazaki, Y. Ishida, T. Suzuki, K. Oda, M. Nagao, Y. Yamaguchi-Iwai, and T. Kambe Zinc Transport Complexes Contribute to the Homeostatic Maintenance of Secretory Pathway Function in Vertebrate Cells J. Biol. Chem., June 30, 2006; 281(26): 17743 - 17750. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Kong, P. Abidi, F. B. Kraemer, J.-D. Jiang, and J. Liu In vivo activities of cytokine oncostatin M in the regulation of plasma lipid levels J. Lipid Res., June 1, 2005; 46(6): 1163 - 1171. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Zhang, M. Lin, P. Abidi, G. Thiel, and J. Liu Specific Interaction of Egr1 and c/EBP{beta} Leads to the Transcriptional Activation of the Human Low Density Lipoprotein Receptor Gene J. Biol. Chem., November 7, 2003; 278(45): 44246 - 44254. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. S. Kapoor, C. Golden, B. Atkins, and K. D. Mehta pp90RSK- and protein kinase C-dependent pathway regulates p42/44MAPK-induced LDL receptor transcription in HepG2 cells J. Lipid Res., March 1, 2003; 44(3): 584 - 593. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Liu, F. Zhang, C. Li, M. Lin, and M. R. Briggs Synergistic Activation of Human LDL Receptor Expression by SCAP Ligand and Cytokine Oncostatin M Arterioscler. Thromb. Vasc. Biol., January 1, 2003; 23(1): 90 - 96. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Nakahara, H. Fujii, P. R. Maloney, M. Shimizu, and R. Sato Bile Acids Enhance Low Density Lipoprotein Receptor Gene Expression via a MAPK Cascade-mediated Stabilization of mRNA J. Biol. Chem., September 27, 2002; 277(40): 37229 - 37234. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Zhang, T. E. Ahlborn, C. Li, F. B. Kraemer, and J. Liu Identification of Egr1 as the oncostatin M-induced transcription activator that binds to sterol-independent regulatory element of human LDL receptor promoter J. Lipid Res., September 1, 2002; 43(9): 1477 - 1485. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
