Induction of Low Density Lipoprotein Receptor (LDLR)
Transcription by Oncostatin M Is Mediated by the Extracellular
Signal-regulated Kinase Signaling Pathway and the Repeat 3 Element of
the LDLR Promoter*
Cong
Li,
Fredric B.
Kraemer,
Thomas E.
Ahlborn, and
Jingwen
Liu
From the Department of Veterans Affairs Palo Alto Health Care
System, Palo Alto, California 94304
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ABSTRACT |
Oncostatin M (OM) activates the transcription of
the human low density lipoprotein receptor (LDLR) in HepG2 cells
through a sterol-independent mechanism. Our previous studies showed
that mutations within the repeat 3 sequence of the LDLR promoter
significantly decreased OM activity on LDLR promoter luciferase
reporter constructs that contain the sterol responsive element-1
(repeat 2) and Sp1 binding sites (repeats 1 and 3). In this study, we
investigated the signal transduction pathways that are involved in
OM-induced LDLR transcription. In HepG2 cells, OM induced a rapid
increase in LDLR mRNA expression, with increases detected at 30 min
and maximal induction at 1 h. This OM effect was not blocked by
protein synthesis inhibitors, inhibitors of p38 kinase,
phosphatidylinositol 3-kinase, or c-Jun N-terminal kinase, but OM
activity was completely abolished by pretreating cells with inhibitors
of the extracellular signal-regulated kinase (ERK) kinase (mitogen/ERK
kinase (MEK)). To investigate whether the repeat 3 sequence of the LDLR
promoter is the OM-responsive element that converts ERK activation at
the promoter level, three luciferase reporters, pLDLR-TATA containing only the TATA-like elements of the promoter, pLDLR-R3 containing repeat
3 and the TATA-like elements, and pLDLR-234 containing repeats 1, 2, 3 and the TATA-like elements were constructed and transiently transfected
into HepG2 cells. OM had no effect on the basal promoter construct
pLDLR-TATA; however, including a single copy of repeat 3 sequence in
the TATA vector (pLDLR-R3) resulted in a full OM response. The activity
of OM on pLDLR-R3 was identical to that of pLDLR-234. Importantly, the
ability of OM to increase luciferase activities in both pLDLR-R3- and
pLDLR-234-transfected cells was blocked in a dose-dependent
manner by inhibition of MEK. These results demonstrate that the
mitogen-activated protein kinase MEK/ERK cascade is the essential
signaling pathway by which OM activates LDLR gene transcription and
provide the first evidence that the repeat 3 element is a new
downstream target of ERK activation.
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INTRODUCTION |
The transcription of the human low density lipoprotein receptor
(LDLR)1 gene is under the
control of intracellular cholesterol and other non-sterol mediators.
Cholesterol regulates LDLR transcription through a negative feedback
mechanism (1, 2). When cellular cholesterol levels rise, LDLR
transcription is reduced. When cellular cholesterol storage is
depleted, LDLR transcription is activated. The promoter region and the
cis-acting elements that are responsible for the basal and the
cholesterol-controlled transcription of LDLR have been localized to
three GC-rich imperfect 16-bp direct repeats (3-5). The three repeats
lie within 100 bp upstream of the transcriptional start site. Repeats 1 and 3 contain Sp1 binding sites that support the basal transcriptional
activity of LDLR. Interference of Sp1 binding to either repeat severely
decreases basal transcription. Cholesterol regulation is mediated
through a 10-bp sequence (5'-ATCACCCCAC-3') within repeat 2 designated sterol responsive element-1 (SRE-1) (6, 7). Under low intracellular cholesterol conditions, the SRE-1-binding proteins SREBP1 and SREBP2
bind to the SRE-1 sequence and interact synergistically with Sp1 in
repeat 3, leading to the activation of LDLR transcription (8-11). In
addition to the SRE-1 site, another cis-acting element, designated FP1,
was recently described that is located upstream of the repeat 1 sequence (-145 to -126) (12). The FP1 site participates in LDLR
transcription only under conditions of sterol depletion.
In addition to cholesterol and its derivatives, LDLR
transcription is also subject to regulation by physiological non-sterol mediators, including growth hormone (13, 14), hepatocyte growth factor
(15), cytokines (16), and some pharmaceuticals (17). The increased
transcription of the LDLR gene induced by these agents has been
ascribed to activation of protein kinase C, protein kinase A, and an
increase in intracellular Ca2+ (18, 19). However, the
cis-acting elements in the LDLR promoter and the transcription factors
that are responsible for regulation by cholesterol-independent pathways
have not been elucidated.
Our laboratory has been involved in characterizing the mechanism by
which the cytokine oncostatin M (OM) activates LDLR transcription. OM,
a member of the interleukin-6 family of cytokines, is a strong inducer
of LDLR transcription in HepG2 cells and in mouse liver (20-23). The
mechanism of OM induction is clearly sterol-independent, because we
have demonstrated that mutations within the SRE-1 site that totally
abolished cholesterol regulation had no effect on OM inducibility of
LDLR transcription. Instead, mutations within the repeat 3 sequence
severely impaired OM activity (22).
In order to identify and characterize the trans-acting factors that
mediate OM-induced LDLR transcription by interacting with the repeat 3 sequence, in the present studies, we examined the OM-elicited signal
transduction pathways and specific kinases that are involved in LDLR transcription.
Two major signaling pathways can be activated by OM and its related
cytokines interleukin-6 (IL-6) and leukemia inhibitory factor (LIF):
the JAK/signal transducer and activator of transcription (STAT) pathway
(24) and the Ras-mitogen-activated protein (MAP) kinase pathway
(25-27). Binding of OM to its cell surface receptors activates JAK
kinases (JAK 1, JAK 2, and tyk), which, in turn, convert latent
cytoplasmic transcription factors, known as STATs, into activated forms
by tyrosine phosphorylation. The tyrosine-phosphorylated STATs (STAT 1, 3, and 5b) form homo- or heterodimers and translocate into the nucleus,
where they bind to their recognition sequences. This signaling pathway
is responsible for OM-induced transcription of the
cyclin-dependent kinase inhibitor p21 (28), aromatase P450
(29), and genes coding for acute phase proteins (30, 31). However, the
STAT pathway does not appear to be directly involved in OM-mediated
LDLR transcription because no STAT binding sites exist in the
OM-responsive region of the LDLR promoter (
142 to +35). Furthermore,
cotransfection of STAT 3 or STAT 5b expression vectors with LDLR
promoter reporter constructs did not alter basal or OM-inducible
promoter activity (22). Therefore, in the present studies, we focused
our investigation on the MAP kinase pathway.
Our findings in this study demonstrate that LDLR transcription is also
regulated by the MAP kinase cascade. By using specific inhibitors that
block each signaling pathway, we demonstrate that inhibition of ERK
activation by inactivating its upstream kinase, MEK, totally prevented
OM-induced LDLR mRNA expression. We also demonstrate that the
activity of OM on the LDLR promoter is mediated through the repeat 3 element. More importantly, we show that the inhibition of ERK
activation totally abolished OM activity on the LDLR promoter. These
data for the first time localize an ERK responsive cis-acting element
in the LDLR promoter and provide the first evidence that the repeat 3 sequence is a novel downstream target of MAP kinase activation.
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MATERIALS AND METHODS |
Cells and Reagents--
The human hepatoma cell line HepG2 was
obtained from American Type Culture Collection (Manassas, VA) and was
cultured in Eagle's minimum essential medium supplemented with 10%
fetal bovine serum (Summit Biotechnology, Fort Collins, CO). Purified
human recombinant OM was provided by Bristol-Myers Squibb (Princeton,
NJ). Cholesterol and 25-hydroxycholesterol, anisomycin, cycloheximide,
puromycin, emetine, and the PI 3-kinase inhibitor wortmannin were
purchased from Sigma. The MEK inhibitor U0126 was obtained from DuPont
Merck Pharmaceutical Co. The MEK inhibitor PD98059 was purchased from New England Biolabs (Beverly, MA). The p38 kinase inhibitor SB-203580 was obtained from SmithKline Beecham Pharmaceuticals (King of Prussia, PA). The rabbit polyclonal antibodies to detect inactive ERK2
(C-14, catalog no. sc-154) were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). The rabbit polyclonal antibodies (V803A) against
activated ERK1 and ERK2 were obtained from Promega, Inc. (Madison, WI).
Plasmid Vectors and Oligonucleotides--
The plasmid
pLDLR-234 was constructed by subcloning a 177-bp fragment of the LDLR
promoter obtained by HindIII digestion of pLDLR-CAT 234 (3)
into HindIII-digested pGL3-basic vector (Promega). To
construct the pLDLR-TATA vector, a double-stranded oligonucleotide corresponding to LDLR promoter sequence +13 to -36
(TCGAGtagaaacctcacattgaaatgctgtaaatgacgtgggccccgagtgcaatA) was synthesized with XhoI and HindIII sites
(italic) at the 5' and 3' ends, respectively, and was cloned between
the XhoI and HindIII sites of pGL3-basic. The
vector pLDLR-R3 was constructed by cloning of a double-stranded
oligonucleotide containing the LDLR promoter sequence from +13 to -52
with SacI and HindIII sites (italic) at the 5'
and 3' ends
(CaaactcctccccctgctagaaacctcacattgaaatgctgtaaatgacgtgggccccgagtgcaatA) into pGL3-basic vector. The repeat 3 sequence is underlined.
RNA Isolation and Northern Blot Analysis--
Cells were lysed
in Ultraspec RNA lysis solution (Biotecxs Laboratory, Houston, TX), and
total cellular RNA was isolated according to the vendor's protocol.
Approximately 15 µg of each total RNA sample was used to analyze the
LDLR mRNA as described previously. The RNA blots were first
hybridized to a 0.84-kilobase 32P-labeled human LDLR probe
(22) and then stripped and reprobed with a human GAPDH probe to ensure
that equivalent amounts of RNA were being loaded. Differences in
hybridization signals of Northern blots were quantitated by the Bio-Rad
Fluro-S MultiImager system. Densitometric analysis of autoradiographs
in these studies included various exposure times to ensure linearity of signals.
Western Blot Analysis--
HepG2 cells in 60-mm culture dishes
were scraped into 0.2 ml of cold lysis buffer (20 mM HEPES,
pH 7.4, 30 mM p-nitrophenyl phosphate, 10 mM NaF, 10 mM MgCl2, 2 mM EDTA, 5 mM dithiothreitol, 0.1 mM Na3VO4, 0.1 mM
Na2MoO4, 10 mM sodium
-glycerophosphate, 10 nM okadaic acid, 10 nM
cypermethrin, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml
aprotinin, 1 µg/ml leupeptin, and 1.25 µg/ml pepstatin) and
disrupted by repeated passages through a 26 gauge needle. Extracts were
centrifuged for 30 min at 10,000 × g and stored at
80 °C. Samples of 30 µg of soluble protein per lane were
electrophoresed through a 12% SDS-polyacrylamide gel and transferred
to a nitrocellulose membrane. The membranes used to probe the inactive
ERK1 and ERK2 were first blocked in 5% powered milk and 0.05% Tween
20 in phosphate-buffered saline for 2 h, and then incubated with
the same blocking buffer containing anti-ERK antibody at the
concentration of 1 µg/ml overnight at 4 °C. The antibodies against
inactive ERK primarily recognize ERK2. The membranes used to probe
activated ERK1 and ERK2 were blocked in TBST buffer (20 mM
Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20) containing 1%
bovine serum albumin for 2 h and then incubated in TBST buffer
containing 0.1% bovine serum albumin and antibody at a 1:5000 dilution
overnight at 4 °C. Detection was performed using a horseradish
peroxidase-conjugated secondary anti-rabbit antibody and enhanced
chemiluminescence (ECL kit, Amersham Pharmacia Biotech).
Transient Transfection Assays--
HepG2 cells were transiently
transfected with plasmid DNA by the method of calcium phosphate
coprecipitation (22). To demonstrate sterol-independent regulation,
cells transfected with pLDLR-234 vector were cultured in media
containing 10% fetal bovine serum and cholesterol (10 µg/ml, plus 1 µg/ml 25-hydroxycholesterol). For the studies of kinase inhibitors,
24 h after termination of transfection, inhibitors at the
indicated concentrations were added 1 h before 50 ng/ml OM was
added. After 4 h of OM treatment, cells were washed twice with
phosphate-buffered saline and lysed with 150 µl of reporter lysis
buffer (Promega). The luciferase assay (70 µl of lysate/sample) was
conducted with Promega luciferase assay reagents, and the luciferase
activity was measured in a luminometer (Promega, model TD-20/20).
-Galactosidase activity (50 µl of lysate) was measured according
to standard methods. Absolute luciferase activity was normalized
against
-galactosidase activity to correct for transfection
efficiency. Triplicate wells were assayed for each transfection
condition, and at least three independent transfection assays were
performed for each reporter construct.
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RESULTS |
The kinetics of OM-induced LDLR gene expression in HepG2 cells was
examined by Northern blot analysis. Total RNA was isolated from cells
that were treated with 50 ng/ml OM for different periods from 30 min to
48 h. OM induced a rapid increase in LDLR mRNA level. This
increase was detected as early as 30 min after OM addition to the
cells, reached a maximal level (3.4-fold of control) at 1 h, and
slowly declined to baseline at 48 h (Fig.
1). Results from five separate
experiments, after normalization with GAPDH, showed that stimulation of
cells with OM for 1 h increased LDLR mRNA to an average of
4.2-fold (4.2 ± 1.2, S.D.), as compared with unstimulated
cells.

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Fig. 1.
Kinetics of LDLR mRNA expression in HepG2
cells treated with OM. HepG2 cells cultured in Eagle's minimum
essential medium containing 10% fetal bovine serum were incubated with
50 ng/ml OM for different times as indicated. Total RNA was isolated,
and 15 µg per sample was analyzed for LDLR mRNA by Northern blot.
The membrane was stripped and hybridized to a human GAPDH probe. The
fold induction of LDLR mRNA level with OM treatment normalized to
GAPDH mRNA over untreated cells was as follows: 30 min, 1.7; 1 h, 3.4; 2 h, 3.0; 6 h, 2.8; 8 h, 2.2; 24 h, 1.4;
32 h, 1.3; 48 h, 0.94. The blot shown is representative of
two separate kinetic studies.
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The rapid action of OM on LDLR mRNA expression suggests that OM may
activate LDLR transcription in the absence of new protein synthesis. To
investigate this issue, HepG2 cells were first incubated with the
protein synthesis inhibitors, anisomycin (12.5 µg/ml), cycloheximide
(10 µg/ml), puromycin (150 µg/ml), and emetine (10 µg/ml) for
1 h prior to addition of OM to the cells. Analysis of protein
synthesis with labeled [3H]leucine indicated that more
than 99% of protein synthesis occurring in HepG2 cells was blocked by
these inhibitors at the doses used. Fig.
2 shows that the protein synthesis
inhibitors had different effects on the basal LDLR mRNA levels. The
normalized LDLR mRNA level was slightly decreased in cycloheximide
treated cells (71% of control), but it was significantly increased in
anisomycin-treated (258% of control) and emetine-treated (183% of
control) cells. However, the OM-induced up-regulation of LDLR mRNA
expression was not inhibited by any of the inhibitors; instead, LDLR
mRNA was further increased. This result confirmed our speculation
that activation of LDLR transcription by OM does not require new
protein synthesis.

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Fig. 2.
Protein synthesis inhibitors have no
inhibitory effect on OM activation of LDLR gene expression. HepG2
cells were either untreated or treated with 50 ng/ml OM for 1 h in
the presence or the absence of protein synthesis inhibitors. The
inhibitors were added 1 h prior to OM addition. Total RNA was
isolated and LDLR mRNA level was analyzed by Northern blot. The
membrane was stripped and hybridized to a human GAPDH probe. The
normalized LDLR mRNA levels (% of control) were as follows:
control, 100; Me2SO (DMSO), 88; anisomycin
(Aniso), 258; cycloheximide (CHX), 71; puromycin
(Puro), 110; emetine (Emetin), 183; OM, 294; OM + anisomycin, 800; OM + cycloheximide, 443; OM + puromycin, 696; OM + emetine, 833.
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Three kinase pathways have been shown to be activated by OM and its
related cytokines: JAK/STAT pathway (24, 30), MAP kinase pathway
(25-27), and PI 3-kinase (32). Because our previous studies already
excluded the role of activated STAT in LDLR transcription (22), we
focused the present investigation on PI 3-kinase and the MAP kinases.
In mammalian cells, extracellular signals are transduced through three
separate MAP kinase cascades, including MAP kinase (also known as
extracellular signal-regulated kinase (ERK) (33)), the c-Jun N-terminal
kinase (JNK) (34), and P38 kinase (35).
To determine which kinase pathway is involved in OM-induced LDLR
mRNA expression, HepG2 cells were stimulated with 50 ng/ml OM for
1 h in the presence or the absence of MEK inhibitors U0126 and
PD98059, p38 kinase inhibitor SB-203580, and PI 3-kinase inhibitor wortmannin. Total RNA was isolated and the level of LDLR mRNA was
analyzed by Northern blot. The result showed that the MEK inhibitors,
U0126 and PD90586, were extremely effective in preventing OM-induced
LDLR mRNA expression. The majority of the OM activity was inhibited
by U0126 at 1 µM and PD98059 at 10 µM. In
contrast, wortmannin at concentrations up to 100 nM, which
has been shown to completely inhibit PI 3-kinase activity (32), had no
effect. The p38 kinase inhibitor SB-203580 did not affect OM activity at its effective concentration (IC50 = 0.6 µM) (36) but moderately lowered OM activity (30%) at 25 µM (Fig. 3).

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Fig. 3.
Effects of different kinase inhibitors on OM
inducibility of LDLR gene expression. HepG2 cells were treated
with MEK inhibitors U0126 (U), PD98059 (PD), p38
kinase inhibitor SB-203580 (SB), and the PI 3-kinase
inhibitor wortmannin (wort) at different doses for 1 h
prior to 1 h of treatment with OM at a concentration of 50 ng/ml
and analyzed for LDLR mRNA and GAPDH mRNA by Northern blot. The
blot shown is representative of three separate experiments.
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The possible role of the third MAP kinase cascade, JNK, in OM-induced
LDLR transcription was examined by using curcumin. Curcumin has been
shown to inhibit the activations of JNK and ERK at different concentrations (37). The JNK pathway is more sensitive to curcumin with
an IC50 of 5-10 µM. At higher
concentrations, activation of ERK by MEK is inhibited with an
IC50 of 20 µM. Treatment of cells with up to
25 µM curcumin lowered the OM-induced increase in LDLR
mRNA only 35% (Fig. 4). This small
effect noted at high concentrations of curcumin suggests that the
inhibitory activity of curcumin was more likely mediated through its
effect on ERK activation rather than JNK activation.

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Fig. 4.
OM-induced LDLR gene expression was not
effectively blocked by curcumin. OM at 50 ng/ml concentration was
added to HepG2 cells that were either untreated or pretreated for
1 h with curcumin at indicated doses. Total RNA was isolated after
1 h of OM treatment and analyzed for LDLR and GAPDH mRNAs by
Northern blot. The figure shown is representative of two separate
experiments. The % inhibition of curcumin on OM activity was as
follows: 2 µM, 13%; 10 µM, 19.3%; 25 µM, 34.9%.
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The compound U0126 is a newly developed inhibitor that specifically
inhibits MEK (38). It strongly inhibits the activity of MEK1 and MEK2
(IC50 = 1 µM) without detectable effects on
other kinases, such as PKC, Raf, ERK, and JNK. Because our data showed a higher potency of U0126 than PD98059 (39) on OM-stimulated LDLR
mRNA expression, the dose-dependent inhibitory effect
of U0126 on OM activity was further examined. Fig.
5 shows a representative Northern blot.
U0126 inhibited OM activity with an IC50 of 0.5-1 µM, with complete inhibition achieved at 5 µM and higher. This dose range is in excellent agreement
with its reported effect on MEK. The basal level of LDLR mRNA was
slightly decreased by the compound. These results together demonstrate
that inhibition of MEK activity totally prevented OM induction of LDLR
transcription, whereas inhibitors of p38 kinase and JNK kinase only
partially inhibited OM activity at doses far exceeding their
IC50s. Based upon these data, we conclude that the MAP
kinase MEK/ERK cascade is the major signaling pathway through which OM
activates LDLR transcription.

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Fig. 5.
The dose-dependent effects of MEK
inhibitor U0126 on OM induction of LDLR gene expression. U0126 was
added to HepG2 cells at the indicated concentrations for 1 h
before OM was added. Total RNA was isolated after 1 h of OM
treatment. The blot shown (A) is representative of three
separate Northern blots. B, the normalized LDLR mRNA
levels (% of control) were as follows: control, 100; OM, 550;
0.05 µM U0126 (U) + OM, 491; 0.1 µM U + OM, 420; 0.5 µM U +OM, 354; 1 µM U +OM, 258; 5 µM U +OM, 108; 10 µM U +OM, 75.
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MAP kinases ERK1 and ERK2 are the only known substrates of MEK1 and
MEK2 (40). MEK activates ERK by inducing phosphorylation on both
threonine and tyrosine residues of ERK. Inhibition of MEK kinase
activity will directly block ERK activation. Western blot analysis
using antibody that detects inactive ERK and antibody that only
recognizes the double-phosphorylated, activated ERK was conducted to
examine OM-induced activation of ERK. As shown in Fig.
6, neither OM nor U0126 treatments
altered the level of expression of inactive ERK1 and ERK2. In contrast,
the detectable level of activated ERK1 and ERK2 was rapidly increased
by OM stimulation. A 3.7-fold increase was detected at 5 min, reached a
maximal level (4.2-fold of control) at 20 min, and slowly declined to
baseline after 60 min. Complete inhibition of OM-induced ERK activation was demonstrated by using 10 µM U0126 (Fig. 6,
+). This result documents that OM activates both ERK1 and ERK2 and
that this activation immediately precedes OM-induced LDLR
transcription.

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Fig. 6.
Time course for activation of MAP kinases
ERK1 and ERK2 by OM. HepG2 cells cultured in Eagle's minimum
essential medium containing 0.5% fetal bovine serum were stimulated
with 50 ng/ml OM in the absence ( ) or the presence (+) of MEK
inhibitor U0126 (10 µM). At the indicated times, the
cells were scraped into lysis buffer and cell extracts were prepared.
Soluble proteins (30 µg/lane) were applied to SDS-PAGE. Detections of
nonphosphorylated and phosphorylated ERK were performed by
immunoblotting as described under "Materials and Methods."
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Our next goal was to determine the promoter region and the cis-acting
element that serves as the functional downstream target of the
OM-activated MEK/ERK cascade. Previously, by deletion analysis, we
localized the OM response to the LDLR promoter region from -142 to +35
(pLDLR-234), which contains repeats 1, 2, and 3 and the TATA-like
elements. Mutagenesis studies of each repeat showed that OM activity
was only diminished when repeat 3 was mutated (22). To prove that
repeat 3 is the response element that mediates OM effects, in the
present studies, we constructed two luciferase reporter vectors that
contain LDLR promoter TATA-like sequences with or without repeat 3. PLDLR-TATA was made by insertion of the LDLR promoter sequence from
-36 to +13 relative to the transcription start site into the
pGL3-basic luciferase vector. PLDLR-R3 contains the LDLR promoter
sequence from -52 to +13. The effect of OM on pLDLR-TATA and pLDLR-R3
was then compared with its effect on pLDLR-234. These reporters, along
with the
-galactosidase expression vector pRSV-
-galactosidase,
were transiently transfected into HepG2 cells and luciferase activity
and
-galactosidase activity were measured. Fig.
7A illustrates a
representative experiment that shows the basal promoter activities and
the response to OM of the three reporter vectors. PLDLR-TATA produced a
low level of luciferase activity and showed no response to OM.
Including a single copy of repeat 3 with the TATA sequence (pLDLR-R3)
increased the basal level of luciferase activity 3-5-fold and,
importantly, created a promoter that was responsive to OM. OM increased
luciferase activity in pLDLR-R3-transfected cells to an extent similar
to that observed in pLDLR-234-transfected cells. Fig. 7B
summarizes the results from over 10 independent transfections and shows
that OM increased pLDLR-234 and pLDLR-R3 promoter activities 3.3-fold and 3.4-fold, respectively, without affecting pLDLR-TATA. These data
provide strong evidence that the OM effect on the LDLR promoter is
mediated through the repeat 3 sequence.

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Fig. 7.
Repeat 3 element of the LDLR promoter
mediates OM activity. The LDLR promoter luciferase reporter
constructs pLDLR-TATA, pLDLR-R3, and pLDLR-234 were transiently
transfected into HepG2 cells along with a -galactosidase expression
vector, pRSV- -galactosidase. Twenty hours after transfection, cells
were stimulated with 50 ng/ml OM for 4 h. Luciferase expression
was normalized to -galactosidase activity to correct for variations
in transfection efficiency. A, basal and OM-induced
luciferase activity of the three promoter constructs. B,
effects of OM on the three promoter constructs. The normalized
luciferase activity of transfected cells that were untreated is
expressed as 100%. The data (mean ± S.E.) shown were derived
from 10-12 independent transfection assays. Solid columns,
without OM; checked columns, with OM.
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We next examined whether blocking ERK activation by inhibition of MEK
could affect the ability of OM to increase the activity of the LDLR
promoter. HepG2 cells were transfected with pLDLR-234 or pLDLR-R3.
Twenty hours after transfection, U0126 was added to the cells at
different doses for 1 h, followed by OM. U0126 produced a
dose-dependent inhibition of OM stimulation of both the
full promoter (pLDLR-234) and the minimum promoter containing a single
copy of repeat 3 (pLDLR-R3) (Fig. 8). The
dose of U0126 that effectively blocked OM-induced LDLR promoter
activity is in a range similar to that of inhibition of OM-induced LDLR
mRNA expression. These data suggest that an ERK-responsive
transcription activator is involved in LDLR transcription through
interaction with the repeat 3 sequence.

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Fig. 8.
Dose-dependent inhibition of
OM-induced LDLR promoter activity by the MEK inhibitor U0126.
HepG2 cells were transiently transfected with LDLR promoter constructs
pLDLR-234 and pLDLR-R3. Transfected cells were stimulated with 50 ng/ml
OM for 4 h in the presence of U0126 at the indicated
concentrations. U0126 was added to the transfected cells 1 h prior
to OM addition. The normalized luciferase activity of transfected cells
that were untreated is expressed as 100%. The data (mean ± S.D.)
shown are representative of five independent transfection experiments
in which triplicate wells were transfected for each condition.
U, U0126.
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Finally, to investigate whether the repeat 3 element in the LDLR
promoter only mediates OM signaling or whether it is used by other
members of the IL-6 family of cytokines, the activities of IL-6 and LIF
on pLDLR-234 and pLDLR-R3 were examined and compared with OM in the
presence or the absence of the MEK inhibitor U0126. All the cytokines
at their maximally effective concentrations increased LDLR promoter
activity, and their activities were inhibited by U0126 (Fig.
9). The abilities of these cytokines to
increase LDLR promoter activity was in the order OM
IL-6
LIF,
which is consistent with their abilities to activate MAP kinase in
HepG2 cells (25).

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Fig. 9.
Activation of LDLR promoter activity by IL-6,
LIF, and OM. HepG2 cells were transiently transfected with LDLR
promoter constructs pLDLR-234 and pLDLR-R3. Transfected cells were
stimulated with 100 ng/ml IL-6, 50 ng/ml LIF, or 50 ng/ml OM for 4 h in the absence or the presence of 25 µM U0126. The data
(mean ± S.D.) shown are representative of three independent
transfection experiments. C, control. Solid
columns, without U0126; checked columns, with
U0126.
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DISCUSSION |
Cytokines bind to their cell surface receptors and initiate
intracellular signaling by activating members of the Janus kinase family of tyrosine kinase. Activated JAK can initially activate two
separate pathways, the STAT pathway and the Ras pathway. Activated Ras
has an essential role in the activation of Raf kinase, which directly
phosphorylates and activates the ERK upstream kinases MEK1 and MEK2.
Activated MEK, in turn, activates ERK1 and ERK2 by inducing
phosphorylation of threonine and tyrosine residues in ERK. Because it
has been previously reported that OM activates Ras and Raf (27,
41-43), we were interested in examining the downstream effector of
this signaling cascade that might play a role in the stimulation of
LDLR transcription. By using inhibitors that block different signal
pathways, we show that the MEK inhibitors U0126 and PD98059 are
effective in blocking OM-induced LDLR transcription, whereas inhibitors
of PI 3-kinase, JNK, and p38 kinase had little or no effect. The
observed small inhibitory effects of p38 kinase inhibitor SB-203580 at
25 µM on OM-induced LDLR mRNA expression was due to
its nonspecific effects on MEK, as the OM-induced ERK activation
detected by Western blot was also inhibited by SB-203580 at 25 µM (data not shown). The IC50 (0.5-1
µM) of U0126 on OM activity is very similar to that
reported for its inhibition of MEK enzyme activity. These data provide
convincing evidence that the MEK/ERK cascade is the essential signaling
pathway by which OM activates LDLR transcription.
The involvement of ERK activation in OM-induced LDLR transcription is
also supported by the tight association of these two biological events.
Activation of ERK by OM was detected within 5 min, reaching a maximum
at 12-20 min and declining to baseline by 60 min. This process was
immediately followed by an increase in LDLR mRNA expression. LDLR
mRNA levels started to rise after 30 min of exposure to OM, reached
a maximal level at 1 h, and slowly decreased afterward. The
closely linked kinetics of ERK activation and LDLR transcription
suggest that these two events elicited by OM are mechanistically related.
Establishment of the essential role of ERK activation in OM-induced
LDLR transcription raises a critical question: which cis-acting element
in the LDLR promoter is the downstream target of ERK activation? Previously, we have localized the OM response to the LDLR promoter region from -142 to +35 (pLDLR-234), which contains repeats 1, 2, and
3 and the TATA-like elements. Mutations within the repeat 3 sequence of
pLDLR-234 significantly lowered OM activity (22). In this study, we
further demonstrate that OM activity is mediated through the repeat 3 sequence. OM had no effect on the basal LDLR promoter (pLDLR-TATA,
36
to +13) that contains the TATA-like elements. Placing the 16-bp repeat
3 sequence 5' to the basal promoter (pLDLR-R3,
52 to +13), as is
found in the native promoter configuration, generated the same OM
response as that seen with the longer promoter fragment (pLDLR-234).
Importantly, the OM-induced responses of pLDLR-234 and pLDLR-R3 were
both prevented by the MEK inhibitor U0126. These results identify for
the first time a region in the LDLR promoter that responds to MAP
kinase activation and demonstrate that the repeat 3 element is a new
downstream target of the MEK/ERK cascade.
Several cis-acting elements that mediate the effects of the IL-6 type
cytokines on gene transcription have been described. These include
nuclear factor-IL-6 (44), STAT binding site (24), and Ap1 (41, 45). In
this study, we demonstrate that OM, IL-6, and LIF are all capable of
inducing LDLR transcription through the repeat 3 sequence, although
their relative potencies differ. These data suggest that the repeat 3 sequence contains a novel responsive element that mediates the effects
of OM and its related cytokines on transcriptions of a subset genes.
Upon activation, ERK translocates to the nucleus, where it
phosphorylates a number of transcription factors, including members of
the Ets family, ELK, c-Jun, and c-Myc (46-48). Phosphorylation of ELK
by ERK potentiates the DNA binding of ELK and the formation of a
ternary complex at the serum response element of the c-fos promoter, resulting in an immediate activation of transcription of the
c-fos gene (49). Interestingly, comparison of the published DNA recognition sequences of the known ERK substrates, such as SRF or
c-Myc, with the repeat 3 sequence (AAACTCCTCCCCCTGC) does not reveal
any significant sequence homology.
Currently, Sp1 and the Sp1-related protein, Sp3, are the only
transcription factors that have been shown to bind to the repeat 3 sequence. Sp1 was shown to be phosphorylated by a
DNA-dependent protein kinase, but this phosphorylation did
not affect the extent and specificity of DNA binding or
Sp1-dependent transcription (50). On the other hand, a
recent study showed that Sp1 in HL-60 leukemia cells was phosphorylated
by protein kinase A, and the Sp1 DNA binding and trans-activating
activities were also stimulated by protein kinase A (51). We have
considered the possibility that Sp1 is phosphorylated by ERK or an ERK
substrate upon OM stimulation, resulting in a increased transcriptional
activity of LDLR gene. However, we could not detect any OM-induced
changes in Sp1 binding by gel shift assay or in the ratio of
hyperphosphorylated versus the hypophosphorylated Sp1 by
Western blot analysis (22). Furthermore, the MEK inhibitor U0126 at
concentrations that totally blocked ERK activation had no effect on Sp1
DNA binding activity (data not shown). At present, it is uncertain
whether Sp1 is phosphorylated by ERK or downstream kinases at specific
residues without changing Sp1 DNA binding activity. This
phosphorylation might somehow alter the affinity of Sp1 to interact
with other transcription factors or cofactors. Alternatively, an
unknown transactivator might be a direct or indirect substrate of ERK.
Upon activation by OM, this factor could either associate with Sp1 as a
cofactor or directly bind to the repeat 3 sequence, thereby stimulating
LDLR transcription. These important issues are currently under investigation.
Recently, Kumar et al. (52) reported that IL-1
-induced
LDLR transcription was mediated through ERK activation. However, comparison of the characteristics of IL-1
with OM on LDLR
transcription demonstrates that these two cytokines regulate LDLR
transcription through different mechanisms. First, the kinetics of OM
in inducing LDLR mRNA expression is much faster than that of
IL-1
. OM activity reached a maximum at 1 h and slowly declined
afterward, whereas a significant increase in LDLR mRNA level
induced by IL-1
was not seen until 4 h and levels continued to
increase after that. Second, cholesterol has no effect on OM induction
of LDLR mRNA expression or on LDLR promoter activity, but
cholesterol totally abolished the IL-1
-induced increase in LDLR
mRNA expression. Third, unlike IL-1
, the activity of which on
LDLR mRNA was potentiated by SB-203580 at concentrations of 1-5
µM, the OM-induced LDLR mRNA expression was not
significantly affected by SB-203580 at 1-10 µM and was
slightly inhibited at 25 µM, suggesting that p38 kinase
may be involved in IL-1
signaling, but it does not play a role in
OM-induced LDLR transcription. Furthermore, the effect of IL-1
on
the LDLR promoter was examined within a reporter construct containing a
1563-bp fragment of the LDLR promoter. Therefore, it is not known what
region of the LDLR promoter was responsible for mediating the IL-1
effect and, critically, whether the IL-1
-responsive element is the
same cis-acting element that responds to ERK activation. These striking
differences, as well as the involvement of ERK activation in both
OM-induced and IL-1
-induced LDLR transcription, suggest that ERK
activation might be involved both in cholesterol-dependent and cholesterol-independent regulation of LDLR transcription.
In conclusion, we have demonstrated that the MAP kinase MEK/ERK cascade
is the essential signaling pathway by which OM activates LDLR gene
transcription. The repeat 3 sequence of the LDLR promoter not only
functions as an Sp1 binding site that controls basal transcription but
also is an OM-responsive element that converts ERK activation at the
LDLR promoter level. These results provide critical clues for further
characterizing the transcriptional complex that controls LDLR
transcription independent of the SRE-1/SREBP pathway.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Kay Klausing at Ligand
Pharmaceuticals and Dr. Michael R. Briggs at SmithKline Beecham for
interesting discussions, Dr. James M. Trjaskos, Cristine Tahaka, and
Jia-Sheng Yan at DuPont Merck for providing the MEK inhibitor U0126,
Dr. John C. Lee at SmithKline Beecham for providing the p38 kinase
inhibitor SB-203580, and Raphael Streiff for reviewing the manuscript.
 |
FOOTNOTES |
*
This work was supported by a Merit Review from the
Department of Veterans Affairs and by Grant 98-218 from the American
Heart Association Western States Affiliate.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: 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{at}icon.palo-alto.med.va.gov.
 |
ABBREVIATIONS |
The abbreviations used are:
LDLR, low density
lipoprotein receptor;
ERK, extracellular signal-regulated kinase;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
IL, interleukin;
JNK, c-Jun N-terminal kinase;
LIF, leukemia inhibitory factor;
MAP, mitogen-activated protein;
MEK, MAPK/ERK kinase;
OM, oncostatin M;
SRE-1, sterol response element-1;
SREBP, SRE-1-binding protein;
STAT, signal transducer and activator of transcription;
PI, phosphatidylinositol;
bp, base pair(s).
 |
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