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INTRODUCTION |
Scavenger receptor class B, type I
(SR-BI),1 is a high density
lipoprotein receptor that mediates both the influx (1-3) and efflux
(4, 5) of cholesterol between high density lipoprotein and cells.
Prominent expression of SR-BI has been observed in the liver and
steroidogenic tissues (1, 2, 6, 7) and more recently in the macrophage
and atherosclerotic vessel wall (2, 4, 8, 9). Although the contribution
of SR-BI in the vessel wall to atherogenesis is not known, hepatic
overexpression of SR-BI reduces atherosclerosis in low density
lipoprotein receptor-deficient mice fed a high fat diet. Because this
effect is associated with reduced plasma high density lipoprotein, it
has been suggested that SR-BI enhances hepatic uptake of high density
lipoprotein cholesterol and thereby suppresses atherogenesis (10, 11). Consistent with this, crossing of SR-BI knockout mice with apoE knockout mice accelerates the onset of atherosclerosis (12), again
suggesting an atheroprotective role for SR-BI.
A variety of stimuli have been demonstrated to regulate SR-BI
expression. The hormones estrogen and adrenocorticotropic hormone have
been observed to alter SR-BI expression (7, 13-15). In addition,
modified low density lipoprotein has been shown to increase SR-BI in
human monocyte-derived macrophages (8), whereas high cholesterol diet
lowers SR-BI expression in rat liver parenchymal cells (15).
Pro-inflammatory mediators such as lipopolysaccharide (LPS) have also
been shown to down-regulate the mRNA and protein levels of SR-BI in
the monocyte and macrophage (16). Despite a number of studies
demonstrating regulation of SR-BI, relatively little is known about the
basic mechanisms involved. Recent promoter studies have shown that
members of the Sp1 transcription factor family are essential for
transcription of the rat SR-BI gene in mouse Leydig tumor cells (17).
It has also been shown that the sterol response element-binding protein
activates transcription of the rat SR-BI promoter in variety of cell
lines (18) and that steroidogenic factor-1 binds to and activates the
human SR-BI promoter in mouse adrenocortical cells (6). More recently, it was shown that the ligand-activated peroxisome
proliferator-activated receptor increases SR-BI expression in human
monocytes and macrophages (9).
The observation that pro-inflammatory stimuli such as LPS repress SR-BI
expression has led to the hypothesis that inflammatory mediators
contribute to atherogenesis by compromising cholesterol efflux from
vascular macrophages. LPS is known to signal through Toll-like
receptor-4 (19) and to trigger the activation of nuclear factor-
B
(NF-B) plus several MAPKs, the ERK, p38, and JNK proteins (20-24).
In addition, LPS has been shown to activate p21-activated kinase-1
(PAK1) in the macrophage, and PAK1 activation triggers nuclear
accumulation of NF-B (25).
PAK1, a member of the PAK family of serine/threonine kinases (26), is
activated by the Rho family GTP-binding proteins Rac1 and Cdc42 (27,
28). Activation of these GTPases is induced by guanine nucleotide
exchange factors, which catalyze the exchange of GDP for GTP (29). In
addition to activation, guanine nucleotide exchange factors may
regulate downstream signaling events of Rac1 and Cdc42 (30). These
downstream signaling events include activation of many of the same
pathways induced by LPS such as the JNK and p38 kinases (31-33).
Although Rac1 and Cdc42 have multiple effectors, the Rac1- and
Cdc42-mediated increase in p38 activity is dependent on PAK1 (32).
Thus, there is accumulating evidence in the literature that the PAK1
pathway plays an important role in LPS-mediated signal transduction.
Many studies have suggested a role for PAK1 in cytoskeletal
reorganization and cell motility (34-37), and several physiologic signals such as thrombin, insulin, and epidermal growth factor have
been reported to activate PAKs (28, 38, 39). However, the role of PAK1
in transcriptional regulation is not well understood. In this work, we
studied the role of the Cdc42/Rac/PAK1 pathway in the
LPS-induced regulation of the SR-BI promoter in macrophage RAW 264.7 cells.
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MATERIALS AND METHODS |
In Vivo Studies--
Male C57BL/6 mice were injected
intraperitoneally with 10 mg/kg LPS; and 48 h later, macrophages
were harvested from the peritoneal cavity of vehicle- or LPS-treated
animals. The cells were washed several times with cold RPMI 1640 medium
(serum-free), and cell lysates were processed for Western blotting.
Cell Culture Studies--
RAW 264.7 cells were grown to 80%
confluency in 100-mm dishes in Dulbecco's modified Eagle's medium
(DMEM; Life Technologies, Inc.) containing 10% fetal bovine serum.
Growth medium was removed and replaced with DMEM containing 10%
lipoprotein-deficient serum, and the cells were incubated for an
additional 24 h. LPS (1 µg/ml) was added directly to fresh
medium (DMEM containing 10% lipoprotein-deficient serum), and the
cells were incubated for an additional 1, 3, 6, or 8 h and used
for total RNA preparation or for an additional for 6, 12, 24, and
36 h and used for protein extract preparation.
mRNA Preparation and Quantification--
Total RNA was
prepared using the QIAGEN RNeasy mini kit, and DNase was treated
according to the manufacturer's protocol (Ambion Inc.). mRNA
expression was analyzed using real-time quantitative PCR on an
Applied Biosystems Prism 7700 sequence detection system. Primer/probe sequences used for mouse SR-BI were as follows: SR-BI forward primer, 5'-AATGACAACGACACCGTGTCC-3'; reverse primer,
5'-TGCGACTTGTCAGGCTGG-3'; and probe,
5'-FAM-CGTGGAGAACCGCAGCCTCCATTTAMRA-3', where FAM
(6-carboxyfluorescein) and TAMRA (6-carboxytetramethylrhodamine) are
fluorescent labels as described by the vendor. Final quantification was
done using the comparative CT method according to
the manufacturer's protocol (catalog no. 4304671, PE Biosystems), and
data are reported as mRNA expression relative to
glyceraldehyde-3-phosphate dehydrogenase internal control transcription.
Western Blotting--
Cell protein extracts were prepared by
removing the growth medium from cell cultures, washing the cells with
cold phosphate-buffered saline, and immediately lysing them with
radioimmune precipitation assay lysis buffer (50 mM
Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.25%
sodium deoxycholate, and protease inhibitors (Complete protease
inhibitor mixture, Roche Molecular Biochemicals)). Whole cell lysates
were centrifuged at 10,000 × g for 10 min to remove cell debris. Protein concentration was determined in the supernatants using the Bradford assay (49). Lysates were solubilized in
Laemmli sample buffer, and 40 µg of protein was loaded onto each lane of an 8-18% Tris/glycine gel (Novex) and subjected to
electrophoresis. After electrophoresis, separated proteins were
transferred to nitrocellulose membranes and immunoblotted with a 1:1500
dilution of a rabbit anti-SR-BI polyclonal antibody (catalog number
400-104, Novus Biologicals) or a 1:1000 dilution of a mouse
anti-glyceraldehyde-3-phosphate dehydrogenase monoclonal antibody
(clone 6C6, Advanced Immunochemical) to verify equal amounts of
protein loaded. After extensive washing with phosphate-buffered saline
and 0.2% Tween 20, the blots were incubated with horseradish
peroxidase-conjugated secondary antibodies (1:2000 dilution), and
detection of immune complexes was carried out with the ECL Western blot
detection system (Amersham Pharmacia Biotech).
Plasmid Constructs--
A 739-base pair fragment (
896 to
157) and a 407-base pair fragment (564 to 157) of the human SR-BI
promoter were generated as outlined in the Advantage-GC genomic
polymerase chain reaction kit (CLONTECH) using
human genomic DNA as the template with the following primers: forward
primers with NheI linkers (underlined), 5'-CCGGGCTAGCCAGTTGGAGCACATGGTCAGAATGCAAG (primer
739) and 5'-CGCGGCTAGCAGAGGAGGAGAGGGAGGAGGAGGGAAAAG (primer
407); and the reverse primer containing an XhoI linker (underlined), 5'-GGCGCTCGAGGGTGGCCAGTGGTTTTATGCCCCATCG. The
polymerase chain reaction products were gel-purified and directionally
cloned into the pGL3-Basic vector (Promega) with NheI and
XhoI digestion. 5'-Deletion constructs were generated from
the 739-base promoter by linearization with NheI, followed
by Bpu1102 (413 to 157), ApaI (255 to
157), or CspI (199 to 157) digestion. The 5'-flanking sequence was numbered according to the start codon (+1) as demonstrated by Cao et al. (6). The shortened constructs were then
gel-purified and religated. All SR-BI construct sequences were
verified. The constitutively active Cdc42 Q61L and dominant-negative
Cdc42 T17N expression constructs were a gift from Dr. Ian Macera
(University of Virginia). The constitutively active RacV12 and
dominant-negative RacN17 expression constructs were a gift from Onyx
Pharmaceuticals, Inc. (Richmond, CA). All PAK1 expression constructs
have been previously described (35, 36) and were provided by Dr.
Jonathan Chernoff (Fox Chase Cancer Center, Philadelphia, PA). The
pFB-Neo-NIK construct (provided by Jeffrey Marine, Pfizer) was
generated by inserting 3 kilobase pairs of NIK coding sequence
between the BamHI and NotI sites of the pFB-Neo
retroviral vector. The p65, pRSV-
gal, and NF-B response
element constructs were kindly provided by Dr. Joseph Menetski (Pfizer).
Cell Transfections--
The murine macrophage RAW 264.7 cell
line was obtained from American Type Culture Collection (Manassas, VA)
and maintained in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 300 µg/ml
L-glutamine. Cells were seeded overnight at 200,000 cells/well in 24-well tissue culture plates (Costar Corp.). The medium
was then replaced with 0.5 ml/well phenol red-free DMEM supplemented
with 5% charcoal/dextran-treated bovine serum (Hyclone Laboratories).
Cells were transfected in triplicate for 6 h with 100 µl of
Opti-MEM mixtures (Life Technologies, Inc.) containing a total of 0.5 µg of experimental plasmid, 0.05 µg of pRSV-gal
normalization plasmid, and 1 µl of LipofectAMINE 2000 (Life
Technologies, Inc.). One ml of phenol red-free DMEM supplemented with
5% charcoal/dextran-treated bovine serum was added to each well after
6 h of transfection, and the cells were then treated with LPS (1 µg/ml; Escherichia coli serotype 0111:B4, Sigma) or saline
as indicated for 16 h. In cotransfection experiments, controls
were cotransfected with empty pGL3-Basic vector to keep total DNA
concentrations constant (empty pGL3-Basic vector luciferase values were
confirmed to read at background levels). Luciferase and
-galactosidase samples were prepared with the luciferase assay
system (Promega) and the Galacto-Star kit (Tropix Inc.), respectively.
Luciferase and -galactosidase activities were then measured in a
microplate luminometer (EG&G Berthold).
Nuclear Extracts--
RAW 264.7 cells were treated with 1 µg/ml LPS or saline vehicle for 16 h. Cells were then rinsed
with cold phosphate-buffered saline and lysed for 5 min in hypotonic
buffer containing 0.2% Nonidet P-40, 20 mM HEPES, 20 mM NaF, 1 µM Na3VO4,
1 µM phosphate buffer, 1 µM EGTA, 1 µM dithiothreitol, 0.5 µM
phenylmethylsulfonyl fluoride, 0.125 µM okadaic acid, and
Complete protease inhibitor mixture (one tablet/50 ml). Cells were then
scraped and centrifuged at 16,000 × g for 20 s.
Nuclear proteins were extracted from the pellet in hypotonic buffer
containing 0.5 M NaCl and 25% glycerol with gentle rocking
for 30 min, followed by centrifugation at 16,000 × g
for 20 min. Nuclear proteins contained in the supernatant were
quantitated with the BCA protein assay kit (Pierce) and frozen at
80 °C.
Electrophoretic Mobility Shift Assays--
Four double-stranded
oligonucleotide probes spanning the SR-BI promoter from 564 to 413
were synthesized and labeled with T4 polynucleotide kinase using
[
-32P]ATP (3000 Ci/mmol) as the donor. Labeled probes
were incubated with 10 µg of nuclear extract protein for 30 min at
room temperature. Samples were then separated on a 0.5× Tris
borate/EDTA and 5% acrylamide gel for 1 h at 300V. Gels were
dried and developed with Hyperfilm MP (Amersham Pharmacia Biotech).
Further analysis of the LPS-responsive region was conducted as
described above with three shorter overlapping double-stranded
oligonucleotide probes (A (497 to 477), C (487 to 466) and B
(476 to 456)) of the SR-BI promoter. Competition assays were
conducted with a 10-100-fold molar excess of unlabeled probes A, B,
and C. Sp1 and AP-1 consensus sequence probes were obtained from
Promega. Double-stranded MZF-1 consensus sequence probes were
synthesized (Life Technologies, Inc.) as follows (sense strands shown):
MZF-1A, 5'-GATCTAAAAGTGGGGAGAAAA; and MZF-1B,
5'-GATCCGGCTGGTGAGGGGGAATCG. Competition assays with transcription
factor-binding site probes were conducted with a 100-fold molar excess
of unlabeled probe.
Statistical Analysis--
The unpaired t test
(two-tailed) was used to compare mean experimental values with the
corresponding control values as described in the figure legends.
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RESULTS |
LPS Inhibits SR-BI Expression in Vitro and in Vivo--
LPS
down-regulates SR-BI expression in human monocytes and macrophages
in vitro (16). These data were confirmed using the macrophage-like RAW 264.7 cells. LPS (1 µg/ml) treatment of these cells resulted in a time-dependent decrease in SR-BI
mRNA levels, with peak reductions at 6 h post-treatment (Fig.
1A). Western blot analysis of
RAW 264.7 cells treated with LPS also indicated a dramatic reduction in
SR-BI protein levels (Fig. 1B). In vivo confirmation of these results was obtained using peritoneal macrophages from LPS-treated C57BL/6 mice. Intraperitoneal injection of these animals with LPS (10 mg/kg) resulted in nearly complete elimination of
SR-BI protein in peritoneal macrophages as determined by Western blot
analysis, whereas it had very minimal effects on the levels of the
-actin control (Fig. 1C). Based on these findings, we conclude that LPS has a powerful negative effect on SR-BI expression in
macrophage cells both in vitro and in vivo.
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Fig. 1.
LPS down-regulates macrophage SR-BI
expression in vitro and in vivo.
A, regulation of SR-BI expression in RAW 264.7 cells. Total
RNA was isolated from mouse macrophages incubated with 1 µg/ml LPS
for 1, 3, 6, or 8 h. Data are expressed as the relative mRNA
expression of SR-BI/glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), where expression at 0 h was arbitrarily set at
1. Values represent the means ± S.E. of triplicate
determinations. B, Western blot analysis of SR-BI protein
expression. Whole cell lysates were prepared from RAW 264.7 cells
incubated with LPS (1 µg/ml) for 6, 12, 24, or 36 h.
C, whole cells lysates of peritoneal macrophages from mice
treated with 1 µg/ml LPS for 2 days. Glyceraldehyde-3-phosphate
dehydrogenase immunoblotting confirmed equal amounts of protein.
mwm, molecular weight marker.
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LPS Inhibits SR-BI Promoter Activity--
LPS has been
demonstrated to down-regulate SR-BI mRNA levels in human monocytes
and macrophages (16); however, the effects of LPS on the
transcriptional activity of the SR-BI promoter have not been studied.
To evaluate the effect of LPS on SR-BI promoter activity, RAW 264.7 cells were transfected with the SR-BI 896/157 promoter construct
and treated for 16 h with LPS (1 µg/ml) or saline as a control.
As shown in Fig. 2, the combined results from six independent experiments each performed in triplicate demonstrate that LPS treatment resulted in a significant
(p < 0.001) reduction in SR-BI 896/157 promoter
activity. To delineate the LPS-responsive region, shortened promoter
constructs were generated and transfected into RAW 264.7 cells. As
shown in Fig. 3, LPS treatment resulted
in a significant decrease in the activity of the 896/157 and
564/157 promoter constructs; however, LPS did not alter the
activity of the shorter promoter constructs. These results indicate
that the LPS-responsive region lies between 564 and 413 of the
promoter.
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Fig. 2.
LPS inhibits SR-BI promoter activity.
RAW 264.7 cells were transiently transfected with the SR-BI 896/157
promoter cloned into the pGL3-Basic luciferase reporter vector and
treated for 16 h with LPS (1 µg/ml) or saline as a control.
Transfection efficiency was normalized with a pRSV-gal plasmid.
Shown are the means ± S.E. of six independent experiments, each
performed in triplicate. *, p < 0.001.
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Fig. 3.
The LPS-responsive region lies
between 564 and 413 of
the SR-BI promoter. The 5'-deletion SR-BI 413/157,
255/157, and 199/157 promoter constructs were generated from
the 896/157 promoter construct with restriction enzymes as
described under "Materials and Methods." Constructs were
transiently transfected into RAW 264.7 cells and treated for 16 h
with LPS (1 µg/ml) or saline as a control. Transfection efficiency
was normalized with a pRSV-gal plasmid. Means ± S.E. are shown
from a representative of three independent experiments, each performed
in triplicate. LPS treatment significantly decreased the activity of
the 896/157 and 564/157 promoters (p < 0.05). Lucif/BGal, luciferase/-galactosidase.
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The Serine/Threonine Kinase PAK1 Inhibits SR-BI Promoter
Activity--
Given that LPS has been demonstrated to activate PAK1 in
RAW 264.7 cells (25) and that PAKs can activate important
transcriptional regulatory cascades such as NF-B, p38 MAPK, and JNK
(25, 32, 33), the ability of PAK1 to down-regulate the SR-BI promoter was tested. As shown in Figs. 4 and 6,
cotransfection of RAW 264.7 cells with the constitutively active PAK1
construct (T423E) and the SR-BI 896/157 promoter construct resulted
in a significant reduction in promoter activity. Cotransfection of the
kinase-deficient PAK1 construct (K299R) also significantly decreased
the activity of the promoter (Figs. 4 and 6). These results demonstrate
that PAK1-mediated down-regulation of the SR-BI promoter is independent of PAK1 kinase activity. No additional decrease in promoter activity was observed in PAK-transfected cells when treated with LPS (data not
shown), suggesting that PAK1 mediates the LPS-induced decrease in
promoter activity.
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Fig. 4.
PAK1 inhibits SR-BI promoter activity
independent of kinase activity. RAW 264.7 cells were cotransfected
with the SR-BI 896/157 promoter construct and the constitutively
active (ca) or kinase-deficient (kd) expression
construct for 16 h. Transfection efficiency was normalized with a
pRSV-gal plasmid. Shown are the means ± S.E. of normalized
luciferase values from three independent experiments, each performed in
triplicate (p < 0.02).
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Rac and Cdc42 Inhibit the SR-BI Promoter--
To
provide further evidence that the Cdc42/Rac/PAK1 pathway is involved in
regulation of the SR-BI promoter, we tested the ability of known
upstream activators of PAK1 to mimic the PAK1 response. It is well
established that the small G-proteins Rac and Cdc42 activate PAK1 (27).
To evaluate the role of these G-proteins in the regulation of the SR-BI
promoter, RAW 264.7 cells were cotransfected with constitutively active
or dominant-negative G-protein expression constructs and the SR-BI
896/157 promoter construct. As demonstrated in Fig.
5, expression of the constitutively active forms of Cdc42 (Q61L) and Rac (RacV12) significantly inhibited promoter activity. As in the PAK1 transfectants, no additional decrease
in promoter activity was observed when constitutively active Cdc42- or
Rac-transfected cells were treated with LPS or when cells were
transfected with both active G-protein and PAK1 constructs (data not
shown). These findings suggest that Cdc42 and Rac mediate the
LPS-induced down-regulation of the SR-BI promoter. Furthermore, as
shown in Fig. 5, expression of the dominant-negative Cdc42 T17N mutant
resulted in a 2-fold increase (p < 0.005) in the
activity of the promoter, whereas cotransfection of the
dominant-negative RacN17 mutant resulted in a smaller yet significant
increase (p < 0.05) in the activity of the promoter.
These dominant-negative G-protein constructs exert their negative
effect by trapping the guanine nucleotide exchange factors, which are
necessary for the disassociation of GDP and subsequent GTP binding that
activate these G-proteins (29). These results suggest that endogenous active Cdc42 and Rac inhibit the SR-BI promoter under basal conditions. In addition, when cells that were cotransfected with the
dominant-negative Cdc42 or Rac constructs were treated with LPS, the
response to LPS was blunted. Although LPS treatment resulted in a
reduction in promoter activity compared with the dominant-negative
constructs alone, this decrease was not significantly different from
that in the control cells transfected with the SR-BI promoter alone (Fig. 5).
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Fig. 5.
Rac and Cdc42 inhibit SR-BI promoter
activity. RAW 264.7 cells were cotransfected with the
constitutively active (ca) or dominant-negative
(d) Cdc42 or Rac expression construct and the SR-BI
896/157 promoter construct. LPS (1 µg/ml) was added as indicated.
Shown are the means ± S.E. of normalized luciferase values from
three independent experiments, each performed in triplicate at 16 h post-transfection. Expression of the constitutively active forms of
Cdc42 and Rac significantly inhibited promoter activity
(p < 0.005).
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Rac and Cdc42 Are Required for PAK1-mediated Down-regulation of
SR-BI Promoter Activity--
To evaluate the role of these G-proteins
in PAK1-mediated down-regulation of the promoter, double mutant PAK1
constructs containing mutations in both the kinase domain (K299R) and
the G-protein-binding domain (H83L and H86L) were cotransfected with
the promoter construct. As shown in Fig.
6, PAK1-mediated decreases in SR-BI
promoter activity were attenuated under both the kinase-deficient and
constitutively active PAK1 conditions when the G-protein-binding domain
was also mutated. These results demonstrate that PAK1-mediated
decreases in SR-BI promoter activity are dependent on a functional
G-protein-binding domain and suggest that the Cdc42- and Rac-induced
decreases in promoter activity are due at least in part to interactions
with PAK1.
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Fig. 6.
PAK1 inhibition of SR-BI promoter activity
requires the GTPase-binding domain. RAW 264.7 cells were
transiently cotransfected with the SR-BI 896/157 promoter construct
and kinase-deficient and constitutively active double mutant PAK1
constructs containing mutations in both the kinase domain and the
G-protein-binding domain (PAK1 kd/GBDm and PAK1
ca/GBDm, respectively). Shown are the means ± S.E. of
normalized luciferase values from three independent experiments, each
performed in triplicate at 16 h post-transfection. Promoter
activity was significantly higher in the double mutant constructs
compared with the kinase mutant constructs (p < 0.005).
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NF-B Is Not Involved in the LPS-mediated Down-regulation of the
SR-BI Promoter--
It is well established that LPS activates NF-B
in the monocyte/macrophage (40, 41). More recently, PAK1 activity has been demonstrated to be required for activation of NF-B in RAW 264.7 cells (25). To evaluate the role of NF-B in the LPS-induced down-regulation of the SR-BI promoter, RAW 264.7 cells were
cotransfected with a p65 expression construct. As shown in Fig.
7B, expression of p65 resulted
in an 18-fold increase in an NF-B response element-luciferase construct. However, cotransfection of the p65 expression construct had
minimal effects on the activity of SR-BI promoter constructs (Fig.
7A). To further confirm that activation of the NF-B
pathway did not alter SR-BI promoter activity, an upstream activator of NF-B, NIK, was coexpressed with the SR-BI promoter. As shown in Fig.
7D, overexpression of NIK resulted in a 5-fold increase in
the activity of the NF-B response element-luciferase construct. However, as in the p65 experiments, NIK had no effect on SR-BI promoter
activity (Fig. 7C). These results suggest that activation of
the NF-B pathway does not alter SR-BI transcriptional activity.
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Fig. 7.
Activation of NF-B
does not regulate SR-BI promoter activity. RAW 264.7 cells were
transiently cotransfected with SR-BI promoter constructs and the p65
expression construct (A) or the NIK expression construct
(C) for 16 h. Cotransfection control experiments with
the B response element (KBRE) with p65 expression and NIK
expression are shown in B and D, respectively.
Shown are representative results from three separate experiments, each
performed in triplicate. Lucif/Bgal,
luciferase/-galactosidase.
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LPS Inhibits Transcription Factor Binding to an MZF-1-like Element
in the SR-BI Promoter--
To further delineate the LPS-responsive
region of the SR-BI promoter (564 to 413) (Fig. 2), four
double-stranded oligonucleotide probes spanning the LPS-responsive
region were synthesized and labeled for gel shift analysis. As shown in
Fig. 8A, DNA-protein complexes
were observed with each of the four probes (GS1-4). However, GS-3
(497 to 456) revealed a prominent DNA-protein complex under control
conditions, which was inhibited in the LPS-treated cells. GS-3 was then
further divided into three probes (A, B, and C) and evaluated by gel
shift analysis. As shown in Fig. 8B, the protein-binding
region was confined to probe B, spanning 476 to 456. As observed in
GS-3 binding, treatment of cells with LPS inhibited protein binding to
probe B. This binding was shown to be specific, as competition
experiments with increasing concentrations of unlabeled probe B blocked
formation of the complex, whereas increasing concentrations of
unlabeled probes A and C had no effect on the DNA-protein complex (Fig.
8C). Transcription factor data base analysis of probe B
revealed two putative cis-elements: a heat shock factor
(HSF) element and an MZF-1 element (Fig.
9A). To determine whether
these putative sites were mediating protein binding to probe B, point
mutations at consensus sites were made and evaluated by gel shift
analysis. As depicted in Fig. 9A, a point mutation of the
HSF element (M1) did not alter protein binding, whereas a point
mutation of the MZF-1 element (M2) blocked protein binding. These
results are supported by analysis of probe C, which contained the HSF
element, but not the MZF-1 element, and did not demonstrate DNA-protein
interaction. However, two different consensus sequences for MZF-1 as
identified by Morris et al. (42) failed to compete for
binding with probe B (Fig. 9B), suggesting that MZF-1 is not
a component of the DNA-protein complex. In addition, AP-1 and
Sp1 consensus probes also failed to block DNA-protein interaction with probe B (Fig. 9C). To confirm that the
MZF-1-like element was involved in the LPS response, the M2 point
mutation was introduced in the SR-BI 896/157 promoter construct.
Transient transfection experiments with the M2 construct demonstrated a 30% decrease in the LPS-mediated repression of the promoter. Together, these results suggest that LPS inhibits the binding of a novel transcription factor that binds to an MZF-1-like element and activates transcription of the human SR-BI promoter.
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Fig. 8.
Gel shift analysis of the LPS-responsive
region. A, nuclear extracts from RAW 264.7 cells
treated for 16 h with LPS or saline as a control (C)
were incubated with four double-stranded oligonucleotide probes
spanning the LPS-responsive region (GS1-4) as described under
"Materials and Methods." GS-3 (497 to 456) demonstrated a
strong difference in DNA-protein complex formation, with decreased
complex observed in the LPS-treated cells (arrow).
B, GS-3 was further divided into three probes (A, B, and C)
and evaluated by gel shift analysis. The protein-binding region was
confined to probe B, spanning 476 to 456; and LPS inhibited protein
binding to probe B. C, competition experiments with
increasing concentrations of unlabeled probe B blocked formation of the
complex, whereas increasing concentrations of unlabeled probes A and C
had no effect on the DNA-protein complex. *B, labeled probe
alone.
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Fig. 9.
Novel transcription factor-binding site in
the LPS-responsive region. A, a putative HSF element
and an MZF-1 element were identified in the LPS-responsive region (GS-3
probe B). Point mutations at consensus sites in these elements were
made and evaluated by gel shift analysis using control nuclear
extracts. Mutation of the HSF element (M1) did not alter protein
binding, whereas mutation of the MZF-1 element (M2) blocked protein
binding. B, a 100-fold molar excess of unlabeled consensus
sequences for MZF-1 (MZF-1A and MZF-1B) failed to compete for binding
with probe B. C, a 100-fold molar excess of unlabeled AP-1
and Sp1 consensus probes also failed to block DNA-protein
interaction with probe B. C, control.
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DISCUSSION |
Previous studies have documented SR-BI expression in human
monocytes and macrophages (2, 8, 9) and in the murine macrophage cell
line RAW 264.7 (4). It has also been shown that LPS down-regulates
SR-BI mRNA and protein levels in monocytes and macrophages (16).
The results in this study confirm these in vitro findings
and demonstrate for the first time that LPS down-regulates SR-BI
protein levels in macrophages in vivo. The finding in this
study that LPS decreases SR-BI promoter activity in RAW 264.7 cells
provided a system to begin elucidating the signaling pathways involved
in LPS regulation of the SR-BI promoter in macrophage cells.
Although LPS signaling is complex and not completely understood, it has
been shown to involve activation of PAK1 and to lead to nuclear
accumulation of NF-B in RAW 264.7 cells (25). The results presented
in this study show that PAK1 inhibits SR-BI promoter activity and
suggest that LPS-mediated reduction in SR-BI promoter activity is
mediated by the PAK1 pathway. Importantly, PAK1 inhibition of the SR-BI
promoter was shown to be independent of PAK1 kinase activity since
expression of either constitutively active or kinase-deficient PAK1
resulted in a similar decrease in promoter activity. Several studies
have demonstrated kinase-independent effects of PAK1 on cytoskeletal
regulation (36, 37, 43); however, the kinase-independent effect of PAK1
on transcriptional regulation observed in this study is novel. Recent
studies showed that PAK1 up-regulates the vascular endothelial
growth factor promoter in MCF-7 cells. The vascular endothelial growth
factor promoter results provide additional evidence that PAK1 can alter transcriptional regulation; however, this regulation was shown to be
dependent on the kinase activity of PAK1 (44). Recent studies provide
evidence that the kinase-independent effects of PAK1 are due to
protein-protein interactions that occur upon recruitment of PAK1 to the
plasma membrane. The kinase-independent effect of PAK1 on neurite
outgrowth in PC-12 cells requires targeting of PAK1 to the plasma
membrane and is dependent on structural features in both the N- and
C-terminal domains of the molecule (43). In addition, PAKs have been
demonstrated to interact with a variety of proteins at the plasma
membrane such as the Cool/Pix proteins (45, 46). Our results support
the previous findings of kinase-independent effects of PAK1 and
furthermore demonstrate that these effects can alter transcriptional
regulatory mechanisms.
The importance of protein-protein interactions for PAK1 activity was
also documented in this study. The results show that the PAK1-mediated
reduction in SR-BI promoter activity was dependent on Rac and Cdc42
since PAK1 mutants unable to bind these GTPases had significantly less
activity than the PAK1 mutants with intact GTPase-binding domains.
Although Cdc42 and Rac are known to trigger the kinase activity of PAK1
(27), our results indicate that these GTPases have an additional role
in mediating the effects of PAK1 on SR-BI transcriptional regulation
perhaps through recruitment of PAK1 to the plasma membrane. Additional
evidence that Rac and Cdc42 were intimately involved in the
down-regulation of SR-BI promoter activity was shown by the significant
decreases in promoter activity in cells transfected with the
constitutively active GTPases. Furthermore, the dominant-negative Rac
and Cdc42 proteins blocked the LPS-mediated reduction in SR-BI promoter
activity and elevated the basal activity of the promoter. Taken
together, these results strongly suggest a role for Cdc42, Rac, and
PAK1 in the LPS-mediated decrease in SR-BI promoter activity. The
kinase-independent effects shown in this study in conjunction with the
observations noted by others suggest a model for PAK1 "activation"
in which the function of PAK1 is determined by both the kinase activity
of the enzyme and, perhaps more importantly, the assembly of signaling complexes.
The observation that LPS inhibits SR-BI promoter activity suggests that
two basic mechanisms may be involved. LPS either results in a loss of
transcriptionally active DNA-protein interactions or leads to formation
of repressive DNA-protein complexes. Gel shift analyses of the
LPS-responsive region as defined by 5'-deletion constructs suggested
that LPS inhibited binding of transcriptional activators to the SR-BI
promoter rather than leading to formation of repressive DNA-protein
interactions. Gel shift experiments narrowed the putative
LPS-responsive region of the human SR-BI promoter to 476 to 456.
Importantly, the steroidogenic factor-1 site previously demonstrated to
regulate human SR-BI promoter activity in adrenocortical cells (6) is
located at positions 218 to 212 and does not appear to be a major
mediator of basal transcriptional activity in RAW 264.7 cells, as
significant decreases in basal activity were observed in the construct
containing the steroidogenic factor-1 site (255 to 157). Together,
these observations indicate that transcriptional regulation of SR-BI is
cell type-dependent.
Transcription factor data base analysis of the putative LPS-responsive
region indicated that an MZF-1-like element was involved; and indeed, a
point mutation in this element at position 464 mimicked the
LPS-induced decrease in protein binding to the region. However, MZF-1
consensus probes were unable to compete for binding, suggesting that
the MZF-1 protein was not a component of the DNA-protein complex
inhibited by LPS. Given that this LPS-responsive region is G-rich,
characteristic of Sp1-binding sites, and that Sp1 has been implicated
in transcription of the rat SR-BI promoter (17), we tested the
possibility that the LPS-responsive region was binding the Sp1 protein.
However, gel shift competition assays with an Sp1 consensus probe
indicated that Sp1 was not a member of the protein complex disrupted by
LPS. In addition, experiments ruled out roles for AP-1 and NF-B in
the LPS-mediated reduction in promoter activity. Together, these
results suggest that a novel transcription factor binds to 476 to
456 of the human SR-BI promoter and drives basal transcription in the
macrophage. In addition, these results suggest that LPS disrupts
binding of this factor, thereby inhibiting SR-BI transcription. Indeed,
a point mutation in the MZF-1-like element, although not abolishing the LPS response, did reduce the LPS response, suggesting that the LPS
effect is due in part to the MZF-1-like element. Further experiments are needed to determine the nature of the factor binding to the MZF-1-like element and to decipher additional LPS-responsive regions in
the SR-BI promoter.
Although the extracellular milieu in an atherosclerotic plaque is
complex, thus exposing foam cells to a multitude of stimuli, recent
studies in platelets provide further evidence that PAK1 may be an
important physiologic regulator of SR-BI in the atherosclerotic vessel.
The coagulation protease, thrombin, has been shown to activate PAK1 in
platelets (38). In addition, ligation of the thrombin
protease-activated receptor (PAR-1) is known to activate Cdc42
and Rac in platelets (47). Importantly, the thrombin receptor has been
detected in macrophages (48), and thrombin is likely present in the
atherosclerotic vessel wall. These observations raise the possibility
that thrombin-induced activation of PAK1 may be an important
physiologic mechanism of SR-BI regulation in the foam cell and warrant
further exploration into the role of PAK1 activity in cholesterol
homeostasis in the atherosclerotic vessel wall.
In summary, we have documented that LPS and the PAK1 pathway
down-regulate human SR-BI promoter activity in the macrophage. These
results support the hypothesis that inflammatory mediators exacerbate
foam cell formation via down-regulation of the SR-BI receptor in the macrophage.
,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Cardiovascular
Therapeutics Dept., Pfizer Global Research and Development, 2800 Plymouth Rd., Ann Arbor, MI 48105. Tel.: 734-622-1585; Fax:
734-622-3135; Email: Thomas.Hullinger@Pfizer.com.
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