Original Contributions |
From the Gladstone Institute of Cardiovascular Disease and Cardiovascular Research Institute (M.M., R.E.P.), and the Departments of Pediatrics (M.M.) and Pathology (R.E.P.), University of California, San Francisco, and the Department of Medicine (C.K.G.), University of California, San Diego.
Correspondence to Robert E. Pitas, PhD, Gladstone Institute of Cardiovascular Disease, PO Box 419100, San Francisco, CA 94141-9100. E-mail rpitas{at}gladstone.ucsf.edu
| Abstract |
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Key Words: scavenger receptor activating protein-1 c-Junactivating kinase phorbol ester oxidative stress
| Introduction |
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DNA footprint analyses have identified 6 sites in the SR-A genomic sequence, from -246 to +46 bp relative to the transcriptional start site, that have potential significance for SR-A expression.6 A composite AP-1/ets binding element located between 67 and 50 bp is critical for SR-A expression during PMA-induced macrophage differentiation.6 Macrophage-specific expression is conferred by an Spi-1/pu.1 binding site located at 198 to 185 bp.6 While the proximal 245 bp of the SR-A promoter are sufficient to confer reporter gene expression,6 the addition of a transcription enhancer element at 4.1 to 4.5 kb increases basal expression and confers maximal induction by PMA in macrophages in cell culture.22 These promoter elements also target in vivo expression of a reporter gene to macrophage-derived foam cells within atherosclerotic lesions of transgenic mice.23
Here we report the results of studies to identify promoter and enhancer elements that are important for the regulation of SMC SR-A expression induced by PMA and reactive oxygen. We demonstrate that the composite AP-1/ets element is critical to the upregulation of SR-A promoter activity in SMCs. Furthermore, the specific c-Jun/AP-1 amino-terminal activating kinase (JNK) is induced in SMCs by the same conditions that increase SR-A expression. A CCAAT/enhancer binding protein (C/EBP) binding element is also necessary for full promoter activation in SMCs. These data demonstrate that redox-sensitive transcriptional control accounts for the induction of SR-A expression in SMCs under conditions that generate oxidative stress. Finally, we show that reactive oxygen can induce THP-1 monocytes to differentiate into macrophages and concomitantly induces macrophage SR-A receptor expression.
| Methods |
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Electromobility Shift Assays (EMSAs)
Nuclear extracts from rabbit and human SMCs, treated with either
100 nmol/L PMA or a combination of 100 µmol/L
H2O2 and 10 µmol/L
vanadate for 0.5, 1, 3, 6, 12, and 18 hours, were prepared by high-salt
microextraction26 and compared with nuclear
extracts from untreated (control) cells. For each reaction, the salt
concentration was adjusted to 100 mmol/L NaCl. Nuclear proteins (5
µg, unless otherwise specified) were preincubated at 37°C for 10
minutes with nonspecific inhibitor (1 µg of poly-dI/dC).
In control samples, a 50-fold molar excess of either unlabeled AP-1 or
AP-2 binding site oligonucleotide was added during the
preincubation. Binding patterns were evaluated in detail for the
AP-1/ets site at 67 to 50 bp and an additional hypersensitive site
at 44 to 21 bp. For the initial AP-1/ets time-course reactions, a
32P-labeled AP-1 consensus binding site
oligonucleotide (Promega), labeled by T4
polynucleotide kinase phosphorylation, was
used as a probe. After a preincubation period, the probe (10 000 cpm)
was added to each reaction mix, and the mixture was incubated at room
temperature for 20 minutes. Reactions were stopped with gel loading
buffer (at a final concentration of 10% glycerol), and the complexes
were resolved on 6% polyacrylamide gels. Subsequent EMSAs with
AP-1positive extracts were performed with the actual sequence of the
human SR-A promoter AP-1/ets binding element (67 to 50 bp) or
mutants of either the AP-1 or ets element, respectively, as previously
described.22 For supershift analyses, 200
ng of specific c-Jun, Jun-B, fos, or ets1/2 antibodies (Santa Cruz
Biotechnologies) was added to the protein extract incubations after
addition of the probe.
For additional binding studies, the human SR-A sequence of the 44- to 21-bp binding element plus flanking sequences was used as a probe. Competition was performed with unlabeled double-stranded oligonucleotide of the same SR-A sequence and with unlabeled C/EBP consensus wild-type (WT) and mutant binding elements. For supershift analyses, performed as above, 200 ng of specific antibodies for C/EBPß and GADD153 (Santa Cruz Biotechnologies) was used.
Endogenous JNK Kinase Assays
A pGEX4T-3 expression construct containing the amino-terminal 79
amino acids of c-Jun was kindly provided by Dr Silvio Gutkind (National
Institutes of Health, Bethesda, Md). The resulting fusion protein was
purified from bacterial lysates with the aid of glutathione-Sepharose
beads (Pharmacia), quantified after Commassie blue staining of
SDSpolyacrylamide gel electrophoresis gels by reference to
known concentrations of BSA, and used as the substrate for the kinase
assays.
Confluent human aortic SMCs were serum starved for 2 hours and then
incubated with agonists at 37°C. At specific time points, cells were
washed with PBS and lysed at 4°C in a buffer containing 25
mmol/L HEPES, pH 7.5, 0.3 mol/L NaCl, 1.5 mmol/L
MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L DTT,
20 mmol/L ß-glycerophosphate, 1 mmol/L vanadate, 1% Triton
X-100, 100 µg/mL PMSF, 20 µg/mL aprotinin, and 20 µg/mL
leupeptin. Cell lysates were rocked for 3 hours at 4°C in the
presence of 10 µg glutathione S-transferase (GST)c-Jun79
fusion protein bound to glutathione-agarose beads. Beads were washed
3x with PBS containing 1% Nonidet P-40 and 2 mmol/L vanadate and
once with 100 mmol/L Tris, pH 7.5, and 0.5 mol/L LiCl. Samples
were then resuspended in 30 µL of kinase reaction buffer containing 1
µCi [
-32P]ATP per reaction and 20
µmol/L of unlabeled ATP. After 20 minutes at 30°C, the reactions
were terminated by the addition of 10 µL of SDS gel loading buffer
(100 mmol/L Tris-HCl, pH 6.8, 200 mmol/L DTT, 4% SDS, 0.2%
bromophenol blue, and 20% glycerol), heated at 95°C for 5 minutes,
and separated by electrophoresis on 10% SDS acrylamide
gels.
Plasmid Constructs and Transfection Assays
The WT human SR-A promoter sequences from 245 to +46 bp alone
or with the 400-bp PMA-responsive enhancer element (4.5 to 4.1 kb)
were cloned upstream of the firefly luciferase cDNA in the expression
vector
5'PSV2 luciferase.22 Promoter mutants
were created by site-directed mutagenesis of the positive
transcriptional elements corresponding to the Spi-1/pu.1 site at 198
to 185 bp, the AP-1 and ets components of the composite AP-1/ets
element at 67 to 50 bp, and the sequence between 44 and 21 bp
(a newly identified C/EBP element), as previously
detailed.6 C/EBP site and AP-1/ets site
concatemer expression constructs were created by cloning 3 consecutive
sites, respectively, in tandem in front of a minimal human prolactin
promoter (69 to +33 bp) upstream of
5'PSV2 luciferase. Human SMCs
were transiently transfected by using 400 µg/mL DEAE-dextran and 1.0
µg of DNA per 35-mm plate at 50% confluence. Cells were treated in
duplicate with agonists 24 to 30 hours after transfection and harvested
for determination of luciferase activity 16 hours after treatment.
THP-1 and U937 cells were transfected by electroporation as previously
described.27 Low levels of background luciferase
activity were subtracted before calculating the relative luciferase
activity for treated versus untreated cells. All values were normalized
for the total cellular protein. An extensive series of studies has been
performed with ß-actinß-galactosidase reporter gene constructs as
internal standards. There was no evidence that ß-actin and SR-A
promoters influenced each other. Duplicate internal ß-galactosidase
control transfections were used to confirm comparable transfection
efficiencies in different experiments.
THP-1 Monocyte Differentiation Studies
THP-1 cells (106) were plated in 35-mm
wells in RPMI without serum and with or without PMA (100 nmol/L) or the
combination of H2O2
(100 µmol/L) and vanadate (10 µmol/L) and incubated at
37°C. After 48 hours, the medium was removed. Cells in suspension
(presumably undifferentiated) were counted, washed with PBS,
resuspended in RPMI containing 10% FBS, and incubated with
DiI-acetylated (Ac)LDL (5 µg/mL) overnight. The adherent cells in
their original plates were also washed with PBS and incubated with
fresh RPMI containing 10% FBS and 5 µg/mL DiI-AcLDL. After
incubation with DiI-AcLDL, adherent cells were lifted with a cell
scraper in 1x PBS. Cells were collected by
centrifugation, resuspended in PBS3%
paraformaldehyde, and analyzed with a
fluorescence-activated cell sorter (FACS) (cytometer
model 440, Becton Dickinson) for the uptake of DiI-AcLDL, as previously
described.7
| Results |
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30%, suggesting that basal levels of
AP-1 binding and activity in resting SMCs may account for some level of
basal transcription. Mutation of either the ets half of this composite
site or mutation of the C/EBP binding element located between 44 and
21 bp had no effect on basal promoter activity.
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AP-1 and C/EBP Elements Are Required to Induce SR-A Promoter
Activity in SMCs
Wild-type and mutant SR-A promoter and luciferase reporter
constructs were subsequently evaluated in the presence of treatments
known to upregulate the native SR-A gene in
SMCs.14 Both PMA and reactive oxygen
significantly increased transcriptional activity of the wildtype (WT)
SR-A promoter (Figure 1
). Mutation of the Spi-1/pu.1 element did not
significantly affect responsiveness of the promoter to PMA or oxidative
stress. Consistent with the critical role previously
demonstrated for the AP-1/ets element in SR-A gene induction in THP-1
macrophages, mutation of either the AP-1- or ets-binding
sequence within the composite site virtually abolished upregulation of
the SR promoter by either treatment in SMCs (Figure 1
). Interestingly,
despite the importance of the AP-1/ets element within the basal SR-A
promoter for maximal induction of SMC SR-A activity, the inclusion of
the 400-bp upstream enhancer element with its additional AP-1/ets
element did not affect the levels of induction by either treatment. In
contrast, the enhancer element increased SR-A induction by PMA 4- to
5-fold in macrophages.22
Another notable difference between SR-A regulation in
macrophages and SMCs relates to the C/EBP-binding element at
44 to 21 bp. Initially identified by DNase footprinting studies
with THP-1 macrophages and mouse macrophage p388D1 cell
extracts, this element exhibited no significant role in either basal or
inducible SR-A gene expression in
macrophages.6 However, mutation of this
site consistently reduced inducibility of SR-A expression in
SMCs by PMA or reactive oxygen by
50% (Figure 1
). These sequences
showed only low basal activity when spliced in front of a minimal
prolactin promoter in a luciferase reporter construct and were
minimally upregulated in SMCs by treatment with either PMA or reactive
oxygen (data not shown). This suggests that the factors binding to this
sequence serve a positive regulatory function in SMCs by cooperating
with AP-1 proteins.
Although the binding element at 44 to 21 bp exhibits only 60%
identity with the human C/EBP consensus sequence, it closely resembles
a C/EBP consensus site identified in a series of avian retroviral
long-terminal repeats28 (Figure 2A
). We evaluated the binding of proteins
within SMC nuclear extracts to the 44- to 21-bp sequence by EMSA
and revealed a tripartite binding pattern. Treatment with either PMA or
reactive oxygen only minimally increased the binding (Figure 2B
). The
binding appeared to be specific for the C/EBP family of transcription
factors. It was completely inhibited by competition with WT C/EBP
consensus binding site oligonucleotide but unaffected
by a mutant C/EBP site oligonucleotide. The ability of
a specific antibody to C/EBPß to shift the binding activity in all 3
bands suggests that this isoform accounts for most, if not all, of the
observed binding activity (Figure 2C
). Variable band intensities in
the complex binding pattern are attributed to the differential
translational and posttranslational processing characteristic of the
C/EBPß trans-activator
protein.29
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PMA or Reactive Oxygen Increases AP-1 Binding in Human
SMCs
Because the AP-1/ets binding element at 67 to 50 bp is
critical for the induction of SMC SR-A expression (Figure 1
), we
examined the induction of AP-1-binding activity in SMC nuclear extracts
isolated at baseline and on induction with either PMA or reactive
oxygen. Marked induction of AP-1 binding to the consensus AP-1 element
was seen in human SMCs after treatment. The levels of AP-1 binding
after PMA treatment were higher than those after treatment with
H2O2 and vanadate. A 4-hour
exposure of the gel containing samples treated with PMA (Figure 3
, left) is compared with an overnight
exposure of the gel containing samples treated with reactive oxygen
(Figure 3
, right). This results in greater intensity of AP-1 binding at
baseline for the longer exposure. A significant increase in AP-1
binding was apparent at 1 hour in human cells treated with PMA and by 2
hours after treatment with reactive oxygen. Levels returned to baseline
by 18 hours in reactive oxygentreated cells but appeared to be
sustained after PMA treatment. The binding activity was specific. In
duplicate samples of extracts taken at 2 hours after treatment, binding
activity was completely inhibited by a 50-fold molar excess of
unlabeled AP-1-binding site but was unaffected by a 50-fold molar
excess of the unrelated unlabeled AP-2-binding site.
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Because the human SR-A promoter AP-1-binding element differs slightly
from the consensus sequence, EMSAs were repeated with the human SR-A
gene AP-1/ets sequence as the probe (Figure 4
). The nuclear extract isolated 2 hours
after treatment with 100 µmol/L
H2O2 and 10 µmol/L
vanadate formed a specific complex with the native promoter AP-1/ets
element, which was completely inhibited with a 50-fold molar excess of
the unlabeled WT promoter element. There was no inhibition by unlabeled
oligonucleotide in which the AP-1 half of the composite
site had been mutated. However, a 50-fold molar excess of unlabeled
oligonucleotide in which the ets component of the
composite AP-1/ets site was mutated competed for the binding as
effectively as the WT sequence. Consistent with these
competition studies, the AP-1 mutant/WT ets site, when
labeled as the probe, formed no complex, whereas the WT AP-1/ets mutant
site as the probe gave a complex similar to the WT promoter element.
These studies confirm that AP-1 accounts for all of the observed
binding activity at the AP-1/ets site.
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The AP-1-binding activity was partially supershifted by addition of
c-Jun antibody, suggesting that this is a principal component of the
observed AP-1-binding activity (Figure 4
). Antibodies to Jun-B or fos
had no effect on the binding complex, nor did an antibody that
recognizes both ets1 and ets2 (data not shown). Taken together, these
data suggest that treatment of SMCs with either PMA or reactive oxygen
induces c-Jun/AP-1 binding. An ets protein likely cooperates with AP-1
on this composite element in vivo owing to the effect of the mutation
in the ets site on the response to PMA and reactive oxygen. The
relevant protein may be present in much lower amounts than AP-1
factors or may be degraded during extract preparation.
Both PMA and Reactive Oxygen Species Activate JNK
Because c-Jun is a component of the AP-1-binding activity
identified in SMCs, we evaluated the possibility that it was regulated
posttranslationally by H2O2
and vanadate. c-Jun is activated by
phosphorylation of serines at positions 63 and 73 in
the amino-terminal activation domain by a specific kinase,
JNK.30 JNK is itself activated by
threonine and tyrosine
phosphorylation.31
Endogenous JNK activity, as indicated by the ability of
treated SMC extracts to phosphorylate a c-Jun substrate
fusion protein, increased 4-fold 15 minutes after treatment with 100
nmol/L PMA (Figure 5
). The activity began
to decrease at 30 minutes and had nearly returned to baseline by 60
minutes. In a separate experiment, 100 µmol/L
H2O2 alone induced even
higher levels of JNK activity: a 10-fold increase at 15 minutes, which
was sustained at a lower level (4-fold) at 30 minutes and returned to
baseline by 60 minutes. Vanadate (10 µmol/L) alone had no
appreciable effect. Consistent with previously reported
synergism of H2O2 with
vanadate on SR-A induction in SMCs,14 the
combination of 10 µmol/L vanadate and 100 µmol/L
H2O2 increased JNK activity
11-fold at 15 minutes, increasing to 12-fold at 30 minutes, and falling
more slowly back to baseline by 90 minutes.
|
Oxidative Stress Induces THP-1 Monocyte-to-Macrophage
Differentiation and SR-A Gene Expression
The SR-A gene is constitutively expressed at high levels in
macrophages. Macrophage internalization of oxidized
lipoproteins is thought to contribute to cholesterol
engorgement and foam cell formation in atherosclerotic
lesions.1 Because the induction of SMC SR-A
expression is mediated in part by reactive
oxygen,14 we next investigated whether oxidative
stress per se could be linked to SR-A gene expression in the
macrophage. Treatment of THP-1 cells with the combination of
10 µmol/L vanadate and 100 µmol/L
H2O2 for 48 hours induced
up to 50% of monocytes to become adherent. Approximately two thirds of
the adherent cells actively took up DiI-AcLDL, as reflected in a
62-fold increase in mean fluorescence compared with control
cells (Figure 6
). PMA treatment was
somewhat more effective. Up to 90% of the cells treated for 48 hours
with PMA became adherent, of which 80% manifested increased DiI-AcLDL
uptake, reflected in a 70-fold increase in mean fluorescence
(Figure 6
). There was no uptake of DiI-AcLDL in nonadherent cells (data
not shown). The monocyte-to-macrophage differentiation and the
increase in uptake of DiI-AcLDL were correlated with specific SR-A
transcriptional upregulation. Because the AP-1/ets element is critical
for receptor expression in both SMCs and macrophages, this site
was concatemerized in front of a minimal prolactin promoter and
luciferase reporter gene. When this construct was transfected into
THP-1 cells, treatment with either PMA or a combination of 10
µmol/L vanadate and 100 µmol/L
H2O2 significantly
increased luciferase activity (Figure 6c
). The relative fold induction
in macrophages by PMA is twice that seen with
H2O2 and vanadate, as
previously observed in SMCs with the full SR-A promoter (Figure 1
).
Similar results were observed in a second monocyte cell line, U937
(data not shown). Taken together, these data establish that reactive
oxygen species exert a potent regulatory function in the expression of
the SR-A gene in macrophages as well as SMCs.
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| Discussion |
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In macrophages, AP-1 and ets are downstream targets of the macrophage colony-stimulating factor (M-CSF) receptor, which regulates macrophage proliferation and differentiation by coupling to components of several signal transduction pathways, including the Ras/Raf/mitogen-activated protein (MAP) kinase cascade. Mutations of the AP-1/ets-binding sites in the SR-A promoter and upstream enhancers abolish transcriptional responses to M-CSF in transgenic mice, indicating that these sites are of critical importance for the regulation of SR-A gene expression in vivo (F. Guidez and C.K. Glass, unpublished observations, 1997). The treatment of resident peritoneal macrophages with M-CSF has been associated with "priming" the oxidative burst.33 Although M-CSF receptors are not expressed by normal vascular SMCs, high-affinity binding of M-CSF with the resultant increase in tyrosine phosphorylation has been demonstrated in intimal SMCs isolated from a rabbit model of atherosclerosis.34 Increased tyrosine phosphorylation is associated with increased intracellular oxidative stress.35 H2O2 activates receptor tyrosine kinasemediated signaling events directly in vascular SMCs, leading to Ras activation.36 The current studies suggest that activation of Ras by oxidative stress may mimic receptor-dependent signaling in both SMCs and macrophages and is sufficient to activate the SR-A gene in both cell types. This promiscuous activation of the MAPK pathway could potentially contribute to cholesterol accumulation in atherosclerotic lesions.
SMC SR-A expression has been demonstrated in rabbit atherosclerotic lesions.13 We have demonstrated that SMC SR-A expression in stimulated rabbit SMCs is 3-fold higher than in human SMCs.14 Low levels of SR-A gene expression make its detection difficult in human lesions but do not preclude relevance of the SR-A in vivo in a disease process that evolves over decades. Furthermore, our studies demonstrate the existence of an oxidative stressresponse pathway in SMCs that results in the activation of AP-1 proteins and is likely to be relevant to the expression of multiple inflammatory SMC genes. Finally, we extended our analysis of the oxidative stress pathway to a human monocytic leukemia cell line, which has been a model for the study of SR-A gene expression in macrophages.
The AP-1-binding element within the proximal SR-A promoter is essential
for induction of SR-A gene activity in both macrophages and
SMCs. AP-1 has long been known to mediate gene induction by
PMA.37 AP-1 transcriptional activity has since
been found to be induced by numerous growth factors and
cytokines. Many of the factors that induce AP-1
activity38 have been independently shown to
induce intracellular oxidative stress, including
PMA,14 39 platelet-derived growth
factor,40 transforming growth
factor-ß,41 tumor necrosis factor-
, and
interleukin-1.42 These factors have also been
associated with the induction of SMC SR-A
activity.7 12 13 Our previous observation, that
direct treatment of either human or rabbit SMCs with reactive oxygen
species was sufficient to induce SR-A
expression,14 led us to hypothesize that reactive
oxygen serves as a common denominator in the mediation of SR-A
upregulation by numerous factors in atherosclerotic lesions. Duplicate
copies of an AP-1/ets composite element closely resembling the sequence
between 67 and 50 bp of the human SR-A promoter have been
identified between 981 and 769 bp of the intercellular adhesion
molecule-1 gene promoter. Consistent with our observations,
this site has also been identified as an
H2O2-responsive
element.43
c-Jun contributes significantly to the observed AP-1-binding activity
induced in SMCs by either PMA or reactive oxygen treatment (Figure 4
).
Although c-Jun expression is rapidly induced by extracellular signals,
its posttranslational activity is tightly regulated by protein
phosphorylation.30 PKC activation
decreases phosphorylation of c-Jun by casein kinase II
at sites that negatively regulate its
activity,44 45 and we have recently shown that
PKC plays a role in SMC SR-A upregulation.14
However, this alone only partially accounts for c-Jun activation. c-Jun
phosphorylation by JNK (the specific c-Junactivating
kinase) at 2 serines within the c-Jun trans-activation
domain is required for maximal transcriptional
activity.46 47 48 JNK, a serine/threonine kinase,
is itself activated by dual threonine/tyrosine
phosphorylation.31 The activation
of JNK by several forms of cellular stress, including UV irradiation,
heat shock, and inflammatory cytokines, has resulted in the
description of the JNK subgroup of MAPKs as
"stress-activated" protein kinases.49
The potent induction of JNK activation by UV light strongly suggests
that oxidative stress mediates its activation, as UV exposure causes
lipid peroxidation and decreases the level of reduced glutathione in
cells.50 The UV response, like the response to
oxidative stress, is characterized by the induction of tyrosine kinase
activity via the inhibition of tyrosine
phosphatases.51 The sensitivity of protein
tyrosine phosphatases to reactive oxygen species has been attributed to
a critical conserved cysteine residue that is vulnerable to oxidative
modification.52 JNK activation in turn results
from unopposed tyrosine kinase activity. Consistent with this
scheme, reactive oxygen species were recently directly implicated in
JNK activation by cytokines.53 Our
current results demonstrate that the same conditions associated with
increased AP-1 binding and SR-A gene expression within SMCs also
activate JNK, providing the first direct link between this
stress-activated MAPK and SR-A regulation.
In addition to AP-1/ets factors, the functional significance of an
additional site within the basal promoter at 44 to 21 bp has led to
the identification of cooperative involvement of C/EBPß in SMC SR-A
transcription. Although the 44- to 21-bp element conforms only
loosely to the consensus C/EBP-binding site, it closely resembles
elements identified within avian retroviral long-terminal repeats that
were found to bind C/EBP specifically (Figure 2A
). The C/EBP family is
composed of at least 3 major members designated as
, ß, and
,
characterized by the presence of a conserved domain in the
carboxyl-terminal region of the protein. C/EBP isoforms are implicated
in regulating processes relevant to cellular proliferation,
differentiation, and expression of cell typespecific
genes.54 Although C/EBP
expression is
restricted to the liver and adipose tissues, C/EBPß and
are
widely expressed. We demonstrate in this report that a tripartite
protein-DNA interaction involving the SR-A promoter sequences at 44
to 21 bp can be attributed to C/EBPß (Figure 2
). The intron-less
gene of the C/EBPß trans-activator protein
encodes a single mRNA, which in turn results in several related
proteins,29 which may explain the complex binding
pattern observed. The various isoforms have been attributed to
differential initiation and inhibition of translation at specific AUG
sites within the mRNA.55 Various prooxidants,
including lipopolysaccharide,
cytokines,56 and heavy
metals,57 have been associated with increased
expression, phosphorylation, and binding of C/EBPß,
with resultant induction of several acute-phase genes. There is
precedent for coordinate regulation by adjacent AP-1- and C/EBP-binding
elements in the promoter of another redox-sensitive gene. A 268-bp
promoter fragment that mediates basal level and inducer-dependent
activation of the mouse HO-1 gene contains 2 AP-1 sites with 2
C/EBP-binding sites directly upstream. Site-directed mutagenesis of 1
or more of the C/EBP and AP-1 sites revealed that each of the 4
elements is required for optimal activity of the enhancer
fragment.57
The enhancement of gene expression by oxidative stress is likely to represent a protective physiological response. Activated macrophages produce high levels of superoxide via NADPH oxidase activity, which results in a variety of reduced oxygen metabolites that are potent bactericidal and tumoricidal agents. Yet the macrophages themselves survive. At least 2 other gene products induced in both macrophages and SMCs by oxidative stress are involved in mediating important cytoprotective responses to free-radical generation, HO-1, and mouse stress-inducible 23-kDa protein (MSP23).58 59 In the case of HO-1, it has been speculated that gene induction leads to a reduction in the cellular pool of heme and heme-containing proteins, thereby removing potential prooxidant catalysts.60 Furthermore, the end product of the heme degradation pathway mediated by HO-1 is bilirubin, which has intrinsic antioxidant properties.61 MSP23 has recently been shown to belong to a new type of thiol-specific antioxidant family, which protects glutamine synthetase from inactivation by a mixed metal-thiol oxidation. HO-1 and MSP23 appear to be part of a coordinate antioxidant cellular response.59 Upregulation of SR-A activity by oxidative stress may also be consistent with cellular self-defense. Innate immune defense systems, like the SR-A, use cell-surface proteins to recognize toxic substances according to their particular carbohydrate structures.62 The fact that bacterial lipopolysaccharide is a ligand for SR-A,63 as well as the recent demonstration of increased susceptibility to infection in the SR-A knockout mouse model,64 supports a protective physiological role for this receptor. Oxidatively modified LDL in atherosclerotic lesions contributes to vascular oxidative stress65 66 and to the induction of numerous inflammatory genes that contribute to atherosclerosis.67 Oxidized LDL also increase SR-A gene expression in SMCs (preliminary data not shown). In the context of excess oxidized lipoproteins, however, the "protective" removal of these proinflammatory cytotoxins by SRs appears to represent a 2-edged sword, which results in foam cell formation and atherogenesis.
| Acknowledgments |
|---|
Received December 29, 1997; accepted March 27, 1998.
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