From Discovery Research, AtheroGenics, Inc., Alpharetta, Georgia 30004 and ¶ Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77025
Received for publication, April 2, 2002, and in revised form, September 9, 2002
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ABSTRACT |
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Atherosclerotic lesions preferentially develop in
areas of the vasculature exposed to nonlaminar blood flow and low fluid shear stress, whereas laminar flow and high fluid shear stress are
athero-protective. We have identified a set of genes including NAD(P)H:quinone oxidoreductase-1 (NQO1), heme oxygenase-1 (HO-1), ferritin (heavy and light chains), microsomal epoxide hydrolase, glutathione S-transferase, and Vascular endothelial cells are exposed to a tangential shearing
force resulting from the flow of blood over the lumenal surface of the
vessel wall (1). The nature and magnitude of this fluid shear stress
play a key role in the maintenance of vascular integrity and in the
development of vascular diseases. For example, the nonrandom
distribution of atherosclerotic lesions is due at least in part to
local alterations in hemodynamic forces impinging on the vasculature
(2-4). At sites vulnerable to lesion formation such as branch points,
bifurcations, and curvatures, unidirectional laminar flow is disturbed,
with areas characterized by complex flow patterns such as nonlaminar
flow and flow reversal. In contrast, lesion-protected areas of the
vasculature are characterized by more uniform laminar flow patterns
with relatively high levels of fluid shear stress (2-4). It is now
well accepted that areas of the vasculature exposed to nonlaminar or
oscillatory flow have a predilection for the development of
atherosclerotic lesions, whereas relatively high levels of steady
laminar flow are athero-protective.
Differential regulation of endothelial gene expression by shear stress
may be involved in the focal localization of early atherosclerotic
lesions (5). For example, pro-atherogenic genes such as
platelet-derived growth factor and monocyte chemoattractant protein-1
are transiently up-regulated by shear stress, peak at 1.5 h, and
are followed by sustained down-regulation (6, 7). These initial
responses of cultured endothelial cells to applied shear stress may
reflect in vivo responses to the temporal and spatial
changes of shear stress resulting from nonlaminar flow. Prolonged
exposure of endothelial cells to oscillatory flow results in
up-regulation of vascular cell adhesion molecule-1
(VCAM-1),1 intercellular
adhesion molecule-1, and E-selectin (8), suggesting that oscillatory
flow imparts a proinflammatory phenotype in endothelial cells. In
contrast, physiological levels of laminar flow are anti-inflammatory and anti-adhesive. Prolonged exposure of murine endothelial cells to
laminar flow results in down-regulation of the expression of VCAM-1,
intercellular adhesion molecule-1, and E-selectin (9). We reported that
chronic laminar flow suppresses interleukin-1 The antioxidant response element (ARE), also referred to as the
electrophile response element (EpRE), is a cis-acting
regulatory element with a core sequence of 5'-RTGACNNNGC-3' (13,
14). The ARE is present in the 5'-flanking regions of several genes encoding enzymes involved in the phase II metabolism of xenobiotics and
antioxidant proteins including glutathione S-transferase
(GST) (15), NAD(P)H:quinone oxidoreductase (NQO1) (16), and
glucuronosyltransferase (15). Activation of the ARE results from
cellular exposure to insults that induce oxidative stress, including
reactive oxygen species (ROS), ionizing radiation, and a variety of
chemical entities including electrophilic compounds, lipid peroxides,
antioxidants, Michael reaction acceptors, redox-cycling polyaromatic
hydrocarbons, and quinones (13, 15). The ability of phase II enzymes to conjugate redox-cycling chemicals is an important protective mechanism against electrophile and oxidative toxicity (13). Although this antioxidant defense mechanism has been studied extensively as a hepatic
detoxification mechanism, it has also been suggested that the ARE
pathway may contribute to antioxidant defenses via induction of other
ARE-regulated antioxidant proteins such as HO-1 (17) and ferritin (18).
There is a considerable body of evidence that suggests that HO-1 plays
an important protective role in the pathogenesis of a variety of
diseases mediated via oxidant stress and may serve as a therapeutic
target (19). For example, targeted expression of HO-1 prevents the
pulmonary inflammatory and vascular responses to hypoxia (20).
Induction of HO-1 in vascular cells suppresses oxidized low density
lipoprotein-induced monocyte transmigration and inhibits
atherosclerotic lesion formation in low density lipoprotein receptor
knockout mice (21, 22). In addition, ferritin, an iron sequestrant, is
up-regulated in response to oxidative stress (18). Overexpression of
ferritin protects endothelial cells from oxidant-mediated cytolysis
(23).
Through the analysis of subtraction libraries, we have identified
numerous genes whose expression is significantly increased by prolonged
exposure to laminar flow as compared with static culture in human
aortic endothelial cells (HAEC). In the present study, we report that
exposure of endothelial cells to laminar flow activates ARE-driven
transcriptional activity and increases the expression of a set of
ARE-regulated genes. These genes include NQO1, HO-1, microsomal epoxide
hydrolase, GST, ferritin, and Cell Culture--
HAEC were obtained from Clonetics, Inc. (San
Diego, CA) and cultured in EGM-2 growth medium (Clonetics,
Inc.). Cells were used between passages 5 and 9. Human
microvascular endothelial cells (HMEC) were described previously (24)
and were cultured in modified MCDB 131 (Invitrogen) supplemented with
10% fetal bovine serum and EGM Singlequots (Clonetics, Inc.). All
cells were maintained at 37 °C under 5% CO2.
Recombinant Plasmid--
To construct p3xARE/Luc and
p3xmutARE/Luc, tandem repeats of double-stranded oligonucleotides
spanning the ARE of the human NQO1 promoter,
5'-CAGTCACAGTGACTCAGCAGAATC-3' (for wild type ARE) and 5'-CAGTCACAG
TGACTCATAAGAATC (for mutated ARE, the underlined sequences
represent mutation of conserved nucleotides GC to TA in the ARE), were
introduced into the SacI and BglII sites of PGL3
promoter plasmid (Promega Corp.) (25).
pNQO1CAT1.55 contains coordinates
LNCX-Nrf2 (an expression vector containing the full-length
murine Nrf2 gene) and LNCX-Nrf2R (containing murine
Nrf2 gene in an antisense orientation) have been described
previously (28). To generate a dominant negative Nrf2 mutant, a
DNA fragment that contains the Cap'nCollar homology region and the
basic leucine-zipper domain (amino acids 399-598) was amplified by PCR
reaction using the following primers:
5'-ATGTCACCAGCTCAAGGGCACAGTGC-3' (forward primer) and
5'-CCATCCTCCCGAACCTAGTT-3' (reverse primer). The amplified fragment was cloned into the NheI and XhoI sites
of pcDNA3 to generate pcDNA3DN-Nrf2. To construct NQO1
and Keap1/INrf2 (a cytoplasmic inhibitor of Nrf2)
mammalian expression vectors, full-length human NQO1 and
Keap1/INrf2 cDNA were generated by RT-PCR and cloned into
the HindIII and XbaI sites of pcDNA3 to
generate pcDNA3NQO1 or pcDNA3Keap1/INrf2, respectively.
p85VCAM/CAT is a chimeric reporter gene containing coordinates Flow System--
The flow system used has been previously
described (8, 12). Briefly, HAEC or HMEC were seeded onto
gelatin-coated glass slides and grown overnight before exposure to
flow. Cells were exposed to either static conditions (cells maintained
on glass slides in a 150-cm2 tissue culture dish) or
exposed to flow conditions. The glass plate containing the monolayer
was inserted into a parallel-plate flow chamber that was installed in a
closed loop flow system. For laminar flow experiments, endothelial
monolayers were subjected to laminar flow with shear stress of +20
dyn/cm2 or +5 dyn/cm2. For oscillatory flow
experiments, the endothelial monolayers were exposed to a very low mean
shear stress (less than 0.5 dyn/cm2) with a superimposed
instantaneous oscillatory shear stress that cycled between +5 and Subtraction Libraries--
Suppression subtraction hybridization
libraries were prepared from total RNA collected from HAEC exposed to
laminar flow or static cultures for 48 h using the
Clontech PCR-SelectTM Subtraction kit
(Clontech, Palo Alto, CA). This method for creating subtraction libraries is based on the method first described by von
Stein et al. (30) and Mueller et al. (31). In
general, this method "subtracts" both low and high abundance
mRNA populations that are in common between two RNA samples while
at the same time equalizing and enriching for differentially expressed
sequences. After construction of the subtracted libraries that were
enriched for sequences expressed preferentially in the laminar flow
population or the static population, the PCR-Select Differential
Screening kit (Clontech) was used to rapidly screen
for clones that were differentially expressed. Approximately 800 clones
from each of the laminar flow-enriched and static-enriched libraries
were hybridized to probes created from each library. Individual clones
demonstrating a differential pattern of hybridization were selected for
DNA sequence analysis. Clone identities were determined by BLAST
analysis of the GenBankTM DNA sequence data base.
Gene Expression Analysis--
Oligonucleotide primer pairs were
designed to amplify an ~500-base pair fragment from the 3' end of
each mRNA (Table I). Total RNA was
collected from HAEC using Trizol (Invitrogen). 3 µg of total RNA was
reverse-transcribed into cDNA by reverse transcriptase (Invitrogen), and the level of each gene product was measured by
semi-quantitative end-point RT-PCR analysis. Cycling conditions were
95 °C initial denaturation for 5 min, 26 cycles of 94 °C denaturation (20 s), 55 °C annealing (30 s), and 72 °C extension (20 s), followed by a 7-min extension at 72 °C. After amplification, 15 µl of the sample was electrophoresed on a 1.5% agarose gel and
stained by ethidium bromide. Relative band intensities were determined
by densitometry using the Bio-Rad Quantity ONE software. For each
primer pair, serial dilutions of each cDNA were assayed, and a
dilution that resulted in linear amplification was used for further
experiments. Each sample was assayed at least three times from the same
RNA sample, and results are expressed as relative levels compared with
Western Blot Analysis--
HAEC were lysed for 30 min on ice in
1 ml of a lysis buffer containing 0.5% Nonidet P-40, 50 mM
Hepes, pH 7.3, 150 mM NaCl, 2 mM EDTA, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM sodium
orthovanadate, and 1 mM NaF. Protein samples (15 µg) were
subjected to electrophoresis on 10% SDS-PAGE gels and transferred to a
nitrocellulose membrane. Antibody-bound protein bands were then
visualized via horseradish peroxidase-dependent
chemiluminescence (Amersham Biosciences). Rabbit anti-NQO1 antibody was
described previously (32). Mouse monoclonal antibody against
Transfection and Promoter Activity Assays--
HMEC were grown
to 60-70% confluence in 6-well plates and transfected with various
plasmids as indicated in figure legends using SuperFect transfection
reagent according to manufacturer's instructions (Qiagen, Inc). For
flow experiments, HMEC were grown to 60% confluence on gelatin-coated
glass plates. HMEC were transiently transfected with the indicated
plasmids using SuperFect reagents. 24 h after transfection, cells
were exposed to either laminar flow (shear stress = 20 dyn/cm2) or static conditions for 24 h. For
determination of CAT activity, cell extracts were prepared from HMEC by
rapid freeze-thaw in 0.25 M Tris, pH 8.0. 5 µg of protein
per sample was then incubated with 5 µCi of 14C-labeled
chloramphenicol (Amersham Biosciences) and 5 µg of
n-butyryl coenzyme A (Amersham Biosciences) for various
times. The acetylated chloramphenicol forms were extracted using a 2:1
mixture of 2,6,10,14-tetramethylpentadecane:xylenes and subjected to
centrifugation (33). The organic phase was removed and counted to
determine CAT activity. Plasmids pRL-TK or pRL-SV40 (Renilla luciferase
constitutively expressed under the control of the thymidine kinase or
SV40 promoter, respectively) were co-transfected in all samples and
were used to normalize for transfection efficiency. Renilla luciferase
activity was measured using a luciferase reporter assay system
according to the manufacturer's instructions (Promega). All CAT
activities were normalized against the Renilla luciferase activity.
Laminar Flow Increases the Expression of ARE-containing
Genes--
To identify genes that were differentially expressed by
exposure to laminar flow, we constructed cDNA subtraction libraries from HAEC kept in static culture or subjected to laminar flow (shear
stress = 20 dyn/cm2) for 48 h. Differentially
expressed clones from each enriched library were identified by
differential hybridization, and several hundred clones from each
library were selected for DNA sequencing. DNA sequence analysis
identified many genes that were relatively abundant in the laminar
flow-enriched library compared with the static-enriched library.
Analysis of the promoters of a subset of these genes including ferritin
(heavy and light chains), NQO1, HO-1, GST, microsomal epoxide
hydrolase, and
To confirm our results from the subtraction libraries that laminar flow
induces the expression of these genes, mRNA levels were further
evaluated by semi-quantitative RT-PCR. As shown in Fig.
1, all of the genes were induced,
although to varying degrees, by exposure to laminar flow for 48 h.
These observations confirm that exposure of HAEC to laminar flow for
48 h results in increased expression of ARE-containing
cytoprotective genes compared with static controls.
To characterize the role of fluid flow in the regulation of
ARE-mediated gene expression, we focused on the NQO1 and HO-1 genes.
The effects of time, flow rates, and types of flow were evaluated for
their contribution to NQO1 gene expression. As shown in Fig.
2A, the expression of NQO1 in
response to laminar flow increased progressively from 12 to 48 h.
Similarly, the expression of HO-1 in response to laminar flow increased
progressively from 6 to 48 h (Fig. 2B). In contrast,
the levels of NQO1 and HO-1 mRNA in static cultures remained
relatively constant throughout the time course (Fig. 2). As shown in
Fig. 3, the expression of NQO1 in
response to laminar flow at 48 h was also dependent on the levels
of shear stress. As compared with static cultures, laminar flow with
shear stress of 5 dyn/cm2 increased the NQO1 mRNA
levels ~7-fold. Increasing the shear stress to 20 dyn/cm2
resulted in an ~14-fold increase in NQO1 mRNA levels at 48 h. These data demonstrate that higher levels of shear stress result in
greater expression of NQO1 in endothelial cells.
Several endothelial cell genes are differentially regulated in response
to either laminar or nonlaminar flow. Laminar flow is believed to
confer an anti-inflammatory, anti-oxidant phenotype to the vasculature,
whereas nonlaminar flow or oscillatory flow imparts a greater oxidant
stress and a more pro-inflammatory phenotype. We compared the effects
of oscillatory flow and laminar flow on NQO1 gene expression at 48 h. As shown in Fig. 3, although exposure of HAEC for 48 h to
oscillatory flow with shear stress of ±5 dyn/cm2 induced
approximately a 3-fold increase in NQO1 mRNA levels, the magnitude
of increase was smaller compared with the more than 7-fold increase in
HAEC treated with laminar flow of the same magnitude of shear stress (5 dyn/cm2). These results suggest that not only the flow
pattern (laminar versus oscillatory) but also the absolute
level of shear stress differentially regulates NQO1 gene expression
in endothelial cells.
We further examined the effects of laminar or oscillatory flow on NQO1
protein levels by Western blot analysis. As shown in Fig.
4, exposure of HAEC for 48 h to
oscillatory flow (shear stress = ±5 dyn/cm2) resulted
in an ~2-fold increase in NQO1 protein level compared with static
culture. Exposure of HAEC to laminar flow (shear stress = 20 dyn/cm2) induced approximately a 6-fold increase in NQO1
protein compared with static culture. These data confirm the results
observed with NQO1 mRNA levels (Fig. 3B), demonstrating
that physiological levels of laminar flow are a potent activator of
ARE-regulated genes such as NQO1.
Laminar Flow Activates ARE-mediated Transcriptional Activity in
Endothelial Cells--
Because laminar flow induces the expression of
a set of genes that contain the ARE regulatory element, we hypothesized
that the ARE or ARE-like sequences may represent a novel and important cis-acting element that confers inducible expression in
response to shear stress. To confirm that the ARE functions as a shear stress-responsive transcriptional control element, we transiently transfected HMEC with p3xARE/Luc and subjected cells to laminar flow
(shear stress = 20 dyn/cm2) for 24 h. This
reporter gene contains three copies of the ARE from the NQO1 enhancer
linked to a SV40 promoter. HMEC were used because HAEC are relatively
refractory to transient transfection. Similar to HAEC, laminar flow
induced the expression of ARE-regulated genes in HMEC (data not shown).
As shown in Fig. 5A, laminar
flow treatment of HMEC resulted in a dramatic increase in the
ARE-driven promoter activity. In contrast, mutation of the conserved GC
nucleotides to TA abolished laminar flow-induced ARE-driven promoter
activity. These data demonstrate that the ARE is a newly identified
shear stress response element in endothelial cells.
To further determine the role of the ARE in laminar flow-induced gene
expression, we used transient transfection assays with the wild type
and ARE mutated NQO1 or HO-1 promoters. The data shown in Fig.
5B demonstrate that exposure of HMEC for 24 h to laminar flow resulted in substantial activation of the wild type NQO1
promoter. In contrast, when HMEC were transfected with pNQO1CAT1.55mARE (containing point mutations of the ARE in the NQO1 promoter), no
activation was observed by laminar flow (Fig. 5B).
Similarly, exposure of HMEC for 48 h to laminar flow activated
wild type HO-1 promoter pHO-1Luc4.0 (Fig. 5C). However,
mutation of the ARE in the HO-1 promoter abolished laminar flow-induced
ARE-driven HO-1 promoter activity (Fig. 5C). These
observations directly demonstrate that laminar flow activation of the
NQO1 and HO-1 genes in endothelial cells occurs at the transcriptional
level. More importantly, activation of NQO1 and HO-1 by laminar flow requires a functional ARE element. Cumulatively, these data indicate that the ARE transcriptional element is an important mediator of
signals generated in endothelial cells by exposure to shear stress.
Nrf2 Mediates Laminar Flow-induced Expression of NQO1 in
Endothelial Cells--
Nrf2 is a Cap'n'Collar and leucine
zipper-containing transcriptional factor and plays a critical role in
ARE-mediated gene expression (34, 35). However, the role of Nrf2
in endothelial cell gene expression is unknown. To investigate whether
Nrf2 can activates the ARE-driven promoter in endothelial cells,
p3xARE/Luc was transfected with an expression vector encoding
Nrf2 into HMEC. As shown in Fig.
6A, co-expression of
Nrf2 in HMEC activated the ARE-driven promoter activity. When
Nrf2 was co-transfected with the p3xmutARE/Luc, a mutated ARE
version of the ARE-driven promoter, no activation was observed by
Nrf2. To examine whether Nrf2 can activate NQO1
transcription through an ARE-dependent mechanism in
endothelial cells, pNQO1CAT1.55 or ARE mutant pNQO1CAT1.55mARE was
co-transfected along with an expression vector encoding Nrf2 into HMEC. As shown in Fig. 6B, co-expression of Nrf2
in HMEC activated wild type NQO1, but not ARE mutant NQO1, gene
transcription from the reporter construct. These data are consistent
with the known role of Nrf2 in ARE-dependent
activation of NQO1 in other cell types (28) and demonstrate for the
first time a role of Nrf2 in ARE-mediated transcriptional
activation in endothelial cells.
To examine whether Nrf2 mediates laminar flow-induced activation
of NQO1 in endothelial cells, pNQO1CAT1.55 was co-transfected with
LNCX-Nrf2R (an expression vector encoding antisense Nrf2) or a dominant negative Nrf2 mutant pcDNA3DN-Nrf2 into
HMEC. As shown in Fig. 7A,
co-transfection with antisense Nrf2 suppressed laminar
flow-induced activation of NQO1 gene expression. Similarly, co-transfection with dominant negative Nrf2 suppressed both
basal and laminar flow-induced activation of Nrf2 gene
transcription (Fig. 7B), suggesting Nrf2 plays an
important role in laminar flow-mediated activation of NQO1 gene
expression.
Keap1/INrf2 Inhibits Laminar Flow-induced Activation of NQO1
Gene Expression--
Recent studies suggest that Nrf2 activity
is normally repressed through its localization in the cytoplasm by
binding to an inhibitor protein called Keap1 (36). The rat homologue of
Keap1 was recently cloned and named INrf2 (37). We will refer to
this protein as Keap1/INrf2. It has been suggested that the
Nrf2-Keap1/INrf2 interaction may constitute a cytoplasmic
sensor for oxidative stress (36). Electrophilic agents release
Nrf2 from its complex with Keap1/INrf2, allowing the
translocation of Nrf2 from the cytoplasm to the nucleus to
activate transcription (36, 37). To further examine the role of
Nrf2 and to determine whether Keap1/INrf2 plays a role in
regulating laminar flow-induced ARE-mediated transcriptional activation
in endothelial cells, we evaluated laminar flow-inducible activation of
the NQO1 gene in the presence and absence of Keap1/INrf2. As
shown in Fig. 7C, overexpression of Keap1/INrf2 in
HMEC inhibited both basal and laminar flow-mediated activation of NQO1
promoter activity. However, in the presence of Keap1/INrf2,
laminar flow was still able to increase NQO1 gene transcription
although to a lesser degree than vector only transfected cells. These
data suggest that under these experimental conditions there was perhaps an insufficient sequestration of Nrf2 by Keap1/INrf2
under the laminar flow condition. Cumulatively, these data demonstrate
that the Nrf2-Keap1/INrf2 pathway regulates
ARE-dependent transcription, and this pathway is involved
in regulation of laminar flow-induced ARE transcriptional activation in
endothelial cells.
NQO1 and Nrf2 Modulate TNF- Many studies suggest that laminar flow may protect the vasculature
from early atherosclerotic lesions; however, the underlying mechanisms
are still unclear. In the present study, we used an in vitro
model of HAEC exposed to physiological levels of laminar flow (shear
stress = 20 dyn/cm2), which approximate the average
wall shear stress typically encountered in lesion-protected areas of
the vasculature. In addition, we have chosen to examine the biological
effects of fluid flow in our model system at 48 h, because we
believe that exposure to shear stress for relatively long periods are
more relevant to the in vivo flow conditions. We found that
the expression of a set of cytoprotective and antioxidant genes was
coordinately induced in endothelial cells upon exposure to laminar
flow. These genes contain ARE or ARE-like transcriptional regulatory
element(s) in their promoters. For the first time, we demonstrated that
the ARE represents a new transcriptional element responsive to shear stress in endothelial cells. Functional experiments demonstrate that
activation of ARE-driven genes through the transcriptional factor
Nrf2 or via overexpression of one of these cytoprotective genes,
NQO1, represses the TNF- Previous studies showed that fluid shear stress regulates endothelial
gene expression through various cis-elements including the
12-O-tetradecanoylphorbol-13-acetate-responsive element
(38), the shear stress response element (39), NF- In contrast, the ARE represents a new shear stress response element
that coordinately regulates cytoprotective and athero-protective genes
in endothelial cells. These proteins, such as GST, NQO1, The present study demonstrates that Nrf2 is required for laminar
flow-induced NQO1 gene expression in endothelial cells. Nrf2 plays a critical role in ARE-mediated gene expression and in the regulation of biological responses to oxidative stress. For example, cells from Nrf2 knockout mice are deficient in their ability to induce anti-oxidative stress genes and as a result are highly sensitive
to oxidative stress-induced cell death (34, 35). In
Nrf2-deficient macrophages, a number of anti-oxidative stress genes could no longer be induced by electrophilic or ROS-generating agents (53). Nrf2 is normally maintained in the cytoplasm
through its interaction with Keap1/INrf2 (36, 37). Upon
stimulation by oxidative stress, Nrf2 is believed to dissociate
from Keap1/INrf2 and translocate to the nucleus where it binds
to the ARE (36).
It has been suggested that the Nrf2-Keap1/INrf2
interaction may constitute a cytoplasmic sensor for oxidative stress
(54). Exposure of endothelial cells to fluid flow stimulates the
generation of ROS through the activation of NAD(P)H oxidase (55, 56). It is possible that the induction of ARE-regulated genes in response to
laminar flow is a compensatory response to elevated levels of ROS. De
Keulenaer et al. (12) demonstrate that the flow patterns differentially modulate endothelial cell redox state. Steady laminar flow induced a transient increase in superoxide production that returned to base line after 24 h, whereas oscillatory flow induced a higher and sustained increase in superoxide production. However, we
observe a continual increase in ARE gene expression up to 48 h
after exposure to laminar flow, suggesting that it is not solely the
"burst" of ROS after exposure to laminar flow that induces ARE
expression. The present study shows that laminar flow is more potent in
the activation of ARE-mediated gene expression compared with
oscillatory flow. In addition, the magnitude of fluid flow-induced expression of the ARE-mediated NQO1 gene is dependent on the levels of
shear stress. A high level of laminar flow (shear stress = 20 dyn/cm2) induces significantly higher expression of
ARE-mediated genes than a low level of laminar flow (shear stress = 5 dyn/cm2) and oscillatory flow (shear stress = ±5
dyn/cm2). These data suggest that laminar flow may use
specific mechanoreceptor(s) to activate the Nrf2/ARE pathway;
however, additional experiments are needed to determine if laminar
flow-mediated ARE activation occurs directly through biomechanical
stimulation or indirectly via modulation of ROS.
It is well accepted that redox-sensitive regulation of inflammatory
gene expression in vascular cells contributes to the pathogenesis of
atherosclerosis (57). ROS such as H2O2
stimulates monocyte chemoattractant protein-1 gene expression in
vascular smooth muscle cells (58). Inflammatory stimuli-activated
endothelial expression of monocyte chemoattractant protein-1 and VCAM-1
is inhibited by antioxidants such as pyrrolidine dithiocarbamate (29,
46). Furthermore, inhibition of superoxide generation in endothelial cells suppressed cytokine-induced VCAM-1 gene expression (59). These
studies suggest that increasing intracellular antioxidant capacity may
be therapeutically beneficial for chronic inflammatory diseases such as
atherosclerosis by controlling redox-sensitive vascular gene
expression. In this report, we demonstrate that co-expression of
Nrf2 or NQO1 can suppress cytokine-induced VCAM-1 gene
expression. Presumably, overexpression of Nrf2 coordinately induces expression of multiple ARE-containing genes in endothelial cells and functions to reduce cytokine-induced oxidant signals that
modulate redox-sensitive gene expression such as VCAM-1. Our data
suggest that increased expression of the ARE-mediated genes has a
potential athero-protective effect and is anti-inflammatory in
endothelial cells. These data suggest that laminar shear stress induces
a protective phenotype to the cells in part by activating the
expression of genes that function to protect endothelial cells from
oxidative stress. These data further confirm the notion that physiological levels of shear stress are essentially
anti-inflammatory and athero-protective.
NQO1 (and its homologue NQO2) are ubiquitous flavoproteins found in
eukaryotes. NQO1 catalyzes the two electron reductive metabolism of
toxic quinones and their derivatives and converts them to hydroxyl
quinones for elimination (60). The obligatory two-electron reduction of
quinones catalyzed by NQO1 competes with the one-electron reduction by
other detoxification enzymes that generate unstable semiquinones that
undergo redox cycling in the presence of molecular oxygen, leading to
the formation of reactive oxygen species (16). Another major function
of NQO1 is to maintain coenzyme Q (ubiquinone) in the antioxidant form (ubiquinol) in membranes (60). Coenzyme Q is a lipid-soluble constituent of membranes and possesses "membrane-stabilizing" activity and inhibition of membrane lipid peroxidation. Ubiquinols can
react with oxygen radicals and, thus, prevent direct damage to
biomolecules and initiation of lipid peroxidation (61). Thus, NQO1 has
the potential to protect against oxidative stress and lipid
peroxidation in the vascular wall. We show that overexpression of NQO1
inhibits the TNF- The discovery that the ARE represents a unique transcriptional element
in response to physiologically athero-protective laminar flow in
endothelial cells extends our understanding of the role of this
important transcriptional regulatory network and the importance of
ARE-regulated genes in vascular biology and diseases. Increased expression of antioxidant and cytoprotective genes via activation of
the ARE likely contributes to the athero-protective and
anti-inflammatory effects of laminar flow. Modulation of endothelial
ARE activity may represent a new approach for the prevention and
treatment of atherosclerosis and other inflammatory diseases.
-glutamylcysteine
synthase, whose expression is induced by exposure to prolonged
physiological levels of steady laminar flow (shear stress = 20 dyn/cm2) in endothelial cells (EC). These genes contain an
antioxidant response element (ARE) or ARE-like transcriptional
regulatory sequence in their promoters and generally function to
protect cells against oxidant stress. We demonstrate that exposure of EC to laminar flow activates ARE-mediated transcriptional activity. Mutation of the ARE from either the NQO1 or HO-1 promoter abolished laminar flow-induced NQO1 and HO-1 transcriptional activation. Expression of antisense Nrf2 (a transcriptional factor for ARE), a dominant negative Nrf2, or the cytoplasmic inhibitor of
Nrf2 (Keap1/INrf2) inhibited laminar flow-induced NQO1
promoter activation in EC. In addition, expression of NQO1 or
Nrf2 inhibited tumor necrosis factor-
-induced activation of
VCAM-1 (vascular cell adhesion molecule-1) gene expression in EC. These
data define the ARE as a novel endothelial shear stress response
element. Furthermore, laminar flow activation of antioxidant genes via an ARE-dependent transcriptional mechanism may represent a
novel athero-protective and anti-inflammatory mechanism in the vasculature.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-induced VCAM-1 gene
expression in endothelial cells (1). Similarly, exposure of endothelial
cells to fluid flow inhibits monocyte adherence and VCAM-1 expression
induced by TNF-
or oxidized low density lipoprotein in endothelial
cells (10). In addition, genes encoding the antioxidants manganese
superoxide dismutase, Cu,Zn superoxide dismutase, heme oxygenase-1
(HO-1), and endothelial nitric-oxide synthase are demonstrated to be
up-regulated by laminar flow but not by oscillatory flow (11, 12).
-glutamylcysteine synthase. We further
demonstrate that the ARE is the cis-acting element in the
promoter of the NQO1 and HO-1 genes that mediates laminar flow-induced
gene expression. Laminar flow-mediated, ARE-dependent NQO1
gene regulation in endothelial cells involves the transcriptional factor NF-E2-related factor 2 (Nrf2). Furthermore, co-expression of NQO1 or Nrf2 inhibited TNF-
-induced activation of VCAM-1
gene expression. These data suggest that laminar flow may exert
athero-protective effects through a coordinated increase in expression
of ARE-regulated intracellular antioxidant genes in endothelial cells.
Therefore, this newly described laminar flow-regulated pathway may
contribute to the modulation of oxidation-reduction (redox)-sensitive
processes that result in vascular dysfunction, including the expression of inflammatory gene products.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1550 to +110 of the human NQO1 gene
cloned upstream of the chloramphenicol acetyltransferase (CAT) reporter
gene (26). To generate a mutated ARE in the NQO1 promoter,
pNQO1CAT1.55mARE, the conserved GC nucleotides in the ARE element (13,
14) were mutated to TA using GeneTailor mutagenesis kit according to
manufacturer's instruction (Invitrogen). The PCR primers used
in the mutagenesis were
5'-CGCAGTCACAGTGACTCATAAGAATCTGAGCC-3' (forward primer, the
underlined TA represents GC to TA mutation) and
5'-TGAGTCACTGTGACTGCGAATTTGGAAGGC-3' (reverse primer). To generate the
human HO-1 promoter construct, pHO-1Luc4.0, a DNA fragment containing
4.0 kilobases of the human HO-1 promoter region was PCR-amplified from
genomic DNA (Promega) using specific primers described previously (27)
and cloned into the MluI and XhoI sites of PGL3
basic plasmid (Promega). To generate a mutated ARE in the HO-1
promoter, pHO-1Luc4.0mARE, the conserved GC nucleotides in the inverted
ARE element were mutated to TA using the GeneTailor mutagenesis kit
according to the manufacturer's instructions (Invitrogen). The PCR
primers used in the mutagenesis were 5'-GGCGGATTTTGC
TAGATTTTATTGAGTCACCA-3' (forward primer, the underlined AT
represents GC to TA mutation) and 5'-TGTTTCCCTTCCGCCTAAAACGATCTAAAA-3'
(reverse primer).
85 to
+12 of the human VCAM-1 promoter linked to the CAT gene (29).
5
dyn/cm2 at a periodicity of 1 Hz. This small mean flow
component was included to ensure adequate media exchange over the
endothelial monolayer. After the indicated times, RNA or protein
samples were collected.
-actin.
DNA sequences for primer pairs used in RT-PCR analysis
-tubulin was purchased from Santa Cruz Biotechnology Inc. Relative
band intensities were determined by densitometry using the Bio-Rad
Quantity One software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glutamylcysteine synthase revealed a common
transcriptional regulatory feature. Each of these genes contains an ARE
or ARE-like transcriptional regulatory element(s) in its promoter and
is known to be up-regulated by oxidative stress. Furthermore, these
genes function as antioxidants and protect cells from oxidative stress.
Table II shows the sequence alignment of the ARE or ARE-like regulatory elements from the laminar
flow-inducible genes compared with the consensus ARE sequence, RTGACNNNGC (13, 14). These observations provide the first suggestion that the AREs may mediate laminar flow-induced gene expression in endothelial cells.
DNA sequence alignment of AREs from laminar flow regulated genes
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Fig. 1.
Effects of laminar flow on the expression of
ARE-mediated genes in endothelial cells. The genes examined
include ferritin (heavy (H) and light (L)
chains), quinone oxidoreductase-1 (NQO1), heme oxygenase-1
(HO-1), microsomal epoxide hydrolase (mEH), GST,
and -glutamylcysteine synthase (
-GCS). HAEC grown on
glass plates were kept in static conditions or subjected to laminar
flow with shear stress at 20 dyn/cm2 (LF) for
48 h. Relative mRNA levels were determined by
semi-quantitative RT-PCR and normalized to
-actin levels. Values are
the means ± S.D., n = 3. *, p < 0.05 compared with static culture.
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Fig. 2.
Temporal effects of laminar flow on NQO1 and
HO-1 mRNA levels in endothelial cells. HAEC were kept in
static culture or subjected to laminar flow with shear stress at 20 dyn/cm2 (LF) for the indicated times. Relative
NQO1 (A) and HO-1 (B) mRNA levels were
determined by semi-quantitative RT-PCR and normalized to -actin
levels. Values are the means ± S.D., n = 3. *,
p < 0.05 compared with static culture.
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Fig. 3.
Shear stress-dependent effects on
NQO1 mRNA expression in endothelial cells. HAEC were kept in
static culture or subjected to oscillatory flow with shear stress
at ± 5 dyn/cm2 (OF) or laminar flow with
shear stress at 5 or 20 dyn/cm2 (LF) for 48 h. Relative NQO1 mRNA levels were determined by semi-quantitative
RT-PCR and normalized by -actin gene levels. Values are means ± S.D., n = 3. *, p < 0.05 compared
with static culture; +, p < 0.05 compared with the
oscillatory flow (±5 dyn/cm2)-treated group.
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Fig. 4.
Effects of laminar or oscillatory flow on
NQO1 protein expression in endothelial cells. HAEC were kept in
static condition or exposed to laminar flow with shear stress at 20 dyn/cm2 (LF) or oscillatory flow with shear
stress at ±5 dyn/cm2 (OF) for 48 h.
Proteins were subjected to electrophoresis and Western blot analysis
using antibodies against NQO1 or -tubulin. Two independent
experiments showed similar results.
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Fig. 5.
A, laminar flow activates
ARE-driven promoter activity in endothelial cells. HMEC were
transfected with 5 µg of p3xARE/Luc or p3xmutARE/Luc (containing the
mutated ARE). B and C, laminar flow-induced NQO1
and HO-1 gene expression is mediated via the ARE. B, HMEC
were transfected with 5 µg of pNQO1CAT1.55 (NQO1/CAT) or
pNQO1CAT1.55mARE (containing the mutated ARE in NQO1 promoter;
NQO1mARE/CAT). C, HMEC were transfected with 5 µg of pHO-1Luc4.0 (HO-1/Luc) or pHO-1Luc4.0mARE
(containing the mutated ARE in NQO1 promoter in HO-1 promoter;
HO-1mARE/Luc). These cells were also transfected with 0.5 µg of pRL-SV40 for normalization of transfection efficiency. After a
24-h recovery, cells were kept in static culture or subjected to
laminar flow with shear stress at 20 dyn/cm2
(LF) for 24 h for the ARE-driven promoter and NQO1
promoter or 48 h for HO-1 promoter. Cell extracts were harvested,
and luciferase assays or CAT assays were performed. Results are
expressed as fold changes over unstimulated control. Values are the
means ± S.D., n = 4. *, p < 0.05 compared with wild type promoter construct in static culture.
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Fig. 6.
Nrf2 activates ARE-driven promoter and
NQO1 gene transcription in endothelial cells. A, HMEC
cultured in 6-well plates were transfected with 1 µg of p3xARE/Luc or
p3xmutARE/Luc plus 1 µg of LNCX-Nrf2 or parental vector.
B, HMEC cultured in 6-well plates were transfected with 1 µg of pNQO1CAT1.55 (NQO1/CAT) or pNQO1CAT1.55mARE
(NQO1mARE/CAT) plus 1 µg of LNCX-Nrf2 or parental
vector. 24 h after transfection, cell extracts were harvested, and
luciferase assays or CAT assays were performed. Results are expressed
as fold change over unstimulated control. Values are means ± S.D., n = 4. *, p < 0.05 compared with
cells with wild type promoter construct co-transfected with parental
vector.
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Fig. 7.
A, antisense Nrf2 suppresses
laminar flow (LF)-induced NQO1 promoter activation. HMEC
cultured on glass plates were transfected with 2 µg of pNQO1CAT1.55
and 4 µg of LNCX-Nrf2R (containing antisense Nrf2
cDNA; AS-Nrf2) or parental vector
(Vector). B, dominant negative Nrf2
inhibits laminar flow-induced NQO1 promoter activation. HMEC cultured
on glass plates were transfected with 5 µg of pNQO1CAT1.55 and 5 µg
of pcDNA3DN-Nrf2 (encoding a dominant negative Nrf2;
DN-Nrf2) or parental vector (Vector).
C, Keap1/INrf2 suppresses laminar flow-induced NQO1
promoter activation. HMEC grown on glass plates were transfected with 2 µg of pNQO1CAT1.55 and 4 µg of pcDNA3Keap1/INrf2 or
parental vector. These cells were also transfected with 0.5 µg of
pRL-SV40 for normalization of transfection efficiency. After a 24-h
recovery, cells were kept in static culture or subjected to laminar
flow with shear stress at 20 dyn/cm2 for 24 h. Cell
extracts were harvested, and 5 µg of total cellular protein was used
for CAT assays. Results are expressed as fold change over unstimulated
control. Values are the means ± S.D., n = 4. *,
p < 0.05 compared with vector-transfected cells kept
in static culture; +, p < 0.05 compared with
Keap1/INrf2-transfected cells kept in static culture for
panel C.
-mediated VCAM-1 Gene
Expression--
Cytokine-activated VCAM-1 gene expression is regulated
by an oxidation-reduction (redox)-sensitive mechanism in endothelial cells (29). We hypothesized that the coordinate induction of ARE-containing antioxidant genes through activation of Nrf2 may be one mechanism whereby fluid flow may modulate redox-sensitive gene
expression in endothelial cells. Because TNF-
-inducible expression
of the VCAM-1 promoter is sensitive to redox modulation (29), we used
the VCAM-1 promoter and transient transfection in HMEC as a readout for
expression of a redox-sensitive inflammatory gene. We sought to
determine whether activation of the ARE pathway via overexpression of
Nrf2 or expression of NQO1 would modulate cytokine-inducible
VCAM-1 gene expression. As shown in Fig.
8, TNF-
induced a significant increase
in VCAM-1 promoter activity. Expression of either NQO1 or Nrf2
in HMEC dramatically inhibited TNF-
-induced activation of the VCAM-1
promoter. These observations provide support for the notion that
activation of ARE-containing genes may modulate redox-sensitive
inflammatory gene expression in endothelial cells.
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Fig. 8.
Effects of expression of NQO1 or Nrf2
on TNF- -induced activation of VCAM-1 promoter
activity. HMEC cultured in 6-well plates were transfected with 1 µg of p85VCAM-1/CAT plus 1 µg of pcDNA3NQO1 (NQO1),
LNCX-Nrf2 (Nrf2) or pcDNA3-LacZ
(Vector). These cells were also transfected with 0.5 µg of
pRL-TK for normalization of transfection efficiency. After a 24-h
recovery, cells were treated with or without TNF-
for 16 h,
cell extracts were harvested, and 5 µg of protein was used for CAT
assays. Values are means ± S.D., n = 4. *,
p < 0.05 compared with control group.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-induced expression of the redox-sensitive adhesion molecule, VCAM-1, in endothelial cells. These experiments provide a novel molecular mechanism that help explain the
athero-protective and antioxidant nature of laminar flow. Activation of
the ARE and ARE-regulated anti-oxidant and cytoprotective genes by
laminar flow likely contributes to defensive mechanisms to protect
against oxidant-mediated endothelial dysfunction and redox-sensitive
inflammatory gene expression.
B (40-42), and an early growth response-1 recognition element (40, 43). For example, the
shear stress response element is found in the promoter region of
platelet-derived growth factor-B chain and intercellular adhesion
molecule-1 gene (39, 44), and the early growth response-1 binding site
is required for the activation of platelet-derived growth factor-A
chain gene by shear stress (40, 43). Platelet-derived growth factor is
a potent mitogen and chemotactic agent for smooth muscle cells.
Activation of
12-O-tetradecanoylphorbol-13-acetate-responsive element is
responsible for shear stress-induced monocyte chemoattractant protein-1, a potent chemotactic agent for monocytes (38). NF-
B is
involved in the up-regulation of a variety of immune and inflammatory genes including VCAM-1 (29, 45, 46). It is notable that these shear
stress-regulated cis elements are commonly found in the
promoters of pro-atherogenic or pro-inflammatory genes.
-glutamylcysteine synthase, HO-1, and the light and heavy chains of
ferritin share a common role as antioxidants (13, 23). HO-1 catalyzes
the rate-limiting reaction in heme degradation to CO and biliverdin, a
potent antioxidant (47). CO may influence vessel tone, mitogenesis, and
inflammatory responses in the vasculature (48). Recently, Hayashi
et al. (49) demonstrated that HO-1 attenuates
leukocyte-endothelial cell adhesion in vivo through the
action of bilirubin, a product from biliverdin (49). Overexpression of
HO-1 has been shown to inhibit atherosclerotic lesion formation in low
density lipoprotein receptor knock-out mice and in Watanabe heritable
hyperlipidemic rabbits (22, 50). Ferritin is another cytoprotective
protein. Ferritin binds free iron and prevents it from participating in
iron-catalyzed free radical reactions and the generation of oxidative
stress. The regulation of ferritin gene expression in response to
oxidative stress is mediated via two AREs in the ferritin promoter
(18). GST catalyzes the S-conjugation of glutathione with
reactive species such as electrophilic compounds (51).
-Glutamylcysteine synthase catalyzes the rate-limiting reaction in
the synthesis of glutathione, which is an efficient ROS scavenger (52).
Because ROS contribute to the pathogenesis of atherosclerosis by
stimulating inflammatory gene expression and promoting smooth muscle
cell proliferation, activation of ARE-regulated antioxidant proteins
and enzymes may help to counterbalance the biological effects of ROS
and to maintain intracellular redox homeostasis. Therefore, shear
stress-mediated activation of this antioxidant defense pathway via the
ARE defines a new athero-protective mechanism in the vascular
endothelium. Conversely, extrapolation of our in vitro
findings suggest that decreased levels of these ARE-regulated
cytoprotective genes may be present in vivo at areas of low
shear stress, which would be more predisposed to oxidative damage and
the activation of redox-sensitive inflammatory signals.
-induced expression of VCAM-1. These data support
the notion that NQO1 may protect against cytokine-induced oxidant
stress and may contribute to the regulation of expression of
redox-sensitive inflammatory genes in the vasculature.
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FOOTNOTES |
---|
* 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.
Contributed equally to this work.
§ To whom correspondence should be addressed: Discovery Research, AtheroGenics, Inc., 8995 Westside Pkwy., Alpharetta, GA 30004. Tel.: 678-336-2711; Fax: 678-393-8616; E-mail: xchen@atherogenics.com.
Published, JBC Papers in Press, October 4, 2002, DOI 10.1074/jbc.M203161200
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ABBREVIATIONS |
---|
The abbreviations used are:
VCAM-1, vascular
cell adhesion molecule-1;
ARE, antioxidant response element;
CAT, chloramphenicol acetyltransferase;
GST, glutathione
S-transferase;
HAEC, human aortic endothelial cells;
HMEC, human microvascular endothelial cells;
HO-1, heme oxygenase-1;
INrf2, cytosolic inhibitor of Nrf2;
NQO1, NAD(P)H:quinone
oxidoreductase-1;
Nrf2, NF-E2-related factor-2;
ROS, reactive
oxygen species;
TNF-, tumor necrosis factor-
;
EpRE, electrophile
response element;
RT, reverse transcriptase.
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