© 1999 American Heart Association, Inc.
Original Contribution |
Nitric Oxide Regulates Shear Stress–Induced Early Growth Response-1
Expression via the Extracellular Signal–Regulated Kinase Pathway in Endothelial Cells
From the Cardiovascular Division (J.J.C., B.S.W., L.W.L., D.L.W.), Institute of Biomedical Sciences, Academia Sinica, and Department of Chemical Engineering (H.J.H.), National Taiwan University, Taipei, Taiwan, ROC.
Correspondence to Dr Danny Ling Wang, Cardiovascular Division, Institute of Biomedical Sciences, Academia Sinica, Taipei, 11529 Taiwan, ROC. E-mail lingwang{at}ibms.sinica.edu.tw
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Abstract |
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Abstract—Endothelial cells (ECs) subjected to shear stress constantly release nitric oxide (NO). The effect of NO on shear stress–induced endothelial responses was examined. ECs subjected to shear stress induced a transient and shear force–dependent increase in early growth response-1 (Egr-1) mRNA levels. Treatment of ECs with an NO donor, S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholinosydnonimine (SIN-1), inhibited this shear stress–induced Egr-1 expression. Conversely, an NO synthase inhibitor to ECs, NG-monomethyl-L-arginine, augmented this Egr-1 expression. NO modulation of Egr-1 expression was demonstrated by functional analysis of Egr-1 promoter activity using a chimera containing the Egr-1 promoter region (–698 bp) and reporter gene luciferase. In contrast to the enhanced promoter activity after NG-monomethyl-L-arginine treatment, shear stress–induced Egr-1 promoter activity was attenuated after ECs were treated with an NO donor. ECs cotransfected with a dominant negative mutant of Ras (RasN17), Raf-1 (Raf301), or a catalytically inactive mutant of extracellular signal–regulated kinase (ERK)–2 (mERK) inhibited shear stress–induced Egr-1 promoter activity. NO modulation of the signaling pathway was shown by its inhibitory effect on shear stress–induced ERK1/ERK2 phosphorylation and activity. This inhibitory effect was further substantiated by the inhibition of NO on both the shear stress–induced transcriptional activity of Elk-1 (an ERK substrate) and the promoter activity of a reporter construct containing serum response element. NO-treated ECs resulted in a reduction of binding of nuclear proteins to the Egr-1 binding sequences in the platelet-derived growth factor-A promoter region. These results indicate that shear stress–induced Egr-1 expression is modulated by NO via the ERK signaling pathway in ECs. Our findings support the importance of NO as a negative regulator in endothelial responses to hemodynamic forces.
Key Words: Egr-1 • endothelial cell • nitric oxide • shear stress • signaling pathway
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vascular endothelial cells (ECs) are constantly exposed to hemodynamic forces, including shear stress imposed by blood flow. Shear stress exerts significant influences on ECs, many of which reflect alterations in gene expression.1 2 A number of pathophysiologically relevant genes, such as the platelet-derived growth factor (PDGF)-B chain,3 4 intercellular adhesion molecule-1 (ICAM-1),5 6 7 8 and monocyte chemotactic protein-1 (MCP-1),9 10 are activated by shear stress. The induction of genes in ECs exposed to shear stress is believed to involve the activation of a variety of transcription factors, including nuclear factor-
B
(NF-B) and activator protein-1
(AP-1).4 10 11 Khachigian et al12 recently
demonstrated that early growth response-1 (Egr-1), an immediate-early
gene, is activated by shear stress in ECs. This
shear-activated Egr-1 acts as a transcription factor that
interacts with the overlapping binding elements for Egr-1 and Sp1 in
the promoter region of PDGF-A and subsequently induces its gene
expression. Similar consensus binding sites for Egr-1 and Sp-1 are also
present in the promoter of various genes such as PDGF-B,
transforming growth factor-ß1, and tissue
factor, the expression of which is also affected by shear stress and
cytokines.13 14 15 Thus, Egr-1 induction has been
indicated as a common theme in vascular injury.13 Despite intensive studies on the effects of fluid shear stress on ECs, the detailed mechanisms that transmit the mechanical stimuli to intracellular signaling still remain largely unclear. Various signals, including calcium mobilization,16 inositol triphosphate,17 18 G protein,19 20 and cyclic GMP (cGMP),20 have been shown to be activated by shear stress. Recent studies have demonstrated that signaling-pathway extracellular signal–regulated kinase (ERK) is activated by shear force.2 21 Li et al,22 however, indicated that shear stress primarily activates the c-Jun N-terminal kinase (JNK) pathway. The detailed mechanisms particularly regarding the synergism and cross talk among different signaling pathways thus remain undefined. Our recent studies demonstrated that reactive oxygen species (ROS) are induced by shear stress or cyclic strain and consequently act as second messengers to stimulate the expression of various genes, including MCP-1, c-fos, and ICAM-1.8 23 24 25 This hemodynamic force–induced gene expression is inhibited after ECs are pretreated with an antioxidant.8 23 24 25
Nitric oxide (NO), a relaxing factor derived from
endothelium via the activation of
endothelial NO synthase (eNOS), plays a protective role
during atherogenesis.26 27 28 29 30 Shear stress to ECs increases
eNOS mRNA levels and NO production.19 28 31 32
This released NO modulates various gene expressions, including
MCP-1,29 vascular cell adhesion molecule-1
(VCAM-1),33 34 35 36 and ICAM-136 in cells exposed
to various stimuli. Frangos and Bao37 further demonstrated
that NO regulates shear stress–induced PDGF-A and MCP-1 gene
expressions in ECs. However, the detailed mechanism by which NO
modulates endothelial responses to chemical or
mechanical stimuli remains unclear. NO may exert its effect by
modulating the intracellular redox status via suppressing ROS
levels29 or by triggering redox-sensitive
mechanisms36 in ECs. NO may modulate the VCAM-1 expression
via the elevation of cGMP.33 A recent study35
demonstrated that cytokine-induced VCAM-1 expression was
mediated by NO via the inhibition of NF-B activation. The ERK
signaling pathway has been suggested to be involved in the
inhibitory effect on smooth muscle cell proliferation by
NO.38 NO may inhibit the Ras/Raf/ERK pathway through the
activation of cGMP-dependent protein kinase.39 For the
induction of Egr-1 in ECs by shear stress, the signaling pathway
involving ERK has been demonstrated.40 Moreover, NO
inhibits Egr-1 expression in cytokine-treated
macrophages.41 Because NO plays a role by
inhibiting key events that promote atherogenesis, and Egr-1 induction
is involved in the vascular injury, we postulate that shear
stress–induced transient expression of Egr-1 is mediated by NO via the
inhibition of the ERK signaling pathway. The present study clearly
indicates that NO down-regulates shear stress–induced Egr-1 expression
via the inhibition of the ERK pathway in ECs. This inhibition
consequently results in a reduction of binding of nuclear Egr-1
proteins to the corresponding binding sequences in the promoter region
of PDGF-A. Our findings thus support the notion that NO serves as a
negative regulator in endothelial responses to
hemodynamic forces.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials
The Egr-1 cDNA probe was a gift from Dr Y.J. Shyy (University of California at San Diego). The catalytically inactive mutant of ERK2 (mERK) was a gift from Dr R.J. Davis, University of Massachusetts Medical School (Worcester, MA). RasN17, RasL61, and Raf310 were previously described.42 43 The Elk-1 trans-reporting system and serum response element (SRE) cis-reporting system were obtained from Stratagene (catalog numbers 219005 and 219079, respectively). S-Nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine (SIN-1), and NG-monomethyl-L-arginine (L-NMMA) were purchased from Calbiochem. All other chemicals, of reagent grade, were obtained from Sigma.
EC Culture
ECs were isolated from human umbilical cords as previously
described.44 ECs were grown in Petri dishes for 3 days and
then seeded onto glass slides (75x38 mm, Corning) to reach
confluence. The culture medium was then exchanged with medium that was
identical except that it contained only 2% FBS, and the cells were
further incubated 24 hours before the experiment.
Flow Apparatus
The slide with cultured ECs was mounted in a parallel-plate flow
chamber, which has been characterized and described in detail
elsewhere.45 The chamber was connected to a perfusion loop
system, kept in a constant-temperature–controlled enclosure, and
maintained at pH 7.4 by continuous gassing with a mixture of 5%
CO2 in air. The flow channel width (w) was 1 cm,
and the channel height (h) was 0.025 cm. The Reynolds number, defined
by the average inlet velocity and the channel height, was 30. The fluid
shear stress generated on the ECs by flow was calculated as 20
dyne/cm2, using the formula 
=6
µQ/wh2, where is the
shear stress, µ is the dynamic viscosity of the perfusate,
and Q is the flow rate. In some experiments, ECs were
pretreated with SNAP (100 µmol/L) or SIN-1 (100 µmol/L)
for 30 minutes or L-NMMA (250 µmol/L) for 1 hour. These ECs were
then subjected to shear flow in the presence of the same reagent. The
static control cells were incubated and changed to new culture medium
while the experimental cells were placed under flow conditions.
RNA Isolation and Northern Blot Analysis
Total RNA was isolated from ECs by the guanidine
isothiocyanate/phenochloroform method as previously
described.8 The RNA (10 µg/lane) was separated by
electrophoresis on a 1% agarose formaldehyde gel and transferred onto
a nylon membrane (Nytran, Schleicher & Schuell, Inc) by a vacuum
blotting system (VacuGene XL, Pharmacia). After hybridization with the
32P-labeled cDNA probes, the membrane was washed
with 1 SSC containing 1% SDS at room temperature for 30 minutes and
then exposed to x-ray film at -70°C. Autoradiographic
results were analyzed by using a densitometer (Computing
Densitometer 300S, Molecular Dynamics).
Reporter Gene Construct, DNA Plasmids, Transfection, and
Luciferase Assay
The Egr-1 promoter construct (Egr-1-Luc) contains 698 bp of
Egr-1 5'-flanking DNA linked to the firefly luciferase reporter gene of
plasmid pGL2 (Promega, Inc). This fragment of the Egr-1 promoter
contains multiple SREs.46 The Elk-1
trans-reporting system contains plasmids
GAL4/ELK1-(307–428) and GAL4-Luc. GAL4/ELK1-(307–428) encodes the
fusion protein of the GAL4 DNA binding domain fused to the activation
domain of Elk-1. GAL4-Luc is a chimeric construct consisting of 5
copies of the GAL4 binding sequence and the luciferase reporter. The
SRE cis-reporting system contains plasmid pSRE-Luc, which
consists of 5 repeats of the SRE. DNA plasmids were transfected into
bovine aortic ECs (BAECs) at their 60% confluence level by using the
lipofectamine method (GIBCO-BRL). The pSV-ß-galactosidase plasmid was
cotransfected to normalize the transfection efficiency. After
transfection, cells were incubated with DMEM (GIBCO) containing 10%
FBS overnight and then seeded onto slides. The medium of the cultured
BAECs was exchanged with medium that was identical except that it
contained only 0.5% FBS, and the cells were further incubated
overnight before being subjected to shear flow treatment. Luciferase
activity was measured by using the Biotec assay system (Promega).
ß-Galactosidase activity was assayed by adding the substrate
o-nitrophenyl-ß-D-galactopyranoside
to 20 µL of cell lysate and incubating at 37°C before
recording at 420 nm.
Assay of ERK Activity and Phosphorylation
ERK activity was assayed according to the method previously
described.22 Briefly, ECs were lysed with buffer
containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and a
protease inhibitor mixture (PMSF, aprotinin, and sodium
orthovanadate). Cells were disrupted by repeated aspiration through a
21-gauge needle. The same amount of protein from each sample was
incubated with anti–ERK1/ERK2 antibody (Santa Cruz Biotechnology) for
2 hours at 4°C with gentle shaking. The immune complex was then
incubated with protein A/G agarose for 1 hour. This agarose-bound
immune complex was then incubated with kinase reaction buffer
containing myelin basic protein (MBP). The kinase reaction was carried
out for 20 minutes at 30°C in buffer containing 0.3 mg/mL MBP,
50 µmol/L ATP, and 1 µCi [
-32P]ATP.
The reaction was stopped by adding an equal volume of sample buffer
containing SDS and boiling for 3 minutes. The samples were
electrophoresed on a 15% polyacrylamide gel. After drying, the
gel was exposed to x-ray film. For detection of ERK
phosphorylation in sheared ECs, the cell lysates were
collected and boiled. Total cell lysates (100 µg of protein) were
separated by SDS-PAGE (12% running, 4% stacking) and transferred onto
a polyvinylidene fluoride membrane (Immobilon P, 0.45-µm pore
size). The membrane was then incubated with anti–active ERK1/ERK2
antibody (Promega Inc). Immunodetection was performed by using the
Western-Light chemiluminescent detection system (Tropix, Inc).
Electrophoretic Mobility Shift Assay (EMSA)
To prepare nuclear protein extracts, ECs were washed with cold
PBS and then immediately removed by scraping in PBS. After
centrifugation of the cell suspension at 2000 rpm, the
cell pellets were resuspended in cold buffer A (containing, in
mmol/L, KCl 10, EDTA 0.1, DTT 1, and PMSF 1) for 15 minutes. The cells
were lysed by adding 10% NP-40 and then centrifuged at 6000
rpm to obtain pellets of nuclei. The nuclear pellets were resuspended
in cold buffer B (containing, in mmol/L, HEPES 20, EDTA 1, DTT 1,
and PMSF 1, and 0.4 mol/L NaCl), vigorously agitated, and then
centrifuged. The supernatant containing the nuclear proteins
was used for the EMSA or stored at -70°C until used. Double-stranded
oligonucleotides (30 bp) containing the Egr-1 binding
site in the proximal region of the PDGF-A promoter were prepared as
described.12 13 14 . The oligonucleotides
were end labeled with [-32P]ATP. Extracted
nuclear proteins (10 µg) were incubated with 0.1 ng
32P-labeled DNA for 15 minutes at room
temperature in 25 µL binding buffer containing 1 µg poly(dI-dC). In
the antibody supershift assay, anti–Egr-1 antibody (1 µg, Santa Cruz
Biotechnology) was incubated with the mixture for 10 minutes at room
temperature followed by the addition of the labeled probe. The mixtures
were electrophoresed on 5% nondenaturing polyacrylamide gels.
Gels were dried and imaged by autoradiography.
Statistical Analysis
Results are expressed as mean±SEM. Significance was determined
by using the Student t test, and the level of statistical
significance was defined as P<0.05.
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Results |
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Shear Stress Induces a Transient and Force-Dependent Increase in Egr-1 mRNA Levels in ECs
To examine the effects of shear stress on Egr-1 gene expression in ECs, cultured ECs were exposed to shear stress of 20 dyne/cm2 for 0.5, 1, 2, 3, and 6 hours, and their Egr-1 mRNA expression was determined by Northern blot analysis. As shown in Figure 1A
, ECs subjected to
30 minutes of shear flow significantly increased their Egr-1 mRNA
levels as compared with the Egr-1 mRNA levels in static controls. The
Egr-1 mRNA levels in ECs remained at an elevated level for 1 hour and
then returned to the basal level 3 hours after the onset of shear
stress. The shear stress–induced Egr-1 expression was shear force
dependent (Figure 1B). There was no apparent increase of Egr-1
mRNA levels in ECs exposed to shear stress of 3
dyne/cm2 for 30 minutes. However, ECs exposed to
shear stress of 10 dyne/cm2 for 30 minutes
significantly increased their Egr-1 mRNA expression. As shear stress
increased to 40 or 100 dyne/cm2, the Egr-1 levels
were further augmented. These results suggest that shear
stress–induced Egr-1 gene expression in ECs is time and shear force
dependent.
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NO Modulates Shear Stress–Induced Egr-1 Gene Expression in
ECs
Shear stress to ECs increases the production and release
of NO.19 28 To explore whether NO modulates the induction
of Egr-1 gene in sheared ECs, ECs were preincubated with an NO donor,
ie, SNAP or SIN-1, for 30 minutes before being subjected to shear flow
in the presence of the NO donor. As shown in Figure 2A, SNAP treatment of ECs at a
concentration of 100 or 400 µmol/L significantly suppressed
shear stress–induced Egr-1 mRNA expression. Similarly, treatment of
ECs with another NO donor, SIN-1 (100 µmol/L), also
significantly attenuated shear stress–induced Egr-1 expression. In
separate experiments, ECs were pretreated with L-NMMA, an NO synthase
(NOS) inhibitor, for 1 hour and then subjected to flow in
the presence of the agent. In contrast to the inhibitory
effect by the NO donor, L-NMMA treatment of ECs at a concentration of
250, 500, or 1000 µmol/L significantly augmented shear
stress–induced Egr-1 mRNA levels (Figure 2B). L-NMMA treatment
had no effect on Egr-1 mRNA expression in static control cells (data
not shown). These results suggest that shear stress–induced Egr-1
expression is modulated by NO. Thus, shear stress–induced NO may serve
as a negative regulator for shear stress–induced Egr-1 expression.
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To further determine whether the modulation of NO in shear
stress–induced Egr-1 expression is a transcriptional event, an Egr-1
promoter construct containing the Egr-1 promoter region (–698 bp) and
the reporter gene luciferase were transiently transfected into ECs. As
shown in Figure 3, ECs exposed to 6 hours
of flow significantly increased Egr-1 promoter activity by 
2.4-fold
compared with static cells. The addition of both SNAP and SIN-1 to ECs
completely abolished this increased Egr-1 promoter activity.
Conversely, treatment of ECs with L-NMMA enhanced this promoter
activity. Pretreatment of ECs with KT5823, a cGMP-dependent protein
kinase inhibitor, did not interfere with the
inhibitory effect of NO on shear stress–induced Egr-1
promoter activity. This finding indicates that the effect of NO on
Egr-1 induction by shear stress is not mediated via the cGMP-dependent
protein kinase pathway. ECs treated with PMA (100 µg/L), as a
positive control, greatly increased their Egr-1 promoter activity.
These results together suggest that NO modulation of Egr-1 induction by
shear stress involves transcriptional regulation.
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Shear Stress–Induced Egr-1 Gene Expression Is Mediated via the
Ras/Raf/ERK Pathway
Egr-1 induction by shear stress reportedly involves activation of
the ERK signaling pathway.40 To further confirm the
involvement of this ERK pathway, we used the dominant negative mutants
of Ras (RasN17) and Raf-1 (Raf301) and a catalytically inactive mutant
of ERK2 (mERK), all of which are associated with the Ras/Raf/ERK
pathway, to examine the effect of these mutants on the induction of
Egr-1 by shear stress. As shown in Figure 4, ECs that were cotransfected with the
empty vector control PSR
revealed no effect on shear
stress–induced Egr-1 promoter activity. However, cotransfection of the
cells with RasN17, Raf301, or mERK resulted in a significant inhibition
in shear stress–induced Egr-1 promoter activity. Consistently,
ECs treated with a specific inhibitor to
mitogen-activated protein kinase kinase (MEK), ie,
PD98059, attenuated shear stress–induced Egr-1 promoter activity. In
contrast, ECs cotransfected with a dominant positive mutant of Ras
(RasL61) or MEK1 greatly increased their Egr-1 promoter activity. These
results confirm that the Ras/Raf/ERK signaling pathway is involved in
shear stress–induced Egr-1 gene expression in ECs.
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NO Regulates Shear Stress–Induced ERK
Phosphorylation and Activity in ECs
Given that we showed that the ERK signaling pathway is involved in
shear-induced Egr-1 expression and that NO modulates shear-induced
Egr-1 expression, we further investigated whether NO modulates the
activation of ERK in sheared ECs. We first examined the ERK1/ERK2
phosphorylation in ECs exposed to shear stress of 20
dyne/cm2 in the presence of the NO donor or NOS
inhibitor. As shown in Figure 5A, shear stress to ECs for 10 minutes
induced rapid phosphorylation of ERK1/ERK2 by 5.6-fold.
ECs preincubated with an NO donor, SNAP or SIN-1, significantly
inhibited shear stress–induced ERK1/ERK2
phosphorylation by 60% or 70%, respectively. In
separate experiments, L-NMMA treatment of ECs augmented shear
stress–induced ERK1/ERK2 phosphorylation by 55% above
that by shear stress only, whereas it showed no effect in static
control cells (Figure 5B). NO modulation of the ERK signaling
pathway was further elucidated by its inhibitory effect on
shear stress–induced ERK activity. ECs exposed to shear stress rapidly
induced ERK activity by 6.7-fold, as indicated by an increase of
32P-labeled phosphorylation of
MBP by ERK (Figure 5C). Treatment of ECs with SNAP or SIN-1
significantly attenuated this ERK activation by 55% or 67%,
respectively. In contrast, L-NMMA treatment of ECs enhanced this ERK
activation by 70% (Figure 5D). L-NMMA treatment had no effect
on ERK activity in static control cells. ECs treated with PMA, as a
positive control, remarkably increased their ERK activity by 13-fold.
Taken together, these findings imply that NO modulates shear-induced
Egr-1 gene expression via its inhibitory effect on the ERK
signaling pathway in ECs.
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NO Regulates Shear Stress–Induced Transcriptional Activity of
Elk-1 and the Promoter Activity of a Reporter Gene Construct
Containing SRE
NO modulation of shear stress–induced ERK
phosphorylation and activity in ECs led us to speculate
that NO may regulate the transcriptional activity of the ternary
complex factor proteins such as Elk-1, an ERK1/ERK2 substrate. To test
this hypothesis, the plasmid GAL4/ELK1-(307–428), which encodes the
GAL4 DNA binding domain fused to the C-terminal activation domain of
Elk-1, was cotransfected with GAL4-Luc into ECs to test their shear
inducibility. As shown in Figure 6A, shear stress caused a 5.4-fold increase in luciferase activity. This
induction, however, was significantly inhibited after ECs were treated
with an NO donor (SNAP or SIN-1). Conversely, L-NMMA treatment
augmented this shear stress–induced transcriptional activity of Elk-1.
As a positive control, ECs cotransfected with a plasmid pFC-MEK1, an
Elk-1 upstream kinase MEK1 expression vector, dramatically increased
the transcriptional activity of Elk-1. The activated Elk-1 has
been recognized as a transcriptional factor that cooperatively
interacts with the serum response factor and binds to the SRE in the
promoter region of various genes such as c-fos and Egr-1 to
induce their expression.47 In the present study,
a reporter gene construct, pSRE-Luc, containing 5 repeats of SRE was
transfected into cells to test the shear inducibility of the construct.
As shown in Figure 6B, ECs subjected to 3 hours of flow induced
a 2.7-fold increase in promoter activity. Not surprisingly, treatment
of cells with an NO donor (SNAP-1 or SIN-1) or NOS
inhibitor (L-NMMA) down-regulated or up-regulated this
shear stress–induced promoter activity, respectively. These results
indicate that NO inhibits shear stress–induced ERK activity followed
by a decrease of transcriptional activity of Elk-1 and SRE binding in
the promoter region of Egr-1.
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NO Regulates Shear-Induced Egr-1 Binding to the Promoter Region
of PDGF-A
Activation of Egr-1 by shear stress contributes to an induction of
PDGF-A in sheared ECs.12 The promoter region of PDGF-A
consists of multiple Egr-1 binding sites, and the increase of this
binding enhances PDGF-A gene expression.12 13 14 To
elucidate the functional role of NO in modulating shear-induced Egr-1
expression, we further investigated whether this NO effect results in
an inhibition of binding of extracted nuclear proteins to the proximal
region of PDGF-A promoter. We used the Egr-1 binding sequences in the
PDGF-A promoter region for an EMSA. As shown in Figure 7, when nuclear proteins extracted from
sheared ECs were incubated with oligonucleotides
corresponding to the Egr-1 binding sequences, increased binding
activity occurred. SNAP or SIN-1 treatment of ECs caused an attenuation
of this binding activity. Conversely, L-NMMA treatment of the ECs
augmented this binding activity. This binding was obviously specific to
Egr-1, because it was abolished by coincubation of nuclear proteins
with 20-fold unlabeled oligonucleotide. This
specificity was further substantiated by the supershifting in gel
mobility of the Egr-1-oligonucleotide complex after
preincubation of nuclear proteins with Egr-1 antibody. These results
indicate that NO inhibits the Egr-1 protein induced by shear stress
that subsequently results in a decrease of the expression of later
genes, including PDGF-A, which requires Egr-1 binding for its
induction.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NO, in addition to being a potent vasodilator, inhibits the expression of a number of pathophysiologically relevant genes, such as MCP-1,29 37 ICAM-1,36 VCAM-1,33 34 35 36 and PDGF-A,37 induced by chemical or mechanical stimulation. During atherogenesis, the loss of endothelium-derived NO and the accompanying expression of atherogenesis-related genes are early events.26 27 NO also acts as a negative regulator of vascular smooth muscle proliferation in response to a remodeling stimulus.30 Thus, a primary defect in NO production can promote abnormal remodeling and facilitate pathological changes in vessel walls.
Shear stress increases the production of NO by increasing eNOS
expression in ECs.19 28 31 32 One of our recent studies
demonstrated that shear stress to ECs for 15 minutes rapidly increases
NO release as detected by electron paramagnetic resonance (L.W.L.,
unpublished observation, 1999). NO release from sheared ECs
decreases monocyte adhesion to ECs.28 It is conceivable
that endogenous NO may exert its effect on signal
transduction followed by transcriptional events that ultimately alter
gene expression. Among the effects of NO on cells, the elevation of
cGMP, change of redox status, and inhibition of NF-B activation are
involved.29 33 35 36 NO inhibits the induction of ERK by
growth factors and subsequently suppresses smooth muscle cell
proliferation.38 NO may exert its effects through the
activation of cGMP-dependent protein kinase.39 The ERK
signaling pathway is also involved in shear stress–induced Egr-1
expression.40 Our present study clearly indicates that
shear stress–induced Egr-1 expression is mediated through the ERK
pathway and the EC-derived NO inhibits the ERK pathway in shear-treated
ECs. Several lines of evidence support this notion. First, NO donors
attenuated shear stress–induced Egr-1 mRNA levels in ECs. Second, ECs
treated with an NOS inhibitor conversely enhanced shear
stress–induced Egr-1 gene expression. This result indicates that
endogenous NO plays a role in modulating shear
stress–induced Egr-1 expression. Third, addition of an NO donor
inhibited and an NOS inhibitor to ECs augmented shear
stress–induced Egr-1 promoter activity. Fourth, when ECs were
cotransfected with a dominant negative mutant of ERK, Raf, or Ras,
shear stress–induced Egr-1 promoter activity was abolished. This
confirms the involvement of the Ras/Raf/ERK pathway in shear-induced
Egr-1 expression. However, this shear-induced ERK
phosphorylation and activity were inhibited after ECs
were treated with an NO donor. Fifth, the inhibitory effect
of NO was further substantiated by the suppression of NO on
shear-induced transcriptional activity of Elk-1, a downstream substrate
of ERK. Sixth, NO attenuated shear stress–induced promoter activity of
a reporter construct containing SRE, which has been shown to be
responsible for the Egr-1 induction by shear stress.40
Finally, the inhibitory effect of NO on Egr-1 expression
was confirmed by a reduction of binding of nuclear Egr-1 proteins to
the corresponding binding sequences in the promoter of PDGF-A.
In addition to the ERK pathway, shear stress to ECs also induces JNK, another mitogen-activated protein kinase.2 22 48 The JNK pathway is involved in the induction of various endothelial genes, including MCP-1.22 Whether ERK and JNK signaling pathways act synergistically or have cross talk in response to mechanical forces that lead to gene induction remains to be clarified. Shear stress exerts differential effects on ERK and JNK in ECs.2 48 Shear stress activates ERK in a time- and force-dependent manner. In contrast, JNK activity is induced by a low shear force (0.5 dyne/cm2) but with a delayed and prolonged response extending from 30 minutes to 4 hours after flow application.48 Consistent with ERK activation, present data indicate that shear stress rapidly induces Egr-1 gene expression in a transient manner, in which the induction peaks at 30 minutes and decreases afterward. These data are consistent with that transient expression reported by Khachigian et al,12 although BAECs rather than human umbilical vein ECs were used in their study. Moreover, ECs exposed to shear stress at <10 dyne/cm2 did not significantly induce their Egr-1 expression. These observations imply that the activation of ERK, rather than JNK, is critical for shear stress–induced Egr-1 expression. This is in agreement with the observation by Schwachtgen et al,40 who showed that the activation of Egr-1 by shear stress involves Elk-1 but not c-jun activity. We recently demonstrated that Egr-1 induced by cyclic strain is mediated primarily via the ERK pathway.49 The present study further supports the notion that the ERK pathway plays a key role in shear stress–induced Egr-1 expression in ECs.
The present study implies that NO regulates shear stress–induced
Egr-1 expression in ECs via the inhibition of the ERK signaling
pathway. However, the exact target molecule(s) of this NO modulation
has not been defined. NO activates guanylate
cyclase to produce cGMP, which affects various targets, including
cGMP-dependent protein kinase.50 The Ras/Raf/ERK signaling
pathway is inhibited by the cGMP-dependent protein kinase via the
phosphorylation of c-Raf kinase on
Ser43.39 However, NO inhibits the activation of NF-B
via non–cGMP-dependent mechanisms.34 36 In the
present study, ECs treated with KT5823, a cGMP-dependent protein
kinase inhibitor, did not attenuate the
inhibitory effect of NO on shear-induced Egr-1 promoter
activity. This implies that the inhibitory effect of NO on
Egr-1 induction in sheared ECs is not mediated via the cGMP-dependent
protein kinase.
The protection effect of NO has been suggested to be caused by the
attenuation of intracellular oxidative stress in cells exposed to
various stimuli, including reperfusion.51 52 Evidence
suggests that NO may reduce intracellular ROS, including superoxide
anion, via direct action on the NADPH oxidase.53 Khan et
al36 suggested that such an inhibitory effect
is responsible for the NO modulation of cytokine-induced
VCAM-1 expression in ECs. The inhibition of NO on MCP-1 induction by
cytokines or oxidized lipoproteins is mediated via the
suppression of the superoxide levels and NF-B
activity.29 Egr-1 is activated by
ROS.54 55 Furthermore, ROS modulate various signaling
pathways, including ERK.56 We previously demonstrated that
shear stress to ECs increases intracellular ROS levels, and this
increased ROS is involved in shear stress–induced ICAM-1 and
c-fos expression.8 25 Whether shear
induces endothelial Egr-1 expression as a result of
increased ROS levels remains to be determined. However, our previous
study demonstrated that the Egr-1 promoter region contains a common SRE
that is shared by cyclic strain as well as
H2O2
stimulation.49 Sheared ECs pretreated with NO, however,
resulted in a decrease of superoxide levels (H.J.H., unpublished
observation, 1999). Thus, the intracellular levels of NO and ROS
and the consequence of their interplay may affect signaling pathways
and then determine the endothelial responses under
hemodynamic conditions. The precise molecular
mechanism(s) by which NO modulates endothelial
responses to mechanical forces remains a complicated issue that
warrants further investigation.
Initial studies by Resnick et al3 defined a shear
stress–responsive element in the PDGF-B promoter region that was
required for its induction by shear stress. Later studies4
indicated that NF-B is the responsible transcriptional factor
binding to the shear stress–responsive element. Shyy et
al10 identified another shear-responsive element in MCP-1
gene that corresponds to the activator protein-1 binding
site. Recent studies have suggested that the overlapping consensus
binding elements for Sp1 and Egr-1 in the promoter of various genes may
be crucial for shear inducibility.12 15 For the Egr-1
induction in sheared ECs, the SRE is the responsible element for Egr-1
expression.40 The transcriptional factors, including
Elk-1, cooperatively interact with the serum response factor and
bind to the SRE in the promoter region of various genes, including
c-fos and Egr-1, and trigger their gene
expression.47 The present study demonstrated that
NO regulates Egr-1 expression by inhibiting Elk-1 transcriptional
activity, which results in a decrease of SRE activity as
demonstrated by the promoter activity of a reporter construct
containing 5 repeats of SRE. Taken together, our data support the
notion that the decrease of SRE activation by NO contributes to the
inhibition of Egr-1 expression in NO-treated ECs.
In summary, the present study demonstrates that shear stress–induced Egr-1 expression is inhibited by NO via the inhibition of the ERK signaling pathway in ECs. This Egr-1 inhibition consequently leads to a decrease of expression of later genes, including PDGF-A. Because ECs under shear stress constantly produce greater NO levels, this released NO thus plays a very important role in protecting cells from flow- or inflammation-induced gene expression. These results emphasize the importance of NO as a negative regulator for endothelial responses under hemodynamic conditions. The understanding of the molecular mechanisms of NO effects in vascular walls is crucial for the therapeutic implications of NO on vascular disorders associated with atherosclerosis, hypertension, and ischemia/reperfusion-induced vascular injuries.
|
Acknowledgments |
|---|
This work was supported in part by Grant NSC 86-2314-B001-004-M26 from the National Science Council, Taiwan, ROC.
Received February 1, 1999; accepted May 10, 1999.
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References |
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