1 Department of Cardiovascular
Biomechanics and 2 Institute of
Medical Electronics, To explore the mechanism of shear stress-induced downregulation
of vascular cell adhesion molecule 1 (VCAM-1) expression in murine
endothelial cells (ECs), we examined the effect of shear stress on
VCAM-1 gene transcription and assessed the
cis-acting elements involved in this
phenomenon. VCAM-1 mRNA expression was downregulated at the
transcriptional level as defined by nuclear run-on assay and transient
transfection of VCAM-1 promoter-luciferase gene constructs. The
luciferase assay on the VCAM-1 deletion mutants revealed that the
cis-acting element is contained
between
vascular endothelial cells; vascular cell adhesion molecule 1; nuclear factor activator protein-1; c-jun
VASCULAR ENDOTHELIAL CELLS (ECs) regulate the tonus of
vessels by releasing various smooth muscle relaxing and contracting substances and display antithrombotic activity by producing
anticoagulant and fibrinolytic mediators. They also actively interact
with other cells via the growth factors, adhesion molecules, and
extracellular matrix that they synthesize and secrete. In addition to
their wide variety of functions, ECs are characterized by constant
exposure to blood flow. This means that wall shear stress, a mechanical force generated by flowing blood, directly stimulates ECs. Studies in
the last decade have shown that ECs change their morphology and
functions in response to shear stress and that their responses are
closely associated with blood flow-dependent phenomena such as
angiogenesis, vascular remodeling, and atherosclerosis (1).
Recently, it has also become apparent that shear stress affects the
gene expression of many bioactive molecules in ECs. For instance, shear
stress upregulates gene expression of tissue plasminogen activator
(tPA) (5), platelet-derived growth factor (PDGF)-A (6),
nitric oxide synthase (NOS) (16), intercellular adhesion molecule 1 (ICAM-1) (15), transforming growth factor- We recently demonstrated that fluid shear stress decreases cell surface
expression of vascular cell adhesion molecule 1 (VCAM-1) in cultured
murine lymph node venule ECs, leading to suppression of their
adhesiveness to lymphocytes, and that VCAM-1 mRNA levels were also
found to be downregulated by shear stress (2, 18). However, the GAGACC
sequence (the PDGF-B SSRE) is not encoded within the VCAM-1 promoter,
and the molecular mechanism of downregulation of VCAM-1 gene expression
by shear stress, including the
cis-elements and transcription factors
involved, remains unclear. To investigate this negative regulatory
mechanism, in this study, we cloned the genomic VCAM-1 gene from the
cultured murine ECs and examined the direct effect of shear stress on
VCAM-1 gene transcription by both nuclear run-on assay and luciferase
assay using a reporter gene consisting of the VCAM-1 promoter coupled
to luciferase. By deletion analysis and gel shift assay, we also
localized a cis-acting element in the
promoter that is critical for negative regulation by shear stress.
Cell culture.
ECs from lymph node venules of C57BL/6 mice (Japan SLC, Hamamatsu,
Japan) were provided by M. Miyasaka (University of Osaka, Osaka,
Japan). Methods of isolation and characterization of the cells have
been described previously (27). Cells were cultured in Dulbecco's
modified Eagle's medium (DMEM; GIBCO, Grand Island, NY) containing
20% fetal calf serum (GIBCO), 10 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 2 mM L-glutamine,
1 mM sodium pyruvate, 100 µM 2-mercaptoethanol, 1%
(vol/vol) 100× nonessential amino acids (Flow Laboratories, Irvine, Scotland), 100 U/ml penicillin, and 100 µg/ml streptomycin and passaged with 0.05% trypsin/2 mM EDTA in phosphate-buffered saline
(PBS) when grown confluently. The cells had the configuration of a
homogeneous monolayer resembling a cobblestone pavement and the ability
to take up fluorescent acetylated low-density lipoprotein labeled with
1,1'-dioctadecyl
1-1-3,3,3',3'-tetramethylindocarbocyanine perchorate (Biomedical Technologies, Stoughton, MA). Cells in their
fourth to seventh passages were plated on a glass plate coated with
collagen (0.1 mg/ml) (Cellmatrix I-A, Nitta Gelatin, Osaka, Japan) for
use in the experiment.
Flow-loading apparatus.
To apply controlled levels of shear stress to cultured cells, we used
the same parallel plate type of flow chamber described previously (2).
One side of the chamber was formed by a coverslip on which ECs were
cultured. The other side was machined from a polymethacrylate plate.
These two flat surfaces were held ~200 µm apart by a silicone
rubber gasket. The chamber had an entrance and an exit for the medium,
and the entrance was connected to a reservoir by a silicone tube. The
medium was perfused by a roller/tube pump (ATTO, Tokyo, Japan). The
entire circuit was placed in an automatic
CO2 incubator, and the
flow-loading experiments were performed at 37°C in an atmosphere of
95% room air-5% CO2.
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
694 and
329 bp upstream from the transcription
initiation site. Gel shift assay using overlapping oligonucleotide
probes of this region showed that oligonucleotides containing a double
AP-1 consensus sequence (TGACTCA) formed distinct complexes with
nuclear proteins extracted from shear-stressed cells. Mutation of
either one or both of two AP-1 consensus sequences completely abolished
the ability of the promoter to respond to shear stress. These results suggest that fluid shear stress downregulates the transcription of the
VCAM-1 gene via an upstream
cis-element, a double AP-1 consensus
sequence, in murine lymph node venule ECs.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
1 (TGF-
1) (17),
monocyte chemotactic protein-1 (MCP-1) (23), C-type natriuretic peptide
(CNP) (19), and both the Cu/Zn- and Mn-dependent isoforms of superoxide
dismutase (SOD) (8, 26), whereas it downregulates gene expression of
angiotensin converting enzyme (ACE) (21). Shear stress up- or
downregulates the expression of genes encoding PDGF-B (11, 14, 20),
endothelin (12, 22, 30), and thrombomodulin (TM) (13, 25), depending on the experimental conditions used. In terms of a molecular mechanism for
the regulation of endothelial gene expression by shear stress, Resnick
et al. (20) first identified a
cis-acting shear stress-responsive element (SSRE) in the 5'-promoter region of the PDGF-B gene. A core-binding sequence within the SSRE (GAGACC) is also present in other
genes that respond to shear stress, including the genes for tPA, NOS,
ICAM-1, TGF-
1, MCP-1, TM, CNP, SOD, and ACE. This suggests a general
mechanism for regulation of endothelial gene transcription by shear
stress. It was later demonstrated, however, that
cis-acting elements other than the
GAGACC sequence were involved in the induction of TGF-
1 and MCP-1
genes by shear stress (17, 24). The PDGF-A gene, which does not contain
GAGACC in its promoter, also responds to shear stress. Thus the
cis-acting elements required for shear
stress responsiveness appear to be diverse.
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
,
dyn/cm2) on the EC layer was
calculated by the formula
= 6µQ/a2b,
where µ is the viscosity of the perfusate (0.0094 P at 37°C), Q
is flow volume (ml/s), and a and
b are cross-sectional dimensions of
the flow path. Because the maximum Reynold's number corresponding to
the highest flow rate used in this study was ~40, we assumed that the
flow was laminar.
RNA isolation and reverse transcriptase-polymerase chain reaction.
Isolation of total RNA from murine ECs was accomplished by the acid
guanidinium thiocyanate-phenol-chloroform extraction method. Reverse
transcription of messages for VCAM-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were carried out using Moloney murine leukemia virus reverse transcriptase (GIBCO, Gaithersburg, MD). The obtained cDNA samples were then coamplified by using 12-24 cycles of
polymerase chain reaction (PCR) with sense and antisense primers for
VCAM-1 and GAPDH, AmpliTaq DNA polymerase (Perkin Elmer-Cetus, Norwalk, CT), and [-32P]dCTP
(Du Pont, Wilmington, DE). Each temperature cycle consisted of 95°C
for 1 min, 60°C for 2 min, and 72°C for 3 min. After the PCR
reactions, the amplification product was fractionated by
electrophoresis on 8.0% polyacrylamide gel. The radioactivity of each
band on the gel was measured with a GS363 molecular imager system
(Bio-Rad Laboratories, Richmond, CA).
Isolation of murine genomic VCAM-1 gene.
Genomic DNA was extracted from cultured murine ECs with a TurboGen
genomic DNA isolation kit (Invitrogen, San Diego, CA). Sau3AI-digested
fragments (~20 kb) of the genomic DNA were ligated to the EMBL3
arms (Stratagene, La Jolla, CA) and packaged with Gigapack II packaging
extracts (Stratagene). The packaged
-phage vectors were transfected
into XL1-Blue MRA strain (Takara Biomedicals, Kyoto, Japan).
DNA sequencing. Template DNA was mixed with a terminator ready reaction mixture containing dye-labeled terminators and AmpliTaq DNA polymerase (PRISM ready reaction terminator cycle sequencing kit; Perkin Elmer) and synthetic oligonucleotide primers prepared with an Oligo 100 DNA synthesizer (Beckman Instruments, Fullerton, CA). PCR was then carried out for 25 cycles in a thermal cycler (Perkin Elmer thermal cycler model 9600). The PCR products were then electrophoresed on 4.25% polyacrylamide gel, and base sequences were automatically determined by an Applied Biosystems 373 DNA sequencer (Perkin Elmer).
Nuclear run-on transcription assay.
ECs were washed with ice-cold PBS, scraped, and pelleted by
centrifugation at 1,500 revolutions/min (rpm) for 5 min. The cell pellet was resuspended in 1 ml NP-40 lysis buffer [10 mM
tris(hydroxymethyl)aminomethane (Tris) · HCl, pH
7.4, 10 mM NaCl, 3 mM MgCl2, 0.5%
(vol/vol) NP-40], incubated for 5 min on ice, and centrifuged at
3,000 rpm for 5 min. The nuclear pellet was washed once with 1 ml NP-40
lysis buffer and centrifuged again at 3,000 rpm for 5 min. Nuclei were resuspended in 100 µl of 50 mM Tris · HCl, pH 8.3, 5 mM MgCl2, 0.1 mM EDTA, and 40%
(vol/vol) glycerol and frozen in liquid
N2. The nuclei were thawed and
reacted in 100 µl reaction buffer consisting of 10 mM
Tris · HCl, pH 8.0, 5 mM
MgCl2, 300 mM KCl, 0.5 mM ATP, 0.5 mM CTP, 0.5 mM GTP, and 3.7 MBq
[-32P]UTP (~111
TBq/mmol) for 30 min at 30°C. The
32P-labeled RNA was precipitated
with trichloroacetic acid and purified with phenol-chloroform
extraction. Plasmids containing murine VCAM-1 or human GAPDH fragment
(Clontech) were linearized by restriction enzyme digestion and
denatured at 95°C. The DNA was spotted onto nylon membranes and
fixed with 0.4 N NaOH. Radiolabeled RNA was adjusted to 2.5 × 106
counts · min
1 · ml
1
in a hybridization solution [5× SSPE, 50% formamide, 0.1%
Denhardt's solution, and 0.1% sodium dodecyl sulfate (SDS)] and
hybridized to DNA immobilized on nylon membranes for 48 h at 42°C.
Blots were washed twice in 2× SSPE and 0.1% SDS for 15 min at
42°C, once in 1× SSPE and 0.1% SDS for 30 min at 42°C,
and twice in 0.1× SSPE and 0.1% SDS for 15 min at room
temperature. Autoradiograms of the membranes were obtained using a
GS363 molecular imager system (Bio-Rad).
Construction of reporter plasmids. To determine the transcriptional activity of the VCAM-1 gene, we used reporter plasmids containing the murine VCAM-1 promoter linked to the luciferase gene. A series of deletions was created through the 5'-flanking sequences of the VCAM-1 gene by restriction enzyme digestion and subcloned into the luciferase reporter vector (pGL2-enhancer vector; Promega, Madison, WI). The following deletion constructs were generated.
pGLVCAM-1 (Transfection and luciferase assays.
Murine ECs were seeded on a coverslip at a density of 1 × 104
cells/cm2. The cells were
transfected with constructs using Transfectam (Biosepra, Malborough,
MA). The pSV--galactosidase vector (Promega) was cotransfected to
monitor transfection efficiency. After incubation at 37°C for 24 h,
the cells were then either exposed to shear stress or incubated under
static conditions. The cells were washed twice with PBS, and 250 µl
of lysis buffer (included in Promega's luciferase assay kit) were
added. After 15 min at room temperature, the lysates were centrifuged
at 12,000 rpm for 2 min. Luciferase activity was determined using 20 µl of the clarified lysate and 100 µl of luciferase assay substrate
(Promega) in a Berthold Lumat LB9501 luminometer (Wildbad, Germany).
The level of
-galactosidase was assayed in parallel by adding the
o-nitrophenyl
-D-galactopyranoside to the
cell lysate and incubation at 37°C for 1 h. The absorbance at 420 nm was measured by microplate reader (Bio-Rad model 3550). The
luciferase activity of each sample was normalized to that of
-galactosidase before calculating the values reported in
RESULTS.
Gel shift assay.
To prepare nuclear extracts, control (static) or shear-stressed (3.5 dyn/cm2 for 24 h) murine EC
monolayers containing ~6 × 107 cells were washed with PBS and
suspended in hypotonic buffer [20 mM HEPES-KOH, pH 7.9, 5 mM KCl,
8 mM MgCl2, 0.5 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride (PMSF); Sigma], and sediments were resolved in extraction buffer [20 mM
HEPES-KOH, pH 7.9, 25% (vol/vol) glycerol, 0.5 M NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.5 mM DTT,
0.5 mM PMSF, 0.5 µg/ml pepstatin A, and 1.3 µg/ml
spermidine]. After the centrifugation for 30 min at 21,000 rpm,
the supernatants were then dialyzed against buffer [20 mM HEPES-KOH, pH 7.9, 10% (vol/vol) glycerol, 50 mM KCl, 0.5 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF], and the nuclear extracts obtained were
kept frozen at 80°C until analysis.
Site-directed mutagenesis.
Mutations were generated at AP-1 binding sites in the VCAM-1 gene
promoter by the Kunkel method of site-directed mutagenesis. Single-stranded template DNA (ssDNA) was prepared by growing
bacteriophage M13K07 (Takara) in a strain of
Escherichia coli, CJ236 (Takara) that
had been transfected with the construct pGLVCAM-1 (1.8 luc). Oligonucleotides corresponding to the wild-type sequence with specific
mutations at sites indicated by asterisks in Fig.
6A were synthesized in an Oligo 100 DNA synthesizer and phosphorylated with
T4 polynucleotide kinase. The
mutagenic oligonucleotides and ssDNA were annealed, and the
polymerization/extension reaction was performed using
E. coli DNA ligase (Takara) and
T4 DNA polymerase (Takara). The
double-stranded DNAs obtained were transfected into JM109, and their
sequences were confirmed. Luciferase assay was carried out in the
murine ECs transfected with these mutagenized DNAs, which were cultured
under static conditions or exposed to shear stress.
Statistical analysis. The results are expressed as means ± SD. The mean values obtained in the control and experimental groups were analyzed for significant differences by analysis of variance and the Bonferroni modification of the t-test. Differences were considered statistically significant at P < 0.05.
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RESULTS |
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Shear stress dependency of the flow-induced decrease in VCAM-1 mRNA levels. Total RNA was extracted from murine ECs exposed to shear stress (1.5 dyn/cm2) for 6 h, and changes in VCAM-1 mRNA levels were determined by reverse transcription-PCR. VCAM-1 mRNA levels decreased in response to flow loading, which agreed quite well with our previous work (2). To determine whether the flow-induced decrease in VCAM-1 mRNA levels is a specific response for shear stress, we carried out flow-loading experiments using two perfusates having different viscosities. The high-viscosity medium (DMEM with 5% dextran) or low-viscosity medium (DMEM alone) was perfused through the circuit, and changes in VCAM-1 mRNA levels were examined. VCAM-1 mRNA levels decreased as the flow velocity (shear rate) increased, but the decrease was greater at higher viscosity and higher shear stress (Fig. 1A). On the other hand, the curves for VCAM-1 mRNA levels plotted against shear stress formed a single line (Fig. 1B), indicating that the flow-induced decrease in VCAM-1 mRNA levels is shear stress dependent rather than shear rate dependent.
|
Sequence of the murine VCAM-1 promoter.
We cloned the murine genomic VCAM-1 gene from the cultured murine ECs
used in the present study. The promoter sequence 1,801 bp from
the transcription initiation site is shown in Fig.
2. The transcription initiation site was
determined by primer extension analysis. The sequences TGGGTTTCCC at
73 bp and AGGGATTTCCC at
68 bp are identical to the
consensus sequence (GGGR[C/A/T]TYYCC) for the NF
B
binding site. The sequence TGACTCA at both
481 and
461 bp
perfectly matches the AP-1 consensus sequence (tumor promoting agent-response element). A sequence (CGTCA) with homology to cAMP response element (CRE) was identified at position
1420.
|
Direct effect of shear stress on VCAM-1 gene transcription. To determine whether shear stress directly influences VCAM-1 gene transcription, we performed a nuclear run-on transcription assay. Nuclei were prepared from static control or shear-stressed ECs, and transcription was allowed to continue in the presence of [32P]UTP. Purified radiolabeled RNA was hybridized to cDNA immobilized on nylon membranes. Although high levels of transcription of the VCAM-1 gene could be detected in static control cells, after 24-h exposure to shear stress, transcription was decreased (Fig. 3A). Densitometry of individual spots of interest revealed that the density of VCAM-1 mRNA signal in shear-stressed cells decreased to 68 ± 1.4% (SD) of that in static controls (n = 3, P < 0.001). The transcription of GAPDH mRNA did not change significantly after exposure of the cells to shear stress. The density of shear-stressed cells was 91.4 ± 5.5% (SD) of that of static controls (n = 3, P = NS).
|
Localization of the shear stress-responsive regions of the VCAM-1
promoter.
The fact that shear stress suppresses VCAM-1 gene transcription
suggests the presence of cis-acting
regions within the VCAM-1 promoter that are responsive to shear stress.
To localize these regions, a series of nested 5'-deletion
mutations of the VCAM-1 promoter coupled to luciferase was generated
(Fig.
4A) and
transfected into murine ECs. The transfected cells were then exposed to
shear stress (3.5 dyn/cm2) for
24 h, and their luciferase activity was measured. As shown in Fig.
4B, the cells transfected with
constructs 3.7,
1.8,
1.1, or
0.7 luc showed
a marked decrease in luciferase transcription in response to shear
stress, whereas transfection of construct
0.3 luc containing 329 bases of the promoter abolished responsiveness to shear stress,
although not completely. These findings indicate that the shear
stress-responsive regions are located between
694 and
329
bp of the 5'-flanking region of the murine VCAM-1 gene.
|
Oligonucleotides bearing the AP-1 binding sites interact with
nuclear proteins from ECs exposed to shear stress.
To examine the presence of transcriptional factors that bind to the
shear stress-responsive regions, we performed gel shift assays in which
nuclear extracts from the static or shear-stressed cells were incubated
with each of 21 radiolabeled oligonucleotides synthesized based on the
sequences of 694 and
328 bp upstream from the
transcription initiation site of the VCAM-1 gene. Three oligonucleotides bearing the transcription factor AP-1 binding sites
(
472 to
443 bp,
492 to
463 bp,
and
481 to
452 bp) were able to form distinct complexes
with nuclear protein derived from either static or shear-stressed
murine ECs (Fig.
5A). The quantity of complexes was much higher in the nuclear extracts from the
shear-stressed cells. Addition of a 100-fold excess of unlabeled
cognate completely inhibited the formation of the complex. The presence
of unrelated DNA, i.e., Epstein-Barr virus nuclear antigen-1 DNA
(EBNA-1 DNA, Pharmacia Biotech), did not reduce the specific binding
between the oligonucleotides and nuclear protein. To establish the
identity of the nuclear protein that bound these three
oligonucleotides, we used antibodies to
c-jun and
c-fos peptides. The protein-DNA
complexes were partially eliminated by the presence of the antibody to
c-jun, but not by the antibody to
c-fos (Fig.
5B). None of the antibodies against
CREB-1, CREM-1, ATF-2, or RAR-
1
affected the protein-DNA complexes (data not shown). These results
suggest that AP-1, i.e.,
c-jun/c-jun
homodimer, was, at least in part, involved in the shear-induced
downregulation of murine VCAM-1 gene transcription.
|
AP-1 consensus elements are essential for shear-mediated downregulation of reporter gene expression. To test whether the AP-1 binding sites are critical for the VCAM-1 gene response to shear stress, we constructed several chimeric genes with site-specific mutagenesis at the AP-1 binding sites and assayed luciferase in the murine ECs transfected with these mutants. Mutant-1 and mutant-2, which had a mutated distal and proximal AP-1 binding site, respectively, and mutant-3, in which both sites were mutated, abolished the downregulation of VCAM-1 gene transcription in response to shear stress seen in the wild-type gene (Fig. 6A). Gel shift assays, which were performed with oligonucleotides bearing the mutated AP-1 consensus sequences as a labeled probe and nuclear extracts from shear-stressed cells, showed the complete disappearance of the protein-DNA complexes formed by the oligonucleotide containing native AP-1 binding site sequences (Fig. 6B). These findings indicate that a double AP-1 consensus element is responsible for the VCAM-1 gene response to shear stress in murine lymph node venule ECs.
|
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DISCUSSION |
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Our previous study demonstrated that laminar flow suppresses the adhesiveness of murine lymph node venule ECs to lymphocytes by downregulating the expression of VCAM-1 at both the mRNA and cell surface protein levels (2, 18). The present study confirmed that downregulation of VCAM-1 mRNA is a specific response to shear stress and not to flow velocity or shear rate. When cells are exposed to fluid flow, two distinct effects can occur: 1) mechanical stimulation of the cells by the direct force of flow, shear stress; and 2) an indirect effect of flow that changes the concentration of agonists at the endothelial surface. In regard to this latter effect, if certain agonists, e.g., adenine nucleotides, bradykinin, and angiotensin, are removed rapidly by degradation at the cell surface, a diffusion boundary layer will exist near the cell surface, and this layer will become thinner as flow velocity increases, i.e., flow will increase the amount of such agonists reaching the cell surface, and consequently modulate cell functions, flow velocity or shear rate dependently. To differentiate the direct and indirect effects of flow, we performed flow-loading experiments with two perfusates of different viscosity. The results demonstrated that the decrease in VCAM-1 mRNA levels occurred to a greater extent at higher viscosity or higher shear stress at a given flow velocity, indicating that the VCAM-1 mRNA response is shear stress dependent rather than shear rate dependent.
Our nuclear run-on assay clearly demonstrated that the downregulation
of murine VCAM-1 gene expression by shear stress is transcriptionally
mediated. Luciferase assay using the reporter gene containing the
promoter of a cloned murine VCAM-1 gene also revealed that
transcriptional activity was markedly suppressed by shear stress.
Although it is unclear whether shear stress alters the stability of
VCAM-1 mRNA, it seems certain that suppression of transcription is
involved. When bovine fetus aortic ECs were transfected with the same
reporter gene, the basal transcription level was markedly lower than
that of murine ECs, but suppression of murine VCAM-1 gene transcription
occurred in response to shear stress. This indicates that there is a
mechanism that specifically enhances basal transcription of the VCAM-1
gene in murine lymph node venule ECs, but that murine and bovine ECs
may possess common transcriptional factors that can be activated by
shear stress. In vivo, lymph node venule ECs, a common site of
leukocyte emigration, constitutively express abundant VCAM-1 on their
cell surface, and display high adhesiveness for lymphocytes. Thus the
inhibitory effect of shear stress on VCAM-1 expression might be
noticeable in such ECs expressing VCAM-1 at high basal levels. There
are certain other situations in which shear stress exerts an inhibitory effect on VCAM-1 expression. Varner et al. (28) showed that preconditioning human umbilical vein ECs with shear stress inhibits cytokine (interleukin-1)-induced VCAM-1 gene expression. Walpola et
al. (29) observed that when blood flow was decreased by surgical manipulations in rabbit carotid arteries in vivo, endothelial VCAM-1
expression greatly increased, indicating that at physiological levels
of shear stress blood flow might have been suppressing VCAM-1
expression in the presurgical arteries.
By analyzing the 5'-promoter of the murine VCAM-1 gene, we found
that the cis-acting element responding
to shear stress is present between 694 and
329 bp
upstream from the transcription initiation site that contains two AP-1
consensus sequences, TGACTCA. Gel shift assay showed that
oligonucleotides bearing a single or double AP-1 consensus element were
able to form distinct complexes with nuclear proteins from
shear-stressed cells, suggesting that shear stress increases nuclear
protein binding to the AP-1 consensus sequences. Furthermore,
site-specific mutations indicate that both the proximal and distal AP-1
consensus element are critical for negative regulation of VCAM-1
transcription by shear stress. The molecular mechanism of
shear-mediated gene regulation involving AP-1 is also seen in the MCP-1
gene, whose promoter contains two AP-1 binding sites, but there are
some differences between the two (24). Shear stress upregulates
transcription of the MCP-1 gene, and only one of the two AP-1 binding
sites is shear responsive. AP-1 consensus elements have been known to
be capable of acting as either transcriptional enhancers or silencers
(3).
The products of the nuclear protooncogenes
c-fos and
c-jun form protein dimers that exhibit
powerful binding activity to AP-1 consensus elements on gene promoters.
Several reports indicate that shear stress increases
c-fos and
c-jun at both protein and mRNA levels
in ECs (7, 10). The present gel shift assay revealed a marked increase
of proteins that specifically bind to oligonucleotides with AP-1
consensus sequence in the nuclei of shear-stressed cells. We carried
out supershift assay to examine whether
c-fos and
c-jun are involved in the protein-DNA
complexes. The results demonstrated that antibody to
c-jun peptide, which recognizes both
phosphorylated and unphosphorylated forms of
c-jun peptide, partially inhibited the
formation of the protein-DNA complexes, whereas antibody to c-fos had no effect on the binding
reaction. This suggests that c-jun
dimers may be involved, at least in part, but there are other proteins
that can form these complexes. The increase in the amount of
protein-DNA complexes detected by gel shift assay does not necessarily
mean that the same phenomenon occurs in vivo, i.e., the AP-1 binding
sites in VCAM-1 promoter are occupied by proteins. From evidence now at
hand, it seems difficult to tell which is more important for the
regulation of murine VCAM-1 gene transcription by shear stress, the
increase in the amount of transcriptional factors, or modifications of
the factors, e.g., phosphorylation. Recently, transcription factor
NFB has been found to be activated by shear stress and to interact
with the PDGF-B SSRE, leading to upregulation of PDGF gene expression
in bovine ECs (9). There are two NF
B binding sites in the proximal
region of murine VCAM-1 promoter, but interestingly, these sites are
not implicated in the VCAM-1 gene response to shear stress. Therefore,
further studies identifying cis-acting
elements and transcription factors involved in a variety of EC
responses to shear stress are needed to clarify the molecular mechanism
for shear-mediated regulation of endothelial gene expression. Such
studies will also provide us with new insights into blood
flow-dependent phenomena such as angiogenesis, vascular remodeling, and
atherosclerosis.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Michael A. Gimbrone, Jr., Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, for advice and criticism.
![]() |
FOOTNOTES |
---|
This work was partly supported by Grants-in-Aid for Scientific Research and for Scientific Research on Priority Areas from the Japanese Ministry of Education, Science, and Culture, a research grant for cardiovascular diseases from the Japanese Ministry of Health and Welfare, and research funds from Tsumura.
Address for reprint requests: J. Ando, Dept. of Cardiovascular Biomechanics, Faculty of Medicine, Univ. of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
Received 4 November 1996; accepted in final form 30 June 1997.
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