(Received for publication, May 31, 1996, and in revised form, November 1, 1996)
From the Department of Pathology, Division of Molecular and Cellular Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294
Shear stress differentially regulates production
of many vasoactive factors at the level of gene expression in
endothelial cells that may be mediated by mitogen-activated protein
kinases, including extracellular signal-regulated kinase (ERK) and
N-terminal Jun kinase (JNK). Here we show, using bovine aortic
endothelial cells (BAEC), that shear stress differentially regulates
ERK and JNK by mechanisms involving Gi2 and pertussis toxin
(PTx)-insensitive G-protein-dependent pathways,
respectively. Shear activated ERK with a rapid, biphasic time course
(maximum by 5 min and basal by 30-min shear exposure) and force
dependence (minimum and maximum at 1 and 10 dyn/cm2 shear
stress, respectively). PTx treatment prevented
shear-dependent activation of ERK1/2, consistent with a
Gi-dependent mechanism. In contrast, JNK
activity was maximally turned on by a threshold level of shear force
(0.5 dyn/cm2 or higher) with a much slower and prolonged
time course (requiring at least 30 min to 4 h) than that of ERK.
Also, PTx had no effect on shear-dependent activation of
JNK. To further define the shear-sensitive ERK and JNK pathways,
vectors expressing hemagglutinin epitope-tagged ERK (HA-ERK) or HA-JNK
were co-transfected with other vectors by using adenovirus-polylysine
in BAEC. Expression of the mutant i2(G203), antisense
G
i2 and a dominant negative Ras (N17Ras) prevented
shear-dependent activation of HA-ERK, while that of
i2(G204) and antisense
i3 did not.
Expression of a G
/
scavenger, the carboxyl terminus of
-adrenergic receptor kinase (
ARK-ct), and N17Ras inhibited
shear-dependent activation of HA-JNK. Treatment of BAEC
with genistein prevented shear-dependent activation of ERK
and JNK, indicating the essential role of tyrosine kinase(s) in both
ERK and JNK pathways. These results provide evidence that 1)
Gi2-protein, Ras, and tyrosine kinase(s) are upstream
regulators of shear-dependent activation of ERK and 2) that
shear-dependent activation of JNK is regulated by
mechanisms involving G
/
, Ras, and tyrosine kinase(s).
Endothelial cells lining the inner vessel wall are in direct
contact with flowing blood, which generates a frictional force, hemodynamic shear stress, acting on the surface of the endothelium. Hemodynamic shear stress controls vascular tone, vessel wall
remodeling, interaction of blood cells with endothelium, coagulation,
and fibrinolysis (1). The focal pattern of atherosclerotic lesions in
areas of low and/or unstable shear stress further highlights the
importance of shear stress in the atherogenic process (2, 3).
Endothelial cells play a key role in shear-dependent
vascular changes, sensing shear stress by an unidentified
mechanoreceptor(s) followed by production of autocrine and paracrine
factors (1). For example, hemodynamic shear stress selectively and
differentially regulates production of intercellular adhesion
molecule-1, vascular cell adhesion molecule-1, platelet-derived growth
factor-B, basic fibroblast growth factor, transforming growth factor
-1, tissue plasminogen activator, endothelial nitric oxide synthase,
and endothelin at the level of gene expression (4, 5, 6, 7, 8, 9, 10, 11, 12, 13). Resnick
et al. (6) identified a conserved cis-acting shear stress
response element with the 6-base pair core binding sequence within the
5
promoter region of the platelet-derived growth factor-B chain.
Interestingly, this shear stress response element has been identified
in other shear-sensitive genes including intercellular adhesion
molecule-1, tissue plasminogen activator, and transforming growth
factor
-1, suggesting its role in shear-dependent
regulation of these genes. Furthermore, NF-
B has been shown to bind
to the shear stress response element (15). Shear stress induces
biphasic expression of c-Fos, c-Jun, and c-Myc as well as activation of
DNA binding activities of NF-
B and transcription factor activator
protein 1 (AP-1)1 (14, 16). These immediate
early response genes and transcription factors are likely to be
involved in the regulation of shear-dependent gene
expression.
Mitogen-activated protein kinase family members including ERK and JNK
(also known as stress-activated protein kinase), have been proposed to
be important signaling components linking extracellular stimuli to
cellular responses including cellular growth, differentiation, and
metabolic regulation (reviewed in Refs. 17 and 18). Mitogen-activated protein kinases can be activated by various external stimuli such as
growth factors (nerve and epidermal growth factors), ligands acting on
G-protein-coupled receptors (2-adrenergic agonists and
lysophosphatidic acid) and physical stresses (ultraviolet radiation and
hyperosmolarity) (17, 18). Recently, shear stress also has been shown
to stimulate ERK in endothelial cells (19, 20). Tseng et al.
(19) showed that GDP
S treatment and inhibition of protein kinase C
blocked shear-dependent ERK activation in fetal BAEC.
In this study we examined the effect of shear stress on JNK and ERK in
adult BAEC. Shear stress activated ERK and JNK with markedly different
time courses and force dependence. Contrary to the previous report
(19), shear-stimulated activation of ERK is regulated by mechanisms
involving a PTx-sensitive i2, Ras, and a tyrosine
kinase(s). On the other hand, shear-dependent activation of
JNK is not PTx-sensitive but is regulated by G
/
, Ras, and
pathways dependent on tyrosine kinase(s).
BAEC harvested from descending thoracic aortas were maintained (37 °C, 5% CO2) in a growth medium (DMEM (1 g/liter glucose; Life Technologies, Inc.) containing 20% fetal calf serum (FCS; Atlanta Biologicals) without antibiotics) (21). Cells used in this study were between passages 5 and 10. For shear experiments, 1 million cells/glass slide (75 × 38 mm; Fisher) were seeded in growth medium. The next day, the medium was changed to a starvation medium (phenol red-free DMEM containing 0.5% FCS and 25 mM HEPES) and incubated for 1-2 days. Where indicated, PTx (0.1 µg/ml; List Biologicals) was added 18 h prior to shear exposure.
Plasmids, Adenovirus, and TransfectionAntisense
i2 and
i3 vectors were prepared by using
the EcoRI fragments of
i2 and
i3 (kind gift of Dr. R. Reed (Johns Hopkins University))
and EcoRI-cut pcDNA3 (Invitrogen) as described (22). Two
Gi2
mutants containing point mutations at
Gly203
Thr (
i2(G203)) and
Gly204
Ala (
i2(G204)) inserted into pCW1
were kind gift of Dr. G. L. Johnson (University of Colorado) (23).
Vectors expressing N17Ras and
ARK-ct fused to CD8 in pcDNA3 (24)
and HA-ERK2 and HA-JNK1 in pSR
(25) were kindly provided by Dr. S. Gutkind (National Institute of Dental Research) and Dr. A. Lin
(University of Alabama at Birmingham), respectively. Endotoxin-free
DNAs used in all transfection experiments were prepared by using a
maxiprep kit following the manufacturer's instruction (Quiagen).
For transfection studies, BAEC (2.5 × 105 cells/glass
plate) were grown overnight in the growth medium and washed in Hanks' buffered salt solution just prior to transfection using the method of
adenovirus conjugated to polylysine (AdpL) as described (26). Briefly,
a replication-defective adenovirus d11014 (kind gift of Dr. D. T. Curiel (University of Alabama at Birmingham)) was cross-linked to
polylysine (Sigma) using
1-ethyl-3-(3-dimethylaminopropyl carbodiimide-HCL), and small aliquots
(1 × 1011 particles/ml) were stored at 80 °C
(26). AdpL was conjugated to DNA on the day of transfection by
incubating 100 µl of HEPES-buffered saline (150 mM NaCl,
20 mM Hepes, pH 7.3), 50 µl of AdpL, and 2 µg of
plasmid DNA per glass plate for 30 min at room temperature followed by
an additional 30-min incubation at room temperature with 2 µg of
polylysine in 100 µl of HEPES-buffered saline. AdpL-DNA conjugate
mixed with 2 ml of DMEM containing 0.5% FCS was added to the cells.
One hour later, 10 ml of DMEM containing 10% FCS was added to the
cells and incubated overnight. The next day, medium was changed to
fresh DMEM containing 10% FCS, incubated for 24 h, and
serum-starved overnight in the starvation medium. Under identical
conditions, the transfection efficiency of
-galactosidase DNA in
pCMV (ATCC) ranged from 25 to 40% (data not shown) as determined by a
5-bromo-4-chloro-3-indoyl
-D-galactoside staining method in 4% paraformaldehyde fixed monolayers (27).
The glass slide containing a BAEC monolayer was assembled into a parallel plate shear chamber forming a flow channel (220-µm height × 2.5-cm width × 6.2-cm length) between the monolayer and fabricated polycarbonate plate as described (28). Non-pulsatile, laminar shear stress was controlled by changing the flow rate of the starvation medium delivered to the cells using the constant head flow loop or a syringe pump (KD Scientific) as described (21, 28).
Preparation of Cytosol and Soluble LysatesFollowing shear
exposure, cells were washed in ice-cold phosphate-buffered saline,
scraped in a 0.5-ml extraction buffer (50 mM
-glycerophosphate, pH 7.33, 1.5 mM EGTA, 0.1 mM vanadate, 1 mM dithiothreitol, 10 µg/ml
leupeptin, 2 µg/ml pepstatin, 1 mM benzamidine, and 0.1 mM phenylmethylsulfonyl fluoride) and immediately frozen at
80 °C until cell fractionation. Cell extracts were sonicated and
centrifuged for 1,000 × g for 10 min, and the supernatants were further centrifuged at 20,000 × g
for 30 min to separate crude cytosolic fractions (cytosol). For
preparation of detergent-soluble lysates, shear-exposed cells were
scraped in the extraction buffer containing 1% Triton X-100,
solubilized for 1 h, and centrifuged at 20,000 × g for 30 min. The entire fractionation and solubilization
procedures were performed at 4 °C. Protein content of each sample
was measured by using a Bio-Rad DC assay kit.
Soluble lysates were resolved on 10% SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Millipore) and probed with antibodies specific to phosphotyrosine (Upstate Biotechnology Inc., Lake Placid, NY), ERK1/2 (Upstate Biotechnology), or phosphorylated forms of ERK1/2 (Ref. 29; New England Biolabs). Goat anti-mouse (or rabbit) IgG conjugated to alkaline phosphatase was used as secondary antibody and developed either colorimetrically using a Bio-Rad kit or by a chemiluminescent detection method as suggested by the manufacturer (New England Biolabs).
Mitogen-activated Protein Kinase AssaysCytosol or soluble
lysates (5 µg each) were used to phosphorylate myelin basic protein
(MBP) (Sigma) and c-Jun (amino acids 5-89) fused to
glutathione S-transferase (GST-c-Jun) (30). Briefly, each
substrate was phosphorylated in buffer A (50 mM
-glycerophosphate, pH 7.33, 2.5 mM EGTA, 0.1 mM vanadate, 20 mM MgCl2, 1 mM dithiothreitol, 70 µM ATP, and 2,000 cpm/pmol [
-32P]ATP) for 5 min at 30 °C. MBP
phosphorylation was stopped either by spotting aliquots to P-81 filter
papers followed by washing and counting (filter assay) as described
(31) or by boiling in Laemmli sample buffer followed by 12.5%
SDS-PAGE, Coomassie staining, and autoradiography. MBP phosphorylation
activity was linear for at least 10 min at 30 °C when less than 10 µg of cytosolic protein was used (data not shown). Phosphorylation of
GST-c-Jun was terminated by boiling in Laemmli sample buffer. Boiled
samples were resolved by 12.5% SDS-PAGE followed by Coomassie staining and autoradiography. Phosphorylated bands were cut and counted in a
scintillation counter. For "in-gel kinase" assays, cytosols (20 µg) were resolved in a 12.5% SDS-PAGE minigel containing immobilized MBP (0.4 mg/ml) followed by phosphorylation of renatured kinases and
autoradiography as described (31). For immunocomplex assays, antibodies
specific for ERK1/2 (Upstate Biotechnology, catalog number 06-182),
JNK1 (Pharmingen, clone G151-333), and HA (Boehringer Mannheim) were
incubated with the soluble lysates (50-100 µg) for 1 h at
4 °C, followed by an additional 1-h incubation with Protein
A-agarose (for ERK and HA antibodies) or Protein G-agarose (for JNK1
antibody) beads. The immunocomplex was washed four times in the
extraction buffer containing 1% Triton X-100 and twice in buffer C (20 mM HEPES, pH 7.6, 20 mM MgCl2, 20 mM
-glycerophosphate, 20 mM
p-nitrophenyl phosphate, 0.1 mM vanadate, and 2 mM dithiothreitol). The washed immunocomplexes were
incubated in 20 µl of buffer C containing either MBP or GST-c-Jun (5 µg each) and 50 µCi of [
-32P]ATP for 20 min at
30 °C followed by SDS-PAGE, autoradiography, and quantitation of
radioactivity incorporated into each band in a scintillation
counter.
Immunoblot studies of
BAEC lysates with a phosphotyrosine antibody showed that exposure of
cells to shear stress (0, 5, 10, and 20 dyn/cm2 for 5 min
each) increased tyrosine phosphorylation of 42-44-kDa bands (p44/42)
in a shear intensity-dependent manner (Fig.
1A). Additional immunoblot studies showed
that an ERK antibody (Upstate Biotechnology) recognized ERK1 (p44) and
ERK2 (p42) in both control and shear-stimulated BAEC (data not
shown).
To test directly whether shear stress stimulated ERK activity, cytosols obtained from BAEC exposed to shear stress were used in an in vitro phosphorylation assay using MBP as a substrate (filter assay; Fig. 1B). Exposure of BAEC to shear stress (5-20 dyn/cm2) for 5 min stimulated ERK activity by 2-3-fold (Fig. 1B). The minimum shear intensity required to stimulate ERK was 1 dyn/cm2. At 10 dyn/cm2 of shear intensity, MBP phosphorylation activity reached a maximal level and did not change further when shear was increased to 20 dyn/cm2. To determine the molecular weight and specificity of the shear-stimulated MBP kinase activity (Fig. 1B), an in-gel kinase assay using immobilized MBP was performed. As shown in Fig. 1C, the electrophoretic mobilities of the shear stress-dependent MBP kinase activities were 44/42 kDa, which are consistent with the molecular masses of ERK1 and ERK2, respectively. The stimulation of p44/42 phosphorylation activity by shear exposure (5 dyn/cm2 for 5 min) was higher (4-7-fold increase compared with no flow controls when determined by the densitometric measurements of autoradiograms obtained from four independent shear stress studies) than those determined by filter assays (2-3-fold; Fig. 1B). This difference is most likely due to the presence of non-ERK activity contributing to the higher basal MBP phosphorylation activity, thus lowering the relative -fold stimulation, in the filter assay. Indeed, there was another kinase activity with a molecular mass of 66 kDa (p66) phosphorylating MBP as shown in the autoradiogram obtained from the in-gel kinase assay (Fig. 1C). However, the phosphorylation activity of p66 was not changed by shear stress. The identity of ERK was further confirmed by an immunocomplex assay (Fig. 1D). ERK was immunoprecipitated by an ERK antibody (cross-reacting with both ERK1 and 2) from the lysates of control and shear-exposed BAEC. Subsequent in vitro phosphorylation of the immunocomplex using MBP as a substrate showed that shear stress stimulated ERK activity compared with control (Fig. 1D). Together, these results demonstrate that ERK was the shear-dependent kinase activity that catalyzed the phosphorylation of MBP.
Shear Stimulates ERK and JNK in Different Time-dependent MannersShear stimulation of ERK
activity was monitored as a function of shear exposure time (Fig.
2). Shear-induced (5 dyn/cm2) ERK activation
was transient, reaching a maximum by 5 min and declining to control
levels by 30 min of shear exposure (Fig. 2, closed circles).
In parallel studies, the effect of shear stress on JNK activity was
also determined using GST-c-Jun as a substrate. Although shear
stimulated JNK activity in a time-dependent manner (Fig. 2,
open circles), the time course was markedly different from
the time course for ERK activation. Shear-stimulated JNK activation
required 30 min of shear exposure and increased further at 60 min.
These results showed that shear activation of ERK clearly precedes that
of JNK.
Shear Stress Stimulates JNK1 in a Time- and Threshold Force-dependent Manner
Shear force- and
time-dependent activation of JNK was further characterized
(Fig. 3). When exposed for 30 min to increasing intensities of shear stress (0-20 dyn/cm2), maximum
stimulation of JNK activity occurred at the lowest shear force tested
(0.5 dyn/cm2) in BAEC (Fig. 3, A and
B). In contrast to the force-dependent activation of ERK (Fig. 1B), this result suggests that JNK
is regulated by an "on or off" mechanism in response to the
sustained increase in shear force. Shear-dependent
activation of JNK was detectable after 30 min, reached a maximum by
4 h, and returned to basal level after 17 h of shear onset
using 5 dyn/cm2 (Fig. 3, C and D).
PTx Inhibits Shear-dependent Activation of ERK but Not JNK
Previous studies have shown that heterotrimeric G proteins regulate both ERK and JNK pathways in other cell types. Furthermore, G-proteins have been shown to mediate many shear-dependent signaling pathways in endothelial cells by PTx-sensitive and -insensitive pathways (8, 28, 32, 33). Therefore, we tested whether Gi-proteins regulate shear-stimulated ERK and/or JNK activation using PTx.
Treatment with PTx completely prevented shear-dependent
phosphorylation of ERK1/2 stimulated by 2-, 5-, and 10-min shear
exposure (Fig. 4, B and D, PTx
groups). Essentially the same inhibitory effect of PTx on
shear-dependent activation of ERK was also shown by
in vitro MBP phosphorylation assay of cytosols (25 ± 7% of control, n = 6, p < 0.0001, 5 or 10 dyn/cm2 of shear stress for 5 min, data not shown). On the
other hand, cholera toxin (CTx), an activator of s, had
no effect on shear-dependent activation of ERK (Fig. 4,
B and D, CTx groups). PTx treatment of BAEC using
the conditions employed here resulted in maximal ADP-ribosylation of
Gi-proteins as evidenced by complete inhibition of in
vitro PTx-catalyzed [32P]ADP-ribosylation (Fig.
4C). The rapid and transient changes in the phosphorylation
status of ERK1/2 were not due to changes in the amount of these enzymes
as shown in Fig. 4A.
Unlike ERK, PTx had no significant effect (p 0.18, n = 3 or 4) on shear-stimulated JNK1 activation at any
time points examined in this study (Fig. 5). CTx
treatment also had no significant effect on JNK1 activation by 30 and
60 min of shear (Fig. 5). However, at 5 min of shear, CTx stimulated
JNK activation of from 89.8 ± 17% of control (n = 4, no toxin group) to 197.7 ± 50% of control
(n = 3, cholera toxin group, p = 0.02)
(Fig. 5, A and B).
To determine the identity of the PTx substrate and the
role of Ras regulating shear-sensitive ERK, BAEC were co-transfected with HA-ERK and i constructs or N17Ras with the AdpL
method. Transfection of BAEC by this method did not have any
significant effect on shear-dependent activation of
endogenous ERK and JNK (data not shown). Shear stress was able to
stimulate the activity of HA-ERK by 3-4-fold in the co-transfected
BAEC (see pcDNA3 groups, Fig. 6, A and
B). Expression of
i2(G203) and antisense
i2, but not
i2(G204) and antisense
i3,
prevented shear activation of HA-ERK. These results showed that the
i2(G203) produces an inhibitory phenotype and
i2(G204) is a null mutant similar to that found in
previous studies (23). The antisense studies showed the specific nature
of
i2 compared with the
i3 construct
(Fig. 6A). Furthermore, expression of N17Ras prevented
shear-sensitive ERK activation (Fig. 6B) suggesting the
involvement of Ras as an upstream regulator.
G
Previous studies have shown that JNK can be regulated
by heterotrimeric G-proteins, 12,
13,
q, or G
/
, in a Ras- and Rac-dependent pathway in other cell types (24, 34, 35). Since dominant negative
mutants of
12,
13, and
q
have not been available, we decided to block G
/
to test the role
of heterotrimeric G-proteins and to use N17Ras in co-transfection
studies with HA-JNK. Expression of
ARK-ct inhibits
shear-dependent activation of JNK by 60% compared with the
HA-JNK/pcDNA3 control (Fig. 7) suggesting the
involvement of heterotrimeric G-proteins as one of its upstream
regulators. Furthermore, expression of N17Ras completely prevented
shear-dependent activation of JNK, showing its Ras-dependence (Fig.
7).
Tyrosine Kinase(s) Regulate Shear-dependent Activation of ERK and JNK
Tyrosine kinases have been shown to regulate G-protein-dependent activation of both ERK and JNK (36, 37). Therefore, we pretreated BAEC with genistein to inhibit tyrosine kinases. Genistein treatment completely blocked shear-dependent activation of both ERK and JNK, indicating tyrosine kinase involvement in both pathways.
Shear stress regulates vessel wall function and structure by the
mechanisms that include expression of multiple genes in endothelial cells (4, 5, 6, 7, 8, 9, 10, 11, 12, 13). Focal development patterns of atherosclerotic lesions in
the areas of low and/or disturbed shear stress support the importance
of shear stress in the pathogenesis of atherosclerosis (2, 3). In this
study we demonstrated the differential mechanisms regulating the
activities of ERK and JNK in endothelial cells in response to shear
stress. ERK activation was rapid and biphasic (peaks at 5 min and
returns to basal by 30 min of shear) and required higher shear force
(insensitive to 0.5 dyn/cm2 and maximum by 10 dyn/cm2) compared with JNK activation (see Figs.
1B and 3A). In comparison, JNK activation
required longer shear exposure (30 min) and showed a slower time course
(peaks at 4 h and returns to basal at 17 h) (Fig.
3C) than ERK activation. Moreover, JNK activity was
activated to the maximal level by shear force as low as 0.5 dyn/cm2. This study also showed that PTx prevents
shear-activated ERK, but not JNK. Further studies identified
i2, Ras, and a tyrosine kinase(s) as upstream regulators
of shear-dependent ERK activity. We also showed evidence
supporting the role of G
/
, Ras, and a tyrosine kinase(s) in
shear-dependent activation of JNK. These results indicate
that arterial endothelial cells, which are in direct contact with shear
stress, respond to this physiologic mechanical stimulus through
differential G-protein-dependent mechanisms leading to
activation of ERK and JNK.
Activation of ERK and JNK in endothelial cells by shear stress may result in the selective phosphorylation and activation of transcription factors leading to selective gene regulation events. ERK activation has been reported to result in phosphorylation of ternary complex factor/Elk, which in turn triggers induction of c-Fos and subsequent stimulation of AP-1 activity (18, 38, 39). Activated JNK has been shown to phosphorylate c-Jun, which in turn induces its own gene expression and subsequent increase in AP-1 activity (18, 31, 25, 40, 41, 42, 43). In endothelial cells, shear stress has been shown to induce biphasic increases in mRNA levels of c-Fos and c-Jun (14), which could have resulted in increased DNA binding activities of AP-1 (16). Interestingly, shear-dependent activation of ERK (5 min) and JNK (30 min to 4 h) precedes the first (20 min) and second phase (60-90 min of shear) of the AP-1 activation, respectively (Figs. 2 and 3 and Ref. 16). Although not directly proven, this temporal relationship suggests possible roles of ERK and JNK as upstream regulators of AP-1 activity.
Our study strongly indicates that shear-dependent
activation of ERK and JNK is regulated by signaling pathways involving
two different heterotrimeric G-proteins (most likely to be
Gi2 protein for ERK and q or
12/
13 for JNK). Several lines of evidence support our conclusion. First, the effect of PTx on
shear-dependent ERK activation (Fig. 4) points to
i2 and/or
i3 as its regulator, since BAEC
are known to express these two
i-proteins but not
i1 and
o (44). Second, co-transfection
studies with
i mutants and antisense
i constructs further
demonstrate the specific effect of
i2 as an activator of
ERK (Fig. 6). Mutations of Gly residues within the invariant sequence
(Asp-Val-Gly203-Gly204-Gln) of all
G
-subunits have been shown to produce dominant negative or null
phenotypes, when expressed in cells, depending upon cell types and
effector systems studied (45, 46, 28). In our study, only
i2(G203) inhibited shear-dependent ERK
activation while
i2(G204) was without any effect. This
specific effect of
i2(G203) is not likely due to a
simple scavenging of G
/
, since both
i2(G203) and
i2(G204) mutants retain the ability to bind G
/
,
although they lose guanine nucleotide binding capabilities (45). Winitz
et al. (23) showed that the expression of
i2(G203), but not
i2(G204), inhibits
cPLA2 and speculated that Gly203 mutation may interfere
with the conformational change required for its interaction with a
specific effector. Similar mutations of
s corresponding
to Gly203 and Gly204 also resulted in a
dominant negative and a null phenotype, respectively, in
vivo (46). The specific effect of antisense
i2, but
not antisense
i3 provides further support for the
essential role of
i2 in shear-dependent
activation of ERK. Third, the inhibitory effect of
ARK-ct, a
G
/
scavenger (47), on shear-dependent JNK activation
suggests the role of heterotrimeric G-proteins in its pathway. Many
examples of G-protein-dependent activation of ERK and JNK
have been documented in other cell types. For example, ERK can be
activated by Gi-coupled receptor agonists, presumably by
stimulating the release of
/
-subunits of Gi-proteins
and subsequent activation of Ras-dependent or -independent
cascades (47, 48, 49, 50, 51, 52). G
/
and constituitively active mutants of
12,
13, and
q have been
shown to activate JNK through Ras and Rac-1-dependent
pathways (24, 34). Since PTx and CTx had no significant effect on
shear-dependent activation of JNK (Fig. 5),
12/
13 and
q classes of
G-proteins are the possible candidates. Last, if ERK and JNK were
stimulated by the same G-protein, PTx should have blocked shear
stimulation of both ERK and JNK, and we found this was not the case
(Fig. 4 and 5).
Tyrosine kinases, such as Pyk2 and Src-related kinases, have been shown
to be essential intermediates providing links between G-proteins and
signaling pathways of ERK and JNK (36, 37). In our system, genistein
blocked shear stimulation of ERK and JNK (Fig. 8),
although the identity of the tyrosine kinase(s) remains to be
determined. A recent study by Ishida et al. (53) provides
further evidence that shear stress increases tyrosine phosphorylation
of many as yet unidentified molecules. Identifying the tyrosine
kinase(s) will greatly enhance the understanding of molecular events
occurring in response to shear. One of the potential downstream
effector molecules that can be activated by tyrosine kinases includes
Shc leading to the recruitment and activation of the Grb2, mSOS, and
Ras pathway (37). This pathway may be important in shear stress
signaling, since we showed that Ras plays an essential role in
shear-dependent activation of ERK and JNK (Figs. 6 and 7).
Although we have not addressed the exact sequence of events,
shear-dependent activations of ERK and JNK do involve
similar signaling components including heterotrimeric G-proteins and
tyrosine kinase(s) acting on Ras-dependent pathways as
described by previous investigators in other
G-protein-dependent ERK and JNK pathways (36, 37,
47, 48, 49, 50, 51, 52).
Endothelial cells are believed to possess a flow-sensing system(s), mechanoreceptor(s) (1). Based on the evidence provided in the present study, one possibility is that there is one mechanoreceptor that can activate two different classes of G-proteins leading to ERK and JNK pathways. Another alternative is that shear stress may be mediated by two different mechanoreceptors, one coupled to Gi-proteins and the other coupled to non-Gi-proteins directly or indirectly.
Recently, shear-dependent activation of ERK has been
reported (19, 20). Tseng et al. (19) also reported that
GDPS completely inhibits shear stress stimulation of ERK activity in
BAEC. While their results of shear-dependent activation of
ERK are in general agreement with ours, these authors concluded that
shear-dependent ERK activation was not inhibited by PTx
treatment of fetal BAEC (12 dyn/cm2 for 5 min). This
discrepancy of PTx sensitivity may be due to the different cell types
used (fetal versus adult BAEC). Other possibilities include
subtle differences in cell culture and starvation conditions, and use
of DMEM containing 0.5% FCS for 1-2 days in our studies
versus Hanks' buffered salt solution for an unspecified length of time (19). A similar difference has been reported previously
that shear-dependent production of NO/cGMP was
PTx-sensitive in BAEC but not in human umbilical endothelial cells (28,
32).
What is the physiological and pathological importance of the shear-dependent activation of ERK and JNK? The remarkably different shear time- and force-dependent responses of ERK and JNK suggest two possibilities that could occur in vivo. First, a relatively large change of shear stress (amplitude) even for a few minutes may be required to activate ERK. Second, even a very small change in shear stress level could activate JNK, however, if the change is sustained for longer than 30 min (duration). ERK regulates cellular growth and metabolic responses in response to many growth factors and other stimuli acting on G-protein-coupled receptors (17, 18). However, the role of JNK is less well characterized. JNK can be activated by growth factors, cellular stresses, and G-protein-coupled receptors, which can induce growth, as well as apoptotic responses, probably depending upon cell environment and types (25, 54, 55). It remains to be determined whether ERK and JNK pathways are directly responsible for the apparent beneficial effect of shear stress on vessel wall, focal patterns of atherosclerotic lesion development, and vascular remodeling (1). Unlike arterial endothelial cells, which are constantly exposed to shear stress, endothelial cells used in our studies were grown in static conditions and exposed to shear stress only during shear experiments ("static-to-shear" in vitro model). Therefore, extrapolation of our results to in vivo situations needs to be confirmed in vivo. This kind of "static-to-shear" in vitro system has been extremely valuable to demonstrate specific phenomena and signaling pathways induced by shear stress that are potentially antiatherogenic as well as proatherogenic responses (1). It is likely that coordinated responses in ERK and JNK may lead to differential expression of multiple shear-sensitive genes. Dysregulation or loss of "check and balance" between ERK and JNK may contribute to the pathogenesis of atherosclerosis.
In summary, our study demonstrates that shear stress differentially regulates ERK and JNK through two different G-proteins acting on Ras-dependent pathways. Future investigations defining both the upstream and downstream signaling events involved in shear-dependent activation of ERK and JNK will be important to understanding the role of shear stress on endothelial physiology as well as vascular diseases such as atherosclerosis.
We thank Drs. A. Kraft and C. Franklin for
GST-c-Jun, Dr. R. Reed for i2 and
i3, Dr.
G. L. Johnson for
i2(G203) and
i3(G204), Dr. S. Gutkind for N17Ras and
ARK-ct, Dr. A. Lin for HA-ERK2 and
HA-JNK1 vectors, and Dr. D. Curiel and M. Lee for adenovirus. We also
thank Dr. V. Darley-Usmar for critical reading of this manuscript and
C. Abbot for technical help.