J Appl Physiol
91: 1574–1581, 2001.
Protein kinase B/Akt activates c-Jun NH2-terminal kinase
by increasing NO production in response to shear stress
YOUNG-MI GO,1,* YONG CHOOL BOO,1,* HEONYONG PARK,1 MATTHEW C. MALAND,1
RAKESH PATEL,2 KIRKWOOD A. PRITCHARD, JR.,3 YASUSHI FUJIO,4
KENNETH WALSH,4 VICTOR DARLEY-USMAR,2 AND HANJOONG JO1
1
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology
and Emory University, Atlanta, Georgia 30322; 2Department of Pathology, Center for Free
Radical Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294;
3
Division of Pediatric Surgery, Department of Surgery, Medical College of Wisconsin,
Milwaukee, Wisconsin 53226; and 4Division of Cardiovascular Research, St. Elizabeth’s
Medical Center and Tufts University School of Medicine, Boston, Massachusetts 02135
Received 25 July 2000; accepted in final form 8 June 2001
the dragging force generated
by blood flow, are sensed by the endothelium. The
important role of shear stress in vessel wall biology has
been highlighted by the findings that the arterial regions of low and unstable shear stress are prone to the
development of atherosclerotic plaques, whereas those
of laminar and relatively high levels of shear force are
protected (25, 43). The underlying mechanisms by
which shear stress prevents or promotes atherosclerosis have been intensely studied.
Shear stress triggers a variety of biochemical and
physical changes in cell structure and function, such as
regulation of vascular tone and diameter, inflammatory responses, hemostasis, and vessel wall remodeling
(6). Mediators of these responses include intracellular
ions (Ca2⫹ and K⫹), vasoactive molecules [nitric oxide
(NO) and superoxide], growth factors, and adhesion
molecules (6). Transcriptional regulation of plateletderived growth factors, endothelial NO synthase
(NOS) (eNOS), Cu/Zn superoxide dismutase, cyclooxygenase-2, and other shear-sensitive genes have also
been well described (4, 20, 33, 37, 39). Induction of the
mechanosensitive genes is likely mediated by transcription factors (e.g., nuclear factor-␬B, activator protein-1, early growth response-1, c-jun, c-fos, and c-myc)
(19, 24, 28) that are regulated by upstream signaling
molecules, including the family of mitogen-activated
protein kinases, extracellular signal regulated kinase
(ERK), c-Jun NH2-terminal kinase (JNK), and big mitogen-activated protein kinase-1 (3, 23, 36, 42).
Shear stress activates ERK and JNK by two different signaling pathways involving both common as well
as unique upstream signaling molecules. ERK is rapidly (within 5 min) and transiently activated by G␣i2,
Src, focal adhesion kinase, protein kinase C⑀, and Rasdependent pathways in the caveolae (21, 23, 31, 32, 40).
On the other hand, JNK is activated slowly (requires
60 min of shear exposure for maximum activation) by
pertussis toxin (PTx)-insensitive G protein (G␣?), G␤␥,
G protein-sensitive phosphatidylinositide 3-kinase
* Y.-M. Go and Y. C. Boo contributed equally to this work.
Address for reprint requests and other correspondence: H. Jo, Georgia Tech-Emory Biomedical Engineering Dept., Emory Univ., 303D
WMB, Atlanta, GA 30322 (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
endothelial cells; mitogen-activated protein kinase; atherosclerosis; endothelial nitric oxide synthase; mechanosensing
CHANGES IN SHEAR STRESS,
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8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society
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Go, Young-Mi, Yong Chool Boo, Heonyong Park,
Matthew C. Maland, Rakesh Patel, Kirkwood A. Pritchard, Jr., Yasushi Fujio, Kenneth Walsh, Victor Darley-Usmar, and Hanjoong Jo. Protein kinase B/Akt activates c-Jun NH2-terminal kinase by increasing NO production
in response to shear stress. J Appl Physiol 91: 1574–1581,
2001.—Laminar shear stress activates c-Jun NH2-terminal kinase (JNK) by the mechanisms involving both nitric oxide (NO)
and phosphatidylinositide 3-kinase (PI3K). Because protein kinase B (Akt), a downstream effector of PI3K, has been shown to
phosphorylate and activate endothelial NO synthase, we hypothesized that Akt regulates shear-dependent activation of
JNK by stimulating NO production. Here, we examined the role
of Akt in shear-dependent NO production and JNK activation
by expressing a dominant negative Akt mutant (AktAA) and a
constitutively active mutant (AktMyr) in bovine aortic endothelial cells (BAEC). As expected, pretreatment of BAEC with the
PI3K inhibitor (wortmannin) prevented shear-dependent stimulation of Akt and NO production. Transient expression of
AktAA in BAEC by using a recombinant adenoviral construct
inhibited the shear-dependent stimulation of NO production
and JNK activation. However, transient expression of AktMyr
by using a recombinant adenoviral construct did not induce
JNK activation. This is consistent with our previous finding
that NO is required, but not sufficient on its own, to activate
JNK in response to shear stress. These results and our previous
findings strongly suggest that shear stress triggers activation of
PI3K, Akt, and endothelial NO synthase, leading to production
of NO, which (along with O2⫺, which is also produced by shear)
activates Ras-JNK pathway. The regulation of Akt, NO, and
JNK by shear stress is likely to play a critical role in its
antiatherogenic effects.
MECHANOACTIVATION OF JNK BY AKT-DEPENDENT NO PRODUCTION
MATERIALS AND METHODS
Cell culture. BAEC harvested from descending thoracic
aortas were maintained (37°C, 5% CO2) in a growth medium
[DMEM (1 g/l glucose; Gibco) containing 20% FCS (Atlanta
Biologicals) without antibiotics] (23). BAEC used in this
study were between passages 5 and 10.
Shear stress studies and preparation of lysates. Cells were
exposed to laminar shear stress by using a parallel-plate
shear chamber (23) or a cone-and-plate viscometer (7), as
modified for a 100-mm culture dish (34). For the parallelplate shear studies, 1 million cells per glass slide (75 ⫻ 38
mm; Fisher Scientific) were seeded in growth medium. For
the cone-and-plate studies, BAEC were grown to confluency
in the growth medium in 100-mm tissue culture dishes (FalJ Appl Physiol • VOL
con). Both systems have been widely used to impose laminar
shear stress on endothelial cells. Exposure of endothelial
cells to laminar shear stress using either system induces
activation of ERK, JNK, and Akt to a similar degree over a
similar time course. The glass slide containing a BAEC
monolayer was assembled into a parallel-plate shear chamber forming a flow channel (220 ␮m height ⫻ 2.5 cm width ⫻
6.2 cm length) between the monolayer and fabricated polycarbonate plate as described previously (23). Nonpulsatile,
laminar shear stress was controlled by changing the flow rate
of the shear medium (phenol red-free DMEM containing
0.5% FCS and 25 mM HEPES) delivered to the cells, by using
the constant-head flow loop as described previously (22).
After treatment, BAEC were washed in ice-cold PBS and
lysed in 0.25 ml lysis buffer (20 mM HEPES, pH 7.6, 10 mM
␤-glycerophosphate, 20 mM p-nitrophenyl phosphate, 0.1
mM vanadate, 2 mM dithiothreithol, and 1% Triton X-100)
for JNK assays. Cell lysates were clarified by spinning at
20,000 g for 15 min at 4°C. Protein content of each sample
was measured by using a Bio-Rad DC kit.
NO assay. The parallel-plate system requires ⬃30 ml of
medium using cells grown in 21.4 cm2 of surface area,
whereas the cone-and-plate system needs ⬃10 ml of medium
using cells grown in 10-cm dish (78.5-cm2 surface area).
These differences in medium volume and cell numbers result
in a ⬎10-fold increase in the concentration of NO accumulating in the medium in the cone-and-plate system, compared
with the parallel plate system. Because this increase in
concentration of NO makes the measurement of nitrite easier, the cone-and-plate system was used for this study. A
confluent BAEC monolayer grown in a 100-mm tissue culture
dish was exposed to laminar shear stress by rotating KrebsRinger carbonate buffer (in mM: 25 NaHCO3, pH 7.4, 118
NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, and 11
glucose) with a cone in a 5% CO2 incubator at 37°C. The cone
had a fixed 0.5° angle and rotated at 6.7 revolutions/s (7, 34).
To measure accumulation of NO in the medium, a 1-ml
sample was collected, replaced with fresh medium, and kept
dark on ice until NO assay. After shear exposure, cells were
washed with ice-cold PBS and scraped in a lysis buffer to
measure the amount of protein. A fluorescent assay using
2,3-diaminonaphthalene was used to measure nitrite, because it accounts for ⬎90% of total NO metabolite accumulating in the medium in response to shear stress (17, 41).
Adenoviral infections. BAEC grown to ⬃75% confluency on
100-mm dishes (for cone-and-plate experiments) or glass
slides (for parallel-plate experiments) were infected with
recombinant adenovirus encoding for the ␤-galactosidase
(␤-gal) (adenovirus-␤-gal) or hemagglutinin (HA) epitopetagged Akt mutants AktAA (a dominant-negative construct
that contains mutations at Thr308 and Ser473 to Ala308 and
Ala473, respectively) or AktMyr (a constitutively active construct that contains a myristoylation site fused to the amino
terminus) (13). One hour after the infection in serum-free
DMEM, cells were incubated overnight in fresh, complete
medium. Infection of BAEC with adenovirus-␤-gal at 50
plaque forming unit (pfu)/cell resulted in expression of ␤-gal
in 70–80% (n ⫽ 4) of cells as determined by immunohistochemical staining, as described previously (23).
Western blot analysis of Akt phosphorylation. To determine
the phosphorylation status of Akt, aliquots (20 ␮g) of the
lysates were resolved on a 7.5% SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) (23). The PVDF membrane was probed with Akt
antibodies specific for phosphorylated Thr308 or phosphorylated Ser473 (New England Biolabs). An Akt antibody detecting the total amount of Akt (New England Biolabs) was also
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(PI3K)␥, and Ras-dependent mechanisms (16, 23). In
addition, unlike the ERK pathway, the shear-dependent activation of JNK requires production of both NO
and superoxide, possibly by forming the reaction product peroxynitrite, which may act as a signaling mediator (17).
Although it is well known that exposure of endothelial cells to shear stress stimulates production of NO
from eNOS, both in cultured cells and in intact vessels
(26, 35), the molecular mechanisms by which shear
stress regulates NO production have not been clearly
elucidated. It is clear, however, that the mechanisms of
NO production from eNOS in response to mechanical
forces are quite different from that induced by Ca2⫹mobilizing humoral stimuli such as bradykinin (26).
For example, regulation of NO production from eNOS
in response to shear stress and stretching involves
Ca2⫹/calmodulin independent mechanisms (5, 11, 12,
26). Recent studies have shown that the shear-sensitive, Ca2⫹/calmodulin-independent production of NO is
mediated by mechanisms dependent on PI3K and protein kinases (9, 11, 12, 14). Interestingly, shear stress
activates Akt (9), which is the major target of PI3K,
raising a possibility that Akt could be directly regulating eNOS activity.
Akt (also known as protein kinase B) is a Ser/Thr
protein kinase that has been implicated in mediating
numerous biological responses, including inhibition of
apoptosis and regulation of metabolism (18). The activation of Akt by PI3K requires both the phosphoinositide products of PI3K as well as phosphorylation of
Thr308 and Ser473 of Akt (1, 2).
The eNOS can be phosphorylated and activated by
humoral ligands, such as vascular endothelial growth
factor, or overexpressing a constitutively active AktMyr
mutant (9, 14). Although it has been clearly demonstrated that PI3K is an essential signaling molecule in
the shear-dependent NO production (9, 15), it has not
been reported whether dominant-negative Akt constructs can prevent the shear-dependent activation of
NO and its downstream JNK pathway.
Here we directly addressed whether Akt plays a key
role in shear-dependent stimulation of NO production
and JNK activation by transiently expressing a dominant-negative Akt mutant AktAA or a constitutively
active mutant AktMyr in bovine aortic endothelial cells
(BAEC).
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MECHANOACTIVATION OF JNK BY AKT-DEPENDENT NO PRODUCTION
RESULTS
Shear stress stimulates phosphorylation of Akt in a
time- and force-dependent manner. To determine the
effect of shear stress on the status of Akt activation,
phosphorylation of Akt was examined by Western blot
analysis using the Akt antibodies specific for phosphorylated Thr308 or phosphorylated Ser473 while using a
total Akt (Akt) antibody as a control for equal loading.
Exposure of BAEC to shear stress (10 dyn/cm2) stimulated phosphorylation of both Thr308 and Ser473 of Akt
in a time-dependent manner (Fig. 1A). In addition,
shear exposure stimulated phosphorylation of both
Thr308 and Ser473 of Akt with an almost identical time
course without changing the total amount of Akt (Fig.
1A). Phosphorylation of Akt at both residues (Thr308
and Ser473) was evident as early as 2 min after the
onset of shear stress. Maximal activation was achieved
by 5-min shear exposure and remained elevated for
30–60 min (Fig. 1A). Next, BAEC were subjected to
increasing magnitudes of shear force for 20 min, and
cell lysates were examined for phosphorylation of Akt
as above. Onset of all levels of shear force examined in
the present study (ⱖ0.5 dyn/cm2) significantly increased phosphorylation of Akt compared with that of
the stationary controls (P ⬍ 0.05, n ⫽ 3–4) (Fig. 1B).
The maximum activation of Akt observed at 20 dyn/
cm2 was not significantly different from that induced
by 0.5, 5, or 10 dyn/cm2 (P ⬎ 0.1, n ⫽ 3–4), suggesting
that Akt phosphorylation is regulated in a threshold
force-dependent manner.
To determine whether phosphorylation of Akt is mediated by PI3K, shear-dependent phosphorylation of
Akt was examined in BAEC that were pretreated with
a PI3K inhibitor, wortmannin. As shown in Fig. 1B,
pretreatment with wortmannin (100 nM) completely
prevented phosphorylation of Akt induced by shear
stress (10 dyn/cm2 for 20 min). This result shows that
PI3K is a critical upstream regulator of Akt in response
to shear stress. The time and force dependencies of Akt
activation shown here are also consistent with the
pattern of PI3K␥ activation induced by shear stress
(16).
PI3K and Akt are upstream regulators of shear stressdependent NO production. To determine whether PI3K
activates its downstream target Akt, which then stimulates NO production in response to the mechanical
stimulation, we first determined the effect of wortmannin on shear-dependent formation of NO. Exposure of
BAEC to shear stress induced a biphasic formation of
NO with an initial rapid burst of NO accumulation
(0–5 min, also known as the first phase), followed by a
substantially slower rate of NO production (the second
phase) (Fig. 2A), as shown previously (5, 26). Treatment of BAEC with wortmannin inhibited shear-dependent NO production by ⬎70–90% of control at all times
studied (5–60 min of shear) (Fig. 2A). This result
Fig. 1. Shear stress stimulates phosphorylation of protein kinase B (Akt) in a time- and
threshold force-dependent manner. Confluent
bovine aortic endothelial cells (BAEC) were
exposed to stationary control or laminar
shear stress (10 dyn/cm2) for time periods
indicated (A) or to increasing shear force levels for 10 min (B), and cell lysates were prepared. In some experiments, cells were pretreated with 100 nM wortmannin (WT) for 10
min (B; E) before static or shear exposure.
Activation of Akt was determined by Western
blot analysis of cell lysates with antibodies
specific for phosphorylated Thr308 (pT-Akt),
phosphorylated Ser473 (pS-Akt), or total Akt
(Akt). Densitometry was performed to quantitate phosphorylated Akt bands. The data
representing means ⫾ SE (n ⫽ 3–5) are expressed as percent maximum activation of
Akt in response to shear stress in each experiment, and the maximum was defined as
100%.
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used as a control. To detect HA-tagged AktAA, cell lysates
were used for Western blot analysis with a HA antibody
(Roche). Goat anti-rabbit or anti-mouse IgG conjugated to
alkaline phosphatase was used as secondary antibody and
developed by a chemiluminescence detection method (23).
JNK assays. After shear exposure, cell lysates (100 ␮g)
were incubated with an antibody specific for JNK1 (Pharmingen) for 1 h at 4°C, followed by an additional 1-h incubation
with protein G agarose beads. The immune complex was
washed and incubated in the presence of c-Jun peptide
(amino acids 5–89) fused to glutathione S-transferase and
[32P]ATP as described previously (23). The reaction products
were resolved by 10% SDS-PAGE and transferred to a PVDF
membrane, the autoradiogram was obtained, and radioactivity incorporated into each band was quantified by scintillation counting. The membrane was then probed with a polyclonal antibody to JNK to monitor equal loading of
immunoprecipitated JNK in each experiment.
Statistical analysis. Statistical analysis was performed
with the use of Student’s t-test. The P ⬍ 0.05 based on at
least three or more independent experiments was considered
to be statistically significant.
MECHANOACTIVATION OF JNK BY AKT-DEPENDENT NO PRODUCTION
1577
Fig. 2. An inhibitor of phosphatidylinositide 3-kinase or a dominant-negative AktAA mutant inhibits shear stressdependent nitric oxide (NO) production.
A: confluent BAEC were pretreated with
vehicle (none) or 100 nM WT for 10 min
before being exposed to stationary control or laminar shear stress (17 dyn/
cm2) for 1 h in Krebs-Ringer buffer as
shear medium. B: BAEC were infected
with adenovirus-␤-galactosidase (␤-gal)
as a control or adenovirus-hemagglutinin (HA)-AktAA mutant. One day after
the infection, cells were exposed to stationary control or laminar shear stress
as in A. During shear or static exposure,
medium (1 ml) was collected and replenished with fresh buffer at indicated time
points (0–1 h) to determine the accumulation of nitrite in the medium. Values are
means ⫾ SE (n ⫽ 3 in A, and n ⫽ 4 in B).
versely affect NO production from BAEC in response to
shear stress.
Akt is an upstream regulator of the shear stressdependent activation of JNK. Previously, our laboratory has shown that PI3K regulates the shear-dependent activation of JNK (16). To determine whether Akt
regulates shear-dependent activation of JNK, BAEC
were infected with increasing concentrations of adenovirus-AktAA (0–50 pfu/cell) or adenovirus-␤-gal as a
control. As shown in Fig. 3B, shear-dependent activation of JNK was inhibited by expressing the dominantnegative AktAA mutant in a concentration-dependent
manner, reaching a maximum ⬃50% inhibition compared with ␤-gal controls (Fig. 3B). Because the AktAA
is tagged with an HA epitope, the increasing levels of
expression of AktAA were demonstrated by Western
blot using a HA antibody (marked as HA-AktAA in Fig.
3B). In contrast, BAEC infected with adenovirus-␤-gal
had no effect on shear-dependent JNK activation, even
at 50 pfu/cell (Fig. 3A). We could not determine the
effect of expressing higher concentrations of adenoviFig. 3. Expression of a dominant-negative AktAA mutant
inhibits shear-dependent activation of c-Jun NH2-terminal kinase (JNK). BAEC were infected with increasing
amounts of adenovirus-␤-gal (A) or adenovirus-HA-AktAA
(B) as indicated [0–50 plaque-forming units (pfu)/cell].
The next day, cells were exposed to a static condition or
laminar shear stress (10 dyn/cm2) for 1 h, and cell lysates
were prepared. Cell lysates were incubated with a JNK1
antibody and protein G-agarose, and the JNK activity of
the immune complex was determined by phoshorylation
of glutathione S-transferase (GST)-c-Jun (GST-cJun⬃P).
The reaction product was separated by SDS-PAGE and
transferred to a polyvinylidene difluoride (PVDF) membrane. Representative autoradiograms demonstrating
phosphorylation of GST-c-Jun obtained from the PVDF
membranes are shown (top). The membranes were then
probed further with an antibody to JNK to show the
amount of immunoprecitated JNK1 in each lane (JNK1).
B: cell lysates were also analyzed by Western blot to show
the expression levels of AktAA mutant (tagged with a HA
epitope) by using a HA antibody (HA-AktAA). Radioactivity incorporated into GST-c-Jun bands was determined by
scintillation counting. Values are means ⫾ SE (n ⫽ 3).
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confirms the previous findings (9, 15) that PI3K is a
critical regulator of the shear-sensitive NO production.
Because Akt is only one of the many downstream
effectors of PI3K, the above result alone does not prove
whether this protein kinase is responsible for activation of eNOS in response to shear stress. To examine
whether shear-dependent production of NO is regulated
by activation of Akt, BAEC were infected with recombinant adenovirus expressing a dominant-negative adenovirus-AktAA mutant (50 pfu/cell). As a control,
BAEC were infected with recombinant adenovirus-␤gal. When BAEC were infected with the adenovirusAktAA, shear-dependent NO production was virtually
prevented, showing that phosphorylation and/or activation of Akt plays a crucial and predominant role in
the mechanosensitive production of NO (Fig. 2B). In
contrast, shear-dependent production of NO in BAEC
that were infected with adenovirus-␤-gal was virtually
identical to that in noninfected BAEC (compare open
circles in Fig. 2, A and B). This result demonstrated
that the adenoviral infection procedure did not ad-
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MECHANOACTIVATION OF JNK BY AKT-DEPENDENT NO PRODUCTION
with 100 ng/ml of the toxin for 16 h and then exposed
to shear as indicated. As shown in Fig. 5, PTx did not
have any significant effect on the NO production and
Akt activation in response to shear stress, whereas it
inhibited the shear-dependent ERK activation as previously reported (23). These results suggest that Gi/o
proteins are not involved in the PI3K, Akt, and NO
pathway.
NO is not the upstream regulator of Akt activation in
response to shear stress. Next we examined whether
activation of Akt in response to shear stress is regu-
rus-HA-AktAA (100–200 pfu/cell) in this study, because
they resulted in changes in cell morphology and increase in basal JNK activity by more than twofold
compared with adenovirus-␤-gal control (data not
shown). These results may be due in part to the essential survival functions of Akt in endothelial cells.
Akt activation alone is not sufficient for JNK activation. To test whether Akt activation alone is sufficient
for JNK activation, BAEC were infected with recombinant adenovirus AktMyr that expresses a constitutively
active and HA-tagged form of Akt. The expression of
AktMyr was confirmed by Western blot with antibodies
specific for HA, total Akt, and phosphorylated Ser473Akt. As shown in Fig. 4, expression of AktMyr expression alone did not stimulate JNK phosphorylation.
This result, taken together with that shown in Fig. 3,
demonstrates that Akt activity is necessary but not
sufficient for JNK activation in response to shear
stress. Previously, we have shown that NO is essential
in the shear-dependent activation of JNK (17), but NO
alone is not sufficient to activate JNK. Therefore, it is
not surprising to find that AktMyr, presumably acting
on the NO pathway, alone does not activate JNK.
Shear-dependent Akt activation and NO production
are insensitive to PTx. There has been conflicting literature regarding the role of PTx-dependent G proteins
in the shear-dependent NO production (27, 30). We
have shown previously that shear-dependent activation of the JNK pathway is regulated by PTx-insensitive G proteins (23). Because we also showed that NO
is an upstream regulator in shear activation of JNK
pathway, we speculated that PTx-sensitive G proteins
would not be involved in shear-dependent NO production (23). To examine whether the shear-dependent NO
production is sensitive to PTx, BAEC were pretreated
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Fig. 4. Expression of a constitutively active Akt mutant (AktMyr)
induces endothelial NO synthase (NOS) phosphorylation. BAEC
infected with adenovirus-HA-AktMyr as indicated and adenovirus-␤gal were used as a control. Two days after the infection, whole cell
lysates were prepared and analyzed by Western blot analysis using
antibodies specific for HA (to detect HA-AktMyr), total Akt (to detect
both endogenous Akt and transfected AktMyr), and pS-Akt. Activation of JNK was determined by Western blot using an antibody
specific for phospho-JNK (P-JNK) and total JNK (as a loading control). As a positive control for JNK activation, the cell lysates from
BAEC exposed to shear stress (10 dyn/cm2) for 1 h, as described in
the legend of Fig. 3, were used. Note that overexpression of AktMyr
does not result in activation of JNK.
Fig. 5. Pertussis toxin (PTx) does not inhibit shear-dependent NO
production and Akt phoshorylation. Confluent BAEC were pretreated with or without PTx (100 ng/ml for 18 h) and then exposed to
shear stress (17 dyn/cm2) for the time periods indicated (A and B) or
15 min (C). A: BAEC pretreated with vehicle or PTx were exposed to
static or laminar shear stress for 60 min in Krebs-Ringer buffer.
Accumulating levels of nitrite in the medium were determined as
described in Fig. 2 legend. B and C: whole cell lysates were used for
Western blot analyses using antibodies specific for pS-Akt and phospho-extracellular signal regulated kinase-1/2 (p-ERK1/2). Note that
treatment with PTx inhibits phosphorylation of ERK1/2 but had no
significant effects (ns) on phosphorylation of Akt and NO production
in response to shear. Values are means ⫾ SE (n ⫽ 4 in A, n ⫽ 3 in B
and C).
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MECHANOACTIVATION OF JNK BY AKT-DEPENDENT NO PRODUCTION
lated by NO-dependent mechanisms. For this study,
shear-dependent production of NO was inhibited by
treating BAEC with the NOS inhibitors 1 mM N Gnitro-L-arginine (17) and 1 mM N G-nitro-L-arginine
methyl ester (data not shown). Neither N G-nitro-Larginine nor N G-nitro-L-arginine methyl ester had any
effect on the shear-dependent phosphorylation of Akt
(Fig. 6). This result further supports the fact that Akt
is an upstream regulator of NO production, but NO is
not an upstream regulator of Akt in response to shear
stress.
DISCUSSION
Fig. 6. Inhibitors of NOS do not affect shear-dependent phosphorylation of Akt. Confluent BAEC were pretreated with vehicle or NOS
inhibitors [1 mM N G-nitro-L-arginine (L-NNA) or N G-nitro-L-arginine methyl ester (L-NAME)] for 60 min and then exposed to shear
stress (17 dyn/cm2) for 30 min. Whole cell lysates were used in
Western blot analysis using antibodies specific for pS-Akt and total
Akt. Values are means ⫾ SE (n ⫽ 3).
J Appl Physiol • VOL
based on the following evidence: 1) the PI3K inhibitors
inhibit shear-dependent activation of Akt and production of NO (9, 15); 2) the PI3K inhibitors inhibit sheardependent phosphorylation of eNOS-Ser1179, which
corresponds to the conserved Akt phosphorylation site
(15); 3) overexpression of the constitutively active
AktMyr mutant can increase NO production (9, 14); and
4) overexpression of AktAA inhibits phosphorylation of
eNOS on unknown Ser residues (1). These findings
clearly demonstrate that the shear-dependent activation of eNOS is regulated by the PI3K-dependent
mechanisms. However, the direct evidence supporting
the role of Akt in the shear-dependent NO production
from eNOS has not been provided until the present
study. PI3K activates the phosphoinositide-dependent
kinase-1, which in turn phosphorylates and activates a
group of enzymes that mediate the PI3K effect. The
downstream effectors of PI3K include Akt, protein kinase A, protein kinase C, serum-glucocorticoid-dependent kinase, and p70-S6-kinase (38). Our present result using AktAA now provides a further support for the
role of Akt in shear-dependent stimulation of NO production.
Our laboratory has previously shown that shear
stress stimulates JNK activity by the mechanisms dependent on PTx-insensitive G protein(s) (23). Our laboratory also showed that shear stress rapidly and transiently stimulates PI3K␥, which in turn activates the
JNK pathway by mechanisms that were not clear at
the time (16). More recently, our laboratory also
showed that the shear-dependent activation of JNK is
prevented by blocking NO production using NOS inhibitors (17). Therefore, both PI3K and NO have been
implicated in JNK activation in response to shear
stress. However, it was not clear how the signal from
PI3K is transmitted to NO production and JNK activation. The present study demonstrates that Akt is a
signaling mediator that links shear-dependent activation of PI3K to the NO production and JNK activation
pathways. Several lines of evidence support this conclusion: 1) the PI3K inhibitor wortmannin prevented
shear-dependent phosphorylation of Akt and sheardependent NO production (Fig. 1); and 2) expression of
AktAA blocks shear-dependent production of NO as
well as activation of JNK (Fig. 3).
It was noted that expression of AktAA did not completely inhibit the shear-dependent JNK activation
(Fig. 3). One reason may be due to transfection efficiency (70–80%) of AktAA contributing to its partial
effect, because BAEC were transfected when ⬃75%
confluent and 70–80% of the total cell population was
transfected by ␤-gal staining. Another reason may be
due in part to the limitation in the infection of AktAA in
BAEC. In our hands, the infection of cells with AktAA
using ⬎100 pfu/cell caused cytotoxic effects (based on
cell morphology) and increased basal JNK activity by
more than twofold (data not shown). Considering the
essential role of Akt in cell survival, this may not be
unexpected. Another possibility is that there may be
additional pathways that contribute to the shear-dependent activation of JNK. Consistent with this notion,
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In this study, we demonstrate that transient expression of AktAA inhibits shear-dependent stimulation of
NO production and JNK activation. This finding resolves two important issues in understanding the role
of Akt in the shear-dependent stimulation of NO production and JNK activation in endothelial cells.
The eNOS is known as a Ca2⫹/calmodulin-dependent
form of NOS (29). Indeed, most humoral ligands, including bradykinin, acetylcholine, and ATP, stimulate
NO production from eNOS by raising the level of intracellular Ca2⫹, which forms Ca2⫹-calmodulin complex (29). Unlike Ca2⫹-mobilizing hormones, however,
shear stress stimulates production of NO from eNOS
by a mechanism that does not require a maintained
intracellular Ca2⫹ level or calmodulin (5, 11, 26). The
phosphorylation of eNOS-Ser1179 by the PI3K/Akt
pathway has been suggested as the underlying mechanisms whereby shear stress stimulates NO production in a Ca2⫹/calmodulin-insensitive manner (9, 15).
However, the proof of this hypothesis needs further
work.
Akt has been considered as the protein kinase responsible for NO production in response to shear stress
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1580
MECHANOACTIVATION OF JNK BY AKT-DEPENDENT NO PRODUCTION
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