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J Appl Physiol 91: 1574-1581, 2001;
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Vol. 91, Issue 4, 1574-1581, October 2001

Protein kinase B/Akt activates c-Jun NH2-terminal kinase by increasing NO production in response to shear stress

Young-Mi Go1,*, Yong Chool Boo1,*, Heonyong Park1, Matthew C. Maland1, Rakesh Patel2, Kirkwood A. Pritchard Jr.3, Yasushi Fujio4, Kenneth Walsh4, Victor Darley-Usmar2, and Hanjoong Jo1

1 Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30322; 2 Department 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 4 Division of Cardiovascular Research, St. Elizabeth's Medical Center and Tufts University School of Medicine, Boston, Massachusetts 02135


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 O<UP><SUB>2</SUB><SUP>−</SUP></UP>, 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.

endothelial cells; mitogen-activated protein kinase; atherosclerosis; endothelial nitric oxide synthase; mechanosensing


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CHANGES IN SHEAR STRESS, 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 platelet-derived 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-kappa 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 Galpha i2, Src, focal adhesion kinase, protein kinase Cepsilon , and Ras-dependent 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 (Galpha ?), Gbeta gamma , G protein-sensitive phosphatidylinositide 3-kinase (PI3K)gamma , 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).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 parallel-plate 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 (Falcon). 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 µm 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 beta -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 Krebs-Ringer 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 beta -galactosidase (beta -gal) (adenovirus-beta -gal) or hemagglutinin (HA) epitope-tagged 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-beta -gal at 50 plaque forming unit (pfu)/cell resulted in expression of beta -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 µg) 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 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 µg) 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (>= 0.5 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.


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  		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; open circle ) 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%.

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 PI3Kgamma activation induced by shear stress (16).

PI3K and Akt are upstream regulators of shear stress-dependent 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 confirms the previous findings (9, 15) that PI3K is a critical regulator of the shear-sensitive NO production.


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  		Fig. 2.   An inhibitor of phosphatidylinositide 3-kinase or a dominant-negative AktAA mutant inhibits shear stress-dependent 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-beta -galactosidase (beta -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).

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-beta -gal. When BAEC were infected with the adenovirus-AktAA, 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-beta -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 adversely affect NO production from BAEC in response to shear stress.

Akt is an upstream regulator of the shear stress-dependent 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-beta -gal as a control. As shown in Fig. 3B, shear-dependent activation of JNK was inhibited by expressing the dominant-negative AktAA mutant in a concentration-dependent manner, reaching a maximum ~50% inhibition compared with beta -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-beta -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 adenovirus-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-beta -gal control (data not shown). These results may be due in part to the essential survival functions of Akt in endothelial cells.


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  		Fig. 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-beta -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).

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 Ser473-Akt. 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.


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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-beta -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.

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 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.


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  		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).

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 regulated by NO-dependent mechanisms. For this study, shear-dependent production of NO was inhibited by treating BAEC with the NOS inhibitors 1 mM NG-nitro-L-arginine (17) and 1 mM NG-nitro-L-arginine methyl ester (data not shown). Neither NG-nitro-L-arginine nor NG-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.


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  		Fig. 6.   Inhibitors of NOS do not affect shear-dependent phosphorylation of Akt. Confluent BAEC were pretreated with vehicle or NOS inhibitors [1 mM NG-nitro-L-arginine (L-NNA) or NG-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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 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 shear-dependent 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 PI3Kgamma , 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 shear-dependent 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 beta -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, expression of AktMyr alone failed to induce JNK activation (Fig. 4), suggesting that Akt is required, but not sufficient, for the JNK activation by shear stress. In support of this, our laboratory has shown previously that shear activation of JNK requires a concomitant production of NO and O<UP><SUB>2</SUB><SUP>−</SUP></UP> and their reaction product peroxynitrite (17).

There has been conflicting literature regarding the role of PTx-dependent G proteins in the shear-dependent NO production (27, 30). Our present study shows that shear-dependent activation of NO production is not mediated by PTx-sensitive G proteins (Fig. 5), and this result is consistent with our previous finding that shear-dependent activation of the JNK pathway is regulated by PTx-insensitive G proteins (23).

Another supporting evidence that Akt is an upstream regulator of NO production in response to shear stress is provided in this study using the NOS inhibitors. Inhibition of NO production had no effect on Akt phosphorylation in response to shear stress (Fig. 6).

In summary, we provide the direct evidence that shear stress stimulates JNK by activating the cascade of PI3K, Akt, and NO production from eNOS. Laminar shear stress regulates vessel wall remodeling, has antiatherogenic properties, and potently inhibits apoptosis induced by proatherogenic stimuli or serum depletion (6, 8). Activation of PI3Kgamma , Akt, NO, and JNK is likely to play an essential role in the antiatherogenic effect of shear stress. Defining the mechanisms by which these mechanosensitive molecules regulate the function and structure of endothelial cells needs further studies.


    FOOTNOTES

* 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: hanjoong.jo{at}bme.gatech.edu).

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.

Received 25 July 2000; accepted in final form 8 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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