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Originally published In Press as doi:10.1074/jbc.M009698200 on May 31, 2001

J. Biol. Chem., Vol. 276, Issue 32, 30359-30365, August 10, 2001
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Akt Down-regulation of p38 Signaling Provides a Novel Mechanism of Vascular Endothelial Growth Factor-mediated Cytoprotection in Endothelial Cells*

Jean-Philippe GrattonDagger §, Manuel Morales-RuizDagger §||, Yasuko Kureishi**, David FultonDagger , Kenneth Walsh**, and William C. SessaDagger DaggerDagger

From the Dagger  Department of Pharmacology and Molecular Cardiobiology Program, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536 and the ** Division of Cardiovascular Research, St. Elizabeth's Medical Center of Boston, Boston, Massachusetts 02135

Received for publication, October 24, 2000, and in revised form, May 30, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Vascular endothelial growth factor (VEGF) utilizes a phosphoinositide 3-kinase (PI 3-kinase)/Akt signaling pathway to protect endothelial cells from apoptotic death. Here we show that PI 3-kinase/Akt signaling promotes endothelial cell survival by inhibiting p38 mitogen-activated protein kinase (MAPK)-dependent apoptosis. Blockade of the PI 3-kinase or Akt pathways in conjunction with serum withdrawal stimulates p38-dependent apoptosis. Blockade of PI 3-kinase/Akt also led to enhanced VEGF activation of p38 and apoptosis. In this context, the pro-apoptotic effect of VEGF is attenuated by the p38 MAPK inhibitor SB203580. VEGF stimulation of endothelial cells or infection with an adenovirus expressing constitutively active Akt causes MEKK3 phosphorylation, which is associated with decreased MEKK3 kinase activity and down-regulation of MKK3/6 and p38 MAPK activation. Conversely, activation-deficient Akt decreases VEGF-stimulated MEKK3 phosphorylation and increases MKK/p38 activation. Activation of MKK3/6 is not dependent on Rac activation since dominant negative Rac does not decrease p38 activation triggered by inhibition of PI 3-kinase. Thus, cross-talk between the Akt and p38 MAPK pathways may regulate the level of cytoprotection versus apoptosis and is a new mechanism to explain the cytoprotective actions of Akt.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Programmed cell death, or apoptosis, is a common cellular response to stress caused by environmental challenges that contribute to inflammation and tissue homeostasis disorders (1). The local balance between pro- and anti-apoptotic stimuli will determine the fate of each individual cell. In vascular endothelial cells, which line all the blood vessels of the body, tight regulation of apoptotic and survival signals contributes to physiological and pathological processes such as angiogenesis, vascular homeostasis, and ischemic diseases (2).

Vascular endothelial growth factor (VEGF),1 an angiogenic cytokine secreted by a variety cells, functions as a survival factor for endothelial cells in vivo and in vitro (3, 4). This endothelial cell-specific growth factor can also act as a mitogen and a chemoattractant (5). VEGF binds to a family of receptor tyrosine kinases, leading to the activation of various signal transducers including the phosphoinositide 3-kinases (PI 3- kinase)/Akt signaling pathway (6), the mitogen-activated protein kinases (MAP kinases) (7, 8), the c-Jun N-terminal protein kinase (9) and stress-activated protein kinase-2 or p38 MAP kinase (10). Activation of the PI 3-kinase/Akt pathway plays a central role in the survival promoting properties of VEGF (3). PI 3-kinase catalyzes the phosphorylation of the inositol ring of phosphatidylinositol (PtdIns) lipids at the D-3 position producing PtdIns(3,4)P2 and PtdIns(3,4,5)P3. Direct binding of Akt to the membrane phosphoinositide products of the PI 3-kinase reaction, through its pleckstrin homology domain, permits phosphorylation by a phosphoinositide-dependent kinase (PDK1). PDK1 is also activated by phosphoinositol lipids and phosphorylates Akt at Thr-308, resulting in autophosphorylation of Ser-473 (11), thus increasing Akt catalytic activity toward a variety of diverse substrates (12). Akt is an important regulator of various cellular processes including metabolism, cell survival, migration, and nitric oxide release (13, 14).

In contrast to the anti-apoptotic signals of the PI 3-kinase/Akt pathway, VEGF also stimulates the activation of the stress-activated serine/threonine protein kinase, p38 MAPK, which appears to be an important modulator of the proapoptotic program in various cell types including endothelial cells. Thrombospondin-1 (15), oxidative stress, tumor necrosis factor-alpha and TL-1, a tumor necrosis factor-like cytokine (16), induce a p38-dependent apoptosis or signaling in endothelial cells. p38 MAP kinase is also involved in cytokine production (17), transcriptional regulation (18), and cytoskeletal reorganization (10). The p38 family of MAP kinases are composed of four members, p38alpha , -beta , -gamma , and -delta (19). Dual phosphorylation by the upstream MAP kinase kinase 6 (MKK6) of the characteristic Thr-Gly-Tyr motif is required for p38 activation (20). This activation is blocked by the pyridinyl imidazole inhibitors of p38, SB203580 and SB202190, which inhibit p38alpha and -beta but not p38gamma or -delta (21). Each p38 may also be activated by MKK3, with the exception of p38beta (20, 21). Some of the downstream substrates of p38 are involved in different signal transduction pathways like those related with changes in morphology and actin organization (MAP kinase-activated protein kinase 2; Ref. 22), and gene transcription (activating transcription factor 2 (ATF2; Refs. 21, 23, and 24) and the myocyte-enhancer factor 2C (Ref. 25)).

Together these data indicate that VEGF is capable of eliciting biochemical responses that either enhance cell survival (PI 3-kinase/Akt) or might lead to cell death (p38 MAPK). However the activation of p38 MAPK by VEGF in endothelial cells has only been studied in the context of cellular motility and actin reorganization; furthermore, VEGF is mostly known as an anti-apoptotic angiogenic growth factor. In this paper we reveal that blockade of PI 3-kinase or Akt signaling attenuates VEGF-stimulated MAPK/ERK kinase kinase 3 (MEKK3) phosphorylation and increases p38 signaling, thus enhancing apoptosis. On the other hand, overexpression of constitutively active Akt enhances MEKK3 phosphorylation and down-regulates p38 activation. These data suggest that cross-talk between PI 3-kinase/Akt and p38 signaling pathways contributes to the balance of pro- versus anti-apoptotic signaling that occurs in endothelial cells.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Reagents-- Bovine aortic endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs) were cultured as described (9, 26).

Hoechst 33342 was purchased from Molecular Probes (Eugene, OR). Cell culture media and supplies were from Life Technologies, Inc. (Beverly, MA). PI 3-kinase inhibitors LY294002 and wortmannin and p38 kinase inhibitor SB203580 were from Calbiochem (La Jolla, CA). Bovine serum albumin (fatty acid-free) and tetracycline were purchased from Sigma and CLONTECH (Palo Alto, CA) respectively.

Western Blotting-- BAECs and HUVECs were washed twice with ice-cold PBS, and total cell lysates were prepared by scraping the cells in lysis buffer (50 mM Tris-HCl, 0.1 mM EDTA, 0.1 mM EGTA, 1% (v/v) Nonidet P-40, 0.1% SDS, 0.1% deoxycholic acid, 20 mM NaF, 1 mM NaPP, 1 mM sodium vanadate, 1 mM Pefabloc, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). Lysates were rotated for 1 h at 4 °C, and the insoluble material removed by centrifugation at 12,000 × g for 10 min at 4 °C. Equal amounts of the denatured proteins were loaded, separated on a 10% sodium dodecyl sulfate-polyacrylamide gel (Mini Protean II, Bio-Rad), and transferred to a nitrocellulose membrane. Membranes were blocked by incubation in Tris-buffered saline (10 mM Tris, pH 7.5, 100 mM NaCl) containing 0.1% (v/v) Tween 20 and 5% (w/v) nonfat dry milk for 2 h, followed by 2 h of incubation, at room temperature, with rabbit polyclonal anti-phospho-p38 MAP kinase (Thr-180/Tyr-182) or anti-p38 MAP kinase antibodies (New England Biolabs, Beverly, MA), rabbit polyclonal anti-phospho-MKK3/MKK6 (Ser-189/Ser-207) or anti-MKK3 antibodies (New England Biolabs), rabbit polyclonal anti-phospho-Akt-Ser-473 or anti-Akt antibodies (New England Biolabs), mouse monoclonal anti-HA-12CA5 (Roche Molecular Biochemicals) or mouse monoclonal anti-Myc antibody (Invitrogen, Carlsbad, CA). The filters were washed extensively in Tris-buffered saline, containing 0.1% (v/v) Tween, before incubation for 1 h with goat anti-mouse or donkey anti-rabbit horseradish peroxidase-conjugated secondary antibodies. Membranes were then washed and developed using enhanced chemiluminescence substrate (ECL, Amersham Pharmacia Biotech).

p38 MAP Kinase Assay-- HUVECs were washed twice with ice-cold PBS, and total cell lysates were prepared by scraping the cells in lysis buffer (50 mM Tris-HCl, 0.1 mM EDTA, 0.1 mM EGTA, 1% (v/v) Nonidet P-40, 0.1% SDS, 0.1% deoxycholic acid, 20 mM NaF, 1 mM Na4P2O7, 1 mM Na3VO4, 1 mM Pefabloc, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). p38 MAP kinase assay was performed by using a commercial kit (New England Biolabs). Immunocomplex kinase assays using anti-phospho-p38 (Thr-180/Tyr-182) immunoprecipitates were performed at 30 °C for 30 min using 2 µg of ATF-2 fusion protein and 200 µM cold ATP in 50 µl of kinase buffer (25 mM Tris, pH 7.5, 5 mM beta -glycerol phosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, 10 mM MgCl2). The reaction were terminated with SDS sample buffer, and the products were resolved by 10% SDS-PAGE. Phosphorylation of ATF-2 at Thr-71 was measured by Western blot using phospho-ATF-2 (Thr-71) antibody.

Adenoviral Constructs-- beta -Galactosidase, HA-tagged inactive phosphorylation mutant Akt (AA-Akt), and C-terminal HA-tagged constitutively active Akt (myr-Akt) were generated as described previously (27, 28). Adenovirus expressing Myc-tagged Rac1 dominant negative (Rac1-DN) under control of tetracycline repressor elements and a minimal cytomegalovirus promoter (tet-mCMV), and adenovirus constitutively expressing a tetracycline represor-Vp16 fusion protein, which was required to activate expression of the tet-mCMV were generated as described previously (29). HUVECs or BAECs were infected with adenovirus (m.o.i. of 100) containing the beta -galactosidase, AA-Akt, myr-Akt, Rac1-DN, or tetracycline represor-VP16 for 12 h. The virus was removed and cells left to recover for 12 h in complete medium. These conditions resulted in uniform expression of the transgenes in close to 95% of the cells (determined by infection with beta -galactosidase, followed by staining for beta -galactosidase activity) and equal expression of Akt proteins, based on Western blotting. To avoid Rac1-DN expression in HUVECs infected with Rac1-DN and tetracycline repressor-VP16, cells were supplemented with tetracycline (1 µg/ml).

Apoptosis Assay-- HUVECs were grown on 12-well tissue culture plates and were subjected to serum and growth factor withdrawal (1% fatty acid-free BSA). Following treatment, floating and trypsin-detached HUVECs were pooled and washed once with ice-cold PBS and fixed in 70% cold ethanol. After fixation, cells were stained in a PBS propidium iodide (50 µg/ml), RNase A (100 µg/ml), and 0.05% Triton X-100 for 45 min. DNA content of HUVECs was analyzed by fluorescent activated cell sorting (FACSort, Becton Dickinson). At least 10,000 events were analyzed, and the percentage of cells in the sub-G1 population was calculated. Aggregates of cell debris at the origin of the histogram were excluded from the analysis of sub-G1 cells, as indicated in the legends to Figs. 2 and 4. In each experiment, serum-grown as well as serum-deprived HUVECs were used as controls and compared with cells treated with either VEGF or the different drugs.

Alternatively, cells were analyzed for the appearance of pyknotic nuclei. Confluent, serum-starved HUVECs, cultured in 12-well plates, were treated as indicated in the legend of Figs. 2 and 4. Following removal of culture media, 3.7% formaldehyde in PBS was added to the culture. After fixation for 20 min, cells were washed in PBS twice and stained with Hoechst 33342 (5 µg/ml; 20 min) and washed with PBS. Cells were examined with a Nikon Diaphot microscope. Percentage of pyknotic nuclei was determined by counting four different fields (32× objective) per well. At least 200 nuclei/well were counted.

Phosphorylation of MEKK3 in Intact Cells and Kinase Activity-- BAECs were placed into phosphate-free DMEM with or without 10% of 0.15 M NaCl-dialyzed (for removal of phosphate contamination in FBS) fetal bovine serum supplemented with 100 µCi/ml [32P]orthophosphate overnight prior to stimulation with VEGF. In some samples, cells were infected with adenovirus encoding dominant-negative Akt or constitutively active Akt at an m.o.i. of 100 for 24 h, or followed by incubation with LY294002 (100 nM) for 4 h prior to stimulation with VEGF. After 10 min of stimulation with VEGF, cells were washed with Tris-buffered saline and harvested with lysis buffer (radioimmune precipitation buffer plus 20 mM NaF, 1 mM Na4P2O7, 1 mM Na3VO4). The lysate was subjected to immunoprecipitation using mouse monoclonal anti-MEKK3 antibody (Transduction Laboratories). Radiolabeled phosphate incorporation into each protein was visualized after SDS-PAGE (10%) by autoradiography.

For the MEKK3 kinase activity, BAECs were harvested with lysis buffer (20 mM Tris, pH 7.4, 137 mM NaCl, 1% Nonidet P-40, and 10% glycerol plus protease and phosphatase inhibitors as above). The lysate was subjected to immunoprecipitation using the above anti-MEKK3 antibody for 2 h. The protein A-precipitated complex was washed twice with lysis buffer and twice with the kinase assay buffer (20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.1 mM Na3VO4). The kinase assay was performed using 25 µl of the protein A-complexed MEKK3 and 10 µl of glutathione-agarose-bound GST-MKK3 as a substrate. The kinase assay was preformed in a total of 50 µl of kinase assay buffer for 20 min at 30 °C in presence of 2 mM dithiothreitol, 100 µM cold ATP, and 10 µCi of [gamma -32P]ATP. Reaction was stopped by addition of Laemmeli buffer and resolved on a 10% SDS-PAGE, visualized by Coomassie staining followed by autoradiography.

Statistical Analysis-- Data are expressed as means ± S.E. Statistical differences were measured by two-way analysis of variance followed by Bonferroni's post hoc test. p value of <0.05 was considered as significant.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To investigate the cross-talk between the PI 3-kinase and the p38 MAP kinase signaling pathways, we studied the effects LY294002, a known PI 3-kinase inhibitor, on the phosphorylation state of p38 MAPK. Fig. 1A shows that treatment of serum-starved HUVECs with LY294002 (10 µM) induces a time-dependent increase in p38 MAPK phosphorylation. The addition of 10% FBS to the media reduces the phosphorylation state of p38 (Fig. 1B, top two panels, lane 1) and LY294002 (10 µM; 5 h) induces an increase in p38 phosphorylation even in the presence of serum (lane 2). Again, in the absence of serum, basal p38 phosphorylation is enhanced and LY294002 further stimulates p38 phosphorylation (lanes 3 and 4). In contrast, in the presence or absence of serum, LY294002 reduces the phosphorylation levels of Akt, a downstream component of the of PI 3-kinase pathway (Fig. 1B, bottom two panels). Identical results were obtained in serum-deprived COS cells treated with LY294002 (data not shown). We then monitored the effects of PI 3-kinase inhibition on serum withdrawal-induced apoptosis in HUVECs (Fig. 1C shows a representative experiment out of four individual experiments and Fig. 1D the summary of theses experiments). Under control conditions with serum, ~4% of HUVECs were hypodiploid based on fluorescent activated cell sorting analysis of propidium iodide-stained cells and LY294002 did not significantly increase this population when serum was present (Fig. 1D) consistent with low level p38 activation in the presence of serum (see Fig. 1B, lane 2). In the absence of serum, the level of apoptosis significantly increased (p < 0.05, n = 4) and this effect is enhanced by PI 3-kinase inhibition (p < 0.05, comparing lane 4 to lanes 3 and 1, n = 4). The increase in apoptosis and the stimulatory effects of LY294002 on cell death correlates with the increase of p38 phosphorylation in serum-deprived HUVECs.


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  		Fig. 1.   The activation of p38, mediated by the inhibition of PI 3-kinase pathway, induces apoptosis in endothelial cells. A, serum-starved HUVECs were incubated with or without 10 µM LY294002 at 37 °C. Phospho-p38 and p38 protein content was analyzed at different times by Western blot of total cell lysates (50 µg). B, HUVECs were treated for 5 h with or without 10 µM LY294002 at 37 °C either in the absence or in the presence of serum. Cell lysates were analyzed by Western blotting (50 µg) with antibodies to phospho-p38, p38, phospho-Akt, or Akt. C, representative figures of the determination of apoptosis by propidium iodide staining and flow cytometry of HUVECs treated with or without LY294002 (10 µM) for 6 h in presence or in absence of serum. For each panel, the percentage of HUVECs with hypodiploid (apoptotic) DNA content is indicated (M1). Panel D shows the average percentage of apoptosis of four experiments shown in C. *, p < 0.05 determined by two-way ANOVA followed by Bonferroni's post hoc test.

In order to verify that the increase in p38 MAP kinase activity is participating in the proapoptotic effects produced by PI 3-kinase inhibition, we studied the effect of the p38 inhibitor SB203580 (25 µM) on wortmannin-induced apoptosis in HUVECs. Wortmannin is a structurally distinct PI 3-kinase inhibitor (30). Fig. 2B shows that serum deprivation (12 h) induces the appearance of pyknotic nuclei, a marker of apoptosis, when compared with serum-grown HUVECs (from 5.2 ± 1.0% in presence of 10% FBS to 20.6 ± 1.6% in absence of serum; p < 0.05). The number of pyknotic nuclei are significantly reduced when HUVECs are incubated with the known survival factor VEGF (40 ng/ml) (from 20.6 ± 1.6% to 14.3 ± 0.6% in presence of VEGF; p < 0.05 versus no serum). Inhibition of PI 3-kinase by wortmannin (100 nM) in serum-free conditions induces significant apoptosis (40.9 ± 2.1%, p < 0.05 versus no serum). The inhibitor of p38 MAP kinase isoforms alpha  and beta , SB203580 (25 µM) alone did not further increase cell death but reduces apoptosis in HUVECs treated with wortmannin (from 40.9 ± 2.1% to 29.1 ± 2.9% in presence of SB203580; p < 0.05). Collectively, the data in Figs. 1 and 2 demonstrate that inhibition of PI 3-kinase stimulates p38 MAPK-mediated proapoptotic pathways.


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  		Fig. 2.   Inhibition p38 MAP kinase prevents appearance of pyknotic nuclei induced by inhibition of PI 3-kinase in HUVECs. A, representative pictures of HUVECs incubated in the absence or presence of serum (10%), VEGF (40 ng/ml), wortmannin (WM, 100 nM), or SB203580 (25 µM) for 12 h and DNA stained with Hoechst 33258 (5 µg/ml) and observed under a fluorescence microscope to determine chromatin appearance. B, effects of serum, VEGF, wortmannin (WM), SB203580, or SB203580 plus wortmannin on the frequency of HUVECs with pyknotic nuclei as described under "Experimental Procedures." Data are shown as the mean ± S.E. (n = 3). *, p < 0.05 determined by two-way ANOVA followed by Bonferroni's post hoc test.

Next, we wanted to examine the relationship between the p38 MAP kinase and PI 3-kinase pathways in HUVECs treated with VEGF. VEGF (40 ng/ml) stimulated the phosphorylation of p38 MAPK and its upstream activators MAP kinase kinases 3 and 6 (MKK3, MKK6) in serum-deprived HUVECs (Fig. 3, lanes 1 and 2, top and bottom panels). LY294002 induces activation of p38 (top panel, lane 3) as well as MKK3 and 6 (bottom panel, lane 3). VEGF stimulation following LY294002 treatment of HUVECs produces a slight but consistent further activation of p38 MAPK and MKK3 and MKK6 (lane 4). We also monitored apoptosis under the exact same conditions (Fig. 4A shows a representative experiment out of four). As expected, in the absence of serum, VEGF significantly reduces the number of pyknotic nuclei compared with serum-starved cells (Fig. 4A, p < 0.05, n = 6, comparing lane 2 to lane 3). Treatment of cells with SB203580 did not influence the cytoprotective effects of VEGF (lane 4), whereas LY294002 augmented apoptosis compared with serum withdrawal (compare lane 2 to lane 5) and blunted the anti-apoptotic actions of VEGF (lane 6). Interestingly, the addition of VEGF to LY294002-treated cells results in further apoptosis compared with LY294002-treated cells alone (lanes 5 and 6). However, when HUVECs are treated with VEGF and LY294002 to maximally increase cell death, SB203580 significantly reduces the level of apoptosis (compare lane 7 to lanes 5 and 6). Similar data were obtained using propidium iodide staining of hypodiploid cells as an index of apoptosis, i.e. SB203580 reduces the level of apoptosis triggered by the combination of VEGF and LY294002 (Fig. 4B, compare middle and bottom histograms on right side; these data are representative of four experiments). Fig. 4C shows that serum depletion increases kinase activity of p38 MAPK, as monitored by in vitro phosphorylation of recombinant ATF-2, and that inhibition of PI 3-kinase increases p38 activity. SB203580 abolishes this increase in p38 activity. These data imply that VEGF uses a PI 3-kinase pathway for endothelial cell protection and that the p38-driven death pathway becomes important when the PI 3-kinase/Akt pathway is blocked. However, in the absence of PI 3-kinase blockade, VEGF-stimulated p38 signaling leading apoptosis is likely offset by other prosurvival mechanisms.


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  		Fig. 3.   Requirement of MKK3/MKK6 phosphorylation for VEGF and PI 3-kinase inhibition-dependent activation of p38 in endothelial cells. HUVECs were serum-starved in DMEM containing 1% BSA in the absence or presence of VEGF (40 ng/ml) or LY294002 (10 µM). At the end of the treatment, protein lysates (50 µg) from each condition were used to examine the abundance of phospho-p38, p38, phospho-MKK3/6, or MKK3 proteins. Numbers below the panels indicate relative levels based on densitometry. Essentially identical results were obtained in another three experiments.


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  		Fig. 4.   VEGF-induced p38 activation in a PI 3-kinase/Akt-deficient background is associated with the induction of apoptosis in endothelial cells. A, HUVECs were treated with VEGF (40 ng/ml) for 6 h in absence or in presence of LY294002 (10 µM; 5-h pretreatment) and SB203580, and the frequency of cells with pyknotic nuclei was counted using the Hoechst 33258 DNA stain as described under "Experimental Procedures." Data are shown as the mean ± S.E. (n = 6). *, p < 0.05 determined by two-way ANOVA followed by Bonferroni's post hoc test. B, serum-deprived HUVECs were treated as described in A. For each panel, the percentage of HUVECs with hypodiploid (apoptotic) DNA content is indicated (M1) as determined by propidium iodide staining and flow cytometry. Similar results were obtained in three additional experiments. C, serum-starved HUVECs were treated for 5 h with or without LY294002 (10 µM) or SB203580 (10 µM) before immunoprecipitation of endogenous proteins and in vitro kinase assay. Cells extracts were incubated overnight with immobilized p38 (Thr-180/Tyr-182) monoclonal antibody. After extensive washing, the kinase reaction was performed in the presence of 100 µM cold ATP and 2 µg of ATF-2 fusion protein. Phosphorylation of ATF-2 at Thr-71 was measured by Western blot using phospho-ATF-2 (Thr-71) antibody. Identical results were obtained in another three experiments.

To test the involvement of Akt, a serine/threonine kinase downstream of PI 3-kinase, in the cross-talk with the p38 pathway, we used adenoviral mediated gene transfer to endothelial cells. Fig. 5A shows that infection of HUVECs with the activation-deficient mutant of Akt (AA-Akt, HA-tagged) modestly increases the basal and LY294002-stimulated phosphorylation levels of p38 MAPK and more strongly activates its upstream activators MKK3 and MKK6 compared with control cells infected with a beta -galactosidase adenovirus (Fig. 5A, lane 1 versus lane 3). Infection of HUVECs with a constitutively activated form of Akt (myr-Akt, HA-tagged) markedly reduces p38 and MKK3/6 phosphorylation levels. Furthermore, myr-Akt prevents the increase in p38 MAPK phosphorylation induced by LY294002. In panel B, VEGF-induced increases in p38 MAPK phosphorylation are also blunted by adenovirus transduction of HUVECs with myr-Akt. The data demonstrate that Akt is the downstream effector of PI 3-kinase mediating the cross-talk between the PI 3-kinase and p38 pathways.


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  		Fig. 5.   Akt modulates VEGF and inhibition of PI 3-kinase-dependent activation of p38. A, HUVECs were infected with adenoviruses for beta -galactosidase, HA-tagged AA-Akt and myr-Akt. After 12 h in complete medium, cells were serum-starved and treated with or without LY294002 (10 µM) for 1 h. Cells were harvested, and cell lysates (50 µg) were subjected to immunoblot analysis with antibodies specific for phospho-p38, phospho-MKK3/MKK6, p38, MKK3, or HA. B, cells were infected with either beta -galactosidase or myr-Akt adenoviruses. Cells were serum-starved and stimulated with VEGF (40 ng/ml) for 10 min. Cell lysates (50 µg) were subjected to immunoblot analysis with antibodies specific for phospho-p38, p38, or HA. Identical results were obtained in another two experiments.

In order to identify a potential substrate for Akt phosphorylation that would lead to the negative regulation of the p38 signaling pathway, we investigated the putative role of the upstream activator of MKK6 (31), MEKK3, and the small GTPase protein Rac1 (32) in this signaling pathway. In Fig. 6A, we examined MEKK3 phosphorylation in BAECs in response to Akt activation by VEGF or via infection with myr-Akt. VEGF or serum stimulated the incorporation of 32P into immunoprecipitated MEKK3 (compare lanes 1, 2, and 6). Both conditions induced Akt phosphorylation in these cells (data not shown). VEGF- or serum-dependent incorporation of 32P was completely abolished when cells were treated with LY294002 or infected with the AA-Akt adenoviruses (compare lanes 2, 3, and 4 for VEGF and lanes 6, 7, and 8 for serum-induced phosphorylation of MEKK3, respectively). Furthermore, robust phosphorylation of MEKK3 was observed when BAECs were transduced with constitutively active form of Akt (myr-Akt). Next we examined the relationship between Akt-mediated phosphorylation of MEKK3 and its kinase activity. As seen in Fig. 6B, infection of BAECs with myr-Akt decreased MEKK3 kinase activity using GST-MKK3 as a substrate (left panel). Conversely, infection with adenoviral AA-Akt or treatment of BAECs with LY294002 enhanced MEKK3 activity. Collectively, these data suggest that Akt-mediated phosphorylation of MEKK3 may account for the decrease in p38 activation.


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  		Fig. 6.   PI 3-kinase/Akt-dependent phosphorylation of MEKK3, but not RAC1, results in decreased kinase activity. A, BAECs were placed into phosphate-free DMEM with or without 10% fetal bovine serum supplemented with 100 µCi/ml [32P]orthophosphate overnight prior to stimulation with VEGF. In some samples, cells were infected with AA-Akt adenovirus or myr-Akt at an m.o.i. of 100 for 24 h, or followed by incubation with LY294002 (100 nM) for 4 h prior to stimulation with VEGF. After 10 min of stimulation with VEGF, the lysate was subjected to immunoprecipitation using mouse monoclonal anti-MEKK3 antibody. Radiolabeled phosphate incorporation into each protein was visualized after SDS-PAGE (10%) by autoradiography. The same samples were probed with anti-MEKK3 antibody (bottom) to determine its level of expression. B, BAECs were infected with beta -galactosidase, myr-Akt, or AA-Akt adenovirus at an m.o.i. of 100 and serum starved for either 16 h (left panel) or 6 h (right panel) in presence or absence of 10 µM LY294002. MEKK3 was immunoprecipitated as described under "Experimental Procedures," and kinase activity was measured using GST-MKK3 as a substrate. Coomassie staining shows equal loading of GST-MKK3 and autoradiography indicated the amount of incorporated 32P in the substrate with densitometric analysis (Dens. ratio) below. Independent experiments show that MEKK3 can be quantitatively immunoprecipitated in all the above conditions (data not shown). These experiments were repeated twice with similar results obtained. In C, HUVECs were infected with inactive Myc-tagged N17-Rac adenovirus, under the control of a tetracycline repressor-VP16 fusion protein. After 12 h in complete medium, cells were serum-starved and treated with or without LY294002 (10 µM) for 5 h. Cell lysates were resolved by 10% SDS-PAGE and immunoblotted with antibodies against phospho-p38, p38, Myc, and VP16. Note that N17-Rac1 protein expression is absent in cells treated with tetracycline (1 µg/ml). Similar results were obtained in another three experiments.

Finally, we investigated the role of the small GTPase Rac1 in the LY294002-induced activation of p38 MAPK since there is evidence that Akt can regulate Rac1 activity (33) and that Rac1 may lie upstream of p38 in certain signaling systems (32). Adenoviral overexpression of a dominant negative form of Rac1, RacN17, under the control of the tetracycline repressor fusion protein VP16, does not prevent the phosphorylation of p38 MAPK induced by treatment of the cells with the PI 3-kinase inhibitor LY294002 (Fig. 6C). Identical results were obtained in COS cells transiently transfected with RacN17 (data not shown). Together, these data suggest that Akt phosphorylates MEKK3 leading to a negative regulation of the p38 MAPK pathway in a Rac1-independent manner.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this article, we provide evidence for the first time about the existence of cross-talk between the p38 MAP kinase and PI 3-kinase/Akt pathways. We demonstrate that inhibition of PI 3-kinase reduces Akt-dependent phosphorylation of MEKK3. The decrease in phosphorylation results in stimulation of MKK3/MKK6 and p38, an effect that occurs independent of the Rac/Pak pathway. This effect is blocked by the expression of a constitutively active form of Akt, which phosphorylates MEKK3 and down-regulates its activity toward the substrate MKK3. Surprisingly, VEGF-induced p38 activation in a PI 3-kinase/Akt-deficient background is associated with the induction of apoptosis in endothelial cells, an effect blocked by the p38 MAPK inhibitor, SB203580. Our results are consistent with previous studies showing that inhibition of PI 3-kinase/Akt (27) or activation of p38 MAPK stimulates apoptosis in endothelial cells (16) and inhibits angiogenesis (15).

Precedence for cross-talk between the PI 3-kinase/Akt pathway and two different sets of MAP kinases has been established by previous studies with ERK (34, 35) and the c-Jun N-terminal kinases (JNK) (36) pathways. In both cases, Akt inhibits the activation of these proteins in response to extracellular signaling. In the context of ERK down-regulation, two recent reports demonstrate that Akt may regulate Raf-1 activity. Rommel et al. (35) demonstrated that Akt can complex with Raf-1, producing inhibition of MEK catalytic activity. These authors demonstrated that this interaction was only present in myotubes cells but not in myoblasts, thus regulating muscle hypertrophy. Consistent with these results, Zimmermann et al. (34) showed that Akt antagonizes Raf-1 activity by direct phosphorylation of serine 259 in its regulatory domain. In this context, certain MAPK kinase kinases have been identified as components of the p38 signaling pathways. MEKK3, an upstream activator of p38 (31, 37), has a putative Akt phosphorylation sites at Ser166. In this paper, we have shown that the activation of Akt or expression of the constitutively active Akt (myr-Akt) in endothelial cells correlates with an increase in MEKK3 phosphorylation. Increased MEKK3 phosphorylation reduces its kinase activity toward MKK3. The 32P incorporation in MEKK3 is completely abolished by the infection with adenoviral dominant negative Akt construct and this results in increased MEKK3 activity. This suggests that Akt may negatively regulate the MKK3/MKK6-p38 pathway by phosphorylating MEKK3 in a manner analogous to that seen in the Raf-MEK-ERK signaling pathways (34). Although we show that phosphorylation can reduce MEKK activity, it is possible that phosphorylation of MEKK3 may lead to the recruitment of 14-3-3, which interacts in a phosphospecific manner with other Akt substrates including Bad (38), Forkhead transcription factor (39), and Raf (34, 35). However, we cannot discount the possibility that Akt may phosphorylate others upstream regulators of MKK3/6 such as MEKK5 (ASK1) (40), which also possesses a putative Akt phosphorylation site at serine 83, or that Akt may regulate other pathways that are important for cell survival and may affect p38 activity. For this reason, further studies will be required to identify the molecular basis of this cross-talk between the PI 3-kinase/Akt and the MEKK3-MKK3/MKK6-p38 pathway.

VEGF is an important survival factor for endothelial cells during vasculogenesis and angiogenesis. VEGF stimulates the PI 3-kinase/Akt pathway, resulting in the inhibition of apoptosis. The mechanisms of VEGF-stimulated PI 3-kinase-dependent cytoprotection may occur through enhanced expression of anti-apoptotic proteins including Bcl-2, A1 (4), and the IAP proteins survivin (41) and XIAP (42). Several direct targets of the PI 3-kinase/Akt signaling pathway have been identified that may underlie the ability of this regulatory cascade to promote survival. These Akt substrates include BAD, caspase 9, transcription factors of the forkhead family, and IKK, which regulates the NF-kappa B transcription factor (39, 43-45). However, there is no evidence that these substrates mediate the survival effects of VEGF. VEGF also activates p38 MAPK, shown previously to be involved in endothelial cell migration (10). In the present study, VEGF stimulates p38 phosphorylation but p38 signaling does not contribute to the anti-apoptotic effects of VEGF, since SB203580 does not influence the levels of VEGF-induced cytoprotection (Fig. 3). However, if the PI 3-kinase system is blocked, in the absence or presence of VEGF, p38 activation is greater than seen with VEGF alone. Under these conditions, we unmask a proapoptotic effect of VEGF that appears to be mediated via p38 activation. This suggests that the level of PI 3-kinase/Akt activity will dictate whether this survival factor can trigger apoptosis and that down-regulation of MKK3/6-p38 pathway may also contribute to the anti-apoptotic properties of Akt activation. Parallel activation of survival and death pathways is a common feature for some growth factors (i.e. platelet-derived growth factor) or cytokines (i.e. tumor necrosis factor-alpha ). It could be argued that the anti-apoptotic properties of VEGF signaling to Akt observed in our experiments can be also related with the inhibition of JNK activity by Akt, since MEKK3 is also known to activate the JNK pathway and because Akt is known to affect JNK signaling (30, 35). However, the fact that SB203580 (an inhibitor of p38 but not JNK MAPK) reduces the level of apoptosis produced in HUVECs treated with both VEGF and LY294002 to levels observed in cells treated with VEGF only (Fig. 4, compare lanes 3-7) supports the idea that the cross-talk between Akt and p38 plays a role in VEGF-induced cell survival. Recent evidence, showing impaired vasculogenesis in MEKK3 knockout embryos without alterations in the expression of VEGF and angiopoeitin-1 or their receptors, suggests that MEKK3 is an important downstream signal of these angiogenic factors consistent with our data (46).

In summary, VEGF can stimulate MKK3/MKK6/p38 and PI 3-kinase/Akt pathways that are physiologically linked to apoptosis and survival in endothelial cells, respectively. It has been suggested that the balance of endothelial apoptosis and survival participates in variety of cardiovascular diseases and cancers (2). Understanding the mechanisms by which the MEKK3-MKK3/6-p38 and PI 3-kinase/Akt pathways regulate endothelial apoptosis may contribute to the development of novel therapeutics that could regulate the pro- and anti-apoptotic balance in such disease states.

    ACKNOWLEDGEMENTS

We are indebted to Dr. Anton Bennett for helpful discussions, to Dr. G. Garcia-Cardeña for kindly providing Rac1-DN and tetracycline represor-VP16 adenoviruses, and to Dr. Roger J. Davis for the GST-MKK3 expression vector.

    FOOTNOTES

* This work was supported in part by National Institute of Health Grants HL 61371 and HL 64793 (to W. C. S.), T32HL10183 (to D. F.), and AR 40197, HL 50692, AG 15052, and HD23681 (to K. W.) and by a grant-in-aid from the American Heart Association (to W. C. S.).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.

§ Both authors contributed equally to this work.

Supported by a fellowship from the Canadian Institutes of Health Research.

|| Supported by Ministerio de Educación y Cultura Grant EX99-38446345 and a grant from the Asociación Española para el Estudio del Hígado, Spain.

Dagger Dagger Established investigator of the American Heart Association. To whom correspondence should be addressed. Tel.: 203-737-2291; Fax: 203-737-2290; E-mail: william.sessa@yale.edu.

Published, JBC Papers in Press, May 31, 2001, DOI 10.1074/jbc.M009698200

    ABBREVIATIONS

The abbreviations used are: VEGF, vascular endothelial growth factor; HUVEC, human umbilical vein endothelial cell; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; MKK, mitogen-activated protein kinase kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase; MEKK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase kinase; PI 3-kinase, phosphoinositide 3-kinase; ERK, extracellular signal-regulated kinase; DN, dominant negative; PBS, phosphate-buffered saline; BAEC, bovine aortic endothelial cell; ANOVA, analysis of variance; HA, hemagglutinin; m.o.i., multiplicity of infection; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; PtdIns, phosphatidylinositol.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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