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Uncorrected Version. Published on March 5, 2007 as DOI:10.1189/jlb.0406256

Dissociation between the translocation and the activation of Akt in fMLP-stimulated human neutrophils— effect of prostaglandin E2 Chantal Burelout,* Paul H. Naccache,*,† and Sylvain G. Bourgoin*,‡,1 *Centre de Recherche en Rhumatologie-Immunologie, Centre de Recherche du CHUL, and De´partements de † Me´decine et ‡d’Anatomie-Physiologie, Universite´ Laval, Que´bec, Canada

Abstract: PGE2 and other cAMP-elevating agents are known to down-regulate most functions stimulated by fMLP in human polymorphonuclear neutrophils. We reported previously that the inhibitory potential of PGE2 resides in its capacity to suppress fMLP-stimulated PI-3K␥ activation via the PGE2 receptor EP2 and hence, to decrease phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] formation. Akt activity is stimulated by fMLP through phosphorylation on threonine 308 (Thr308) and serine 473 (Ser473) by 3-phosphoinositide-dependent kinase 1 (PDK1) and MAPK-AP kinase (APK)-APK-2 (MAPKAPK-2), respectively, in a PI-3K-dependent manner. Despite the suppression of fMLP-induced PI3K␥ activation observed in the presence of PGE2, we show that Akt is fully phosphorylated on Thr308 and Ser473. However, fMLP-induced Akt translocation is decreased markedly in this context. PGE2 does not affect the phosphorylation of MAPKAPK-2 but decreases the translocation of PDK1 induced by fMLP. Other cAMP-elevating agents such as adenosine (Ado) similarly block the fMLP-induced PI-3K␥ activation process but do not inhibit Akt phosphorylation. However, Akt activity stimulated by fMLP is down-regulated slightly by agonists that elevate cAMP levels. Whereas protein kinase A is not involved in the maintenance of Akt phosphorylation, it is required for the inhibition of Akt translocation by PGE2. Moreover, inhibition of fMLP-stimulated PI-3K␦ activity by the selective inhibitor IC87114 only partially affects the late phase of Akt phosphorylation in the presence of PGE2. Taken together, these results suggest that cAMP-elevating agents, such as PGE2 or Ado, are able to induce an alternative mechanism of Akt activation by fMLP in which the translocation of Akt to PI(3,4,5)P3-enriched membranes is not required prior to its phosphorylation. J. Leukoc. Biol. 81: 000 – 000; 2007.

invading microorganisms, release of proteolytic enzymes, cytokines, and chemokines, and generation of reactive oxygen species. Attracted by chemotactic substances from endogenous origin, such as leukotriene B4 (LTB4), IL-8, and the anaphylatoxin C5a, or from bacterial origin, such as fMLP, PMNs migrate rapidly toward the site of infection, where they perform their bactericidal activity. Their physiological functions are down-regulated by agonists such as adenosine (Ado) or PGE2, which raise the intracellular level of cAMP. PGE2 is an active metabolite derived from arachidonic acid under the action of cyclooxygenases (COX-1/2) and PGE synthases. Several effector cells of the innate immune response such as monocytes/ macrophages, PMNs, or dendritic cells (DCs) are important sources of endogeneous PGE2, which is synthesized following the activation of the inducible COX-2 under inflammatory conditions [1]. Several PMN functional responses to fMLP are inhibited by PGE2, including motility and chemotaxis [2], production of superoxide anions [3], as well as the release of cytotoxic enzymes and LTB4 [4, 5]. PGE2 has also been shown to delay constitutive apoptosis in PMNs [6, 7]. The physiological activities of PGE2 are mediated via G-protein-linked seven transmembrane domain receptors classified into four subtypes, EP1–EP4, according to their structure and coupling to distinct signaling pathways [8]. EP2 and EP4 stimulate adenylyl cyclase, whereas EP1 triggers intracellular calcium release. The various EP3 isoforms generated by alternative splicing are connected to cAMP or to calcium metabolism. Pharmacological studies have shown that the inhibitory effects of PGE2 on fMLP-induced superoxide production, enzyme release, and chemotaxis are mediated via the EP2 receptor in human PMNs [9, 10]. Akt, a serine/threonine protein kinase belonging to the “AGC” superfamily of kinases, plays critical roles in the regulation of cell survival and proliferation [11, 12]. Its conserved structure contains an N-terminal pleckstrin homology (PH) domain, which binds phosphoinositides with high affinity, a central catalytic domain, and a C-terminal regulatory domain,

Key Words: cAMP 䡠 PI-3K 䡠 adenosine

INTRODUCTION Polymorphonuclear neutrophils (PMNs) play a predominant role in the innate host immune defense through phagocytosis of 0741-5400/07/0081-0001 © Society for Leukocyte Biology

1 Correspondence: Centre de Recherche en Rhumatologie-Immunologie, Centre de Recherche du CHUL, 2705 Boul. Laurier, Room T1-49, Sainte-Foy, Que´bec, Canada G1V 4G2. E-mail: [email protected] Received April 6, 2006; revised November 3, 2006; accepted February 1, 2007. doi: 10.1189/jlb.0406256

Journal of Leukocyte Biology Volume 81, June 2007

Copyright 2007 by The Society for Leukocyte Biology.

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characterized by the presence of a hydrophobic motif, which mediates interactions with signaling molecules [11]. The PH domain of Akt binds phosphatidylinositol 3,4-diphosphate [PI(3,4)P2] and PI 3,4,5-triphosphate [PI(3,4,5)P3] specifically, resulting from PI-3K activity. Upon activation of PI-3K by various agonists (growth factors, insulin), Akt is recruited to the plasma membrane, where it undergoes phosphorylation on two sites: threonine 308 (Thr308) in the kinase domain by the phosphoinositide-dependent kinase-1 (PDK1) and serine 473 (Ser473) in the C-terminal domain by a PDK2, the nature of which may vary according to the cell type. These two phosphorylation steps are necessary and sufficient for full activation of Akt [11], and Akt activity can be stimulated in PMNs by chemoattractants such as fMLP [13] and has been shown to contribute to chemotaxis and to the respiratory burst [14]. In fMLP-stimulated neutrophils, MAPK-AP kinase (APK)-APK-2 (MAPKAPK-2), a p38 substrate, phosphorylates the Ser473 of Akt in a PI-3K-dependent manner and thus, fulfills the role of PDK2 [15]. We reported previously that PGE2 interferes with the fMLPsignaling pathway at the PI-3K␥ level in human PMNs. Indeed, PGE2 abolished the stimulation of the p110␥ activity induced by fMLP and consequently decreased PI(3,4,5)P3 formation. This inhibitory effect of PGE2 is mediated via EP2 receptors. Subsequently, the translocation to membranes of several components downstream of PI-3K [Tec kinases, Rho and Arf GTPases, and protein kinase C (PKC)] is impaired severely [16]. In the present study, we further investigated the consequences of the inhibition of the fMLP-induced PI-3K␥ activity by PGE2, and we focused specially on the effect of PGE2 on Akt activation induced by fMLP, as Akt is a major downstream target of PI-3K.

MATERIALS AND METHODS Reagents fMLP, wortmannin, and cytochalasin B (CB) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Dextran T-500 was purchased from Pharmacia Biotech (Dorval, Que´bec, Canada) and Ficoll-Paque, from Wisent (St-Bruno, Que´bec, Canada). Ado deaminase (ADA) was purchased from Roche Diagnostics (Laval, Que´bec, Canada), and diisopropylfluorophosphate (DFP), from Serva (Heidelberg, Germany). PGE2 and 9-oxo-11␣, 16S-dihydroxy-17-cyclobutyl-prosta-5Z,13E-dien-1-oic acid (CAY10399) were purchased from Cayman Chemical (Ann Arbor, MI, USA). Forskolin was obtained from Biomol Research Laboratory (Plymouth Meeting, PA, USA). 2-p-(2Carboxyethyl)phenethylamino-5⬘-N-ethylcarboxiamido Ado hydrochloride (CGS-21680) and chloro-2-(2-furyl) [1, 2, 4]triazolo[1,5-c]quinazolin-5-amine (CGS-15943) were obtained from RBI (Natick, MA, USA). H-89 was purchased from Calbiochem (La Jolla, CA, USA). IC87114 was a gift from Dr. Tom Crabbe (UCB, Slough, UK). The Akt kinase assay kit was purchased from Cell Signaling Technology (Beverly, MA, USA; #9840).

Antibodies The mouse mAb against phospho-p38 (Thr180/Tyr182; #9216) and the rabbit polyclonal antibody (pAb) against p38 (#9212), MAPKAPK-2 (#3042), phospho-MAPKAPK-2 (Thr222; #3044), phospho-MAPKAPK-2 (Thr334; #3041), PDK1 (#3062), Akt (#9272), phospho-Akt (Ser473; #9271), phospho-Akt (Thr308; #9275), phospho-Bad (Ser136; #9295), and the phospho-glycogen synthase kinase (GSK)-3␣/␤ (Ser21/9; #9331) were obtained from Cell Signaling Technology. The mouse-immobilized mAb against Akt (1G1) was purchased from Cell Signaling Technology (#9279). The antiflotillin-1 mouse mAb

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was obtained from BD Biosciences (Mississauga, ON, Canada; #610820). The anti-p110␥ pAb was raised in rabbits as described previously [17].

Isolation of human neutrophils Venous blood was collected from healthy adult volunteers in isocitrate anticoagulant solution. PMNs were separated as described previously [18]. Briefly, whole blood was centrifuged at 180 g for 10 min, and the resulting platelet-rich plasma was discarded. Leukocytes were obtained following erythrocyte sedimentation in 2% Dextran T-500. Mononuclear cells were removed by centrifugation on Ficoll-Paque cushions, and contaminating erythrocytes in the neutrophil pellets were removed by a 20-s hypotonic lysis in water. PMNs were resuspended in HBSS, pH 7.4, containing 1.6 mM Ca 2⫹ but no Mg2⫹.

Immunoblotting analysis of whole cell lysates PMNs (2⫻107 cells/ml) were preincubated at 37°C with 1 mM DFP for 10 min. The cells were preincubated for 10 min with 0.1 U/ml ADA to eliminate endogenous Ado and EP2 receptor agonists (PGE2 and CAY10399), the A2A receptor agonist CGS-21680, antagonist CGS-15943, forskolin, or an equal volume of diluent (Me2SO) as a control. When indicated, kinase inhibitors (H-89, wortmannin, or IC87114) were added prior to PGE2. PMNs were stimulated with 100 nM fMLP for the indicated times, and the reaction was stopped by transferring 100 ␮l of the cell suspensions to an equal volume of boiling 2⫻ Laemmli sample buffer (SB; 1⫻ is 62.5 mM Tris-HCl, pH 6.8, 4% SDS, 5% ␤-ME, 8.5% glycerol, 2.5 mM orthovanadate, 10 mM paranitrophenylphosphate, 10 ␮g/ml leupeptin, 10 ␮g/ml aprotinin, 0.025% bromophenol blue) and boiled for 7 min. The samples were then subjected to 7.5–20% gradient SDS-PAGE and transferred to Immobilon polyvinylidene difluoride (PVDF) membranes. Immunoblotting was performed using the indicated antibodies and revealed with the HRP-conjugated secondary antirabbit or antimouse antibodies (1/20,000) and the Renaissance detection system (NEN, Perkin Elmer Life Sciences, Boston, MA, USA).

Translocation assays PMNs (4⫻107 cells/ml) were treated with 1 mM DFP for 10 min at room temperature. The cell suspensions were centrifuged, and the cells were resuspended in HBSS at 107 cells/ml. PMNs were warmed for 5 min at 37°C and preincubated for 10 min with 0.1 U/ml ADA to eliminate endogeneous Ado. When indicated, PGE2 or the EP2 receptor agonist CAY10399, CGS-21680 (A2A receptor agonist), CGS-15943 (A2A receptor antagonist), forskolin, or an equal volume of diluent (Me2SO) as a control was added to the cell suspensions at the same time as ADA. CB (10 ␮M) was added 5 min prior to stimulation with fMLP. PMNs were stimulated with 100 nM fMLP at 37°C for 30 s. Incubations were stopped by diluting the cell suspensions fivefold with icecold HBSS, and total membrane proteins were collected as described previously [18]. Briefly, cell suspensions were centrifuged as indicated and resuspended in 1 ml ice-cold KCl-HEPES relaxation buffer (100 mM KCl, 50 mM HEPES, 5 mM NaCl, 1 mM MgCl2, 0.5 mM EGTA, 2.5 ␮g/ml aprotinin, 2.5 ␮g/ml leupeptin, and 2.5 mM PMSF, pH 7.2). Cell suspensions were sonicated for 20 s and centrifuged for 7 min at 1000 g. Unbroken cells and nuclei were discarded, and the supernatants were ultracentrifuged at 180,000 g for 45 min. Membrane pellets were washed once and resuspended in a small volume of buffer A containing 0.25 M Na2HPO4, 0.3 M NaCl, 2.5% SDS, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, and 2.5 mM PMSF, and samples were assayed for protein content with the Coomassie brilliant blue protein assay (Pierce, Rockford, IL, USA). Protein samples (30 ␮g) were resolved on an 8% SDSPAGE and transferred to Immobilon PVDF membranes (Millipore Corp., Bedford, MA, USA). Immunoblotting analyses were performed using antip110␥ (1/1000), anti-Akt (1/1000), or anti-PDK1 (1/1000) antibodies. The PVDF membranes were reprobed with an antiflotillin-1 antibody as a control for equal protein loading. Proteins were revealed with HRP-conjugated secondary antirabbit or antimouse antibody and the Renaissance detection system (NEN, Perkin Elmer Life Sciences) as described above.

Akt immunoprecipitation and kinase assay PMN suspensions (1 ml at 2⫻107 cells/ml) were warmed at 37°C in the presence of 1 mM DFP and preincubated with 0.1 U/ml ADA for 5 min of preincubation. H-89 (10⫺5 M; or an equal volume of Me2SO) was added for an

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additional 5 min and 10⫺5 M PGE2 or CGS-21680 (or an equal volume of Me2SO), for another 10 min incubation. PMNs were then stimulated with 100 nM fMLP (or Me2SO as a nonstimulated control), and the reaction was stopped by a quick centrifugation of the cell suspensions. Cell pellets were lysed by adding 1 ml ice-cold lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM paranitrophenylphosphate, 1 mM orthovanadate, 1 mM PMSF, and 10 ␮g/ml aprotinin/leupeptin) and were left 10 min on ice. The cell lysates were centrifuged at 12,000 g for 10 min at 4°C. Supernatants (30 ␮l) were kept aside to ensure that the same amounts of Akt were present in cell lysates. Akt was immunoprecipitated in the clarified lysates (900 ␮l) with 20 ␮l immobilized Akt antibody bead slurry for 2 h at 4°C. The beads were collected and washed twice with cold lysis buffer and twice with kinase buffer (25 mM Tris, pH 7.5, 10 mM MgCl2, 5 mM ␤-glycerophosphate, 2 mM DTT). The bead pellets were suspended in 50 ␮l kinase buffer supplemented with 1 ␮l 10 mM ATP and 1 ␮g GSK-3 fusion protein as a substrate and incubated at 30°C for 25 min. The reaction was stopped by adding 25 ␮l 3⫻ Laemmli SB. Proteins were separated on 7.5–20% gradient SDS-PAGE and transferred to Immibilon PVDF membranes. Immunoblottings were performed with anti-Akt (1/1000) and antiphospho-GSK-3 (1/1000) antibodies.

Statistics The paired Student’s t-test was used for data analysis.

RESULTS Effect of PGE2 on fMLP-induced Akt phosphorylation Akt activation stimulated by various agonists is PI-3K-dependent, and its phosphorylation on Ser473 and Thr308 residues is most often considered as an index of PI-3K activation. As we reported previously that PGE2, acting via EP2 receptors, inhibited the stimulation of PI-3K␥ activity induced by fMLP in human PMNs, we examined the effect of this eicosanoid and of the EP2-selective agonist CAY10399 on the fMLP-stimulated phosphorylation status of Akt. The data presented in Figure 1 show that unexpectedly, the stimulation of the phosphorylation of Akt on Ser473 and Thr308 induced by fMLP was maintained when PMNs were incubated with PGE2 or CAY10399 prior to stimulation. The level of Akt phosphorylation on both residues was decreased even slightly at 5 and 10 min after fMLP stimulation, thus indicating that PGE2 could weakly modify the Akt phosphorylation time-course induced by fMLP. In PMNs

incubated with PGE2 alone up to 40 min, we did not observe the phosphorylation of Akt.

Effect of a PKA inhibitor on the phosphorylation of Akt in the presence of PGE2 The binding of PGE2 to EP2 receptors stimulates the formation of cAMP by adenylyl cyclase [19]. The inhibition of the fMLPstimulated production of superoxide anions by PGE2 in human PMNs is mediated via the EP2 receptors and the cAMP/PKA pathway [9]. Moreover, we observed that the inhibition of the translocation of the p110␥ catalytic subunit to membranes by the EP2 pathway was PKA-dependent (unpublished results). It has also been reported that PKA stimulated Akt activity in a PI-3K-independent manner in 293-EBNA and COS7 cell lines [20, 21]. To examine the role of PKA in the stimulation of Akt phosphorylation induced by fMLP in the presence of PGE2, we examined the effects of the PKA pharmacological inhibitor H-89 on this response. The data presented in Figure 2 show that preincubation of PMNs with H-89 did not significantly modify the phosphorylation of Akt induced by fMLP. Therefore, in our experimental model, PKA does not appear to be involved, directly or indirectly, in the phosphorylation of Akt.

PGE2 inhibits the translocation of PDK1 and Akt to membranes— effect of H-89 PDK1 and Akt translocate rapidly to the plasma membrane in response to PI-3K activation. This membrane localization has been shown to be necessary for the phosphorylation of Akt by PDK1 and for its full activation [22, 23]. As, on the one hand, PGE2 decreased the fMLP-induced formation of PI(3,4,5)P3, and conversely, the PH domains of Akt and PDK1 display a high affinity for polyphosphoinositides [PI(3,4)P2 and PI(3,4,5)P3], which render them sensitive to variations in the concentration of polyphosphoinositides at the plasma membrane, we therefore examined the effect of PGE2 on the translocation of Akt and PDK1 induced by fMLP. As expected, we observed that when PMNs were incubated in the presence of PGE2 prior to stimulation by fMLP, the amounts of membraneassociated Akt and PDK1 were decreased dramatically in a dose-dependent manner as compared with the fMLP-stimulated controls (Fig. 3A).

Fig. 1. Effect of PGE2 and CAY10399 on fMLP-induced Akt phosphorylation. PMNs were preincubated for 10 min with 10⫺5 M PGE2 or 10⫺5 M CAY10399 and stimulated with 100 nM fMLP for the times indicated. The cell lysates were analyzed as described in Materials and Methods. The membranes were probed with antiphospho (P within circle)-Ser473 Akt, antiphospho-Thr308 Akt, or anti-Akt antibodies. The data shown are representative of four independent experiments. IB, Immunoblots.

Burelout et al. PGE2 inhibits the translocation of Akt but not its phosphorylation

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Fig. 2. Effect of H-89 on fMLP-induced Akt phosphorylation. PMNs were preincubated for 5 min with 10⫺5 M H-89 and for 10 additional min with 10⫺5 M PGE2 and stimulated with 100 nM fMLP for the times indicated. The cell lysates were analyzed as described in Materials and Methods. The membranes were probed with antiphospho-Ser473 Akt, antiphospho-Thr308 Akt, or anti-Akt antibodies. The immunoblots shown are representative of four independent experiments.

To examine the role of PKA in the inhibition of Akt translocation by PGE2, we performed the translocation assays in the presence of the PKA inhibitor, H-89. As shown in Figure 3B, the inhibitory effect of PGE2 and of the EP2 agonist CAY10399 was abolished almost completely when PMNs were preincubated with H-89 (Lanes 6 vs. 9 and Lanes 7 vs. 10). This latter result indicates that PKA is involved in the decreased translocation of Akt to membranes induced by PGE2 via the EP2 receptor.

Effect of PGE2 on the p38-MAPKAPK2-Akt pathway MAPKAPK-2, a p38 substrate, has been shown to phosphorylate Akt on Ser473 in human PMNs in a PI-3K-dependent

manner and therefore, to fulfill the PDK2 function in this cell type [15]. We have thus examined the effect of PGE2 and the EP2 agonist CAY10399 on the phosphorylation of MAPKAPK-2 on Thr222 and Thr334 mediated by p38 and the phosphorylation of p38 as well. Our data show that fMLP induced the phosphorylation of p38 and of MAPKAPK-2 on Thr222 and Thr334 and that none of these phosphorylations were affected by PGE2 (Fig. 4). Thus, PGE2 inhibited the fMLP-stimulated PI-3K␥ activity and subsequently, the formation of PI(3,4,5)P3 but affected neither the phosphorylation of p38 nor that of its substrate MAPKAPK-2. These latter results are similar to those observed with the phophorylation of Akt in the presence of PGE2.

Fig. 3. Effect of PGE2 on fMLP-induced Akt and PDK1 translocation. (A) PMNs were preincubated for 10 min with 10⫺5 M PGE2 and stimulated with 100 nM fMLP for 30 s. (B) PMNs were preincubated for 5 min with 10 ␮M H-89 and for 10 additional min with 10⫺6 M PGE2 or CAY10399 and then stimulated with 100 nM fMLP for 30 s. The membrane fractions were prepared as described in Materials and Methods and were analyzed by immunoblotting with anti-Akt or anti-PDK1 antibodies and antiflotillin-1 antibody as a control for total protein loading. Densitometric analyses are expressed as percentages of fMLP-stimulated controls and shown as means of three immunoblots obtained from three independent experiments.

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Fig. 4. Effect of PGE2 and CAY10399 on p38 and MAPKAPK-2 phosphorylation. Neutrophils were preincubated for 10 min with 10⫺5 M PGE2 or 10⫺5 M CAY10399 and stimulated with 100 nM fMLP for the times indicated. The cell lysates were analyzed as described in Materials and Methods. (A) The membranes were probed with antiphospho-p38 and anti-p38 antibodies. (B) The membranes were probed with antiphospho-MAPKAPK-2 Thr 222, antiphospho-MAPKAPK-2 Thr334, or anti-MAPKAPK-2 antibodies. The immunoblots shown are representative of three independent experiments.

Effect of cAMP-elevating agents on the translocation of p110␥, Akt, and PDK1 induced by fMLP All agents that elevate intracellular cAMP (forskolin, permeable cAMP analogs, phosphodiesterase inhibitors, Ado) share the ability to inhibit PMN functions stimulated by fMLP as well as to delay apoptosis. The inhibitory effect of endogenous Ado on the fMLP-stimulated phospholipase D (PLD) pathway in human PMNs is mediated via the A2A receptors, which stimulate an increase of intracellular cAMP [24]. We thus wondered if these agents have the same effect as PGE2 on the PI-3K␥-Akt signaling pathway. First, the effect of Ado and of the adenylyl cyclase activator forskolin were tested on the translocation to membranes of p110␥, PDK1, and Akt in fMLP-stimulated PMNs. Cells were preincubated with ADA to suppress the inhibitory effect of Ado, with ADA and the A2A receptor agonist CGS-21680, the A2A receptor antagonist CGS-15943, or with forskolin prior stimulation with fMLP. When 0.1 U/ml ADA was added to cell suspensions, the amounts of membrane-associated p110␥, PDK1, and Akt were increased markedly as compared with the fMLP-stimulated control (Fig. 5, Lanes 6 and 7), thereby suggesting an inhibitory role for endogenously released Ado in the fMLP-induced recruitment to membranes of these kinases. Conversely, in the presence of ADA and the A2A receptor agonist (CGS-21680), the amounts of p110␥, PDK1, and Akt translocated to membranes were decreased considerably (Fig. 5, Lane 8, as compared with Lane 7) and were com-

parable with those observed in the absence of ADA (i.e., in the presence of Ado in the suspensions, Lane 6). Similarly to the addition of ADA to the PMN suspensions, the A2A receptor antagonist CGS-15943 increased the recruitment of p110␥, PDK1, and Akt to the membranes (Lane 9, as compared with Lane 6). Taken together, these results indicate that Ado, acting via A2A receptors, dramatically diminishes the fMLP-induced translocation of p110␥, PDK1, and Akt to the membranes. Forskolin, a direct activator of adenylyl cyclase, which elevates intracellular levels of cAMP, also decreased the amounts of membrane-associated p110␥, PDK1, and Akt (Lane 10).

fMLP-induced Akt activation is maintained in the presence of cAMP-elevating agents As Ado and forskolin negatively regulate the translocation of p110␥, Akt, and PDK1 induced by fMLP, we wondered if these substances were also able to modulate Akt phosphorylation. PMNs were incubated with ADA alone or with ADA and CGS-21680 or with forskolin prior to stimulation with fMLP. Neither the degradation of extracellular Ado by ADA nor the binding of the selective agonist CGS-21680 to the A2A receptor modified the phosphorylation of Akt on Ser473 or Thr308 (Fig. 6). Similarly, forskolin did not affect the phosphorylation of Akt. Thus, as is the case for PGE2, cAMP-elevating agents inhibit the translocation of p110␥, Akt, and PDK1 to membranes but not the phosphorylation of Akt.


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Fig. 5. Effect of cAMP-elevating agents on fMLP-induced p110␥ translocation. PMNs were preincubated for 10 min with ADA alone (0.1 U/ml) or ADA plus CGS-21680 (10⫺6 M), CGS-15943 (10⫺6 M), or forskolin (10⫺5 M) prior to stimulation with 100 nM fMLP. The membrane fractions were prepared as described in Materials and Methods and were analyzed by immunoblotting with anti-p110␥, anti-Akt, or anti-PDK1 antibodies. Antiflotillin-1 immunoblot is shown as a control for total protein loading. Densitometric analyses are expressed as percentages of fMLP-stimulated samples and are shown as means of three immunoblots obtained from three independent experiments.

Effect of PGE2 and of the A2A receptor agonist on Akt kinase activity The data presented above suggest that in the presence of cAMP-elevating agonists, such as PGE2 or Ado, Akt is phosphorylated on Thr308 and Ser473 in response to fMLP, although the mechanism of translocation is impaired severely by these agonists. As the phosphorylation of Akt on both residues is considered as its main mechanism of activation, the amount of phosphorylated Akt is often presented as an indicator of Akt activity in cells. We thus wondered whether in the presence of cAMP-elevating agonists, the kinase activity of Akt was cor-

related with its phosphorylation. Using the Akt kinase assay kit, Akt was immunoprecipitated in PMNs preincubated with PGE2 or CGS-21680 and stimulated with fMLP (30 s). The kinase activity of Akt was monitored using the phosphorylation of a GSK-3 fusion protein, an exogenous substrate. The amount of phosphorylated GSK-3 was measured by immunoblotting with an antiphospho-GSK-3␣/␤ (Ser21/9) antibody. The results of these experiments, illustrated in Figure 7, show that Akt, immunoprecipitated from fMLP-stimulated but not from control PMNs, strongly phosphorylates GSK-3. PGE2 and CGS-21680 slightly decrease the amounts of GSK-3 fusion protein phos-

Fig. 6. Effect of cAMP-elevating agents on fMLP-induced Akt phophorylation. PMNs were preincubated for 10 min with ADA alone (0.1 U/ml) or ADA plus CGS-21680 (10⫺6 M) or forskolin (10⫺5 M) prior to stimulation with 100 nM fMLP. The cell lysates were analyzed as described in Materials and Methods. Membranes were probed with antiphospho-Ser473 Akt, antiphospho-Thr308 Akt, or anti-Akt antibodies. The data shown are representative of three independent experiments.

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PI-3K␦ activity could be involved in the phosphorylation of Akt and compensate for the inhibition of PI-3K␥ in the presence of PGE2. To address this question, PMNs were preincubated with IC87114 prior to stimulation by fMLP in the presence or absence of PGE2, and the amounts of phosphorylated Akt were determined by immunoblotting and densitometric analyses. The results of these studies are depicted in Figure 8. As shown above (Fig. 1), stimulation of PMNs by fMLP led to a rapid increase in the phosphorylation levels of Akt (on Thr308 and Ser473). This response was little affected by PGE2. However, here again, the phosphorylation of Akt in the presence of PGE2 seemed to decrease somewhat faster than in fMLP-stimulated, control cells. Preincubation of cells with the PI-3K␦ inhibitor (IC87114) did not decrease the stimulation of the phosphorylation of Akt, although like PGE2, it led to a more rapid decrease in the level of phosphorylation than in fMLPstimulated control cells (most evident at the 5-min time-point; P⫽0.03, Lanes 4 vs. 12 for Thr308 phosphorylation), an effect that was additive with that of PGE2 (P⫽0.0132, Lanes 8 vs. 16). These data indicate that PI-3K␦ is not involved directly in the initiation of the fMLP-induced stimulation of the phosphorylation of Akt but that it plays a role in the late phase of the latter (i.e., the phosphorylation observed at 5 min of stimulation), and PI-3K␦ is, at least in part, involved in the maintenance of the Ser473 phosphorylation after 5 min of stimulation by fMLP in the presence of PGE2.

Fig. 7. Enzymatic activity of Akt in fMLP-stimulated PMNs. PMN suspensions (2⫻107 cell/ml) were preincubated with 0.1 U/ml ADA (5 min), with 10 uM H-89 for an additional 5 min, and with 10⫺5 M PGE2 or CGS-21680 for 10 min. After stimulation with 100 nM fMLP, Akt was immunoprecipitated in cell lyates as described in Materials and Methods. The Akt kinase assay was carried out for 25 min using a GSK-3 fusion protein as substrate. Proteins were separated with SDS-PAGE, and the level of GSK-3 fusion protein was monitored using an antiphospho-GSK-3 antibody. Membranes were also probed with an anti-Akt antibody to show the amounts of immunoprecipitated (IP) Akt. Densitometric analyses of the phospho-GSK-3 immunoblots are shown. The data shown are representative of two independent experiments. NS, Not significant.

phorylated by immunoprecipitated Akt from fMLP-stimulated PMNs. To examine the role of PKA in the fMLP-stimulated Akt activity in the presence of PGE2, PMNs were also preincubated with H-89 prior to addition of PGE2 to cell suspensions and stimulation. In these experimental conditions, we observed a slight increase in the amounts of phosphorylated GSK-3 fusion protein, whether the PMNs were incubated with PGE2 or not. Taken altogether, these data indicate that PGE2 and CGS21680 diminished Akt activity only slightly and that PKA slightly down-regulates Akt activity stimulated by fMLP.

Effect of a PI-3K␦-specific inhibitor on the fMLPinduced phosphorylation of Akt in the presence or the absence of PGE2 A recently developed, selective inhibitor of the PI-3K␦ isoform (IC87114) has been shown to decrease PI(3,4,5)P3 formation and to partially inhibit several functions stimulated by fMLP in human PMNs [25, 26]. We thus wondered if fMLP-stimulated

DISCUSSION In a previous study, we showed that the main signaling mechanism accounting for the inhibitory effect of PGE2 on the fMLP signaling in PMNs was the inhibition of the PI-3K␥ activity via the EP2 receptor [16]. As a consequence of the decreased amount of PI(3,4,5)P3 produced, we observed that the recruitment to membranes of several factors that play a major role in the activation of PLD, i.e., Arf and Rho GTPases, PKC␣, and Tec kinase, was reduced dramatically. Given the high dependency of Akt activation on PI-3K, we expected that in the presence of PGE2, which inhibits fMLP-induced PI-3K␥ activation and the formation of its product PI(3,4,5)P3, the translocation of Akt would be decreased greatly and that subsequently, the activation of Akt through phosphorylation would be inhibited, at least partially. Quite unexpectedly and to the contrary, we found that the fMLP-induced phosphorylation of Akt on Ser473 and Thr308 was not decreased significantly by PGE2. Only minor changes in the kinetics of Akt phosphorylation were observed. Nevertheless and as expected, the translocation of Akt to membranes was diminished dramatically in the presence of PGE2 or the EP2 receptor agonist CAY10399. However, whereas Akt was phosphorylated fully in the presence of PGE2, its activity stimulated by fMLP was reduced slightly. Therefore, it seems that in the presence of PGE2, an unusual mode of activation of Akt is induced, characterized by a dissociation between the translocation and the phosphorylation of Akt on Ser473 and Thr308. In PMNs, Akt Ser473 is phosphorylated by MAPKAPK-2, a substrate of p38, which is activated by phosphorylation on Thr222 and Thr334. The phosphorylation of Akt on Ser473 by


7

Fig. 8. Effect of IC87114 on fMLP-induced Akt phosphorylation in the presence of PGE2. PMN suspensions containing 0.1 U/ml ADA were preincubated for 10 min with 10 ␮M IC87114 or 100 nM wortmannin (Wort) and for 10 additional min with 10⫺5 M PGE2 prior to stimulation with 100 nM fMLP for the times indicated. Cell lysates were analyzed as described in Materials and Methods. (A) The membranes were probed with antiphospho-Ser473 Akt, antiphospho-Thr308 Akt, or anti-Akt antibodies. (B) Densitometric analyses of the immunoblots are shown. The data shown are representative of five independent experiments.

MAPKAPK-2 as well as the phosphorylation of p38 have been reported to be inhibited by wortmannin and thus, to depend on PI-3K activation [15]. Nevertheless, we observed that the phosphorylation of Akt on Ser473 is not affected when the fMLPinduced PI-3K␥ activity is inhibited by PGE2. The persistence of this phosphorylation is supported by the evidence that the phosphorylation of p38 and MAPKAPK-2 stimulated by fMLP is similarly not inhibited in the presence of PGE2. The interconnection between the MAPK and the cAMP signaling pathways may vary considerably in a cell- and stimulus-dependent manner [27, 28]. In fMLP-stimulated PMNs, we observed no inhibitory effect of PGE2 on the MAPK pathway, and this result was confirmed in our laboratory by the fact that other cAMPelevating agents, such as the A2A receptor agonist CGS-21680, did not affect the phosphorylation of p38 in TNF-␣-primed and fMLP-stimulated PMNs [29]. Although the phosphorylation of Akt by MAPKAPK-2 has been reported to depend on PI(3,4,5)P3 formation [15], p38 and MAPKAPK-2 are cytosolic kinases, which do not possess PH domain and need not relocate to the plasma membrane to be activated. We could therefore hypothesize that the p38/MAPKAPK-2/Akt phosphorylation cascade could occur in the cytosol in the presence of 8


PGE2. An argument in favor of this hypothesis relies on the observation that Ser473 phosphorylation depends on PI-3K activity but in a manner that does not involve Akt membrane localization [30, 31]. The phosphoinositides generated by PI-3K bring Akt and PDK1 in close proximity at the plasma membrane and thus, allow Akt phosphorylation on Thr308 by PDK1 [22, 23, 32]. In this study, we report that although the formation of PI(3,4,5)P3 is decreased significantly, and in turn, the relocalization at the membrane of Akt and PDK1 is impaired in the presence of PGE2, the phosphorylation of Akt on Thr308 is not affected. This suggests that PGE2 renders the fMLP-stimulated activation of Akt independent of PI-3K␥ and promotes a different pattern of activation in which Thr308 phosphorylation does not require the translocation of PDK1 and Akt. Indeed, several alternative mechanisms of Akt activation, which are independent of PI-3K and PDK1, have been described [33, 34]. Amongst them, PKA has been reported to stimulate Akt activity in a PI-3K-independent manner and through a mechanism that results in Akt phosphorylation on Thr308. As PGE2 stimulates the cAMP/PKA pathway via EP2 receptors, we hypothesized that PKA could promote Thr308 Akt phosphorylation. http://www.jleukbio.org

However, the PKA inhibitor H-89 had no effect on Akt phosphorylation on Thr308 or on Ser473. Therefore, PKA cannot be considered as a mediator of Akt phosphorylation in our model. Nevertheless, we showed that the inhibition of Akt translocation by PGE2 is mediated by PKA in fMLP-stimulated PMNs, as is the case for p110␥ (C. Burelout, unpublished data). Moreover, PKA is also involved in the slight down-regulation of fMLP-stimulated Akt activity observed in the presence of PGE2. A direct effect of PKA on Akt cannot be considerered as a mechanism of regulation, as Akt is not phosphorylated by PKA in vitro or in cells [20, 21]. Although PKA affects both events (the translocation of Akt and the regulation of its activity), we observed that the magnitude of the inhibitory effect was much higher on Akt translocation than on Akt activity (⬃50% and 10 –20% of the fMLP-induced responses, respectively). One possible role of PKA in the regulation of Akt activity by PGE2 could reside in its capacity to inhibit PI-3K␥ and Akt translocation and hence, the binding of phosphoinositides to the Akt PH domain, as it has been reported that this binding could result in a conformational change that influences Akt activity [35, 36]. The inhibition of Akt by the cAMP/PKA pathway has already been reported elsewhere [37, 38]. These studies showed that this inhibitory pathway affected upstream regulators of Akt, such as PI-3K and PDK1, and resulted in a concomitant decrease in the levels of Akt phosphorylation and activity. The inhibitory mechanism involved in our experimental model is therefore different, as the inhibition of the translocation of Akt mediated by PKA is not coupled to the inhibition of its phosphorylation Another putative kinase, which could be involved is the Ca2⫹/calmodulin-dependent protein kinase kinase (CaM-KK), which has been shown to promote cell survival by phosphorylating Akt on Thr308 [34]. We cannot exclude that this signaling pathway could account, at least in part, for Thr308 phosphorylation, as CaM-KK is expressed in human PMNs, and the calmodulin inhibitor W7 diminishes the phosphorylation of Akt on Thr308 and Ser473 [39], and PGE2 decreases only the late influx of calcium triggered by fMLP [16, 40]. Akt activity stimulated by growth factors can also be regulated by tyrosine phosphorylation on Tyr474 [41] or on Tyr315 by Src [42, 43]. These tyrosine-phosphorylation events have no major impact on the serine/threonine phosphorylation of Akt but require PI-3K activation. A regulation of Akt activity by tyrosine phosphorylation is thus not likely to take place in the presence of PGE2, as PI-3K␥ is inhibited. The fact that PGE2 suppressed the translocation of Akt induced by fMLP but not its phosphorylation also suggests that Akt could be activated by PDK1, directly in the cytosol. PDK1 is a cytosolic kinase, which shows a high constitutive activity and is able to phosphorylate most of its substrates (such as PKCs or p70S6K) in the cytosol, independently of PI-3K activity [44]. Therefore, we can envision a possible molecular mechanism induced by PGE2, which would permit the formation of a signaling complex, enabling the interaction of Akt with PDK1 (or another, as-yet-unidentified kinase) in the cytosol. It has been reported already that in heat-shocked cells, Akt can be activated through the formation of a protein complex in a PI-3K-independent manner [45]. Several Akt binding partners or adaptor proteins, which can modulate Akt or PDK1

activity, have been described [46]. Amongst them, TCL1, an oncoprotein that activates Akt by oligomerization, the heatshock proteins Hsp27 and Hsp90, or the more recently discovered ArgBP2␥ [46] are putative candidates, which could be involved in a possible mechanism to be explored in further detail. Another point, which needs to be clarified, is the relative contribution of the different PI-3K isoforms to the fMLPinduced Akt activation pathway. The generation of PI-3K␥deficient mice has defined a predominant role for this isoform in the polarization and the directional migration of murine PMNs in a fMLP gradient [47– 49]. Another consequence of the genetic deletion of PI-3K␥ was the partial [50] or almost complete disappearance [47] of the Ser473 Akt phosphorylation. In human PMNs, PI-3K␥ (p101/p110␥) is activated rapidly in response to fMLP [17], and the first burst of PI(3,4,5)P3 produced allows the activation of the class Ia p110␦, which is associated with the p85 regulatory subunit [25]. The recent development of pharmacologic inhibitors selective for the various PI-3K isoforms has brought experimental evidence for the involvement of PI-3K␦ in several functional responses (i.e., chemotaxis, superoxide anion production, cytotoxic enzyme release) stimulated with fMLP in human PMNs [26]. Our data, obtained with the PI-3K␦-selective inhibitor (IC87114), suggest that this isoform modulates only the late phosphorylation of Akt (5 min of stimulation by fMLP) and could also partially account for the persistence of Ser473 Akt phosphorylation (5 min), observed in the presence of PGE2. However, PI-3K␦ does not seem to be involved in the early steps of phosphorylation of Akt stimulated by fMLP or in the maintenance of Akt phosphorylation in the presence of PGE2 (1 min of stimulation by fMLP). These data are consistent with the model proposed by Sadhu et al. according to which PI-3K␦ is involved in the amplification loop regulating the formation of PI(3,4,5)P3 but only in late signaling events. This latter result also suggests that the alternative mechanism of Akt activation observed in the presence of PGE2 is mainly independent of PI-3K activation. Several studies have reported that Akt activity can be stimulated by a mechanism independent of PI-3K activation in stress-stimulated cells, therefore strengthening the pertinence of our observations [45, 51] It is of interest to note that PGE2 alone has been shown to induce the phosphorylation of Akt in various cell types including DCs [52–55]. This increased phosphorylation of Akt was always related to the activation of PI-3K and in some cases, was mediated via EP2 receptors [54, 55]. Nevertheless, these findings do not apply to our model, as PGE2 alone was not able to promote the phosphorylation of Akt in PMNs. cAMP is the major second messenger formed after EP2 receptor stimulation. We thus wondered whether Ado, which stimulates cAMP formation via A2A receptors [56, 57], and the adenylyl cyclase activator forskolin affected the PI-3K␥/Akt pathway in a manner similar to PGE2. Our data indicate that the elevation of intracellular cAMP induced by various stimuli inhibited the translocation of p110␥ as well as Akt and PDK1 to membranes. However, the phosphorylation of Akt was not suppressed in the presence of these cAMP-elevating agents. Therefore, these results suggest that the elevation of intracellular cAMP promotes a mechanism of activation of Akt, which


9

Fig. 9. Model of Akt signaling pathway in the presence of PGE2. (A) fMLP stimulates the formation of PI(3,4,5)P3 (PIP3) by PI-3K␥ and the translocation of PDK1 and Akt, which allows the phosphorylation of Akt on Thr308 (T.308) by PDK1 and on Ser473 (S.473) by MAPKAPK-2 at the plasma membrane. PIP2, PI(3,4)P2. (B) In the presence of PGE2 (or other cAMPelevating agents), the translocation of PI-3K␥ is inhibited and as a result of the reduced formation of PI(3,4,5)P3, that of Akt and PDK1 as well. However, the phosphorylation of Akt on Thr308 and Ser473 triggered by fMLP is not inhibited by PGE2 and hence, occurs in the cytosol. The activation of PKA by PGE2 mediates the inhibition of p110␥, Akt, and PDK1 translocation but does not influence Akt phosphorylation.

does not require a translocation step in fMLP-stimulated PMNs. However, as is the case with PGE2, the A2a agonist reduced Akt activity slightly, further suggesting a common mechanism of activation of Akt mediated through EP2 or A2A receptors. A physiological role for the persistence of Akt phosphorylation in the presence of PGE2 remains to be found. On the one hand, cAMP-elevating agents delay apoptosis in PMNs [7, 58, 59]. Conversely, the antiapoptotic role of Akt in various cellular types is well established. Therefore, it is tempting to speculate that the antiapoptotic function of PGE2 and other cAMP-elevating agents could be mediated through the alternative mode of Akt activation reported in this study. Nevertheless, such a conclusion could be erroneous, as fMLP (or IFN-␤) stimulates Akt activity without delaying apoptosis in PMNs, indicating that the fMLP-induced activation of Akt is not linked to a prosurvival response [60], and we have verified that PGE2 did not affect cell viability significantly when PMNs were stimulated with fMLP (unpublished data). Amongst the known Akt substrates, p47phox (a NADPH complex component) [14, 61], GSK-3 [62], and the FOXO transcription factors [63] have been shown to be phosphorylated in response to fMLP in PMNs. Using a specific Akt inhibitor, we found that GSK-3 is not a substrate of Akt in fMLP-stimulated PMNs (data not shown). As Akt activity stimulated by fMLP seems to be involved primarily in the respiratory burst activity and in chemotaxis in PMNs [64], the exposure of cells to PGE2 or Ado could help maintain these cellular functions at a minimal level. In summary, our data show that although PI(3,4,5)P3 formation by fMLP-activated PI-3K␥ is decreased in the presence of PGE2 or other cAMP-elevating agents, Akt phosphorylation is not affected, whereas its activity is down-regulated slightly. In this experimental model, fMLP-induced Akt phosphorylation does not require its translocation to membranes, thereby suggesting that cAMP-elevating agents alter compartmentalization of fMLP-induced Akt signaling. Taken together, these observations lead us to hypothesize that cAMP-elevating agents induce an alternative mechanism for Akt activation in PMNs stimulated with fMLP. This mechanism is represented schematically in Figure 9 and needs further characterization. 10


ACKNOWLEDGMENTS This work was supported by grants from the Canadian Institutes of Health Research (CIHR). We thank Dr. Tom Crabbe from UCB (Slough, UK) for kindly supplying the PI-3K␦selective inhibitor IC87114. We are grateful to Drs. Nicolas Flamand and Caroline Gilbert for their helpful comments.

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