Neutrophil activation by fMLP regulates FOXO (forkhead) transcription factors by multiple pathways, one of which includes the binding of FOXO to the survival factor Mcl-1

Neutrophil activation by fMLP regulates FOXO (forkhead)transcription factors by multiple pathways, one of whichincludes the binding of FOXO to the survival factor Mcl-1

Lisa J. Crossley1

Center For Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and PainMedicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts

Abstract: Activation signals from bacterial stim-uli set into motion a series of events that alter theabbreviated lifespan of neutrophils. These studiesshow that the bacterial chemoattractant, formyl-Met-Leu-Phe (fMLP), promotes the phosphoryla-tion/inactivation of the FOXO subfamily of fork-head transcription factors (FKHR, FKHR-L1, andAFX) through the phosphatidylinositol-3-kinase/Akt (protein kinase B) and the RAS mitogen-acti-vated protein kinase pathways. Furthermore,fMLP stimulation causes the inducible expressionof the prosurvival Bcl-2 family member Mcl-1,which then binds to a complex containing FKHR.These studies show that fMLP-stimulated neutro-phils coordinate the regulation of FOXO transcrip-tion factors and the survival factor Mcl-1, a mech-anism that may allow neutrophils to alter theirsurvival. J. Leukoc. Biol. 74: 583–592; 2003.

Key Words: phosphatidylinositol-3-kinase (PI-3K) � Akt � mito-gen-activated protein kinase (MAPK) signaling � Rsk

INTRODUCTION

Neutrophils are first responders in an organism’s rapid assaulton infectious pathogens. Through genetically conserved recep-tors, neutrophils recognize chemoattractants, lipid products,and the molecular patterns present on the surface of bacteria,viruses, and fungi [1]. The formyl peptide, formyl-Met-Leu-Phe(fMLP), is an example of a bacterial product that is recognizedby neutrophils. fMLP, upon binding to its heterotrimeric Gprotein-coupled receptor, initiates signaling cascades that ac-tivate multiple pathways [2]. These pathways include the mi-togen-activated protein kinase (MAPK) and phosphatidylino-sitol 3-kinase (PI-3K) cascades, which are important for thedevelopment of the functional responses of neutrophils ininflammation (e.g., the respiratory burst, transmigration, andphagocytosis) [3–6].

Lipopolysaccharide (LPS) and granulocyte macrophage-col-ony stimulating factor (GM-CSF) are inflammatory stimuli thathave been shown to inhibit neutrophil apoptosis, whereas thesurvival of neutrophils in response to chemoattractants (e.g.,fMLP or C5a) has not been established [7–9]. A recent studyhas suggested that signals arising from fMLP or C5a may

predominate in directing the responses of neutrophils that havemigrated to the final site of an infection [10]. fMLP and LPSactivate different receptors within neutrophils. The activationof heterotrimeric G protein-coupled receptors by fMLP versusthe activation of Toll receptors by LPS [11, 12] will most likelyresult in different nuclear signaling responses. These studiesreveal a mechanism through which neutrophils present in sitesof inflammation respond to fMLP activation of G protein-coupled receptors via signaling responses that regulate a familyof nuclear transcription factors shown to function in pathwaysthat affect survival.

A pathway that regulates the survival of many cells is theMAPK cascade. fMLP-mediated activation of MAPK (e.g.,p42/p44 MAPK or the stress-activated kinases p38 MAPK andc-jun NH2-terminal kinase) within neutrophils leads to a cas-cade of events. Signaling through p42/p44 MAPK is propa-gated through the family of p90 kDa ribosomal S6 kinases (p90Rsk) [13, 14], which undergoes phosphorylation and thenactivates transcription factors [e.g., cyclic AMP response ele-ment-binding protein (CREB), inhibitor of �B�/nuclear factor-�B, c-fos] through phosphorylation [15–17]. p90 RSK exists inthree isoforms, Rsk-1, -2, and -3. The absence of Rsk-2 inhumans causes a severe neurologic disease, the Coffin-Lowrysyndrome, and alters glycogen metabolism in the muscle ofknockout mice [18, 19]. Additionally, I have previously shownthat in neutrophils, Rsk-2 phosphorylates and inactivates gly-cogen synthase kinase 3, an event that may also improve theneutrophil’s survival [20, 21]. Rsk-2 not only inactivates pro-apoptotic substrates but is involved in the up-regulation ofprosurvival Bcl-2 family members through its phosphorylationand activation of CREB [22–24].

fMLP stimulation also activates PI-3K [25], which thenleads to the activation of the serine/threonine kinase, Akt, alsocalled protein kinase B (PKB) [26]. Once activated by phos-phorylation on two residues [serine (Ser) 473, threonine (Thr)308], Akt translocates into the nucleus and phosphorylates itssubstrates [27]. Akt’s effect on cell-survival responses is me-

1 Correspondence: Center For Experimental Therapeutics and ReperfusionInjury, Department of Anesthesiology, Perioperative and Pain Medicine, Brighamand Women’s Hospital, Harvard Medical School, Thorn Building, Room 703, 75Francis Street, Boston, MA 02115. E-mail: [email protected]

Received January 15, 2003; revised May 15, 2003; accepted May 27, 2003;doi: 10.1189/jlb.0103020.

Journal of Leukocyte Biology Volume 74, October 2003 583

diated by the regulation of the FOXO subfamily of forkheadtranscription factors, which are conserved in nematodes andmammals (for reviews, see refs. [28, 29]).

In the nematode, the FOXO transcription factor Daf-16, wasfound to function in a pathway that regulates the lifespan of theworm [30]. In mammals, there exist three Daf-16 homologousproteins, FOXO1 [Forkhead (FKHR)], FOXO3a [FKHR-like 1(FKHR-L1)], and FOXO4 [ALL1 fused gene from chromosomeX (AFX)]. The FOXO proteins are characterized by the pres-ence of a 110-amino acid DNA-binding domain that forms awinged helix structure [31]. FOXO transcription factors areinvolved in the regulation of the immune system. Studiessuggest that FOXO may affect cell-cycle progression and thesurvival of hematopoietic cell lineages through the regulationof the cyclin-dependent kinase inhibitor p27kip1 and of Bcl-2family members [32]. Recently, FOXO proteins have beenshown to regulate the expression of the tumor necrosis factor(TNF)-related apoptosis-inducing ligand (TRAIL) [33], a TNFfamily member that can accelerate the rate of apoptosis inneutrophils [34].

Few studies have examined the regulation of FOXO tran-scription factors in activated neutrophils. The delineation ofthe regulation of FOXO in other cells has revealed that phos-phorylation of FOXO through PI-3K facilitates cellular survival[26, 28]. Additionally, in a mouse model of endotoxemia, theregulation of several transcriptional factors by PI-3K-mediatedphosphorylation was shown to critically regulate the rate ofneutrophil apoptosis [35]. Therefore, I hypothesized that theactivation of PI-3K signaling by fMLP in neutrophils wouldalso mediate the phosphorylation of FKHR as a mechanismthrough which neutrophils might regulate their survival. InfMLP-stimulated neutrophils, I found that the FOXO transcrip-tion factors undergo phosphorylation. Using selective chemicalinhibitors of the MAPK and PI-3K pathways, I determined thatFOXO is a target of Rsk and Akt activation in fMLP-stimulatedneutrophils.

Mcl-1, a Bcl-2 family member [36], was shown to have animportant role in mediating the increased survival of neutro-phils and myeloid cells activated by cytokines [37, 38]. Addi-tionally, Mcl-1 is a point of convergence for the PI-3K/AKTsignaling pathway and activated CREB [24]. We found that inthe ex vivo model of elicited neutrophils, Mcl-1 was bound toa complex containing FKHR.

We propose that fMLP stimulation affected the regulation ofthe FOXO transcription factors and the survival factor Mcl-1.Understanding the regulation of FOXO and Mcl-1 within neu-trophils may provide insight into how these cells regulate theirsurvival when responding to inflammatory signals.

MATERIALS AND METHODS

Reagents

fMLP, protein A 4� agarose beads, glutathione-linked agarose beads, dimethylsulfoxide (DMSO), and other chemicals and reagents were purchased fromSigma Chemical Co. (St. Louis, MO) unless otherwise noted. The inhibitorsPD90859, U1026, U1024, LY294002, and wortmannin were purchased fromCalbiochem (San Diego, CA). Total FKHR, phospho-FKHR (Ser 256), phos-pho-FKHR (Thr24)/FKHR-L1 (Thr32), phospho-AKT (Ser473), and phospho-

Rsk (Ser 380) rabbit polyclonal antibodies raised to the human proteins werepurchased from Cell Signaling Technology (CST; Beverly, MA). Total AKT waspurchased from CST and Promega (Madison, WI). Goat polyclonal Rsk-2(C-19) was from Santa Cruz Biotechnology (Santa Cruz, CA). Mcl-1 monoclonalantibodies (mAb) raised to the human protein were obtained from TransductionLaboratories (Lexington, KY) and Oncogene Research Products (Boston, MA).The glutathionine S-transferase (GST) polyclonal antibody was purchased fromUpstate (Charlottesville, VA). The GST–FKHR-L1 fusion proteins and GSTprotein were kind gifts from the laboratory of Dr. Michael Greenberg (Chil-dren’s Hospital, Boston, MA).

Preparation of guinea pig peritoneal neutrophils

Guinea pig peritoneal neutrophils were freshly prepared using the publishedmethod of Badwey and Karnovsky [39]. Briefly, guinea pig neutrophils wereharvested 18 h after intraperitoneal injection of 30 ml 12% casein (w/v) in0.9% NaCl, and the peritoneal cavity was washed twice with 0.9% NaCl. Theperitoneal cells were gently pelleted, and the erythrocytes were lysed inhypertonic saline. The neutrophils were washed and resuspended in 0.9%NaCl. The cells were kept on ice and used within 2–3 h. This method typicallyyielded �1 � 109 cells that are �90% polymorphonuclear leukocytes.

Human neutrophil isolation

Neutrophils were freshly isolated from the whole blood of human volunteersusing the method of Colgan et al. [40]. Briefly, blood obtained by venipuncturewas anticoagulated with acid-citrate-dextrose, purified by aspiration of thebuffy coat after centrifugation (400 g for 20 min at 25°C) to remove plasma andmononuclear cells. Erythrocytes were removed by a 2% gelatin gradient-sedimentation protocol followed by lysis of remaining erythrocytes in coldNH4Cl buffer. The cells were more than 90% neutrophils and were used within4 h of isolation.

Cell stimulation and lysis

Neutrophils (1.5�107) were suspended in a modified Dulbecco’s phospate-buffered saline (PBS) medium [135 mM NaCl, 2.7 mM KCl, 16.2 mMNa2HPO4, 1.47 mM KH2PO4, containing 0.9 mM CaCl2, 0.5 mM MgCl2 (pH7.35), containing 7.5 mM D-glucose] for 10 min at 37°C. Stimulation withfMLP (1.0 �M) was for various times. For experiments evaluating the involve-ment of signaling pathways for which specific chemical inhibitors were avail-able, the inhibitors were preincubated with the neutrophils for 30 min beforethe addition of stimuli.

After the completion of reactions, the cells were lysed by the addition of 5�sodium dodecyl sulfate (SDS) lysis buffer (125 mM Tris-HCl, pH 7.5, 5% SDS,10 mM EGTA, 50% glycerol, 5 mM sodium pyrophosphate, 5 mM sodiumfluoride, 0.5% bromophenol blue). The following reagents were freshly addedto the SDS lysis buffer: 10% ß-mercaptoethanol, 2 mM phenylmethylsulfonylfluoride (PMSF), and 5 mM sodium orthovanadate. The samples were quicklymixed by vortexing, boiled for 5 min, centrifuged, and kept on ice until loadingonto gels.

SDS-polyacrylamide gel electrophoresis (PAGE)and immunoblotting

Equal aliquots of the samples were loaded onto polyacrylamide gels (rangingbetween 7.5 and 15% w/v), and the proteins were transferred to Immobilon-Pmembranes [41]. The membranes were blocked 1 h at room temperature with5% milk in 20 mM Tris-HCl (pH 7.4) containing 250 mM NaCl. The blockingbuffer was removed. The membranes were then incubated with one of thefollowing antibodies: phoshoFKHR256 (1/1000 dilution), Mcl-1 (1/1000),Rsk-2 (C-19; 1/500), GST (1/1000), or the control antibodies for these proteinsfor 1 h at room temperature or overnight at 4°C in Tris-buffered saline/Tween20 [TBST; 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% Tween-20(vol/vol)] and 5% (wt/vol) bovine serum albumin or milk as recommended bythe manufacturer. Following incubation with primary antibodies, the mem-branes were washed (3�10 min/wash) with TBST. Incubation with a secondaryAb, which was diluted in TBST with 5% milk, was conducted for 1 h; forpolyclonal antibodies, goat anti-rabbit immunoglobulin G (IgG)-horseradishperoxidase (HRP) conjugate, 1:10,000 dilution; for phosphoFKHR, 1:3000;total FKHR, 1:5000 dilution; for GST, 1:2000; for mAb, goat anti-mouseIgG-HRP conjugate; for Mcl-1, 1:2000 dilution. Membranes were washed for

584 Journal of Leukocyte Biology Volume 74, October 2003 http://www.jleukbio.org

40 min in TBST and rinsed with distilled and deionized water. The activity ofthe HRP was visualized by incubating the membranes for 20 min at roomtemperature in an enhanced chemiluminescence detection system (Pierce,Rockford, IL) followed by autoradiography.

Immunoprecipitation

Neutrophils (3�107 cells) were stimulated for various times and then lysed byincubation on ice for 15 min in a lysis buffer [50 mM Tris (pH 8.0), 1% NonidetP-40 (NP-40), 10% glycerol, 150 mM NaCl, 1 mM Na3VO4, 10 mM NaF, 1 mMEDTA] containing several protease inhibitors (5 �g/ml aprotinin, 5 �g/mlleupeptin, 2 �g/ml pepstatin, 2 mM/ml PMSF). The lysates were rotated at 4°Cfor 30 min, and the supernatents were collected after centrifugation. Thelysates were precleared with Protein A/G sepharose beads for 15 min, and thesupernatents were collected after centrifugation. For RSK and FKHR immu-noprecipitation, 4.0 �g RSK or FKHR antibody was added to the neutrophillysates and incubated 15 min on ice followed by 2 h rotating at 4°C. Theimmune complexes were then washed extensively. After washing extensivelyover 30 min, the complexes were loaded on SDS-PAGE and were subjected toprocedures described for immunoblotting.

Protein–protein association assays

Plasmids containing GST–FKHR-L1 fusion proteins or GST alone were inoc-ulated into BL21 Escherichia coli. The bacteria were grown overnight inselection media. The next day, the cultures were amplified, and proteinexpression was induced using 1 mM isopropylthiogalactoside for 4 h. Thecultures were collected by centrifugation, resuspended in cold PBS lysis buffercontaining 100 mM EDTA, 1% Triton X-100, and protease inhibitors (10�g/ml aprotinin, 1 mM PMSF). The lysates were sonicated on ice for 1 min andcollected by centrifugation at 16,000 g for 30 min. The fusion proteins wereincubated with glutathione agarose beads, washed extensively, and resus-pended in PBS lysis buffer containing 0.02% Na azide. The GST fusion proteincomplexes were used in pull-down assays using the methods described inimmunoprecipitation.

In vitro kinase assay

Cells (3�107) were stimulated for the times indicated. The cells were dis-rupted on ice for 15 min in lysis buffer [50 mM Tris (pH 8.0), 1% NP-40, 150mM NaCl, 5 mM EDTA, 10 �g/ml leupeptin, 5 �g/ml pepstatin, 10 �g/mlaprotinin, 2 mM Na orthovanadate, 10 mM NaF, 1 mM PMSF]. The lysateswere centrifuged at 4°C for 20 min at 11,000 g, and the supernatents werecollected. Rsk was immunoprecipitated from the neutrophil extracts with aRSK-2 goat polyclonal antibody (C-19; Santa Cruz Biotechnology), and themixture was incubated on a rotating platform for 1 h at 4°C. The RSKimmunoprecipitates were then complexed to Protein G sepharose (SigmaChemical Co.) for an additional 2 h at 4°C. The Rsk immune complex waspelleted and washed five times in kinase buffer [50 mM Hepes (pH 7.4), 20mM MgCl2, 5 mM NaF, 20 mM � glycerophosphate, 1 mM NaVO4]. For in vitrokinase assays, the RSK complex was resuspended in 40 �l kinase buffer andwarmed to 30°C for 15 min. Purified FKHR-L1 (1 �g) and 200 �M adenosine5�-triphosphate (5 �l) were added to the reaction, which was run for anadditional 30 min at 30°C. The reaction was terminated by the addition of anequal volume of SDS sample buffer and was boiled at 95°C for 5 min, and thesamples were collected after centrifugation. The proteins were resolved on a7.5% polyacrylamide gel and analyzed by Western blotting using an antibodythat recognized phosphospecific FKHR.

RESULTS

fMLP-stimulated neutrophils regulate targets ofPI-3K, which are implicated in cellular survival

AKT undergoes prolonged phosphorylation/activation inresponse to fMLP

We first assessed AKT phosphorylation on Ser 473 duringstimulation of neutrophils with fMLP. AKT phosphorylation onSer 473 was minimal in unstimulated cells (Fig. 1a, lanes a,

b, j, and k). Stimulation of cells with fMLP led to the phos-phorylation of AKT on Ser 473 within 15 s, and the maximalphosphorylation was observed between 0.5 and 3 min. AKTphosphorylation was sustained during the period of stimulationfor up to 45 min (Fig. 1a; and data not shown). Total Akt isshown in the bottom portion of Figure 1a and reveals lessprotein during the periods of peak phosphorylation of Akt (Fig.1a, lanes e–g and i). This may represent that the antibody isless sensitive to the phosphorylated form of Akt, or it may bea result of the process of stripping the membrane.

FOXO transcription factors undergo phosphorylation infMLP-stimulated neutrophils

FKHR is expressed in neutrophils and becomes phosphory-lated at Ser 256 with fMLP stimulation (Fig. 1b). In thisexperiment, phosphorylation of FKHR was detected at 15 s,and the peak of phosphorylation was observed between 0.5 and3 min. A sustained level of phosphorylation was observedthroughout the period of stimulation [Fig. 1b, lanes c–i, FKHR(Ser256)]. The phospho-specific FKHR (Ser 256) antibody alsoallowed the detection of other FOXO family members, phos-phorylated FOXO4 (AFX, arrow in upper portion) andFOXO3a (FKHR-L1, top, pentagon). The basal level of AFXphosphorylation was higher than that of the other isoforms, butAFX exhibited enhanced phosphorylation at 0.5–1 min ofstimulation. FKHR-L1 migrates at �100 kD (Fig. 1b, top,pentagon). FKHR-L1 is also present in neutrophils and be-comes phosphorylated upon fMLP stimulation of neutrophils.FKHR-L1, similar to FKHR, was phosphorylated on Ser 253within 15 s, and the phosphorylation was sustained above basallevels. To determine the specificity of the phosphospecificFKHR antibody in detecting other FOXO proteins by Westernanalysis, the presence of these proteins was compared inneutrophils of two different species, guinea pigs and humans(Fig. 1c). Neutrophils obtained from guinea pigs or humanswere stimulated for 3, 5, and 10 min or incubated in thepresence of the control (DMSO) for 10 min. fMLP stimulationled to intense phosphorylation of a 70–75 kD FKHR proteinduring 3–10 min of fMLP stimulation in guinea pig and humanneutrophils (Fig. 1c, arrow). With DMSO incubation, the phos-phorylation of FKHR was not present in human neutrophils,whereas basal activity was detected in the guinea pig neutro-phils. A hyperphosphorylated species of FKHR, which mi-grates at approxiamtely 81 kD, is also detected in the guineapig neutrophils. The hyperphosphorylated species was alsoseen in human neutrophil lysates in prolonged autoradio-graphic exposures of the data (data not shown). The 100-kDFKHR-L1 protein is detected in the guinea pig neutrophillysates. Two controls verify the presence of this protein: serum-treated NIH/3T3 mouse fibroblast cell line lysate and 293human kidney cell lysate, which was transfected withFKHR-L1 (Fig. 1c, arrowhead). An intensely phosphorylatedband detected in the human neutrophils most likely representsAFX, but a control protein to identify this band was notavailable.

FOXO phosphorylation occurs through PI-3K- andMAPK-mediated pathways

The signaling pathways that contribute to the phosphorylationof FKHR and FKHR-L1 were determined using selective

Crossley fMLP-stimulated neutrophils regulate FOXO and Mcl-1 585

chemical inhibitors. Neutrophils were stimulated with fMLP inthe absence or presence of chemical inhibitors of PI-3K (wort-mannin and LY294002) or MAPK kinase (MEK; U1026 andPD90859). Wortmannin (200 nM) markedly decreased phos-phorylation of FKHR and FKHR-L1 (Fig. 2a, lane d) at Ser256 and Ser 253 to levels below those observed without inhib-itors (Fig. 2a, lanes c, g, and j). Phosphorylation at these siteswas also decreased, albeit to a lesser extent, with a differentinhibitor of PI-3K, LY294002 (50 �M). Inhibition of PI-3Kwith LY294002 caused greater inhibition of FKHR-L1 thanthat of FKHR (Fig. 2a, lane h). It is interesting that thephosphorylation of FKHR was also dependent on MAPK sig-naling. Inhibition of MEK with U1026 (10 �M) decreasedphosphorylation of FKHR (Fig. 2a, lane e) on Ser 256 but didnot affect the basal phosphorylation at this site. The controlcompound U1024 (50 �M), used at a concentration five timeshigher than that for U1026, did not affect FKHR phosphory-lation (Fig. 2a, lane f, and compare lanes g and j). A differentinhibitor of MEK, PD90859, also inhibited the phosphorylationof FKHR, similar to that seen with U1026. A representative

experiment of three independent studies is shown. The resultsof these experiments are summarized in Figure 2b.

Rsk-2 catalyzes the phosphorylation of FKHR-L1 in vitro

The observations shown in Figure 2a suggested that AKT andMAPK cascades mediated FKHR and FKHR-L1 phosphoryla-tion on Ser 256 and Ser 253. The FKHR Ser 256 phosphory-lation site is a good consensus site for RSK kinases (for areview, see ref. [42]). This led us to test whether RSK-2 wasable to catalyze the phosphorylation of FKHR-L1. Rsk 2 wasimmunoprecipitated from control (DMSO) or fMLP-stimulatedneutrophils. The immunoprecipitated RSK-2 was used in an invitro kinase reaction with purified FKHR-L1. We found thatRSK-2 catalyzed the phosphorylation of FKHR-L1. The phos-phorylation of FKHR-L1 at Ser 253 was monitored throughWestern blotting using the phosphospecific FKHR (Ser 256)antibody that also detects phosphorylated FKHR-L1 (Fig. 2c).Time-dependent phosphorylation of FKHR-L1 revealed aslower migrating hyperphosphorylated species (shaded arrow,

Fig. 1. The kinetics of phosphorylation of AKTand the FKHR members that are targets of Akt inneutrophils: FKHR, FKHR-L1, and AFX duringfMLP stimulation. (a) Akt phosphorylation duringfMLP stimulation. Neutrophils were stimulatedwith fMLP (1 �m) for the times indicated or incu-bated with a control (DMSO). The lysates wereloaded on a 7.5% SDS-PAGE, subjected to West-ern-blotting procedures, and probed with an anti-body that recognizes AKT phosphorylated on Ser473. This figure was representative of two experi-ments. The membrane was then stripped andprobed with an antibody that recognizes total Akt.(b) FOXO members become phosphorylated infMLP-stimulated neutrophils. The blot from theexperiment in a was stripped and reprobed with anantibody that specifically recognized FKHR phos-phorylated on Ser 256. The forkhead (Ser 256)antibody also detects other FKHR family membersin neutrophils (FKHR-L1, top, pentagon, andAFX, arrow in upper portion). Total FKHR proteinis also denoted. (c) Detection of FOXO proteins inguinea pig and human neutrophils. The specificityof the antibody was determined by the detection ofFOXO proteins in the neutrophils of different spe-cies. Guinea pig or human neutrophils were stim-ulated for the indicated times with fMLP (1 �m) orincubated with the control (DMSO) for 10 min. Thelysates were loaded on a 7.5% SDS-PAGE, sub-jected to Western-blotting procedures. Controlslysates were included, consisting of the 3T3 mousefibroblast cell line and a 293 cell line containingoverexpressed FKHR-L1. The membrane wasprobed with the phosphospecific FKHR antibody(Ser 256). The phosphorylated FOXO proteins,FKHR-L1, FKHR, and AFX, which are detectedin guinea pig and human neutrophil lysates, aredesignated by an arrowhead, arrow, and pentagon(at right), respectively.

586 Journal of Leukocyte Biology Volume 74, October 2003 http://www.jleukbio.org

top, ppFKHR-L1) initially detected at 3 min, but maximalactivity was noted at 5 min of stimulation (Fig. 2c, lanes b andc). A barely detected, slower migrating hyperphosphorylatedspecies of FKHR is seen at 3–5 min (Fig. 2c, lanes b and c, andshaded arrow, ppFKHR). High basal phosphorylation of Rskoccurs in the unstimulated condition with DMSO (Fig. 2c, lanea). A protein that migrates at �80 kD (Fig. 2c, shaded penta-gon) undergoes intense phosphorylation. This protein, found ona Coomassie-stained gel of the purified FKHR-L1 protein, mostlikely represents a bacterial contaminant or breakdown prod-uct. The blots were stripped and reprobed for total FKHR-L1to determine protein loading.

Activated neutrophils coordinate the timing and location ofMcl-1: a survival factor Mcl-1 is rapidly induced

fMLP leads to robust activation of PI-3K- and MAPK-mediatedcascades. Mcl-1, a Bcl-2 family member, has been shown tofunction downstream of both pathways [24]. Therefore, I exam-ined neutrophils at different times of fMLP stimulation for theexpression of the survival factor Mcl-1. In Figure 3a, Mcl-1 isdetected in neutrophils from guinea pigs (upper) and humans(lower). An increase in the level of Mcl-1 expression is de-tected between 1 and 3 min of fMLP stimulation, and a declinein expression was detected at 10 min (Fig. 3a, upper). In aseparate experiment, Mcl-1 expression in fMLP-stimulated

Fig. 2. Determination of the signaling pathways thatcontribute to fMLP-mediated phosphorylation ofFKHR in neutrophils. (a) PI-3K and MAPK signalingpathways contribute to the FOXO phosphorylationduring fMLP stimulation. Neutrophils were preincu-bated for 30 min with DMSO (control) or with inhib-itors of MEK, U1026, the inactive control U0124, orPD90859, or with inhibitors of PI-3K, wortmannin orLY294002. Stimulation was then conducted withfMLP, and DMSO was included in control conditionsfor the times indicated. Neutrophils were lysed, andthe proteins were resolved by SDS-PAGE. Analysisby Western blotting was conducted using a phospho-specific FKHR antibody that detected phosphoryla-tion of FKHR on Ser 256. The bottom arrow denotesthe phosphorylated FKHR, whereas the pentagon(top) represents phosphorylated FKHR-L1, a fork-head family member. The membrane was stripped andreprobed with an antibody that specifically recognizedAKT when phosphorylated on Ser 473. The mem-brane was washed extensively and then reprobed withan antibody that recognizes Rsk when phosphorylatedon Ser 380. The blot was again washed extensivelyand probed with an antibody to total FKHR. (b)Summary of FOXO phosphorylation. Summary ofthree separate experiments in which FKHR phos-phorylation area was measured using NIH image 1.62

retrieved from the public NIH domain. (c) Rsk-2 from fMLP-stimulated neutrophils can phosphorylate FKHR and FKHR-L1 in an in vitro kinase experiment.RSK-2 was immunoprecipitated from neutrophils stimulated with fMLP (lanes b–f) or incubated with the control, and DMSO (lane a) was used an in vitro kinasereaction with FKHR-L1 protein, which was purified from E. coli-induced expression of GST–FKHR-L1. The shaded arrows (from top to bottom) representhyperphosphorylated FKHR-L1 and FKHR, respectively, and the arrow represents p90 Rsk (pp90Rsk). The shaded pentagon shows a bacterial-contaminatingprotein derived from the bacterial expression and purification of the FKHR-L1 protein. The blot was stripped and probed with an antibody that recognizes totalFKHR-L1. This experiment is a representative of three separate experiments.

Crossley fMLP-stimulated neutrophils regulate FOXO and Mcl-1 587

Fig. 3. Mcl-1 protein expression is induced rapidly inneutrophils stimulated with fMLP. (a) Mcl-1 protein ex-pression in fMLP-stimulated neutrophils. Neutrophils(2�106) were stimulated with fMLP (1 �M) or incubatedwith DMSO (the control). Using Western-blotting proce-dures, Mcl-1 was detected as a 42/40-kDa protein with amAb that recognized total Mcl-1. This experiment isrepresentative of three separate experiments. To deter-mine protein loading, the membrane was stripped andreprobed for expression of total FKHR. In the lowerportion of Figure 3a, 1 � 106 human neutrophils werestimulated with fMLP (1 �m) for the times indicated.After Western-blotting procedures were performed, Mcl-1was detected as a 42/40-kDa protein within human neu-trophils when using the mAb that recognized total Mcl-1.(b) GST–FKHR-L1 protein association assay. In a repre-sentative experiment of the GST–FKHR-L1 protein asso-ciation assay, lysates of 3 � 107 neutrophils were stim-ulated and incubated with GST–FKHR-L1 that had beencoupled to glutathione-linked agarose beads. The immunecomplexes were resolved on a 15% gel and probed byWestern blotting with a polyclonal antibody that recog-nizes phosphospecific FKHR. The blots were washedextensively and reprobed with a mAb that recognizes totalMcl-1. The arrow and arrowhead demonstrate phosphor-ylated FKHR and FKHR-L1, which are recognized by thephosphospecific FKHR antibody. The Mcl-1 antibodyrecognizes a single band (middle arrow) that migrates atapproximately 42 kD. The blots were again washed ex-tensively and probed with an antibody that recognizedtotal AKT. (c) A control experiment was conducted inwhich the lysates of 3 � 107 neutrophils were stimulatedwith fMLP for 10 min or incubated with DMSO for 10 min.The lysates were then incubated with GST–FKHR-L1,GST–FKHR-L1 (triple mutant), GST alone, or glutathione(GSH) beads in a pull-down assay. The immune com-plexes were resolved on a 10% gel and probed with amAb to detect Mcl-1. To visualize the GST proteinsloaded in each condition, the membrane was washedextensively, Western blotting procedures were performed,and the membrane was probed with an antibody thatrecognized GST. A company-provided mouse macrophagelysate is included as a positive control to identify Mcl-1.(d) FKHR interacts with Mcl-1 in vivo. This is a repre-sentative experiment in which a polyclonal FKHR anti-body coupled to protein A agarose was used in an immu-noprecipitation assay with 3 � 107 neutrophils that werestimulated with fMLP or incubated with the controlDMSO. After SDS-PAGE, the proteins in the immunecomplexes were resolved on a 10% gel and probed with amAb that recognizes Mcl-1. An arrow denotes a 42-kDprotein that is detected after 10 min of stimulation withfMLP, which is not present in neutrophil lysates incu-bated with DMSO or in lysates in which IgG is substitutedfor FKHR. To determine the amount of immunoprecipi-tated protein present, the membrane was washed exten-sively and detected with an antibody to total FKHR.

588 Journal of Leukocyte Biology Volume 74, October 2003 http://www.jleukbio.org

neutrophils was detected at 1 h, whereas in unstimulatedneutrophils, only basal levels of Mcl-1 were detected (data notshown). We repeated this experiment in human neutrophilsand showed that Mcl-1 is detected as a 42/40-kD protein forwhich the expression increases during fMLP stimulation be-tween 1 and 10 min (Fig. 3a, lower).

Mcl-1 binds to a complex containing FKHR in vitro

As PI-3K and MAPK pathways regulate FKHR and Mcl-1, Inext tested in stimulated neutrophils whether FKHR associ-ated with Mcl-1. In an experiment using neutrophils stimulatedfor 1–10 min or unstimulated neutrophils (DMSO), I performeda GST pull-down assay. The immune complexes were exam-ined for the presence of proteins that associated with FKHR-L1, using a GST–FKHR-L1 and a phosphorylation-defectiveconstruct (for construct descriptions, see ref. [28]). We testedwhether Mcl-1 was contained in the immune complex withGST–FKHR-L1. In Figure 3b, Mcl-1 was found to coassociatewith GST–FKHR-L1 in a manner that did not depend onstimulation. Lower amounts of Mcl-1 were observed with theGST–FKHR-L1 (triple mutant). The lower recovery of Mcl-1with the triple mutant GST–FKHR-L1 may have occurredbecause of the inability of Akt-dependent kinases within theneutrophil lysate to phosphorylate the mutant FKHR-L1. Theblots were then washed extensively and probed with an anti-body that detected total AKT (bottom arrow). To rule out thenonspecific binding of proteins to the complex, the membranewas stripped and reprobed with an antibody to total MAPK.Total MAPK was not present in the immune complex (data notshown). The membrane then washed extensively and probed forBcl-2, a prosurvival family member. Bcl-2 was not found in thecomplex (data not shown). A control experiment was performedto determine whether the binding of Mcl-1 to the immunecomplex was secondary to the presence of GST in the fusionprotein. Neutrophils were stimulated for 10 min or incubatedfor 10 min with the control (DMSO) and were used in apull-down assay with GST–FKHR-L1, the GST-FKHR-L1 (tri-ple mutant), GST alone, or glutathione beads alone (Fig. 3c).The presence of Mcl-1 in the immune complex was thendetermined. This experiment reveals that the binding of Mcl-1depends on the presence of the FOXO protein, FKHR-L1, inthe immune complex. A slight change in the mobility of Mcl-1is detected in stimulated neutrophils as compared with un-stimulated neutrophils (Fig. 3c, lane B). The mobility shiftmight suggest phosphorylation of Mcl-1 or a Mcl-1 protein ofhigher molecular density. The amount of Mcl-1 protein in theFOXO complex appears to be greater when the three Aktphosphorylation sites are intact (Fig. 3c, lanes B and I com-pared with lanes C and D; GST–FKHR-L1 protein, comparedwith the GST–FKHR-L1 triple mutant). To estimate proteinloading, the GST proteins present in each condition werevisualized after Western blotting and detection with a GSTantibody (Fig. 3c, bottom panel). The protein staining revealsthat the GST–FKHR-L1 and the GST–FKHR-L1 (triple mu-tant) proteins are present at similar levels, whereas a greateramount of the GST protein was present (Fig. 3c, bottom panel).The macrophage lysate included as a positive control for theidentity of Mcl-1 was overexposed to visualize the protein (Fig.3c, lane J).

To determine if the association between FKHR and Mcl-1was found in stimulated neutrophils in vivo, two experimentswere conducted in which FKHR was immunoprecipitated, andthe immune complexes were detected for the presence ofcoimmunoprecipitated Mcl-1. In a representative experiment, aprotein of low abundance that migrates at �42 kD was seen at10 min of fMLP stimulation. With DMSO incubation for 10 minor with mouse IgG, the 42-kD protein was not present (Fig. 3d,upper arrow). A protein of unknown identity migrates above 52kD, is also detected with the Mcl-1 antibody, and showsincreased abundance during fMLP stimulation (Fig. 3d, arrow-head). The reverse experiment was also conducted in whichMcl-1 was immunoprecipitated, and FKHR was detected afterWestern-blotting procedures. As a result of technical difficul-ties and the low recovery of immunoprecipitated Mcl-1, I couldnot conclusively determine that an interaction with FKHR waspresent.

DISCUSSION

This study shows that FOXO transcription factors are presentin neutrophils and undergo rapid phosphorylation in the pres-ence of bacterial chemoattractants that may be found duringinfectious processes.

We show that fMLP stimulation targets the FOXO transcrip-tion factors leading to their phosphorylation through PI-3K-and through Rsk-2-mediated pathways. Prolonged MAPK andRSK activation may facilitate the induction by CREB of genesthat affect the survival of cells [22, 23]. Furthermore in neu-trophils stimulated with fMLP, the activation of PI-3K and thephosphorylation of the downstream kinase AKT are pathwaysthrough which cell survival of a wide variety of cells is regu-lated (for reviews, see refs. [26, 43]). We propose that bothpathways converge on FKHR (FOXO1), FKHR-L1 (FOX03a),and AFX (FOX04) in neutrophils and promote their phosphor-ylation. The phosphorylation of FOXO family members pro-motes survival in neurons and other hematopoietic cells [28,44]. In this study, there is evidence that FKHR becomesphosphorylated in neutrophils in response to fMLP, an eventthat may mediate survival responses within these cells.

Early studies suggested that PI-3K/PKB activation was themajor route through which the phosphorylation of FKHR on Ser256 or on homologous sites in other family members occurred[28, 45–49]. Recently, other kinases, such as the serum andglucocorticoid-induced kinases, a PI-3K-dependent kinase,and casein kinase 1, have been shown to preferentially phos-phorylate FKHR on sites other than those phosphorylated byPKB/AKT [50, 51]. These findings show that Rsk-2 can phos-phorylate FKHR-L1 in vitro (Fig. 2c) and that inhibition ofMAPK cascades with specific inhibitors suppresses the phos-phorylation of FKHR in vivo (Fig. 2a). These results suggestthat PI-3K and MAPK contribute to the phosphorylation ofFKHR. MAPK-mediated phosphorylation of FKHR may alsotarget sites other than those phosphorylated by PKB/AKT. Inoverexpression studies, Rsk did not catalyze the phosphoryla-tion of FKHR on PKB-targeted residues (24, 256, 319) in cellsstimulated with insulin-like growth factor-1 or the phosphory-lation of FKHR-L1 on Ser 316 in cells stimulated with insulin

Crossley fMLP-stimulated neutrophils regulate FOXO and Mcl-1 589

[48, 50]. These findings suggest that Rsk-2 can phosphorylateFOXO family members in neutrophils. This implies that fMLPstimulation activates kinases downstream of MAPK other thanRsk, which may participate in assuring that FKHR is phos-phorylated.

As PI-3K- and MAPK-activated Rsk have been implicatedin cellular survival, I examined stimulated neutrophils for theexpression of Mcl-1, which is a target of both pathways [24].Mcl-1 is a Bcl-2 family member that has prosurvival propertiesin hematopoietic cells [52]. Mcl-1 was expressed constitutivelyin elicited neutrophils, but an increase in expression of Mcl-1protein was detected during fMLP stimulation (Fig. 3a). Thetranscriptional up-regulation of Mcl-1 has previously beensuggested to correlate with phosphorylation via the MAPK/extracellular-regulated kinase pathway, whereas an increase intranslation of Mcl-1 has been shown to depend on the PI-3K/AKT pathway [53, 54]. The early increase in Mcl-1 expressiondetected at 3–5 min in this study may represent phosphoryla-tion or stabilization of Mcl-1 protein (Fig. 3a). Additionally, Ifound that the expression of Mcl-1 was greater at 1 h instimulated neutrophils, whereas in unstimulated neutrophils,Mcl-1 expression had declined to basal levels (data not shown).This finding is consistent with the time course of Mcl-1 ex-pression in a leukemia cell line, ML-1 [55]. It has beensuggested that the neutrophil’s survival is dependent on theinducible expression of Mcl-1 [56]. These findings suggest thatthe pathways activated with fMLP stimulation of neutrophilsmay induce the expression of Mcl-1.

FKHR and Mcl-1 may be regulated not only by PI-3K andMAPK signaling but also in response to oxidative stresses thatresult as a consequence of the neutrophil’s production ofreactive oxygen species [57, 58]. In response to the respiratoryburst that is activated by fMLP, neutrophils might relocalizeFKHR from the nucleus to the cytoplasm. In immunocyto-chemical studies, FKHR was localized in the cytoplasm andwas colocalized with AKT within neutrophils (unpublished).

We also demonstrated that FOXO proteins were bound in acomplex that contained Mcl-1 in neutrophils in in vitro assays(Fig. 3, b and c). Furthermore, these experiments revealed thatan association between FKHR and Mcl-1 was present in vivo(Fig. 3d). The design of these studies does not allow us todetermine whether the association between FOXO and Mcl-1involves direct binding between the proteins or whether theassociation is indirect. The association between FKHR andMcl-1 is likely to occur in the cytoplasm, as phosphorylation ofFKHR allows it to be escorted out of the nucleus by the 14-3-3scaffolding protein [59, 60].

We have provided additional evidence that in neutrophilsactivated by the bacterial chemoattractant fMLP, signalingthrough PI-3K and MAPK pathways leads to the phosphoryla-tion of FOXO family members. Additionally, fMLP stimulationup-regulates the expression of Mcl-1, which is then able tobind a complex that contains FOXO proteins, a mechanismthrough which neutrophils may sequester FOXOs in the cyto-plasm and promote their survival.

Most studies have found that fMLP stimulation does notdelay apoptosis within neutrophils [61, 62]. Studies using theantiapoptotic factor GM-CSF have shown that Janus tyrosinekinase–signal transducer and activator of transcription and

PI-3K pathways cooperate in human neutrophils to mediate thesurvival response through Mcl-1 [63, 64]. These studies haveshown that fMLP stimulation of neutrophils leads to the acti-vation of parallel pathways, which allow neutrophils to regulatethe function of FOXO transcription factors and the survivalfactor Mcl-1. The phosphorylation of the FOXO family mem-bers may result in the inhibition downstream targets of FOXOsuch as TRAIL or p27kip1, factors shown to promote apoptosisin neutrophils and other hematopoietic cells [34, 65]. Theinduction of Mcl-1 that occurs with fMLP stimulation mayparticipate in maintaining mitochondrial function, organellesthat have recently been shown to regulate the multiple func-tions of neutrophils [66]. Future studies will be required todetermine the requirement for FOXOs in these functions inneutrophils. In summary, these studies have delineated a path-way, whereby fMLP stimulation regulates FOXO and Mcl-1,factors that are then found in a complex. The regulation ofFOXOs and Mcl-1 is a mechanism through which fMLP stim-ulation may alter neutrophils’ survival and function duringinflammatory conditions.

ACKNOWLEDGMENTS

These studies were supported by grants from the NationalInstitutes of Health, K08 NS01922 (L. J. C.) and DK50015(John Badwey, Center for Experimental Therapeutics andReperfusion Injury, Department of Anesthesiology, Periopera-tive and Pain Medicine, Brigham and Women’s Hospital, Bos-ton, MA). I thank Drs. Anne Brunet and Michael Greenberg forkindly providing reagents. I also thank Drs. Paul Allen, JohnBadwey, and Anne Brunet for critical reading of the manu-script and stimulating discussions. This research was con-ducted in the laboratory of and with the continuing support ofDr. John A. Badwey.

REFERENCES

1. Medzhitov, R., Janeway Jr., C. (2000) Innate immune recognition: mech-anisms and pathways. Immunol. Rev. 173, 89–97.

2. Haribabu, B., Richardson, R. M., Verghese, M. W., Barr, A. J., Zhelev,D. V., Snyderman, R. (2000) Function and regulation of chemoattractantreceptors. Immunol. Res. 22, 271–279.

3. Mocsai, A., Jakus, Z., Vantus, T., Berton, G., Lowell, C. A., Ligeti, E.(2000) Kinase pathways in chemoattractant-induced degranulation of neu-trophils: the role of p38 mitogen-activated protein kinase activated by Srcfamily kinases. J. Immunol. 164, 4321–4331.

4. Rane, M. J., Carrithers, S. L., Arthur, J. M., Klein, J. B., McLeish, K. R.(1997) Formyl peptide receptors are coupled to multiple mitogen-activatedprotein kinase cascades by distinct signal transduction pathways: role inactivation of reduced nicotinamide adenine dinucleotide oxidase. J. Im-munol. 159, 5070–5078.

5. Della Bianca, V., Grzeskowiak, M., Dusi, S., Rossi, F. (1993) Transmem-brane signaling pathways involved in phagocytosis and associated activa-tion of NADPH oxidase mediated by Fc gamma Rs in human neutrophils.J. Leukoc. Biol. 53, 427–438.

6. Coffer, P. J., Geijsen, N., M’rabet, L., Schweizer, R. C., Maikoe, T.,Raaijmakers, J. A., Lammers, J. W., Koenderman, L. (1998) Comparisonof the roles of mitogen-activated protein kinase kinase and phosphatidyl-inositol 3-kinase signal transduction in neutrophil effector function. Bio-chem. J. 329, 121–130.

7. Yamamoto, C., Yoshida, S., Taniguchi, H., Qin, M. H., Miyamoto, H.,Mizuguchi, Y. (1993) Lipopolysaccharide and granulocyte colony-stimu-

590 Journal of Leukocyte Biology Volume 74, October 2003 http://www.jleukbio.org

lating factor delay neutrophil apoptosis and ingestion by guinea pigmacrophages. Infect. Immun. 61, 1972–1979.

8. Colotta, F., Re, F., Polentarutti, N., Sozzani, S., Mantovani, A. (1992)Modulation of granulocyte survival and programmed cell death by cyto-kines and bacterial products. Blood 80, 2012–2020.

9. Lee, A., Whyte, M. K., Haslett, C. (1993) Inhibition of apoptosis andprolongation of neutrophil functional longevity by inflammatory mediators.J. Leukoc. Biol. 54, 283–288.

10. Heit, B., Tavener, S., Raharjo, E., Kubes, P. (2002) An intracellularsignaling hierarchy determines direction of migration in opposing chemo-tactic gradients. J. Cell Biol. 159, 91–102.

11. Le, Y., Murphy, P. M., Wang, J. M. (2002) Formyl-peptide receptorsrevisited. Trends Immunol. 23, 541–548.

12. Yang, R. B., Mark, M. R., Gurney, A. L., Godowski, P. J. (1999) Signalingevents induced by lipopolysaccharide-activated Toll-like receptor 2.J. Immunol. 163, 639–643.

13. Sturgill, T. W., Ray, L. B., Erickson, E., Maller, J. L. (1988) Insulinstimulated MAP2 kinase phosphorylates and activates ribosomal proteinS6 kinase. Nature 334, 715–718.

14. Jones, S. W., Erikson, E., Blenis, J., Maller, J. L., Erikson, R. L. (1988)A Xenopus ribosomal protein S6 kinase has two apparent kinase domainsthat are each similar to distinct protein kinases. Proc. Natl. Acad. Sci. USA85, 3377–3381.

15. Xing, J., Ginty, D. D., Greenberg, M. E. (1996) Coupling of the Ras-MAPKpathway to activation by RSK2, a growth factor-regulated CREB kinase.Science 273, 959–963.

16. Schouten, G. J., Vertegaal, A. C., Whiteside, S. T., Israel, A., Toebes, M.,Dorsman, J. C., van der Eb, A. J., Zantema, A. (1997) IkappaB alpha is atarget for the mitogen-activated 90 kDa ribosomal S6 kinase. EMBO J.16, 3133–3144.

17. Chen, R. H., Abate, C., Blenis, J. (1993) Phosphorylation of the c-Fostransrepression domain by mitogen-activated protein kinase and 90-kDaribosomal S6 kinase. Proc. Natl. Acad. Sci. USA 90, 10952–10956.

18. Trivier, E., De Cesare, D., Jacquot, S., Pannetier, S., Zackai, E., Young, I.,Mandel, J. L., Sassone-Corsi, P., Hanauer, A. (1996) Mutations in thekinase Rsk-2 associated with Coffin-Lowry syndrome. Nature 384, 567–570.

19. Dufresne, S. D., Bjorbaek, C., El-Haschimi, K., Zhao, Y., Aschenbach,W. G., Moller, D. E., Goodyear, L. J. (2001) Altered extracellular signal-regulated kinase signaling and glycogen metabolism in skeletal musclefrom p90 ribosomal S6 kinase 2 knockout mice. Mol. Cell. Biol. 21,81–87.

20. De Mesquita, D. D., Zhan, Q., Crossley, L., Badwey, J. A. (2001) p90-RSKand Akt may promote rapid phosphorylation/inactivation of glycogensynthase kinase 3 in chemoattractant-stimulated neutrophils. FEBS Lett.502, 84–88.

21. Pap, M., Cooper, G. M. (1998) Role of glycogen synthase kinase-3 in thephosphatidylinositol 3-kinase/Akt cell survival pathway. J. Biol. Chem.273, 19929–19932.

22. Bonni, A., Brunet, A., West, A. E., Datta, S. R., Takasu, M. A., Greenberg,M. E. (1999) Cell survival promoted by the Ras-MAPK signaling pathwayby transcription-dependent and -independent mechanisms. Science 286,1358–1362.

23. Riccio, A., Ahn, S., Davenport, C. M., Blendy, J. A., Ginty, D. D. (1999)Mediation by a CREB family transcription factor of NGF-dependentsurvival of sympathetic neurons. Science 286, 2358–2361.

24. Wang, J. M., Chao, J. R., Chen, W., Kuo, M. L., Yen, J. J., Yang-Yen,H. F. (1999) The antiapoptotic gene Mcl-1 is up-regulated by the phos-phatidylinositol 3-kinase/Akt signaling pathway through a transcriptionfactor complex containing CREB. Mol. Cell. Biol. 19, 6195–6206.

25. Ding, J., Vlahos, C. J., Liu, R., Brown, R. F., Badwey, J. A. (1995)Antagonists of phosphatidylinositol 3-kinase block activation of severalnovel protein kinases in neutrophils. J. Biol. Chem. 270, 11684–11691.

26. Datta, S. R., Brunet, A., Greenberg, M. E. (1999) Cellular survival: a playin three Akts. Genes Dev. 13, 2905–2927.

27. Andjelkovic, M., Alessi, D. R., Meier, R., Fernandez, A., Lamb, N. J.,Frech, M., Cron, P., Cohen, P., Lucocq, J. M., Hemmings, B. A. (1997)Role of translocation in the activation and function of protein kinase B.J. Biol. Chem. 272, 31515–31524.

28. Brunet, A., Bonni, A., Zigmond, M. J., Lin, M. Z., Juo, P., Hu, L. S.,Anderson, M. J., Arden, K. C., Blenis, J., Greenberg, M. E. (1999) Aktpromotes cell survival by phosphorylating and inhibiting a Forkheadtranscription factor. Cell 96, 857–868.

29. Burgering, B. M., Kops, G. J. (2002) Cell cycle and death control: long liveForkheads. Trends Biochem. Sci. 27, 352–360.

30. Lin, K., Dorman, J. B., Rodan, A., Kenyon, C. (1997) daf-16: An HNF-3/forkhead family member that can function to double the life-span ofCaenorhabditis elegans. Science 278, 1319–1322.

31. Kaufmann, E., Knochel, W. (1996) Five years on the wings of fork head.Mech. Dev. 57, 3–20.

32. Stahl, M., Dijkers, P. F., Kops, G. J., Lens, S. M., Coffer, P. J., Burgering,B. M., Medema, R. H. (2002) The forkhead transcription factor FoxOregulates transcription of p27Kip1 and Bim in response to IL-2. J. Im-munol. 168, 5024–5031.

33. Modur, V., Nagarajan, R., Evers, B. M., Milbrandt, J. (2002) FOXOproteins regulate tumor necrosis factor-related apoptosis inducing ligandexpression. Implications for PTEN mutation in prostate cancer. J. Biol.Chem. 277, 47928–47937.

34. Renshaw, S. A., Parmar, J. S., Singleton, V., Rowe, S. J., Dockrell, D. H.,Dower, S. K., Bingle, C. D., Chilvers, E. R., Whyte, M. K. (2003)Acceleration of human neutrophil apoptosis by TRAIL. J. Immunol. 170,1027–1033.

35. Yang, K. Y., Arcaroli, J., Kupfner, J., Pitts, T. M., Park, J. S., Strasshiem,D., Perng, R. P., Abraham, E. (2003) Involvement of phosphatidylinositol3-kinase gamma in neutrophil apoptosis. Cell. Signal. 15, 225–233.

36. Kozopas, K. M., Yang, T., Buchan, H. L., Zhou, P., Craig, R. W. (1993)MCL1, a gene expressed in programmed myeloid cell differentiation, hassequence similarity to BCL2. Proc. Natl. Acad. Sci. USA 90, 3516–3520.

37. Moulding, D. A., Quayle, J. A., Hart, C. A., Edwards, S. W. (1998) Mcl-1expression in human neutrophils: regulation by cytokines and correlationwith cell survival. Blood 92, 2495–2502.

38. Chao, J. R., Wang, J. M., Lee, S. F., Peng, H. W., Lin, Y. H., Chou, C. H.,Li, J. C., Huang, H. M., Chou, C. K., Kuo, M. L., Yen, J. J., Yang-Yen,H. F. (1998) Mcl-1 is an immediate-early gene activated by the granulo-cyte- macrophage colony-stimulating factor (GM-CSF) signaling pathwayand is one component of the GM-CSF viability response. Mol. Cell. Biol.18, 4883–4898.

39. Badwey, J. A., Karnovsky, M. L. (1986) NaDH oxidase from guinea pigpolymorphonuclear leukocytes. Methods Enzymol. 132, 365–368.

40. Colgan, S. P., Dzus, A. L., Parkos, C. A. (1996) Epithelial exposure tohypoxia modulates neutrophil transepithelial migration. J. Exp. Med.184, 1003–1015.

41. Towbin, H., Staehelin, T., Gordon, J. (1979) Electrophoretic transfer ofproteins from polyacrylamide gels to nitrocellulose sheets: procedure andsome applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354.

42. Frodin, M., Gammeltoft, S. (1999) Role and regulation of 90 kDa ribo-somal S6 kinase (RSK) in signal transduction. Mol. Cell. Endocrinol. 151,65–77.

43. Cantley, L. C. (2002) The phosphoinositide 3-kinase pathway. Science296, 1655–1657.

44. Dijkers, P. F., Medema, R. H., Pals, C., Banerji, L., Thomas, N. S., Lam,E. W., Burgering, B. M., Raaijmakers, J. A., Lammers, J. W., Koender-man, L., Coffer, P. J. (2000) Forkhead transcription factor FKHR-L1modulates cytokine-dependent transcriptional regulation of p27(KIP1).Mol. Cell. Biol. 20, 9138–9148.

45. Biggs III, W. H., Meisenhelder, J., Hunter, T., Cavenee, W. K., Arden,K. C. (1999) Protein kinase B/Akt-mediated phosphorylation promotesnuclear exclusion of the winged helix transcription factor FKHR1. Proc.Natl. Acad. Sci. USA 96, 7421–7426.

46. Nakae, J., Park, B. C., Accili, D. (1999) Insulin stimulates phosphoryla-tion of the forkhead transcription factor FKHR on serine 253 through aWortmannin-sensitive pathway. J. Biol. Chem. 274, 15982–15985.

47. Kops, G. J., de Ruiter, N. D., De Vries-Smits, A. M., Powell, D. R., Bos,J. L., Burgering, B. M. (1999) Direct control of the Forkhead transcriptionfactor AFX by protein kinase B. Nature 398, 630–634.

48. Rena, G., Guo, S., Cichy, S. C., Unterman, T. G., Cohen, P. (1999)Phosphorylation of the transcription factor forkhead family member FKHRby protein kinase B. J. Biol. Chem. 274, 17179–17183.

49. Tang, E. D., Nunez, G., Barr, F. G., Guan, K. L. (1999) Negative regulationof the forkhead transcription factor FKHR by Akt. J. Biol. Chem. 274,16741–16746.

50. Brunet, A., Park, J., Tran, H., Hu, L. S., Hemmings, B. A., Greenberg,M. E. (2001) Protein kinase SGK mediates survival signals by phosphor-ylating the forkhead transcription factor FKHRL1 (FOXO3a). Mol. Cell.Biol. 21, 952–965.

51. Rena, G., Woods, Y. L., Prescott, A. R., Peggie, M., Unterman, T. G.,Williams, M. R., Cohen, P. (2002) Two novel phosphorylation sites onFKHR that are critical for its nuclear exclusion. EMBO J. 21, 2263–2271.

52. Zhou, P., Qian, L., Bieszczad, C. K., Noelle, R., Binder, M., Levy, N. B.,Craig, R. W. (1998) Mcl-1 in transgenic mice promotes survival in aspectrum of hematopoietic cell types and immortalization in the myeloidlineage. Blood 92, 3226–3239.

53. Domina, A. M., Smith, J. H., Craig, R. W. (2000) Myeloid cell leukemia1 is phosphorylated through two distinct pathways, one associated withextracellular signal-regulated kinase activation and the other with G2/M

Crossley fMLP-stimulated neutrophils regulate FOXO and Mcl-1 591

accumulation or protein phosphatase 1/2A inhibition. J. Biol. Chem. 275,21688–21694.

54. Schubert, K. M., Duronio, V. (2001) Distinct roles for extracellular-signal-regulated protein kinase (ERK) mitogen-activated protein kinases andphosphatidylinositol 3-kinase in the regulation of Mcl-1 synthesis. Bio-chem. J. 356, 473–480.

55. Townsend, K. J., Trusty, J. L., Traupman, M. A., Eastman, A., Craig, R. W.(1998) Expression of the antiapoptotic MCL1 gene product is regulated bya mitogen activated protein kinase-mediated pathway triggered throughmicrotubule disruption and protein kinase C. Oncogene 17, 1223–1234.

56. Moulding, D. A., Akgul, C., Derouet, M., White, M. R., Edwards, S. W.(2001) BCL-2 family expression in human neutrophils during delayed andaccelerated apoptosis. J. Leukoc. Biol. 70, 783–792.

57. Nemoto, S., Finkel, T. (2002) Redox regulation of forkhead proteinsthrough a p66shc-dependent signaling pathway. Science 295, 2450–2452.

58. Inoshita, S., Takeda, K., Hatai, T., Terada, Y., Sano, M., Hata, J.,Umezawa, A., Ichijo, H. (2002) Phosphorylation and inactivation of my-eloid cell leukemia 1 by JNK in response to oxidative stress. J. Biol.Chem. 277, 43730–43734.

59. Rena, G., Prescott, A. R., Guo, S., Cohen, P., Unterman, T. G. (2001)Roles of the forkhead in rhabdomyosarcoma (FKHR) phosphorylation sitesin regulating 14-3-3 binding, transactivation and nuclear targetting. Bio-chem. J. 354, 605–612.

60. Brunet, A., Kanai, F., Stehn, J., Xu, J., Sarbassova, D., Frangioni, J. V.,Dalal, S. N., DeCaprio, J. A., Greenberg, M. E., Yaffe, M. B. (2002) 14-3-3transits to the nucleus and participates in dynamic nucleocytoplasmictransport. J. Cell Biol. 156, 817–828.

61. Brach, M. A., deVos, S., Gruss, H. J., Herrmann, F. (1992) Prolongationof survival of human polymorphonuclear neutrophils by granulocyte-mac-rophage colony-stimulating factor is caused by inhibition of programmedcell death. Blood 80, 2920–2924.

62. Verploegen, S., van Leeuwen, C. M., van Deutekom, H. W., Lammers,J. W., Koenderman, L., Coffer, P. J. (2002) Role of Ca2/calmodulinregulated signaling pathways in chemoattractant induced neutrophil ef-fector functions. Comparison with the role of phosphotidylinositol-3 ki-nase. Eur. J. Biochem. 269, 4625–4634.

63. Epling-Burnette, P. K., Zhong, B., Bai, F., Jiang, K., Bailey, R. D., Garcia,R., Jove, R., Djeu, J. Y., Loughran Jr., T. P., Wei, S. (2001) Cooperativeregulation of Mcl-1 by Janus kinase/STAT and phosphatidylinositol 3-ki-nase contribute to granulocyte-macrophage colony-stimulating factor-de-layed apoptosis in human neutrophils. J. Immunol. 166, 7486–7495.

64. Sakamoto, C., Suzuki, K., Hato, F., Akahori, M., Hasegawa, T., Hino, M.,Kitagawa, S. (2003) Antiapoptotic effect of granulocyte colony-stimulatingfactor, granulocyte-macrophage colony-stimulating factor, and cyclic AMPon human neutrophils: protein synthesis-dependent and protein synthesis-independent mechanisms and the role of the Janus kinase-STAT pathway.Int. J. Hematol. 77, 60–70.

65. Dijkers, P. F., Birkenkamp, K. U., Lam, E. W., Thomas, N. S., Lammers,J. W., Koenderman, L., Coffer, P. J. (2002) FKHR-L1 can act as a criticaleffector of cell death induced by cytokine withdrawal: protein kinaseB-enhanced cell survival through maintenance of mitochondrial integrity.J. Cell Biol. 156, 531–542.

66. Fossati, G., Moulding, D. A., Spiller, D. G., Moots, R. J., White, M. R.,Edwards, S. W. (2003) The mitochondrial network of human neutrophils:role in chemotaxis, phagocytosis, respiratory burst activation, and com-mitment to apoptosis. J. Immunol. 170, 1964–1972.

592 Journal of Leukocyte Biology Volume 74, October 2003 http://www.jleukbio.org