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Originally published online as doi:10.1189/jlb.0103020 on July 15, 2003

Published online before print July 15, 2003
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(Journal of Leukocyte Biology. 2003;74:583-592.)
© 2003 by Society for Leukocyte Biology

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

Lisa J. Crossley1

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

1Correspondence: Center For Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Harvard Medical School, Thorn Building, Room 703, 75 Francis Street, Boston, MA 02115. E-mail: crossley{at}zeus.bwh.harvard.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation signals from bacterial stimuli set into motion a series of events that alter the abbreviated lifespan of neutrophils. These studies show that the bacterial chemoattractant, formyl-Met-Leu-Phe (fMLP), promotes the phosphorylation/inactivation of the FOXO subfamily of forkhead transcription factors (FKHR, FKHR-L1, and AFX) through the phosphatidylinositol-3-kinase/Akt (protein kinase B) and the RAS mitogen-activated protein kinase pathways. Furthermore, fMLP stimulation causes the inducible expression of the prosurvival Bcl-2 family member Mcl-1, which then binds to a complex containing FKHR. These studies show that fMLP-stimulated neutrophils coordinate the regulation of FOXO transcription factors and the survival factor Mcl-1, a mechanism that may allow neutrophils to alter their survival.

Key Words: phosphatidylinositol-3-kinase (PI-3K) • Akt • mitogen-activated protein kinase (MAPK) signaling • Rsk


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophils are first responders in an organism’s rapid assault on infectious pathogens. Through genetically conserved receptors, 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 recognized by neutrophils. fMLP, upon binding to its heterotrimeric G protein-coupled receptor, initiates signaling cascades that activate multiple pathways [2 ]. These pathways include the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI-3K) cascades, which are important for the development of the functional responses of neutrophils in inflammation (e.g., the respiratory burst, transmigration, and phagocytosis) [3 4 5 6 ].

Lipopolysaccharide (LPS) and granulocyte macrophage-colony stimulating factor (GM-CSF) are inflammatory stimuli that have been shown to inhibit neutrophil apoptosis, whereas the survival of neutrophils in response to chemoattractants (e.g., fMLP or C5a) has not been established [7 8 9 ]. A recent study has suggested that signals arising from fMLP or C5a may predominate in directing the responses of neutrophils that have migrated to the final site of an infection [10 ]. fMLP and LPS activate different receptors within neutrophils. The activation of heterotrimeric G protein-coupled receptors by fMLP versus the activation of Toll receptors by LPS [11 , 12 ] will most likely result in different nuclear signaling responses. These studies reveal a mechanism through which neutrophils present in sites of inflammation respond to fMLP activation of G protein-coupled receptors via signaling responses that regulate a family of nuclear transcription factors shown to function in pathways that affect survival.

A pathway that regulates the survival of many cells is the MAPK cascade. fMLP-mediated activation of MAPK (e.g., p42/p44 MAPK or the stress-activated kinases p38 MAPK and c-jun NH2-terminal kinase) within neutrophils leads to a cascade of events. Signaling through p42/p44 MAPK is propagated through the family of p90 kDa ribosomal S6 kinases (p90 Rsk) [13 , 14 ], which undergoes phosphorylation and then activates transcription factors [e.g., cyclic AMP response element-binding protein (CREB), inhibitor of {kappa}B{alpha}/nuclear factor-{kappa}B, c-fos] through phosphorylation [15 16 17 ]. p90 RSK exists in three isoforms, Rsk-1, -2, and -3. The absence of Rsk-2 in humans causes a severe neurologic disease, the Coffin-Lowry syndrome, and alters glycogen metabolism in the muscle of knockout mice [18 , 19 ]. Additionally, I have previously shown that in neutrophils, Rsk-2 phosphorylates and inactivates glycogen synthase kinase 3, an event that may also improve the neutrophil’s survival [20 , 21 ]. Rsk-2 not only inactivates proapoptotic substrates but is involved in the up-regulation of prosurvival Bcl-2 family members through its phosphorylation and activation of CREB [22 23 24 ].

fMLP stimulation also activates PI-3K [25 ], which then leads to the activation of the serine/threonine kinase, Akt, also called protein kinase B (PKB) [26 ]. Once activated by phosphorylation on two residues [serine (Ser) 473, threonine (Thr) 308], Akt translocates into the nucleus and phosphorylates its substrates [27 ]. Akt’s effect on cell-survival responses is mediated by the regulation of the FOXO subfamily of forkhead transcription factors, which are conserved in nematodes and mammals (for reviews, see refs. [28 , 29 ]).

In the nematode, the FOXO transcription factor Daf-16, was found to function in a pathway that regulates the lifespan of the worm [30 ]. In mammals, there exist three Daf-16 homologous proteins, FOXO1 [Forkhead (FKHR)], FOXO3a [FKHR-like 1 (FKHR-L1)], and FOXO4 [ALL1 fused gene from chromosome X (AFX)]. The FOXO proteins are characterized by the presence of a 110-amino acid DNA-binding domain that forms a winged helix structure [31 ]. FOXO transcription factors are involved in the regulation of the immune system. Studies suggest that FOXO may affect cell-cycle progression and the survival of hematopoietic cell lineages through the regulation of the cyclin-dependent kinase inhibitor p27kip1 and of Bcl-2 family members [32 ]. Recently, FOXO proteins have been shown to regulate the expression of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) [33 ], a TNF family member that can accelerate the rate of apoptosis in neutrophils [34 ].

Few studies have examined the regulation of FOXO transcription factors in activated neutrophils. The delineation of the regulation of FOXO in other cells has revealed that phosphorylation of FOXO through PI-3K facilitates cellular survival [26 , 28 ]. Additionally, in a mouse model of endotoxemia, the regulation of several transcriptional factors by PI-3K-mediated phosphorylation was shown to critically regulate the rate of neutrophil apoptosis [35 ]. Therefore, I hypothesized that the activation of PI-3K signaling by fMLP in neutrophils would also mediate the phosphorylation of FKHR as a mechanism through which neutrophils might regulate their survival. In fMLP-stimulated neutrophils, I found that the FOXO transcription factors undergo phosphorylation. Using selective chemical inhibitors of the MAPK and PI-3K pathways, I determined that FOXO is a target of Rsk and Akt activation in fMLP-stimulated neutrophils.

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

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


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
fMLP, protein A 4ß agarose beads, glutathione-linked agarose beads, dimethyl sulfoxide (DMSO), and other chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted. The inhibitors PD90859, U1026, U1024, LY294002, and wortmannin were purchased from Calbiochem (San Diego, CA). Total FKHR, phospho-FKHR (Ser 256), phospho-FKHR (Thr24)/FKHR-L1 (Thr32), phospho-AKT (Ser473), and phospho-Rsk (Ser 380) rabbit polyclonal antibodies raised to the human proteins were purchased from Cell Signaling Technology (CST; Beverly, MA). Total AKT was purchased from CST and Promega (Madison, WI). Goat polyclonal Rsk-2 (C-19) was from Santa Cruz Biotechnology (Santa Cruz, CA). Mcl-1 monoclonal antibodies (mAb) raised to the human protein were obtained from Transduction Laboratories (Lexington, KY) and Oncogene Research Products (Boston, MA). The glutathionine S-transferase (GST) polyclonal antibody was purchased from Upstate (Charlottesville, VA). The GST-FKHR-L1 fusion proteins and GST protein were kind gifts from the laboratory of Dr. Michael Greenberg (Children’s Hospital, Boston, MA).

Preparation of guinea pig peritoneal neutrophils
Guinea pig peritoneal neutrophils were freshly prepared using the published method of Badwey and Karnovsky [39 ]. Briefly, guinea pig neutrophils were harvested 18 h after intraperitoneal injection of 30 ml 12% casein (w/v) in 0.9% NaCl, and the peritoneal cavity was washed twice with 0.9% NaCl. The peritoneal cells were gently pelleted, and the erythrocytes were lysed in hypertonic 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 typically yielded ~1 x 109 cells that are >90% polymorphonuclear leukocytes.

Human neutrophil isolation
Neutrophils were freshly isolated from the whole blood of human volunteers using the method of Colgan et al. [40 ]. Briefly, blood obtained by venipuncture was anticoagulated with acid-citrate-dextrose, purified by aspiration of the buffy coat after centrifugation (400 g for 20 min at 25°C) to remove plasma and mononuclear cells. Erythrocytes were removed by a 2% gelatin gradient-sedimentation protocol followed by lysis of remaining erythrocytes in cold NH4Cl buffer. The cells were more than 90% neutrophils and were used within 4 h of isolation.

Cell stimulation and lysis
Neutrophils (1.5x107) were suspended in a modified Dulbecco’s phospate-buffered saline (PBS) medium [135 mM NaCl, 2.7 mM KCl, 16.2 mM Na2HPO4, 1.47 mM KH2PO4, containing 0.9 mM CaCl2, 0.5 mM MgCl2 (pH 7.35), containing 7.5 mM D-glucose] for 10 min at 37°C. Stimulation with fMLP (1.0 µM) was for various times. For experiments evaluating the involvement of signaling pathways for which specific chemical inhibitors were available, the inhibitors were preincubated with the neutrophils for 30 min before the addition of stimuli.

After the completion of reactions, the cells were lysed by the addition of 5x 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 sodium fluoride, 0.5% bromophenol blue). The following reagents were freshly added to the SDS lysis buffer: 10% ß-mercaptoethanol, 2 mM phenylmethylsulfonyl fluoride (PMSF), and 5 mM sodium orthovanadate. The samples were quickly mixed by vortexing, boiled for 5 min, centrifuged, and kept on ice until loading onto gels.

SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting
Equal aliquots of the samples were loaded onto polyacrylamide gels (ranging between 7.5 and 15% w/v), and the proteins were transferred to Immobilon-P membranes [41 ]. The membranes were blocked 1 h at room temperature with 5% milk in 20 mM Tris-HCl (pH 7.4) containing 250 mM NaCl. The blocking buffer was removed. The membranes were then incubated with one of the following antibodies: phoshoFKHR256 (1/1000 dilution), Mcl-1 (1/1000), Rsk-2 (C-19; 1/500), GST (1/1000), or the control antibodies for these proteins for 1 h at room temperature or overnight at 4°C in Tris-buffered saline/Tween 20 [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 by the manufacturer. Following incubation with primary antibodies, the membranes were washed (3x10 min/wash) with TBST. Incubation with a secondary Ab, which was diluted in TBST with 5% milk, was conducted for 1 h; for polyclonal antibodies, goat anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase (HRP) conjugate, 1:10,000 dilution; for phosphoFKHR, 1:3000; total FKHR, 1:5000 dilution; for GST, 1:2000; for mAb, goat anti-mouse IgG-HRP conjugate; for Mcl-1, 1:2000 dilution. Membranes were washed for 40 min in TBST and rinsed with distilled and deionized water. The activity of the HRP was visualized by incubating the membranes for 20 min at room temperature in an enhanced chemiluminescence detection system (Pierce, Rockford, IL) followed by autoradiography.

Immunoprecipitation
Neutrophils (3x107 cells) were stimulated for various times and then lysed by incubation on ice for 15 min in a lysis buffer [50 mM Tris (pH 8.0), 1% Nonidet P-40 (NP-40), 10% glycerol, 150 mM NaCl, 1 mM Na3VO4, 10 mM NaF, 1 mM EDTA] containing several protease inhibitors (5 µg/ml aprotinin, 5 µg/ml leupeptin, 2 µg/ml pepstatin, 2 mM/ml PMSF). The lysates were rotated at 4°C for 30 min, and the supernatents were collected after centrifugation. The lysates were precleared with Protein A/G sepharose beads for 15 min, and the supernatents were collected after centrifugation. For RSK and FKHR immunoprecipitation, 4.0 µg RSK or FKHR antibody was added to the neutrophil lysates and incubated 15 min on ice followed by 2 h rotating at 4°C. The immune complexes were then washed extensively. After washing extensively over 30 min, the complexes were loaded on SDS-PAGE and were subjected to procedures described for immunoblotting.

Protein-protein association assays
Plasmids containing GST-FKHR-L1 fusion proteins or GST alone were inoculated into BL21 Escherichia coli. The bacteria were grown overnight in selection media. The next day, the cultures were amplified, and protein expression was induced using 1 mM isopropylthiogalactoside for 4 h. The cultures were collected by centrifugation, resuspended in cold PBS lysis buffer containing 100 mM EDTA, 1% Triton X-100, and protease inhibitors (10 µg/ml aprotinin, 1 mM PMSF). The lysates were sonicated on ice for 1 min and collected by centrifugation at 16,000 g for 30 min. The fusion proteins were incubated with glutathione agarose beads, washed extensively, and resuspended in PBS lysis buffer containing 0.02% Na azide. The GST fusion protein complexes were used in pull-down assays using the methods described in immunoprecipitation.

In vitro kinase assay
Cells (3x107) were stimulated for the times indicated. The cells were disrupted on ice for 15 min in lysis buffer [50 mM Tris (pH 8.0), 1% NP-40, 150 mM NaCl, 5 mM EDTA, 10 µg/ml leupeptin, 5 µg/ml pepstatin, 10 µg/ml aprotinin, 2 mM Na orthovanadate, 10 mM NaF, 1 mM PMSF]. The lysates were centrifuged at 4°C for 20 min at 11,000 g, and the supernatents were collected. Rsk was immunoprecipitated from the neutrophil extracts with a RSK-2 goat polyclonal antibody (C-19; Santa Cruz Biotechnology), and the mixture was incubated on a rotating platform for 1 h at 4°C. The RSK immunoprecipitates were then complexed to Protein G sepharose (Sigma Chemical Co.) for an additional 2 h at 4°C. The Rsk immune complex was pelleted and washed five times in kinase buffer [50 mM Hepes (pH 7.4), 20 mM MgCl2, 5 mM NaF, 20 mM ß glycerophosphate, 1 mM NaVO4]. For in vitro kinase assays, the RSK complex was resuspended in 40 µl kinase buffer and warmed to 30°C for 15 min. Purified FKHR-L1 (1 µg) and 200 µM adenosine 5'-triphosphate (5 µl) were added to the reaction, which was run for an additional 30 min at 30°C. The reaction was terminated by the addition of an equal volume of SDS sample buffer and was boiled at 95°C for 5 min, and the samples were collected after centrifugation. The proteins were resolved on a 7.5% polyacrylamide gel and analyzed by Western blotting using an antibody that recognized phosphospecific FKHR.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
fMLP-stimulated neutrophils regulate targets of PI-3K, which are implicated in cellular survival
AKT undergoes prolonged phosphorylation/activation in response to fMLP
We first assessed AKT phosphorylation on Ser 473 during stimulation of neutrophils with fMLP. AKT phosphorylation on Ser 473 was minimal in unstimulated cells (Fig. 1a , lanes a, b, j, and k). Stimulation of cells with fMLP led to the phosphorylation of AKT on Ser 473 within 15 s, and the maximal phosphorylation was observed between 0.5 and 3 min. AKT phosphorylation was sustained during the period of stimulation for up to 45 min (Fig. 1a ; and data not shown). Total Akt is shown in the bottom portion of Figure 1a and reveals less protein during the periods of peak phosphorylation of Akt (Fig. 1a , lanes e–g and i). This may represent that the antibody is less sensitive to the phosphorylated form of Akt, or it may be a result of the process of stripping the membrane.



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  		Figure 1. The kinetics of phosphorylation of AKT and the FKHR members that are targets of Akt in neutrophils: FKHR, FKHR-L1, and AFX during fMLP stimulation. (a) Akt phosphorylation during fMLP stimulation. Neutrophils were stimulated with fMLP (1 µm) for the times indicated or incubated with a control (DMSO). The lysates were loaded on a 7.5% SDS-PAGE, subjected to Western-blotting procedures, and probed with an antibody that recognizes AKT phosphorylated on Ser 473. This figure was representative of two experiments. The membrane was then stripped and probed with an antibody that recognizes total Akt. (b) FOXO members become phosphorylated in fMLP-stimulated neutrophils. The blot from the experiment in a was stripped and reprobed with an antibody that specifically recognized FKHR phosphorylated on Ser 256. The forkhead (Ser 256) antibody also detects other FKHR family members in neutrophils (FKHR-L1, top, pentagon, and AFX, arrow in upper portion). Total FKHR protein is also denoted. (c) Detection of FOXO proteins in guinea pig and human neutrophils. The specificity of the antibody was determined by the detection of FOXO proteins in the neutrophils of different species. Guinea pig or human neutrophils were stimulated for the indicated times with fMLP (1 µm) or incubated with the control (DMSO) for 10 min. The lysates were loaded on a 7.5% SDS-PAGE, subjected to Western-blotting procedures. Controls lysates were included, consisting of the 3T3 mouse fibroblast cell line and a 293 cell line containing overexpressed FKHR-L1. The membrane was probed with the phosphospecific FKHR antibody (Ser 256). The phosphorylated FOXO proteins, FKHR-L1, FKHR, and AFX, which are detected in guinea pig and human neutrophil lysates, are designated by an arrowhead, arrow, and pentagon (at right), respectively.

 
FOXO transcription factors undergo phosphorylation in fMLP-stimulated neutrophils
FKHR is expressed in neutrophils and becomes phosphorylated at Ser 256 with fMLP stimulation (Fig. 1b) . In this experiment, phosphorylation of FKHR was detected at 15 s, and the peak of phosphorylation was observed between 0.5 and 3 min. A sustained level of phosphorylation was observed throughout the period of stimulation [Fig. 1b , lanes c–i, FKHR (Ser256)]. The phospho-specific FKHR (Ser 256) antibody also allowed the detection of other FOXO family members, phosphorylated FOXO4 (AFX, arrow in upper portion) and FOXO3a (FKHR-L1, top, pentagon). The basal level of AFX phosphorylation was higher than that of the other isoforms, but AFX exhibited enhanced phosphorylation at 0.5–1 min of stimulation. FKHR-L1 migrates at ~100 kD (Fig. 1b , top, pentagon). FKHR-L1 is also present in neutrophils and becomes phosphorylated upon fMLP stimulation of neutrophils. FKHR-L1, similar to FKHR, was phosphorylated on Ser 253 within 15 s, and the phosphorylation was sustained above basal levels. To determine the specificity of the phosphospecific FKHR antibody in detecting other FOXO proteins by Western analysis, the presence of these proteins was compared in neutrophils of two different species, guinea pigs and humans (Fig. 1c) . Neutrophils obtained from guinea pigs or humans were stimulated for 3, 5, and 10 min or incubated in the presence of the control (DMSO) for 10 min. fMLP stimulation led to intense phosphorylation of a 70–75 kD FKHR protein during 3–10 min of fMLP stimulation in guinea pig and human neutrophils (Fig. 1c , arrow). With DMSO incubation, the phosphorylation of FKHR was not present in human neutrophils, whereas basal activity was detected in the guinea pig neutrophils. A hyperphosphorylated species of FKHR, which migrates at approxiamtely 81 kD, is also detected in the guinea pig neutrophils. The hyperphosphorylated species was also seen in human neutrophil lysates in prolonged autoradiographic exposures of the data (data not shown). The 100-kD FKHR-L1 protein is detected in the guinea pig neutrophil lysates. Two controls verify the presence of this protein: serum-treated NIH/3T3 mouse fibroblast cell line lysate and 293 human kidney cell lysate, which was transfected with FKHR-L1 (Fig. 1c , arrowhead). An intensely phosphorylated band detected in the human neutrophils most likely represents AFX, but a control protein to identify this band was not available.

FOXO phosphorylation occurs through PI-3K- and MAPK-mediated pathways
The signaling pathways that contribute to the phosphorylation of FKHR and FKHR-L1 were determined using selective chemical inhibitors. Neutrophils were stimulated with fMLP in the absence or presence of chemical inhibitors of PI-3K (wortmannin and LY294002) or MAPK kinase (MEK; U1026 and PD90859). Wortmannin (200 nM) markedly decreased phosphorylation of FKHR and FKHR-L1 (Fig. 2a , lane d) at Ser 256 and Ser 253 to levels below those observed without inhibitors (Fig. 2a , lanes c, g, and j). Phosphorylation at these sites was also decreased, albeit to a lesser extent, with a different inhibitor of PI-3K, LY294002 (50 µM). Inhibition of PI-3K with LY294002 caused greater inhibition of FKHR-L1 than that of FKHR (Fig. 2a , lane h). It is interesting that the phosphorylation of FKHR was also dependent on MAPK signaling. Inhibition of MEK with U1026 (10 µM) decreased phosphorylation of FKHR (Fig. 2a , lane e) on Ser 256 but did not affect the basal phosphorylation at this site. The control compound U1024 (50 µM), used at a concentration five times higher than that for U1026, did not affect FKHR phosphorylation (Fig. 2a , lane f, and compare lanes g and j). A different inhibitor of MEK, PD90859, also inhibited the phosphorylation of FKHR, similar to that seen with U1026. A representative experiment of three independent studies is shown. The results of these experiments are summarized in Figure 2b .



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  		Figure 2. Determination of the signaling pathways that contribute to fMLP-mediated phosphorylation of FKHR in neutrophils. (a) PI-3K and MAPK signaling pathways contribute to the FOXO phosphorylation during fMLP stimulation. Neutrophils were preincubated for 30 min with DMSO (control) or with inhibitors of MEK, U1026, the inactive control U0124, or PD90859, or with inhibitors of PI-3K, wortmannin or LY294002. Stimulation was then conducted with fMLP, and DMSO was included in control conditions for the times indicated. Neutrophils were lysed, and the proteins were resolved by SDS-PAGE. Analysis by Western blotting was conducted using a phospho-specific FKHR antibody that detected phosphorylation of FKHR on Ser 256. The bottom arrow denotes the phosphorylated FKHR, whereas the pentagon (top) represents phosphorylated FKHR-L1, a forkhead family member. The membrane was stripped and reprobed with an antibody that specifically recognized AKT when phosphorylated on Ser 473. The membrane was washed extensively and then reprobed with an antibody that recognizes Rsk when phosphorylated on Ser 380. The blot was again washed extensively and probed with an antibody to total FKHR. (b) Summary of FOXO phosphorylation. Summary of three separate experiments in which FKHR phosphorylation 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 kinase reaction with FKHR-L1 protein, which was purified from E. coli-induced expression of GST-FKHR-L1. The shaded arrows (from top to bottom) represent hyperphosphorylated FKHR-L1 and FKHR, respectively, and the arrow represents p90 Rsk (pp90Rsk). The shaded pentagon shows a bacterial-contaminating protein derived from the bacterial expression and purification of the FKHR-L1 protein. The blot was stripped and probed with an antibody that recognizes total FKHR-L1. This experiment is a representative of three separate experiments.

 
Rsk-2 catalyzes the phosphorylation of FKHR-L1 in vitro
The observations shown in Figure 2a suggested that AKT and MAPK cascades mediated FKHR and FKHR-L1 phosphorylation on Ser 256 and Ser 253. The FKHR Ser 256 phosphorylation site is a good consensus site for RSK kinases (for a review, see ref. [42 ]). This led us to test whether RSK-2 was able to catalyze the phosphorylation of FKHR-L1. Rsk 2 was immunoprecipitated from control (DMSO) or fMLP-stimulated neutrophils. The immunoprecipitated RSK-2 was used in an in vitro kinase reaction with purified FKHR-L1. We found that RSK-2 catalyzed the phosphorylation of FKHR-L1. The phosphorylation of FKHR-L1 at Ser 253 was monitored through Western blotting using the phosphospecific FKHR (Ser 256) antibody that also detects phosphorylated FKHR-L1 (Fig. 2c) . Time-dependent phosphorylation of FKHR-L1 revealed a slower migrating hyperphosphorylated species (shaded arrow, top, ppFKHR-L1) initially detected at 3 min, but maximal activity was noted at 5 min of stimulation (Fig. 2c , lanes b and c). A barely detected, slower migrating hyperphosphorylated species of FKHR is seen at 3–5 min (Fig. 2c , lanes b and c, and shaded arrow, ppFKHR). High basal phosphorylation of Rsk occurs in the unstimulated condition with DMSO (Fig. 2c , lane a). A protein that migrates at ~80 kD (Fig. 2c , shaded pentagon) undergoes intense phosphorylation. This protein, found on a Coomassie-stained gel of the purified FKHR-L1 protein, most likely represents a bacterial contaminant or breakdown product. The blots were stripped and reprobed for total FKHR-L1 to determine protein loading.

Activated neutrophils coordinate the timing and location of Mcl-1: a survival factor Mcl-1 is rapidly induced
fMLP leads to robust activation of PI-3K- and MAPK-mediated cascades. Mcl-1, a Bcl-2 family member, has been shown to function downstream of both pathways [24 ]. Therefore, I examined neutrophils at different times of fMLP stimulation for the expression of the survival factor Mcl-1. In Figure 3a , Mcl-1 is detected in neutrophils from guinea pigs (upper) and humans (lower). An increase in the level of Mcl-1 expression is detected between 1 and 3 min of fMLP stimulation, and a decline in expression was detected at 10 min (Fig. 3a , upper). In a separate experiment, Mcl-1 expression in fMLP-stimulated neutrophils was detected at 1 h, whereas in unstimulated neutrophils, only basal levels of Mcl-1 were detected (data not shown). We repeated this experiment in human neutrophils and showed that Mcl-1 is detected as a 42/40-kD protein for which the expression increases during fMLP stimulation between 1 and 10 min (Fig. 3a , lower).



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  		Figure 3. Mcl-1 protein expression is induced rapidly in neutrophils stimulated with fMLP. (a) Mcl-1 protein expression in fMLP-stimulated neutrophils. Neutrophils (2x106) were stimulated with fMLP (1 µM) or incubated with DMSO (the control). Using Western-blotting procedures, Mcl-1 was detected as a 42/40-kDa protein with a mAb that recognized total Mcl-1. This experiment is representative of three separate experiments. To determine protein loading, the membrane was stripped and reprobed for expression of total FKHR. In the lower portion of Figure 3a, 1 x 106 human neutrophils were stimulated with fMLP (1 µm) for the times indicated. After Western-blotting procedures were performed, Mcl-1 was detected as a 42/40-kDa protein within human neutrophils when using the mAb that recognized total Mcl-1. (b) GST-FKHR-L1 protein association assay. In a representative experiment of the GST-FKHR-L1 protein association assay, lysates of 3 x 107 neutrophils were stimulated and incubated with GST-FKHR-L1 that had been coupled to glutathione-linked agarose beads. The immune complexes were resolved on a 15% gel and probed by Western blotting with a polyclonal antibody that recognizes phosphospecific FKHR. The blots were washed extensively and reprobed with a mAb that recognizes total Mcl-1. The arrow and arrowhead demonstrate phosphorylated FKHR and FKHR-L1, which are recognized by the phosphospecific FKHR antibody. The Mcl-1 antibody recognizes a single band (middle arrow) that migrates at approximately 42 kD. The blots were again washed extensively and probed with an antibody that recognized total AKT. (c) A control experiment was conducted in which the lysates of 3 x 107 neutrophils were stimulated with 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 complexes were resolved on a 10% gel and probed with a mAb to detect Mcl-1. To visualize the GST proteins loaded in each condition, the membrane was washed extensively, Western blotting procedures were performed, and the membrane was probed with an antibody that recognized GST. A company-provided mouse macrophage lysate is included as a positive control to identify Mcl-1. (d) FKHR interacts with Mcl-1 in vivo. This is a representative experiment in which a polyclonal FKHR antibody coupled to protein A agarose was used in an immunoprecipitation assay with 3 x 107 neutrophils that were stimulated with fMLP or incubated with the control DMSO. After SDS-PAGE, the proteins in the immune complexes were resolved on a 10% gel and probed with a mAb that recognizes Mcl-1. An arrow denotes a 42-kD protein that is detected after 10 min of stimulation with fMLP, which is not present in neutrophil lysates incubated with DMSO or in lysates in which IgG is substituted for FKHR. To determine the amount of immunoprecipitated protein present, the membrane was washed extensively and detected with an antibody to total FKHR.

 
Mcl-1 binds to a complex containing FKHR in vitro
As PI-3K and MAPK pathways regulate FKHR and Mcl-1, I next tested in stimulated neutrophils whether FKHR associated with Mcl-1. In an experiment using neutrophils stimulated for 1–10 min or unstimulated neutrophils (DMSO), I performed a GST pull-down assay. The immune complexes were examined for the presence of proteins that associated with FKHR-L1, using a GST-FKHR-L1 and a phosphorylation-defective construct (for construct descriptions, see ref. [28 ]). We tested whether Mcl-1 was contained in the immune complex with GST-FKHR-L1. In Figure 3b , Mcl-1 was found to coassociate with GST-FKHR-L1 in a manner that did not depend on stimulation. Lower amounts of Mcl-1 were observed with the GST-FKHR-L1 (triple mutant). The lower recovery of Mcl-1 with the triple mutant GST-FKHR-L1 may have occurred because of the inability of Akt-dependent kinases within the neutrophil lysate to phosphorylate the mutant FKHR-L1. The blots were then washed extensively and probed with an antibody that detected total AKT (bottom arrow). To rule out the nonspecific binding of proteins to the complex, the membrane was stripped and reprobed with an antibody to total MAPK. Total MAPK was not present in the immune complex (data not shown). The membrane then washed extensively and probed for Bcl-2, a prosurvival family member. Bcl-2 was not found in the complex (data not shown). A control experiment was performed to determine whether the binding of Mcl-1 to the immune complex was secondary to the presence of GST in the fusion protein. Neutrophils were stimulated for 10 min or incubated for 10 min with the control (DMSO) and were used in a pull-down assay with GST-FKHR-L1, the GST-FKHR-L1 (triple mutant), GST alone, or glutathione beads alone (Fig. 3c) . The presence of Mcl-1 in the immune complex was then determined. This experiment reveals that the binding of Mcl-1 depends on the presence of the FOXO protein, FKHR-L1, in the immune complex. A slight change in the mobility of Mcl-1 is detected in stimulated neutrophils as compared with unstimulated neutrophils (Fig. 3c , lane B). The mobility shift might suggest phosphorylation of Mcl-1 or a Mcl-1 protein of higher molecular density. The amount of Mcl-1 protein in the FOXO complex appears to be greater when the three Akt phosphorylation sites are intact (Fig. 3c , lanes B and I compared with lanes C and D; GST-FKHR-L1 protein, compared with the GST-FKHR-L1 triple mutant). To estimate protein loading, the GST proteins present in each condition were visualized after Western blotting and detection with a GST antibody (Fig. 3c , bottom panel). The protein staining reveals that the GST-FKHR-L1 and the GST-FKHR-L1 (triple mutant) proteins are present at similar levels, whereas a greater amount of the GST protein was present (Fig. 3c , bottom panel). The macrophage lysate included as a positive control for the identity of Mcl-1 was overexposed to visualize the protein (Fig. 3c , lane J).

To determine if the association between FKHR and Mcl-1 was found in stimulated neutrophils in vivo, two experiments were conducted in which FKHR was immunoprecipitated, and the immune complexes were detected for the presence of coimmunoprecipitated Mcl-1. In a representative experiment, a protein of low abundance that migrates at ~42 kD was seen at 10 min of fMLP stimulation. With DMSO incubation for 10 min or with mouse IgG, the 42-kD protein was not present (Fig. 3d , upper arrow). A protein of unknown identity migrates above 52 kD, is also detected with the Mcl-1 antibody, and shows increased abundance during fMLP stimulation (Fig. 3d , arrowhead). The reverse experiment was also conducted in which Mcl-1 was immunoprecipitated, and FKHR was detected after Western-blotting procedures. As a result of technical difficulties and the low recovery of immunoprecipitated Mcl-1, I could not conclusively determine that an interaction with FKHR was present.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study shows that FOXO transcription factors are present in neutrophils and undergo rapid phosphorylation in the presence of bacterial chemoattractants that may be found during infectious processes.

We show that fMLP stimulation targets the FOXO transcription factors leading to their phosphorylation through PI-3K- and through Rsk-2-mediated pathways. Prolonged MAPK and RSK activation may facilitate the induction by CREB of genes that affect the survival of cells [22 , 23 ]. Furthermore in neutrophils stimulated with fMLP, the activation of PI-3K and the phosphorylation of the downstream kinase AKT are pathways through which cell survival of a wide variety of cells is regulated (for reviews, see refs. [26 , 43 ]). We propose that both pathways converge on FKHR (FOXO1), FKHR-L1 (FOX03a), and AFX (FOX04) in neutrophils and promote their phosphorylation. The phosphorylation of FOXO family members promotes survival in neurons and other hematopoietic cells [28 , 44 ]. In this study, there is evidence that FKHR becomes phosphorylated in neutrophils in response to fMLP, an event that may mediate survival responses within these cells.

Early studies suggested that PI-3K/PKB activation was the major route through which the phosphorylation of FKHR on Ser 256 or on homologous sites in other family members occurred [28 , 45 46 47 48 49 ]. Recently, other kinases, such as the serum and glucocorticoid-induced kinases, a PI-3K-dependent kinase, and casein kinase 1, have been shown to preferentially phosphorylate FKHR on sites other than those phosphorylated by PKB/AKT [50 , 51 ]. These findings show that Rsk-2 can phosphorylate FKHR-L1 in vitro (Fig. 2c) and that inhibition of MAPK cascades with specific inhibitors suppresses the phosphorylation of FKHR in vivo (Fig. 2a) . These results suggest that PI-3K and MAPK contribute to the phosphorylation of FKHR. MAPK-mediated phosphorylation of FKHR may also target sites other than those phosphorylated by PKB/AKT. In overexpression studies, Rsk did not catalyze the phosphorylation of FKHR on PKB-targeted residues (24, 256, 319) in cells stimulated with insulin-like growth factor-1 or the phosphorylation of FKHR-L1 on Ser 316 in cells stimulated with insulin [48 , 50 ]. These findings suggest that Rsk-2 can phosphorylate FOXO family members in neutrophils. This implies that fMLP stimulation activates kinases downstream of MAPK other than Rsk, which may participate in assuring that FKHR is phosphorylated.

As PI-3K- and MAPK-activated Rsk have been implicated in cellular survival, I examined stimulated neutrophils for the expression of Mcl-1, which is a target of both pathways [24 ]. Mcl-1 is a Bcl-2 family member that has prosurvival properties in hematopoietic cells [52 ]. Mcl-1 was expressed constitutively in elicited neutrophils, but an increase in expression of Mcl-1 protein was detected during fMLP stimulation (Fig. 3a) . The transcriptional up-regulation of Mcl-1 has previously been suggested to correlate with phosphorylation via the MAPK/extracellular-regulated kinase pathway, whereas an increase in translation of Mcl-1 has been shown to depend on the PI-3K/AKT pathway [53 , 54 ]. The early increase in Mcl-1 expression detected at 3–5 min in this study may represent phosphorylation or stabilization of Mcl-1 protein (Fig. 3a) . Additionally, I found that the expression of Mcl-1 was greater at 1 h in stimulated 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 expression in a leukemia cell line, ML-1 [55 ]. It has been suggested that the neutrophil’s survival is dependent on the inducible expression of Mcl-1 [56 ]. These findings suggest that the pathways activated with fMLP stimulation of neutrophils may induce the expression of Mcl-1.

FKHR and Mcl-1 may be regulated not only by PI-3K and MAPK signaling but also in response to oxidative stresses that result as a consequence of the neutrophil’s production of reactive oxygen species [57 , 58 ]. In response to the respiratory burst that is activated by fMLP, neutrophils might relocalize FKHR from the nucleus to the cytoplasm. In immunocytochemical studies, FKHR was localized in the cytoplasm and was colocalized with AKT within neutrophils (unpublished).

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

We have provided additional evidence that in neutrophils activated by the bacterial chemoattractant fMLP, signaling through PI-3K and MAPK pathways leads to the phosphorylation of FOXO family members. Additionally, fMLP stimulation up-regulates the expression of Mcl-1, which is then able to bind a complex that contains FOXO proteins, a mechanism through which neutrophils may sequester FOXOs in the cytoplasm and promote their survival.

Most studies have found that fMLP stimulation does not delay apoptosis within neutrophils [61 , 62 ]. Studies using the antiapoptotic factor GM-CSF have shown that Janus tyrosine kinase-signal transducer and activator of transcription and PI-3K pathways cooperate in human neutrophils to mediate the survival response through Mcl-1 [63 , 64 ]. These studies have shown that fMLP stimulation of neutrophils leads to the activation of parallel pathways, which allow neutrophils to regulate the function of FOXO transcription factors and the survival factor Mcl-1. The phosphorylation of the FOXO family members may result in the inhibition downstream targets of FOXO such as TRAIL or p27kip1, factors shown to promote apoptosis in neutrophils and other hematopoietic cells [34 , 65 ]. The induction of Mcl-1 that occurs with fMLP stimulation may participate in maintaining mitochondrial function, organelles that have recently been shown to regulate the multiple functions of neutrophils [66 ]. Future studies will be required to determine the requirement for FOXOs in these functions in neutrophils. In summary, these studies have delineated a pathway, whereby fMLP stimulation regulates FOXO and Mcl-1, factors that are then found in a complex. The regulation of FOXOs and Mcl-1 is a mechanism through which fMLP stimulation may alter neutrophils’ survival and function during inflammatory conditions.


   ACKNOWLEDGEMENTS
 
These studies were supported by grants from the National Institutes of Health, K08 NS01922 (L. J. C.) and DK50015 (John Badwey, Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Boston, MA). I thank Drs. Anne Brunet and Michael Greenberg for kindly providing reagents. I also thank Drs. Paul Allen, John Badwey, and Anne Brunet for critical reading of the manuscript and stimulating discussions. This research was conducted in the laboratory of and with the continuing support of Dr. John A. Badwey.

Received January 15, 2003; revised May 15, 2003; accepted May 27, 2003.


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