Involvement of phosphatidylinositol 3-kinase γ in neutrophil apoptosis

Cellular Signalling 15 (2003) 225 – 233 www.elsevier.com/locate/cellsig

Involvement of phosphatidylinositol 3-kinase g in neutrophil apoptosis Kuang-Yao Yang a, John Arcaroli b, John Kupfner b, Todd M. Pitts b, Jong Sung Park b, Derek Strasshiem b, Reury-Perng Perng a, Edward Abraham b,* a

Chest Department, Taipei Veterans General Hospital, School of Medicine, National Yang-Ming University, Taipei, Taiwan b Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, Mail Code C-272, 4200 East Ninth Ave, Denver, CO 80262, USA Received 27 March 2002; accepted 7 August 2002

Abstract Although phosphoinositide 3-kinases (PI3-K) are known to participate in anti-apoptotic pathways, their importance in modulating neutrophil apoptosis in vivo has not been examined. In these studies, we used neutrophils from mice lacking the PI3-Kg isoform (PI3Kg / ) to determine the role that PI3-Kg occupies in neutrophil apoptosis under in vivo conditions. We found that neutrophil apoptosis under basal and LPS-stimulated conditions was increased in PI3-Kg / mice compared to that present in control PI3Kg+/+ animals. Neutrophils from PI3-Kg / mice demonstrated decreased amounts of active, serine 473 phosphorylated Akt, phosphorylated CREB, and diminished nuclear translocation of NF-nB. Levels of the CREB-dependent anti-apoptotic protein Mcl-1 and of the NF-nB-dependent anti-apoptotic mediator Bcl-xL were significantly decreased in PI3-Kg / neutrophils. In contrast, PI3Kg / neutrophils contained diminished amounts of phosphorylated, inactive forms of the pro-apoptotic mediators, Bad, FKHR, and GSK-3h. These results demonstrate that PI3-Kg directly participates in multiple in vivo pathways involved in regulating neutrophil apoptosis. D 2003 Elsevier Science Inc. All rights reserved. Keywords: AKT; BCL-2; MCL-1; CREB; Bad

1. Introduction Phosphoinositide 3-kinases (PI3-K) and the downstream serine/threonine kinase Akt/protein kinase B have a central role in modulating neutrophil activation, chemotaxis, and apoptosis [1– 7]. PI3-Ks catalyze the addition of a phosphate molecule to the three positions of the inositol ring of phosphoinositides (PtdIns). Four different lipid products are generated by the different PI3-K members: the singly phosphorylated form PtdIns-3-P; the doubly phosphorylated forms PtdIns-3,4-P2 and PtdIns-3,5-P2; and the triply phosphorylated form PtdIns-3,4,5-P3. Akt is the best characterized target of PtdIns-3,4-P2 and PtdIns-3,4,5-P3 [1,4,5,8]. The pleckstrin homology (PH) domain of Akt binds phosphoinositides, with activation of Akt dependent on phosphorylation of threonine-308 and serine-473.

* Corresponding author. Tel.: +1-303-315-3397; fax: +1-303-315-5632. E-mail address: [email protected] (E. Abraham).

These phosphorylation events are modulated by phosphatidylinositol-dependent kinases (PDK1 and PDK2), following binding of phosphoinositides to the Akt PH domain [8]. PI3-K is a heterodimeric complex, comprising a p110 catalytic subunit, of which there are four characterized isoforms (a, h, g, and y). The type IA PI3-Ks, p110a, p110h, and p110y, associate with the p85 family of regulatory subunits, but type IB p110g binds to a p101 adaptor molecule. Whereas type IA PI3-Ks are activated by interaction with tyrosine-phosphorylated molecules, p110g is activated by engagement of G-protein-coupled receptors. In addition, recent data also demonstrate that PI3-Kg can be activated by pathways independent of G-proteins such as those initiated by exposure of neutrophils to LPS [9]. Cell-based assays, using wortmannin or LY294002 as potent, but not isoform-specific inhibitors of PI3-K, demonstrated that PI3-K has a pivotal role in neutrophil activation [10 – 16]. In particular, such studies showed that PI3-K, through phosphorylating and activating Akt, occupies a central position in modulating neutrophil

0898-6568/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. PII: S 0 8 9 8 - 6 5 6 8 ( 0 2 ) 0 0 0 6 3 - 3

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chemotaxis and respiratory burst. Activation of the PI3-K/ Akt pathway also has potent anti-apoptotic effects through (a) preventing release of cytochrome c from mitochondria; (b) phosphorylating and thereby preventing activation of the pro-apoptotic Bcl-2 family member Bad; (c) increasing transcription of the anti-apoptotic proteins Mcl-1 and Bcl-2 through activating the transcriptional regulatory factor CREB; (d) inhibiting the death protease caspase9; and (e) activating NF-nB, resulting in enhanced transcription of anti-apoptotic genes such as Bcl-xL, A1, and A20 [17 – 24]. PI3-Kg appears to have an important role in modulating neutrophil functions in vivo. Recent studies in mice lacking functional PI3-Kg showed that neutrophils from these animals are unable to produce PtdIns-3,4,5-P3 and activate Akt when stimulated with G-protein-coupled receptor agonists such as fMLP, C5a, or IL-8 [25 – 27]. In PI3-Kg knockout mice, neutrophil respiratory burst was severely diminished, migration of neutrophils toward chemokines in vitro and in vivo was reduced, and clearance of Escherichia coli or S. aureus from the peritoneum was impaired [25 – 27]. Additionally, nuclear translocation of NF-nB and expression of proinflammatory cytokines, such as MIP-2 and TNF-a, produced by exposure of neutrophils to LPS was reduced in PI3-Kg / cells compared to PI3-Kg+/+ controls [9]. Endotoxemia induced neutrophil accumulation in the lungs, activation of NF-nB in lung neutrophils, and lung injury also were reduced in PI3-Kg / mice compared to PI3-K+/+ controls [9]. Although PI3-K is known to participate in anti-apoptotic pathways, the importance of PI3-K-dependent events in modulating neutrophil apoptosis in vivo has not been examined. As discussed above, in vivo neutrophil functions are clearly affected when the PI3-Kg isoform is absent. In the present experiments, we specifically examined the role that PI3-Kg plays in neutrophil apoptosis under basal and activated in vivo conditions. These experiments demonstrate that PI3-Kg, presumably through its effects on Akt activation, directly participates in multiple pathways involved in regulating neutrophil apoptosis.

2. Materials and methods 2.1. Mice Male C57/BL6 (PI3Kg+/+) mice, 8– 12 weeks of age, were purchased from Harlan Sprague –Dawley (Indianapolis, IN). PI3Kg / mice on a C57/BL6 background were bred in the University of Colorado Health Sciences Center animal facility. The homozygous state for the PI3Kg / mice was confirmed by PCR analysis of mouse tail-derived DNAs. The mice were kept on a 12-h light/dark cycle with free access to food and water. All experiments were conducted in accordance with institutional review boardapproved protocols.

2.2. Materials Isoflurane was obtained from Abbott Laboratories (Chicago, IL). E. coli 0111:B4 endotoxin, collagenase, and Dnase were obtained from Sigma (St. Louis, MO). RPMI 1640/25 mM HEPES/L-glutamine was obtained from BioWhittaker Products (Walkersville, MD), while foetal bovine serum (FBS) and penicillin/streptomycin were purchased from Gemini Bioproducts (Calabasas,CA). Percoll was purchased from Amersham-Pharmacia (Piscataway, NJ). BCA Protein Assay Reagent was purchased from Pierce (Rockford, IL). The Annexin V – FITC Apoptosis Detection Kit was obtained from R&D Systems (Minneapolis, MN). Custom Cocktail antibodies and columns for neutrophil isolation were purchased from Stem Cell Technologies (Vancouver, BC). 2.3. Model of endotoxemia The model of endotoxemia induced lung injury was used as reported previously [28 – 30]. Mice received an i.p. injection of LPS at dose of 1 mg/kg in 0.1 ml phosphate buffered saline (PBS). This dose of LPS produces acute neutrophilic alveolitis, histologically consistent with acute lung injury [29,31,32]. 2.4. Isolation of neutrophils Lung or bone marrow neutrophils were purified from intraparenchymal pulmonary or bone marrow cell suspensions. To obtain the bone marrow cell suspension, the femur and tibia of a mouse were flushed with 5 ml RPMI 1640/ penicillin/streptomycin and the cells passed through a glass wool column. Lung neutrophils were isolated from intraparenchymal pulmonary cell suspensions, prepared as previously described by our laboratory [28 – 30]. In brief, the chest of the mouse was opened and the lung vascular bed flushed with 2 –3 ml of chilled (4 jC) PBS injected into the right ventricle. Lungs were then excised, avoiding the paratracheal lymph nodes and thymus, and washed twice in RPMI 1640/25 mM HEPES/L-glutamine supplemented with penicillin/streptomycin. The excised lungs were minced finely, and the tissue pieces placed in RPMI 1640 medium containing 5% FBS, 20 U/ml collagenase, and 1 Ag/ml DNase. Following incubation for 60 min at 37 jC, any remaining intact tissue was disrupted by passage through a 21-gauge needle. Tissue fragments and the majority of dead cells were removed by rapid filtration through a glass wool column, and cells were collected by centrifugation. The cell pellets from the intraparenchymal pulmonary or bone marrow cell suspensions were resuspended in RPMI 1640/5% FCS and then incubated with 10 Al of primary antibodies specific for cell surface markers F4/80, CD4, CD45R, CD5, and TER119 for 15 min at 4 jC. This custom cocktail (Stem Cell Technologies) is specific for T and B

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cells, RBC, monocytes and macrophages. After 15-min incubation, 100 Al of anti-biotin tetrameric antibody complexes was added, and the cells incubated for 15 min at 4 jC. Following this, 60 Al of colloidal magnetic dextran iron particles was added to the suspension and incubated for 15 min at 4 jC. The entire cell suspension was then placed onto a column, surrounded by a magnet. The T cells, B cells, RBC, monocytes, and macrophages were captured in the column, allowing the neutrophils to pass through by negative selection methods. The neutrophil suspension was then layered on 50% Percoll, centrifuged at 3000 rpm for 15 min, and the neutrophil layer was collected. Viability, as determined by trypan blue exclusion, was consistently greater than 98%. Neutrophil purity, as determined by Wright’s stained cytospin preparations, was greater than 99%. 2.5. EMSA Nuclear extracts were prepared as previously described [29,30,33,34]. Isolated bone marrow neutrophils were incubated for 15 min in buffer A (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl, pH 7.9). After the cytoplasm was removed from the nuclei by 15 passages through a 25-gauge needle, the nuclei was collected by centrifugation at 600  g for 6 min at 4 jC. The nuclear pellet was incubated on ice for 15 min in buffer C (20 mM HEPES [pH 7.9], 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol), after which the extract was centrifuged at 4 jC for 10 min at 12,000  g. The supernatant was collected, divided into aliquots, and stored at 86 jC. Protein concentration was determined by using the Coomassie-Plus Protein Assay Reagent (Pierce) standardized to bovine serum albumin, according to the manufacturer’s protocol. Nuclear translocation of NF-nB was determined as described previously by our laboratory [29,30,33 – 35]. The nB DNA sequence of the immunoglobulin gene was used. Synthetic double-stranded sequences (with enhancer motifs underlined) were fill in labeled with a-32P-dATP using Sequenase DNA polymerase: nB: 5V-TTTTCGAGCTCGGGACTTTCCGAGC-3V 3V-GCTCGAGCCCTGAAAGGCTCGTTTT-5V DNA binding reaction mixtures of 20 Al contained 10 Ag nuclear extract, 10 mM Tris – HCl pH 7.5, 50 mM EDTA, 0.5 mM dithiothreitol, 1 mM MgCl2, 4% glycerol, 0.08 Ag poly (dI –dC)
poly (dI –dC), and 0.7 fmol 32P-labeled double-stranded oligonucleotide. After the samples were incubated at room temperature for 20 min, they were loaded onto a 4% polyacrylamide gel (acrylamide/bisacrylamide 80:1, 2.5% glycerol in Tris-borate-EDTA) and run at 10 V/ cm. Each gel was then dried and subjected to autoradiography.

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2.6. Western blot analysis Whole cell extracts from bone marrow neutrophils were collected from cells denatured in ice cold lysis buffer (50 mM HEPES, 150 mM NaCl, 10% Glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 1 mM Na3 vanadate, 10 mM Na pyrophosphate, 10 mM NaF, 300 AM p-nitrophenyl phosphate, 1 mM phenylmethylsulfonyl fluoride, 10 Ag/ml leupeptin, 10 Ag/ml aprotinin, pH 7.3) for 15 min. The protein concentration of each sample was assayed using the BCA protein assay kit standardized to bovine serum albumin, according to manufacturer’s protocol. For Western blots, 70 Ag of protein from whole cell or nuclear extracts was loaded on a 10% Tris – HCl SDS polyacrylamide gel. Protein was electrotransferred to a nitrocellulose membrane and then blocked with 5% nonfat dry milk, 20 mM Tris buffered saline, with 0.1% Tween. After blocking, the membrane was incubated overnight at 4 jC with a rabbit polyclonal specific primary antibody: antiA20 (Imgenex, CA), anti-A1, anti-Bcl-2, anti-Bcl-xL, antiMcl-1, anti-NF-nB p65, anti-NF-nB p50 (Santa Cruz Biotechnology, CA), anti-phospho-Akt (Ser-473), anti-Akt, anti-phospho-FKHR (Ser-256), anti-FKHR, anti-phosphoBad (Ser136), anti-Bad, anti-phospho-GSK-3h (Ser 9) (Cell Signaling Technology, Beverly, MA), anti-GSK-3h (BD Biosciences), and anti-serine 133-phosphorylated CREB, anti-CREB (Upstate Biotechnology, Lake Placid, NY), using a dilution of 1:1000 followed by horseradish peroxidase-coupled secondary antibody at a dilution of 1:2000. After washing five times, bands were detected using chemiluminescence Western blotting detection reagents (ECL Detection System, Amersham-Pharmacia). Densitometry was performed using chemiluminescence system and analysis software (BioRad, Hercules, CA). 2.7. Flow cytometry Annexin V assays were performed using the manufacturer’s protocol (R&D Systems). Briefly, intraparenchymal pulmonary or bone marrow neutrophil populations were collected, counted, and 5  105 cells resuspended in the binding buffer. Propidium iodide and fluorescein-conjugated annexin V were added, and the reaction was stopped after 15 min. Samples were analysed using a BeckmanCoulter XL (Coulter, Miami, FL). Gates were adjusted to include the neutrophil population using forward scatter-side scatter plots, and 10,000 events in this gate were analysed. For analysis of apoptosis, results are presented as the percentage of cells stained for annexin V, but not for propidium iodide. 2.8. Statistical analysis To limit variability and provide appropriate controls for each experimental condition, the entire group of animals

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Fig. 1. Decreased amounts of serine 473 phosphorylated p-Akt and serine 133 phosphorylated CREB (p-CREB) are present in PI3-Kg / vs. PI3-Kg+/+ neutrophils. Additionally, PI3-Kg / neutrophils demonstrate reduced nuclear translocation of NF-nB (using EMSA) as well as decreased nuclear concentrations of the p65 and p50 subunits of NF-nB (as detected with Western blots) compared to PI3-Kg+/+ neutrophils. Representative gels are shown in (A). The histograms (B) show the ratio of phosphorylated to total Akt in total cellular extracts as well as amounts of nuclear p50, p65, and serine 133 phosphorylated CREB (p-CREB) in neutrophils obtained from three to six separate groups of PI3-Kg / or PI3-Kg+/+ mice, with six mice in each group. *p < 0.05 compared with levels in PI3-Kg+/+ neutrophils.

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was prepared and studied at the same time. For each experimental condition, mice in all groups had the same birth date and had been housed together. Separate groups of mice (n = 3 – 9 per group) were used for Western blotting and flow cytometry analysis. Data from three to six groups of mice were combined for calculating means F S.E.M. and statistically significant differences, using the Student’s t-test for comparisons between two groups. p < 0.05 was considered significant.

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3. Results 3.1. Involvement of PI3-Kc in neutrophil apoptosis Increased percentages of apoptotic cells were present among bone marrow neutrophils from PI3-Kg / mice compared to control PI3-Kg+/ + mice. In the PI3Kg+/ + animals, 2.1 F 0.1% of bone marrow neutrophils were apoptotic vs. 6.1 F 1.3% in PI3-Kg / mice ( p < 0.01).

Fig. 2. Decreased amounts of the phosphorylated inactive forms, but unchanged total levels of the pro-apoptotic proteins Bad, Forkhead (FKHR), and GSK-3h, are present in PI3-Kg / compared to PI3-Kg+/+ neutrophils. Representative Western gels are shown in (A). The histograms in (B) show the ratio of phosphorylated to total protein for Bad, FKHR, and GSK-3h in neutrophils obtained from three to six separate groups of PI3-Kg / or PI3-Kg+/+ mice, with six mice in each group. *p < 0.05 compared with levels in PI3-Kg+/+ neutrophils.

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The absence of PI3-Kg also affected neutrophil apoptosis in extra-medullary sites. Among lung neutrophils from PI3Kg+/ + mice, the percentage of apoptotic cells was 31.5 F 5.1%. In contrast, in PI3-Kg / mice, 60.0 F 15.0% of lung neutrophils were apoptotic ( p < 0.05 vs. PI3-Kg+/+).

To determine if these PI3-Kg-dependent alterations in neutrophil apoptosis continued to be present in neutrophils elicited to an inflammatory focus, we administered endotoxin to control and PI3-Kg knockout mice and then examined apoptosis among neutrophils that accumulate in

Fig. 3. Levels of anti-apoptotic proteins in PI3-Kg / and PI3-Kg+/+ neutrophils. Representative Western gels are shown in (A). The histograms in (B) show amounts of the anti-apoptotic mediators in neutrophils obtained from three to six separate groups of PI3-Kg / or PI3-Kg+/+ mice, with six mice in each group. *p < 0.05 compared with levels in PI3-Kg / neutrophils.

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the lungs. In control PI3-Kg+/ + mice, 15.9 F 5.4% of the endotoxemia elicited lung neutrophils were apoptotic. In contrast, the percentage of apoptotic lung neutrophils after endotoxemia was significantly increased to 44.9 F 4.5% ( p < 0.001 vs. PI3-Kg+/ + controls). 3.2. PI3-Kc contributes to the activation of Akt, NF-nB, and CREB in neutrophils All four isoforms of PI3-K appear to be capable of resulting in the phosphorylation and activation of Akt, the kinase immediately downstream of PI3-K [6]. To determine the relative importance of PI3-Kg in modulating Akt activation in neutrophils, we examined levels of serine 473 phosphorylated Akt among bone marrow neutrophils from PI3Kg / and PI3-Kg+/+ mice (Fig. 1A and B). Increased amounts of phosphorylated Akt were found in PI3-Kg+/+ neutrophils ( p < 0.05 vs. PI3-Kg / ), indicating that under basal conditions PI3-Kg is involved in Akt activation. Because Akt has anti-apoptotic properties due, at least in part, to enhancing expression of anti-apoptotic mediators under the regulatory control of the transcriptional factors CREB and NF-nB [17 –24], we postulated that the decrease in Akt activation in PI3-Kg / neutrophils would be associated with diminished activation of these transcriptional factors. To examine this issue, we determined amounts of transcriptionally active serine 133 phosphorylated CREB as well as of NF-nB translocating to the nucleus in neutrophils from PI3-Kg / and PI3-Kg+/+ mice (Fig. 1A and B). As shown in Fig. 1, NF-nB activation was decreased in PI3-Kg / bone marrow neutrophils compared to those from control PI3-Kg+/+ mice ( p < 0.05). In PI3-Kg / neutrophils, there was diminished nuclear localization of both p65 and p50 subunits of NF-nB compared to that found in PI3-Kg+/+. Similarly, activation of CREB, as determined by amounts of the serine 133 phosphorylated form, was decreased in PI3-Kg / neutrophils ( p < 0.05 vs. levels in PI3-Kg+/+ neutrophils). 3.3. PI3-Kc is involved in modulating pro- and antiapoptotic mediators in neutrophils In the above experiments, we found diminished activation of Akt, NF-nB, and CREB in neutrophils from PI3-Kg / mice. In addition to participating in activation of NF-nB and CREB, Akt also participates in other anti-apoptotic pathways including phosphorylation and inactivation of the pro-apoptotic factors Bad, Forkhead (FKHR), and GSK-3h [36 – 41]. Probably reflecting decreased PI3-Kg-dependent activation of Akt, phosphorylated levels of these pro-apoptotic factors were diminished in PI3-Kg / neutrophils compared to those present in control, PI3-Kg+/+ cells (Fig. 2). Both NF-nB and CREB are involved in regulating the transcription of anti-apoptotic mediators. In particular, antiapoptotic proteins under the regulatory control of NF-nB include members of the Bcl-2 family, such as A1 and Bcl-

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xL, as well as the zinc finger protein A20 [7 –14]. CREB is involved in modulating transcription of Mcl-1, a Bcl-2 family member important in neutrophil survival [22]. Expression of Bcl-2, which also has anti-apoptotic properties, appears to be regulated by both NF-nB and CREB. As shown in Fig. 3, levels of Bcl-xL and Mcl-1 were decreased in neutrophils from PI3-Kg / mice. In contrast, there were no differences in the expression of Bcl-2, A1, or A20 between PI3-Kg / and PI3-Kg+/+ neutrophils.

4. Discussion The present studies show that the PI3-Kg isoform exerts significant anti-apoptotic effects in neutrophils under both basal and stimulated conditions. In these experiments, decreased levels of the active, serine 473 phosphorylated form of Akt were present in unstimulated PI3-Kg / neutrophils. Akt occupies a central role in several antiapoptotic pathways, including activation of the transcriptional factors CREB and NF-nB, that then are directly involved in regulating expression of anti-apoptotic proteins such as Mcl-1 and Bcl-xL [7 – 14]. Consistent with the importance of Akt in these intracellular events, diminished phosphorylation of CREB and nuclear translocation of NFnB, as well as decreased expression of Mcl-1 and Bcl-xL, were found in PI3-Kg / neutrophils. In addition to its effects in activating NF-nB- and CREBdependent transcription of anti-apoptotic mediators, Akt also participates in anti-apoptotic pathways through phosphorylating and inactivating several important pro-apoptotic proteins such as Bad, Forkhead (FKR), and GSK-3h [36 – 41]. In the present experiments, decreased amounts of the phosphorylated forms of these pro-apoptotic mediators were found in PI3-Kg / neutrophils. Bad is a pro-apoptotic member of the Bcl-2 family [36] that binds to both Bcl-2 and Bcl-xL, thereby inhibiting their anti-apoptotic effects. Akt-mediated phosphorylation of Bad on serine 136 results in cytoplasmic sequestration of Bad through formation of Bad/14-3-3 protein complexes and also liberates Bcl-2 and Bcl-xL to enhance cell survival [37,42]. Similarly, recent studies have shown that Akt can phosphorylate all three isoforms of the pro-apoptotic transcriptional factor Forkhead, i.e. FKHR, AFX, and FKHRL1, resulting in sequestration of these proteins through interaction with 14-3-3 [38,39,43,44]. Recent reports show that Akt-dependent phosphorylation of GSK-3h results in improved cell survival, presumably through interfering with GSK-3hdependent alterations of mitochondrial function [40,41]. The presence of consistent alterations in multiple downstream events under the regulatory control of Akt suggests that the primary mechanism by which PI3-Kg modulates neutrophil apoptosis is through activation of Akt, rather than through affecting independent events further downstream. Levels of the anti-apoptotic proteins Bcl-xL and Mcl-1 were decreased in PI3-Kg / neutrophils. Both Bcl-xL

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and Mcl-1 are members of the Bcl-2 family and exert their anti-apoptotic effects, at least in part, through binding to and inactivating the pro-apoptotic factor Bax [42,43]. However, while expression of Bcl-xL is regulated by NF-nB [44], that of Mcl-1 depends on phosphorylation and activation of CREB [22]. The parallel changes in levels of Bcl-xL and Mcl-1 in PI3-Kg / neutrophils therefore indicate that the observed decreases in phosphorylation of CREB and nuclear translocation of NF-nB in the absence of PI3-Kg in neutrophils are functionally significant because they are associated with diminished expression of proteins under the regulatory control of each of these transcriptional factors. Despite the fact that nuclear translocation of NF-nB was decreased in PI3-Kg / neutrophils, several anti-apoptotic factors previously described to be under the regulatory control of this transcriptional factor did not demonstrate uniform decreases in expression. In particular, levels of A1, A20, and Bcl-2 were similar in PI3-Kg / and PI3-Kg+/+ neutrophils. These results would suggest that transcriptional factors other than NF-nB, and not affected by PI3-Kg or Akt, are involved in modulating the expression of these antiapoptotic molecules. Consistent with this, the promoter regions for A1, A20, and Bcl-2 have been shown to contain binding sites for transcriptional factors in addition to NF-nB [21,45,46]. In previous studies examining the role of NF-nB in modulating neutrophil apoptosis, we found that inhibition of its nuclear translocation did not completely prevent endotoxemia-induced decreases in lung neutrophil apoptosis. In those experiments, expression of Mcl-1, an antiapoptotic factor under the transcriptional regulatory control of CREB, appeared to be involved in inhibiting neutrophil apoptosis under the in vivo conditions examined. In vitro experiments, including those utilizing overexpression or specific blockade of Mcl-1, have also demonstrated that Mcl-1 is a major anti-apoptotic factor in neutrophils [42,43,47,48]. The present results, showing that PI3-Kginduced decreases in Mcl-1 expression were directly associated with increased neutrophil apoptosis, are also consistent with a central anti-apoptotic role for Mcl-1. Previous reports using PI3-Kg / mice showed that neutrophil chemotaxis and activation, including oxidative burst, in response to agonists interacting with G-proteincoupled receptors, were dependent on PI3-Kg [25 – 27]. Additionally, recent evidence has shown that PI3-Kg also plays a role in other pathways leading to neutrophil activation such as those dependent on toll-like receptors (TLR) and initiated by LPS exposure. For example, phosphorylation of Akt and nuclear translocation of NF-nB were decreased in PI3-Kg / neutrophils cultured with endotoxin as compared to PI3-Kg+/+ cells [9]. In a model of endotoxemia-induced acute lung injury similar to that used in the present experiments, the number of neutrophils accumulating in the lungs of PI3-Kg / mice was reduced compared to those found in PI3-Kg+/+ controls [9]. While decreases in neutrophil chemotaxis dependent on

PI3-Kg / have been postulated as a mechanism explaining the diminished numbers of pulmonary neutrophils after endotoxemia, the present experiments suggest an additional explanation. Because apoptotic neutrophils are rapidly cleared from the lungs by macrophage-dependent and other mechanisms, accelerated reduction of neutrophil numbers would be expected to occur under conditions when greater percentages of neutrophils are apoptotic, as from what occurs when the PI3-Kg isoform is lacking. Although previous studies [1– 7] using isoform nonspecific inhibitors of PI3-K had shown that these kinases had important anti-apoptotic effects in multiple cell types, the present studies extend those findings by demonstrating a fundamental role for the PI3-Kg isoform in modulating neutrophil apoptosis in vivo. These experiments show that PI3-Kg is involved in regulating neutrophil apoptosis not only under basal conditions, but also when neutrophils are involved in acute inflammatory responses such as those due to endotoxemia. Such results, as well as previous studies that demonstrated a central role for PI3-Kg in modulating neutrophil chemotaxis and oxidative burst [25 –27], underline the importance of the PI3-Kg isoform in multiple in vivo pathways involved in neutrophil activity and survival.

Acknowledgements This work was supported in part by National Institutes of Health grants HL 62221 and PO1 HL68743 (E. Abraham).

References [1] Rameh LE, Cantley LC. J Biol Chem 1999;274:8347 – 50. [2] Cross RG, Scheel-Toellner D, Henriquez NV, Deacon E, Salmon M, Lord JM. Exp Cell Res 2000;256:34 – 41. [3] Wymann MP, Sozzani S, Altruda F, Mantovani A, Hirsch E. Immunol Today 2000;21:260 – 4. [4] Datta SR, Brunet A, Greenberg ME. Genes Dev 1999;13:2905 – 27. [5] Kandel ES, Hay N. Exp Cell Res 1999;253:210 – 29. [6] Khwaja A. Nature 1999;401:33 – 4. [7] Coffer PJ, Geijsen N, M’Rabet L, Schweizer RC, Maikoe T, Raaijmakers JA, et al. Biochem J 1998;329:121 – 30. [8] Toker A, Newton AC. J Biol Chem 2000;275:8271 – 5. [9] Yum HK, Arcaroli J, Kupfner J, Shenkar R, Penninger JM, Sasaki T, et al. J Immunol 2001;167:6601 – 8. [10] Naccache PH, Levasseur S, Lachance G, Chakravarti S, Bourgoin SG, McColl SR. J Biol Chem 2000;275:23636 – 42. [11] Manna SK, Aggarwal BB. FEBS Lett 2000;473:113 – 8. [12] Rane MJ, Coxon PY, Powell DW, Webster R, Klein JB, Ping P, et al. J Biol Chem 2001;276:3517 – 23. [13] Klein JB, Rane MJ, Scherzer JA, Coxon PY, Kettritz R, Mathiesen JM, et al. J Immunol 2000;164:4286 – 91. [14] Thelen M, Difichenko SA. Ann NY Acad Sci 1997;832:368 – 82. [15] Pellegatta F, Chierchia SL, Zocchi MR. J Biol Chem 1998;273: 27768 – 71. [16] Nishioka H, Hiriuchi H, Arai H, Kita T. FEBS Lett 1998;441:63 – 6. [17] Madrid LV, Wang CY, Guttridge DC, Schottelius AJG, Baldwin AS, Mayo MW. Mol Cell Biol 2000;20:1626 – 38. [18] Jones RG, Parsons M, Bonnard M, Chan VSF, Yeh WC, Woodget JR, et al. J Exp Med 2000;191:1721 – 33.

K.-Y. Yang et al. / Cellular Signalling 15 (2003) 225–233 [19] Romashkova JA, Makarov SS. Nature 1999;401:86 – 90. [20] Ardeshna KM, Pizzey AR, Devereux S, Khwaja A. Blood 2000;96: 1039 – 46. [21] Pugazhenthi S, Nesterova A, Sable C, Heidenreich KA, Boxer LM, Heasley LE, et al. J Biol Chem 200;275:10761 – 6. [22] Wang JM, Chao JR, Chen W, Kuo ML, Yen JJ, Yang-Yen HF. Mol Cell Biol 1999;19:6195 – 206. [23] Liu Q, Sasaki T, Kozieradzki I, Wakeham A, Itie A, Dumont DJ, et al. Genes Dev 1999;13:786 – 91. [24] Ward C, Chilvers ER, Lawson MF, Pryde JG, Fujihara S, Farrow SN, et al. J Biol Chem 1999;274:4309 – 18. [25] Sasaki T, Irie-Sasaki J, Jones RG, Oliveira-dos-Santos AJ, Stanford WL, Bolon B, et al. Science 2000;287:1040 – 6. [26] Li Z, Jiang H, Xie W, Zhang Z, Smrcka AV, Wu D. Science 2000;287: 1046 – 9. [27] Hirsh E, Katanaev VL, Garianda C, Azzolino O, Pirola L, Silengo L, et al. Science 2000;287:1049 – 53. [28] Parsey MV, Tuder RM, Abraham E. J Immunol 1998;160:1007 – 13. [29] Abraham E, Carmody A, Shenkar R, Arcaroli J. Am J Physiol, Lung Cell Mol Physiol 2000;279:L1137 – 45. [30] Abraham E, Arcaroli J, Shenkar R. J Immunol 2000;166:522 – 30. [31] Faggioni R, Gatti S, Demitri MT, Delgado R, Echtenacher B, Gnocchi P, et al. J Lab Clin Med 1994;123:394 – 9. [32] Gatti S, Faggioni R, Echtenacher B, Ghezzi P. Clin Exp Immunol 1993;191:456 – 61. [33] Shenkar R, Abraham E. Am J Respir Cell Mol Biol 1997;16: 145 – 52. [34] Shenkar R, Schwartz MD, Terada LS, Repine JE, McCord J, Abraham E. Am J Physiol 1996;270:L729 – 35.

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[35] Le Tulzo Y, Shenkar R, Kaneko D, Moine P, Fantuzzi G, Dinarello CA, et al. J Clin Invest 1997;99:1516 – 25. [36] Del Peso L, Gonzalez-Garcia M, Page C, Herrera R, Nunez G. Science 1997;278:687 – 9. [37] Pastorino JG, Tafani M, Farber JL. J Biol Chem 1999;274:19411 – 6. [38] Kops GJ, de Ruiter ND, De Vries-Smits AM, Powell DR, Bos JL, Burgering BM. Nature 1999;398:630 – 4. [39] Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, et al. Cell 1999;96:857 – 68. [40] Pap M, Cooper GM. J Biol Chem 1998;273:19929 – 32. [41] Beitner-Johnson D, Rust RT, Hsieh TC, Millhorn DE. Cell Signal 2001;13:23 – 7. [42] Zhou P, Qian L, Bieszczad CK, Noelle R, Binder M, Levy NB, et al. Blood 1998;92:3226 – 39. [43] Sato T, Hanada M, Bodrug S, Irie S, Iwama N, Boise LH, et al. Proc Natl Acad Sci U S A 1994;91:9238 – 42. [44] Lee HH, Dadgostar Q, Cheng J, Shu J, Cheng G. Proc Natl Acad Sci U S A 1999;96:9136 – 41. [45] Wang CY, Guttridge DC, Mayo MW, Baldwin AS. Mol Cell Biol 1999;19:5923 – 9. [46] Hu X, Yee E, Harlan JM, Wong F, Karsan A. Blood 1998;92: 2759 – 65. [47] Zhou P, Qian KM, Kozopas KM, Craig RW. Blood 1997;89: 630 – 43. [48] Epling-Burnette PK, Zhong B, Bai F, Jiang K, Bailey RD, Garcia R, et al. J Immunol 2001;166:7486 – 95.