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• W2000444833 abstract "The second messenger molecule cyclic AMP dramatically modulates the apoptotic program in a wide variety of cells, accelerating apoptosis in some and delaying the rate of apoptosis in others. Human neutrophil apoptosis, a process that regulates the fate and numbers of these potentially histotoxic cells in inflammatory sites, is profoundly delayed by the cell-permeable analog of cyclic AMP, dibutyryl-cAMP. We have investigated the mechanisms underlying cyclic AMP-mediated delay of neutrophil apoptosis, and we show that cyclic AMP inhibits loss of mitochondrial potential occurring during constitutive neutrophil apoptosis. Furthermore, we demonstrate that cyclic AMP also suppresses caspase activation in these inflammatory cells. Despite increasing protein kinase A activity, this kinase is unlikely to mediate the effect of cyclic AMP on apoptosis because blockade of protein kinase A activation did not influence the survival effects of cyclic AMP. Further investigation of the signaling mechanism demonstrated that the delay of apoptosis is independent of phosphoinositide 3-kinase and MAPK activation. Our results suggest cyclic AMP delays neutrophil apoptosis via a novel, reversible, and transcriptionally independent mechanism. We show that proteasome activity in the neutrophil is vitally involved in this process, and we suggest that a balance of pro-apoptotic and anti-apoptotic proteins plays a key role in the powerful ability of cyclic AMP to delay neutrophil death. The second messenger molecule cyclic AMP dramatically modulates the apoptotic program in a wide variety of cells, accelerating apoptosis in some and delaying the rate of apoptosis in others. Human neutrophil apoptosis, a process that regulates the fate and numbers of these potentially histotoxic cells in inflammatory sites, is profoundly delayed by the cell-permeable analog of cyclic AMP, dibutyryl-cAMP. We have investigated the mechanisms underlying cyclic AMP-mediated delay of neutrophil apoptosis, and we show that cyclic AMP inhibits loss of mitochondrial potential occurring during constitutive neutrophil apoptosis. Furthermore, we demonstrate that cyclic AMP also suppresses caspase activation in these inflammatory cells. Despite increasing protein kinase A activity, this kinase is unlikely to mediate the effect of cyclic AMP on apoptosis because blockade of protein kinase A activation did not influence the survival effects of cyclic AMP. Further investigation of the signaling mechanism demonstrated that the delay of apoptosis is independent of phosphoinositide 3-kinase and MAPK activation. Our results suggest cyclic AMP delays neutrophil apoptosis via a novel, reversible, and transcriptionally independent mechanism. We show that proteasome activity in the neutrophil is vitally involved in this process, and we suggest that a balance of pro-apoptotic and anti-apoptotic proteins plays a key role in the powerful ability of cyclic AMP to delay neutrophil death. protein kinase A dibutyryl cyclic AMP Dulbecco's modified Eagle's medium granulocyte macrophage-colony-stimulating factor 5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazocarbocyaniniodide mitogen-activated protein kinase phosphoinositide 3-kinase phosphate-buffered saline prostaglandin E2 fluorescein isothiocyanate The neutrophil is a terminally differentiated phagocytic cell that plays a key role in first line defense against invading bacteria. Neutrophils are rapidly recruited to inflamed sites in response to infection and, following phagocytosis of the invading organism, release a variety of toxic granule contents into the phagolysosome containing the engulfed microorganisms (1Haslett C. Savill J.S. Meagher L. Curr. Opin. Immunol. 1989; 2: 10-18Crossref PubMed Scopus (117) Google Scholar, 2Rossi A.G. Hellewell P.G. Hellewell P.G. Williams T.J. Immunopharmacology of Neutrophils. Academic Press, London1994: 223-243Google Scholar). The neutrophil normally has a short life span, and senescent neutrophils must be prevented from releasing their cytotoxic cell contents into the surrounding milieu because such liberation will lead to local tissue damage. To avoid this undesirable and inappropriate response, the neutrophil undergoes a regulated process of programmed cell death or apoptosis (3Savill J.S. Wyllie A.H. Henson J.E. Walport M.J. Henson P.M. Haslett C. J. Clin. Invest. 1989; 83: 865-875Crossref PubMed Scopus (1336) Google Scholar, 4Rossi A.G. Haslett C. Lenfant C. Lung Biology in Health and Disease. 112. Marcel Dekker, Inc., New York1998: 9-24Google Scholar, 5Haslett C. Am. J. Respir. Crit. Care Med. 1999; 160 (suppl.): 5-11Crossref Scopus (441) Google Scholar), allowing shutdown of secretory capacity (6Whyte M.K.B. Meagher L.C. MacDermot J. Haslett C. J. Immunol. 1993; 150: 5124-5134PubMed Google Scholar) and phagocytic removal of the intact effete cell by a mechanism that does not incite an inflammatory response (7Meagher L.C. Savill J.S. Baker A. Fuller R.W. Haslett C. J. Leukocyte Biol. 1992; 52: 269-273Crossref PubMed Scopus (247) Google Scholar, 8Fadok V.A. Bratton D.L. Konowal A. Freed P.W. Westcott J.Y. Henson P.M. J. Clin. Invest. 1998; 101: 890-898Crossref PubMed Scopus (2537) Google Scholar, 9Liu Y. Cousin J.M. Hughes J. Van Damme J. Seckl J.R. Haslett C. Dransfield I. Savill J. Rossi A.G. J. Immunol. 1999; 162: 3639-3646PubMed Google Scholar).The execution of the apoptotic program generally involves the activation of a family of cysteine proteases, collectively referred to as the caspases, that are ultimately responsible for the structural dismantling of the cell (10Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2429) Google Scholar, 11Hengartner M.O. Nature. 2000; 407: 770-776Crossref PubMed Scopus (6211) Google Scholar). In addition, the mitochondria play a central role through their ability to integrate anti-apoptotic or pro-apoptotic signals from Bcl-2 family members with coordinated activation of downstream caspases and nucleases (12Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 13Kroemer G. Biochem. Soc. Symp. 1999; 66: 1-15Crossref PubMed Scopus (182) Google Scholar). In many cell types it has been documented that apoptosis is accompanied by an early dissipation of the mitochondrial transmembrane potential (ΔΨm) with increased permeability of the outer mitochondrial membrane allowing release of apoptosis-inducing factors such as cytochrome c (12Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 13Kroemer G. Biochem. Soc. Symp. 1999; 66: 1-15Crossref PubMed Scopus (182) Google Scholar, 14Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4254) Google Scholar). Neutrophils are thought to contain very few mitochondria, and it has not yet been fully established whether they have the capacity to play a functional role in regulation of neutrophil apoptosis (15Pryde J.G. Walker A. Rossi A.G. Hannah S. Haslett C. J. Biol. Chem. 2000; 275: 33574-33584Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 16Simon H.U. Immunol. Rev. 2001; 179: 156-162Crossref PubMed Scopus (102) Google Scholar).Neutrophils undergo constitutive apoptosis during in vitroculture and exhibit the classic changes associated with apoptosis including cytoplasmic condensation, internucleosomal cleavage of DNA by endogenous endonucleases, and exposure of phosphatidylserine on the outer leaflet of the plasmalemma (3Savill J.S. Wyllie A.H. Henson J.E. Walport M.J. Henson P.M. Haslett C. J. Clin. Invest. 1989; 83: 865-875Crossref PubMed Scopus (1336) Google Scholar). Although the apoptotic program in neutrophils is an intrinsic cell process, the rate of apoptosis can be altered dramatically by a number of agents (17Ward C. Dransfield I. Chilvers E.R. Haslett C. Rossi A.G. Trends Pharmacol. Sci. 1999; 20: 503-509Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). In particular, we and others (18Rossi A.G. Cousin J.M. Dransfield I. Lawson M.F. Chilvers E.R. Haslett C. Biochem. Biophys. Res. Commun. 1995; 217: 892-899Crossref PubMed Scopus (127) Google Scholar, 19Parvathenani L.K. Buescher E.S. Chacon-Cruz E. Beebe S.J. J. Biol. Chem. 1998; 273: 6736-6743Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 20Ottonello L. Gonella R. Dapino P. Sacchetti C. Dallegri F. Exp. Hematol. 1998; 26: 895-902PubMed Google Scholar) have shown that elevated levels of the second messenger cyclic AMP can prolong neutrophil longevity by delaying apoptosis.The cyclic AMP-dependent signaling transduction pathway is a multienzyme cascade that regulates a diverse array of biological processes. Specific ligation of appropriate G-protein-coupled receptors followed by adenylate cyclase activation leads to the production of cyclic AMP. Cyclic AMP then binds to cytoplasmic protein kinase A, a tetrameric structure composed of two regulatory (R) and two catalytic (C) subunits, resulting in dissociation of the C subunits and subsequent phosphorylation of target proteins (21Daniel P.B. Walker W.H. Habener J.F. Annu. Rev. Nutr. 1998; 18: 353-383Crossref PubMed Scopus (212) Google Scholar). Although most components of this signaling cascade are well characterized, the molecular mechanisms underlying cyclic AMP-mediated modulation of apoptosis remain to be elucidated. The signaling mechanism(s) used by cyclic AMP to control these events is (are) likely to be complex and cell type-specific. For example, in contrast to the profound delay in the engagement of the apoptotic process in neutrophils (18Rossi A.G. Cousin J.M. Dransfield I. Lawson M.F. Chilvers E.R. Haslett C. Biochem. Biophys. Res. Commun. 1995; 217: 892-899Crossref PubMed Scopus (127) Google Scholar, 19Parvathenani L.K. Buescher E.S. Chacon-Cruz E. Beebe S.J. J. Biol. Chem. 1998; 273: 6736-6743Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 20Ottonello L. Gonella R. Dapino P. Sacchetti C. Dallegri F. Exp. Hematol. 1998; 26: 895-902PubMed Google Scholar), cyclic AMP elevation induces apoptosis in thymocytes (22McConkey D.J. Orrenius S. Jondal M. J. Immunol. 1990; 145: 1227-1230PubMed Google Scholar) and leukemic cell lines (23Lanotte M. Riviere J.B. Hermouet S. Houge G. Vintermyr O.K. Gjertsen B.T. Doskeland S.O. Cell. Physiol. 1991; 146: 73-80Crossref Scopus (106) Google Scholar, 24Kiefer J. Okret S. Jondal M. McConkey D.J. J. Immunol. 1995; 155: 4525-4528PubMed Google Scholar). The signaling mechanism determining this ability to differentially influence apoptosis in diverse cell types remains to be elucidated.In the present study we show that elevated cyclic AMP inhibits activation of caspase-3 and loss in mitochondrial potential (ΔΨm) when neutrophils are aged in vitro,i.e. effects that appear to be associative rather that causative. Although we could demonstrate that cyclic AMP rapidly elevates endogenous PKA1activity in cultured neutrophils, blockade of PKA activation did not influence the observed delay in neutrophil apoptosis induced by cyclic AMP elevation. We also show that cyclic AMP elevation delays neutrophil apoptosis via a transcriptionally independent and reversible pathway, which does not require PI 3-kinase and MAPK activity. Together these data point to a novel mode of action for the major retardation of neutrophil apoptosis induced by cyclic AMP elevation.EXPERIMENTAL PROCEDURESGranulocyte Isolation and CultureNeutrophils were purified from the peripheral blood of normal donors by dextran sedimentation (Sigma) followed by centrifugation on discontinuous PercollTM (Amersham Pharmacia Biotech) gradients as described previously (25Haslett C. Guthrie L.A. Kopaniak M.M. Johnston Jr., R.B. Henson P.M. Am. J. Pathol. 1995; 119: 101-110Google Scholar, 26Ward C. Chilvers E.R. Lawson M.F. Pryde J.G. Fujihara S. Farrow S.N. Haslett C. Rossi A.G. J. Biol. Chem. 1999; 274: 4309-4318Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). Only neutrophil preparations with a purity of >98% were used. Cells were cultured in flat-bottomed Falcon flexible wells (Becton Dickinson, Oxford, UK) at 37 °C in a 5% CO2 atmosphere at a concentration of 5 × 106/ml in Iscove's modified Dulbecco's medium (Life Technologies, Inc.) supplemented with 100 units/ml penicillin/streptomycin (Life Technologies, Inc.) and 10% (v/v) autologous serum. As an index of necrosis, cell membrane integrity was assessed by the ability of cells to exclude the vital dye trypan blue (Sigma). Under all experimental conditions, greater than 99% of the cells consistently excluded trypan blue.Assessment of Granulocyte ApoptosisMorphologyCells were cyto-centrifuged, fixed in methanol, stained with Diff-QuikTM Gamidor Ltd. (Abingdon, Oxon, UK), and counted using oil immersion microscopy to determine the proportion of cells with distinctive apoptotic morphology (3Savill J.S. Wyllie A.H. Henson J.E. Walport M.J. Henson P.M. Haslett C. J. Clin. Invest. 1989; 83: 865-875Crossref PubMed Scopus (1336) Google Scholar, 26Ward C. Chilvers E.R. Lawson M.F. Pryde J.G. Fujihara S. Farrow S.N. Haslett C. Rossi A.G. J. Biol. Chem. 1999; 274: 4309-4318Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). At least 500 cells were counted per slide with the observer blinded to the experimental conditions. The results were expressed as the mean percent apoptosis ± S.E.Annexin V BindingA separate and independent assessment of apoptosis was performed by flow cytometry using annexin V binding (annexin V-FLUOS, Roche Molecular Biochemicals) to measure phosphatidylserine exposure on the surface of apoptotic cells. A working solution of annexin V-FLUOS was made from stock annexin V-FLUOS (0.1 μg/μl) diluted 1:3000 in Hanks' balanced salt solution (Sigma) supplemented with 2.5 mm CaCl2. Neutrophils (20 μl of 5 × 106/ml) were added to 200 μl of the working solution of annexin V-FLUOS before being assessed by flow cytometry on a FACSCalibur (Becton Dickinson, Oxford, UK) and analyzed on associated CellQuest (Becton Dickinson) software. All experiments were performed at least three times unless otherwise indicated.Measurement of PKA ActivityPKA activity was measured using Promega's SignaTECTTM cAMP-dependent Protein Kinase (PKA) Assay System, which utilizes biotinylated Kemptide (LRRASLG), a peptide substrate derived from the in vivo substrate pyruvate kinase. Neutrophils (5 × 106 cells) were preincubated with control buffer or 10 μm H89 (Calbiochem) for 1 h in PBS with Ca2+/Mg2+ (or for 19 h in DMEM Iscove's with 10% autologous serum) at 37 °C before being stimulated with 0.2 mm Bt2cAMP or 1 μm PGE2 (both from Sigma) for 30 min at 37 °C. Following one wash in ice-cold PBS, neutrophils were resuspended in 0.5 ml of cold extraction buffer (25 mmTris-HCl, pH 7.4, 0.5 mm EDTA, 0.5 mm EGTA, 10 mm β-mercaptoethanol, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride, and 1% Triton X-100 (Sigma)). The lysates were centrifuged (5 min at 4 °C; 14,000 × g) and the supernatants retained. The PKA reaction mixture consisting of 5 μl of 5× PKA Assay Buffer, 5 μl of cyclic AMP (0.025 mm), 5 μl of PKA-biotinylated peptide substrate (0.5 mm), 5 μl of [γ-33P]ATP mixture (5 μl of 0.5 mm ATP and 0.05 μl of [γ-33P]ATP (3,000 Ci/mmol) 10 μCi/μl) was mixed gently and preincubated at 30 °C for 5 min (Promega, Southampton, UK). A control reaction without substrate was performed to determine background counts. The PKA activity reaction was initiated by adding 5 μl of the lysates to the reactants and incubated at 30 °C for 5 min. The reaction was terminated by adding 12.5 μl of Termination Buffer to each sample (Promega, Southampton, UK). Aliquots (10 μl) from each terminated reaction sample were spotted onto prenumbered SAM2TM membrane squares (Promega, Southampton, UK). The SAM2TM membrane squares containing the spotted samples were then washed 1 time for 30 s with 200 ml of 2 m NaCl (Sigma) followed by 3 washes for 2 min with 200 ml of 2 m NaCl and then 4 washes for 2 min with 200 ml of 2 m NaCl in 1% H3PO4. Finally the Membrane squares were quickly washed in deionized water before being allowed to dry. PKA activity was measured by scintillation counting.Measurement of Mitochondrial DissipationChanges in mitochondrial potential were measured in neutrophils following stimulation using JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazocarbocyaniniodide (Molecular Probes), a cationic dye that exhibits potential dependent accumulation in mitochondria indicated by a fluorescence emission shift from green (525 nm) to red (590 nm) (27Reers M. Smith T.W. Chen L.B. Biochemistry. 1991; 30: 4480-4486Crossref PubMed Scopus (869) Google Scholar). Mitochondrial depolarization is therefore indicated by a decrease in the red/green fluorescence intensity ratio. JC-1 (10 μg/ml) was diluted in PBS from stock JC1 (5 mg/ml in Me2SO) and added to neutrophils (1 × 106/ml) for 10 min at 37 °C. Neutrophil mitochondria labeled with JC-1 were examined by confocal fluorescent microscopy together with TO-PRO-3 (1 μm) (Molecular Probes) (28Van Hooijdonk C.A Glade C.P. Van Erp P.E. Cytometry. 1994; 17: 185-189Crossref PubMed Scopus (76) Google Scholar) to assess neutrophils with necrotic morphology. Alternatively, neutrophils labeled with JC-1 were assessed by flow cytometric analysis using FACSCalibur (Becton Dickinson, Oxford, UK) and analyzed on associated CellQuest (Becton Dickinson) software. Non-apoptotic neutrophils were removed using immunomagnetic separation with sheep anti-mouse IgG-Dynabeads (Dynabeads M-450, Dynal, Mersyside, UK) coated with the murine anti-neutrophil antibody 3G8 (anti-CD16; a gift from Dr. J. Unkeless, Mount Sinai Medical School, New York). Cells were mixed with washed antibody-coated magnetic beads on a rotary mixer at 4 °C for 20 min, and the beads removed magnetically by two 3-min stationary magnetic contacts (Dynal Magnetic Particle Concentrator, MPC-1) to yield an apoptotic neutrophil preparation. After purification, the apoptotic neutrophils were labeled with JC-1 as described previously.Western BlottingHuman neutrophils (5 × 106/ml) were cultured with or without Bt2cAMP (0.2 mm) at 37 °C for various time points as detailed under “Results.” Cytoplasmic extracts were then prepared from equivalent numbers of cells (10 × 106 cells). To minimize problems with proteolysis, lysates were prepared using methods normally used for electrophoretic mobility shift assay preparations (26Ward C. Chilvers E.R. Lawson M.F. Pryde J.G. Fujihara S. Farrow S.N. Haslett C. Rossi A.G. J. Biol. Chem. 1999; 274: 4309-4318Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 29Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9143) Google Scholar) with the addition of 1 mm phenylmethylsulfonyl fluoride. Samples were loaded onto a 12.5% Tris-HCl polyacrylamide mini-gel under reducing conditions and transferred to nitrocellulose membrane (Amersham Pharmacia Biotech) at 60 V for 1 h before overnight incubation at 4 °C with an antibody specific to caspase-3 (catalog number 65906E, PharMingen). After washing, blots were incubated with donkey anti-rabbit horseradish peroxidase conjugate (Amersham Pharmacia Biotech) diluted 1:2000 and developed using a commercial chemiluminescence detection system (ECL,Amersham Pharmacia Biotech).Further MaterialsFurther specific materials were obtained as follows: (Rp)-8-Br-cAMPS, PD98059, SB203580, and cycloheximide (Calbiochem); lactacystin and epoxomicin (Affiniti, Mamhead, UK); and LY294002 (New England Biolabs, Hertfordshire, UK).Statistical AnalysisStatistical analysis was performed using the Student'st test or by analysis of variance with comparisons between groups made using the Newman-Keuls procedure. Differences were considered significant when p < 0.05.DISCUSSIONHuman neutrophils undergo apoptosis, a process that is centrally important in the resolution of inflammation. It has been shown previously that cyclic AMP is an important regulator of neutrophil apoptosis (18Rossi A.G. Cousin J.M. Dransfield I. Lawson M.F. Chilvers E.R. Haslett C. Biochem. Biophys. Res. Commun. 1995; 217: 892-899Crossref PubMed Scopus (127) Google Scholar, 19Parvathenani L.K. Buescher E.S. Chacon-Cruz E. Beebe S.J. J. Biol. Chem. 1998; 273: 6736-6743Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 20Ottonello L. Gonella R. Dapino P. Sacchetti C. Dallegri F. Exp. Hematol. 1998; 26: 895-902PubMed Google Scholar), yet little is known of the signaling mechanism by which by cyclic AMP controls neutrophil cell death. The studies herein have established that cyclic AMP acts upstream of caspase-3 activation to inhibit the apoptotic pathway in neutrophils. For the first time, it was also demonstrated that neutrophils contain a small but significant number of mitochondria, which exhibit a loss of membrane potential during constitutive apoptosis, which can be delayed by cyclic AMP elevation. We are currently investigating whether loss of mitochondrial membrane potential occurs before other indices of apoptosis in neutrophils, such as phosphatidylserine exposure and nuclear condensation. This would help ascertain whether loss of mitochondrial potential during neutrophil apoptosis, shown to trigger apoptosis in other cell types, has a similar function in neutrophils and whether Bt2cAMP can directly affect loss of mitochondrial potential to delay neutrophil apoptosis.It has been suggested that PKA plays an important role in cyclic AMP-mediated delay of neutrophil apoptosis (18Rossi A.G. Cousin J.M. Dransfield I. Lawson M.F. Chilvers E.R. Haslett C. Biochem. Biophys. Res. Commun. 1995; 217: 892-899Crossref PubMed Scopus (127) Google Scholar, 19Parvathenani L.K. Buescher E.S. Chacon-Cruz E. Beebe S.J. J. Biol. Chem. 1998; 273: 6736-6743Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 20Ottonello L. Gonella R. Dapino P. Sacchetti C. Dallegri F. Exp. Hematol. 1998; 26: 895-902PubMed Google Scholar). It is known that cyclic AMP analogs, which selectively activate type I PKA, attenuate neutrophil apoptosis, compared with analogs that preferentially activate type II PKA suggesting that that type I PKA is necessary and sufficient to mediate the cyclic AMP-induced delay in human neutrophil apoptosis (19Parvathenani L.K. Buescher E.S. Chacon-Cruz E. Beebe S.J. J. Biol. Chem. 1998; 273: 6736-6743Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). We suggest, alternatively, that PKA activation by cyclic AMP is not responsible for the major apoptosis-retarding influences of cyclic AMP in neutrophils. Indeed, we have demonstrated directly that cyclic AMP elevation in neutrophils stimulates an increase in PKA activity, which is blocked by pharmacological inhibitors. Importantly, however, blockade of PKA was not sufficient to reverse the anti-apoptotic effect of cyclic AMP, implying that this molecule has little or no role in the cyclic AMP signaling pathway responsible for delay of neutrophil apoptosis.Previous publications (18Rossi A.G. Cousin J.M. Dransfield I. Lawson M.F. Chilvers E.R. Haslett C. Biochem. Biophys. Res. Commun. 1995; 217: 892-899Crossref PubMed Scopus (127) Google Scholar, 19Parvathenani L.K. Buescher E.S. Chacon-Cruz E. Beebe S.J. J. Biol. Chem. 1998; 273: 6736-6743Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) have implicated a role for PKA in cyclic AMP regulation of neutrophil apoptosis using concentrations of H89 greater than 10 μm. The specificity of H89 at these concentrations is questionable, and it has been published (47Davies S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Crossref PubMed Scopus (3919) Google Scholar) that H89 may inhibit several other kinases, some with potency similar to or greater than that for PKA. We propose that failure to directly measure PKA activity together with the use of high and possibly nonspecific concentrations of H89 could have led to misinterpretation of previous data. We have demonstrated that 10 μm H89 is sufficient to block PKA activity for extended culture periods and is active in the presence of autologous serum. The failure therefore of both H89 and (Rp)-8-Br-cAMPS, a highly specific inhibitor of PKA, to reverse cyclic AMP-mediated delay of neutrophil apoptosis points to a novel signaling pathway used by cyclic AMP to inhibit neutrophil apoptosis, which is independent of PKA activation.There have been a few studies reporting PKA-independent effects of cyclic AMP; however, little has been elucidated of the alternative signaling pathways downstream of cyclic AMP. Pharmacological blockade of the MAPK and PI 3-kinase signaling cascades in this study suggest that neither of these signaling pathways are likely to be important in the cyclic AMP-mediated delay of neutrophil apoptosis. There has been interest in the discovery that cyclic AMP can bind specifically to and activate small guanine nucleotide exchange factors which, when bound by cyclic AMP, activate the small Ras-like GTPase, Rap1 (48DeRooij J. Zwartkruis F.J. Verheijen M.H. Cool R.H. Nijman S.M. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1602) Google Scholar, 49Kawasaki H. Springett G.M. Mochizuki N. Toki S. Nakaya M. Matsuda M. Housman D.E. Graybiel A.M. Science. 1998; 282: 2275-2279Crossref PubMed Scopus (1165) Google Scholar). The biological function of Rap1 is still unclear, but it has been proposed that activation of this small GTPase may feed into MAPK signaling pathways (50Bos J.L. EMBO J. 1998; 17: 6776-6782Crossref PubMed Scopus (286) Google Scholar). As an approach to establishing if Rap1 has a role in cyclic AMP-mediated delay of neutrophil apoptosis, we have blocked Rap1 activity using the Clostridium sordellii lethal toxin, which has been reported to inhibit specifically the small GTPases Rap1, Ras, and Rac (51Popoff M.R. Chaves-Olarte E. Lemichez E. von Eichel-Streiber C. Thelestam M. Chardin P. Cussac D. Antonny B. Chavrier P. Flatau G. Giry M. de Gunzburg J. Boquet P. J. Biol. Chem. 1996; 271: 10217-10224Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Furthermore, we have tested GGTI-286, a geranylgeranyltransferase inhibitor, which blocks geranylgeranylation required by Rap1 to achieve its mature, biologically active form (52Lerner E.C. Qian Y. Hamilton A.D. Sebti S.M. J. Biol. Chem. 1995; 270: 26770-26773Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Thus our preliminary experiments suggest that Rap1 is not involved in cyclic AMP-mediated delay of neutrophil apoptosis (data not shown): however, this area of research is still under investigation. Our studies are in accord with a very recent publication that demonstrates that cyclic AMP-dependent inhibition of interleukin-5 from human T lymphocytes is not mediated by PKA or by the Rap1 signaling pathway (53Staples K.J. Bergmann M. Tomita K. Houslay M.D. McPhee I. Barnes P.J. Giembycz M.A. Newton R. J. Immunol. 2001; 167: 2074-2080Crossref PubMed Scopus (67) Google Scholar).Regulation of neutrophil apoptosis is thought to depend on the balance between pro-apoptotic and anti-apoptotic death factors expressed in the cell (17Ward C. Dransfield I. Chilvers E.R. Haslett C. Rossi A.G. Trends Pharmacol. Sci. 1999; 20: 503-509Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 54Akgul C. Moulding D.A. Edwards S.W. FEBS Lett. 2001; 487: 318-322Crossref PubMed Scopus (400) Google Scholar). Neutrophils contain death regulator proteins, including Bax and Bad, and also express some members of the anti-apoptotic family such as Mcl-1 and Bcl-xL but not Bcl-2 (17Ward C. Dransfield I. Chilvers E.R. Haslett C. Rossi A.G. Trends Pharmacol. Sci. 1999; 20: 503-509Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 54Akgul C. Moulding D.A. Edwards S.W. FEBS Lett. 2001; 487: 318-322Crossref PubMed Scopus (400) Google Scholar). It has been proposed that neutrophil longevity may be prolonged by the synthesis of anti-apoptotic proteins such as Mcl-1 (55Moulding D.A. Quale J.A. Hart C.A. Edwards S.W. Blood. 1998; 92: 2495-2502Crossref PubMed Google Scholar). However, it is unlikely that cyclic AMP effects are mediated by such a mechanism in the retardation of neutrophil apoptosis since we have demonstrated that cyclic AMP-mediated delay of neutrophil apoptosis does not require gene transcription. Furthermore, “wash out” experiments have revealed that retardation of neutrophil apoptosis is rapidly lost when Bt2cAMP is removed from culture, even after incubation periods that should permit new protein synthesis.Together, these data suggest a mechanism whereby cyclic AMP does not stimulate production of a survival protein but may alternatively induce post-transitional modifications in the neutrophil to promote survival. One potential mechanism for cyclic AMP-mediated retardation of neutrophil apoptosis may involve cyclic AMP specifically targeting a death protein(s) to the proteasome for degradation. We have demonstrated that blockade of proteasome activity results in a dramatic loss of the pro-survival effect of cyclic AMP. We speculate that cyclic AMP may be involved in the post-translational modification of a death protein, which targets the neutrophil proteasome. If cyclic AMP stimulation is removed or proteasome activity is blocked, then the accumulation of a death protein(s) would be predicted to permit the constitutive death pathway of neutrophils to be reconstituted. Further characterization of proteasome activity in this signaling pathway and possible death protein targets of cyclic AMP are currently under investigation.In conclusion, cyclic AMP delays neutrophil apoptosis via a novel, reversible, and transcriptionally independent mechanism. Our results contest the dogma th" @default.
• W2000444833 created "2016-06-24" @default.
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• W2000444833 date "2001-11-01" @default.
• W2000444833 modified "2023-09-30" @default.
• W2000444833 title "Cyclic AMP Regulation of Neutrophil Apoptosis Occurs via a Novel Protein Kinase A-independent Signaling Pathway" @default.
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