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-mediated neutrophil apoptosis involves Ly-GDI, a Rho GTPase regulator
Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Medical Faculty of the Charité, Humboldt University of Berlin, Germany
Correspondence: Ralph Kettritz, M.D., Division of Nephrology, Franz Volhard Clinic, Wiltbergstrasse 50, 13122 Berlin, Germany.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-mediated PMN apoptosis by means of 2-D
gel electrophoresis and western blotting. Proteins were sequenced with
electrospray ionization mass spectrometry. Apoptosis was quantitated by
flow cytometry. We detected a cluster of acidic, high molecular-weight
proteins that were only tyrosine phosphorylated when TNF--treated
PMN interacted with fibronectin. Sequence analysis revealed that one of
these proteins was Ly-GDI, a regulator of Rho GTPases. Fibronectin
increased the TNF--induced Ly-GDI cleavage, yielding a 23-kD
fragment. At 8 h, intact Ly-GDI was decreased to 33% on
fibronectin, compared with 69% on PolyHema (P<0.05).
Inhibition of tyrosine phosphorylation prevented phosphorylation of
Ly-GDI, fibronectin-accelerated Ly-GDI cleavage, and
fibronectin-accelerated apoptosis in TNF--treated PMN. We found that
Ly-GDI cleavage was dependent on caspase-3 activation and that
caspase-3 inhibition decreased apoptosis. We conclude that tyrosine
phosphorylation of Ly-GDI, followed by increased caspase-3-mediated
Ly-GDI cleavage, is a signaling event associated with accelerated
TNF--mediated apoptosis on fibronectin.
Key Words: apoptosis • human neutrophils • matrix • Ly-GDI • caspases
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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[12
] and delayed by interleukin
(IL)-8 [13
]. Interestingly, TNF--mediated PMN
apoptosis was accelerated by the interaction with ECM substances, such
as fibronectin [14
]. These results suggested that
different apoptotic stimuli interact at the signal-transduction level.
Our earlier study suggested that the accelerating effect of matrix
proteins on TNF--mediated PMN apoptosis also involved tyrosine
phosphorylation [14
]. However, whether TNF- and
matrix proteins use different signaling pathways to enhance apoptosis
is unknown. We have now identified lymphoid-specific guanosine
diphosphate (GDP) dissociation inhibitor (Ly-GDI), a regulator of
Rho-proteins, as a substrate for matrix-induced tyrosine
phosphorylation. Ly-GDI was progressively cleaved in TNF--treated
PMN. This effect was accelerated on fibronectin and blocked by
inhibiting tyrosine phosphorylation. The time course of the Ly-GDI
cleavage paralleled the induction of apoptosis. Ly-GDI cleavage was
mediated by caspase-3, and its inhibition decreased the apoptotic
response to TNF-. This matrix-dependent apoptosis pathway may be
important for controlling local inflammation after PMN migration into
inflamed tissue. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was from Genzyme (Rüsselsheim, Germany).
Antiphosphotyrosine antibody PY20 (Transduction Laboratories,
Lexington, KY), goat polyclonal antibody to Ly-GDI, and horseradish
peroxidase-labeled anti-mouse and anti-goat antibodies (Santa Cruz,
Heidelberg, Germany) were purchased. PMN were isolated from heparinized whole blood from healthy donors, described previously [12 ]. The protocol included red blood cell sedimentation by Plasmagel followed by Ficoll-Hypaque density-gradient centrifugation and hypotonic red cell lysis. Cells were resuspended at 107/ml in RPMI 1640 supplemented with 2 mM glutamine and penicillin/streptomycin. Trypan blue exclusion was used to determine cell viability.
PMN (250 µl) at 107/ml RPMI without fetal calf serum
(FCS) were pipetted into 12-well tissue-culture plates (TPP-Company,
Munich, Germany), coated with PolyHema (50 mg/ml) to achieve
nonadherent conditions [15
] or with fibronectin (10
µg/cm2) to achieve adherent conditions. RPMI 1640 (245
µl) containing 20% heat-inactivated FCS was added, followed by 5
µl of TNF- (20 ng/ml) or an equal volume of carrier protein
containing PBS solution (0.5% BSA). When indicated, cells were
preincubated with 5 µl of inhibitor (or diluent control) for 20 min
on ice before the stimulation with TNF-. Samples were incubated at
37°C in 5% CO2. HL-60 cells were cultured in RPMI
1640/10% FCS supplemented with 2 mM glutamine and
penicillin/streptomycin. Experiments were performed during the
exponential phase of cell growth. Apoptosis was induced by treating
HL-60 with 0.15 µM of the DNA topoisomerase I inhibitor CAM for up to
4 h. All experiments were carried out in duplicate.
Flow cytometry was used to measure DNA content in ethanol-permeabilized cells at the single cell level, as described previously [12 ]. Briefly, freshly isolated or cultured cells were spun at 200 g for 5 min at 4°C and resuspended in PBS containing 0.5 mM ethylenediaminetetraacetate (EDTA). Chilled 95% ethanol was added to a final concentration of 70%, and the cells were stored at -20°C for 1–2 days. Cells were pelleted (200 g, 5 min, 4°C) and resuspended in 250 µl PBS/0.5 mM EDTA/1% BSA. PBS (250 µl) containing 200 µg DNase-free RNase and 500 µl PBS containing 50 µg propidium iodide were added. After 6–8 h in the staining mixture at 4°C, cells were analyzed using a fluorescence-activated cell sorter (FACscan) (Becton Dickinson, Heidelberg, Germany), and 10,000 events per sample were collected in listmode using Lysis II software for data acquisition and analysis.
For the 2-D gel electrophoresis, samples were harvested after 1 h, pelleted, weighted, and stored at -70°C. The frozen cell pellet was thawed in a protease inhibitor mix, according to Klose and Kobalz [16 ]. Cells were lysed in 9 M urea before 2% (v/v) carrier ampholytes (Servalyt pH 2-4, Serva, Heidelberg, Germany) and 70 mM dithiothreitol (DTT) were added. The protein lysate was separated by centrifugation (20 min, 70,000 rpm, 20°C), and the protein concentration of the clear supernatant was determined by amino acid analysis (Aminoacid-analyzer, Sykam, Germany). Protein (100 µg) was electrophoretically separated using a small, 2-D, gel electrophoresis technique (gel size: 6.5x8.5x0.15 cm), adapted from Klose and Kobalz [16 ]. Isoelectric focusing tube gels were used in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in the second dimension (first dimension: 75 min 100 V, 75 min 200 V, 75 min 400 V, 75 min 600 V, 10 min 1000 V, 5 min 1000 V; second dimension: 5 min 35 V, 10 min 55 V, 15 min 100 V, 60 min 150 V). For analytical studies, gels were silver stained or for western blotting, immediately transferred onto the polyvinylidene difluoride (PVDF) membrane. For preparative studies, the gels were stained by Coomassie Blue. For protein identification, the protein spot of interest was excised, cleaved with trypsin (Promega, Madison, WI), and followed by peptide extraction, as previously described [17 ]. The peptide solution was analyzed by nanoelectrospray mass spectrometry in a Q-Tof (Micromass, Manchester, UK) equipped with a Z-spray ion source. The analyte solution was pipetted in a nanospray borosilicate glass capillary, and the mass spectrum was measured using the time of flight (TOF) analyzer. Tandem mass spectrometry was then used to select precursor ions to be fragmented in the high-efficiency collisions cell to determine the amino acid sequences. The spectra of seven selected peptides were obtained by tandem mass spectrometry (MS/MS). The protein was identified by peptide mass fingerprinting (PMF). The peptide ion mass maps obtained by ESI-MS were searched using the program MS-FIT (UCSF mass spectrometry facility, San Francisco, CA). The obtained peptide ion-mass maps were compared with the theoretical peptide masses using SWISSPROT. The determined amino acid sequences obtained by tandem mass spectrometry were compared with the amino acid sequences of the identified protein to confirm identification.
To detect tyrosine phosphorylation by western blotting, proteins were electrophoretically separated as described above and transferred onto the PVDF membrane at 2.5 mA/cm2 for 1 h. The membrane was blocked with 1% BSA/TBS-T solution overnight at 4°C, followed by washing in TBS-T (137 mM NaCL, 20 mM Tris-HCL, pH 7.5, 0.2% Tween 20). The membrane was incubated with the antiphosphotyrosine monoclonal antibody (mAb) PY 20 (1:2000 dilution) for 3 h at reverse transcription (RT), washed, and incubated in goat anti-mouse immunoglobulin G (IgG) conjugated with horseradish peroxidase (1:1000) for 1 h at RT. After washing, the membrane was developed in a chemiluminescence substrate (NEN Life Science Products, Boston, MA) and exposed to X-ray film.
To study expression of Ly-GDI by western blotting, PMN were lysed in a solution containing 20 mM Tris, pH 8.0, 138 mM NaCl, 10% glycerol, 2 mM EDTA, 1% Triton X-100, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 0.1 mM quercetin, 5 mM iodoacetamid, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Supernatant was recovered by centrifugation, and 30 µg protein per lane was loaded on a 12% SDS-PAGE and blotted onto a PVDF membrane by semidry technique. Lysate from human BL-60 lymphoma cells treated with anti-IgM antibodies was a gift from Rickers et al. [18 ] and was also loaded onto the gel. This treatment resulted in Ly-GDI cleavage yielding a 23-kD fragment. The membrane was blocked in 5% nonfat dry milk/TBS-T solution overnight at 4°C, washed, and incubated with 1:1000 diluted goat anti-Ly-GDI antibody for 1.5 h at RT. The membrane was incubated with a horseradish peroxidase-conjugated secondary antibody (1:10,000) for 1 h at RT and developed as described above. Densitometric analysis of intact Ly-GDI was performed by using scanned X-ray films and the NIH-image program.
For the statistical analysis, a general linear model was calculated with two different surfaces (fibronectin and PolyHema) and two different caspase inhibitors (DEVD and YVAD) at increasing concentrations (1, 10, 25, and 100 µM), as within-subject effects. The significance of experimental variation, as well as variable interactions, was tested with analysis of variance. In case of significant effects, nested submodels were calculated with post-hoc Scheffé tests to allow for multiple testing. The same procedure was performed to compare the effects on the cleavage of intact Ly-GDI. The Wilcoxon rank-sum test was used to test differences in distributions between groups. For paired groups, the centered signed rank statistic was used to test if the differences were unequal to 0. Data are presented as mean ± SEM. For these calculations, statistical analysis was carried out using the commercially available statistics program SPSS (SPSS Inc., Chicago, IL).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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on a fibronectin-coated surface, PolyHema, or
fibronectin without stimulation with TNF-. Samples were harvested
after 1 h, and 2-D gels were performed. All experiments were
performed in triplicate. The results (Fig. 1
) demonstrate a cluster of acidic, high molecular-weight,
tyrosine-phosphorylated proteins. The number of tyrosine phosphorylated
spots was upregulated in TNF--treated PMN on fibronectin (panel A),
whereas less tyrosine phosphorylation was seen in cells treated with
TNF- on PolyHema (panel B) or in untreated controls on fibronectin
(panel C). Among distinct spots, the indicated spot (arrow) was only
tyrosine phosphorylated in TNF--treated PMN on fibronectin and was
also identified in the corresponding Coomassie gel (unpublished
results). The spot was selected for those reasons. Preincubation with
genistein resulted in a marked decrease in tyrosine-phosphorylation,
including the indicated spot (panel D). Following tryptic in-gel
digestion of this indicated protein spot, the peptide mixture was
analyzed with nanoelectrospray mass spectrometry. The spectrum of the
peptide mixture is shown in Figure 2
. The protein was unequivocally identified as Ly-GDI with peptide
mass fingerprinting and using the MS/MS spectra of the labeled (*)
peptides (spectra not shown). The spectrum of the peptide mixture
contained exclusively Ly-GDI peptides. However, we were not able to
detect phosphorylated peptides with mass spectrometric methods. We used
a calibrated 2-D gel to show that Ly-GDI runs as a 27-kD spot
with a pI of 5.1.
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-induced PMN apoptosis. PMN from the
same preparation were cultured in the presence of TNF- on PolyHema,
fibronectin, or fibronectin without TNF- stimulation. Samples were
harvested at 2 h intervals for 8 h, and SDS-PAGE followed by
western blotting using an anti-Ly-GDI-specific antibody was performed.
All PMN showed a 27-kD band representing intact Ly-GDI (Fig. 3
). After 2 h, a 23-kD cleavage product was detected in
TNF--treated PMN on fibronectin (panel A) and PolyHema (panel B) but
not in non-TNF--stimulated controls (panel C). A 19-kD fragment was
constitutively expressed in PMN. Cleavage of intact Ly-GDI was greater
in TNF--treated cells on fibronectin. We next verified these results
in five separate experiments using optical densitometry (Fig. 4
). The decrease in intact Ly-GDI began between 1 and 2 h of
TNF--treatment on both surfaces and was significant at 4, 6, and
8 h. However, fibronectin induced a stronger cleavage of Ly-GDI
than PolyHema (P<0.05).
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-induced Ly-GDI cleavage and apoptosis. TNF--stimulated PMN
of the same preparation were cultured for up to 8 h in the
presence or absence of 50 µM genistein on fibronectin or PolyHema.
Samples were harvested at the indicated time points, and Ly-GDI was
analyzed by western blotting. The fibronectin-accelerated Ly-GDI
cleavage in TNF--treated PMN was clearly inhibited by genistein
(Fig. 5A
). Moreover, genistein significantly blocked the
fibronectin-accelerated apoptosis in TNF--treated PMN (Fig. 5
, panel
B).
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in human neutrophils. PMN on
fibronectin were pretreated with the caspase-3 inhibitor, z-DEVD-cmk,
in the presence of TNF- (Fig. 6
). PMN without TNF- and without z-DEVD-cmk showed no Ly-GDI
cleavage, although PMN with TNF-, but without z-DEVD-cmk,
showed clear-cut Ly-GDI cleavage. Ly-GDI cleavage was completely
inhibited by DEVD-cmk in TNF--stimulated PMN. We then performed five
separate experiments to examine the dose-dependent effects of
z-DEVD-cmk and the caspase-1 inhibitor ac-YYAD-cmk on apoptosis in
TNF--stimulated PMN on fibronectin or PolyHema. Preincubation with
z-DEVD-fmk resulted in a dose-dependent inhibition of TNF--mediated
apoptosis on both surfaces at the 8-h timepoint (Fig. 7
). The inhibition was significant on fibronectin and on PolyHema
for z-DEVD-fmk-concentrations of 10, 25, and 100 µM
(P<0.05). In contrast, only the highest dose (100 µM) of
the caspase-1 inhibitor ac-YVAD-cmk (unpublished results) decreased in
the percentage of apoptotic PMN on fibronectin by only 20%. We finally
studied Ly-GDI cleavage in two other models of apoptosis as well as in
PMN treated with inflammatory agents that did not induce apoptosis in
PMN (n=2; Fig. 8
). In a model of constitutive apoptosis, freshly isolated PMN were
cultured for up to 24 h. Samples were harvested at 8 h and
24 h, and Ly-GDI was analyzed by western blotting (Fig. 8
, panel
A). We found that apoptosis was accompanied by Ly-GDI cleavage with
generation of the 23-kD fragment (percent apoptotic cells: 0.7±0.1 at
0 h; 4.2±0.8 at 8 h; 45.1±4.1 at 24 h). In the second
model, human promyelocytic HL-60 cells were stimulated to undergo rapid
apoptosis by the DNA topoisomerase I inhibitor, camptothecin (percent
apoptotic cells: 6.0±1.2 at 2 h; 12.5±1.1 at 3 h; 31.6±4.5
at 4 h). Again, apoptosis was paralleled by Ly-GDI cleavage and
the generation of the 23-kD fragment by 3 h (Fig. 8
, panel B). In
contrast, the inflammatory agents, IL-8 and granuloctye-macrophage
colony-stimulating factor (GM-CSF), did not accelerate PMN apoptosis at
8 h and did not cause Ly-GDI cleavage (percent apoptotic cells:
2.9±1.9 in controls; 1.0±1.4 for IL-8; 1.2±0.1 for GM-CSF; Fig. 8
,
panel C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-stimulated PMN interacted with fibronectin but not when PMN
were cultured on fibronectin without TNF- stimulation or when PMN
were TNF- stimulated on a nonadherent surface. TNF- caused
progressive cleavage of intact Ly-GDI, which was increased on
fibronectin compared with PolyHema. Inhibiting tyrosine phosphorylation
with genistein prevented phosphorylation of Ly-GDI,
fibronectin-accelerated Ly-GDI cleavage, and fibronectin-accelerated
apoptosis in TNF--treated PMN. Cleavage of intact Ly-GDI was
accompanied by the new expression of a 23-kD Ly-GDI fragment, whereas a
19-kD fragment was constitutively expressed. Pretreatment with
z-DEVD-fmk, a caspase-3 inhibitor, blocked the expression of the 23-kD
Ly-GDI fragment and also inhibited apoptosis. These results suggest
that TNF--induced apoptosis in PMN is dependent on caspase-3
activity and that Ly-GDI phosphorylation and cleavage may be important
in the matrix-induced acceleration of PMN apoptosis.
A variety of biologic agents have been shown to alter PMN apoptosis.
However, the underlying intracellular signaling events that delay or
accelerate PMN apoptosis are not well understood. TNF- is an
inflammatory cytokine that causes rapid acceleration of neutrophil
apoptosis. Although TNF- is capable of inducing apoptosis in the
absence of extracellular matrix proteins [11
,
12
, 19
], we showed earlier that fibronectin
accelerates TNF--induced apoptosis by a tyrosine
phosphorylation-dependent mechanism [14
]. The purpose of
this study was to identify targets of tyrosine phosphorylation and to
define their role in TNF--mediated apoptosis accelerated by
fibronectin. During PMN adherence to fibronectin in the presence of
TNF-, we observed increased tyrosine phosphorylation of the 27-kD
protein, Ly-GDI, a guanine nucleotide disssociation inhibitor
preferentially expressed in hematopoietic cells [20
,
21
]. The amino acid sequence of Ly-GDI shows seven
tyrosine residues at positions 24, 48, 107, 125, 130, 146, and 172.
Ly-GDI belongs to a group of proteins that regulate guanine nucleotide
binding to guanosine 5'-triphosphate (GTP)-binding proteins of the Ras
superfamily by inhibiting the dissociation of GDP. Ras proteins are
active when occupied by GTP and inactive when GDP is bound
[22
, 23
]. The specific GTPases that form
stable complexes with Ly-GDI in vivo have not yet been
identified. Ly-GDI is phosphorylated on threonine residues upon
stimulation of cells with PMA [21
, 24
]. The
in vivo consequence of Ly-GDI phosphorylation is not yet
clear; however, phosphorylation may favor recruitment of Ly-GDI
complexes to the cell membrane [24
].
We implicated caspase-3 as a possible mediator for Ly-GDI cleavage in
TNF--stimulated PMN undergoing apoptosis. Caspases, or
Ced-3/ICE-like proteases, are cysteine-containing, aspartate-specific
proteases and are key apoptosis mediators in many cells, including PMN
[25
26
27
]. We are not the first to show that Ly-GDI is a
substrate of caspase-3. Ly-GDI contains a specific caspase-3 cleavage
site with the sequence DELD19S [28
]. Earlier
studies describing Ly-GDI processing by caspases revealed that
caspase-3 cleaves Ly-GDI, yielding a 23-kD fragment [18
,
28
]. Our results show that Ly-GDI cleavage not only
occurs in TNF--induced PMN apoptosis but also in spontaneous PMN
apoptosis and in camptothecin-induced apoptosis of a human
promyelocytic cell line. All three models are dependent on caspase-3
[26
, 29
]. These data suggest that tyrosine
phosphorylation of Ly-GDI is important for fibronectin-accelerated
apoptosis of TNF--treated PMN and that cleavage of Ly-GDI may be an
event found in other caspase-3-dependent apoptosis models, including
TNF--induced PMN apoptosis on nonadherent surfaces, constitutive PMN
apoptosis, and CAM-induced apoptosis of promyelocytic HL-60 cells. The
specificity of Ly-GDI cleavage in apoptosis is underscored by our data
indicating that IL-8 and GM-CSF do not accelerate apoptosis and do not
result in Ly-GDI cleavage. Caspase-1 action, by contrast, results in
the generation of a smaller fragment of 19 kD [30
]. We
found a 19-kD Ly-GDI fragment to be constitutively expressed in
neutrophils, in agreement with a study by Danley et al.
[30
]. Ly-GDI cleavage was increased by PMN interaction
with the extracellular matrix protein fibronectin. The time course of
this cleavage paralleled the induction of apoptosis. We, and others,
have demonstrated that TNF--mediated apoptosis occurs by 2 h
[14
, 19
], and here, we show that
TNF--induced Ly-GDI cleavage becomes detectable between the first
and the second hour. This time course supports the view that cleavage
of Ly-GDI is not a result of overall protein degradation, which is
known to occur in late apoptosis.
Interestingly, inhibiting tyrosine phosphorylation prevented
fibronectin-induced phosphorylation of Ly-GDI, fibronectin-accelerated
Ly-GDI cleavage, and fibronectin-accelerated apoptosis in
TNF--treated PMN. This finding suggests a functional link between
these effects that may provide a mechanism by which fibronectin
accelerates TNF--mediated apoptosis in human PMN. A synergistic
interaction between fibronectin and TNF- has been shown for other
PMN functions, such as respiratory burst and degranulation
[31
, 32
]. Activation of the ß2-integrin
CD11/CD18 [3
], regulation of cAMP [31
,
33
], chloride ion efflux [34
], and
shedding of CD43 [31
] were found to be important
mediators. Moreover, the significance of tyrosine phosphorylation in
the crosstalk between fibronectin and TNF- was described, and
specific protein tyrosine kinases as well as specific phosphorylated
target proteins are currently being defined [35
]. Our
data describe Ly-GDI as a protein that was only tyrosine phosphorylated
in TNF--treated PMN on fibronectin, and we suggest that tyrosine
phosphorylation of Ly-GDI followed by its increased cleavage is an
important intracellular event for fibronectin-accelerated apoptosis in
TNF--stimulated PMN.
Conceivably, the increased 23-kD cleavage product is responsible for the accelerated apoptosis of PMN adherent to matrix proteins. Truncated Ly-GDI lacks the ability to regulate Rho GTPase function [30 ]. Rho GTPases are simple enzymes with complex roles in regulating cell morphology, gene transcription, cell-cycle progression, and apoptosis [36 ]. Ly-GDI binds to Rho and inhibits GDP dissociation from Rho proteins. To explore this relationship further, Ly-GDI-deficient mice have been generated recently [37 ]. These mice demonstrate decreased IL-2 withdrawal apoptosis of lymphnode cells, and dexamethasone-induced apoptosis remained intact. These findings indicate that Rho GTPase regulation by Ly-GDI is important to apoptosis, an observation consistent with our data. Further studies will be necessary to elucidate the role of Ly-GDI in PMN apoptosis.
Received September 1, 1999; revised March 13, 2000; accepted March 15, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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