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INTRODUCTION |
In the absence of specific survival factors, human monocytes
spontaneously undergo apoptosis in 24-48 h (1, 2). In contrast to
cellular necrosis, apoptosis is an energy-requiring, non-inflammatory mechanism of programmed cell death. The apoptotic pathway can be
extrinsic or intrinsic. The extrinsic pathway is regulated by cell
receptors such as Fas, which is in the tumor necrosis factor
receptor family. Once clustered by Fas ligand, Fas activation leads to
the formation of the death-inducing signaling pathway (DISC).
This complex contains the adapter protein Fas-associated death domain
protein and cysteinyl-aspartic acid
protease (caspase)-8 and caspase-10, which can activate
cellular apoptosis. In type I cells, caspase-8 activation directly
activates downstream caspases, resulting in cellular apoptosis. In
contrast, type II cells rely on an amplification loop where caspase-8
cleaves and activates the pro-apoptotic protein Bid, inducing the
release of cytochrome c and Diablo from the mitochondria.
These events lead to caspase-3 activation, which induces the execution
of cellular apoptosis. Moreover, active caspase-3 activates caspase-8,
further amplifying this feedback loop.
In contrast to the Fas receptor system, the intrinsic apoptotic pathway
is initiated by the release of cytochrome c from the mitochondria. Cytochrome c interacts with procaspase-9,
APAF-1, and ATP to liberate active caspase-9. Active caspase-9 cleaves and activates caspase-3, leading to cellular apoptosis. As a family, these caspases exist as inactive procaspase zymogens that require activation by proteolytic or autocatalytic cleavage. Processing and
activation of upstream caspases such as caspase-8 and/or caspase-9 lead
to activation of executioner caspase-3, -6, and -7, which are
responsible for the characteristic changes associated with apoptosis
such as membrane blebbing, nuclear condensation, and DNA fragmentation.
Caspase-3 is a primary executioner caspase and inactivates ICAD
(inhibitor caspase-activated
deoxyribonuclease) to induce the cleavage of DNA into
oligonucleosomal fragments (5). These cysteine proteases are described
as mammalian homologs to Caenorhabditis elegans CED-3
(C.
elegans death gene
3), originally discovered to be involved in the regulation of apoptotic cell death (3, 4).
Nitric oxide promotes cell survival by blocking caspase-3 activation by
S-nitrosylating the cysteine residue in the catalytic site
of caspase-3 (6). In fact, it has been suggested that the binding of
Fas to Fas ligand induces apoptosis by denitrosylating and activating
caspase-3, providing a causal link between nitric oxide and cell
survival (6). Consistent with a key role for nitric oxide effects on
caspase-3 function, S-nitrosylation of Cys-163 by nitric
oxide specifically inhibits caspase-3 enzymatic activity (7). Thus,
S-nitrosylation is widely believed to be the primary
mechanism by which nitric oxide prevents caspase-3 cleavage. Because
certain nitric oxide donors such as S-nitrosoglutathione (GSNO)1 and
S-nitroso-N-acetylpenicillamine (SNAP) promote
S-nitrosylation by transferring nitrosyl groups to protein
thiol residues (8), we speculated that these nitrosothiol donors would
reduce caspase-3 activation and promote survival of human monocytes.
Paradoxically, nitric oxide can also induce apoptosis in certain cells
(9-11). The specific mechanism for this effect is not clear; however,
nitric oxide reacts rapidly with superoxide (O
) to form
peroxynitrite (ONOO
) (12-14). Peroxynitrite can nitrate
tyrosine residues and inhibit tyrosine phosphorylation-mediated
activation events. The monocyte survival factor macrophage
colony-stimulating factor (M-CSF) induces reactive oxygen
species production (15) and relies on the phosphorylation of tyrosine
residues in the cytoplasmic domain of the receptor to activate
signaling pathways. We speculated that nitric oxide donors (such as
1-propamine 3-(2-hydroxy-2-nitroso-1-propylhydrazine) (PAPA) NONOate
and diethylamine (DEA) NONOate) that do not target thiol residues would
interrupt phosphorylation of these tyrosine residues and reduce
monocyte survival induced by M-CSF.
Consistent with these hypotheses, we found that the
nitrosothiol donors GSNO and SNAP inhibited caspase-3 activity and
reduced DNA fragmentation in human monocytes, whereas the nitric oxide donors PAPA NONOate and DEA NONOate did not consistently reduce caspase-3 activation or DNA fragmentation. Homeostasis of blood monocytes by these donors appeared to involve the activation state of
caspase-9 and caspase-3 and correlated to modification of thiol residues by nitric oxide.
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EXPERIMENTAL PROCEDURES |
Materials--
RPMI 1640 medium and Dulbecco's modified
Eagle's medium were purchased from BioWhittaker, Inc. (Walkersville,
MD). Fetal calf serum was obtained from Hyclone Laboratories (Logan,
UT). GSNO and SNAP was obtained from BIOMOL Research Laboratories Inc.
(Plymouth Meeting, PA). PAPA NONOate was purchased from Alexis
Biochemicals (San Diego, CA). DEA NONOate was obtained from Calbiochem.
Recombinant human M-CSF was purchased from R&D Systems (Minneapolis,
MN). The anti-phosphotyrosine antibody used is a 30:30:1 ratio of
clonal antibodies PY72, PY20, and 4G10 obtained from Dr. Bart Sefton (Transduction Laboratories, San Diego, CA) and Upstate Biotechnology, Inc. (Lake Placid, NY). The anti-caspase-3 antibody was purchased from
Upstate Biotechnology, Inc. The caspase inhibitors DEVD-fluoromethyl ketone (FMK) and LEHD-FMK and the fluorogenic substrates
DEVD-aminotrifluoromethylcoumarin (AFC) and LEHD-AFC were obtained from
Enzyme Systems Products (Livermore, CA). The active recombinant human
caspase-3 enzyme was purchased from BioVision (Mountain View, CA). All
other reagents were purchased from Sigma unless indicated otherwise.
Purification of Peripheral Blood Monocytes--
Monocytes were
isolated from buffy coats obtained from the American Red Cross
according to a method described previously (16). For DNA fragmentation
analysis, nitric oxide measurement, and caspase 3-like activity
measurement, monocytes (5 × 106/sample) were treated
in the indicated conditions immediately after monocyte isolation. In
signaling experiments for tyrosine-phosphorylated proteins, monocytes
were resuspended at 10 × 106 cells/ml of RPMI 1640 medium, 10% fetal bovine serum, 10 µg/ml polymyxin B, and 20 ng/ml
recombinant human M-CSF. After 24 h in culture, the cells were
serum-starved for 2 h, treated for 1 h with the indicated
nitric oxide donors, and stimulated for 2 min with 100 ng/ml
M-CSF.
Cytosolic DNA Fragmentation Analysis--
Apoptotic DNA
fragments were purified using DNA isolation kits (Suicide-Track DNA
isolation kit, Oncogene Research Products, Cambridge, MA). DNA
fragments were resolved by 1.6% agarose gel electrophoresis. DNA bands
were visualized by staining with Syber Green (Molecular Probes, Inc.,
Eugene, OR). The DNA fragments were analyzed on a digital gel
documentation system (Gel-Doc 1000, Bio-Rad).
Quantification of Apoptosis--
Apoptosis was measured by using
an annexin V-FITC apoptosis dectection kit according to the
manufacturer's protocol (Pharmingen). Samples were analyzed by a flow
cytometer (FACSCalibur, BD Biosciences).
Nitric Oxide Measurement by Chemiluminescence--
Monocytes
were cultured for 24 h in Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum with the following NO donors at 2 µM to 2 mM concentrations: GSNO, SNAP, DEA
NONOate, and PAPA NONOate. Nitrate and nitrite in the cultured
supernatants were injected into a collection chamber and reduced to
nitric oxide by VCl3 in 1 N HCl. The nitric
oxide reacted with ozone, producing a chemiluminescent signal that
was measured by a nitric oxide analyzer (Sievers NOA 280).
Caspase Enzymatic Activity Measured with AFC--
Monocytes were
collected by centrifugation and washed with KPM buffer (50 mM KCl, 50 mM PIPES, 10 mM EGTA,
1.92 mM MgCl2, 1 mM dithiothreitol
(DTT), 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
cytochalasin B, and a 2 µg/ml concentration of each the following
protease inhibitors: chymostatin, pepstatin, leupeptin, and antipain).
The cell suspension was lysed by four cycles of freezing and thawing.
The cytosolic protein was obtained after centrifugation at 12,000 × g for 20 min at 4 °C. The extracts were incubated with
the fluorogenic substrate DEVD-AFC for caspase-3-like activity or with
LEHD-AFC for caspase-9 activity in a cyto-buffer (10% glycerol,
50 mM PIPES, pH 7, and 1 mM EDTA) containing 1 mM DTT and 20 µM DEVD-AFC as previously
described (16). For experiments using the active recombinant human
caspase-3 enzyme, the NO donors were incubated with the protein at
one-seventh of their half-lives in an attempt to control for the
varying half-lives of the different nitric oxide donors, except GSNO.
Because of its extremely long half-life, GSNO was incubated with
recombinant caspase-3 at 1/242 of its half-life. DTT was omitted
from KPM buffer to prevent denitrosylation of recombinant human
caspase-3. DTT was added to the indicated samples for 15 min prior to
measurement of caspase-3 activity to reverse S-NO formation.
All conditions were assayed for caspase-3 activity by measuring the
release of free AFC as determined with a Cytofluor 4000 fluorometer
(filters, excitation at 400 nm and emission at 505 nm; PerSeptive
Diagnostics, Ramingham, MA).
Immunoprecipitation and Immunoblotting--
Stimulated samples
were left on ice for 15 min in lysis buffer containing 50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 10 mM NaF, 0.5% deoxycholate, 10 mM EDTA, 0.1%
SDS, and 1% Nonidet P-40. Lysates were centrifuged at 12,000 × g for 10 min at 4 °C. Equivalent amounts of protein were
used for each immunoprecipitation or whole cell lysate as determined by
the Bradford protein assay (Bio-Rad). Immunoprecipitated samples were
incubated with the mouse anti-phosphotyrosine antibody mixture
overnight at 4 °C. After incubation with protein G-agarose beads
(Invitrogen), the immune complexes attached to the beads were washed
three times with lysis buffer. Laemmli sample buffer containing
2-mercaptoethanol was added to the agarose beads and whole cell lysates
and incubated for 5 min at 95 °C. The samples were run on a 10%
SDS-polyacrylamide gel and transferred to nitrocellulose membranes. The
membranes were blocked with 5% bovine serum albumin in 1×
Tris-buffered saline and 1% Tween and probed with the indicated primary antibodies, followed by incubation with the appropriate secondary antibody linked to horseradish peroxidase. The membrane was
evaluated by enhanced chemiluminescence detection (Amersham Biosciences).
High Performance Liquid Chromatography (HPLC) Electrochemical
Measurement of GSH--
Samples were prepared as described (17).
Glutathione was separated using a C18 column and detected
using an HPLC colorimetric electrode array detector (Coularray Detector
Model 5600 with 12 channels, ESA Inc., Chelmsford, MA). The mobile
phase consisted of 50 mM sodium dihydrogen phosphate, 0.5 mM octanosulfonic acid, and 3% acetonitrile at pH 2.7 (18).
Statistical Analysis--
All data are expressed as means ± S.E. Statistical significance was defined as p < 0.05 using analysis of variance with post hoc testing
(Minitab, College Park, PA).
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RESULTS |
Nitric Oxide Supports Survival by a Caspase-3-dependent
Mechanism--
The spontaneous apoptosis of human monocytes appears to
be partly regulated by the activation of caspase-3 (1). GSNO has been
shown to S-nitrosylate the critical cysteine residue in the catalytic domain of caspase-3 to inhibit caspase-3 activation and to
facilitate cell survival (6). Thus, we speculated that nitrosothiol
donors such as GSNO would suppress caspase-3 activation and facilitate
cell survival. The nitrosothiol donor GSNO suppressed DNA fragmentation
events (Fig. 1A) and inhibited
the activation of native caspase-3 (Fig. 1B). Low
concentrations of DEA NONOate and PAPA NONOate also reduced caspase-3
activation in human monocytes (Fig. 1, C and D),
but did not appear to reduce DNA fragmentation. Higher concentrations
of DEA NONOate and PAPA NONOate resulted in caspase-3 activation,
whereas similar concentrations of GSNO did not.

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Fig. 1.
Nitric oxide presentation influences survival
and caspase-3 activation in primary human monocytes. Human
monocytes (5 × 106/condition) were left
non-stimulated (0, ) or were incubated with the nitrosothiol donor
GSNO or the nitric oxide donor PAPA NONOate or DEA NONOate at 0.5, 1.0, and 1.5 mM as indicated for 24 h. M-CSF (+) was used
as a control. A, cytosolic extracts from monocytes incubated
with GSNO were assayed for DNA fragmentation as described under
"Experimental Procedures." B, monocytes were lysed
immediately after isolation (FRESH) or lysed after 24 h
of incubation with GSNO and assayed for caspase-3-like activity using
the fluorogenic substrate DEVD-AFC. Data are expressed as means ± S.E. for four independent experiments. GSNO at 0.5, 1.0, and 1.5 mM suppressed caspase-3 activation compared with
non-stimulated control cells incubated for 24 h. **,
p < 0.001. C, cytosolic extracts from
monocytes incubated with PAPA NONOate or DEA NONOate were assayed for
DNA fragmentation. D, lysates from monocytes treated with
PAPA NONOate or DEA NONOate were assayed for caspase-3 activity as
described above. Data are means ± S.E. for four independent
experiments, with *, p < 0.05 reduction in caspase-3
activity by PAPA NONOate and DEA NONOate compared with the
non-stimulated 24-h control.
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Compared with non-stimulated cells staining positive for annexin
V/propidium iodide, GSNO and SNAP reduced annexin V/propidium iodide staining (p < 0.01 non-stimulated
versus GSNO or SNAP; non-stimulated, 84.8 ± 2.3%;
GSNO, 39.8 ± 10.1%; SNAP, 53.2 ± 0.76%), whereas PAPA
NONOate and DEA NONOate did not (p > 0.4 non-stimulated versus PAPA NONOate or DEA NONOate;
non-stimulated, 84.3 ± 2.3%; PAPA NONOate, 71.52 ± 5.6%;
DEA NONOate, 80.46 ± 6.6%).
PAPA NONOate and DEA NONOate Block M-CSF-induced Monocyte Survival
through Caspase-3 Activation--
Previously, we demonstrated that
M-CSF promotes the survival of human monocytes (15). We next
investigated whether PAPA NONOate or DEA NONOate would reverse the
suppression of caspase-3 and DNA fragmentation promoted by M-CSF in
human monocytes. We found that PAPA NONOate or DEA NONOate
independently induced DNA fragmentation and caspase-3 activation in
M-CSF-stimulated human monocytes (Fig. 2,
A and B). In contrast to the effects of PAPA NONOate or DEA NONOate, GSNO did not promote DNA fragmentation or the
activation of native caspase-3 in parallel samples of M-CSF-stimulated monocytes (Fig. 2, C and D).

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Fig. 2.
PAPA NONOate and DEA NONOate inhibit
M-CSF-induced monocyte survival and activate caspase-3. Monocytes
(5 × 106/condition) were left non-stimulated or were
incubated with the nitric oxide donor PAPA NONOate, DEA NONOate, or
GSNO (0.5, 1.0, and 1.5 mM) as indicated for 30 min and
then incubated without ( ) or with (+) M-CSF (100 ng/ml) for 24 h. A, cytosolic extracts from non-stimulated or
M-CSF-stimulated monocytes treated with the indicated concentrations of
PAPA NONOate or DEA NONOate were assayed for DNA fragmentation.
B, lysates from monocytes prepared as described above were
assayed for caspase-3-like activity using the fluorogenic substrate
DEVD-AFC. Data are expressed as means ± S.E. for four
experiments. Cells left non-stimulated ( ) for 24 h and cells
treated with PAPA NONOate or DEA NONOate in the presence of M-CSF had
no caspase-3 activation versus cells treated with M-CSF
alone or freshly isolated (FRESH). , p < 0.01. C, cytosolic DNA fragmentation was assayed in extracts
of monocytes left non-stimulated or stimulated with M-CSF alone or with
the indicated concentrations of GSNO. D, lysates from
monocytes as cultured above were assayed for caspase-3-like activity.
Data are means ± S.E. from four experiments. Only non-stimulated
cells for 24 h had more caspase-3 activity compared with cells
incubated with M-CSF with or without GSNO. *, p < 0.05.
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Glutathione Levels and Cumulative Nitric Oxide Production over
24 h Are Not Responsible for the Differing Effects on DNA
Fragmentation and Caspase-3 Activation in Human Monocytes--
Because
GSNO is an S-nitrosoglutathione, we first examined the
possibility that enhanced glutathione stores resulting from treatment
with GSNO in monocytes were responsible for differences seen between
GSNO and NONOates. We found that intracellular glutathione levels were
decreased in monocytes treated with PAPA NONOate or DEA NONOate in the
absence of M-CSF (Fig. 3A).
However, replenishing intracellular glutathione with
N-acetylcysteine (Fig. 3B) did not inhibit
caspase-3 activation by these nitric oxide donors and even appeared to
enhance caspase-3 activity in monocytes treated with M-CSF or GSNO
(Fig. 3C). To ensure that the protective effect of GSNO was
not related solely to increased glutathione levels donated by GSNO, we
also used the nitrosothiol donor SNAP, which does not generate
glutathione. Similar to GSNO, SNAP reduced DNA fragmentation over a
24-h period and reduced the activation of native caspase-3 in human
monocytes (Fig. 4, A and
B).

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Fig. 3.
Intracellular glutathione levels are not
responsible for the effects of PAPA NONOate and DEA NONOate.
A, monocytes (5 × 106/condition) were left
non-stimulated (NS) or were stimulated with the nitric oxide
donor GSNO, PAPA NONOate, or DEA NONOate (0.5 mM) for
24 h and measured for GSH. PAPA NONOate or DEA NONOate reduced
intracellular glutathione compared with cells treated with GSNO and the
non-stimulated control. , p < 0.01. B,
monocytes (5 × 106/condition) were treated as
described above without ( ) or with (+) exogenous
N-acetylcysteine (NAC; 20 mM) for
24 h, and glutathione was measured. Only in glutathione levels did
significant differences exist in cells treated with PAPA NONOate or DEA
NONOate in the absence of N-acetylcysteine compared with the
non-stimulated + N-acetylcysteine control. *,
p < 0.05. C, lysates from monocytes (5 × 106/condition) treated as described for B
were assayed for caspase-3-like activity. These data represent three
independent studies and demonstrate that the addition of
N-acetylcysteine increased caspase-3 activity compared with
samples treated with M-CSF and GSNO alone. *, p < 0.05.
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Fig. 4.
The nitrosothiol SNAP also blocks DNA
fragmentation and caspase-3 activity in human monocytes.
A, monocytes (5 × 106) were left
non-stimulated (NS) or were treated with the
Me2SO (DMSO) solvent control and assayed for DNA
fragmentation as described under "Experimental Procedures." Cells
were treated with the nitric oxide donors SNAP and GSNO (20 and 200 µM and 2 mM). B, monocytes were
incubated with the above controls and with 2 mM SNAP or
GSNO. Lysates were assayed for caspase-3 activity by measuring the
caspase-3 substrate DEVD-AFC. These data are representative of two
separate experiments. , p < 0.02 versus
non-stimulated and Me2SO controls.
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We next wanted to determine whether the dichotomous effects of the
nitric oxide donors could be explained by quantitative differences in
nitric oxide release by the donors. We found that, although GSNO
blocked the activation of caspase-3, it produced less nitric
oxide than DEA NONOate and PAPA NONOate measured at 24 h (Table
I). To ensure that the higher amounts of
nitric oxide delivered by DEA NONOate or PAPA NONOate did not
independently injure the cells, lower concentrations of PAPA NONOate
and DEA NONOate were measured at 24 h (Table
II), and they did not consistently prevent the activation of caspase-3 (Fig.
5). Of note, using these lower
concentrations of nitric oxide donors, only SNAP significantly suppressed caspase-3 activity.
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Table I
Nitric oxide production in human monocytes
Monocytes (5 × 106) were incubated for 24 h with the
indicated nitric oxide donors in millimolar concentrations, with the
exception of M-CSF. The Culture medium was measured for nitric oxide
production by ozone chemiluminescence as described under
"Experimental Procedures." Data are expressed as means ± SE
for four independent experiments.
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Table II
Nitric oxide production at lower doses of NO donors in human
monocytes
Monocytes (5 × 106) were incubated for 24 h with the
indicated nitric oxide donors in micromolar concentrations. The culture
medium was measured for nitric oxide production by ozone
chemiluminescence as described under "Experimental Procedures."
Data are expressed as means ± S.E. for two independent
experiments.
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Fig. 5.
DEA NONOate and PAPA NONOate do not reduce
caspase-3 activation at lower nitric oxide concentrations.
Monocytes (5 × 106) were treated with GSNO, SNAP,
DEA-NONOate, and PAPA-NONOate (2-200 µM, as indicated).
Monocytes were also left non-stimulated (NS) or were treated
with the vehicle control Me2SO (DMSO). The
fluorogenic substrate DEVD-AFC released from these lysates was measured
as described under "Experimental Procedures." Data are means ± S.E. from two independent experiments. *, p < 0.05 for SNAP compared with the non-stimulated control.
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Nitric Oxide Donors Suppress Tyrosine Phosphorylation Events
Induced by M-CSF-stimulated Human Monocytes--
We next sought to
explain the mechanism of DEA NONOate and PAPA NONOate suppression of
M-CSF-induced monocyte survival. Because we have shown that M-CSF
induces reactive oxygen species production in monocytes (15), we
speculated that nitric oxide combined with reactive oxygen species
forms peroxynitrite and suppresses tyrosine phosphorylation events in
M-CSF-stimulated monocytes. As predicted, we found that each of the
three nitric oxide donors reduced tyrosine phosphorylation events in
M-CSF-stimulated monocytes (Fig. 6).

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Fig. 6.
Nitric oxide donors suppress tyrosine
phosphorylation events induced by M-CSF-stimulated human
monocytes. A, monocytes (10 × 106/condition) were left non-stimulated ( ) or were
pretreated with the nitric oxide donors GSNO, PAPA NONOate, and DEA
NONOate (0.5 mM) for 1 h prior to stimulating these
samples with M-CSF (100 ng/ml; +) for 2 min. The cells were lysed,
immunoprecipitated, and blotted for tyrosine-phosphorylated proteins
with anti-phosphotyrosine antibodies as described under
"Experimental Procedures." IgG heavy chain (H.C.) and
light chain (L.C.) are labeled. Data are representative of
three independent studies. B, shown are densitometric units
(D.U.) of bands representing tyrosine-phosphorylated
proteins of the blot in A.
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GSNO and SNAP Prevent the Proteolytic Processing of
Caspase-3 to Promote Monocyte Survival--
Because GSNO and DEA
NONOate or PAPA NONOate reduced cellular tyrosine phosphorylation
events in M-CSF-stimulated monocytes, but only GSNO promoted monocyte
survival events, we concluded that GSNO promoted cell survival by
acting downstream of the receptor. In keeping with this hypothesis,
only GSNO and SNAP blocked the cleavage of caspase-3 to its active
17-kDa subunit at 24 h of incubation, whereas cells incubated with
DEA NONOate or PAPA NONOate or incubated alone did not (Fig.
7A). The 17-kDa subunit was
also measured by densitometry (Fig. 7B).

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Fig. 7.
The nitrosothiols GSNO and SNAP can prevent
the formation of caspase-3 cleavage products. A,
monocytes (5 × 106) were freshly isolated
(FRESH) from buffy coats or were isolated and left
non-stimulated (NS) or were treated with 0.5 mM
GSNO, SNAP, DEA-NONOate, or PAPA-NONOate for a 24-h incubation period.
Processed 17-kDa products of caspase-3 were detected using
anti-caspase-3 antibodies according to the immunoblotting procedure
described under "Experimental Procedures." Anti- -actin
antibody was used as an indicator to show equal protein loading.
B, a ratio of the 17-kDa caspase-3 cleavage product to total
-actin protein is represented in densitometric units
(D.U.). DMSO, Me2SO.
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GSNO and SNAP Inhibit the Activity of Active Recombinant
Caspase-3--
In addition to reducing the activation of caspase-3, we
next wanted to determine whether the nitrosothiol donors GSNO and SNAP
could also interrupt the biological activity of active caspase-3. This
prediction was based on the finding that the active cysteine site of
caspase-3, which can be modified via S-nitrosylation, influences the enzymatic activity of caspase-3 (7). To test this
hypothesis, we first determined the kinetics of active recombinant caspase-3 and found that this enzyme spontaneously lost biological activity quickly after 15 min, but stabilized between 30 and 90 min of
incubation (Fig. 8A). Using a
time point of 40 min, we then added the nitric oxide donors
SNAP, PAPA NONOate, and DEA NONOate at one-seventh of their
respective half-lives and GSNO at 1/242 of its half-life. GSNO
and SNAP reduced the enzymatic activity of active recombinant
caspase-3, whereas DEA and PAPA NONOate did not (Fig. 8B).
To determine whether the direct inhibition of caspase-3 by GSNO and
SNAP was due to modification of the sulfhydryl group, the reducing
agent dithiothreitol was added to remove NO modification of thiol
residues by nitric oxide. In the presence of GSNO or SNAP, DTT restored
caspase-3 enzymatic activity, suggesting that GSNO and SNAP modify
active recombinant caspase-3 by S-nitrosylation of the
active caspase (Fig. 8C).

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Fig. 8.
GSNO and SNAP directly inhibit active
recombinant human caspase-3, which is reversible by DTT.
A, the active recombinant human caspase-3 enzyme was
incubated at 37 °C for 0, 15, 30, 60, 75, and 90 min. The
spontaneous loss of enzymatic activity stabilized between 30 and 90 min. B, the nitric oxide donors SNAP, DEA NONOate, and PAPA
NONOate were added at one-seventh of their half-lives, or GSNO was
added at 1/242 of its half-life and incubated with active
recombinant human caspase-3. Caspase-3 activity was determined as
described under "Experimental Procedures." Results are means ± S.D. for three independent experiments. **, p < 0.001 versus non-stimulated recombinant caspase-3 enzyme.
C, because only GSNO and SNAP reduced caspase-3 activity, we
next added dithiothreitol (+) for 15 min to samples of active
recombinant caspase-3 that were incubated with GSNO or SNAP. The
non-stimulated (NS) samples are taken as 100% control. Data
are means ± S.D. for three independent experiments. **,
p < 0.001 versus GSNO + DTT, SNAP + DTT, or
non-stimulated.
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Blocking Caspase-3 and Caspase-9 Activation Reverses Apoptosis
Induced by PAPA NONOate or DEA NONOate in M-CSF-stimulated
Monocytes--
Because GSNO and SNAP appeared to reduce DNA
fragmentation and activation of caspase-3 in human monocytes, we next
wanted to determine whether caspase-3 activation was involved in
promoting DNA fragmentation in PAPA NONOate- and DEA NONOate-treated
monocytes. The caspase-3 inhibitor DEVD-FMK reduced oligonucleosomal
DNA fragmentation in M-CSF-stimulated monocytes treated with DEA
NONOate or PAPA NONOate (Fig. 9). To
evaluate the role of caspase-9 in this pathway, we found that GSNO and
SNAP also suppressed the activation of caspase-9 in human monocytes
(Fig. 10A). Moreover, the
caspase-9-selective inhibitor LEHD-FMK inhibited caspase-3 activation
in monocytes incubated with PAPA NONOate or DEA NONOate (Fig.
10B), suggesting that caspase-3 is regulated by caspase-9 in
this pathway.

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Fig. 9.
Blocking caspase-3 activation with DEVD-FMK
reverses DNA fragmentation induced by PAPA NONOate or DEA NONOate in
M-CSF-stimulated monocytes. Monocytes (5 × 106/condition) were left non-stimulated ( ) or were
stimulated with M-CSF alone (+) or with the indicated concentrations of
PAPA NONOate or DEA NONOate in the presence (+) or absence ( ) of the
caspase-3-selective peptide benzyloxycarbonyl-DEVD-FMK (100 µM) or an equal concentration of Me2SO for
24 h. Cytosolic extracts were assayed for DNA fragments. Data are
representative of three independent studies.
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Fig. 10.
GSNO and SNAP reduce caspase-9 activation,
and blocking caspase-9 activation inhibits caspase-3 activity in
monocytes treated with PAPA NONOate or DEA NONOate. A,
monocytes (5 × 106/condition) were incubated with 1.0 mM GSNO, 1.0 mM SNAP, 0.5 mM PAPA
NONOate, or 0.5 mM DEA NONOate for 24 h. These
concentrations of NO donors gave more equivalent concentrations of NO
at 24 h. Caspase-9-like activity was assayed and is expressed as
means ± S.E. for two studies.  , p = 0.004 versus the non-stimulated (NS) control.
B, monocytes (5 × 106/condition) were left
non-stimulated ( ) or were stimulated with M-CSF treated with PAPA
NONOate or DEA NONOate in the presence (+) or absence ( ) of the
caspase-9-selective inhibitor LEHD-FMK (100 µM) for
24 h and measured for caspase-3-like activity. Me2SO
was added to all conditions as the solvent control for LEHD-FMK.
LEHD-FMK reduced caspase-3-like activity in monocytes treated with
M-CSF and either PAPA or DEA NONOate. Results are expressed as
means ± S.E. for four independent studies. , p < 0.01 versus the non-stimulated control.
|
|
 |
DISCUSSION |
This study was designed to test the hypothesis that the
presentation of nitric oxide determines whether nitric oxide functions to promote cell life or cell death. Incubating human monocytes with the
nitrosothiol donors GSNO and SNAP suppressed the activation of native
caspase-3, reduced the enzymatic activity of active recombinant
caspase-3, and reduced DNA fragmentation in monocytes incubated with or
without M-CSF. In contrast, incubating parallel samples of these
monocytes with the nitric oxide donor PAPA NONOate and DEA NONOate,
which do not target thiol groups, did not consistently suppress the
activation of native caspase-3 or reduce the activity of active
recombinant caspase-3. Furthermore, in the presence of M-CSF, PAPA
NONOate and DEA NONOate induced the activation of native caspase-3 and
also induced cytosolic oligonucleosomal DNA fragmentation. Thus, it
appears that the ability to donate nitric oxide to protein thiol
residues is important in reducing caspase-3 activation and in promoting
the survival of human monocytes.
To support the hypothesis that the interruption of caspase-3 is
important in monocyte survival, we found that the caspase-3 inhibitor
DEVD-FMK blocked DNA fragmentation in monocytes treated with PAPA
NONOate or DEA NONOate in the presence of M-CSF, suggesting that
caspase-3 activation regulates these apoptotic events. Moreover, the
caspase-9-selective inhibitor LEHD-FMK suppressed caspase-3 activation
in monocytes treated with PAPA NONOate or DEA NONOate, suggesting that
caspase-9 activation is involved as the upstream activator of caspase-3
in human monocytes. To confirm that caspase-9 is involved in GSNO- or
SNAP-induced monocyte survival, we found that treating monocytes with
the nitrosothiol donors GSNO and SNAP also suppressed native caspase-9
activity in the absence of M-CSF, suggesting that nitrosothiol donors
interrupt the activation of caspase-9 upstream of caspase-3. These data
suggest that modulating the activity of caspases is a critical
determinant of monocyte survival by the nitrosothiol donors.
We considered the possibility that intracellular glutathione depletion
by PAPA NONOate and DEA NONOate may lead to caspase activation, as
glutathione depletion can induce the release of cytochrome
c, leading to caspase-9 and caspase-3 activation (19). Other
reports suggest that caspase-3 function may be impaired upon depletion
of glutathione (20). Consistent with the possibility that glutathione
depletion by PAPA NONOate and DEA NONOate promotes caspase-3
activity, monocytes treated with PAPA NONOate or DEA NONOate had lower
concentrations of intracellular glutathione than monocytes treated with
GSNO. However, replenishing glutathione stores using
N-acetylcysteine did not reduce caspase-3 activation in
monocytes stimulated with PAPA NONOate or DEA NONOate. Moreover, the
addition of N-acetylcysteine increased the activation of
caspase-3 in GSNO-treated monocytes, suggesting that replenishment of
glutathione is not responsible for the observed differences in cell
survival mediated by the different nitric oxide donors. To confirm that glutathione is not directly responsible for monocyte survival or
apoptosis, we incubated monocytes with the nitrosothiol donor SNAP,
which can S-nitrosylate thiol residues without donating glutathione. SNAP also promoted monocyte survival and blocked the
activation of caspase-3.
We then considered the possibility that quantitative differences in
nitric oxide delivered by the different donors determine cell fate and
caspase-3 and caspase-9 inhibition. We found that the quantity of
nitric oxide produced by GSNO, SNAP, and PAPA NONOate or DEA NONOate
did not correlate with the inhibition of caspase-9 or caspase-3. To
determine the mechanism of caspase-3 inhibition by GSNO or SNAP, we
found that these donors blocked the production of the 17-kDa caspase-3
cleavage product in human monocytes. Because these donors also blocked
caspase-9 activation in human monocytes, we speculated that
nitrosothiol donors inhibit the upstream activation of caspase-3 to
block monocyte apoptosis. Interestingly, using active recombinant
caspase-3, we found that GSNO and SNAP also blocked enzymatically
active caspase-3. We next sought to understand how these nitrosothiol
donors are able to block caspase-3 activity and found that the reducing
agent DTT, which can regenerate disulfide bonds in modified thiol
residues, reversed the effects of these nitrosothiol donors and
restored caspase-3 activity. In composite, these data argue that GSNO
and SNAP reduce caspase-3 activity by both blocking the upstream
activation of caspase-3 by caspase-9 and blocking the biological
activity of enzymatically active caspase-3, likely through
S-nitrosylation.
We next wanted to understand why PAPA NONOate and DEA NONOate did not
promote monocyte survival, even though these donors augment the
N-nitrosylation of proteins. We previously described that
M-CSF stimulation augments the production of reactive oxygen species by
human monocytes (15). We speculated that PAPA NONOate and DEA NONOate
promote caspase-9 and caspase-3 activation by reducing tyrosine
phosphorylation events in monocytes stimulated with M-CSF.
Interestingly, we found that PAPA NONOate, DEA NONOate, and
GSNO each reduced tyrosine phosphorylation events in M-CSF-stimulated monocytes, suggesting that the protective effects of GSNO are downstream of the receptor. We speculated that it is the ability of
GSNO and SNAP to directly suppress caspase-3 activation that differentiates GSNO and SNAP from DEA NONOate or PAPA NONOate in
promoting monocyte survival. However, it is also possible that GSNO and
SNAP influence other signaling events that result in the suppression of
caspase-9 or caspase-3 activation after M-CSF stimulation, and this is
being actively investigated in our laboratory.
It is interesting to note that, in the absence of M-CSF, PAPA NONOate
and DEA NONOate were intermittently able to reduce the activation of
caspase-3. These data are very interesting and suggest either that
there is some targeting of thiol residues by PAPA NONOate or DEA
NONOate or that, perhaps more likely, N-nitrosylation events
may play some role in caspase activation. We are investigating the
mechanism of this observation.
These findings may have relevance to inflammatory human diseases in
which nitric oxide may be produced in a localized milieu. In the
presence of superoxide, nitric oxide may combine to form peroxynitrite
and block tyrosine phosphorylation of proteins. In the absence of being
able to suppress caspase activation, nitric oxide may lead to cellular
apoptosis (21). This pathway may be important in the loss of left
ventricular heart function after acute myocardial infarction, as
inducible nitric-oxide synthase-deficient mice appear to be protected
from cell death more than control animals (22-24). Similarly, nitric
oxide has been implicated in immune destruction of islet cells in the
pancreas, leading to diabetes mellitus (25-27). In contrast, nitric
oxide may also directly nitrosylate protein thiol residues regulating
cell survival through suppressing caspase activation, favoring cell
survival (6, 28). The resulting accumulation of inflammatory cells may
create a burden for the involved organ and precipitate injury.
Interestingly, correlating with the important in vitro
effects of GSNO, a GSNO reductase that is conserved from yeast to man
has been identified recently (29), suggesting that GSNO likely has
important biological functions in vivo. Thus, regulation of
GSNO in mammalian cells appears to be of importance.
In summary, the data generated in this study support the
hypothesis that the presentation of nitric oxide plays an important role in determining cell fate. When SNAP or GSNO was used, each of
which can S-nitrosylate protein residues, nitric oxide
inhibited the activation of caspase-9 and caspase-3 and DNA
fragmentation in human monocytes. Moreover, these donors also
suppressed the activity of active recombinant caspase-3. These data
suggest that GSNO and SNAP can both inhibit the activation of casapse-3
and interfere with the biological activity of enzymatically active recombinant caspase-3, likely via protein S-nitrosylation.
In contrast, the addition of PAPA NONOate or DEA NONOate, which
primarily leads to N-nitrosylation of protein tyrosine
residues in human monocytes, does not consistently reduce caspase-9 or
caspase-3 activation or DNA fragmentation. These data begin to unravel
the molecular details of how nitric oxide may be able to induce both cell survival and cell death. Our data suggest that the presentation of
nitric oxide and the subsequent suppression of caspase-9 and caspase-3
activation determine whether nitric oxide promotes cell survival or
cell death in human monocytes.