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Prepublished online as a Blood First Edition Paper on August 29, 2002; DOI 10.1182/blood-2002-06-1779.
IMMUNOBIOLOGY
From the Department of Clinical and Experimental
Medicine, Pharmacology Section, University of Perugia, and the
Department of Experimental Medicine, University of L'Aquila,
Italy.
Glucocorticoid hormones (GCHs) regulate normal and neoplastic
lymphocyte development by exerting antiproliferative and/or apoptotic
effects. We have previously shown that dexamethasone (DEX)-activated
thymocyte apoptosis requires a sequence of events including interaction
with the glucocorticoid receptor (GR), phosphatidylinositol-specific phospholipase C (PI-PLC), and acidic sphingomyelinase (aSMase) activation. We analyzed the mechanisms of GCH-activated apoptosis by
focusing on GR-associated Src kinase, cytochrome c release, and caspase-8, -9, and -3 activation. We show here that PI-PLC binds to
GR-associated Src kinase, as indicated by coimmunoprecipitation experiments. Moreover, DEX treatment induces PI-PLC
phosphorylation and activation. DEX-induced PI-PLC phosphorylation,
activation, and apoptosis are inhibited by PP1, a Src kinase inhibitor,
thus suggesting that Src-mediated PI-PLC activation is involved
in DEX-induced apoptosis. Caspase-9, -8, and -3 activation and
cytochrome c release can be detected 1 to 2 hours after DEX
treatment. Caspase-9 inhibition does not counter cytochrome c
release, caspase-8 and caspase-3 activation, and apoptosis.
Caspase-8 inhibition counters cytochrome c release,
caspase-9 and caspase-3 activation, and apoptosis, thus suggesting that
caspase-8 inhibitor can directly inhibit caspase-9 and/or that
DEX-induced caspase-8 activation is upstream to mitochondria and can
regulate caspase-3 directly or through cytochrome c release
and the consequent caspase-9/caspase-3 activation. DEX-induced
caspase-8 activation, like ceramide-induced caspase-8
activation, correlates with the formation of Fas-associated death domain protein (FADD)/caspase-8 complex. Caspase-8
activation is countered by the inhibition of macromolecular synthesis
and of Src kinase, PI-PLC, and aSMase activation, suggesting it is downstream in the DEX-activated apoptotic pathway of thymocytes.
(Blood. 2003;101:585-593) Glucocorticoid hormones (GCHs) are used in a number
of inflammatory and autoimmune diseases, in organ transplantation, and in the treatment of leukemia and lymphomas.1-6 GCHs induce
the apoptosis of thymocytes, particularly in stress conditions, when higher levels of circulating hormones are present in the blood. They
also induce cytotoxicity against normal and neoplastic lymphocytes during pharmacologic therapy. However, their therapeutic use is limited
because of several unwanted side effects, which can lead to the
suspension of treatment, and because of resistance, as may occur in
leukemia and lymphoma. Thus, a more profound analysis of the molecular
mechanisms implicated in apoptosis induction appears necessary.
Thymocytes are highly sensitive to GCH-induced apoptosis, and, though
they have been investigated in many studies, the molecular mechanisms
responsible for GCH-induced cell death have not yet been fully
clarified. GCH interaction with the glucocorticoid receptor (GR),
endonuclease activation, Ca++ mobilization, cytochrome
c release, and proteasome and caspase activation have been
shown to participate in GCH-induced apoptosis.7-12
In a previous work13 we showed that activation of
a sequence of events is required for dexamethasone (DEX)-induced
apoptosis of thymocytes. In particular, DEX-induced apoptosis can be
attributed to early (5-15 minutes) ceramide generation due to
activation of acidic sphingomyelinase (aSMase). Moreover, DEX treatment
rapidly (1-5 minutes) activates phosphatidylinositol-specific
phospholipase C (PI-PLC) through a mechanism mediated by protein kinase
C (PKC) activity and induces diacylglycerol (DAG) generation, which
precedes, and is required for, aSMase activation and ceramide
generation.13
Caspase activity plays a crucial role in and is downstream to PI-PLC
and aSMase activation, insofar as inhibition of early ceramide
generation blocks caspase activation and thymocyte death. All these
events, including early PKC and PI-PLC activation, require GCH
interaction with the GR and are countered by GR antagonists such as
mifepristone.13
The GCH/GR interaction is required for GCH-induced genomic and
nongenomic effects, GR release from other components of the cytoplasm
macromolecular complex, including HSP90 and GR-associated Src kinase,
and its translocation to the nucleus.14,15
In the present study, we further analyzed the molecular mechanisms
involved in DEX-induced apoptosis of thymocytes to understand how GCH
activates PI-PLC and to elucidate the role of different caspases in
apoptosis. In particular, we performed experiments aimed at defining
the role of GR-associated Src kinase, the role of activation of
different caspases such as caspase-8, -9, and -3, and the role of
cytochrome c release. Our results indicate that (1)
PI-PLC binds GR-associated Src in the HSP90/GR/Src complex; (2)
GR-associated Src kinase inhibition blocks DEX-induced PI-PLC phosphorylation, activation, and apoptosis; and (3) DEX treatment, like
ceramide treatment, induces Fas-associated death domain protein (FADD)/caspase-8 complex formation and activation of caspase-8, -9, and -3. In addition, we found that caspase-9 inhibition does not
counter cytochrome c release, caspase-8 and caspase-3
activation, or apoptosis. On the other hand, caspase-8 inhibition
counters cytochrome c release, caspase-9 and caspase-3
activation, and apoptosis. We also found that DEX-induced caspase-8
activation is downstream of early events such as Src, PI-PLC, and
aSMase activation and is inhibited by Src, PI-PLC, aSMase, and
macromolecular synthesis inhibitors. These results indicate that a
complex of molecular mechanisms, including GR-associated Src kinase and
caspase-8/caspase-3 activation, is involved in DEX-induced apoptosis of thymocytes.
Cell system and treatments
Apoptosis evaluation
Immunofluorescence staining and nuclear translocation assay To evaluate the nuclear translocation of the GR, thymocytes were processed for immunofluorescence using the paraformaldehyde-saponin procedure.21 Cells (2 × 106/mL) were preincubated for 30 minutes with PP1 (10 µM) or U73122 (2.5 µM); thymocytes were then incubated for 30 minutes with DEX 10 7 M, and the treatment was stopped by the immersion of
samples in methanol at  20°C for 30 seconds.
After extensive washing in phosphate-buffered saline (PBS) with 1% HEPES (N-2-hydroxyethylenepiperazine-N'-2-ethanesulfonic acid), cells were fixed in 4% formaldehyde for 20 minutes on ice, washed again, and incubated at 4°C for 1 hour with blocking buffer (PBS with 3% bovine serum albumin [BSA] and 1% glycine). For staining, cells were incubated for 45 minutes at 4°C with 100 ng polyclonal rabbit anti-GR antibodies (Santa Cruz Biotechnology, CA), in buffer containing 0.1% saponin and were washed and incubated for 45 minutes at 4°C with Texas Red-conjugated goat antirabbit immunoglobulin G (IgG; Molecular Probes, Eugene, OR) in PBS-saponin. Cells were then washed, stuck on slides coated with poly-L-lysine, and mounted in buffered glycerol for fluorescence microscopic analysis. Photographs were taken on a Leitz Dialux 20 microscope (Wetzlar, Germany). PI-PLC activity assay PI-PLC activity was determined in vitro by its capacity to hydrolyze 14C-PI vesicles to generate DAG. Cells were treated for 5 minutes with DEX in the presence or absence of the PC-PLC inhibitor D609 (50 µg/mL), the PI-PLC inhibitor U73122 (2.5 µM), or the Src kinase inhibitor PP1 (10 µM). Treatment was stopped by the immersion of samples in methanol/dry ice (70°C) for 10 seconds,
followed by centrifugation at 4°C in a microfuge. Pellets were then
resuspended in 250 mM Tris
(tris[hydroxymethyl]aminomethane)-HCl buffer, pH 7.4, containing 10 µM phenylmethylsulfonyl fluoride (PMSF), 100 µM
bacitracin, 1 mM benzamidine, 1 µM aprotinin, 10 µM leupeptin, 10 µM pepstatin, and 5 µg/mL soybean trypsin inhibitor. Cells were
lysed by sonication with a cell sonifier. Radiolabeled PI vesicles were
prepared by sonicating (5 minutes, 5W, 80% output) L-3-phosphatidylinositol-1stearoyl-2[14C]arachidonoyl
(NEN Life Science Products, Boston, MA) for the detection of released
DAG through PI-PLC. Vesicles were resuspended at 10 µM in the
reaction buffer (50 mM Tris-HC1, pH 7.4, 5 mM CaCl2, 5 mM
MgCl2, 0.01% fatty acid free-BSA). Whole cell lysate (50-100 µg protein) was added to 250 µL reaction buffer containing the vesicles and incubated at 37°C for 1 hour, and the reaction buffer was stopped by the addition of 250 µL
chloroform/methanol/acetic acid (4:2:1, by volume). To separate the
organic phase from the aqueous phase, 250 µL H20, 250 µL CHCl3, and 100 µL KCl were added, and the mixture
was centrifuged at 4.000 rpm in a microfuge for 5 minutes. The
organic phase was removed, dried under nitrogen, resuspended in 200 µL chloroform, and applied to a silica gel thin-layer chromatography
(TLC) plate (Merck, Darmstadt, Germany), with an automatic
applicator Linomat IV (Camag, Muttens, Switzerland). Samples were
chromatographed in chloroform/methanol/acetic acid/water (100:60:16:8)
to separate the parent phospholipids from the PI-PLC product (ie, DAG).
Authentic standards were chromatographed with the lipid extracts to
locate the compounds of interest by exposure to iodine vapor.
Radioactive spots, as visualized by autoradiography that corresponded
to standards, were scraped from the plate and counted by liquid
scintillation. Radioactive measurements were converted to picomole
product by using the specific activity of substrate. Blank values
obtained from controls lacking cell proteins were subtracted from the
experimental values. PI-PLC activity was expressed as pmol DAG
produced/106 cells.
In vitro aSMase assay Aliquots of 6 × 106 cells/mL were treated for 5 minutes with DEX or U73122 or monensin or any combination of them. Treatment was stopped by the immersion of samples in methanol/dry ice (70° C) for 10 seconds, followed by centrifugation at 4°C in a
microfuge. To measure aSMase, the cells were washed after treatment,
and the pellet was resuspended in 200 µL 0.2% Triton X-100 and was incubated for 15 minutes at 4°C. The cells were sonicated, and the
protein concentration was assayed. Then 50 to 100 µg protein was
incubated for 2 hours at 37°C in a buffer (50 µL final volume) containing 250 mM sodium acetate, 1 mM EDTA (ethylenediaminetetraacetic acid), pH 5.0, and 0.32 µL N-methyl-14C
sphingomyelin (0.04 µCi/mL [0.00148 MBq], specific
activity 56.6 mCi/mM [2094.2 MBq]; Amersham). The reaction was
stopped by the addition of 250 µL chloroform/methanol (2:1, by
volume). Lipids were extracted as described above. The organic phase,
obtained through the different extraction steps, was collected and
washed once with 1 mL chloroform/methanol/water (3:48:47, by volume) to
totally remove free radioactive phosphorylcholine. Aqueous phases were
collected, transferred to scintillation vials, and routinely counted by
liquid scintillation. Counts per minute represented the choline
phosphate generated from sphingomyelin (SM) hydrolysis. The organic
phase was analyzed on TLC plates by using
chloroform/methanol/ammonia hydroxide (7N)/water (85:15:0.5:0.5, by
volume). The hydrolysis of SM was quantitated by autoradiography and
liquid scintillation and was expressed as pmol SM
hydrolysed/106 cells.
Immunoprecipitation and Western blotting of PI-PLC Thymocytes were incubated for 1 hour with the Src inhibitor PP1 (10 µM), PI-PLC inhibitor U73122 (2.5 µM), and PC-PLC inhibitor D609 (50 µg/mL) and were treated alone or with DEX 107
M for 5 minutes. After treatment, cells were harvested, and whole cell
lysates were prepared in an extraction buffer containing 50 mM
Tris-HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 1% Nonidet P-40
(NP-40), 1 mM sodium vanadate, 10 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM PMSF, 10 µg/mL leupeptin, and 2 µg/mL aprotinin.
Phosphotyrosine-containing proteins were immunoprecipitated with
agarose-conjugated 4G-10 antibodies (Upstate Biotechnology, Waltham,
MA), and PI-PLC abundance in the 4G-10 immunoprecipitates was assessed
by Western blot analysis using monoclonal anti-PI-PLC Coimmunoprecipitation of HSP90, GR, Src, PI-PLC, caspase-8, and FADD Thymocytes were treated with DEX 107 M or C2
ceramide for the indicated times, and the lysates were prepared with
RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5%
deoxycholate, 0.1% sodium dodecyl sulfate [SDS], and 5 mM EDTA)
supplemented with 1 mM PMSF, 1 mM sodium vanadate, 10 mM sodium
pyrophosphate, 50 mM sodium fluoride, 10 µg/mL leupeptin, and 2 µg/mL aprotinin. In some experiments, groups were pretreated for 30 minutes with the specific Src inhibitor PP1 (10 µM).22 Proteins, 1 mg in RIPA buffer, were
immunoprecipitated overnight with 3 µg monoclonal anti-FADD antibody
(Upstate Biotechnology), monoclonal anti-caspase-8 antibody (Alexis),
or monoclonal anti-HSP90 antibody (BD PharMingen, San Diego, CA) with
continuous rocking. Antigen-antibody complexes were precipitated with
protein A bound to Sepharose beads (Pharmacia, Piscataway, NJ)
for 2 hours before SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
For coimmunoprecipitation experiments, Western blot analysis was
performed using anti-FADD (Upstate Biotechnology), anti-caspase-8 (Alexis), monoclonal anti-HSP90 (BD PharMingen), polyclonal anti-GR (Santa Cruz Biotechnology), monoclonal anti-Src-1 (Upstate Biotechnology) able to recognize the GR-associated
Src,22 or monoclonal anti-PI-PLC (Upstate
Biotechnology) antibodies.
Determination of cytochrome c release Thymus cells (3 × 107) treated with DEX (107 M) were washed in PBS and resuspended in 500 µL
ice-cold buffer containing 20 mM HEPES, 250 mM sucrose, 2 mM EDTA, 20 µg/mL PMSF, 2 µg/mL leupeptin, and 10 µg/mL aprotinin, pH 7.1. Cells were disrupted on ice by a Teflon homogenizer and were
centrifuged for 5 minutes at 3000g to remove nuclei and
unbroken cells. Supernatants were then centrifuged for 20 minutes at
12 000g to isolate the mitochondrial fraction. For Western
blot detection of cytochrome c, the supernatant from the
last centrifugation and the mitochondrial fractions were subjected to
15% SDS-PAGE. After protein transfer, the membrane was blocked with a
5% solution of BSA in TBST buffer (25 mM Tris-HCl, 137 mM NaCl, 0.05%
Tween 20, pH 7.4) and was incubated for 1 hour at room temperature with
monoclonal antibody against cytochrome c (BD PharMingen).
The membrane was then incubated with monoclonal antibody goat antimouse
coupled to peroxidase (Pierce). Specific protein complexes were
identified using the SuperSignal (Pierce) substrate
chemiluminescence reagent.
Western blotting to evaluate activation of caspase-3, -8, and -9 Cells were washed once with ice-cold PBS and were lysed by incubation for 30 minutes on ice in 100 µL lysis buffer (20 mM Tris-HCl, 0.15 M NaCl, 5 mM EDTA, 100 mM PMSF, 2.5 mM leupeptin, 2.5 mM aprotinin). After centrifugation at 15 000 rpm for 15 minutes, extracted proteins were separated on a 12% or a 15% SDS-polyacrylamide gel and were electrophoretically transferred to a nitrocellulose transfer membrane (Schleicher & Schuell, Keene, NH). Membrane was blocked with TBST (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20) containing 5% skimmed milk for 1 hour at room temperature, and each antibody was applied overnight at 4°C in the same blocking solution. Anti-caspase-3 antibody and anti-caspase-8 antibody were purchased from Santa Cruz Biotechnology, and anti-caspase-9 antibody was purchased from New England Biolabs (Beverly, MA). After incubation, membranes were washed with TBST and incubated for 1 hour with horseradish peroxidase-labeled goat antirabbit (for anti-caspase-8 and -9) or antimouse (for caspase-3) IgG (Pierce). Antigen-antibody complexes were revealed by enhanced chemiluminescence.Statistical analysis All the experiments here shown were repeated at least 3 times. For data analysis, the Student t test was used with the STATPAC Computerized Program, and P < .05 was considered significant.
Role of GR-associated Src kinase in DEX-mediated PI-PLC activation of apoptosis We have previously reported that DEX-induced apoptosis requires a sequence of biochemical events: DEX/GR interaction is followed by a rapid (1-5 minutes) PI-PLC activation that can be inhibited by the specific PI-PLC inhibitor U73122, but not by the PC-PLC inhibitor D609. Moreover, this PI-PLC activation is under the control of PKC activity and is inhibited by PKC inhibitors.13 GR-associated Src kinase, following DEX/GR interaction, is activated and contributes to early nongenomic effects.22We evaluated the role of GR-associated Src kinase in DEX-induced PI-PLC
activation and apoptosis. For that purpose, thymocytes were treated
with PP1, a specific Src kinase family inhibitor that stabilizes the
HSP90/GR/Src cytoplasm complex and inhibits DEX-induced GR-associated
Src activation.19,22 Results indicate that PP1, like the
specific PI-PLC inhibitor U73122, inhibits PI-PLC activation (Figure
1A) and apoptosis (Figure 1B). As
previously reported, D609, a PC-PLC inhibitor, did not inhibit PI-PLC
or DEX-activated apoptosis.13 DEX-induced apoptosis
(Figure 1B) was evaluated 18 hours after DEX treatment, and similar
results were obtained after 12 hours (P < .001 comparing
percentage apoptosis of DEX-treated [52 ± 5] and untreated
[14 ± 2] thymocytes).
As control for PP1 treatment efficacy, we used 2 different approaches. First, we performed coimmunoprecipitation experiments. GCH treatment induces GR and Src release from the cytoplasm macromolecular complex.14,15 Results in Figure 1C confirm previous observations indicating that an HSP90/GR/Src complex exists, that DEX treatment induces GR and Src release from the protein complex, and that release is inhibited by PP1.22 Second, DEX-induced GR nuclear translocation requires the GR release from the cytoplasm macromolecular complex. We evaluated the effect of PP1 treatment on DEX-induced GR nuclear translocation. Results in Figure 1D indicate that the PP1-induced stabilization of the HSP90/GR/Src macromolecular complex was paralleled by an inhibition of GR nuclear translocation. Moreover, the specific PI-PLC inhibitor U73122 did not interfere with GR nuclear translocation. PI-PLC associates with the HSP90/GR/Src macromolecular complex and is phosphorylated after DEX treatment Overall the results illustrated in Figure 1 suggest that GR-associated Src kinase is involved in DEX-mediated PI-PLC activation and apoptosis. We determined whether PI-PLC could reside in the same molecular complex with GR-associated Src and HSP90. Results of a representative experiment, obtained by coimmunoprecipitation with anti-HSP90 antibodies, are shown in Figure 2. Figure 2A shows that GR, Src PI-PLC, and HSP90 coimmunoprecipitated in the protein extract from untreated thymocytes. DEX treatment induced GR, Src, and PI-PLC release at 5 and 30 minutes after treatment. In particular, 5 minutes after DEX treatment, GR was not detectable, whereas PI-PLC and Src were still detectable but were significantly reduced; 30 minutes after DEX treatment GR, PI-PLC, and Src were not detectable (Figure 2A). These results indicate that PI-PLC was in the same macromolecular complex with HSP90, GR, and Src and that DEX treatment induced GR, Src, and PI-PLC release.
We also performed experiments to determine the possible binding of GR, Src, and PI-PLC in untreated and DEX-treated thymocytes. Results in Figure 2B, obtained by coimmunoprecipitation with anti-Src antibodies, showed that Src binds PI-PLC and GR in untreated thymocytes. In contrast, at 30 minutes after DEX treatment Src binds PI-PLC, but not GR. These data confirm that DEX treatment induces GR, Src, and PI-PLC release but does not affect Src/PI-PLC binding. These data further confirm the results reported in Figure 1C, which show DEX treatment induced the release of GR and Src from the HSP90 and which confirm previous reports indicating that Src associates directly with PI-PLC.23 PI-PLC activation is reported to be a consequence of phosphorylation, which is blocked by PP1.24,25 We evaluated whether DEX induces PI-PLC phosphorylation. In the same Src/PI-PLC coimmunoprecipitation experiment, we analyzed the presence of phosphorylated proteins. For that purpose, phosphorylated proteins were immunoprecipitated with agarose-conjugated antiphosphotyrosine monoclonal antibodies, and the presence of PI-PLC was evaluated by anti-PI-PLC monoclonal antibody. Results indicate that phosphorylated PI-PLC was present in DEX-treated thymocytes (Figure 2C). Moreover, treatment with the specific Src inhibitor, PP1, inhibited DEX-induced PI-PLC phosphorylation, suggesting that Src is involved in PI-PLC phosphorylation. As control, we also tested the effect of the specific PI-PLC inhibitor U73122. U73122 inhibited DEX-induced PI-PLC phosphorylation, whereas D609, a specific PC-PLC inhibitor, did not. These results indicate that Src and PI-PLC are in the same protein complex and that GR-associated Src may contribute to DEX-induced PI-PLC phosphorylation and activation. DEX induces caspase activation It has been reported that protease activation is involved in apoptosis.26-29 Two main caspase activation pathways can be considered the pathway activated by death receptors, which includes the sequential activation of caspase-8 and of caspase-3, and the cytochrome c-dependent pathway activated by nonreceptor signals, which includes the sequential activation of caspase-9 and caspase-3.30-33We analyzed the DEX-activated caspase pathway in thymocytes. Western
blot experiments (Figure 3A) indicated
that DEX treatment activates caspase-8 (detectable at 1-2 hours after
treatment), caspase-9, and caspase-3.
Experiments were performed to elucidate the role of caspases in DEX-induced apoptosis and to determine whether the caspase-8/-3 or the caspase-9/-3 pathway is involved. We tested the influence of caspase-8, -9, and -3 inhibitors on apoptosis. Results in Figure 3B indicate the caspase-8 inhibitor inhibits apoptosis but the caspase-9 inhibitor, though able to inhibit caspase-9 activation, does not. As expected, the caspase-3 inhibitor blocks apoptosis. These results are in agreement with previous observations indicating that FADD or caspase-8 inhibition can result in caspase-9 and -3 inhibition.34 As further control, we evaluated the effect of caspase-8, -9, and -3 inhibitors on DEX-induced caspase-8, -9, and -3 activation. The caspase-9 inhibitor completely inhibits caspase-9 but not caspase-8 and only slightly caspase-3; the caspase-3 inhibitor inhibits caspase-3; and the caspase-8 inhibitor inhibits caspase-8, -9, and -3 (Figure 4A-C). It has been previously reported that, as detected by using florigenic
caspase substrates, an order of appearance of caspase activity can
occur so that caspase-9 appears before caspase-8 activity in
DEX-induced thymocytes.35 It has also been reported that
caspase-8 activation can precede caspase-9 in Fas/FADD-dependent and
also in Fas/FADD-independent apoptosis.35,36 We were
unable to find a clear order in caspase-9, -8, and -3 activation
(Figures 3-4), and this difference may be attributed to the different
sensitivities of the assays used to detect caspase activation. These
results suggest DEX treatment activates the caspase-8/-3 and the
caspase-9/-3 pathways. Caspase-9 is dispensable, but caspase-8
activation is required for apoptosis induction.
DEX-induced cytochrome c release: inhibition of caspase-8 counters cytochrome c release It has been reported that DEX induces cytochrome c release that, in turn, mediates caspase-9 activation.37-41 We evaluated the effect of DEX treatment on cytochrome c release. Results show that DEX treatment induces cytochrome c release, already detectable at 2 hours after treatment (Figure 4D).Cytochrome c release is under the control of caspase-8.31,32,41 We evaluated the possible roles of caspase-8 and -9 in the regulation of DEX-induced cytochrome c release in thymocytes. Although the inhibition of caspase-9 did not counter cytochrome c release, caspase-8 inhibition countered cytochrome c release (Figure 4E). These results indicate that in DEX-treated thymocytes, cytochrome c release is under the control of caspase-8, and they explain, at least in part, why caspase-8 inhibition counters DEX-induced caspase-9 activation (Figure 3B). These data suggest caspase-8 is important in DEX-induced thymocyte apoptosis because activated caspase-8 can directly activate caspase-3 and allow caspase-9 activation by regulating DEX-induced cytochrome c release. Moreover, though caspase-9 inhibition does not block apoptosis (Figure 3B) because caspase-8 directly activates caspase-3, caspase-8 inhibition does block apoptosis because direct caspase-3- and caspase-9-mediated caspase-3 activation are inhibited (Figure 4A-C). DEX and ceramide induce FADD/caspase-8 association The above results indicate that DEX activates thymocyte caspase-8. Apoptotic stimuli are reported to induce the association of FADD with caspase-8. This association is necessary for caspase activation and can be detected in the FADD/caspase-8 complex in Fas receptor-dependent and receptor-independent apoptosis.42,43 We previously reported that DEX treatment induces rapid aSMase activation and ceramide generation that precedes caspases activation.13 It has been reported that ceramide can induce caspase-8 and -3 activation in cardiomyocytes.44 We attempted to determine whether DEX and ceramide induce the FADD/caspase-8 complex, and our results showed DEX or ceramide treatment induces FADD/caspase-8 formation in thymocytes (Figure 5A-B). In addition, we evaluated whether ceramide, like DEX, activates caspase-8 in thymocytes. Our results showed that ceramide, when exogenously added to thymocytes, induced caspase-8 activation (Figure 5C). These findings indicate that ceramide contributes to DEX-induced caspase-8 activation in thymocytes, that caspase-8 activation is caused, at least in part, by induction of the FADD/caspase-8 complex, and that DEX-induced ceramide generation is upstream of caspase activation.
DEX-induced caspase-8 activation is countered by PI-PLC, aSMase, macromolecular synthesis, and Src kinase inhibitors Many studies report DEX-induced apoptosis is a macromolecular, synthesis-dependent event.8,45 We have also demonstrated that sequential PI-PLC and aSMase activation precede caspase activation and contribute to DEX-induced apoptosis.13 Results shown in Figure 5 indicate that DEX, like ceramide, activates caspase-8. We assessed the effect of inhibitors of Src, PI-PLC, aSMase, mRNA, and protein synthesis on DEX-induced caspase-8 activation and apoptosis. Results shown in Figure 6 indicate apoptosis is inhibited by macromolecular synthesis inhibitors (Figure 6A) and PI-PLC and aSMase inhibitors (Figure 6B), such as the Src inhibitor PP1 (Figure 1B).
As control we tested the effect of PI-PLC and aSMase inhibitors on aSMase activity. Results in Figure 6C concur with previous data indicating that PI-PLC precedes, and is required for, aSMase activation and ceramide generation and showing that DEX-induced apoptosis is under the control of macromolecular synthesis and PI-PLC/aSMase activation.8-9,13,45,46 We also tested the effect of those inhibitors on DEX-induced caspase-8
activation. Figure 7 shows that Src
kinase, PI-PLC aSMase, and macromolecular synthesis inhibitors all
counter DEX-induced caspase-8 activation, indicating DEX-induced
caspase-8 activation is required for apoptosis induction and is
downstream in the apoptotic signal pathway. In particular, results in
Figure 7B-C indicate that actinomycin D (act-D) and cycloheximide (Chx)
inhibit caspase-8 activation. We have reported that macromolecular
synthesis inhibitors do not counter DEX-induced ProCPP32
cleavage.13 This apparent discrepancy can be attributed to
the different assays used; a colorimetric assay was used for ProCPP32
cleavage detection, whereas caspase activation was evaluated here
through a more specific assay, Western blot immunoassay with specific
antibodies.
The results of the present study provide new information concerning mechanisms of the GCH-mediated apoptotic pathway in thymocytes, and we show that GR-associated Src kinase and caspase-8 activation contribute to DEX-induced apoptosis. GCH-mediated regulation of apoptosis has been described in a number of different cells and tissues, including thymocytes, normal and neoplastic lymphocytes, granulocytes, neurones, epithelial cells, germ cells, and osteoblasts.7-8,46-52 GCH-induced apoptosis in cells of the lymphoid compartment may be partly responsible for its immunosuppressive and anti-inflammatory effects and its antileukemia efficacy in pharmacologic treatments. Transcription-dependent and -independent mechanisms have been described as mediating the GCH-activated death of lymphoma and leukemia cell lines, suggesting that different molecular mechanisms are involved in GCH-mediated apoptosis induction in different cells and tissues.4-6,48,53-55 We have reported that both transcription-dependent and
transcription-independent signals These observations confirm previous findings that DEX-GR interaction is necessary for apoptosis induction, that PI-PLC activation is regulated by phosphorylation, and that DEX-induced PKC activity is required for PI-PLC activation and apoptosis.13,22-25 Moreover, these data suggest that thymocyte PI-PLC phosphorylation and activation are mediated by DEX-induced Src kinase activity. Many studies report GCH activates caspases and suggest caspase activation may play a role in DEX-induced thymocyte apoptosis.9,28,29 Sequential cleavage and activation of caspases is an important mechanism in most apoptosis models. Initiator caspases, including receptor-activated caspase-8 and cytochrome c-dependent caspase-9, activate effector caspases, including caspase-3.33 The results of our experiments in the present study indicate DEX-treatment activates caspase-8, -9, and -3 in thymocytes. Caspase-8 may have a major role; its inhibition blocks apoptosis. The finding that inhibition of caspase-9 does not block apoptosis provides further evidence of the role of caspase-8 activity in DEX-induced thymocyte death. Caspase inhibitors might not be specific.56 Caspase-9 inhibitor does inhibit caspase-9 activation (Figure 3A) but does not inhibit caspase-3 activation and apoptosis (Figure 3B). Caspase-8 inhibitor counters caspase-8 and caspase-9 activation, possibly because of a nonspecific inhibiting activity but possibly also because caspase-8, but not caspase-9, inhibition counters the DEX-induced cytochrome c release (Figure 4D-E) required for caspase-9 activation. These results are in agreement with data from other experimental models suggesting caspase-8 has a major role in apoptosis activation, and this effect is amplified through cytochrome c release and the consequent caspase-9 activation.34,41 Caspase-8 can directly activate caspase-3 or regulate cytochrome c release and, consequently, caspase-9 activation so that, though caspase-9 inhibition leaves the caspase-8/-3 pathway functional, caspase-8 inhibition can counter direct caspase-3 activation, cytochrome c release, and the consequent cytochrome c-dependent activation of the caspase-9/-3 pathway.33,37-41 As a consequence, caspase-8 inhibition can block DEX-induced caspase-9 and caspase-3 activation. These data are in part in agreement with previous results obtained in
caspase-9 null mice.57,58 In fact, it has been reported that DEX treatment of caspase-9 null thymocytes induces detectable Our results in adult thymocytes concur with previous observations indicating that DEX activates caspase-8, that caspase-8 controls cytochrome c release and the consequent caspase-9 activation, and that caspase-8 can be activated without triggering membrane receptors.34,37,43,61,62 This receptor-independent caspase-8 activation may be mediated, in part, through an increase in DEX-induced ceramide levels, and indeed ceramide is reported to activate caspase-8.44,63 The data presented here indicate that exogenous ceramide induces the FADD/caspase-8 complex and caspase activation and that the aSMase inhibitor monensin, like the PI-PLC inhibitor U73122, counters DEX-induced caspase-8 activation. Receptor-mediated caspase-8 activation may be mediated by FADD/caspase complex formation.42,43 Our results show that DEX treatment induces the FADD/caspase-8 complex. A similar effect is obtained with exogenous ceramide. These results confirm previous findings showing FADD/caspase-8 complex formation can be detected when caspase-8 activation occurs in the absence of membrane receptor-mediated apoptotic stimuli.43,64 Ceramide may be involved in DEX-induced FADD/caspase-8 complex formation, caspase-8 activation, and apoptosis. We have previously reported that the inhibition of PI-PLC and/or of the consequent aSMase and ceramide generation counters DEX-induced apoptosis.13 We show that the inhibitors of PI-PLC or aSMase block DEX-induced caspase-8 activation. In conclusion, the results of the present study, together with our
previous observations,13 indicate that a sequence of biochemical events is activated by DEX in thymocytes (Figure
8). Although the experimental results
indicate precise biochemical mechanisms are involved in thymocyte
apoptosis, they may be only a part of a complex array of apoptotic
signals activated by DEX and may not be relevant for other tissues,
including neoplastic cells such as leukemia and lymphoma. This advance
in knowledge of DEX-activated biochemical events may be the basis for
further studies aimed at analyzing the complex pattern of resistance
and susceptibility to GCH treatment. Accurate study of the
GCH-activated biochemical events involved in apoptosis modulation, in
all susceptible tissues, may contribute to a better understanding of
some of the effects responsible for the therapeutic efficacy of GCH
treatment and may be helpful in the development of new pharmacologic
approaches aimed at controlling immune response and neoplastic cell
growth.
Submitted June 26, 2002; accepted August 14, 2002.
Prepublished online as Blood First Edition Paper, August 29, 2002; DOI 10.1182/blood-2002-06-1779.
Supported by Associazione Italiana Ricerca sul Cancro (AIRC), Milan, Italy.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Carlo Riccardi, Department of Clinical and Experimental Medicine, Pharmacology Section, University of Perugia, Via del Giochetto, 06122 Perugia, Italy; e-mail: riccardi{at}unipg.it.
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Abstract
Introduction
for example, PI-PLC activation and the consequent aSMase activation and ceramide generation