Unambiguous indication of the data subject’s wishes

Les objectifs de cette étude ont consisté à étudier la signalisation apoptotique caspase- 8-indépendante du récepteur Fas et en particulier à élucider le rôle de la caspase-10.

Cette étude a été réalisée dans les cellules Jurkat déficientes en caspase-8 (I9-2) traitées par du FasL humain recombinant ou par le FasL murin issu du surnageant de culture de cellules Neuro2A surexprimant FasL. L’apoptose a été appréciée par différents critères tels que l’externalisation des phosphatidylsérine (PS), la perméabilité à l’Iodure de Propidium, la morphologie du noyau, l’activation des caspases et l’hypodiploïdie. L’implication de la caspase-10 dans l’apoptose des cellules I9-2 a été évaluée par l’utilisation du z-AEVD-fmk

ARTICLE 1

Milhas, D., Cuvillier, O., Therville, N., Clave, P., Thomsen, M., Levade, T., Benoist, H., and Segui, B. (2005). Caspase-10 triggers Bid cleavage and caspase cascade activation in FasL- induced apoptosis. J Biol Chem 280(20), 19836-42.

Caspase-10 Triggers Bid Cleavage and Caspase Cascade Activation

in FasL-induced Apoptosis*

Received for publication, December 21, 2004, and in revised form, March 4, 2005 Published, JBC Papers in Press, March 16, 2005, DOI 10.1074/jbc.M414358200

Delphine Milhas, Olivier Cuvillier, Nicole Therville, Patricia Clave´, Mogens Thomsen, Thierry Levade, Herve´ Benoist, and Bruno Se´gui‡

From INSERM U466, Institut Louis Bugnard, BP 84225, 31432 Toulouse Cedex 4, France

In contrast to caspase-8, controversy exists as to the ability of caspase-10 to mediate apoptosis in response to FasL. Herein, we have shown activation of caspase-10, -3, and -7 as well as B cell lymphoma-2-interacting do- main (Bid) cleavage and cytochrome c release in caspase-8-deficient Jurkat (I9 –2) cells treated with FasL. Apoptosis was clearly induced as illustrated by nuclear and DNA fragmentation. These events were in- hibited by benzyloxycarbonyl-VAD-fluoromethyl ke- tone, a broad spectrum caspase inhibitor, indicating that caspases were functionally and actively involved. Benzyloxycarbonyl-AEVD-fluoromethyl ketone, a caspase-10 inhibitor, had a comparable effect. FasL- induced cell death was not completely abolished by caspase inhibitors in agreement with the existence of a cytotoxic caspase-independent pathway. In subpopula- tions of I9 –2 cells displaying distinct caspase-10 expres- sion levels, cell sensitivity to FasL correlated with caspase-10 expression. A robust caspase activation, Bid cleavage, and DNA fragmentation were observed in cells with high caspase-10 levels but not in those with low levels. In vitro, caspase-10, as well as caspase-8, could cleave Bid to generate active truncated Bid (p15). Alto- gether, our data strongly suggest that caspase-10 can serve as an initiator caspase in Fas signaling leading to Bid processing, caspase cascade activation, and apoptosis.

Cell death is an essential process in the regulation of cellular homeostasis. Dysfunction of the mechanisms involved can lead to human diseases such as cancers, autoimmune diseases (in the case of death defect), and neurodegenerative and immune deficiency diseases (in the case of death excess) (reviewed in Refs. 1 and 2). Two types of cell death have been clearly dis- tinguished, apoptosis (programmed cell death) and necrosis (3). Apoptosis can be characterized by typical sets of changes in- cluding plasma membrane blebbing, cellular and chromatin condensation, nuclear fragmentation, and formation of apop- totic bodies. Phosphatidylserine externalization and DNA frag- mentation are biochemical features of apoptosis. Most of these processes are mediated by caspases that cleave and inactivate proteins essential for cell survival (for review, see Ref. 4). In

oncosis. Intermediate forms of programmed cell death, namely apoptosis- and necrosis-like cell death, have been recently de- scribed (for review, see Ref. 5).

Programmed cell death is essential for elimination of self- reactive lymphocytes and down-regulation of the immune re- sponse. Stimulation of Fas receptor (CD95) by FasL (CD95L or CD178) plays a crucial role in inducing lymphocyte pro- grammed cell death. Fas cross-linking triggers activation of both caspase-dependent and -independent pathways (6, 7). Caspase-independent cell death requires receptor-interacting protein (RIP) as an effector molecule, but cytotoxic signaling pathways activated by RIP remain largely unknown (6). Caspase- dependent pathways have been extensively explored (for a re- cent review, see Ref. 8). Oligomerization of Fas by FasL leads to successive recruitment of FADD (9) and initiator caspase-8 (10) and -10 (11). Formation of this complex, termed DISC1_(death-

inducing signaling complex) (12), allows caspase-8 and -10 ac- tivation (10, 11). Both caspases can directly activate effector caspases (13–15). Once activated, effector caspases specifically cleave and inactivate proteins leading to apoptosis. In addition, caspase-8 cleaves Bid, a pro-apoptotic member of the B cell lymphoma-2 superfamily (16, 17). The terminal part of Bid containing a B cell lymphoma-2 homology (BH) 3 domain, namely truncated Bid, translocates to mitochondria and pro- motes cytochrome c release into the cytosol (16, 17). In associ- ation with APAF-1 and pro-caspase-9, another initiator caspase, cytochrome c forms the apoptosome complex leading to the activation of caspase-9 that cleaves and activates effector caspases (18).

In analogy with caspase-8, caspase-10 has two DEDs (death effector domains) and the QACQG sequence containing the catalytic cysteine residue (4). To date, four different caspase-10 splice variants have been identified, caspase-10a (Mch4) (13), -10b (FADD-like ICE or FLICE-2) (11), -10c and -10d (19). In contrast to caspase-10c, caspase-10a, -10b, and -10d have large and small catalytic subunits. Strong homology between caspase-8 and -10 might suggest that both enzymes share similar substrate specificity and biological function. However, in contrast to caspase-10, caspase-8 can cleave receptor-inter- acting protein in FasL-treated Jurkat cells (20).

Jurkat T lymphoma cells contain caspase-8 and -10, and caspase-8 has been shown to be essential for apoptosis induc-

THEJOURNAL OFBIOLOGICALCHEMISTRY Vol. 280, No. 20, Issue of May 20, pp. 19836 –19842, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

as type II cells (22). More recently, Ashkenazi and co-workers (15) reported that endogenous caspase-10 could be recruited to and activated at the DISC level in FasL-treated Jurkat cells. Similar findings were described in activated PBL upon Fas ligation (20). However, the role of caspase-10 in Fas signaling remains controversial. Overexpression of caspase-10 comple- mented caspase-8 deficiency in FasL-treated Jurkat cells in two independent studies (15, 20), but not in another (23). In addition, there is no direct evidence in the literature that caspase-10 is able to cleave Bid and trigger cytochrome c release.

Herein, we have shown that FasL can induce apoptosis of caspase-8-deficient Jurkat cells, which was associated to en- dogenous caspase-10, -3, and -7 activation, as well as Bid cleav- age and cytochrome c release. Cell sensitivity to FasL corre- lated with caspase-10 expression in caspase-8-deficient cells. A caspase-10 inhibitor prevented Bid processing, caspase cascade activation, and apoptosis. In addition, evidence is provided for the first time that Bid is a direct substrate of caspase-10, strongly indicating that, like caspase-8, caspase-10 can lead to activation of the mitochondrial pathway.

EXPERIMENTAL PROCEDURES

Reagents—Final concentrations or dilutions used of the following

reagents are indicated: Z-VAD(OMe)-fmk (40 ␮M) and Z-AE(OMe)-

VD(OMe)-fmk (10 ␮M) were purchased from Bachem (Voisins-Le- Bretonneux, France) and RD systems (Lille, France), respectively; monoclonal anti-FADD (clone A66 –2; 0.5 ␮g/ml), anti-cytochrome c (clone 7H8.2C12; 1␮g/ml), and anti-caspase-8 (clone B9–2; 0.5 ␮g/ml) antibodies were obtained from BD Biosciences; polyclonal anti- caspase-8 (1/5 dilution) was a kind gift from Dr. G. Cohen (Leicester, UK); monoclonal anti-caspase-10 (clone 4C1; 1␮g/ml) was purchased from MBL (Meudon, France); polyclonal anti-caspase-3 (10␮g/ml) was obtained from Dako; polyclonal anti-caspase-7, anti-PARP, and anti- Bid antibodies were purchased from Cell Signaling Technology and used at 1/1000 dilution; monoclonal anti-␤-actin (clone AC-15; 5 ␮g/ml) was obtained from Sigma. Monoclonal anti-CD95 (clone 7C11; 1/5 dilu- tion) and IgM irrelevant antibody (10␮g/ml) coupled to phycoerythrin (PE) were purchased from Immunotech (Marseille, France) and Santa Cruz, respectively. Human recombinant FasL was obtained from Abcys (Paris, France). Alternatively, mouse FasL produced in the supernatant of Neuro-2A cells stably transfected with a plasmid encoding FasL was used (24). Similar data were obtained with mouse and human FasL. Human recombinant caspase-8 (6000 units/mg) and -10 (8000 units/mg) were purchased from Abcys.

Cell Lines—Parental Jurkat T lymphoma cells (clone A3) and derived

cell lines deficient for FADD (clone I2–1) or caspase-8 (clone I9 –2) were kindly provided by Dr. J. Blenis (Boston, MA). Cells were cultured in RPMI containing Glutamax and 10% heat-inactivated fetal calf serum. In limiting dilution experiments, I9 –2 cells were cultured in RPMI medium containing 10% fetal calf serum in 96-well plates. From serial dilutions, five cell cultures were isolated and named I9 –2a, I9 –2b, I9 –2c, I9 –2d, and I9 –2e.

Flow Cytometry Analyses—CD95 cell surface expression was deter-

mined after incubation of cells for 30 min at 4 °C with or without anti-CD95-PE or an irrelevant antibody coupled to PE. To allow study of phosphatidylserine externalization, cells were labeled with Annexin V-fluorescein isothiocyanate (250 ng/ml) and propidium iodide (12.5 ␮g/ml) (Immunotech) for 10 min at 4 °C. To allow study of hypodiploidy, cells were washed in phosphate-buffered saline and permeabilized in ethanol (70%) for 10 min at⫺20 °C. Cells were next incubated for 30 min at 37 °C with RNase (1 mg/ml) and propidium iodide (0.1 mg/ml). Percentage of hypodiploid cells carrying DNA content below cells in G0/G1was quantified by flow cytometry. Analyses were performed on a

FACScan (BD Biosciences) cytometer.

Morphological Analyses—Cells were co-incubated with propidium

iodide (2␮g/ml) (Sigma) and Syto 13 (2.5 ␮M) (Molecular Probes) for 15 min at 37 °C and analyzed under a Leica fluorescence-equipped micro- scope (25). At least 300 cells were examined.

MTT Assay—Viability was evaluated by the tetrazolium-based MTT

assay. Cells were seeded in flat-bottom 96-well plates (106_{cells/ml, 100}

of solubilization buffer (HCl 0.01 N, 10% SDS) and spectrophotometri- cally quantified using an enzyme-linked immunosorbent assay reader (␭ ⫽ 590 nm).

Cell-free System Assay—Mitochondria-free cytosolic protein extracts

(250␮g) from A3 or I9–2 cells were incubated in the presence of one unit (as defined by the manufacturer) of recombinant caspase-8 or -10 for different times up to 90 min at 37 °C in a final volume of 200␮l of buffer containing 50 mMHEPES, pH 7.5, 150 mMNaCl. At the indicated times, reaction was stopped by freezing on dry ice. 20␮g of protein were subjected to SDS-PAGE and Western blotting analysis.

Alternatively, 1␮g of His-tagged mouse recombinant Bid (26) was incubated in the presence of one unit of recombinant caspase at 37 °C in a final volume of 60␮l of buffer containing 50 mMHEPES, pH 7.5, 150 mMNaCl. At the indicated times, 100 ng of protein were immediately

frozen on dry ice and analyzed by Western blotting with anti-His antibody (clone sc8036, Santa Cruz). Alternatively, extracts were sep- arated on 15% SDS-PAGE and stained with Coomassie blue. Of note, mouse and human Bid proteins share 63.5% identity and the LQTD2G caspase cleavage motif (16).

Protein Extraction and Western Blotting Analyses—For total protein

extraction, 5–20⫻ 106_{of cells were lysed for 30 min on ice in a buffer}

containing 50 mMHEPES, pH 7.5, 150 mMNaCl, 10% glycerol, 1% Triton X-100, 0.5% deoxycholate, 1 mMNaVO4, 10 mM␤-glycerophos-

phate, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 ␮g/ml leupeptin, 2␮g/ml pepstatin A, and 10 ␮g/ml aprotinin. Samples were centrifuged at 10,000⫻ g at 4 °C for 10 min. Supernatants were col- lected and protein content determined by the Bradford method (Bio- Rad). Cytosolic protein extracts were prepared from 20⫻ 106

of cells as described previously (27). For Western blot analyses, equal amount of proteins were separated on 15% SDS-PAGE.

RESULTS

FasL Can Trigger Apoptosis in Caspase-8-deficient Jurkat Cells—Caspase-8 has previously been reported to be essential

in anti-Fas-induced Jurkat apoptosis (21). However, two differ- ent groups have recently shown that caspase-8-independent cell death occurs in response to high FasL concentration (6, 7). Both studies reached the conclusion that this caspase-8-inde- pendent cell death is a form of necrosis rather than apoptosis. Herein, we have re-examined caspase-8-independent cell death in Jurkat cells. First, we compared the sensitivity to FasL of parental (clone A3), caspase-8-deficient (clone I9 –2), and FADD-deficient (clone I2–1) Jurkat cell lines (21, 28). Caspase-8 and FADD expression was analyzed by Western blotting to confirm respective protein deficiency in I9 –2 and I2–1 clones (Fig. 1A). Flow cytometry analysis indicated that the various cell lines exhibited similar Fas expression on the cell surface (Fig. 1B). When incubated at a low FasL concen- tration (15 ng/ml), apoptotic features were seen only in A3 cells in agreement with previous reports (21, 28). When a higher FasL concentration (500 ng/ml) was used, caspase-8-deficient cells displayed an apoptotic phenotype with pronounced cellu- lar condensation and nuclear fragmentation (Fig. 1C). Flow cytometry analysis indicated that 54.4_{⫾ 4.4% and 11.5 ⫾ 3.6%} (mean_{⫾ S.D. of three independent experiments) of caspase-8-} deficient cells were scored Annexin-V-positive when incubated in the presence or absence of 500 ng/ml FasL, respectively. FADD-deficient cells completely resisted FasL-induced apopto- sis at all doses tested (up to 500 ng/ml) (Fig. 1C).

FasL Induces Caspase-dependent and Caspase-independent Cell Death in Caspase-8-deficient Jurkat Cells—DNA fragmen-

tation in caspase-8-deficient cells was next assessed by meas- uring hypodiploidy. Although 15 ng/ml FasL did not induce hypodiploidy in these cells, 14.4⫾ 1.6% (mean ⫾ S.D. of three independent experiments) of them displayed DNA fragmenta- tion in response to 500 ng/ml FasL (Fig. 2A). This process was completely abolished by the broad-spectrum caspase inhibitor Z-VAD-fmk, indicating involvement of caspases in DNA frag-

(Fig. 3B and data not shown). As a matter of fact, 45.9⫾ 3.4% (mean⫾ S.D. of three independent experiments) of cells were still Annexin-V-positive in the presence of Z-VAD-fmk and FasL (Fig. 2B). The cell size was reduced as evaluated by flow cytometry (Fig. 2C) and microscopic examination (Fig. 2D). It is of interest that Z-VAD-fmk abrogated nuclear fragmentation, but not nuclear condensation and membrane alterations, as illustrated by propidium iodide uptake (Fig. 2D). Thus, Z-VAD- fmk weakly affected FasL-induced cytotoxicity, but the latter was shifted into cell death without nuclear fragmentation.

FasL Promotes Bid Cleavage, Cytochrome c Release, and Caspase Activation in Caspase-8-deficient Jurkat Cells—

Caspase activation in I9 –2 cells was next analyzed by Western blotting (Fig. 3). Cleavage of caspase-3, -7, and PARP was observed in I9 –2 cells incubated for 16 h with FasL at concen- trations higher than 60 ng/ml (Fig. 3A). Because caspase-10 can be recruited to and activated at the DISC in Jurkat cells (15, 23), we examined caspase-10 expression using a specific anti-caspase-10 antibody (15, 23). The level of pro-caspase-10 clearly decreased in I9 –2 cells incubated with FasL, in a dose- dependent manner. Interestingly, Bid content also decreased in I9 –2 cells treated with 500 ng/ml FasL. The disappearance of caspase-10 and Bid in I9 –2 cells treated with FasL was com-

points (8 and 16 h). This phenomenon might indicate that early caspase activation did not require cytochrome c release. Alter- natively, our assay might not be sensitive enough to detect low amounts of cytochrome c released at early time points. To- gether, our data strongly support the notion that FasL can promote caspase activation, Bid processing, and cytochrome c release even in the absence of caspase-8 and that caspase-10 is likely involved in these events.

Sensitivity to FasL Correlates with Caspase-10 Concentra- tion in I9 –2 Cells—All the above-mentioned experiments indi-

cated that only about half of I9 –2 cells were sensitive to FasL. We hypothesized that I9 –2 cells were heterogeneous and con-

FIG. 1. FasL induces apoptosis in caspase-8-deficient Jurkat

cells. A, 50␮g of total protein extract from parental (A3), caspase-8- deficient (⌬casp-8), and FADD-deficient (⌬FADD) Jurkat cell lines were subjected to SDS-PAGE and Western blotted with anti-caspase-8, anti- FADD, or anti-␤-actin antibodies. B, CD95 expression was analyzed by flow cytometry. Cells were incubated with irrelevant (dotted lines) or anti-CD95 PE-conjugated antibodies (plain lines). Percentages of CD95- expressing cells are indicated. C, cells were incubated for 16 h in the presence or absence of the indicated FasL concentration. Cells were stained with Syto 13 and propidium iodide and analyzed by fluores- cence microscopy. Data are representative of at least three independent experiments.

FIG. 2. Caspase-dependent hypodiploidy and caspase-independ-

ent cell death in FasL-treated caspase-8-deficient Jurkat cells.

Caspase-8-deficient Jurkat cells were incubated for 16 h in the presence or absence of the indicated FasL concentration and Z-VAD-fmk (40␮M). A, cells were harvested, stained with propidium iodide (P.I.), and DNA content was analyzed by flow cytometry. Percentages of hypodiploid cells are indicated. B, alternatively, cells were labeled with Annexin-V-fluores- cein isothiocyanate (Ann.V) and propidium iodide (P.I.) and analyzed by flow cytometry. Percentages of Annexin-V- and propidium iodide-positive cells are indicated. C, size and granularity were analyzed by flow cytom- etry. Percentages of cells displaying a decreased size are indicated. D, cells were labeled with Syto 13 probe and propidium iodide and examined by fluorescence microscopy. Percentages of cells having fragmented or con- densed nuclei were quantified (mean⫾ S.D.). All data are representative of three independent experiments.

Caspase-10 in Fas Signaling

I9 –2a and I9 –2e cells strongly resisted FasL-induced toxicity, I9 –2d cells were highly sensitive and I9 –2b and I9 –2c cells displayed an intermediate response. By comparison, A3 cells that expressed not only caspase-10 but also caspase-8 similarly to I9 –2d cells were more sensitive to FasL than I9 –2d cells (Fig. 6A). Z-VAD-fmk efficiently impaired FasL-induced toxic- ity in I9 –2d cells, but not in I9 –2e cells, suggesting a stronger caspase activation in the high caspase-10-expressing cells (data

were not or were weakly activated in I9 –2a and I9 –2e cells. Caspase-10 was processed in the various cell lines, whereas Bid was cleaved proportionally to caspase-10 content (Fig. 7A). Similarly, FasL-induced hypodiploidy correlated with the amount of caspase-10 and was strongly impaired by Z-VAD- fmk and Z-AEVD-fmk (Fig. 7, B and C). Altogether, our data indicate that caspase-10 can cleave Bid and activate the

FIG. 3. FasL induces caspase activation and Bid processing in

caspase-8-deficient Jurkat cells. A, caspase-8-deficient Jurkat cells

were incubated for 16 h in the presence or absence of the indicated FasL concentration. B, caspase-8-deficient Jurkat cells were incubated for 16 h in the presence or absence of 500 ng/ml FasL and 40␮MZ-VAD- fmk as indicated. A and B, cells were then harvested, and 50␮g of total protein extract were subjected to SDS-PAGE and Western blotted with anti-caspase-10, -7, -3, anti-Bid, anti-PARP, or anti-␤-actin antibodies. Data are representative of three independent experiments.

FIG. 4. Caspase-10 is involved in Bid processing, caspase cas-

cade activation, and apoptosis in caspase-8-deficient Jurkat cells treated by FasL. Caspase-8-deficient Jurkat cells were incu-

bated for 16 h in the presence or absence of 500 ng/ml FasL and 10␮M

Z-AEVD-fmk as indicated. A, 50␮g of total protein extract were sub- jected to SDS-PAGE and Western blotted with anti-caspase-10, anti- Bid, anti-PARP, or anti-␤-actin antibodies. B, hypodiploidy was evalu- ated by flow cytometry. Percentages of hypodiploid cells are indicated. Data are representative of three independent experiments.

FIG. 5. FasL induces cytochrome c release in caspase-8-defi-

cient Jurkat cells. Caspase-8-deficient Jurkat cells were incubated in

the presence or absence of 500 ng/ml FasL for the indicated times. Cells were harvested, and 20␮g of cytosolic protein extract were subjected to SDS-PAGE and Western blotted with anti-caspase-10, -7, anti-cyto- chrome c, anti-PARP, or anti-␤-actin antibodies. Percentages of hypo- diploid cells were determined by flow cytometry. Data are representa- tive of two independent experiments.

FIG. 6. Sensitivity to FasL correlates with caspase-10 expres-

sion in caspase-8-deficient Jurkat cells. A, parental (A3) and var-

ious caspase-8-deficient (I9 –2a, -b, -c, -d, -e) Jurkat cells were incubated for 16 h with or without the indicated FasL concentrations. Cell viabil- ity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- lium bromide assay as the percentage of untreated cells (mean⫾ S.D.,

n⫽ 3). B, 50␮g of parental (A3) and various caspase-8-deficient (I9–2a,

-b, -c, -d, -e) Jurkat cells were subjected to SDS-PAGE and Western

blotted with anti-caspase-10, -8, -3, -7, anti-FADD, or anti-␤-actin an- tibodies. C, CD95 expression was analyzed by flow cytometry. Cells were incubated with irrelevant (dotted lines) or anti-CD95 PE-conju- gated antibodies (plain lines). Percentages of CD95-expressing cells are indicated.

processing leading to cytochrome c release from the mitochon- dria into the cytosol (16, 17). Little is known about caspase-10 substrate specificity. Notably, to our knowledge, it has never been demonstrated that Bid could be a substrate of caspase-10. Thus, we compared the activity of human recombinant caspase-8 and -10 produced in Escherichia coli. A mitochon- dria-free cytosolic protein extract from parental A3 cells was used as a source of substrate and incubated for different times in the presence or absence of recombinant caspases. Substrate cleavage was next evaluated by Western blotting (Fig. 8). Re- combinant caspase-8 and -10 had no or little effect on endoge- nous caspase-8 in agreement with a previous study (15). In contrast to recombinant caspase-8, recombinant caspase-10 triggered an obvious endogenous caspase-10 processing. Both caspase-8 and -10 activated caspase-3 and -7. Interestingly, Bid concentration clearly decreased after 60 min of incubation in both cases indicating that caspase-10, like caspase-8, was ca- pable of cleaving Bid. Caspase-3 and -7 cleavage, as well as Bid

DISCUSSION

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