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IMMUNOBIOLOGY
From Medicine and Biosystemic Science, Kyushu
University Graduate School of Medical Sciences; Institute of Genetic
Information, Kyushu University; and Department of Molecular Immunology,
Medical Institute of Bioregulation, Kyushu University; all of Fukuoka,
Japan.
Caspase-8 (Fas-associating protein with death domain-like
interleukin-1 Apoptosis (programmed cell death) is a strictly
regulated cell suicide mechanism that is morphologically and
biochemically distinct from necrosis and plays a crucial role in the
development, homeostasis, and defense of multicellular
organisms.1-5
The morphologic changes observed in apoptosis, including mitochondrial
damage, nuclear membrane breakdown, DNA fragmentation, chromatin
condensation, and the formation of apoptotic bodies, are conducted by a
specialized cellular mechanism consisting of 3 major components: the
caspases, the Bcl-2 family proteins, and the Apaf-1/Caenorhabditis
elegans cell death protein-4 (CED-4) protein.6,7
Among these components, caspases, which belong to a family of cysteine
proteases, are the core of this mechanism and participate in the
apoptosis cascade that is triggered in response to proapoptotic signals
by cleaving a set of proteins.6 They are synthesized as
inactive proenzymes that have to be activated by proteolytic cleavage
after specific aspartate residues, and once the caspases are activated,
they cleave their substrates, thus resulting in the disintegration of
the cells.6 Among them, caspase-8 is the key enzyme, and
it is presumed to be the apex of the Fas-mediated apoptosis pathway.
Caspase-8 is activated in association with the Fas death-inducing
signaling complex (DISC).8 The binding of Fas ligand to
Fas receptor induces the trimerization of Fas, and the cytoplasmic
region of Fas, containing a death domain (DD), recruits a DD-containing
adaptor molecule, FADD (Fas-associating protein with death
domain).9-11 FADD also contains a DD at its C-terminus and
binds to Fas via interactions between the DDs.9-11 The
N-terminal region of FADD, termed the death-effector domain (DED), is
responsible for downstream signal transduction and binds to
caspase-8.12,13 Caspase-8 carries 2 DEDs in its N-terminal region, through which it binds to FADD.12,13 The Fas
oligomerization induced by the binding of Fas ligand results in the
formation of DISC and the oligomerization of caspase-8. Subsequently,
caspase-8 is fully activated by self-cleavage and thereby cleaves many
cellular substrates.14-17
There are various distinct inhibitory molecules involved in the
regulation of the activation of caspases triggered by proapoptotic signals from death receptors.7 One group of these
inhibitory molecules belongs to a family of viral proteins, FADD-like
interleukin-1 In addition to these endogenous regulatory molecules, various isoforms
of different caspases, such as ICE- We recently reported a novel isoform of caspase-8, named
caspase-8L, generated by the alternative splicing of human caspase-8 gene, from human peripheral blood lymphocytes (PBLs) by reverse transcriptase-polymerase chain reaction (RT-PCR).39 In
the present study, we show evidence for the antiapoptotic activity of
caspase-8L.
Cell culture and media
Antibodies
RNA extraction and reverse transcriptase reaction Total RNA was isolated using the ISOGEN reagent (Nippongene, Tokyo, Japan) following the manufacturer's instructions. The RT reaction was conducted using First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer's instructions.Molecular cloning of caspase-8L cDNA Human caspase-8L complementary DNA (cDNA) and caspase-8a cDNA were cloned by RT-PCR from healthy human PBLs. PBLs were prepared by Lymphocyte Separation Medium (ICN Biomedicals, Aurora, OH) from the heparinized blood of healthy individuals. RT-PCR was conducted using 2 primers, 5'-GGAGTTAGGCAGGTTAGGGG-3' (5'-primer) and 5'-GCGACAGAGCGAGATTCTG-3' (3'-primer).39 After 5 minutes incubation at 94°C, PCR was carried out for 30 seconds at 94°C, 30 seconds at 60°C, and 90 seconds at 72°C for 35 cycles. The resulting PCR products (1723 base pairs [bp]: caspase-8L; 1587 bp: caspase-8a) were cloned into pCR2.1 vector by Original TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced using the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Tokyo, Japan) and the ABI PRISM 310 genetic analyzer (Perkin-Elmer, Norwalk, CT).Expression vectors To generate caspase-8L or -8a expression vectors, RT-PCR was performed as described previously.40 RT-PCR of caspase-8L was conducted using 2 primers, 5'-CGGGGTACCATGGACTTCAGCAGAAATC-3' (5'-primer) and 5'-CGCGGATCCTCCTGTCCATCAGTGCCATA-3' (3'-primer). RT-PCR of caspase-8a was performed using 2 primers, 5'-CGGGGTACCATGGACTTCAGCAGAAATC-3' (5'-primer) and 5'-TCAATCAGAAGGGAAGACAA-3' (3'-primer). Each PCR reaction was carried out for 30 seconds at 94°C, 30 seconds at 60°C, and 90 seconds at 72°C for 35 cycles. Before the cycle, a denaturation step for 5 minutes at 94°C was included. The resulting products were subcloned into pcDNA3 mammalian expression vector (Invitrogen). To obtain N-terminal polyhistidine-tagged (His-tagged) caspase-8L or -8a expression plasmids, pCR2.1 vectors encoding caspase-8L or -8a were digested with BstXI, and the resulting fragments were subcloned into a pcDNA3.1/HisA mammalian expression vector (Invitrogen). Full-length FADD cDNA containing cosmid pAxCAhFADD41 (obtained from RIKEN Gene Bank, the Institute of Physical and Chemical Research, Tsukuba, Japan) was digested with KpnI and BamHI, and FADD cDNA fragment was subcloned into pcDNA3 mammalian expression vector (Invitrogen).In vitro translation In vitro translation was performed using the TNT-Coupled Reticulocyte Lysate Systems (Promega, Madison, WI) in the presence or the absence of Transcend biotinylated transfer RNA (tRNA) (Promega) according to the manufacturer's instruction. In vitro-translated proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to the nitrocellulose membrane (BIO-RAD Laboratories, Hercules, CA). The membrane was blocked with Tris-buffered saline (TBS) containing 0.5% Tween 20 (TBS-T), probed with HRP-conjugated streptavidin, and visualized by Transcend Chemiluminescent Non-Radioactive Translation Detection Systems (Promega).RT-PCR analysis of various caspase-8 isoforms' mRNA expression To determine the expression of caspase-8L, -8a, -8b, and other isoforms of caspase-8 messenger RNA (mRNA) in different tissues or in human tumor cell lines, cDNAs of selected human normal tissues (CLONTECH, Palo Alto, CA), the synthesized cDNAs prepared from human PBLs, or human tumor cell lines were amplified by means of the PCR as described previously.40 PCR was conducted using 2 primers, 5'-TCTGTGCCCAAATCAACAAG-3' (5'-primer) and 5'-GCCACCAGCTAAAAACATTCC-3' (3'-primer). After 5 minutes' incubation at 94°C, PCR was carried out for 30 seconds at 94°C, 30 seconds at 60°C, and 30 seconds at 72°C for 35 cycles. The resulting products were subjected to 1.2% agarose gel electrophoresis and visualized by ethidium bromide staining. As an internal control for RT-PCR, glyceraldehyde-3-phosphate dehydrogenase cDNA was amplified by PCR using 2 primers, 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' (5'-primer) and 5'-CATGTGGGCCATGAGGTCCACCAC-3' (3'-primer) at the same experimental conditions and then were used for normalization.Expression of caspase-8L in COS-7 cells COS-7 cells were transiently transfected with caspase-8L expression vector using LipofectAMINE reagent (Life Technologies) according to the manufacturer's instructions. Forty-eight hours after transfection, a Western blotting analysis was performed to detect caspase-8L protein.Western blot analysis The cells (2.5 × 106 cells) were lysed in 50 µL SDS-PAGE sample buffer (0.625 M Tris-HCl [pH 8.8], 2% SDS, 10% -mercaptoethanol, 10% glycerol, and 0.01% bromophenol blue), and
lysates were boiled for 5 minutes and then analyzed by SDS-PAGE
followed by transfer to nitrocellulose membrane. The membranes were
incubated for 1 hour in phosphate-buffered saline containing 5% nonfat
milk. After incubation, the membranes were blotted using rabbit
anti-caspase-8 polyclonal antibody (BD Pharmingen) or using mouse
anti-caspase-8 monoclonal antibody (MBL) overnight. After washing with
TBS-T, the membranes were incubated with peroxidase-conjugated
secondary antibody for 2 hours and visualized with ECL Western Blotting Detection Reagents (Amersham Pharmacia Biotech).
Ribonuclease protection assay Using RT-PCR, a probe for ribonuclease (RNase) protection was constructed with a sense primer (5'-GGATTTAAACATATTTCCCTGTGG-3') and an antisense primer (5'-TCCATGGGAGAGGATACAGC-3') to clone the 288-bp probe. The resulting PCR product was cloned into the pCR2.1 vector using a TA cloning kit (Invitrogen), and the vector was sequenced to rule out any point mutations. Plasmid DNA was linearized with BamHI, and in vitro transcription was performed using Riboprobe in vitro transcription system (Promega) in the presence of 50 M [ -32P]CTP (cytidine triphosphate) according to
the manufacturer's protocol. The hybridization of probe and sample RNA
and the RNase digestion of hybridized probe and sample RNA were
performed using the RPAII Ribonuclease Protection Assay Kit (Ambio,
Austin, TX) according to the manufacturer's instructions. Briefly,
after gel purification, the 4 × 105 cpm probe was
hybridized to sample total RNA (20 µg) overnight at 42°C. RNase
digestion of hybridized probe and sample RNA was performed using RNase
A (0.25 U/µL) and RNase T1 (10 U/µL) solution at 37°C for 30 minutes. Next, the digestion mixtures were treated with RNase
Inactivation/Precipitation Mixture to inactivate RNase and precipitate
RNA. The precipitated products were loaded on denaturing polyacrylamide
gel. After electrophoresis, the gel was transferred to filter paper,
and autoradiographic exposure to x-ray film was performed overnight.
Cytotoxicity assays MCF-7 cells (3 × 105 cells in 3 cm dishes) were transiently transfected with the cDNAs of the indicated proteins together with the-galactosidase expression vector
(pcDNA3.1/His/LacZ, Invitrogen) using LipofectAMINE reagent (Life
Technologies). In cotransfection assays, 0.5 µg caspase-8a expression
vector plus various amounts of empty vector or caspase-8L expression
vector were transiently transfected with 0.75 µg -galactosidase
expression vector into MCF-7 cells. The cells were rinsed 5 hours after
transfection and were further incubated for 18 hours without any
additional treatment. The extent of cell death was assessed by
determining the percentage of apoptotic cells in morphology in
-galactosidase-positive cells, as described
previously.42
Stable transfection and evaluation of the transfected cells A total of 20 µg linearized empty vector (pcDNA3.1/HisA, Invitrogen) (mock) or 20 µg linearized vector expressing the His-tagged caspase-8L cDNA was transfected into Jurkat cells by electroporation using Gene Pulser apparatus (BIO-RAD Laboratories) at 250 V, 960 microfarads. Cells were immediately plated to prewarmed medium and cultured at 37°C. Two days after transfection, transfected cells were selected in the presence of 2 mg/mL G418 (Sigma, St Louis, MO). The clones were established by limiting dilution, and the expression of the transfected caspase-8L was assessed by RT-PCR. In these clones, 3 clones were used for apoptosis assay by flow cytometry because of its high expression level of caspase-8L.Cell death evaluation by flow cytometry Fas-induced cell death of empty vector-transfected (mock) and caspase-8L-transfected Jurkat cells were evaluated by membrane permeability cell death analysis. Cells (2.0 × 105) were left either untreated or were treated with anti-Fas monoclonal antibody (0.5 µg/mL) for 12 hours. After stimulation, the cells were stained with 40 µg/mL propidium iodide in phosphate-buffered saline containing 2% FCS and 0.05% NaN3 for 5 minutes on ice. Then the percentage of propidium iodide-positive (dead) and -negative (alive) cells was determined with FACScan (Becton Dickinson, San Jose, CA) flow cytometer using CellQuest software (Becton Dickinson).Caspase activity assays Fas-induced caspase-8 catalytic activity in empty vector-transfected (mock) and caspase-8L-transfected Jurkat cells was measured using a Caspase-8 Colorimetric Protease Assay Kit (MBL) according to the manufacturer's instructions. In brief, 1 × 106 cells were left either untreated or were treated with anti-Fas monoclonal antibody (0.5 µg/mL) for 4 hours and were harvested by centrifugation. The cells were lysed by lysis buffer and recentrifuged at 10 000g for 1 minute. Then, 50 µL of the cytosolic extracts (1 mg of protein contained) was diluted in 50 µL of reaction buffer and was added with fluorogenic substrates (IETD-pNA; Ile-Glu-Thr-Asp-P-nitroanilide) and incubated at 37°C for 12 hours. Release of pNA was measured by spectrofluorometry at 400 nm.In vitro binding assays The cDNA of the indicated proteins was in vitro translated, and the proteins were incubated with 50 µL Ni-NTA Magnetic Agarose Beads (QIAGEN, Valencia, CA) overnight at 4°C in protein binding buffer (50 mM NaH2PO4 [pH 8.0], 300 mM NaCl, 0.2 mM phenylmethylsulfonyl fluoride, and 20 mM imidazole) to selectively collect His-tagged proteins. After incubation, the Ni-NTA beads were collected using a magnetic separator and then were washed 3 times with wash buffer (50 mM NaH2PO4 [pH 8.0], 300 mM NaCl, 0.005% Tween 20, 0.2 mM phenylmethylsulfonyl fluoride, and 20 mM imidazole). Next, Ni-NTA beads bearing His-tagged proteins were resuspended in interaction buffer (50 mM NaH2PO4 [pH 8.0], 300 mM NaCl, 0.005% Tween 20, 0.2 mM phenylmethylsulfonyl fluoride, and 20 mM imidazole) and incubated with in vitro-translated FADD or caspase-8L for 2 hours at room temperature. Ni-NTA beads were collected on a magnetic separator. After washing, the bound proteins were eluted by adding 50 µL SDS-PAGE buffer, analyzed by 10% SDS-PAGE, and detected with anti-FADD monoclonal antibody (MBL) or with anti-caspase-8 polyclonal antibody (BD Pharmingen) and HRP-conjugated secondary antibody.In vivo interaction assay of caspase-8L with FADD in MCF-7 cell lines MCF-7 cells (2 × 107) were transiently transfected with 4 µg empty vector (pcDNA3.1/HisA, Invitrogen) or His-tagged caspase-8L expression vector using LipofectAMINE (Life Technologies) according to the manufacturer's instructions. Eighteen hours after transfection, cells were lysed, resuspended in lysis buffer (50 mM NaH2PO4 [pH 8.0], 300 mM NaCl, 10 mM imidazole, and 0.05% Tween 20), and sonicated on ice to lyse cells. After sonication, lysates were cleared by centrifugation at 10 000g for 30 minutes at 4°C , and the supernatants were collected. Next, those supernatants were incubated with 100 µL Ni-NTA Magnetic Agarose Beads overnight at 4°C. After incubation, Ni-NTA beads were collected on a magnetic separator. After washing, the bound proteins were eluted by adding 50 µL SDS-PAGE buffer, analyzed by 10% SDS-PAGE, and detected with anti-FADD monoclonal antibody (MBL) or with anti-HisG antibody and HRP-conjugated secondary antibody.
Molecular cloning of caspase-8L We previously reported a novel isoform of caspase-8, named caspase-8L (caspase-8 long form [GenBank accession number AF 380342]), in human PBLs.39 To clone caspase-8L, we performed RT-PCR on mRNA from human PBL with 2 primers encompassing the initiation and stop codons of caspase-8. In addition to caspase-8a (1587 bp) and caspase-8b (1542 bp), we identified a long amplification product (1723 bp) representing caspase-8L (Figure 1A). Similar results were obtained from an RT-PCR analysis in purified human peripheral T cells and B cells (data not shown). As a result, we cloned caspase-8L and entirely sequenced it. Sequence data concurred with the size of the amplified product and with the published human caspase-8a cDNA sequence, except for the 136 bp insertion of the 5' end sequence of intron 8 as previously described.39 When compared with the consensus sequences at the 5' splice site,43 no difference was observed among the caspase-8L 5' splice site, normal caspase-8a 5' splice site, and the consensus sequences. This 136 bp insertion resulted in a frame shift of the transcript. Thus, this caspase-8L cDNA encoded a putative caspase-8L protein lacking the catalytic domain but retaining 2 N-terminal repeats of the DED (Figure 1B-C). The molecular mass of the predicted protein was estimated to be 32 kd, and the in vitro-translated product migrated as a 32 kd protein on SDS gels (Figure 1D).
Expression of caspase-8L mRNA in normal human tissues and various cell lines We examined the expression of caspase-8L mRNA in a series of normal human tissues by RT-PCR. Caspase-8a and -8b mRNAs were detected in most normal tissues but not in the brain (Figure 2A), and these expression patterns of caspase-8a and -8b were compatible with the results demonstrated by a Northern blotting analysis in previous reports.11,12 The tissue distribution of caspase-8L mRNA was similar to that of caspase-8a and -8b. The ratio of caspase-8L to caspase-8a or -8b varied in these tissues. In PBLs, the caspase-8L expression was most prominent (Figure 2A). An RT-PCR analysis in various cell lines did not demonstrate the expression of caspase-8L mRNA in Jurkat (human T-cell leukemia cells), Daudi (human lymphoblastoid cells), MCF-7 (human breast cancer cells), or Hela (human cervical cancer cells) despite the expression of caspase-8a or -8b mRNA (Figure 2B). To evaluate the expression level of caspase-8L mRNA more precisely, we performed an RNase protection assay on mRNA from human PBLs. The probe was obtained by amplifying a smaller fragment of caspase-8L cDNA in RT-PCR using primers discussed in "Materials and methods." This probe was constructed to include the 3' end 20 bp of the insertion and the 5' end 158 bp of exon 9, and therefore this probe protects fragments of 178 bp (caspase-8L) and 158 bp (caspase-8a) (Figure 3, left). Two fragments were equally evident after hybridization with the labeled probe in human PBLs (Figure 3, lane 3). The longer fragment represented caspase-8L mRNA, and the smaller fragment represented caspase-8a mRNA, respectively. As a negative control, tRNA was hybridized with a probe and digested with RNase, but no fragments were observed (Figure 3, lane 4). These data indicate that in human PBLs caspase-8L mRNA is expressed almost at the same level as caspase-8a.
Caspase-8L protein expression in human PBLs To investigate the expression of caspase-8L protein in human PBLs, at first we tested an anti-human caspase-8 polyclonal antibody directed against the N-terminus of caspase-8 for its capacity to detect caspase-8L. As shown in Figure 4A, this antibody could detect in vitro-translated caspase-8L as well as in vitro-translated caspase-8a (lanes 3 and 4). Thus, we performed immunoblot assay in human PBLs. In PBLs, we detected 2 larger bands (55 kd and 54 kd), representing caspase-8a and -8b, and a 32 kd band (Figure 4B, lane 3). This endogeneous 32 kd protein comigrated with the in vitro-translated caspase-8L (Figure 4B, lane 1) and with recombinant caspase-8L (lane 2). Additionally, this 32 kd band was not detected in Daudi cell lysate (lane 4). The Daudi cell line was used as a negative control for caspase-8L expression, because RT-PCR analysis has shown that caspase-8L mRNA was not detected in Daudi cell line (Figure 2B). These data indicated that this 32 kd band most probably represents caspase-8L. The possibility that this 32 kd band represents other isoforms of caspase-8 that mimic caspase-8L in molecular weight must be denied because this antibody can also recognize such isoforms of caspase-8 (caspase-8f: 32.4 kd; caspase-8g: 30.8 kd, respectively). To exclude this possibility, we conducted an RT-PCR analysis on human PBLs, and we detected only 3 transcripts, representing caspase-8a, -8b, and -8L (Figure 2A). Moreover, we detected no significant transcript even after a long exposure or increased PCR cycle (data not shown), except for those 3 transcripts corresponding to caspase-8a, -8b, and -8L. These results strongly suggest that this endogeneous 32 kd protein represents caspase-8L.
Caspase-8L is not toxic to MCF-7 cells and protects MCF-7 cells from caspase-8-induced apoptosis Because caspase-8L, which carries N-terminal 2 repeats of DED but lacks catalytic domain, structurally resembles c-FLIP (also called Casper/I-FLICE/FLAME-1/CASH/CLARP/MRIT/Usurpin), we expected that caspase-8L might act as an inhibitor of caspase-8. We therefore performed a transient transfection study using an MCF-7 cell line. As shown in Figure 5A, the MCF-7 cells transfected with caspase-8a exhibited cell death (64.18% ± 7.7%). This was accompanied by the production of intermediate forms (p43 and p42) and an active form (p18) of caspase-8a (Figure 5B). In contrast, no death was observed in the cells transfected with either an empty vector or with caspase-8L (10.03% ± 3.97% and 10.65% ± 3.5%, respectively). In vector- or caspase-8L-transfected cells, active form of caspase-8a was not detected (Figure 5B). These data suggest that caspase-8L itself does not exhibit any cytotoxic activity. We next performed a coexpression study of caspase-8a and -8L by titrating the amount of caspase-8L that effectively inhibits caspase-8a-mediated apoptosis (Figure 5C). The coexpression of caspase-8a and an empty vector at various ratios induced similar levels of apoptosis at approximately 60% (Figure 5C, ), while coexpression of caspase-8L
dose-dependently protected cells from apoptosis induced by caspase-8a.
Interestingly, caspase-8L significantly inhibited caspase-8a-mediated
apoptosis by approximately 30% even at a ratio of 0.125 (caspase-8L/caspase-8a) (Figure 5C,  ). These results indicated that
caspase-8L did not exhibit any cytotoxic activity like caspase-8a but
might act as a dominant negative inhibitor of caspase-8a.
Caspase-8L expression protects Jurkat cells from Fas-mediated apoptosis and interferes with Fas-induced caspase-8 protease activity To further investigate the caspase-8L function in the Fas-mediated apoptosis pathway, Jurkat cells were stably transfected with caspase-8L. The transfection of empty vector (mock) or caspase-8L expression vector did not influence the expression level of endogeneous caspase-8a assessed by Western blot analysis (data not shown). We induced cytotoxic activity in mock-transfected or caspase-8L-transfected cells by treating them with anti-Fas monoclonal antibody (Figure 6A-B). In mock-transfected Jurkat cells, the administration of anti-Fas antibody induced cell death effectively (38.33% ± 1.86%), which was compatible with wild-type Jurkat cells. In contrast, caspase-8L-transfected Jurkat cells were less sensitive to Fas stimulation (5.17% ± 0.27%). These data suggest that caspase-8L has a protective effect on the Fas-induced apoptosis pathway by inhibiting Fas-induced caspase-8 catalytic activity. We next determined whether or not the caspase-8 catalytic activity is inhibited in caspase-8L-transfected cells (Figure 6C). Caspase-8 catalytic activity was measured by detecting pNA release as described. In mock-transfected cells, Fas stimulation induced the catalytic activity of caspase-8 (0.138 ± 0.01). In contrast, pNA release was hardly detected in caspase-8L-transfected Jurkat cells (0.01 ± 0.01). Taken together, these results indicate that caspase-8L acts as an antiapoptotic molecule by blocking caspase-8 activation.
Caspase-8L binds to FADD and caspase-8 and interferes with the binding of caspase-8 to FADD To activate caspase-8, FADD must interact with Fas through its DD and also interact with caspase-8 by its DED. To study the possibility that caspase-8L competes with caspase-8 for binding to FADD, preventing caspase-8 activation, we performed in vitro binding assays. Figure 7 shows that in vitro-translated FADD bound to His-caspase-8a (lane 3) and to His-caspase-8L (lane 5), while FADD bound to neither Ni-NTA beads alone nor His-HS1 (lanes 1 and 2). HS1 is a 75 kd intracellular adaptor molecule,44 which is used as a negative control to exclude the possibility of nonspecific binding of FADD to His-tagged proteins. These results demonstrate the specific binding of FADD to caspase-8 or caspase-8L. Interestingly, in the presence of caspase-8L, the interaction of caspase-8a and FADD was abrogated (Figure 7, lane 4). Additionally, in vitro-translated caspase-8L bound to His-caspase-8a directly (Figure 7, lane 8), while caspase-8L bound to neither Ni-NTA beads alone nor His-HS1 (lanes 6 and 7). Taken together, in vitro-translated caspase-8L bound not only FADD but also caspase-8a. Finally, we examined the interaction of caspase-8L and endogeneous FADD in vivo (Figure 8). Endogeneous FADD was detected in the eluate from His-tagged caspase-8L-transfected MCF-7 cells but not from empty vector-transfected cells (Figure 8, upper column), indicating that caspase-8L associates with FADD in vivo. These results and our functional study demonstrated that caspase-8L acts as an endogeneous inhibitor of apoptosis by competing with the caspase-8a/FADD interaction by binding to FADD or to caspase-8a. It is thus strongly suggested that caspase-8L participates in the regulation of apoptosis in vivo.
In this study, we cloned and characterized a novel isoform of caspase-8, named caspase-8L, generated by alternative splicing of the human caspase-8 gene. A functional study demonstrated that caspase-8L is an endogeneous inhibitor of the caspase cascade. Caspase-8L carries N-terminal 2 repeats of DED of the full-length
caspase-8 but lacks a C-terminal half catalytic domain (Figure 1C). Our
transient transfection assays in MCF-7 cells demonstrated that
caspase-8L protected cells from caspase-8-mediated apoptosis in a
dominant negative fashion (Figure 5C). In addition, Jurkat cells that
stably expressed caspase-8L showed resistance to Fas-mediated cell
death and decreased catalytic activity of caspase-8 induced by Fas
stimulation (Figure 6). In in vitro binding assays, we demonstrated
that caspase-8L directly bound to FADD and caspase-8a and also
interferes with the binding of caspase-8a to FADD (Figure 7).
Additionally, we showed direct interaction of caspase-8L and FADD in in
vivo transfection assay (Figure 8). These results strongly suggest that
caspase-8L is a novel antiapoptotic molecule that modulates the
activation of caspase-8, the most important and apical enzyme in the
caspase cascade. Several isoforms of caspase-8 other than caspase-8L
have been previously described.11,12,38 These caspase-8
isoforms have been suggested to function as modulators of the
activation of caspase-8 in Fas-induced apoptosis.12
Caspase-8c (MACH Several structurally and functionally similar molecules have been reported, such as viral proteins, v-FLIPs, and their mammalian homolog, c-FLIPs. In the case of c-FLIPs, there are 2 alternatively spliced forms, c-FLIP-long (c-FLIPL) and c-FLIP-short (c-FLIPS). In addition to the N-terminal 2 repeats of DED, FLIPL possesses a C-terminal domain resembling caspase-8 missing protease activity. The c-FLIPS possesses only 2 DEDs and resembles caspase-8L. Both isoforms bind to FADD or to caspase-8 through DED-DED interaction, which competitively blocked the recruitment of caspase-8 to the DISC.7,19,20,22,23,25 This strict regulation of caspase-8 activation by multiple molecules is conceivable considering the molecular ordering of caspase-8 in caspase cascade. In fact, DED-containing proteins, such as FADD, c-FLIP,
caspase-8d (MACH The dysregulation of apoptosis has been implicated for the pathogenesis of disease such as autoimmunity (eg, systemic lupus erythematosus [SLE]) and cancer.5,47 SLE is an autoimmune disease characterized by the production of autoantibodies by the breakdown of self-tolerance and subsequent immune complex deposition and tissue injury in several organ systems.48-50 In patients with SLE, apoptosis of PBLs has been shown to increase in comparison to that of healthy controls.51 In addition, degenerated proteins during apoptosis have been shown to contribute to the supply of autoantigens.52-55 Taken together, the increased apoptosis of PBL in SLE patients has thus been suggested to be associated with the development of SLE by the increased supply of autoantigens and the production of autoantibodies. We previously reported that the expression of caspase-8L mRNA decreased in the PBLs of patients with SLE,39 thus raising the possibility that the increased sensitivity to apoptosis in PBLs from SLE patients is, at least in part, due to the decreased expression of caspase-8L. In addition, we also demonstrated the decreased expression of caspase-8L mRNA in several tumor cell lines (Figure 3B). In normal physiology, the apoptotic machinery must be strictly controlled at several levels to avoid dysregulated cell death, and this control is exerted by several antiapoptotic proteins. In combination with these findings, it is thus speculated that the decreased expression of antiapoptotic caspase-8L in certain tumor cell lines may correlate with an abnormal response of tumor cells to various proapoptotic and antiapoptotic stimuli. There is supporting evidence demonstrating an involvement of caspases and other apoptosis regulators beyond cell death, such as cell-cycle progression.56 Clearly, much remains to be investigated regarding the contribution of the imbalanced expression of caspase-8L to the pathogenesis of these diseases. In conclusion, caspase-8L is a novel splice variant of caspase-8,
expressed in various normal tissues, including PBLs, but not in several
cancer cell lines. The expression of caspase-8L protein is strongly
suggested in PBLs. In transient transfection assays, caspase-8L could
thus contribute to the prevention of apoptosis by an overexpression of
caspase-8 in a dominant negative manner. Jurkat cells that stably
expressed caspase-8L are resistant to Fas-stimulation, which was
associated with a significantly decreased amount of caspase-8 catalytic
activity. In vitro binding assays showed that caspase-8L bound to FADD
and caspase-8a and prevented the binding of caspase-8 to FADD. The
association of caspase-8L and FADD was confirmed in an in vivo system
using MCF-7 cells transfected with caspase-8L. These results indicate
that caspase-8L is an endogeneous competitive inhibitor of
caspase-8
We thank Dr Hirohumi Hamada for providing cosmid pAxCAhFADD.
Submitted August 29, 2001; accepted February 5, 2002.
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: Takahiko Horiuchi, Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan; e-mail: horiuchi{at}intmed1.med.kyushu-u.ac.jp.
1. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wideranging implications in tissue kinetics. Br J Cancer. 1972;26:239-257[Medline] [Order article via Infotrieve]. 2. Vaux DL, Korsmeyer SJ. Cell death in development. Cell. 1999;96:245-254[CrossRef][Medline] [Order article via Infotrieve]. 3. Nagata S. Apoptosis by death factor. Cell. 1997;88:355-365[CrossRef][Medline] [Order article via Infotrieve]. 4. Salvesen GS, Dixit VM. Caspases: intracellular signaling by proteolysis. Cell. 1997;91:443-446[CrossRef][Medline] [Order article via Infotrieve]. 5. Krammer PH. CD95's deadly mission in the immune system. Nature. 2000;407:789-795[CrossRef][Medline] [Order article via Infotrieve].
6.
Thornberry NA, Lazenbnik Y.
Caspases: enemies within.
Science.
1998;281:1312-1316 7. Budihardjo A, Wang X. Annu Rev Cell Dev Biol. 1999;15:269-290[CrossRef][Medline] [Order article via Infotrieve]. 8. Medema JP, Scaffidi C, Kischkel FC, et al. FLICE is activated by association with the CD95 death-inducing signaling complex (DISC). EMBO J. 1997;16:2794-2804[CrossRef][Medline] [Order article via Infotrieve].
9.
Boldin MP, Varfolomeev EE, Pancer Z, Mett IL, Camonis JH, Wallach D.
A novel protein that interacts with the death domain of Fas/APO1 contains a sequence motif related to the death domain.
J Biol Chem.
1995;270:7795-7798 10. Chinnaiyan AM, O'Rourke K, Tewari M, Dixit VM. FADD, a novel death domain containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell. 1995;81:505-512[CrossRef][Medline] [Order article via Infotrieve].
11.
Chinnaiyan AM, Tepper CG, Seldin MF, et al.
FADD/MORT1 is a common mediator of CD95 (Fas/APO-1) and tumor necrosis factor receptor-induced apoptosis.
J Biol Chem.
1996;271:4961-4965 12. Boldin MP, Goncharov TM, Goltsev YV, Wallach D. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell. 1996;85:803-815[CrossRef][Medline] [Order article via Infotrieve]. 13. Muzio M, Chinnaiyan AM, Kischkel FC, et al. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell. 1996;85:817-827[CrossRef][Medline] [Order article via Infotrieve].
14.
Martin DA, Siegel RM, Zheng L, Lenardo MJ.
Membrane oligomerization and cleavage activates the caspase-8 (FLICE/MACH
15.
Muzio M, Stockwell BR, Stennicke HR, Salvesen GS, Dixit VM.
An induced proximity model for caspase-8 activation.
J Biol Chem.
1998;273:2926-2930 16. Yang X, Chang HY, Baltimore D. Autoproteolytic activation of pro-caspases by oligomerization. Mol Cell. 1998;1:319-325[CrossRef][Medline] [Order article via Infotrieve]. 17. Nagata S. Fas ligand-induced apoptosis. Annu Rev Genet. 1999;33:29-55[CrossRef][Medline] [Order article via Infotrieve]. 18. Thome M, Schneider P, Hofmann K, et al. Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature. 1997;386:517-521[CrossRef][Medline] [Order article via Infotrieve]. 19. Irmler M, Thome M, Hahne M, et al. Inhibition of death receptor signals by cellular FLIP. Nature. 1997;388:190-195[CrossRef][Medline] [Order article via Infotrieve]. 20. Shu HB, Halpin DR, Goeddel DV. Casper is a FADD- and caspase-related inducer of apoptosis. Immunity. 1997;6:751-763[CrossRef][Medline] [Order article via Infotrieve].
21.
Hu S, Vincenz C, Ni J, Gentz R, Dixit VM.
I-FLICE, a novel inhibitor of tumor necrosis factor receptor-1- and CD-95-induced apoptosis.
J Biol Chem.
1997;272:17255-17257
22.
Srinivasula SM, Ahmad M, Ottilie S, et al.
FLAME-1, a novel FADD-like anti-apoptotic molecule that regulates Fas/TNFR1-induced apoptosis.
J Biol Chem.
1997;272:18542-18545
23.
Goltsev YV, Kovalenko AV, Arnold E, Varfolomeev EE, Brodianskii VM, Wallach D.
CASH, a novel caspase homologue with death effector domains.
J Biol Chem.
1997;272:19641-19644
24.
Inohara N, Koseki T, Hu Y, Chen S, Nunez G.
CLARP, a death effector domain-containing protein interacts with caspase-8 and regulates apoptosis.
Proc Natl Acad Sci U S A.
1997;94:10717-10722
25.
Han DK, Chaudhary PM, Wright ME, et al.
MRIT, a novel death-effector domain-containing protein, interacts with caspases and BclXL and initiates cell death.
Proc Natl Acad Sci U S A.
1997;94:11333-11338 26. Rasper DM, Vaillancourt JP, Hadano S, et al. Cell death attenuation by `Usurpin', a mammalian DED-caspase homologue that precludes caspase-8 recruitment and activation by the CD-95 (Fas, APO-1) receptor complex. Cell Death Differ. 1998;5:271-288[CrossRef][Medline] [Order article via Infotrieve].
27.
Deveraux QL, Reed JC.
IAP family proteins 28. Rothe M, Pan MG, Henzel WJ, Ayres TM, Goeddel DV. The TNFR2-TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptotis proteins. Cell. 1995;83:1243-1252[CrossRef][Medline] [Order article via Infotrieve]. 29. Duckett CS, Nava VE, Gedrich RW, et al. A conserved family of cellular genes related to the baculovirus iap gene and encoding apoptosis inhibitors. EMBO J. 1996;15:2685-2689[Medline] [Order article via Infotrieve]. 30. Liston P, Roy N, Tamai K, et al. Suppression of apoptosis in mammalian cells by NAIP and a related family of IAP genes. Nature. 1996;379:349-353[CrossRef][Medline] [Order article via Infotrieve]. 31. Ambrosini G, Adida C, Altieri D. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med. 1997;3:917-921[CrossRef][Medline] [Order article via Infotrieve].
32.
Hauser HP, Bardroff M, Pyrowolakis G, Jentsch S.
A giant ubiquitin-conjugating enzyme related to IAP apoptosis inhibitors.
J Cell Biol.
1998;141:1415-1422
33.
Alnemri ES, Fernandes-Alnemri T, Litwack G.
Cloning and expression of four novel isoforms of human interleukin-1 34. Droin N, Dubrez L, Eymin B, et al. Upregulation of CASP genes in human tumor cells undergoing etoposide-induced apoptosis. Oncogene. 1998;16:2885-2894[CrossRef][Medline] [Order article via Infotrieve].
35.
Droin N, Beauchemin M, Solary E, Bertrand R.
Identification of a caspase-2 isoform that behaves as an endogeneous inhibitor of the caspase cascade.
Cancer Res.
2000;60:7039-7047
36.
Seol DW, Billiar TR.
A caspase-9 variant missing the catalytic site is an endogeneous inhibitor of apoptosis.
J Biol Chem.
1999;274:2072-2076
37.
Srinivasula SM, Ahmad M, Guo Y, et al.
Identification of an endogeneous dominant-negative short isoform of caspase-9 that can regulate apoptosis.
Cancer Res.
1999;59:999-1002
38.
Fernandes-Alnemri T, Armstrong R, Krebs J, et al.
In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains.
Proc Natl Acad Sci U S A.
1996;93:7464-7469 39. Horiuchi T, Himeji D, Tsukamoto H, Harashima S, Hashimura C, Hayashi K. Dominant expression of a novel splice variant of caspase-8 in human peripheral blood lymphocytes. Biochem Biophys Res Commun. 2000;272:877-881[CrossRef][Medline] [Order article via Infotrieve].
40.
Harashima S, Horiuchi T, Hatta N, et al.
Outside-to-inside signal through the membrane TNF- 41. Shinoura N, Yoshida Y, Sadata A, et al. Apoptosis by retrovirus- and adenovirus-mediated gene transfer of Fas ligand to glioma cells: implications for gene therapy. Hum Gene Ther. 1998;9:1983-1993[Medline] [Order article via Infotrieve].
42.
Kumar S, Kinoshita M, Noda M, Copeland NG, Jenkins NA.
Induction of apoptosis by the mouse Nedd2 gene, which encodes a protein similar to the product of the Caenorhabditis elegans cell death gene ced-3 and the mammalian IL-1 43. Padgett RA, Grabowski PJ, Konarska MM, Seiler S, Sharp PA. Splicing of messenger RNA precursors. Annu Rev Biochem. 1986;55:1119-1150[CrossRef][Medline] [Order article via Infotrieve]. 44. Kitamera D, Kaneko H, Miyagoe Y, Ariyasu T, Watanabe T. Isolation and characterization of a novel human gene expressed specifically in the cells of hematopoietic lineage. Nucleic Acids Res. 1989;17:9367-9379[Medline] [Order article via Infotrieve].
45.
Scaffidi C, Medema JP, Krammer PH, Peter ME.
FLICE is predominantly expressed as two functionally active isoforms, caspase-8/a and caspase-8/b.
J Biol Chem.
1997;272:26953-26958
46.
Siegel RM, Martin DA, Zheng L, et al.
Death-effector filaments: novel cytoplasmic structures that recruit caspases and trigger apoptosis.
J Cell Biol.
1998;141:1243-1253 47. Tsokos GC, Wong HK, Enyedy EJ, Nambiar MP. Immune cell signaling in lupus. Curr Opin Rheumatol. 2000;12:355-363[CrossRef][Medline] [Order article via Infotrieve].
48.
Mills JA.
Sytemic lupus erythematosus.
N Engl J Med.
1994;330:1871-1879 49. Kotzin BL. Systemic lupus erythematosus. Cell. 1996;85:303-306[CrossRef][Medline] [Order article via Infotrieve]. 50. Mohan C, Datta SK. Lupus: key pathogenic mechanisms and contributing factors. Clin Immunol Immunopathol. 1995;77:209-220[CrossRef][Medline] [Order article via Infotrieve]. 51. Emlen W, Niebur J, Kadera R. Accelerated in vitro apoptosis of lymphocytes from patients with systemic lupus erythematosus. J Immunol. 1994;152:3685-3692[Abstract]. 52. LeFeber WP, Norris DA, Ryan SR, et al. Ultraviolet light induces binding of antibodies to selected nuclear antigens on cultured human keratinocytes. J Clin Invest. 1984;74:1545-1551[Medline] [Order article via Infotrieve].
53.
Casciola-Rosen LA, Anhalt G, Rosen A.
Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes.
J Exp Med.
1994;179:1317-1330 54. Utz PJ, Hottelet M, Schur PH, Anderson P. Proteins phosphorylated during stress-induced apoptosis are common targets for autoantibody production in patients with systemic lupus erythematosus. J Exp Med. 1997;185:843-854[CrossRef][Medline] [Order article via Infotrieve].
55.
Mevorach D, Zhou JL, Song X, Elkon KB.
Systemic exposure to irradiated apoptotic cells induces autoantibody production.
J Exp Med.
1998;188:387-392 56. Los M, Stroh C, Janicke RU, Engels IH, Schlze-Osthoff K. Caspases: more than just killers? Trends Immunol. 2001;22:31-34[CrossRef][Medline] [Order article via Infotrieve].
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  S. H. Jia, J. Parodo, A. Kapus, O. D. Rotstein, and J. C. Marshall Dynamic Regulation of Neutrophil Survival through Tyrosine Phosphorylation or Dephosphorylation of Caspase-8 J. Biol. Chem., February 29, 2008; 283(9): 5402 - 5413. [Abstract] [Full Text] [PDF]
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 U. Testa and R. Riccioni Deregulation of apoptosis in acute myeloid leukemia Haematologica, January 1, 2007; 92(1): 81 - 94. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
(caspase-1 isoform), caspase-2S,
CASP-2L-Pro (caspase-2 isoforms), caspase-9S, and caspase-9b (caspase-9
isoforms), have been suggested to act as dominant negative, endogeneous
inhibitors of apoptosis.
below the alignment. Caspase-8L, a splice
variant of caspase-8a, is truncated at amino acid residue 276, including C-terminal 8 amino acids different from caspase-8a (italics).
The C-termini of these proteins are denoted by asterisks. (C) Molecular
structures of caspase-8a and -8L are shown schematically. The DEDs are
denoted by the checked areas. The catalytic cysteine residue and amino
acids that were implicated in catalytic activity are indicated by 
namely, an initiator of cascade. Regulation by their own
alternative splicing products might be common in proteins belonging to
the caspase cascade due to the fact that a similar inhibitory mechanism
has been described in caspase-1, -2, and -9 as well.