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Originally published In Press as doi:10.1074/jbc.M311819200 on January 27, 2004

J. Biol. Chem., Vol. 279, Issue 15, 15515-15523, April 9, 2004
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Dimerization and Processing of Procaspase-9 by Redox Stress in Mitochondria*

Iyoko Katoh{ddagger}, Yoshiya Tomimori{ddagger}§, Yoji Ikawa{ddagger}, and Shun-ichi Kurata¶||

From the {ddagger}Ikawa Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 350-0198, the §Molecular Pathophysiology, Graduate School of Allied Health Sciences, and the Department of Biochemical Genetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan

Received for publication, October 28, 2003 , and in revised form, January 26, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We studied the mechanism of intra-mitochondrial death initiator caspase-9 activation by a redox response, in which hydrogen peroxide (H2O2) caused a subtle decrease in the inner membrane potential ({Delta}{psi}m) with little evidence of cytochrome c release. Initiation of the intra-mitochondrial autocleavage of procaspase-9 preceded the onset of caspase cascade induction in the cytosol. Purified mitochondria demonstrated procaspase-9 processing and releasing abilities when exposed to H2O2. Bcl-2 overexpression caused accumulation of the active form caspase-9 in the mitochondria, rendering the cells resistant to the redox stress. Intriguingly, disulfide-bonded dimers of autoprocessed caspase-9 were generated in the mitochondria in the pre-apoptotic phase. Using a substrate-analog inhibitor, dimer formation of procaspase-9 was also detectable inside the mitochondria. Furthermore, thiol reductant thioredoxin blocked the caspase-9 activation step and the cell death induction. Thus, redox stress-responsive thiol-disulfide converting reactions in the mitochondrion seemed to mediate procaspase-9 assembly that allows autoprocessing. This study offers an explanation for the recent observation that Apaf-1-null cells can execute apoptosis, which can be blocked by Bcl-2, and supports the proposition that the cytochrome c-Apaf-1-procaspase-9 complex functions in the caspase amplification rather than in its initiation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The respiratory chain reaction in mitochondria inevitably generates reactive oxygen species (ROS),1 including oxygen radicals and hydrogen peroxide (H2O2). When the ROS production dominates cellular ROS-reducing functions, oxidative stress develops (1), ultimately leading to apoptosis. Various stress signal pathways respond to the oxidative stress (2-5) for activation of genes encoding cell death inducers/suppressors. Paradoxically, mitochondria serve as the reservoir of death signal influxes originating from physiological stresses, nuclear events and membrane receptors, and act as the commander of cell death execution by their nature to release cytochrome c (6-8), an ion carrier associated with complexes III and IV of the respiratory chain.

In the prevailing model, mitochondria need to disrupt the inner membrane potential ({Delta}{psi}m), because cytochrome c is released during the outer membrane permeability transition (PT) (8, 9). The adenine nucleotide translocator (10) and the voltage-dependent anion channel (VDAC) (11-13) are thought to accomplish the cytochrome c translocation under the control of Bcl-2 family proteins (14, 15). However, pro-apoptotic "BH3-only" molecules, tBID and Bik, can release cytochrome c by outer membrane pore formation in cooperation with the "BH1-3" members represented by Bax and Bak without disruption of {Delta}{psi}m (16-18). Furthermore, outer membrane pore opening for short duration time occurs constantly in steady-state intact cells (19). Neither the action of each Bcl-2 family protein nor the mitochondrial mechanism controlling the influx/efflux of various types of proteins, including cytochrome c, has been fully understood (20).

The activation of death initiator caspase-9 requires formation of "apoptosome" comprising cytochrome c, procaspase-9, and cytosolic protein Apaf-1 (21). Apaf-1 binds to cytochrome c using dATP/ATP (22). By the interaction between the caspase-recruitment domain (CARD) of Apaf-1 and the prodomain (also referred to as CARD) of procaspase-9 (23, 24), the procaspase-9 molecules are assembled to homodimers that perform autoprocessing (22, 25). Nevertheless, bacterially expressed procaspase-9 in concentrated solutions are able to self-dimerize and self-process to form an active "homodimer" of mature "two-chain" caspase-9 in the absence of Apaf-1 (25, 26).

Striking results came from the studies of procaspase-2, -3, and -9 existing in mitochondria (27, 28). Although one study suggested migration of cytoplasmically activated caspase-9 into the mitochondria (29), others demonstrated the release of processed caspase-9, -3, and -2 together with cytochrome c from mitochondria in vitro by disruption of {Delta}{psi}m with an uncoupler or in cell death caused by staurosporine (27, 28, 30), implying that the mitochondrial procaspase molecules participate in apoptosis induction. Furthermore, recent reports showed (i) cytoprotective function of Bcl-2 in Apaf-1-null cells (31), (ii) caspase activation in Apaf-1-null or caspase-9-null cells and its blockage by Bcl-2 (32), (iii) apoptosis triggered by caspase-2 (33), and (iv) oxidative stress-mediated, cytochrome-c/Apaf-1-independent caspases-9 activation by a virus infection (34), proposing a revised view that apoptosome functions in amplification of the cytosolic caspase cascade rather than in its initiation (32, 35).

Based on the above notions, we examined the mitochondrial caspase-9 activation in response to hydrogen peroxide (H2O2, 0.2 mM) in human leukemic U937 cells as well as in vitro with mitochondria purified from the cell culture and mouse liver. Although multiple kinase cascades were activated by H2O2 in the culture (2), caspase-9-dependent cell death was induced without newly caused transcription or translation, allowing us to focus on the structurally prepared mechanism. Results indicate that procaspase-9 existing in the mitochondria initiates its autoprocessing in the pre-apoptotic phase, in which homodimerization with trans-molecular disulfide, S-S, linkage is involved. In vitro experiments indicated that mitochondria can activate and release caspase-9 in response to H2O2 as low as 0.2-2 µM by which neither PT nor release of cytochrome c was induced. The mitochondrial redox control system may serve as a sensitive pathway to the cytoplasmic death cascade unless cytochrome c is released by a dramatic structural change in the inner or outer membrane.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Cultures and H2O2 Treatment—U937 (ATCC CRL-1593.2) cell culture and transfection of the p53 expression vector were described previously (2, 36). Cells were refed with fresh medium and equilibrated for 1 h before the experiments (2). Caspase inhibitors, LEHD-CHO and IETD-CHO (Calbiochem), were dissolved in Me2SO, and added to the cultures at 100 µM. Endotoxin-free, recombinant human thioredoxin (TRX) (Calbiochem) was used at 5 µg/ml.

Western Blotting—For sample preparation, cells (106) were washed in ice-cold STEE (0.15 M NaCl, 10 mM Tris-HCl, pH 7.4, 2 mM EDTA, 2 mM EGTA), lysed in 200 µl of SDS-PAGE sample buffer and boiled immediately (2). Separating gel was prepared with 9, 11, 13, or 15% acrylamide. Typically, 100 µg of proteins was loaded in each slot. Biotinylated SDS-PAGE standards (broad range, Bio-Rad) were used for size determination. For the two-dimensional electrophoresis, samples were dissolved in the loading buffer without 2-mercaptoethanol (2-ME). The first dimension 1.0-mm thick gel strip (10% constant gel or 15-20% gradient gel) was equilibrated with 2-ME-containing sample buffer and embedded with 1% agarose on top of the second dimension gel (1.5-mm thick). Protein transfer and immunodetection were performed as described previously (36). Human-specific (#9502) and mouse-specific (#9504) anti-caspase-9 antibodies reactive with the sequences around the cleavage sites and an anti-poly(ADP-ribose) polymerase (PARP) antibody (#9542) were from Cell Signaling Technology. Anti-caspase-3 (H277, sc-7148), anti-cytochrome c (H104, sc-7159), anti-Bcl-2 (C-2, sc-7382), anti-Bcl-XL/S (L-19, sc-1041), and anti-I{kappa}B{alpha} (C-21, sc-371) antibodies were from Santa Cruz Biotechnology. The anti-JNK2 antibody (sc-7345) was cross-reactive with JNK1. An anti-{beta}-actin antibody (AC-15, Sigma) and an anti-Apaf-1 antibody (559863, BD Biosciences) were also used. Blotting membrane was exposed to instant film (FP-3000B, Fuji) with an ECL Mini Camera (Amersham Biosciences).

Cell Staining and Flow Cytometry—For microscopic observation, cells were stained with Hoechst 33342 (ICN Biomedicals) as described (2). Semi-quantitation of {Delta}{psi}m in isolated mitochondria was performed as described (37) with rhodamine 123 (Sigma). Cells stained with 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) (Aldrich) and biotinylated annexin V/streptavidin-allophycocyanin (APC) (BD Biosciences) were analyzed for {Delta}{psi}m alterations and apoptosis induction using FACS Calibur (BD Biosciences).

Cell Fractionation—To obtain mitochondria and cytosol fractions from H2O2-treated cell cultures, washed cells (107) were disrupted with 40 strokes in loosely fitted Dounce homogenizer in 3 volumes of isolation buffer (20 mM Hepes, pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 250 mM sucrose) supplemented with protease inhibitors, including PMSF (0.5 mM), leupeptin (1 µg/ml), aprotinin (1 µg/ml), pepstatin A (1 µg/ml), L-1-chloro-3-(4- tosylamido)-7-amino-2-heptanone hydrochloride, N{alpha}- tosyl-L-lysine chloromethyl ketone (0.1 mM), and E-64 (1 µg/ml). For -2-ME/+2-ME two-dimensional gel electrophoresis, DTT was omitted from the buffer. After two-cycle clarification at 2500 x g for 5 min, supernatant was centrifuged at 12,000 x g for 30 min to obtain cytosol and mitochondrial fractions. The cytosol fraction was concentrated by centrifugal ultrafiltration with Vivaspin 2 (MWCO 5000, Vivascience).

In Vitro Experiments with Purified Mitochondria—Mitochondria for in vitro experiments were purified as described (38). Briefly, U937 or units/Bcl-2 cells (5 x 108) were collected, washed, and disrupted in a glass-Teflon Potter homogenizer in a buffer (0.25 M sucrose, 20 mM HEPES, pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, and 1 mM EGTA) containing protease inhibitors without L-1- tosylamido-2-phenylethyl chloromethyl ketone and E-64. Mouse liver was minced and homogenized in the same way. After centrifugation at 10,000 x g for 10 min at 4 °C, the pellets containing mitochondria were re-suspended and subjected to sedimentation through a Percoll step gradient (18, 30, and 60%) at 9,000 x g, for 15 min at 4 °C. Mitochondrial fraction formed on the 60% cushion was taken and diluted in the buffer for sedimentation. Protease inhibitors were eliminated from the Percoll gradient centrifugation and the subsequent procedures.

Purified mitochondria (wet material, 5 mg) were suspended in 1 ml of buffer (200 mM mannitol, 50 mM sucrose, 1.0% bovine serum albumin, 10 mM HEPES-KOH, pH 7.4) without an inhibitor. After adding succinate (5 mM), the suspension (200 µl) was incubated for 15 min at 25 °C in the presence or absence of H2O2. After incubation, a 1/2 aliquot was centrifuged at 15,000 x g for 5 min at 4 °C to obtain supernatant and pellet fractions. Supernatant was concentrated as described above. The other portion was immediately examined for {Delta}{psi}m with rhodamine 123 using a Hitachi fluorometer as described previously (37).

Caspase Enzyme Activity Assay—Semi-quantification of caspase-9 and caspase-3 enzyme activities was performed with colorimetric substrates, Ac-LEHD-pNA, and Ac-DEVD-pNA (Calbiochem), respectively. Cytosol fractions and supernatants of the H2O2-treated mitochondria were adjusted to the assay buffer containing Tris-HCl (50 mM, pH 7.6), DTT (10 mM), PMSF (0.5 mM), leupeptin (1 µg/ml), aprotinin (1 µg/ml), and pepstatin A (1 µg/ml), and incubated with either of the substrates (0.2 mM) for 10 min at 37 °C. Buffer exchange was performed by repeated membrane filtration and dilution. Mitochondria were disrupted with 0.1% Tween 20 prior to the assay. Absorbance at 405 nm was measured.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cell Death Induction by Caspase-9 Activation—Under the culture conditions with 0.2 mM H2O2, cells with morphological features of apoptosis became detectable by nuclear staining in 3.5 h (Fig. 1A). Apoptotic cells exhibiting nuclear condensation and fragmentation occupied 60% of the culture at 7 h (Fig. 1, A and B), and all cells died within 12 h. In the Western blot analysis, 47-kDa procaspase-9 was dominantly found in the whole lysates of cells incubated with H2O2 for up to 3 h (Fig. 2A). In the samples prepared at 3.5 h, the band of 35-kDa caspase-9 was substantially intensified, indicating induction of the procaspase-9 autoprocessing at Asp-315 (25). The 370-kDa caspase-9 molecules due to the cleavage at Asp-330 by caspase-3 (25) was detectable less efficiently only in the later samples (5 and 7 h), reflecting amplification of the caspase cascade.



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  		FIG. 1.
The onset and progression of H2O2-induced apoptosis detected by microscopic observation and flow cytometry. A, apoptosis induced by 0.2 mM H2O2 in U937 cells. Cells were examined for nuclear condensation and fragmentation by staining with Hoechst 33342 at time points indicated (h). B, graphic presentation of the dying cell population (%) after incubation of U937, U/p53 and U/Bcl-2 cells with 0.2 mM H2O2. It also shows effects of TRX, caspases-9 inhibitor LEHD-CHO and caspase-8 inhibitor IETD-CHO on the U937 cell death, in which cells were pretreated with TRX or an inhibitor for 1 h. C, TNF-{alpha}-induced apoptosis susceptible to IETD-CHO. Cells pretreated (+) or untreated (-) either with LEHD-CHO or with IETD-CHO were cultured in the presence (+) or absence (-) of TNF-{alpha} for 5 h. D, flow cytometric analysis for the status of {Delta}{psi}m and cytoplasmic membrane integrity. Each plot (at 0, 5, or 7 h) displays 104 cells with respect to the emissions from DiOC6(3) and APC-annexin V in vertical and horizontal axes, respectively. Gray centers in dotted areas (red) indicate compaction.

 



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  		FIG. 2.
Caspase-9 induction in the absence of cytosolic translocation of cytochrome c. A, procaspase-9 processing detected by Western blotting with whole cell lysates from U937 and U/p53 cultures. Time courses from 0 to 7 h and from 2.5 to 4.5 h are shown. Positions of procaspase-9 and caspase-9 are marked with arrows. Sizes of the proform and mature forms of capase-9 as well as those of protein standards are denoted in kilodaltons in parentheses. B, inhibition of the H2O2-caused caspase-9 activation by LEHD-CHO. U937 cells pre-treated either with LEHD-CHO or with IETD-CHO were exposed to H2O2 for the periods (h) indicated. C, caspase-3 activation detected by the cleavage of PARP and a decrease in procaspase-3. PARP (116 kDa) and its cleavage products (89 and 24 kDa) are indicated with arrows. D, retention of cytochrome c in the mitochondria during cell death by 0.2 mM H2O2. It shows Western blots with the mitochondrial and cytosol fractions obtained in the time course (in h). Control experiment with 2 mM H2O2 indicates the release of cytochrome c. E, analysis for a p53-stabilizing effect. U/p53 cells incubated with H2O2 (0.2 mM), actinomycin D (5 nM), bleomycin (0.06 units/ml), or etoposide (500 nM) were examined for p53 by Western blotting. Cultures with H2O2 were examined through the time course (h) indicated, and those with a drug at 16 h.

 
U/p53 cells constitutively expressing wild type tumor suppressor p53 underwent apoptosis similarly (Fig. 1B). Although the procaspase-9 cleavage began ~30 min earlier in these cells (Fig. 2A), no other result suggested a mechanistic difference between U937 and U/p53 cells in H2O2-induced cell death. In contrast to actinomycin D (5 nM), bleomycin (0.06 unit/ml), and etoposide (500 nM), with which the p53 protein accumulation was evident at 16 h (Fig. 2E), H2O2 at 0.2 mM did not exert a p53-stabilizing effect either at the onset or during the progression of apoptosis. Despite the general assumption that H2O2 would cause chromosomal damage to stabilize p53 for induction of genes encoding mitochondrially localized proteins (4) and enzymes affecting redox conditions (39), neither DNA damage nor a p53 function was found essential for the death induction under the oxidative conditions.

If thioredoxin (TRX), a small molecular weight thiol-reducing protein, which cells can take up from the medium, was added to the culture 1 h before the H2O2 input, only 10% of the cells were found apoptotic at 7 h (Fig. 1B). TRX suppressed the H2O2-induced cell death effectively as reported (40).

A membrane-permeable caspase-9 inhibitor, LEHD-CHO, effectively protected cells from apoptosis by H2O2, allowing at least 80% of the cells to survive for 7 h (Fig. 1B). In contrast, caspase-8 inhibitor IETD-CHO exerted little effect on the cell death. The procaspase-9 processing induced by H2O2 was certainly inhibited by LEHD-CHO, but not by IETD-CHO (Fig. 2B). In the experiment with TNF-{alpha}, where ~60% of the cells became apoptotic within 5 h, IETD-CHO decreased the dying cell ratio to 10%, but LEHD-CHO did not affect it significantly (Fig. 1C). Consistently, the 8-kDa cleavage product from BID was detectable in the cells exposed to TNF-{alpha}, but not those exposed to H2O2 (data not shown). The cleavage of 116-kDa PARP to the 89- and 24-kDa peptides by caspases-3 (41) was readily detectable at 3.5 h (Fig. 2C), indicating activation of the entire apoptosis cascade. An obvious decrease in procaspase-3 was found at 5 and 7 h, whereas the amount of {beta}-actin was not altered significantly (Fig. 2C, right). Thus, the apoptosis induction was dependent on initiator caspase-9 by which the downstream caspases were induced abruptly.

Apoptosis in the Absence of PT and Release of Cytochrome c—To determine whether the death induction depended on the loss of {Delta}{psi}m, flow cytometry was performed with annexin V-conjugated with allophycocyanin (APC) and 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) (Fig. 1D). A majority of untreated U937 cells were found in the annexin V-negative, DiOC6(3)-positive (death-negative/{Delta}{psi}mnormal) population (93%). After 5 h of the H2O2 treatment, the death-negative cell count decreased to 54%. A newly generated fraction consisting of 25% of the cells displayed an annexin V-positive, DiOC6(3)-hyposensitive (death-positive/{Delta}{psi}mhypo) feature corresponding to an ~100-fold increase in the emission from APC-annexin V and 50% decrease in the emission from DiOC6(3). After 7 h, the death-positive population reached a total of 58% (Fig. 1D), in which only a small fraction (6% of the culture) displayed {Delta}{psi}mlow corresponding to a DiOC6(3) intensity decreased by one order of magnitude. The plots showed that the death-positive/{Delta}{psi}mlow cells arose from the death-positive/{Delta}{psi}mhypo population, suggesting that the large decrease in {Delta}{psi}m occurred as a result cell death.

After subcellular fractionation (Fig. 2D, 0.2 mM H2O2), an intense band of 15-kDa cytochrome c remained n in the mitochondrial fractions throughout the time course. The faint band in the cytosol fractions did not change during the time course to 7 h. However, if a 10-fold increased concentration of H2O2, 2 mM, was applied to the culture, the release of cytochrome c from the mitochondria to the cytosol was evident (Fig. 2D). Thus, cell death by the moderately oxidative culture conditions with 0.2 mM H2O2 occurred without apparent translocation of cytochrome c.

Initiation of the Procaspase-9 Processing in the Mitochondria—Cultures incubated with 0.2 mM H2O2 for 1 or 2 h were examined for mitochondrial and cytosolic procaspase-9 status in the pre-apoptotic phase (Fig. 3A). Approximately 20% of the procaspase-9 molecules existing in the cells were contained in the mitochondria (Fig. 3A, lane 0 h). After incubation with H2O2 for 1 or 2 h, the level of 35-kDa caspase-9 was obviously elevated in the mitochondria (lanes 1 and 2 h), but not in the cytosol fraction, indicating that the caspase-9 priming reaction in the mitochondria preceded the cytoplasmic caspase induction. In the Western blotting with the total cell lysates (Fig. 2A), 35-kDa caspase-9 in the mitochondria (at 1 and 2 h) may have been overwhelmed by the dominantly existing procaspase-9 in the cytosol. Retention of cytochrome c and presence of endogenously expressed Bcl-2 in the mitochondria were confirmed. Apaf-1 was found poorly associated with the mitochondria (Fig. 3A).



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  		FIG. 3.
Intra-mitochondrial procaspase-9 processing in vivo and in vitro. A, detection of the procaspase-9 processing in mitochondria. U937 cells in H2O2 cultures were fractionated into mitochondrial and cytosol fractions at 0, 1, and 2 h. Proteins indicated were analyzed by Western blotting. Five portions of each mitochondrial fraction (5x) were loaded against one portion of the cytosol fraction (1x) containing 100 µg of proteins. B, in vitro detection of the H2O2-induced processing and release of caspase-9 with mitochondria purified from U937 cells. Percoll gradient-purified mitochondria were incubated in a buffer containing H2O2 (0-2 µM). By centrifugation, mitochondrial pellets and supernatant fractions were obtained for protein analyses (left). Five portions of the supernatant (5x) were analyzed in parallel with one portion of the mitochondria (1x). The supernatant fractions were incubated with an unstressed cytosol fraction to examine their abilities to induce cell-free caspase reactions (right). Control experiments with H2O2 (0, 0.2, and 2 µM) were also performed. C, semi-quantification of caspase-9 and -3 activities generated in the mitochondria and cytosol of U937 cells. Enzyme activity at each time point (h) is indicated in relation to the maximum value obtained in the cytosol at 7 h (100%). D, caspase-9 activity in mitochondria exposed to H2O2 and the supernatants. Colorimetric measurements in the fractions were shown in relation to the total caspase-9 activity produced by 2 µM H2O2 without fractionation (100%).

 
By a colorimetric assay with Ac-LEHD-pNA, a synthetic substrate cleavable by caspase-9, a gradual increase in the enzyme activity was detectable in the mitochondrial fractions obtained at 1, 2, and 5 h after the H2O2 input (Fig. 3C). The caspase activity appeared to decrease in the mitochondria obtained at 7 h, probably reflecting the loss of structural integrity as indicated by {Delta}{psi}mlow in the late phase (Fig. 1D). The cytosol fractions at 1 and 2 h did not show an increase in the substrate-cleaving activity, whereas those at 5 and 7 h exhibited its amplification. Casapse-3 activity measured with Ac-DEVD-pNA was also elevated in the cytosol fractions at 5 and 7 h. Thus, supporting the Western blot analyses (Figs. 2A, 3A, and 3B), the biochemical assays indicated intra-mitochondrial procaspase-9 processing in the early phase and cytosolic caspase cascade induction/amplification in the apoptotic phase.

Activation and Release of Caspase-9 by Mitochondria in Vitro—For in vitro experiments, mitochondria were purified from unstressed U937 cultures by Percoll gradient centrifugation. When mitochondria were exposed to a lower concentration (0.2 or 2 µM) of H2O2 for 15 min at 25 °C, 35-kDa caspase-9 was newly generated (Fig. 3B), confirming intra-mitochondrial procaspase-9 autoprocessing. Intriguingly, the 35-kDa product also increased in the supernatants after incubation with H2O2, suggesting release of processed caspase-9 to the outside. Furthermore, the supernatant did induce the processing reaction of procaspase-9 contained in the cytosol fraction from untreated control culture (0 h), if the two fractions were combined and incubated for 10 min at 37 °C (Fig. 3B). Generation of the 35- and 37-kDa forms indicated self-cleavage and hetero-cleavage, respectively. H2O2 (0.2 or 2 µM) per se failed to induce the cell-free caspase reactions in the cytosol fraction. In the enzyme assay (Fig. 3D), an increased caspase-9 activity appeared both in the mitochondria exposed to H2O2 (0.2 or 2 µM) and in the supernatants, indicating that the in vitro produced 35-kDa caspase-9 molecules were enzymatically active.

Retention of cytochrome c and Bcl-2 by the mitochondria was obvious. Approximately a 10% fraction of the procaspase-9 molecules migrated to the supernatant in an H2O2-independent manner, possibly reflecting basal level protein shuttling (19).

The U937-derived mitochondria were examined for changes in {Delta}{psi}m by staining with rhodamine 123, in which {Delta}{psi}m was assessed by quenching of the emission at 534 nm (Fig. 4B). Compared with the absolute loss of {Delta}{psi}m by carbonyl cyanide m-chlorophenylhydrazone (CCCP), the decrease caused by H2O2 (0.2-200 µM) was found to be minor. Thus, the 35-kDa caspase-9 production and release (Fig. 3B) occurred under the subtly declined {Delta}{psi}m condition corresponding to {Delta}{psi}mhypo in cells detected by staining with DiOC6(3) (Fig. 1D). The {Delta}{psi}mhypo status persisted at least for 30 min in vitro.



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  		FIG. 4.
In vitro analyses of mitochondria from mouse hepatocytes and U937 cells for procaspase-9 processing. A, processing and release of caspase-9 by mouse liver mitochondria. After incubation with indicated concentrations of H2O2, proteins contained in the mitochondria (1x) and those in the supernatant (5x) were analyzed. B and C, impairment of {Delta}{psi}m by H2O2 in purified mitochondria from U937 cells (B) and mouse liver (C). Mitochondria incubated with H2O2 at the indicated concentrations or CCCP (0.2 mM) were measured for {Delta}{psi}m with rhodamine 123 (Rh123) as described previously (37). D, analysis of the purified mitochondrial (Mt) and cytosol (Cyt) fractions for their protein components. Lanes "1x Mt" and "1x Cyt" show proteins detectable in equivalent portions of the Mt and Cyt fractions. Lanes "1x Pell" and "5x Sup" show proteins in the Mt pellet (1 portion) and supernatant (5 portions) recovered after incubation of the Mt suspension in the buffer on ice for 15 min. Protein size markers were in lanes M. E, proteinase K treatment of the mitochondria purified from normal U937 cells and those from H2O2-exposed cells. It shows Western blot analyses of proteinase K (PK)-treated and untreated (-) mitochondria for procaspase-9/caspase-9 (left panels) and Tom20 (right panels). Protein size markers are in lane M.

 
Mitochondria purified from thoroughly perfused mouse liver also contained 49-kDa procaspase-9 (Fig. 4A). After incubation with H2O2 (0-200 µM) for 15 min at 25 °C, the 39/37-kDa processed form(s) of caspase-9 appeared in the supernatant. The caspase-9 band intensity increased as the H2O2 concentrations were elevated, indicating enhancement of the procaspase-9 processing by H2O2. The mitochondria from healthy mouse liver poorly retained the newly generated active-form caspase-9 molecules. In contrast to the constantly dissociating feature of procaspase-9 and -3, cytochrome c was kept associated with the mitochondria. Again the decrease in {Delta}{psi}m was minor when the mitochondria was in vitro exposed to H2O2 (0.2-200 µM) (Fig. 4C), indicating the absence of PT.

The mitochondrial and cytosol fractions obtained from U937 and mouse hepatocytes were further examined for their protein components (Fig. 4D). The procaspase-9 molecules existed in the mitochondria and cytosol at a ratio of 1:5 supporting the results shown in Figs. 3A, 3B, and 4A, whereas stress-responsive kinases JNK-2 (p54) and JNK-1 (p46) (2) were localized to the cytosol fractions. Apaf-1 and I{kappa}B{alpha} also appeared in the cytosol but not in the mitochondria that possessed cytochrome c, Bcl-2, or Bcl-XL/S. Furthermore, neither procaspase-9 nor caspase-9 was recovered from the supernatants after incubating the human and mouse mitochondrial suspensions in the fully equipped buffer on ice for 15 min. This excluded the possibility of procaspase-9 carryover from the cytosol and suggested that a mitochondrial activity was required to release the caspase molecules.

To assess whether the H2O2-induced procaspase-9 processing occurred inside or at the surface of the mitochondrion, we performed an experiment in which the surface proteins were digested with proteinase K (Fig. 4E). Tom20, a component of the translocase complex on the outer membrane (42), was tested as a control surface protein, because it is anchored to the outer membrane by an N-terminal peptide and exposes the rest of the body to the cytoplasm. When the mitochondria from H2O2 (1 h)-treated or untreated U937 cells were incubated with proteinase K (100 µg/ml) for 15 min on ice, Tom20 became undetectable by Western blotting (Fig. 4E, right panels) as described (43). In contrast, procaspase-9 in the mitochondrial fractions was protected from degradation (left panels). Furthermore, the mitochondria obtained from H2O2-exposed cells preserved 35-kDa caspase-9 during the proteinase treatment. These results indicate that procaspase-9 existed inside the mitochondria, probably in the intermembrane space, where its autoprocessing took place in response to H2O2.

Accumulation of Caspase-9 in Bcl-2-overexpressing Mitochondria—A Bcl-2 expression vector (9) was introduced into U937 cells to obtain U/Bcl-2. Although parental U937 cells produced a level of Bcl-2, constitutive, 3-fold enhanced Bcl-2 expression was achieved in units/Bcl-2 cells. In nuclear staining, only 10% of the cells displayed morphological features of apoptosis 7 h after exposure to 0.2 mM H2O2 (Fig. 1B). In flow cytometry, cells with the death-positive/{Delta}{psi}mhypo features were not generated (Fig. 5C, 7 h). Instead, an annexin V-negative, DiOC6(3)-hyposensitive (death-negative/{Delta}{psi}mhypo) cluster comprising 24% of the cells appeared below the death-negative/{Delta}{psi}mnormal cluster (49%), indicating survival of U/Bcl-2 cells in the {Delta}{psi}mhypo status (cf. Fig. 1D).



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  		FIG. 5.
Accumulation of caspase-9 in U/Bcl-2 cells. A, suppression of H2O2-induced caspase-9 activation in units/Bcl-2 cells. Whole cell lysates (upper panels) and the mitochondrial and cytosol fractions (lower panels) were prepared from the cultures with 0.2 mM H2O2 in the time course indicated. Procaspase-9, caspase-9, procaspase-3, and Bcl-2 detected by Western blotting are shown. Positions and sizes (in kilodaltons) of the proteins are indicated. B, blockage of the export of processed caspase-9 in vitro. Mitochondria purified from U/Bcl-2 cells were exposed to H2O2 (0, 0.2, or 2 µM). After centrifugation, the pellets containing mitochondria and the supernatants were analyzed for proteins. Procaspae-9, caspases-9, cytochrome c, and Bcl-2 are shown. C, flow cytometric analysis of U/Bcl-2 cells for changes in {Delta}{psi}m and plasma membrane integrity by staining with DiOC6(3) and APC-annexin V, respectively. D, caspase-9 and caspase-3 activity assays in the Mt and Cyt fractions in the time course to 7 h. Results of the colorimetric measurement are displayed as relative activities to the maximum value in the cytoplasm of U937 cells at 7 h as in Fig. 3C. U937 and U/Bcl-2 samples were standardized by protein amounts.

 
When total U/Bcl-2 cell extracts were analyzed, the levels of procaspase-9 and procaspase-3 were found to be maintained without an obvious increase in 37-kDa and 35-kDa caspase-9 for 10 h (Fig. 5A, upper panel), indicating that the cell death suppression by Bcl-2 occurred through prevention of the caspase-9-inducing step. Surprisingly, mitochondria purified from untreated U/Bcl-2 cells contained 35-kDa caspase-9 as well as procaspase-9 in an approximate ratio of 1:3 (Fig. 5A, lower panels, lane 0 h). Intra-mitochondrial amount of the 35-kDa product was increased further by incubation with H2O2 (lanes 1 and 2 h).

Consistently, a level of Ac-LEHD-pNA-cleaving activity was present in the mitochondria from untreated U/Bcl-2 cells, whereas the enzyme activity in an equivalent portion of the cytosol fraction was at the basal level as observed in U937 cells (Fig. 5D). The mitochondrially associated caspase activity was found to increase 3.5-fold after incubation with H2O2 (1-2 h). In contrast to the results with U937 cells (Fig. 3C), the U/Bcl-2 cytosol fraction did not increase the Ac-LEHD-pNA-cleaving activity significantly even at 7 h. Thus, despite the production of 35-kDa active-form caspase-9 in the mitochondria, the caspase amplification reaction did not begin in the cytosol of U/Bcl-2 cells.

In the in vitro experiment with purified Bcl-2-overexpressing mitochondria (Fig. 5B), the constant migration of procaspase-9 to the supernatant occurred less efficiently than that observed in the mitochondria from U937 cells (Figs. 3B). More significantly, 35-kDa caspase-9 was not detectable in the supernatant after incubation with H2O2 (0.2-2 µM), implying that the release of caspase-9 was blocked by the overexpression of Bcl-2. Retention of cytochrome c was also confirmed. Thus, Bcl-2 seemed to prevent the outer membrane traffic of proteins, including active-form capase-9, which is essential for apoptosis induction in response to 0.2 mM H2O2.

Homodimerization of Procaspase-9/Caspase-9—From the notions that biochemical reactions with thiol moieties are often involved in redox controls of proteins (44-46) and that procaspase-9 is dimerized by Apaf-1/cytochorome c for the enzyme activation (25), we tested the possibility that the caspase-9 activation occurs through procaspase-9 dimerization by disulfide bridging in mitochondria. Mitochondrial and cytosolic procaspase-9/caspase-9 status in the pre-apoptotic phase (at 2 h after exposure to H2O2) was analyzed by two-dimensional gel electrophoresis in which the first dimension was run under the non-reducing conditions without 2-mercaptoethanol (-2-ME), and the second dimension with 2-mercaptoethanol (+2-ME).

The Western blot analysis with the mitochondria purified from untreated cells showed a single spot of procaspase-9 (Fig. 6A, left). In mitochondria from the H2O2 culture at 2 h, 35-kDa caspase-9 formed three spots at positions corresponding to 70 kDa in the first dimension (Fig. 6B, left), indicating that the active-form caspase-9 molecules were in dimers with intermolecular disulfide linkage. Only a minor population of 35-kDa caspase-9 migrated to the monomer position. Because the procaspase-9 molecules in the same sample did not show this feature, we speculated that procaspase-9 dimers were once assembled, but immediately converted to the active form, rendering the detection impossible. Supporting the speculation, if cells were pre-treated with reversible inhibitor LEHD-CHO and exposed to H2O2, a small population of the procaspase-9 molecules also formed a spot with a mass of its dimer, 100 kDa, as estimated by the first dimensional size markers (Fig. 6C, left). Procaspase-9 homodimers formed in the transition state may have been immobilized by the interaction with the substrate analog inhibitor, aborting the autocatalytic reaction. The appearance of minor spots around the 100-kDa spot may have reflected involvement of multiple cysteine residues and/or modification/degradation of the inactive complexes. The polymorphism of 35-kDa caspase-9 dimers (Fig. 6B) also appeared in this experiment. LEHD-CHO was not able to inhibit the procaspase-9 priming reaction completely in the mitochondria, although it blocked the caspase-9 inducing reaction (Fig. 2B) to delay apoptosis significantly (Fig. 1B). The tetrapeptide substrate analog inhibitor might be less effective against the autocatalytic reaction than against the substrate-cleaving reaction of processed caspase-9. Without the H2O2 challenge, LEHD-CHO alone did not cause a detectable complex (Fig. 6D, left), indicating that the S-S linkage requires a mitochondrial response to H2O2.



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  		FIG. 6.
Dimer formation of caspase-9 by disulfide linkage. Western blot analysis was performed after the two-dimensional separation under the -2-ME and +2-ME conditions in the first and second dimensions, respectively. A-E, mitochondrial (left panels) and cytosol (right panels) fractions were obtained 2 h after the input of 0.2 mM H2O2. Samples in A-E are as follows: A, control culture; B, cells incubated with 0.2 mM H2O2; C, cells preincubated (for 1 h) with LEHD-CHO and exposed to H2O2; D, incubation with LEHD-DHO only (for 3 h); and E, cells pretreated with TRX (for 1 h) and exposed to H2O2. Sections spanning from 25 to 150 kDa in the first dimension and from 30 to 50 kDa in the second dimension are shown. Positions of procaspase-9 and caspase-9 are marked with an arrowhead. Fractions in F were obtained at 4 h after incubation with 0.2 mM H2O2. G, two-dimensional analysis of procaspase-9 (49 kDa) and caspase-9 (39 and 37 kDa) released from the mouse liver mitochondria exposed to H2O2 (2 µM) in vitro.

 
Furthermore, pretreatment of the cells with TRX abolished the procaspase-9 processing in the mitochondria (Fig. 6E, left), accounting for its cytoprotective effect (Fig. 1B). Neither S-S bonded oligomer formation nor activation of caspase-9 was evident in any cytosol sample in the pre-apoptotic phase (Fig. 6, A-E, right) in agreement with the results obtained by the conventional SDS-PAGE/Western blot analysis of the H2O2 treated cells (Fig. 3A).

At 4 h of the H2O2 treatment, generation of 35- and 37-kDa caspase-9 in the cytoplasm was evident in the two-dimensional analysis (Fig. 6F, right), which was consistent with the time-course experiment (Fig. 2A). However, the cytoplasmically existing caspase-9 molecules were not dimerized by disulfide bonding. After a longer exposure of the Western blot membrane to the film, very faint spots appeared at the position of the dimers (data not shown). In the mitochondria, the ratio of caspase-9 (35-kDa)/procaspase-9 significantly increased, indicating progression of the autocleavage inside the mitochondria (Fig. 6F, left). This agrees with the semi-quantitative assay with Ac-LEHD-pNA (Fig. 3C). Most of the mitochondrial 35-kDa caspase-9 molecules were in disulfide dimers. Notably, a small fraction of procaspse-9 also migrated to the position of the dimers (100 kDa) in the -2-ME gel. Enzymatically essential peptide residues, including the active center cysteine, may have become susceptible when oxidative condition persisted.

The two-dimensional analysis of the materials released from in vitro H2O2 (2 µM)-treated mouse mitochondria also showed the presence of disulfide homodimers of 49-kDa procaspase-9 as well as those of 37-kDa caspase-9 (Fig. 6G). Although 39-kDa caspase-9, the cleavage product by caspase-3, was also contained in the fraction, S-S linkage was not detectable in those molecules. Thus, dimerization and processing of procaspase-9 and release of the active caspase-9 molecules, including the disulfide-bonded dimers occurred in the mitochondria from healthy animals.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The results described above altogether indicate that oxidative stress is a cause of the intra-mitochondrial caspase-9 activation, in which procaspase-9 dimerization is mediated by thiol-disulfide converting reactions. The H2O2-induced intra-mitochondrial procaspase-9 processing and cell death induction were blocked by TRX. A recent report showed that the deletion of Trx-2, a gene encoding mitochondrion-specific TRX, resulted in embryonic lethality by causing massive apoptosis (47), implying critical role of the thiol reducing activity in mitochondria for cell survival. The majority of the autoprocessed molecules were in disulfide-linked dimers within the mitochondria of U937 cells. Furthermore, using a substrate analog inhibitor, we could detect disulfide bridging between the procaspase-9 molecules in the mitochondria of H2O2-exposed cells, implying formation of an S-S bond at the transition state of the autocleavage. The intermolecular covalent bond formation may facilitate the procaspase-9 assembly leading to autoactivation. Interestingly, an artificial fusion protein between the immunoglobulin Fc peptide and procaspase-9 sequences was capable of autoprocessing presumably by dimerization with the spontaneous disulfide linkages between the Fc domains (25), supporting that the procaspase-9 molecules perform autocleavage when two molecules are bought in close proximity to each other.

The catalytic and substrate-recognition domains of procaspase-9/caspae-9 exhibit a self-dimerizing feature in solutions as well as in crystals (25, 26), where no S-S bond has been observed. Procaspase-9 contains 13 cysteines, including the active center cysteine, Cys-287, that needs to be in the reduced form for its enzyme activity (48). The human and mouse procaspase-9 molecules have two conserved cysteines, Cys-12, and Cys-76, in their prodomains folded in six packed {alpha}-helices (23, 49). Cys-12 is located at the edge of the basic concave surface that interacts with the acidic surface of Apaf-1 CARD (49). On the -2-ME gel (Fig. 6, B and C), disulfide-bonded caspases-9 dimers were found in a few spots with slightly different electrophoretic mobilities, implying participation of at least two cysteine residues of each molecule in the trans-molecular covalent bond(s). Mitochondria might have a redox-transducer system as identified in Saccharomyces cerevisiae (50). Alternatively, an oxidative stress-responsive molecular chaperone similar to Hsp33 of bacteria (44) might participate in the S-S bridge formation. In an earlier study, mitochondrion-specific chaperone protein Hsp60 was found to associate with procaspase-3 (27).

Although redox-induced processing seemed to occur only in a minor population of procaspase-9 at the pre-apoptotic stage, once activated and released, the caspase-9 molecules would act as inducers of the amplification reaction in the cytosol as shown in Fig. 3. Caspaset-9 translocated from the mitochondria in a Bcl-2-inhibitable fashion, possibly through VDAC (11), the main pathway for the flux of metabolites between the cytoplasm and the mitochondrial space. Pre-existing 35-kDa caspase-9 in the mitochondria of U/Bcl-2 cells may be the product of the autoprocessing reaction caused by spontaneously developed oxidative microenvironment. Bcl-2 seemed to prevent the outer membrane traffic to some extent under the normal culture conditions. The lack of Bcl-2 expression in adult mouse liver (51) may have allowed the mitochondria to release processed caspase-9 efficiently without trapping the active-form inside (Fig. 5A).

A marked decrease in {Delta}{psi}m became apparent only in the late phase of 0.2 mM H2O2-induced apoptosis in U937 cells (Fig. 1D), indicating that the cell death was not caused by the loss of {Delta}{psi}m. The {Delta}{psi}mhypo status described in this study is distinguished from the inner membrane depolarization that causes PT pore formation. Certainly, cytochrome c was well retained in mitochondria during the cell death. Mitochondrial redox response reactions may be enhanced under the persisting {Delta}{psi}mhypo condition, by which the cell fate either to survive or to die may be eventually determined. Furthermore, the cell death by H2O2 did not include initiator caspase-8, which activates tBID to release cytochrome c by causing dysfunction of mitochondria through the association with the multidomain Bcl-2 family proteins (18). The cytosolic Apaf-1-dependent caspase-9 activation mechanism may be essential in cell death, due to the sudden decrease of {Delta}{psi}m by a functional or structural damage of mitochondria or by the positive suicide mechanism with pro-apoptotic Bcl-2 family proteins.

The disulfide bridge-mediated caspase-9 activation suggested in this study may offer an explanation for various types of Apaf-1-independent, Bcl-2-regulatable cell death (31-34, 52). Proteases with a critical, timely function may take the strategy of self-association followed by autoactivation, as we have learned in retrovirus proteases (53, 54).


   FOOTNOTES
 
* This work was supported by grants-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Dept. of Biochemical Genetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. Tel.: 81-3-5803-5824; Fax: 81-3-5803-0248; E-mail: kushbgen{at}mri.tmd.ac.jp.

1 The abbreviations used are: ROS, reactive oxygen species; H2O2, hydrogen peroxide; Apaf-1, apoptotic protease activating factor-1; {Delta}{psi}m, mitochondrial inner membrane potential; PT, permeability transition; VDAC, voltage-dependent anion channel; CARD, caspase-recruitment domain; CCCP, carbonyl cyanide m-chlorophenylhydrazone; TRX, thioredoxin; 2-ME, 2-mercaptoethanol; DiOC6(3), 3,3'-dihexyloxacarbocyanine iodide; APC, allophycocyanin; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; pNA, p-nitroanilide; Ac, acetyl; TNF, tumor necrosis factor; LEHD-CHO, Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Leu-Glu-His-Asp-CHO; IETD-CHO, Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Ile-Glu-Thr-Asp-CHO; DEVD, Asp-Glu-Val-Asp; LEHD, Leu-Glu-His-Asp. Back


   ACKNOWLEDGMENTS
 
Bcl-2 expression plasmid was kindly supplied by Dr. Y. Tsujimoto, Osaka University through Dr. M. Shirakata, Tokyo Medical and Dental University (TMDU). We also acknowledge Drs. Y. Hara and R. Asakai at TMDU and Dr. K. Machida at RIKEN for discussion and Mr. N. Shirato for electronic filing of Western blot data.



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