Cathepsin B contributes to TNF-α–mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c (original) (raw)
Is cat B translocated to cytosol during treatment of mouse hepatocytes with TNF-α/AcD? In initial experiments, cultured hepatocytes were treated with 28 ng/ml TNF-α plus 0.2 μg/ml AcD for 4 hours, then assayed for intracellular cat B activity in situ using the fluorogenic substrate VLK-CMAC and digitized video microscopy (Figure 1a). As expected, no significant cat B activity was detected in hepatocytes from catB–/– mice. In contrast, a 4.5-fold increase in intracellular hydrolysis of VLK-CMAC was observed in treated catB+/+ mouse hepatocytes compared to untreated cells. Because this substrate is impermeant to lysosomes, these data suggested that cytosolic cat B activity was increased after treatment of mouse hepatocytes with TNF-α/AcD.
Cat B contributes to TNF-α/AcD–induced hepatocyte apoptosis. Isolated hepatocytes from catB+/+ and catB–/– mice were incubated in the absence (control) or presence of TNF-α (28 ng/ml) and AcD (0.2 μg/ml) for up to 12 hours. (a) Intracellular cat B activity was measured fluorometrically in the cells after 4 hours of treatment using the fluorogenic substrate VLK-CMAC and digitized video microscopy, as described in Methods. (b) At the indicated time points, cytosolic extracts were prepared by selective permeabilization with digitonin as described in Methods and subjected to immunoblot analysis using an anti–cat B antiserum. Locations of 30-kDa (p30) and 27-kDa (p27) active fragments of cat B are indicated. Immunoblot analysis of β-actin was performed as a control for protein loading. Cont., control. (c) Cultured McNtcp.24 cells grown on collagen-coated glass coverslips were transfected with the plasmid construct encoding the cat B-GFP fusion protein (control, TNF-α) or double-transfected with the cat B-GFP plasmid and the plasmid encoding the viral protein CrmA (CrmA + TNF-α). Forty-eight hours later, cells were incubated in the absence (control) or presence of TNF-α/AcD at 37°C for 2 hours and transferred to the stage of an inverted confocal microscope. Cat B-GFP fluorescence was imaged as described in Methods.
To determine whether the increase in cytosolic cat B activity was due to release of cat B from lysosomes, we searched for cat B in cytosol by immunoblot analysis (Figure 1b). Cat B active fragments p30 and p27 increased in the cytosol of catB+/+ cells after 8 hours of treatment (lane 3), providing further evidence that TNF-α induces cat B release from lysosomes into the cytosol.
To provide independent evidence for the proposed translocation of cat B from lysosomes to cytosol during TNF-α–induced apoptosis, confocal microscopy was used to visualize directly the cellular compartmentation of expressed cat B-GFP in transfected McNtcp.24 cells (Figure 1c). In untreated cells, the distribution of the cat B-GFP fusion protein was punctate and only localized in the cytosol. After treatment with TNF-α/AcD, the fluorescence was diffusely distributed in the cytoplasm and was also clearly identified in the nuclear region. These data are consistent with translocation of cat B from a vesicular compartment into the cytoplasm during exposure to TNF-α/AcD.
Do proximal caspases promote release of cat B from lysosomes? Because TNFR-1 appears to initiate apoptotic signaling by activating caspases (11, 15), we sought to determine whether proximal caspases could directly promote release of cat B from lysosomes. We addressed this question by (a) transfecting McNtcp.24 cells using CrmA, a specific caspase-8 inhibitor, and assessing the distribution of cat B-GFP; and (b) using a cell-free system to determine whether caspase-8 will directly cause release of cat B from lysosomes. After transfection with CrmA, release of cat B-GFP from lysosomes was inhibited in TNF-α/AcD–treated McNtcp.24 cells (Figure 1c). In the cell-free system, isolated lysosomes were incubated with different concentrations of active recombinant caspase-8 in the presence or in the absence of cytosol. After the lysosomes were sedimented, supernatants were analyzed for cat B (Figure 2a). Active caspase-8 directly induced a release of cat B from lysosome (Figure 2a, lane 2) corresponding to about 26% of the maximum release obtained treating the lysosomes with the detergent Triton X-100 (Figure 2b, lane 6). The release was markedly enhanced to 82% by the presence of cytosol (Figure 2a, lane 3) and was almost completely blocked (6%) by the broad-spectrum caspase inhibitor Z-VAD-fmk (Figure 2a, lane 4), confirming that the catalytic activity of the recombinant caspase-8 was necessary for this phenomenon. Similar results were also observed with another potential proximal caspase, recombinant caspase-2 (data not shown). As a protease control for these experiments, we chose m-calpain, a cytosolic cysteine protease that has been implicated in apoptosis (45) and has been shown to be activated by TNF-α (46). Although addition of calpain to the cell-free system induced release of cat B from lysosomes (Figure 2b, lanes 2 and 4), this release was blocked in the presence of cytosol (Figure 2b, lanes 3 and 5). Thus, the results with calpain were the reverse of those with caspase-8, wherein cytosol potentiated the release of cytochrome c. Likely, the presence of calpastatin in the cytosol inhibits the activity of calpain preventing its proteolytic effects on lysosomes in the cell-free system. Coupled with the CrmA results (Figure 1c), these observations suggest caspase specificity for the observed effects and implicate a proximal caspase or a cytosolic caspase substrate in the TNF-α/AcD–induced release of cat B from lysosomes.
Caspase-8 induces release of cat B from lysosomes. Isolated lysosomes from catB+/+ mouse liver (10 μg protein) were incubated at 37°C with either active recombinant human caspase-8 (20 ng) (a) or rabbit m-calpain (10–25 ng) (b), in the absence or presence of S-100 cytosol fraction (50 μg protein) and the caspase inhibitor Z-VAD-fmk (20 μM). Lysosomes were also treated with 0.1% Triton-X 100 to induce maximal release of cat B (positive control). After 1 hour, lysosomes were pelleted by centrifugation at 15,000 g for 30 minutes. Supernatants were subjected SDS-PAGE on gels containing 10% acrylamide, transferred to nitrocellulose, and probed with anti–cat B antiserum.
Is TNF-α/AcD–induced apoptosis reduced in catB−/− mouse hepatocytes? To determine whether this cytosolic translocation of cat B plays a role in TNF-α–induced hepatocyte apoptosis, we compared the ability of catB+/+ and catB–/– hepatocytes to undergo apoptosis in response to TNF-α + AcD. To quantitate apoptosis, we monitored the nuclear changes by DAPI and the cellular externalization of phosphatidylserine by FITC-annexin V labeling. Both techniques yielded similar results (Figure 3a). Actinomycin D alone did not significantly increase the amount of apoptosis observed in untreated hepatocytes (Figure 3b). In contrast, TNF-α/AcD markedly enhanced catB+/+ hepatocyte apoptosis (Figure 3, a and b). An increased number of apoptotic cells were first detected between 4 and 8 hours after addition of TNF-α and AcD to catB+/+ hepatocytes and progressively increased with time until they comprised 59 ± 6% of the total cell population at 24 hours (Figure 3, a and b). TNF-α/AcD–induced apoptosis was reduced by almost half in catB−/− cells (35 ± 3% vs. 59 ± 6% after 24 hours of treatment; P < 0.05; Figure 3b). A similar decrease was observed when catB+/+ hepatocytes were treated with TNF-α/AcD in the presence of the highly selective cat B inhibitor, CA-074 (Figure 3b). To exclude the possibility that these results are due to a specific effect of actinomycin D in sensitizing cells to TNF-α (47), we performed similar experiments using expression of an IκB superrepressor to block NF-κB survival signals. Cells were infected with an adenovirus that expressed the superrepressor of IκB (Ad5IκB) or an empty control virus (Ad5ΔE1). Compared with controls, TNF-α–induced apoptosis was completely blocked in catB−/− cells (Figure 3c). These results suggest that TNF-α–induced apoptosis is dependent, in part, upon cat B activity.
catB–/– mouse hepatocytes are more resistant to TNF-α–induced apoptosis. Isolated hepatocytes from catB+/+ and catB–/– mice were incubated in the absence (control) or presence of TNF-α and AcD. (a) Apoptosis was quantitated in catB+/+ and catB–/– hepatocytes at different times of incubation after staining with both FITC-annexin V (dotted lines) and DAPI (solid lines). Cells were considered apoptotic if either externalization of phosphatidylserine residues on the plasma membrane or chromatin condensation and nuclear fragmentation occurred. At least 300 cells in six high-power fields were counted by an individual blinded to the experimental conditions. (b) Apoptosis was quantitated in catB+/+ and catB–/– hepatocytes by DAPI staining after 24 hours of incubation in medium lacking (control) or containing either TNF-α and AcD (TNF-α) or AcD alone (AcD). catB+/+ hepatocytes were also treated with TNF-α and AcD after a 30-minute preincubation with CA-074, a pharmacological inhibitor of cat B. Results are representative of at least three independent experiments using cells from three separate isolations and are expressed as mean ± SEM. Data were compared using a one-tail t test. A_P_ < 0.05, catB–/– vs. catB+/+; B_P_ < 0.05, catB+/+ + CA-074 vs. catB+/+. (c) Isolated mouse hepatocytes from catB+/+ (filled bars) and catB–/– (open bars) mice were infected with an adenovirus expressing the IκB-superrepressor (Ad5IκB) or with an empty adenovirus (Ad5ΔE1) as a negative control, and treated with TNF-α (28 ng/ml) for 12 hours. Apoptosis was quantitated after staining with DAPI. Results are representative of three independent experiments performed in triplicate from separate isolations and are expressed as mean ± SEM.
Does cat B deletion alter TNF-α/AcD–induced caspase activation in mouse hepatocytes? Current concepts implicate caspases as an integral part of the cell death machinery in death receptor–mediated apoptosis. The reduced rates of apoptosis in catB−/− hepatocytes suggested that caspase activation might be quantitatively or qualitatively altered by deletion of cat B. To address this possibility, we initially assessed the cleavage of several well-characterized substrates of downstream effector caspases, including PARP; lamins A, B1, and C; and the nucleolar protein B23, by immunoblot analysis. As shown in Figure 4, all of these substrates were completely cleaved in catB+/+ mouse hepatocytes after a 24-hour treatment with TNF-α/AcD (Figure 4, lane 2). In contrast, cleavage of these substrates was markedly diminished in hepatocytes from catB−/− mice (Figure 4, lane 4). These observations suggested that deletion of cat B causes a defect in TNF-α/AcD–induced activation of effector caspases.
Cleavage of PARP, lamins, and B23 after TNF-α/AcD treatment is diminished in catB–/– hepatocytes. Hepatocytes from catB+/+ and catB–/– mice were incubated for 24 hours with medium lacking or containing TNF-α + AcD. Whole-cell lysates were then prepared as described in Methods. Aliquots containing 50 μg protein were subjected to SDS-PAGE on gradient gels containing 5–15% acrylamide, transferred to nitrocellulose, and sequentially blotted for PARP, lamins A and C, lamin B1, or B23. Cleavage of the substrates was detected by the loss of the bands corresponding to the molecular weight of the native protein, and, in the case of B23, by the appearance of a new band (arrow). GRP78 served as a control for protein loading.
In some cell types, caspase-8–initiated apoptosis occurs by a process associated with mitochondrial release of cytochrome c and subsequent activation of caspase-9 (48). To determine whether TNF-α–induced apoptosis involves this pathway in mouse hepatocytes and assess whether cat B was upstream or downstream of mitochondria, the presence of cytosolic cytochrome c and activated caspase-9 was evaluated by immunoblotting. Increased cytochrome c was detectable in the cytosol of catB+/+ mouse hepatocytes beginning 6 hours after addition of TNF-α/AcD (Figure 5a, lane 2). In contrast, no cytochrome c was detectable in the cytosol of catB−/− cells after TNF-α/AcD treatment for up to 24 hours (Figure 5a, lanes 1–4). Consistent with these data, proteolytically activated caspase-9 (36) was readily detectable in treated catB+/+ hepatocytes but not catB−/− cells (Figure 5b, lanes 2 and 4). Likewise, activated caspase-3 was observed in catB+/+ mouse hepatocytes but not catB−/− cells (Figure 5b, lanes 2 and 4).
(a) TNF-α–induced release of cytochrome c into the cytosol is reduced in catB–/– mouse hepatocytes. At the indicated times after addition of medium lacking (control) or containing TNF-α + AcD, cytosol fractions were prepared by selective permeabilization with digitonin as described in Methods. Aliquots containing 20 μg of protein were subjected to SDS-PAGE on gels containing 15% acrylamide, transferred to nitrocellulose, and probed for cytochrome c. Samples from catB+/+ hepatocytes were also probed for cytochrome c oxidase, to exclude a possible mitochondrial contamination in the cytosol. (b) Caspase-9 and caspase-3 are processed in TNF-α/AcD–treated catB+/+ hepatocytes but not in catB–/– hepatocytes. Cells were incubated for 24 hours in medium lacking or containing TNF-α + AcD. After whole-cell lysates were prepared as described in Methods, aliquots containing 50 μg protein were subjected to SDS-PAGE, transferred to nitrocellulose membrane, and analyzed by immunoblot using antisera that recognize only active caspase-9 or active caspase-3 (36). The same blot was probed with sera that recognize procaspase-8 and β-actin to confirm loading and transfer of samples from catB–/– mice.
Taken together, the results in Figure 5 indicate that TNF-α/AcD–induced hepatocyte apoptosis involves mitochondrial release of cytochrome c and activation of caspase-9. These events are reminiscent of type II cells as defined by Scaffidi et al. (49, 50). In such cells, the initial activation of caspase-8 is limited and generally below the level of detection. Release of cytochrome c and activation of caspase-9 amplifies the original signal, leading to activation of downstream caspases (49, 50). Once these downstream caspases are activated, recent results indicate that they can in turn cleave more procaspase-8 (51). Thus, if TNF-α/AcD–treated hepatocytes were behaving like type II cells, we predicted that (a) the amount of activated caspase-8 would be limited until after caspase-9 was activated,and (b) catB–/– cells (which do not activate caspase-9) would not display detectable caspase-8 activation. Further experiments were performed to test these predictions.
Immunoblot analysis demonstrated that TNF-α/AcD treatment of catB+/+ hepatocytes resulted in a time-dependent processing of procaspase-8 (Figure 5b) into the catalytically active subunits of 18–20 kDa (p20) and 10 kDa (p10) (Figure 6). As predicted, detectable processing of procaspase-8 was not evident until 6–8 hours after addition of the cytokine (Figure 6, lanes 2 and 3). Thus, the amount of active caspase-8 required for detection by immunoblot analysis appears to be generated after mitochondrial release of cytochrome c (Figure 5a, lane 2), not by direct activation at the TNFR-1–associated signaling complex. Consistent with this conclusion, catB–/– hepatocytes, which did not release cytochrome c, did not display detectable caspase-8 processing (Figure 5b, lane 4; Figure 6, lanes 6–9).
Caspase-8 activation after TNF-α/AcD treatment is reduced in catB–/– cells. After cells were incubated in medium without (cont.) or with TNF-α + AcD for the indicated lengths of time, cytosolic fractions were prepared. Aliquots containing 40 μg protein were subjected to SDS-PAGE on gels containing 15% acrylamide, transferred to nitrocellulose, and immunoblotted for caspase-8. Processing of caspase-8 was detected by the appearance of the 18- to 20-kDa (p20) and 10-kDa (p10) active fragments. β-Actin served as a control for protein loading. Results are representative of three independent experiments.
Does cat B induce release of cytochrome c from mitochondria? The preceding results place cat B upstream of mitochondria in the TNF-α/AcD–induced apoptotic cascade in mouse hepatocytes. Although more complicated models are possible, the most direct model would have cat B or a substrate of cat B inducing cytochrome c release from mitochondria. To assess this possibility, purified cat B was incubated with isolated mitochondria in the presence and absence of cytosol. At the end of the incubation, the mitochondria were sedimented and the resulting supernatants analyzed for cytochrome c. In the absence of cytosol, active cat B directly induced a moderate release of cytochrome c from mitochondria (Figure 7a, compare lanes 1–3 with lane 7) corresponding to about 8% of the maximum release as obtained treating the mitochondria with the detergent Triton X-100 (Figure 7a, lane 9). However, cytochrome _c_–releasing activity of cat B was fivefold greater in the presence of cytosol (Figure 7a, lanes 4–6). Addition of calpain to the cell-free system did not induce release of cytochrome c from mitochondria, demonstrating that the release is due to cat B activity and not a nonspecific proteolytic effect (Figure 7b). To rule out the possibility that mitochondria from catB–/– might have developed an increased resistance toward cytochrome c release, we performed the same experiments using both mitochondria and cytosolic extracts obtained from catB–/– hepatocytes. As previously observed in catB+/+, active cat B induced release of cytochrome c from the mitochondria in the presence of cytosol (Figure 7c, lane 3), demonstrating that the absence of cytochrome c release in catB–/– is not due to secondary modifications in the mitochondria of these animals. These data imply the existence of a cytosolic cat B substrate that, after proteolytic activation, is capable of causing release of cytochrome c from mitochondria.
Cat B–induced release of cytochrome c from mitochondria is enhanced by cytosol and is not due to a nonspecific proteolytic effect. Isolated mitochondria from catB+/+ mouse liver (25 μg protein) were incubated at 37°C with increasing concentrations of purified recombinant cat B (5–50 ng) (a) or m-calpain (10 ng) (b), in the presence or in the absence of S-100 cytosol fraction (50 μg) as described in Methods. Mitochondria were also treated with 0.1% Triton X-100 to induce maximum release of cytochrome c (positive control). After 1 hour, mitochondria were pelleted by centrifugation at 12,000 g for 5 min. Supernatants were subjected to SDS-PAGE on 15% acrylamide gels, transferred to nitrocellulose, and immunoblotted for cytochrome c. Blots were also probed for cytochrome c oxidase (subunit IV) to exclude mitochondria contamination in the supernatant. (c) Active cat B induces release of cytochrome c from isolated catB–/– mouse liver mitochondria in the presence of cytosol. Isolated mitochondria from catB–/– mouse liver (25 μg protein) were incubated at 37°C with purified recombinant cat B (25 ng), in the presence or in the absence of S-100 cytosol fraction (50 μg) obtained from the same animal. Mitochondria were also treated with 0.1% Triton X-100 to induce maximum release of cytochrome c (positive control). After 1 hour, mitochondria were pelleted by centrifugation at 12,000 g for 5 minutes, and the resulting supernatants were subjected to SDS-PAGE and subsequent immunoblot analysis for cytochrome c as already above. Immunoblot for cytochrome c oxidase (subunit IV) was also performed to exclude mitochondria contamination in the supernatant.
Are catB−/− mice resistant to TNF-α–induced liver damage in vivo? catB+/+ and catB−/− mice were injected with the adenovirus Ad5IκB to block the TNF-α–survival pathways signaling through NF-κB. Twenty-four hours later, TNF-α was administered intravenously. At 2 and 4 hours after TNF-α treatment, serum ALT concentrations were significantly higher in catB+/+ than in catB−/− animals (Figure 8a). Livers from the catB+/+ mice displayed extensive hemorrhagic lesions and numerous clusters of apoptotic cells. In contrast, liver specimens from the catB−/− mice showed minimal damage (Figure 8b). Further studies revealed that TNF-α treatment of the catB+/+ mice was fatal in greater than 80% of animals at 5 hours (n = 6), whereas catB−/− mice universally survived at least 72 hours after TNF-α treatment (n = 4).
catB–/– mice are more resistant to TNF-α–induced liver damage. catB–/– and catB+/+ were injected via tail vein with the adenovirus Ad5IκB (0.35 × 109 pfu/mouse) encoding for an IκB superrepressor. In control experiments, mice were injected with the adenovirus Ad5ΔE1 (0.35 × 109 pfu/mouse in 0.22 ml sterile saline) or with sterile saline (0.22 ml).Twenty-four hours later, each mouse received a dose of 0.5 μg of recombinant mouse TNF-α intravenously. Mice were sacrificed after 2- and 4-hour treatment with TNF-α. (a) Serum alanine aminotransferase (ALT) levels were measured and expressed as mean ± SEM (n = 3). A_P_ < 0.01. ALT values in control samples were < 20 IU/L, except in the Ad5ΔE1-injected mice, in which they were < 750 IU/L at 2 hours and < 1850 IU/L at 4 hours (data not shown). (b) H&E staining of Ad5IκB-injected catB+/+ (left) and catB–/– (right) mouse liver harvested 4 hours after treatment with TNF-α.