TNF-
-mediated lysosomal permeabilization is FAN and caspase 8/Bid dependent
Nate Werneburg,1
M. Eugenia Guicciardi,1
Xiao-Ming Yin,2 and
Gregory J. Gores1
1Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905; and 2Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260
Submitted 15 January 2004
; accepted in final form 29 March 2004
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ABSTRACT
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TNF-
cytotoxic signaling involves lysosomal permeabilization with release of the lysosomal protease cathepsin B (ctsb) into the cytosol. However, the mechanisms mediating lysosomal breakdown remain unclear. Because caspase-8 and factor associated with neutral sphingomyelinase activation (FAN) have been implicated as proximal mediators of TNF-
-associated apoptosis, their role in lysosomal permeabilization was examined. Cellular distribution of ctsb-green fluorescent protein (ctsb-GFP) in a rat hepatoma cell line was imaged by confocal microscopy. ctsb-GFP fluorescence was punctate under basal conditions but became diffuse after treatment with TNF-
/actinomycin D. This cellular redistribution of ctsb-GFP was blocked by transfection with a vector expressing a dominant-negative Fas-associated protein with death domain (
FADD), cytokine response modifier A, or a pharmacological caspase-8 inhibitor, IETD-fmk. Consistent with the concept that caspase 8-mediated apoptosis is also Bid-dependent in hepatocytes, ctsb-GFP release from lysosomes was reduced in hepatocytes from Bid/ mice. Interestingly, transfection with a vector expressing a dominant-negative FAN (
FAN) also blocked ctsb-GFP release and caspase-8 activation. Paradigms that inhibited ctsb-GFP release from lysosomes also reduced apoptosis as assessed by morphology and biochemical criteria. In conclusion, these studies suggest FAN is upstream of caspase-8/Bid in a signaling cascade culminating in lysosomal permeabilization.
cathepsin B; dominant-negative Fas-associated protein with death domain; dominant-negative factor associated with neutral sphingomyelinase activation; green fluorescent protein
TNF-
IS A PLEIOTROPIC CYTOKINE that has been implicated in both liver regeneration and cytotoxicity (27, 29). In regards to liver injury, TNF-
contributes to liver injury in alcoholic liver disease, ischemia-reperfusion injury, acute liver failure of viral etiology, and cholestatic liver diseases (3, 19, 21). Cytotoxic signaling by this ligand is mediated by the TNF receptor-1, which contains a death domain in its cytoplasmic tail (2, 24). Although both oncotic necrosis and apoptosis may be induced after TNF-
binding to this receptor, the cascades culminating in apoptosis appear to predominate (25).
Cell signaling after TNF-R1 oligomerization by its cognate ligand is extremely complex. Indeed, this receptor complex first appears to recruit TNF receptor-associated protein with death domain (TRADD) to its cytoplasmic tail via homotypic interactions between death domains present in both proteins (24, 27). TRADD then recruits receptor-interacting protein (RIP) and TNF receptor-associated factor-2 (TRAF-2) to this membrane-associated complex (27). RIP and TRAF-2 are involved in activating NF-
B, a transcription factor that induces expression of cytoprotective molecules (27). Thereafter, TRADD, RIP, and TRAF-2 leave the receptor complex and within the cytosol recruit Fas-associated protein with death domain (FADD) and procaspase-8 to a cytotoxic signaling complex. In this cytoplasmic complex, caspase-8 is activated by homodimerization of recruited procaspase monomers (12). Active caspase-8 initiates apoptotic cascades via cleavage of Bid, a proapoptotic Bcl-2 homology domain 3 (BH3)-only member of the Bcl-2 family of proteins (15). Another receptor-binding protein is factor associated with neutral sphingomyelinase activation (FAN). Binding of FAN to the TNF-R1 receptor has been associated with ceramide and sphingosine generation, which have also been implicated in cell death signaling (4, 23). Finally, activation of JNK also contributes to cell death during TNF-R1-initiated signaling (16). JNK activation requires TRAF-2, but the further downstream events leading to its activation require the coordinated involvement of several kinases (5). Thus multiple proximal signaling events have been identified in TNF-R1-mediated apoptosis.
The downstream processes culminating in TNF-R1 cytotoxic signaling are as complex as the initiating proximal events and involve the generation of oxygen radicals, mitochondrial dysfunction, caspase cascades, and lysosomal permeabilization. In hepatocytes treated with TNF-R1, lysosomal permeabilization with release of cathepsin B (ctsb), a cysteine protease, into the cytosol has been associated with subsequent mitochondrial dysfunction and caspase-dependent cell death (10, 28). Although ctsb has been shown to participate in hepatocyte apoptosis by TNF-
both in vitro and in vivo (10, 11), the cellular mechanisms responsible for lysosomal permeabilization by TNF-R1 signaling remain incompletely understood. Further insight into the mechanism of lysosomal permeabilization in apoptosis are of scientific and biomedical importance, given the role of this process in liver injury by TNF-
.
The overall objective of this proposal was to further identify the mechanisms by which TNF-
causes lysosomal destabilization. To address this objective, we formulated several questions as follows: 1) Is lysosomal permeabilization caspase-8/Bid and/or FAN dependent? 2) Is there a relationship between blocking lysosomal ctsb release and apoptosis in these paradigms? and 3) Are the signaling pathways implicated in lysosomal permeabilization linked in a mechanistic scheme? The results of this study implicate both FAN and caspase-8 in lysosomal breakdown. FAN appears to function upstream of caspase-8 activation. These observations have implications for the attenuation of TNF-
-mediated liver injury and provide further insight into TNF-R1 cytotoxic signaling.
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MATERIALS AND METHODS
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Cell line and culture.
McNtcp.24 cells, a rat hepatoma cell line, were grown in DMEM supplemented with 10% fetal bovine serum, 100,000 U/l penicillin, 100 mg/l streptomycin, and 100 mg/l gentamycin. This cell line was selected because of its high transfection efficiency [
70% with green fluorescent protein (GFP)]. Bid knock-out (Bid/) mice were generated as previously described (30). Wild-type mice with identical genetic background were used as controls. Animals were cared for using protocols approved by the Mayo Clinic Institutional Animal Care and Use Committee. Mouse hepatocytes were isolated and cultured as previously described in detail by us (7). When cells were treated with TNF-
(28 ng/ml), actinomycin D (AcD; 0.2 µg/ml) was included in the medium to block NF-
B-mediated transcription of cytoprotective genes. AcD is necessary for lysosomal permeabilization, as recent studies have demonstrated (17), showing that NF-
B induces the transcription of the ctsb inhibitor Spi2A. Induction of this inhibitor prevents lysosomal permeabilization.
ctsb-Red fluorescent protein plasmid construction.
The rat ctsb-GFP expression vector described previously (22) was restriction-enzyme digested by using HindIII and BamHI (GIBCO-BRL, Gaithersburg, MD). The extracted cDNA fragment and cut vector were separated by electrophoresis on a 1% agarose gel, and the ctsb insert was purified by using the QIAquick gel extraction kit (Qiagen, Chatsworth, CA). The pDsRed1-N1 fluorescent expression vector from CLONTECH (Palo Alto, CA) and the purified ctsb insert were both cut with the restriction enzymes EcoRI and SmaI (GIBCO-BRL). The cut pDsRed1-N1 vector was treated with calf intestine alkaline phosphatase (Boehringer-Mannheim, Indianapolis IN) to remove the terminal phosphate groups and prevent self-ligation. The purified ctsb cDNA fragment was then ligated into the expression vector using T4 DNA Ligase (Roche Diagnostics, Mannheim, Germany) at 4°C for 16 h. One microliter of the ligation reaction was transformed employing One ShotTOP10 competent cells (Invitrogen, Carlsbad, CA). Transformed competent cells were plated on selective agar plates; colonies were selected and expanded in CIRCLEGROW medium (Bio 101, Carlsbad, CA) containing 30 µg/ml kanamycin. DNA for transfection was purified by using Qiagen Endofree Plasmid DNA Maxikit (Qiagen). To ensure that the plasmid contained the insert, the DNA was restriction digested with EcoRI and SmaI (GIBCO-BRL) and the digested plasmid resolved on a 1% agarose gel to check for the appropriate molecular weight product.
Truncated FAN (
FAN-GFP) plasmid construction.
Primers were designed to produce a truncated form of FAN; the forward primer (5'-GTC CAT GGT ACC ATG GCA TTT GGA AGA CGC CAA G-3') contains a Kpn1 restriction enzyme site and ATG start codon. The reverse primer (5'-GCA CCC GGT ACC TGA TGG AGA GGA AAA GGC ACT-3') contains a Kpn1 restriction site. PCR was performed, and the 689-bp product was separated on a 1% agarose gel and further purified by using the QIAquick gel extraction kit (Qiagen). Both the purified FAN insert and pcDNA3 vector were digested with the Kpn1 restriction enzyme (GIBCO-BRL). The linearized pcDNA3 plasmid was treated with calf intestine alkaline phosphatase (Boehringer-Mannheim). The Kpn1 digested FAN cDNA fragment was then ligated into the pcDNA3 vector using T4 DNA Ligase (Roche Diagnostics) at 4°C for 16 h. One microliter of the ligation reaction was transformed employing One ShotTOP10 competent cells (Invitrogen). Transformed competent cells were plated on selective agar plates, and colonies were selected and expanded in CIRCLEGROW medium (Bio 101) containing 30 µg/ml ampicillin. Vector and insert were sequenced to ensure that FAN insert was in the proper orientation. pEGFP-N1 (CLONTECH) and DN-FAN-pcDNA3 were digested with HindIII and BamH1 (GIBCO). The GFP expression vector was then treated with calf intestine alkaline phosphatase (Boehringer-Mannheim). The restriction enzyme treated DN-FAN-pcDNA3 vector was separated on a 1% agarose gel and the DN-FAN insert was gel purified by using the QIAquick gel extraction kit (Qiagen). The purified DN-FAN insert and GFP vector were ligated by using T4 DNA Ligase (Roche Diagnostics) at 4°C for 16 h. One microliter of the ligation reaction was transformed employing One ShotTOP10 competent cells (Invitrogen). Transformed competent cells were plated on selective agar plates, and colonies were selected and expanded in CIRCLEGROW medium (Bio 101) containing 30 µg/ml kanamycin. DNA for transfection was purified by using Qiagen Endofree Plasmid DNA Maxikit (Qiagen). To ensure that the plasmid contained the correct insert, the DNA was restriction digested with HindIII and BamHI (GIBCO-BRL) and the digested plasmid products were separated on a 1% agarose gel to check for the appropriate molecular weight insert.
ctsb-GFP, ctsb-red fluorescent protein, GFP-truncated FADD,
FAN-GFP and Bid-GFP transfection, LysoTracker Red loading, and fluorescence visualization with confocal microscopy.
The Bid-GFP and GFP-truncated FADD (GFP-
FADD) transfection plasmids have been characterized and described (13, 26). The ctsb-GFP, ctsb-red fluorescent protein (ctsb-RFP), Bid-GFP and GFP-
FADD (7), and
FAN-GFP were transfected into McNtcp.24 cells using Lipofectamine Plus (Invitrogen). Cotransfections with ctsb-RFP and either GFP-
FADD or
FAN-GFP were performed with 1 ml of OptiMEM-1 containing 6 µl Plus reagent, 1 µg/ml ctsb-RFP and GFP-
FADD or
FAN-GFP cDNA, and 6 µl/ml lipofectamine reagent, following the manufacturer's instructions. Confocal microscopy was performed with an inverted Zeiss laser scanning confocal microscope (model LSM S10; Carl Zeiss, Thornwood, NJ), using excitation and emission wavelengths of 488 and 507 nm for GFP and 558 and 583 nm for RFP, respectively. LysoTracker Red DND-99 (Molecular Probes, Eugene, OR) was loaded into primary mouse hepatocytes by incubating the cells in probe-containing medium at a final concentration of 50 nM for 1 h at 37°C. The cellular distribution of LysoTracker Red was visualized by using confocal microscopy at excitation and emission wavelengths of 577 and 590, respectively.
Morphological quantitation of apoptosis.
Apoptosis was quantitated by assessing the characteristic nuclear changes of apoptosis (i.e., chromatin condensation and nuclear fragmentation) using the nuclear binding dye 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma, St. Louis, MO) and fluorescence microscopy using excitation and emission filters of 380 and 430 nm, respectively.
Measurement of caspase-3/-7 activity and caspase-8-like activity.
Measurement of caspase-3/-7 activity in cell cultures was performed by using the Apo-ONE homogeneous caspase-3/-7 assay (Promega, Madison, WI) following the manufacturer's instructions.
For caspase-8-like activity, cells were cultured on 35-mm glass bottom microwell dishes (Mattek Ashland, MA) at a density of 1,000 cells/dish. Intracellular caspase-8-like activity was measured in single cells using the fluorophore-tagged caspase-8 inhibitor, carboxyfluorescein-IETD-fmk (CaspaTag; Intergen, Purchase, NY). Briefly, the commercially supplied lyophilized compound was reconstituted into a 150x stock with DMSO and further diluted into a 30x working solution with PBS. Before the experiment, 10 µl of this solution were added to 290 µl PBS for each dish. The DMEM medium was gently aspirated and replaced with 300 µl of the fluorophore-containing buffer. Cells were incubated at 37°C for 1 h, washed twice with the supplied working solution wash buffer, and mounted on the microscope stage of an inverted fluorescent microscope (Axiovert 35M; Carl Zeiss, Thornwood, NY). Fluorescence was visualized by using excitation and emission wavelengths of 490 and 520 nm, respectively. Digital images were captured by using a cooled charge-coupling device camera (model KAF-1400; Photometrics, Tucson, AZ) and then analyzed and quantitated by using fluorescent imaging software (Metafluor Imaging System, Universal Imaging, West Chester PA). Fluorescence intensity for individual cells was quantitated and the average fluorescent intensity determined for 100 cells per experimental group.
Reagents.
Human recombinant TNF-
and AcD were obtained from Sigma, and the caspase inhibitors IETD-fmk and z-VAD-fmk were from Enzyme Systems Products (Livermore, CA). TNF-related apoptosis-inducing ligand (TRAIL) was obtained from R&D Systems (Minneapolis, MN).
Statistical analysis.
All data represent at least three independent experiments and are expressed as the means ± SD unless otherwise indicated. Differences between groups were compared by using ANOVA for repeated measures and a post hoc Bonferroni test to correct for multiple comparisons.
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RESULTS
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Does TNF-
/AcD-induced ctsb release and apoptosis occur in a FADD/caspase-8-dependent manner?
McNtcp.24 cells were transfected with ctsb-RFP and either cotransfected with GFP-
FADD or pretreated with IETD-fmk, a selective pharmacological caspase-8 inhibitor. The truncated GFP-
FADD possesses a death domain, but lacks death effector domains, and as such, binds to TRADD but blocks the recruitment of procaspase-8 to the protein complex functioning as a dominant-negative protein preventing caspase-8 activation (26). Cells transfected with ctsb-RFP displayed punctate fluorescence consistent with lysosomal localization (Fig. 1); indeed, we have previously reported that fluorescent ctsb proteins colocalize with lysosomal-associated membrane protein-1 confirming the localization of the protein in lysosomes (28). In contrast, cells transfected with ctsb-RFP and treated with TNF-
/AcD manifested diffuse fluorescence consistent with a redistribution of the protease from lysosomes to the cytosol (Fig. 1). Cells either cotransfected with GFP-
FADD or pretreated with IETD-fmk displayed predominantly punctate fluorescence despite treatment with TNF-
/AcD, indicating that these maneuvers prevented ctsb cellular redistribution (Fig. 1A). Because IETD-fmk may also inhibit other caspases beside caspase-8, cells were also transfected with CrmA, a highly selective caspase-1 and -8 inhibitor, to ascertain whether it also blocked lysosomal release of ctsb. Consistent with the IETD-fmk observations, transfection with CrmA also blocked lysosomal release of ctsb (Fig. 1A). Quantitation of punctate vs. diffusely fluorescent (Fig. 1B) cells demonstrated a dramatic reduction in cells with diffuse fluorescence in the GFP-
FADD- or CrmA-transfected cells, and also in the IETD-fmk-treated groups compared with cells treated with TNF-
/AcD alone. Consistent with their ability to prevent ctsb release from lysosomes into the cytosol, TNF-
-induced apoptosis was also markedly decreased by transfection with GFP-
FADD or CrmA, and in cells pretreated with IETD-fmk, compared with cells treated with TNF-
/AcD alone (Fig. 2A). The occurrence of apoptosis was also assessed biochemically by determining downstream caspase-3/-7 activity (Fig. 2B). Caspase-3/-7 activity was also reduced by transfection with GFP-
FADD or CrmA and in cells treated with IETD-fmk (Fig. 2B). The decrease in ctsb release and apoptosis caused by GFP-
FADD, CrmA, and IETD-fmk treatment suggests that TNF-
/AcD-associated lysosomal permeabilization is, in part, FADD/caspase-8-dependent.
Because death receptor-mediated, caspase-8-associated apoptosis in hepatocytes is Bid-dependent (15, 18), the role of Bid in lysosomal permeabilization was examined. LysoTracker Red, a dye that labels acidic vesicles in intact cells, was employed to assess lysosomal integrity in primary mouse hepatocytes. Consistent with our previously published observations, LysoTracker Red was released from acidic vesicles in wild-type mouse hepatocytes (28) but not in hepatocytes isolated form Bid-deficient animals (Fig. 3, A and B). These data place Bid upstream of TNF-R1-associated lysosomal destabilization. Because Bid-mediated mitochondrial permeabilization is associated with its translocation to this organelle, experiments were performed to determine whether Bid also translocates to acidic vesicles. McNtcp.24 cells were transfected with Bid-GFP and acidic vesicles loaded with LysoTracker Red. Bid-GFP fluorescence was heterogeneous in transfected cells but did not colocalize with acidic vesicles under basal conditions. In contrast, Bid-GFP was associated with acidic vesicles in TNF-
/AcD-treated cells (Fig. 4). These data suggest that caspase-8-mediated Bid activation is upstream and contributes to lysosomal destabilization after TNF-
/AcD treatment of hepatocytes.
Is FAN involved in TNF-
/AcD-induced ctsb release from lysosomes and apoptosis?
To determine whether FAN is involved in TNF-
/AcD-induced ctsb release, cells were cotransfected with ctsb-RFP and
FAN-GFP. NH2-terminal truncations of FAN, which only contain the TNF-R1 COOH-terminal binding region of the protein and functions as a dominant-negative protein (1). Transfected cells were treated with TNF-
/AcD and imaged by confocal microscopy. Cells that were cotransfected with Ctsb-RFP and
FAN-GFP displayed punctate fluorescence similar to control cells (Fig. 5, A and B). Quantitation of punctate vs. diffusely fluorescent cells demonstrated a dramatic reduction in cells with diffuse fluorescence in the
FAN-GFP-transfected cells compared with cells treated with TNF-
/AcD alone. Consistent with its ability to prevent ctsb-RFP release from lysosomes into the cytosol, TNF-
-induced apoptosis was also markedly decreased by transfection with
FAN-GFP after treatment with TNF-
/AcD (Fig, 6, A and B). These data suggest FAN is also a requisite for TNF-
signaling through the lysosomal pathway.
Does
FAN-GFP block caspase-8 activation?
Collectively, the above observations implicated both caspase-8 activation and FAN in lysosomal permeabilization after TNF-
/AcD treatment of these cells. However, it was unclear whether these proteins acted in parallel processes converging on lysosomes or whether FAN was necessary for caspase-8 activation. To address this question, caspase-8 catalytic activity was quantitated fluorescently in cells treated with TNF-
/AcD. Measurement of caspase-8 activity was deemed more appropriate than performing relatively insensitive immunoblot analysis for caspase-8 cleavage products, as minimal caspase-8 activity may be required for lysosomal permeabilization. Furthermore, only procaspase-8 homodimerization is necessary for caspase-8 activity within the initial protein complexes (6). Caspase-8-like fluorescent activity was readily observed in TNF-
/AcD-treated cells. This activity was eliminated by IETD-fmk, suggesting that predominantly caspase-8 activity was being measured (data not shown). Transfection with
FAN-GFP also blocked the TNF-
/AcD-associated increase in caspase 8-like activity (Fig. 7). In contrast, transfection with
FAN-GFP did not block apoptosis by TRAIL (100 ng/ml) in the presence of AcD. After 16 h of incubation, the percentage of transfected cells undergoing apoptosis was virtually identical to the one of untransfected cells (Fig. 8). Because TRAIL cytotoxic signaling also requires FADD and caspase-8, but not FAN, these observations indicate that
FAN-GFP does not directly interfere with FADD and/or caspase-8 activities. Collectively, these data suggest FAN recruitment to the TNF-R1 receptor is also required for subsequent caspase-8 activation.
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DISCUSSION
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The principal findings of this study relate to TNF-
cytotoxic signaling involving lysosomes. The results demonstrate that during treatment of a rodent hepatoma cell line with TNF-
/AcD: 1) redistribution of ctsb-GFP from lysosomes into the cytosol is blocked by inhibiting caspase-8 activation or employing a
FAN; 2) genetic deletion of Bid reduces lysosomal permeabilization as assessed by cellular compartmentation of LysoTracker Red; 3) inhibition of lysosomal ctsb release by these maneuvers also is associated with a reduction in cellular apoptosis; and 4) transfection with a
FAN also blocks caspase-8 activation. Taken together, these findings suggest FAN, in concert with caspase-8, contributes to an intracellular signaling cascade resulting in lysosomal permeabilization. Each of these findings and their implications are discussed below.
Loss of lysosomal integrity with subsequent activation of proapoptotic cascades after TNF-
-mediated cytotoxicity has been amply documented (8, 10, 28). Despite its contribution to TNF-
-mediated apoptosis, the mechanisms of lysosomal breakdown remain incompletely understood. Lysosomal permeabilization requires, in part, the intralysosomal protease ctsb (28), an observation confirmed by the finding that NF-
B upregulation of serine protease inhibitor 2A (Spi2A), a potent inhibitor of ctsb, also reduces lysosomal breakdown and apoptosis (17). Because ctsb is thought to be constitutively active within lysosomes, it must require an extralysosomal trigger to promote its own release. Previous studies (10, 14, 28) have implicated caspase-8 and sphingosine as upstream mediators of lysosomal destabilization during TNF-
cytotoxicity. The current data extend these observations by further examining the roles of caspase-8 activation and FAN, a protein necessary for neutral sphingomyelinase-mediated generation of ceramide and therefore sphingosine formation (1), in TNF-
-mediated lysosomal permeabilization. Consistent with prior observations that caspase-8 is necessary for TNF-
-mediated apoptosis, inhibition of caspase-8 markedly reduced lysosomal release of ctsb. However,
FAN was equally effective in this regard. These seemingly disparate observations were reconciled by demonstrating that
FAN also reduces caspase-8 activation. Current models of TNF-
/TNF-R1 signaling suggest caspase-8 activation occurs in a protein complex remote from the receptor (12, 20). Perhaps FAN and/or lipid products downstream of neutral sphingomyelinase activation after FAN's recruitment to the receptor complex are responsible for separation of the TRADD/RIP/TRAF2 complex from the membrane receptor. This disengaging event is necessary for FADD and caspase-8 recruitment to these proteins, and therefore for caspase-8 activation. Further studies are necessary to define the role of FAN in caspase-8 activation.
How caspase-8 activation results in lysosomal permeabilization is of interest. Cleavage of Bid (a proapoptotic, BH3-only member of the Bcl-2 family) by caspase-8 generates truncated Bid (tBid), which translocates to mitochondria and induces release of cytochrome c (15, 18). In hepatocytes, mitochondrial dysfunction by death receptors is Bid-dependent (30). In the present study, lysosomal permeabilization after TNF-R1 ligation was also Bid-dependent and Bid association with acidic vesicles was observed after TNF-
/AcD exposure. Together, these data suggest truncated Bid cleavage may also promote lysosomal destabilization as well as mitochondrial dysfunction. How tBid results in membrane organelle permeabilization is unclear, but maybe simpler to study in single membrane vesicles, such as lysosomes, compared with mitochondria.
The degree of apoptosis suppression seen with
FAN in the present study in this hepatoma cell line (almost 100%) is much greater than that seen with inhibition of ctsb in mouse hepatocytes (
40%). This suggests that FAN may control more than just the lysosomal apoptotic pathway. Indeed, FAN recruitment to the TNF-R1 complex is essential for activation of neutral sphingomyelinase, which is critical for ceramide production by TNF-
(23). Ceramide has been implicated as a trigger for the mitochondrial pathway of apoptosis independent of caspase-8 (9). Thus the ability of
FAN to inhibit apoptosis more strongly than genetic or pharmacological inhibition of ctsb is consistent with its ability to inhibit multiple cytotoxic signaling events.
In summary, these studies further elucidate the cellular mechanisms responsible for activation of the lysosomal pathway by the TNF-
/TNF-R1 signaling complex. The results support a model in which caspase-8 activation and subsequent activation of Bid are necessary for lysosomal permeabilization. Interestingly, these data also implicate FAN, for the first time in this event, a protein that appears to function upstream of caspase-8 activation. Targeting either caspase-8 or FAN would appear to be viable strategies to block activation of this pathway in human liver diseases in which TNF-
plays a critical role in tissue injury. These concepts merit further investigation especially as they relate to in vivo liver injury.
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GRANTS
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This work was supported by National Institutes of Health Grants DK-63947 (to G. J. Gores) and CA-83817 (to X.-M. Yin) and the Mayo and Palumbo Foundations.
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ACKNOWLEDGMENTS
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The secretarial service of Erin Bungum is gratefully acknowledged.
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FOOTNOTES
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Address for reprint requests and other correspondence: G. J. Gores, Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905 (E-mail: gores.gregory{at}mayo.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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