Division of Gastroenterology and Hepatology, Mayo Medical School, Clinic, and Foundation, Rochester, Minnesota 55905
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ABSTRACT |
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Cathepsin B
(Cat B) is released from lysososomes during tumor necrosis factor-
(TNF-
) cytotoxic signaling in hepatocytes and contributes to cell
death. Sphingosine has recently been implicated in lysosomal
permeabilization and is increased in the liver by TNF-
. Thus the
aims of this study were to examine the mechanisms involved in
TNF-
-associated lysosomal permeabilization, especially the role of
sphingosine. Confocal microscopy demonstrated Cat B-green fluorescent
protein and LysoTracker Red were both released from lysosomes after
treatment of McNtcp.24 cells with TNF-
/actinomycin D, a finding
compatible with lysosomal destabilization. In contrast, endosomes
labeled with Texas Red dextran remained intact, suggesting lysosomes were specifically targeted for permeabilization. LysoTracker Red was released from lysosomes in hepatocytes treated with TNF-
or
sphingosine in Cat B(+/+) but not Cat B(
/
) hepatocytes, as assessed
by a fluorescence-based assay. With the use of a calcein release assay
in isolated lysosomes, sphingosine permeabilized liver lysosomes
isolated from Cat B(+/+) but not Cat B(
/
) liver. C6
ceramide did not permeabilize lysosomes. In conclusion, these data
implicate a sphingosine-Cat B interaction inducing lysosomal destabilization during TNF-
cytotoxic signaling.
calcein release assay; cathepsin B-green fluorescence protein; LysoTracker Red; sphingosine
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INTRODUCTION |
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TUMOR NECROSIS
FACTOR- (TNF-
) is a pleiotropic cytokine signaling such
complex and diverse processes as apoptosis, cell growth, and
proinflammatory gene expression (16, 40). The specific
consequences of TNF-
signaling depend on the cell type and cellular
context. For example, in the liver, TNF-
is important in liver
regeneration after a partial hepatic resection and as a cytotoxic agent
in a variety of disease processes (2). TNF-
-mediated cytotoxicity is of considerable importance and is mediated, in part, by
its ability to induce apoptosis. Indeed, anti-TNF-
therapy is currently used in the treatment of human disease (17).
The TNF-
-initiated intracellular cascades interacting to induce cell demise are, therefore, of broad clinical and scientific interest.
TNF- induces apoptosis by oligomerizing the TNFR-1 receptor
(40). The aggregation of this receptor results in the
recruitment of the adaptor protein TRADD to the receptor complex. TRADD
via homotypic interactions between common death domains recruits FADD, which in turn binds procaspase 8. Procaspase 8 undergoes autocatalytic activation through an induced proximity mechanism (34).
Caspase 8 is essential for TNF-
-mediated apoptosis in
fibroblasts (39). This initiator caspase appears to
commence apoptotic cascades via several mechanisms. It may directly
cleave procaspase 3, resulting in activation of this effector caspase,
resulting in apoptosis (26). Also, caspase 8 can
cleave Bid, a BH3 domain-only member of the Bcl-2 protein family
(11, 22). The truncated Bid translocates to mitochondria,
inducing cytochrome c release. Cytosolic cytochrome c forms a complex with Apaf-1 and procaspase 9, resulting in
activation of this initiator caspase (21). This pathway is
referred to as the mitochondrial pathway and is mediated by caspase
9-induced activation of caspase 3 (10). More recently, we
and others (9, 12) have shown that TNF-
-associated
cytotoxic signaling also results in permeabilization of lysosomes
releasing cathepsin B (Cat B) in the cytosol. Cat B then initiates the
mitochondrial pathway of apoptosis (12, 13).
Indeed, TNF-
-mediated apoptosis is markedly attenuated by
alkalinizing acidic vesicles, employing Cat B inhibitors, and in Cat B
knockout mice (9, 12, 13, 23). Cat B gene-deleted animals
are also resistant to TNF-
-mediated liver injury; this in vivo
observation highlights the dominant role of this lysosomal/Cat B
pathway in TNF-
-mediated liver injury (13). Further
information on this pathway will help provide key insights into
TNF-
-mediated signaling and may potentially lead to additional
therapeutic strategies to reduce TNF-
-associated tissue injury.
There are several unresolved issues regarding the lysosomal/Cat B
pathway of TNF--mediated apoptosis. It is unclear whether the vesicle permeabilization is specific for lysosomes or if other vesicles also break down. Likewise, it is unknown if all lysosomes or
if a subpopulation of lysosomes (killer lysosomes) is permeabilized. Finally, the mechanisms of lysosomal permeabilization are unclear. We
addressed these questions using complementary morphological and
biochemical approaches. The results suggest that vesicle
permeabilization is selective for lysosomes, may occur in a
subpopulation of lysosomes, and is, in part, Cat B dependent.
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EXPERIMENTAL PROCEDURES |
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Isolation and culture of mouse hepatocytes and culture of
McNtcp.24 cells.
Cat B knockout [catB(/
)] mice were generated as
reported previously (30). Animals were cared for using
protocols approved by the Mayo Clinic Institutional Animal Care and Use
Committee. Mouse hepatocytes were isolated and cultured as described by
us in detail previously (6). The rat hepatoma McNtcp.24
cell line was cultured as described previously (19). When
cells were treated with TNF-
(28 ng/ml), actinomycin D (AcD, 0.2 µg/ml) was included in the medium to block the NF-
B-mediated
transcription of cytoprotective TNF-
-induced genes.
Lysosome-associated membrane protein-2-cyan fluorescent protein plasmid construction. Both lysosome-associated membrane protein 2-cyan fluorescent protein (LAMP-2-CFP, a generous gift from Dr. M. McNiven, Mayo Clinic), a precise marker for the lysosomal compartment, and pECFP-N1 (Clontech Laboratories, Palo Alto, CA) were cut using EcoR I and Xho I (GIBCO-BRL, Gaithersburg, MD). Both the cDNA fragment and cut vector were separated by electrophoresis on a 1% agarose gel and purified using the QIAquick gel extraction kit (Qiagen, Chatsworth, CA). The cut pECFP-N1 was treated with calf intestine alkaline phosphatase (Boehringer-Mannheim, Indianapolis, IN) to remove the terminal phosphate groups and prevent self-ligation. The purified LAMP-2 cDNA fragment was then ligated in 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 Shot TOP10 competent cells (Invitrogen, Carlsbad, CA). Transformed competent cells were plated on selective agar plates, and colonies were selected and grown up in CIRCLEGROW media (Bio 101, Carlsbad, CA) containing 30 µg/ml kanamycin. DNA for transfection was purified using the Qiagen Endofree Plasmid DNA Maxikit (Qiagen).
Cat B-green fluorescent protein, LAMP-2-CFP transfection, and confocal microscopy. The rat catB-green fluorescent protein (GFP) expression vector described previously (33) and LAMP-2-CFP were transfected into McNtcp.24 cells using Lipofectamine Plus (Invitrogen). Cotransfection with catB-GFP and LAMP-2-CFP was performed with 1 ml of OptiMEM-1 containing 6 µl Plus reagent, 1 µg/ml catB-GFP and LAMP-2-CFP cDNA, and 6 µl/ml lipofectamine reagent, following the manufacturer's instructions. Confocal microscopy was performed with an inverted Zeiss Laser Scanning Confocal Microscope (Zeiss LSM S10; Carl Zeiss, Thornwood, NJ) using excitation and emission wavelengths of 488 and 507 nm for GFP and 433 and 475 nm for CFP, respectively.
LysoTracker and dextran cell loading and confocal microscopy. LysoTracker Red DND-99 (Molecular Probes, Eugene, OR) was loaded into McNtcp.24 cells and mouse hepatocytes by incubating the cells in probe-containing media at a final concentration of 50 nM for 1 h at 37°C. Texas Red dextran (3,000 mol wt; Molecular Probes) was loaded in McNtcp.24 cells by incubating the cells in probe-containing media at a final concentration of 100 µM for 2 h at 37°C. Cells were viewed and counted in 36 random microscopic high-power fields using an inverted laser scanning confocal microscope with emission and excitation wavelengths of 577 and 590 nm, respectively.
Electron microscopy. Cells were fixed in 1% glutaraldehyde and 4% formaldehyde in 0.1 M phosphate buffer, pH 7.2, at 4°C for 15 min. The cells were then rinsed for 30 min in three changes of 0.1 M phosphate buffer, pH 7.2, followed by a 1-h postfix in phosphate-buffered 1% OsO4. After being rinsed in three changes of distilled water for 30 min, the cells were stained with 2% uranyl acetate for 30 min at 60°C. Next, the cells were rinsed in three changes of distilled water, dehydrated in progressive concentrations of ethanol followed by 100% propylene oxide, and embedded in Spurr's resin. Sections (90 nm) were cut on an LKB Ultratome III (Mager Scientific, Dexter, MI), placed on 200-nm mesh copper grids, and stained with lead citrate. Micrographs were taken (model 1200; JEOL, Peabody, MA) at 60 kV.
LysoTracker release fluorescence assay.
To quantify the release of LysoTracker from lysosomes in treated and
untreated cells, LysoTracker-loaded Cat B(/
) and C57/BL6 (+/+)
[Cat B(+/+)] mouse hepatocytes were washed in Krebs-Ringers-HEPES buffer (in mM: 115 NaCl, 20 HEPES, 5 KCl, 2 CaCl2, 1.2 MgSO4, and 1 KH2PO4, pH 7.4) and
then treated in Krebs-Ringers-HEPES buffer containing TNF-
/AcD for
4 h at 37°C. Cells were next permeabilized by adding digitonin
at a final concentration of 20 µM or Triton X-100 at a final
concentration of 1%. Under these conditions, digitonin permeabilizes
the plasma membrane but not lysosomes (see RESULTS). Triton
X-100 permeabilizes all membranes and was used as a control to
ascertain the maximum releasable dye. The cells were permeabilized for
30 min at room temperature. The buffer was removed and centrifuged at
3,000 g for 5 min to remove debris; LysoTracker Red
fluorescence in the supernatant was measured using a fluorometer with
excitation and emission wavelengths of 577 and 590 nm, respectively.
The spontaneous release of LysoTracker Red (blank) was determined by
measuring the fluorescence in the supernatant from untreated,
nonpermeabilized cells and was subtracted from all experimental
samples. LysoTracker Red release from lysosomes was quantitated as a
percentage of basal release using the following equation:
fluorescence (%) = (fluorescence of digitonin-permeabilized, TNF-
-treated cells
blank fluorescence)/(fluorescence of
digitonin-permeabilized, untreated cells
blank
fluorescence) × 100.
Lysosomal isolation.
Lysosomes were isolated from adult male Cat B(+/+) and Cat
B(/
) mice. Briefly, the abdomen of the mouse was opened, the portal
vein was catheterized with a 20-gauge intravenous catheter, and the
liver was flushed with Mg2+- and Ca2+-free
buffer containing (in mM) 115 NaCl, 20 HEPES, 5 KCl, 1 KH2PO4, and 0.5 EGTA (pH 7.4) at a flow rate of
10 ml/min for a period of time long enough to exsanguinate the liver.
The liver was removed rapidly and placed in 10 ml of ice-cold
homogenization buffer (in mM: 70 sucrose, 220 mannitol, 10 HEPES, and 1 EGTA, pH 7.4). Homogenization of the liver was performed using a
motorized Teflon pestle at 1,200 rpm for six to eight strokes. The
suspension was centrifuged at 2,000 g for 12 min at 4°C.
The supernatant was then treated with 2 mM CaCl2 for 10 min
at 37°C; the Ca2+ treatment swells mitochondria, thereby
changing their density so they can be separated easily from lysosomes
(24). This suspension was then layered on an isoosmotic
gradient [4:1:5.5 Percoll-2.5 M sucrose-H2O, with the
addition of 10 mM HEPES (pH 7.4)] in 35-ml quick-seal tubes (Beckman,
Palo Alto, CA) and centrifuged at 55,000 g for 22 min with a
mild deceleration. The bottom 8 ml were collected from each tube and
diluted three times with wash buffer (0.3 M sucrose and 10 mM HEPES, pH
7.4). This suspension was then centrifuged at 20,000 g for
30 min, and the subsequent loose pellet was resuspended and washed two
times in wash buffer. The remaining pellet was resuspended in 250 µl
wash buffer, and protein content was assayed via the Bradford method.
Calcein-AM release assay. A calcein release assay was developed analogous to the approach we previously described employing calcein to assess mitochondrial permeabilization (1). Calcein-AM (5 µM; Molecular Probes) was added to the lysosomal suspension for a 30-min incubation at 37°C in wash buffer. The lysosomes were washed two times and resuspended in 250 µl wash buffer. Lysosomal protein (50 µg) was added to 1.5 ml wash buffer in a 4-ml clear-sided acrylic cuvette, and fluorescence was monitored at 490 nm excitation and 520 nm emission wavelength using a Perkin-Elmer LS50B Luminescence Spectrophotometer (Perkin-Elmer, Foster City, CA). After a 10-min baseline, the appropriate agonist was added, and fluorescence was monitored for an additional 10 min. Triton X-100 (0.1%) was then added to ascertain complete calcein-fluorescence release.
Reagents.
Anti-Fas agonistic antibody Jo2 was from BD Transduction
Laboratories (San Diego, CA). Mouse recombinant TNF-, AcD,
sphingosine, C6 ceramide, 4',6-diamidino-2-phenylindole
(DAPI), Bradford reagent, and all other chemicals were from Sigma
Chemical (St. Louis, MO).
Statistical analysis. All data represent at least three independent experiments and are expressed as means ± SD unless otherwise indicated. Differences between groups were compared 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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TNF-/AcD induces lysosomal permeabilization to Cat B-GFP and
LysoTracker Red.
The rat hepatoma cell line McNtcp.24 was cotransfected with Cat B-GFP
and LAMP-2-CFP (Fig.
1A). Both
Cat B-GFP and LAMP-2-CFP displayed a punctate fluorescent appearance
when viewed by confocal microscopy, consistent with a vesicular
compartmentation of the tagged proteins. Moreover, overlay images
demonstrated virtually complete colocalization of the two fluorescent
proteins. Because LAMP-2 is predominantly present on lysosomal
membranes, these observations suggest Cat B-GFP, like its native
protein, is also targeted to lysosomes. Cat B-GFP is, therefore, an
appropriate expression construct for assessing lysosomal integrity
during apoptosis.
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Endosomes are not permeabilized during TNF-/AcD treatment.
The specificity of vesicle permeabilization was examined by evaluating
endosomal integrity after exposure to TNF-
/AcD. Endosome integrity
was assessed by loading this compartment with Texas Red dextran (3 kDa
dextran) in Cat B- and GFP-transfected cells (Fig.
3). As expected, Texas Red dextran did
not colocalize with Cat B-GFP. Moreover, Texas Red dextran fluorescence
remained punctate despite the redistribution of Cat B-GFP from
lysosomes to the cytosol. These data suggest vesicle permeabilization
during TNF-
/AcD exposure is selective and likely limited to
lysosomes.
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Lysosomal breakdown is promoted by Cat B.
We have previously reported that TNF-/AcD-associated
apoptosis is, in part, Cat B dependent. However, the above data
suggested lysosomal permeabilization was nonspecific and would release
multiple enzymes in the cytosol. Multiple cathepsins (Cat B, D, and L) have also been implicated in apoptosis (32). Based
on these concepts, we presumed Cat B may be mechanistically involved in lysosomal permeabilization. To test this concept, hepatocytes from Cat
B(+/+) and Cat B(
/
) mice were labeled with LysoTracker Red. Cells
were then treated with TNF-
/AcD and scored as either demonstrating
punctate or cytosolic fluorescence (Fig.
4, A and B). Following incubation with TNF-
/AcD, 85% of Cat
B(+/+) cells showed diffuse cytosolic LysoTracker Red fluorescence,
whereas only 20% of Cat B(
/
) cells showed the same pattern.
Consistently, measurement of LysoTracker Red release in the cytosol by
a fluorometric assay confirmed that TNF-
/AcD treatment caused
extensive lysosomal permeabilization in Cat B(+/+) hepatocytes
associated with release of 37% of total LysoTracker (Fig.
4C; P < 0.001, TNF-treated vs. untreated
cells). On the contrary, only minimal lysosomal permeabilization (5%
of total) was observed in Cat B(
/
) hepatocytes, where release of
LysoTracker in the cytosol was not statistically significant compared
with that observed in untreated cells (Fig. 4C;
P = not significant, TNF-treated vs. untreated cells).
Treatment with Fas, whose cell death is not Cat B dependent, did not
induce LysoTracker Red release (Fig. 4C). Thus, in
hepatocytes isolated form wild-type mice, the lysosomes are more
sensitive to permeabilization by TNF-
/AcD than those in hepatocytes
obtained from Cat B-deficient mice.
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Sphingosine induces lysosomal permeabilization in a Cat B-dependent
manner.
TNF- signaling is associated with activation of sphingomyelinase
through an adaptor protein referred to as FAN (factor associated with
neutral sphingomyelinase activation; see Ref. 36).
Sphingomyelinase activation results in the release of ceramide from
membranes, which, in turn, can be converted to sphingosine. Sphingosine
has a detergent-type structure with a polar head and a long lipophilic tail. An amino group within the polar head confers lysosomotropic properties to the molecule, facilitating its intralysosomal
accumulation by proton trapping. Protonation at the hydrophilic end
increases sphingosine detergent capacity and may induce lysosomal
permeabilization/rupture (8). Indeed, sphingosine, but not
ceramide, has recently been reported to cause lysosomal
permeabilization and apoptosis (20). Therefore, we
determined if sphingosine-induced lysosomal permeabilization was Cat B
dependent in sphingosine-treated hepatocytes. With the sue of the
LysoTracker Red assay in cell monolayers, significantly more
LysoTracker Red was released from lysosomes in Cat B(+/+) vs. Cat
B(
/
) hepatocytes (Fig. 4D). These data suggest that TNF-
- and sphingosine-mediated lysosomal permeabilization is Cat B dependent.
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DISCUSSION |
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The principal findings of this study relate to lysosomal
permeabilization during TNF--mediated apoptosis. The results
demonstrate that, during TNF-
treatment of hepatocytes:
1) lysosomes, but not endosomes, undergo a permeabilization
process; 2) lysosomal permeabilization is, in part, Cat B
dependent; and 3) sphingosine duplicates the
TNF-
-associated lysosomal permeabilization, also in a Cat
B-dependent manner both in cells and in a cell-free system. These data
support a model implicating a TNF-
/sphingosine/Cat B/lysosomal
permeabilization signaling cascade. Each of these findings and their
implications are discussed below.
Loss of lysosomal integrity with subsequent activation of
proapoptotic cascades is an emerging paradigm in the field of cell death (3). For example, lysosomal permeabilization or
breakdown has been implicated in apoptosis during oxidative
stress (29, 31), growth factor starvation, Fas activation,
and -tocopheryl- and succinate-mediated apoptosis in Jurkat
T cells (4, 25), 6-hydroxydopamine-associated death of
cultured microglia (37), and apoptosis induced by
the synthetic retinoid CD437 in human leukemia HL-60 cells
(42), in addition to TNF-
- and bile acid-mediated hepatocyte apoptosis (12, 13, 33) and, more
recently, p53-mediated apoptosis of M1-t-p53 myeloid leukemic
cells (41). The current data extend these observations by
examining the selectivity and specificity of the lysosomal
permeabilization process. Lysosomal permeabilization would appear to be
nonselective, since not only is Cat B, a naturally occurring lysosomal
constituent, released, but the structurally unrelated compound
LysoTracker Red is also translocated from lysosomes to the cytosol.
This nonselectivity has mechanistic implications, since it favors
lysosomal destabilization as opposed to a transport process across an
intact membrane, which would be expected to be specific for a single
class of molecules. In contrast to lysosomes, endosomes labeled with a
low-molecular-weight dextran remained intact during TNF-
-induced
hepatocyte apoptosis, supporting the hypothesis that not all
acidic vesicles are permeabilized. This observation suggests lysosomes
are specifically targeted for permeabilization during TNF-
signaling
and that their breakdown is not an epiphenomenon resulting from
uncontrolled intracellular digestion/degradation. Further supporting
this concept was the observation that lysosomal permeabilization does
not occur during Fas-induced apoptosis. Finally, neither
release of Cat B nor LysoTracker Red from lysosomes was complete.
Whether a specific subpopulation of lysosomes is targeted for
permeabilization or whether the release of lysosomal constituents
within an individual lysosome is incomplete remains unclear; the
techniques used in this study cannot distinguish between these two
possibilities. Lysosomal heterogeneity has previously been reported,
supporting the concept that only a subpopulation of lysosomes undergoes
permeabilization during the early stages of apoptosis
(27). Collectively, the current observations support a
model in which lysosomes, but not endosomes, undergo permeabilization during TNF-
treatment of hepatocytes, resulting in a nonspecific translocation of intraorganelle constituents in the cytosol.
Lysosomal permeabilization could be stimulated by exogenous sphingosine
but not ceramide. Sphingosine has been shown to accumulate in the liver
during TNF- treatment and also to permeabilize lysosomes (20). Thus sphingosine is a likely candidate for mediating
lysosomal permeabilization during TNF-
proapoptotic signaling.
Enhanced formation of sphingosine during TNF-
signaling likely is
the result of increased activity of either or both acidic and neutral sphingomyelinase, which has been reported with TNF-
(35). Sphingomyelinase cleaves sphingolipids, generating
ceramide, which is further metabolized to sphingosine by ceramidases
(28). The current data showing lysosomal permeabilization
with sphingosine, but not ceramide, support a role for sphingosine in
the lysosomal permeabilization process. In previous studies employing
cell cultures and cell-free systems, a role for caspase 8 in lysosomal
destabilization was also suggested (12). However, in these
studies, inhibition of caspase 8 by overexpression of the viral protein
CrmA in murine hepatocytes significantly reduced, but did not
completely abolish, TNF-
-induced Cat B release from lysosomes,
suggesting at least another signaling pathway (i.e., sphingosine)
exists and functions in a cooperative manner with caspase 8 to induce
lysosomal permeabilization. The relative contributions, synergies, and
additive effects of these two mediators in causing lysosomal
destabilization will require further study.
The sphingosine-stimulated lysosomal permeabilization was partially Cat
B dependent. Indeed, LysoTracker Red release from lysosomes was
attenuated in TNF--treated, Cat B-deficient hepatocytes. These data
suggest a key mechanistic link between Cat B and sphingosine in
lysosomal destabilization. Sphingosine could potentially stimulate Cat
B activity by binding to this protein and inducing conformational changes, enhancing its catalytic activity within lysosomes. The change
in Cat B activity may permit proteolysis of membrane or intralysosomal
proteins, causing lysosomal destabilization. A precedent for such a
lipid mediator-cathepsin interaction has been demonstrated for ceramide
and cathepsin D (14, 15). Sphingosine could alter the
lipid milieu, changing lysosomal topology and/or composition and
rendering previously concealed proteins open to proteolytic attack.
Limited proteolysis of a transport/import protein could result in pore
formation, as has been described for bacterial proteins, or proteolysis
may release amphipathic peptides, which insert directly in the membrane
or bind to other membrane proteins inducing pore development
(38). Alternatively, a small amount of cytosolic Cat B
could activate cytosolic factors, further inducing lysosomal
destabilization in a feedback loop. Zhao et al. (43, 44)
have recently published evidence for activation of phospholipase
A2 by leaking lysosomal contents. Once activated,
phospholipase A2 may attack mitochondria with consequent
release of cytochrome c and also, in a feedback loop, induce
further lysosomal destabilization. If Cat B is an activator of
phospholipase A2, this would explain the reduced
apoptosis in hepatocytes from catB(
/
) mice when
their lysosomes are partially ruptured by the lysosomotropic detergent
sphingosine. However, this model would not address the findings in the
cell-free system where hepatic lysosomes from catB(
/
)
mice were not permeabilized by sphingosine.
Ferri and Kroemer (7) have suggested that each
intracellular organelle possesses sensors that detect locally active
signals, which in turn result in the organelle emitting signals that
stimulate common apoptotic pathways. We propose that, for
lysosomes, one of the sensors is Cat B, which is capable of responding
to local increases in the lipid milieu, namely sphingosine. The
sphingosine-associated increase in Cat B activity or substrate
availability results in lysosomal destabilization, releasing lysosomal
constituents into the cytosol. After leakage of lysosomal constituents,
various lysosomal proteases within the cytosol are likely capable of
activating common apoptotic cascades. Indeed, a cathepsin L-like
protease has been implicated in caspase activation, Cat B can activate the mitochondrial pathway of apoptosis, and cathepsin D is also proapoptotic (5, 12, 18). The relative role of these
proapoptotic cathepsins may be tissue specific. However, the
release of these proapoptotic lysosomal proteases or
"lysoapoptases" from lysosomes in liver appears to be Cat B
mediated; therefore, Cat B functions as a linchpin enzyme in this
cascade. This working model suggests that targeting sphingomyelinases
and/or ceramidases responsible for sphingosine generation, inhibiting
Cat B activity, or using lysosomal stabilizing agents are all potential
therapeutic maneuvers to inhibit TNF--mediated hepatocyte
apoptosis and merit further investigation as cytoprotective strategies.
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ACKNOWLEDGEMENTS |
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The secretarial assistance of Sara Erickson is gratefully acknowledged. We thank Dr. M.McNiven for providing the LAMP-2-GFP construct.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-41876 (to G. J. Gores), the Palumbo Foundation, and the Mayo Foundation.
Address for reprint requests and other correspondence: G. J. Gores, Mayo Medical School, Clinic, and Foundation, 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.
10.1152/ajpgi.00151.2002
Received 19 April 2002; accepted in final form 29 May 2002.
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