Am J Physiol Gastrointest Liver Physiol 283: G947–G956, 2002;
10.1152/ajpgi.00151.2002.
Tumor necrosis factor-␣-associated lysosomal
permeabilization is cathepsin B dependent
NATHAN W. WERNEBURG, M. EUGENIA GUICCIARDI,
STEVEN F. BRONK, AND GREGORY J. GORES
Division of Gastroenterology and Hepatology, Mayo Medical School,
Clinic, and Foundation, Rochester, Minnesota 55905
Received 19 April 2002; accepted in final form 29 May 2002
calcein release assay; cathepsin B-green fluorescence protein;
LysoTracker Red; sphingosine
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
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: [email protected]).
http://www.ajpgi.org
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
The costs of publication of this article were defrayed in part by the
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0193-1857/02 $5.00 Copyright © 2002 the American Physiological Society
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Werneburg, Nathan W., M. Eugenia Guicciardi,
Steven F. Bronk, and Gregory J. Gores. Tumor necrosis
factor-␣-associated lysosomal permeabilization is cathepsin
B dependent. Am J Physiol Gastrointest Liver Physiol 283:
G947–G956, 2002; 10.1152/ajpgi.00151.2002.—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.
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LYSOSOMAL PERMEABILIZATION IN APOPTOSIS
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.
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 ␮g/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 (BoehringerMannheim, 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 ␮g/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 catBGFP and LAMP-2-CFP was performed with 1 ml of OptiMEM-1 containing 6 ␮l Plus reagent, 1 ␮g/ml catB-GFP and
LAMP-2-CFP cDNA, and 6 ␮l/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 ␮M 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 ␮M 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 digitoninpermeabilized, 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
LYSOSOMAL PERMEABILIZATION IN APOPTOSIS
RESULTS
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.
To determine if lysosomal permeabilization after
TNF-␣/AcD treatment is selective or nonselective, Cat
B- and GFP-transfected cells were coloaded with LysoTracker Red, a fluorescent dye that loads predominantly into lysosomes. Under these conditions, the
cells will ultimately develop the classic morphological
changes of apoptosis; however, for these experiments,
the cells were examined before these morphological
changes to identify early mechanistic events and to
distiguish them from secondary cell death phenomenon. Before TNF-␣/AcD treatment, LysoTracker Red
and Cat B-GFP colocalized to the same vesicular compartment. After treatment, Cat B-GFP fluorescence
largely redistributes to the cytosol (Fig. 1B). LysoAJP-Gastrointest Liver Physiol • VOL
Tracker Red fluorescence also underwent a partial
redistribution from a vesicular to a mixed cytosolic/
vesicular pattern of fluorescence (Fig. 1B). These data
suggest that TNF-␣/AcD-associated lysosomal permeabilization was nonselective, since two structurally unrelated molecules, LysoTracker Red and Cat B-GFP,
were both released from lysosomes into the cytosol.
Supporting this interpretation of the data were the
ultrastructural studies demonstrating lysomal swelling in TNF-␣/AcD-treated cells (Fig. 1C). The nonselectivity of the permeabilization is consistent with lysosomal pore formation or lysosomal destabilization/
rupture as opposed to an active transport process
across an intact membrane.
To ascertain the completeness of Cat B-GFP release
from lysosomes, we selectively permeabilized the
plasma membrane with digitonin. Because it is a cholesterol-solubilizing agent, low concentrations of digitonin permeabilize cholesterol-rich membranes, such
as the plasma membrane, but not cholesterol-poor lysosomal or mitochondrial membranes. A validation of
this approach is shown in Fig. 2. Digitonin (20 ␮M)
selectively permeabilizes the plasma membrane, releasing the cytosolic dye calcein-AM from the cells
without altering the lysosomes, as demonstrated by the
fluorescent pattern of LysoTracker Red (Fig. 2A). Thus
we used this approach in TNF-␣/AcD-treated cells to
study the distribution of Cat B-GFP fluorescence. After
the redistribution of Cat B fluorescence, the cell was
incubated in the presence of digitonin, which caused
the release of cytosolic Cat B-GFP. This approach
showed that lysosomal permeabilization appears to be
incomplete, since a residual population of punctate Cat
B-GFP vesicles remained intact (Fig. 2B). Thus Cat
B-GFP is only partially released from lysosomes after
TNF-␣/AcD treatment.
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.
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
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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 ␮l 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 ␮M; 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 ␮l wash buffer. Lysosomal protein (50
␮g) 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 PerkinElmer LS50B Luminescence Spectrophotometer (PerkinElmer, 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-diamidino2-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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LYSOSOMAL PERMEABILIZATION IN APOPTOSIS
sosomal release assay was developed. In this assay,
lysosomes are loaded with calcein-AM. Endogenous
esterases within lysosomes cleave the esters from calcein trapping the charged form of calcein in lysosomes.
Because of fluorophore stacking and inner filter effects,
calcein fluorescense is quenched within the fluorophore-loaded lysosomes but increases significantly
with release from the lysosomes, as shown in Fig. 5A
with Triton X-100. With the use of this cell-free system,
the ability of sphingosine to induce lysosomal permeabilization was examined in Cat B(⫹/⫹) and Cat
B(⫺/⫺) mouse liver lysosomes. Consistent with the
observations above, calcein release by sphingosine was
also Cat B dependent (Fig. 5B). In contrast, C6 ceramide did not induce lysosomal permeabilization (Fig.
5C). Collectively, these data implicate a sphingosineCat B interaction in lysosomal permeabilization during
TNF-␣ apoptotic signal.
DISCUSSION
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 perme-
Fig. 1. A: cathepsin B (Cat B)-green fluorescent protein (GFP) localizes within lysosomes. After 48 h of cotransfection with the plasmids encoding the fusion proteins Cat B-GFP and lysosome-associated membrane protein-2cyan fluorescent protein (LAMP-2-CFP), a lysosomal-associated membrane protein, McNtcp.24 cells were imaged
by laser scanning confocal microscopy as described in details in EXPERIMENTAL PROCEDURES. Cat B-GFP colocalized
with LAMP-2-CFP as shown in the overlay image, confirming its lysosomal distribution. B: treatment with tumor
necrosis factor (TNF)-␣/actinomycin D (AcD) results in partial permeabilization of lysosomes. Cat B-GFP transiently transfected McNtcp.24 cells were loaded with LysoTracker Red for 1 h to selectively stain the lysosomal
compartment and then were either left untreated (control) or treated with TNF-␣/AcD for 4 h, as described in
EXPERIMENTAL PROCEDURES. Cells were then imaged with an inverted scanning confocal microscope. C: treatment
with TNF-␣/AcD induces lysosomal swelling. McNtcp.24 cells were incubated in the presence and absence (control)
of TNF-␣/AcD for 4 h. Cells were then fixed and viewed by transmission electron microscopy (original magnification
⫻10,000) as described in EXPERIMENTAL PROCEDURES. Arrows designate lysosomes.
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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, TNFtreated 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.
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 detergenttype 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 sphingosinetreated 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.
To further test the Cat B dependence of sphingosineinduced lysosomal permeabilization, a fluorescent ly-
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abilization 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
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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
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Fig. 2. Treatment with digitonin results in selective permeabilization of the plasma membrane. A: McNtcp.24 cells
were incubated in media containing calcein-AM (1 ␮M), 4⬘,6-diamidino-2-phenylindole (DAPI, 5 ␮g/ml), and
LysoTracker Red (50 M) at 37°C for 30 min to visualize the cytosolic compartment, the nuclear compartment, and
the lysosomes, respectively. Probe-containing media were then replaced with buffer C containing 20 ␮M digitonin,
and cells were imaged using an inverted scanning confocal microscope every 10 min at room temperature. At 30
min, both LysoTracker and DAPI fluorescence remained intact, whereas calcein-AM was completely lost, demonstrating a selective, digitonin-induced permeabilization of the plasma membrane. B: Cat B-GFP transiently
transfected McNtcp.24 cells were treated with TNF-␣/AcD for 4 h. Media were then removed and replaced with
buffer C containing 20 ␮M digitonin. Cells were imaged with an inverted scanning confocal microscope after a
30-min incubation with digitonin-containing buffer.
LYSOSOMAL PERMEABILIZATION IN APOPTOSIS
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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
AJP-Gastrointest Liver Physiol • VOL
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
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Fig. 3. Treatment with TNF-␣/AcD does not permeabilize the endosome compartment. Cat B-GFP transiently
transfected McNtcp.24 cells were loaded with Texas Red dextran (3 kDa dextran) at 37°C for 2 h to selectively stain
endosomes. Cells were then either left untreated (control) or treated with TNF-␣/AcD for 4 h and imaged by a
scanning confocal microscope.
G954
LYSOSOMAL PERMEABILIZATION IN APOPTOSIS
Fig. 4. Cat B promotes lysosomal permeabilization. A: Cat B(⫹/⫹)
and Cat B(⫺/⫺) mouse hepatocytes were loaded with LysoTracker
Red and incubated in the absence (control) or presence of TNF-␣/AcD
at 37°C for 4 h. B: cells were then imaged by confocal microscopy and
counted according to their diffuse or punctate appearance. C: LysoTracker Red release was quantified further by fluorometrically measuring the amount released in the cytosol after treatment of the
hepatocytes with TNF-␣/AcD or an agonistic anti-Fas antibody (Jo2).
Cat B(⫹/⫹) and Cat B(⫺/⫺) mouse hepatocytes were loaded with
LysoTracker Red, treated with TNF-␣/AcD or anti-Fas at 37°C for
4 h, and permeabilized with digitonin for 30 min at room temperature as described in Fig. 3. Cells were then centrifuged, and their
supernatant was collected and measured using a fluorometer as
described in EXPERIMENTAL PROCEDURES. Data are expressed as
mean ⫾ SE percent changes in fluorescence above baseline. Baseline
was considered as fluorescence released by LysoTracker Red-loaded,
unpermeabilized, untreated cells. D: LysoTracker Red release was
measured as described in A-C in Cat B(⫹/⫹) and Cat B(⫺/⫺) mouse
hepatocytes after either no treatment (control) or a 3 h-treatment
with 10 ␮M sphingosine at 37°C.
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Fig. 5. Lysosome permeabilization by sphingosine is Cat B dependent. A: lysosomes isolated from Cat B(⫹/⫹) and Cat B(⫺/⫺) were
loaded with calcein-AM in vitro, and fluorescence was monitored
spectrophotometrically as described in EXPERIMENTAL PROCEDURES
before and after the addition of Triton X-100 (0.1%) to measure
complete calcein fluorescence release. arb units, Arbitrary units. B:
lysosomes from Cat B(⫹/⫹) and Cat B(⫺/⫺) loaded with calcein-AM
were treated with increasing concentrations of sphingosine, and
fluorescence in the supernatant was monitored for an additional 10
min as described above. Data are expressed as a percentage of total
calcein-AM release obtained by permeabilization with Triton X-100.
C: Cat B(⫹/⫹) and Cat B(⫺/⫺) lysosomes were loaded with calcein-AM in vitro and treated with either sphingosine (1.67 ␮M) or C6
ceramide (1.67 ␮M) for 10 min. Release of calcein-AM was measured
fluorometrically as described in A.
LYSOSOMAL PERMEABILIZATION IN APOPTOSIS
The secretarial assistance of Sara Erickson is gratefully acknowledged. We thank Dr. M.McNiven for providing the LAMP-2-GFP
construct.
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.
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