Tumor necrosis factor-alpha -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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cathepsin B (Cat B) is released from lysososomes during tumor necrosis factor-alpha (TNF-alpha ) 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-alpha . Thus the aims of this study were to examine the mechanisms involved in TNF-alpha -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-alpha /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-alpha 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-alpha cytotoxic signaling.

calcein release assay; cathepsin B-green fluorescence protein; LysoTracker Red; sphingosine


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

TUMOR NECROSIS FACTOR-alpha (TNF-alpha ) 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-alpha signaling depend on the cell type and cellular context. For example, in the liver, TNF-alpha is important in liver regeneration after a partial hepatic resection and as a cytotoxic agent in a variety of disease processes (2). TNF-alpha -mediated cytotoxicity is of considerable importance and is mediated, in part, by its ability to induce apoptosis. Indeed, anti-TNF-alpha therapy is currently used in the treatment of human disease (17). The TNF-alpha -initiated intracellular cascades interacting to induce cell demise are, therefore, of broad clinical and scientific interest.

TNF-alpha 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-alpha -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-alpha -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-alpha -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-alpha -mediated liver injury; this in vivo observation highlights the dominant role of this lysosomal/Cat B pathway in TNF-alpha -mediated liver injury (13). Further information on this pathway will help provide key insights into TNF-alpha -mediated signaling and may potentially lead to additional therapeutic strategies to reduce TNF-alpha -associated tissue injury.

There are several unresolved issues regarding the lysosomal/Cat B pathway of TNF-alpha -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.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-alpha (28 ng/ml), actinomycin D (AcD, 0.2 µg/ml) was included in the medium to block the NF-kappa B-mediated transcription of cytoprotective TNF-alpha -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-alpha /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: Delta fluorescence (%) = (fluorescence of digitonin-permeabilized, TNF-alpha -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-alpha , 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

TNF-alpha /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.


View larger version (64K):
[in this window]
[in a new window]
 
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-2-cyan 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)-alpha /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-alpha /AcD for 4 h, as described in EXPERIMENTAL PROCEDURES. Cells were then imaged with an inverted scanning confocal microscope. C: treatment with TNF-alpha /AcD induces lysosomal swelling. McNtcp.24 cells were incubated in the presence and absence (control) of TNF-alpha /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.

To determine if lysosomal permeabilization after TNF-alpha /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-alpha /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). LysoTracker 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-alpha /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-alpha /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 µM) 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-alpha /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-alpha /AcD treatment.


View larger version (38K):
[in this window]
[in a new window]
 
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 µM), 4',6-diamidino-2-phenylindole (DAPI, 5 µg/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 µM 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-alpha /AcD for 4 h. Media were then removed and replaced with buffer C containing 20 µM digitonin. Cells were imaged with an inverted scanning confocal microscope after a 30-min incubation with digitonin-containing buffer.

Endosomes are not permeabilized during TNF-alpha /AcD treatment. The specificity of vesicle permeabilization was examined by evaluating endosomal integrity after exposure to TNF-alpha /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-alpha /AcD exposure is selective and likely limited to lysosomes.


View larger version (40K):
[in this window]
[in a new window]
 
Fig. 3.   Treatment with TNF-alpha /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-alpha /AcD for 4 h and imaged by a scanning confocal microscope.

Lysosomal breakdown is promoted by Cat B. We have previously reported that TNF-alpha /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-alpha /AcD and scored as either demonstrating punctate or cytosolic fluorescence (Fig. 4, A and B). Following incubation with TNF-alpha /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-alpha /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-alpha /AcD than those in hepatocytes obtained from Cat B-deficient mice.


View larger version (14K):
[in this window]
[in a new window]
 
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-alpha /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-alpha /AcD or an agonistic anti-Fas antibody (Jo2). Cat B(+/+) and Cat B(-/-) mouse hepatocytes were loaded with LysoTracker Red, treated with TNF-alpha /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 µM sphingosine at 37°C.

Sphingosine induces lysosomal permeabilization in a Cat B-dependent manner. TNF-alpha 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-alpha - and sphingosine-mediated lysosomal permeabilization is Cat B dependent.

To further test the Cat B dependence of sphingosine-induced lysosomal permeabilization, a fluorescent lysosomal 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 sphingosine-Cat B interaction in lysosomal permeabilization during TNF-alpha apoptotic signal.


View larger version (18K):
[in this window]
[in a new window]
 
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 µM) or C6 ceramide (1.67 µM) for 10 min. Release of calcein-AM was measured fluorometrically as described in A.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The principal findings of this study relate to lysosomal permeabilization during TNF-alpha -mediated apoptosis. The results demonstrate that, during TNF-alpha 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-alpha -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-alpha /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 alpha -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-alpha - 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-alpha -induced hepatocyte apoptosis, supporting the hypothesis that not all acidic vesicles are permeabilized. This observation suggests lysosomes are specifically targeted for permeabilization during TNF-alpha 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-alpha 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-alpha treatment and also to permeabilize lysosomes (20). Thus sphingosine is a likely candidate for mediating lysosomal permeabilization during TNF-alpha proapoptotic signaling. Enhanced formation of sphingosine during TNF-alpha signaling likely is the result of increased activity of either or both acidic and neutral sphingomyelinase, which has been reported with TNF-alpha (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-alpha -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-alpha -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-alpha -mediated hepatocyte apoptosis and merit further investigation as cytoprotective strategies.


    ACKNOWLEDGEMENTS

The secretarial assistance of Sara Erickson is gratefully acknowledged. We thank Dr. M.McNiven for providing the LAMP-2-GFP construct.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1.   Botla, R, Spivey JR, Aguilar H, Bronk SF, and Gores GJ. Ursodeoxycholate (UDCA) inhibits the mitochondrial membrane permeability transition induced by glycochenodeoxycholate: a mechanism of UDCA cytoprotection. J Pharmacol Exp Ther 272: 930-938, 1995[Abstract].

2.   Bradham, CA, Plumpe J, Manns MP, Brenner DA, and Trautwein C. Mechanisms of hepatic toxicity. I. TNF-induced liver injury. Am J Physiol Gastrointest Liver Physiol 275: G387-G392, 1998[Abstract/Free Full Text].

3.   Brunk, UT, Neuzil J, and Eaton JW. Lysosomal involvement in apoptosis. Redox Rep 6: 91-97, 2001[ISI][Medline].

4.   Brunk, UT, and Svensson I. Oxidative stress, growth factor starvation and Fas activation may all cause apoptosis through lysosomal leak. Redox Rep 4: 3-11, 1999[ISI][Medline].

5.   Deiss, LP, Galinka H, Berissi H, Cohen O, and Kimchi A. Cathepsin D protease mediates programmed cell death induced by interferon-gamma, Fas/APO-1 and TNF-alpha. EMBO J 15: 3861-3870, 1996[Abstract].

6.   Faubion, WA, Guicciardi ME, Miyoshi H, Bronk SF, Roberts PJ, Svingen PA, Kaufmann SH, and Gores GJ. Toxic bile salts induce rodent hepatocyte apoptosis via direct activation of Fas. J Clin Invest 103: 137-145, 1999[Abstract/Free Full Text].

7.   Ferri, KF, and Kroemer G. Organelle-specific initiation of cell death pathways. Nat Cell Biol 3: E255-E263, 2001[ISI][Medline].

8.   Firestone, RA, Pisano JM, and Bonney RJ. Lysosomotropic agents 1. Synthesis and cytotoxic action of lysosomotropic detergent. J Med Chem 22: 1130-1133, 1979[ISI][Medline].

9.   Foghsgaard, L, Wissing D, Mauch D, Lademann U, Bastholm L, Boes M, Elling F, Leist M, and Jaattela M. Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor. J Cell Biol 153: 999-1009, 2001[Abstract/Free Full Text].

10.   Green, DR, and Reed JC. Mitochondria and apoptosis. Science 281: 1309-1312, 1998[Abstract/Free Full Text].

11.   Gross, A, Yin XM, Wang K, Wei MC, Jockel J, Milliman C, Erdjument-Bromage H, Tempst P, and Korsmeyer SJ. Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-R1/Fas death. J Biol Chem 274: 1156-1163, 1999[Abstract/Free Full Text].

12.   Guicciardi, ME, Deussing J, Miyoshi H, Bronk SF, Svingen PA, Peters C, Kaufmann SH, and Gores GJ. Cathepsin B contributes to TNF-alpha-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c. J Clin Invest 106: 1127-1137, 2000[Abstract/Free Full Text].

13.   Guicciardi, ME, Miyoshi H, Bronk SF, and Gores GJ. Cathepsin B knockout mice are resistant to tumor necrosis factor-alpha-mediated hepatocyte apoptosis and liver injury: implications for therapeutic applications. Am J Pathol 159: 2045-2054, 2001[Abstract/Free Full Text].

14.   Heinrich, M, Wickel M, Schneider-Brachert W, Sandberg C, Gahr J, Schwandner R, Weber T, Saftig P, Peters C, Brunner J, Kronke M, and Schutze S. Cathepsin D targeted by acid sphingomyelinase-derived ceramide. EMBO J 18: 5252-5263, 1999[Abstract/Free Full Text].

15.   Heinrich, M, Wickel M, Winoto-Morbach S, Schneider-Brachert W, Weber T, Brunner J, Saftig P, Peters C, Kronke M, and Schutze S. Ceramide as an activator lipid of cathepsin D. Adv Exp Med Biol 477: 305-315, 2000[ISI][Medline].

16.   Heller, RA, and Kronke M. Tumor necrosis factor receptor-mediated signaling pathways. J Cell Biol 126: 5-9, 1994[ISI][Medline].

17.   Illei, GG, and Lipsky PE. Novel, non-antigen-specific therapeutic approaches to autoimmune/inflammatory diseases. Curr Opin Immunol 12: 712-718, 2000[ISI][Medline].

18.   Ishisaka, R, Utsumi T, Yabuki M, Kanno T, Furuno T, Inoue M, and Utsumi K. Activation of caspase-3-like protease by digitonin-treated lysosomes. FEBS Lett 435: 233-236, 1998[ISI][Medline].

19.   Jones, B, Roberts PJ, Faubion WA, Kominami E, and Gores GJ. Cystatin A expression reduces bile salt-induced apoptosis in a rat hepatoma cell line. Am J Physiol Gastrointest Liver Physiol 275: G723-G730, 1998[Abstract/Free Full Text].

20.   Kagedal, K, Zhao M, Svensson I, and Brunk UT. Sphingosine-induced apoptosis is dependent on lysosomal proteases. Biochem J 359: 335-343, 2001[ISI][Medline].

21.   Li, P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, and Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91: 479-489, 1997[ISI][Medline].

22.   Luo, X, Budihardjo I, Zou H, Slaughter C, and Wang X. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94: 481-490, 1998[ISI][Medline].

23.   Monney, L, Olivier R, Otter I, Jansen B, Poirier GG, and Borner C. Role of an acidic compartment in tumor-necrosis-factor-alpha-induced production of ceramide, activation of caspase-3 and apoptosis. Eur J Biochem 251: 295-303, 1998[Abstract].

24.   Myers, BM, Prendergast FG, Holman R, Kuntz S, and LaRusso NF. Alterations in the structure, physicochemical properties, and pH of hepatocyte lysosomes in experimental iron overload. J Clin Invest 88: 1207-1215, 1991[ISI][Medline].

25.   Neuzil, J, Svensson I, Weber T, Weber C, and Brunk UT. Alpha-tocopheryl succinate-induced apoptosis in Jurkat T cells involves caspase-3 activation, and both lysosomal and mitochondrial destabilisation. FEBS Lett 445: 295-300, 1999[ISI][Medline].

26.   Nicholson, DW. Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ 6: 1028-1042, 1999[ISI][Medline].

27.   Nilsson, E, Ghassemifar R, and Brunk UT. Lysosomal heterogeneity between and within cells with respect to resistance against oxidative stress. Histochem J 29: 857-865, 1997[ISI][Medline].

28.   Ohanian, J, and Ohanian V. Sphingolipids in mammalian cell signalling. Cell Mol Life Sci 58: 2053-2068, 2001[ISI][Medline].

29.   Ollinger, K, and Brunk UT. Cellular injury induced by oxidative stress is mediated through lysosomal damage. Free Radic Biol Med 19: 565-574, 1995[ISI][Medline].

30.   Reinheckel, T, Deussing J, Roth W, and Peters C. Towards specific functions of lysosomal cysteine peptidases: phenotypes of mice deficient for cathepsin B or cathepsin L. Biol Chem 382: 735-741, 2001[ISI][Medline].

31.   Roberg, K, and Ollinger K. Oxidative stress causes relocation of the lysosomal enzyme cathepsin D with ensuing apoptosis in neonatal rat cardiomyocytes. Am J Pathol 152: 1151-1156, 1998[Abstract].

32.   Roberts, LR, Adjei PN, and Gores GJ. Cathepsins as effector proteases in hepatocyte apoptosis. Cell Biochem Biophys 30: 71-88, 1999[Medline].

33.   Roberts, LR, Kurosawa H, Bronk SF, Fesmier PJ, Agellon LB, Leung WY, Mao F, and Gores GJ. Cathepsin B contributes to bile salt-induced apoptosis of rat hepatocytes. Gastroenterology 113: 1714-1726, 1997[ISI][Medline].

34.   Salvesen, GS, and Dixit VM. Caspase activation: the induced-proximity model. Proc Natl Acad Sci USA 96: 10964-10967, 1999[Abstract/Free Full Text].

35.   Schutze, S, Wiegmann K, Machleidt T, and Kronke M. TNF-induced activation of NF-kappa B. Immunobiology 193: 193-203, 1995[ISI][Medline].

36.   Segui, B, Cuvillier O, Adam-Klages S, Garcia V, Malagarie-Cazenave S, Leveque S, Caspar-Bauguil S, Coudert J, Salvayre R, Kronke M, and Levade T. Involvement of FAN in TNF-induced apoptosis. J Clin Invest 108: 143-151, 2001[Abstract/Free Full Text].

37.   Takai, N, Nakanishi H, Tanabe K, Nishioku T, Sugiyama T, Fujiwara M, and Yamamoto K. Involvement of caspase-like proteinases in apoptosis of neuronal PC12 cells and primary cultured microglia induced by 6-hydroxydopamine. J Neurosci Res 54: 214-222, 1998[ISI][Medline].

38.   van der Goot, FG, Lakey J, Pattus F, Kay CM, Sorokine O, Van Dorsselaer A, and Buckley JT. Spectroscopic study of the activation and oligomerization of the channel-forming toxin aerolysin: identification of the site of proteolytic activation. Biochemistry 31: 8566-8570, 1992[ISI][Medline].

39.   Varfolomeev, EE, Schuchmann M, Luria V, Chiannilkulchai N, Beckmann JS, Mett IL, Rebrikov D, Brodianski VM, Kemper OC, Kollet O, Lapidot T, Soffer D, Sobe T, Avraham KB, Goncharov T, Holtmann H, Lonai P, and Wallach D. Targeted disruption of the mouse Caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally. Immunity 9: 267-276, 1998[ISI][Medline].

40.   Wallach, D, Varfolomeev EE, Malinin NL, Goltsev YV, Kovalenko AV, and Boldin MP. Tumor necrosis factor receptor and Fas signaling mechanisms. Annu Rev Immunol 17: 331-367, 1999[ISI][Medline].

41.   Yuan, XM, Li W, Dalen H, Lotem J, Kama R, Sachs L, and Brunk UT. Lysosomal destabilization in p53-induced apoptosis. Proc Natl Acad Sci USA 99: 6286-6291, 2002[Abstract/Free Full Text].

42.   Zang, Y, Beard RL, Chandraratna RA, and Kang JX. Evidence of a lysosomal pathway for apoptosis induced by the synthetic retinoid CD437 in human leukemia HL-60 cells. Cell Death Differ 8: 477-485, 2001[ISI][Medline].

43.   Zhao, M, Brunk UT, and Eaton JW. Delayed oxidant-induced cell death involves activation of phospholipase A2. FEBS Lett 509: 399-404, 2001[ISI][Medline].

44.   Zhao, M, Eaton JW, and Brunk UT. Bcl-2 phosphorylation is required for inhibition of oxidative stress-induced lysosomal leak and ensuing apoptosis. FEBS Lett 509: 405-412, 2001[ISI][Medline].


Am J Physiol Gastrointest Liver Physiol 283(4):G947-G956
0193-1857/02 $5.00 Copyright © 2002 the American Physiological Society