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[Cancer Research 64, 1000-1007, February 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Increase of Nuclear Ceramide through Caspase-3-Dependent Regulation of the "Sphingomyelin Cycle" in Fas-Induced Apoptosis

Mitsumasa Watanabe1, Toshiyuki Kitano1, Tadakazu Kondo1, Takeshi Yabu1, Yoshimitsu Taguchi1,3, Masaro Tashima1, Hisanori Umehara2, Naochika Domae4, Takashi Uchiyama1 and Toshiro Okazaki1

Departments of 1 Hematology and Oncology and 2 Rheumatology and Clinical Immunology, Graduate School of Medicine, 3 Graduate School of Biostudies, Kyoto University, Kyoto, and 4 Department of Medicine, Osaka Dental University, Osaka, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Regardless of the existence of ceramide-related molecules, such as sphingomyelin (SM), neutral sphingomyelinase (nSMase), and SM synthase, in the nucleus, the regulation of ceramide in the nucleus is poorly understood in stress-induced apoptosis. In Fas-induced Jurkat T-cell apoptosis, we found a time- and dose-dependent increase of ceramide content in the nuclear and microsomal fractions. Fas-induced increase of ceramide content in the nucleus also was detected by confocal microscopy using anticeramide antibody. Activation of nSMase and inhibition of SM synthase were evident in the nuclear fraction after Fas cross-linking, whereas nSMase was activated, but SM synthase was not affected, in the microsomal fraction. Pretreatment with D-609, a putative SM synthase inhibitor, enhanced Fas-induced increase of ceramide in the nucleus and induction of apoptosis along with increase of Fas-induced inhibition of nuclear SM synthase. Fas-induced activation of caspase-3 was detected in the nuclear fraction and in whole cell lysate. A caspase-3 inhibitor, acetyl-Asp-Glu-Val-Asp-chloromethyl ketone, blocked not only Fas-induced increases of apoptosis and ceramide content but also Fas-induced activation of nSMase and inhibition of SM synthase in the nuclear fraction. Taken together, it is suggested that the nucleus is a site for ceramide increase and caspase-3 activation in Fas-induced Jurkat T-cell apoptosis and that caspase-3-dependent regulation of the "SM cycle" consisting of nSMase and SM synthase plays a role in Fas-induced ceramide increase in the nucleus.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ceramide has been recognized as an intracellular lipid mediator related to a variety of cell functions, including cell differentiation, secretion, senescence, and apoptosis (1, 2, 3) . A diverse array of stresses, including Fas cross-linking, tumor necrosis factor {alpha}, irradiation, heat shock, and anticancer drugs (4) , were reported to increase intracellular ceramide in induction of apoptosis (4, 5, 6, 7) . Generation of ceramide through many kinds of ceramide-related enzymes, such as sphingomyelinase (SMase), ceramidase, de novo ceramide synthase, and glucosylceramide (GC) synthase, has been shown to play a role in apoptosis. For example, acid SMase-deficient human lymphoblasts from acid SMase-/- mice were found to be resistant to the ionizing radiation-induced apoptosis because of a lack of ceramide generation from sphingomyelin (SM; Ref. 6 ). Although the indispensable role of acid SMase also was suggested in Fas-induced apoptosis (8) , its role still is controversial because Niemann-Pick disease-derived lymphocytes, which show a low activity of acid SMase because of its genetic defect, were reported to be sensitive to Fas treatment (9) . Exogenous neutral sphingomyelinase (nSMase) and overexpression of bacterial nSMase in mammalian cells induced apoptosis with excess of ceramide increase (10) , and the ceramidase inhibitor (1S, 2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol, which increases ceramide by blocking the deacylation of ceramide to sphingosine, enhanced apoptosis in human leukemia HL-60 cells (11) . It also was reported that the activity of GC synthase, which converts ceramide to GC, was more increased in apoptosis-resistant cells than in apoptosis-sensitive cells (12, 13, 14) . The involvement of SM synthase in nuclear factor {kappa}B activation of transformed fibroblast and basic fibroblast growth factor-induced proliferation of primary astrocytes was suggested recently, but the implication of SM synthase has yet to be clarified in Fas-induced apoptosis (15 , 16) .

In terms of the topology of ceramide generation, SM hydrolysis in plasma membrane by nSMase was first reported to be involved in apoptosis (17) , and lysosome, endoplasmic reticulum (ER), and mitochondria then were proposed as possible sites of ceramide generation (18 , 19) .5 Increase of ceramide through activation of nSMase in the nucleus of hepatocytes was reported in the animal model of hepatic vein ligation (20) , and addition of ceramide-treated cell extract was shown to induce nuclear apoptotic events in the reconstituted condition (21) . In addition, the activities of nSMase and SM synthase were found in rat nuclear membrane together with the existence of phosphatidylcholine and diacylglycerol (DAG; Refs. 22, 23, 24, 25 ). These results suggest the existence of the "SM cycle," consisting of nSMase and SM synthase in the nucleus (1) . However, ceramide regulation through the SM cycle in the nucleus still is unknown in Fas-induced apoptosis.

Fas-induced apoptosis is known to show the typical and morphologic changes in the nucleus, such as chromatin condensation and DNA fragmentation. Activation of proteolytic molecules, including caspases, granzyme B, cathepsin D, and histone-associated protease, is reported to play a role in execution of apoptosis (26) . Among them, caspase-3 seems to be critical to nuclear changes in apoptosis because caspase-3-deficient breast cancer cells (MCF-7) underwent cell death without chromatin condensation and DNA fragmentation (27) . In Fas-induced apoptosis, active caspase-3 was shown to translocate from the cytosol to the nucleus and to cleave nuclear proteins, such as lamins and poly(ADP-ribose)polymerases (28, 29, 30) . The translocation of active caspase-3 to the nucleus also was reported in apoptosis of colonic epithelial cells (31) and neuroblastoma cells (32) , and a recombinant, active caspase-3 was shown to induce apoptotic changes of the nucleus in a cell-free system (33) . However, it remains to be clarified whether active caspase-3 is involved in regulation of nuclear ceramide content in Fas-induced apoptosis.

Therefore, we investigate whether and how ceramide generation in the nucleus is regulated in Fas-induced apoptosis and, if this is the case, how Fas-activated caspase-3 is involved in regulation of nuclear ceramide. Our results show that ceramide content in nuclear fraction is increased through activation of nSMase and inhibition of SM synthase in Fas-induced Jurkat T-cell apoptosis. Increase of ceramide in the nucleus by Fas treatment also is shown by confocal microscopy using anticeramide antibody (34) . Activation of nuclear caspase-3 is detected after Fas cross-linking, and the caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-chloromethyl ketone (DEVD-CMK) clearly blocks Fas-induced apoptosis and increase of nuclear ceramide by inhibiting its effects on SM synthase and nSMase in the nucleus. Therefore, we suggest that ceramide content in the nucleus is increased through caspase-3-dependent activation of nSMase and inhibition of SM synthase in the nucleus, namely regulation of the SM cycle in Fas-induced apoptosis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Reagents.
Human leukemia Jurkat T-lymphoid leukemia cells were obtained from Riken (Saitama, Japan) and were maintained in RPMI 1640 medium containing heat-inactivated 10% fetal bovine serum (JRH Biosciences, Lenexa, KS) and kanamycin sulfate (80 ng/ml) at 37°C in a humidified 5% CO2/95% air atmosphere. The cells in exponentially growing phase were resuspended in 2% serum-containing media at a concentration of 3 x 105 cells/ml and then treated. Anti-Fas antibody (CH-11) was purchased from Medical & Biological Laboratories (Nagoya, Japan). C6-NBD ceramide and C6-NBD sphingomyelin were from Matreya, Inc. (Pleasant Gap, PA). Other chemicals, if manufacturer is not mentioned, were obtained from Sigma Chemical Co. (St. Louis, MO).

Fluorescence-Activated Cell Sorter Analysis Using Annexin V Staining.
A total of 3 x 105 cells were harvested and washed with PBS. To detect apoptotic changes in the position of phosphatidylserine, annexin V binding assay was performed using the ApoAlert Annexin V-FITC kit (Clontech, Palo Alto, CA) according to the manufacturer’s protocol. Fluorescence of 488-nm wavelength was measured with a FACScan (Becton Dickinson, Franklin Lakes, NJ). Ten thousand events generally were monitored, and data analysis was performed using Cell Quest software (Becton Dickinson).

Fluorometric Assay of DEVD-MCA Hydrolyzing Activity.
The cells were treated with anti-Fas antibody at various conditions, harvested, and homogenized in lysis buffer containing 10 mM HEPES/NaOH (pH 7.4), 2 mM EDTA, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, 5 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin A, 0.15 units/ml aprotinin, and 50 µg/ml leupeptin. The lysate was centrifuged at 10,000 x g for 10 min at 4°C. The supernatant was collected and used as enzyme source and added to the reaction mixture [10% sucrose, 10 mM HEPES/NaOH (pH 7.4), 5 mM DTT, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and 10 µM acetyl-Asp-Glu-Val-Asp-a-(4-methyl-coumaryl-7-amide) (DEVD-MCA), followed by incubation at 37°C for 1 h. Fluorescence was measured with a microplate reader (MTP-100F; Corona Electric, Ibaragi, Japan) using 360-nm excitation and 450-nm emission filters. Concentrations of 7-amino-4-methylcoumarin liberated as a result of hydrolysis were calculated compared with standard 7-amino-4-methylcoumarin solutions.

Fractionation of Cells.
Subcellular fractionation was performed by the modified method described previously (29 , 35) . Briefly, the cells (5 x 106/ml) were lysed by passing through a 27-gauge needle in a lysis buffer containing 10 mM HEPES/KOH (pH 7.4), 10 mM KCl, 3 mM MgCl2, 1 mM DTT, 0.2 mM spermine, 0.5 mM spermidine, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin A, 0.15 units/ml aprotinin, and 50 µg/ml leupeptin. After incubation on ice for 15 min, the suspension in lysis buffer was loaded onto 1 M sucrose. The nuclear pellet recovered from centrifugation through the sucrose cushion at 1600 x g for 10 min at 4°C was resuspended in 10 mM HEPES/KOH lysis buffer and recentrifuged at 1600 x g for 10 min at 4°C.

Marker Enzyme Assays.
The 5'-nucleotidase activity was determined by using 200 µg of protein in the sample diluted to a total volume of 1.5 ml with assay buffer. The assay buffer contained 2 mM 5'-AMP, 100 mM KCL, 15 mM MgCl2, and 80 mM glycine-NaOH (pH 9.0). The thiamine pyrophosphatase activity was determined by using 200 µg of protein in the sample diluted to a total volume of 1.5 ml with assay buffer. The assay buffer contained 50 mM sodium barbital (pH 9.0), 15 mM CaCl2, and 3.3 mM thiamine PPI (36) . The acid phosphatase activity was determined by using 200 µg of protein in the sample diluted to a total volume of 1.5 ml with assay buffer. The assay buffer contained 50 mM glycerophosphate buffer (pH 5.5; Ref. 37 ). Addition of perchloric acid (5% w/v) after a 15-min incubation at 37°C stopped this enzyme reaction. These mixtures were centrifuged for 5 min at 6000 x g. For phosphate assessment, 400 µl of the supernatants were used.

Before starting the experiments, the quality of nuclei was examined using an electron microscope (Fig. 1A)Citation , and the purity of the nuclear pellet was assessed by comparison of the activities of the marker enzymes for each compartment as follows. Activities of 5'-nucleotidase, thiamine pyrophosphatase, and acid phosphatase, which were recognized as marker enzymes for plasma membrane, Golgi apparatus, and lysosome, respectively, were not observed in our nuclear fraction before and after treatment with anti-Fas antibody (Fig. 1B)Citation . In addition, KDEL, a specific marker protein for ER, was not detected in our nuclear fraction (Fig. 1C)Citation . We additionally examined the difference of protein and DNA levels between, before, and after Fas treatment. The results did not show any significant difference of protein and DNA levels in whole cells or nuclei between, before, and after Fas treatment (Fig. 1, D and E)Citation . These results clearly demonstrate that the purity and quality of nuclei in the nuclear fraction we used are enough to assess their characteristics as described previously (29 , 35) .



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  		Fig. 1. The purity and quality of our nuclear fraction as judged by electron microscopy (EM), marker enzymes, and the recovery rates of DNA and protein after treatment with anti-Fas antibody. The cells were treated with or without 50 ng/ml anti-Fas antibody for 4 h and harvested for the preparation. A, evaluation of structural purity and quality of whole cells and prepared nuclei by EM. B, marker enzymes for plasma membrane (5'-nucleotidase), Golgi apparatus (thiamine pyrophosphatase), and lysosome (alkaline phosphatase) were measured as described in "Materials and Methods." C, after the cells were fractionated into whole homogenate (W), nuclei (N), and lysate (Ly), KDEL (an endoplastic reticulum marker) and ß-tubulin (a cytoskeletal marker) were detected in each fraction by Western blot analysis. DNA (D) and protein (E) were extracted from whole cells or prepared nuclei, and their recovery rates were measured. These results were obtained from at least three different experiments; error bars, 1 SD. N.S., not statistically significant.

 
Ceramide Measurement.
Lipids in the nuclear pellet were extracted by the method of Bligh and Dyer, and ceramide mass measurement using Escherichia coli DAG kinase, which phosphorylates ceramide to ceramide-1-phosphate, was performed as described previously (1) . The solvent system to separate ceramide-1-phosphate and phosphatidic acid on thin-layer chromatography (TLC) plates consists of chloroform, acetone, methanol, acetic acid, and H2O (ratio, 10:4:3:2:1). To calculate ceramide content, the positive spots on TLC plates were measured using the BASE III image analyzer system (Fuji, Tokyo, Japan).

Sphingomyelin Synthase Assay.
The protein of nuclear pellet, suspended in 10 mM HEPES/KOH lysis buffer, was extracted in the presence of 0.1% Triton X-100, and protein concentration was determined by using the Protein Assay kit (Bio-Rad, Hercules, CA) Nuclei in 10 µl of a lysis buffer, corresponding to 50 µg of protein, then were mixed with a reaction buffer [5 mM Tris/HCl (pH 7.5), 500 µM EDTA, 10 µg/ml C6-NBD-ceramide, and 100 µg/ml phosphatidylcholine (total 100 µl)] and incubated at 37°C for 1 h. The reaction was stopped by the addition of 900 µl H2O and 2 ml chloroform/methanol (2:1; v/v), mixed well, and centrifuged. Lower phase was collected, and solvent was evaporated. Aliquots were applied to TLC plates, and GC was resolved using the solvent system, which contained chloroform, methanol, and 12 mM MgCl2 in H2O (65:25:4; v/v). C6-NBD GC and SM were visualize by UV irradiation and measured by a TLC scanner with fluorometer (excitation = 475 nm; emission = 525 nm).

Sphingomyelinase Assay.
The nuclear pellet was lysed in a buffer containing 10 mM Tris/HCl (pH 7.5), 1 mM EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin A, 0.15 units/ml aprotinin, and 50 µg/ml leupeptin. The lysate was centrifuged at 10,000 x g for 10 min at 4°C. The supernatant was collected and used as nuclear extract. Fifty µg of protein were mixed in a reaction buffer [nSMase: 100 mM Tris/HCl (pH 7.5), 10 mM MgCl2, 5 mM DTT, 10 µM C6-NBD-sphingomyelin, and 0.1% Triton X-100 (total 100 µl); acid SMase: 100 mM acetate buffer (pH 5), 10 µM C6-NBD-sphingosine, and 0.1% Triton X-100 (total 100 µl)] and incubated at 37°C for 2 h. The reaction was stopped by the addition of 900 µl H2O and 2 ml chloroform and methanol (2:1; v/v), mixed well, and centrifuged. Lower phase was collected, and solvent was evaporated. Aliquots were applied to TLC plates, and GC was resolved using the solvent system, which contained chloroform, methanol, and 12 mM MgCl2 in H2O (65:25:4; v/v). C6-NBD ceramide was visualized by UV irradiation and measured by a TLC scanner with fluorometer (excitation = 475 nm; emission = 525 nm).

Confocal Microscopy.
The cells were treated with or without 50 ng/ml anti-Fas antibody for 4 h, harvested, cytospun, and fixed in 10% paraformaldehyde in PBS solution at room temperature for 10 min. Fixed cells were permeabilized with 0.4% saponin in Tris-buffered saline and 0.1% Triton X-100, and nonspecific reactive sites were blocked with 10% chicken egg albumin and 10% polyvinylpyrrolidone 25 in Tris-buffered saline and 0.1% Triton X-100 for 1 h. After washing, cells first were incubated for 1 h at 37°C with mouse anticeramide and rabbit anticalnexin antibody (Calbiochem, San Diego, CA) and then with cyanine-3-conjugated antimouse antibody (red) or FITC-conjugated antirabbit (green), respectively, as secondary antibody for 10 min at 37°C (34) .

Statistical Examination.
Statistical significance was calculated using Student’s t test.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Increases of Ceramide Content in the Nuclear Fraction in Fas-Induced Apoptosis.
As judged by annexin V externalization, Fas cross-linking induced apoptosis of Jurkat T cells in a time- and dose-dependent manner (Fig. 2)Citation . The percentages of annexin V-positive cells were increased from 10% to 12%, 23%, and 33% after 2, 4, and 6 h of treatment, respectively, with 50 ng/ml of anti-Fas antibody.



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  		Fig. 2. Fas-induced apoptosis in Jurkat T cells. Jurkat T cells at an initial number of 3 x 105/ml were treated with 0, 25, or 50 ng/ml of anti-Fas antibody and harvested at the indicated times. Apoptotic cells were determined by annexin V externalization method as described in "Materials and Methods." These results were obtained from at least three different experiments; error bars, 1 SD.

 
By treatment with anti-Fas antibody, ceramide content in the nuclear and microsomal fractions was increased in a time- and dose-dependent manner. As shown in Fig. 3Citation , the control levels of ceramide in the microsomal fraction and nuclear fraction were 5 and 6 pmol/nmol phosphate, respectively. Ceramide was hardly detected in the cytosol fraction (data not shown). These approximately equal amounts of ceramide in the nuclear and microsomal fractions also were confirmed in our previous data using anticeramide antibody (34) . After treatment with 50 ng/ml of anti-Fas antibody for 4 h, ceramide levels in the microsomal fraction and nuclear fraction were increased to ~200% and ~330% of the control level, respectively. Because Fas treatment for 2 h could significantly increase ceramide level in the nuclear fraction (Fig. 3BCitation ; P < 0.05) but hardly induced an increase of apoptosis (Fig. 2)Citation , ceramide increase in the nucleus seemed to precede the execution of apoptosis. In addition, higher increasing rates of nuclear ceramide level (approximately fourfold of the control level) than those of microsomal ceramide level (approximately twofold of the control level) were detected, suggesting a different mechanism of ceramide increase in nuclear fraction from that in microsomal fraction.



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  		Fig. 3. Fas-induced increase of ceramide content in the nucleus. Jurkat T cells at an initial number of 3 x 105/ml were treated with 0 or 50 ng/ml anti-Fas antibody and harvested at the indicated times (A and B) or treated with 0, 25, 50, and 100 ng/ml anti-Fas antibody for 6 h (C). The microsomal (A) and the nuclear (B and C) fractions then were isolated, and lipids were extracted by Bligh and Dyer method. Ceramide contents were measured by diacylglycerol kinase method as described in "Materials and Methods." These results were obtained from at least three different experiments; error bars, 1 SD. D, before and after treatment with 50 ng/ml of anti-Fas antibody for 4 h, cells were fractionated, and the nuclei were prepared for confocal microscopy and stained using anticeramide (red) or anticalnexin (green) antibody as described in "Materials and Methods."

 
Increase of nuclear ceramide by anti-Fas antibody also was confirmed by confocal microscopy using antibodies for ceramide and calnexin, which is reported to be a specific marker for ER (38) . Before treatment with 50 ng/ml of anti-Fas antibody, ceramide recognized by anticeramide antibody (red in Fig. 3DCitation ) was weakly detected in the nuclear region under cell-permeabilizing conditions. After Fas treatment for 4 h, ceramide was detected mainly in the nucleus and partially in the perinuclear region, probably in ER because the yellow in Fig. 3DCitation showing the merge image of ceramide (red) and calnexin (green) signal was more intensified after Fas treatment as compared with the no-treatment control. Because the perinuclear region generally corresponds to ER, increase of ceramide in ER seems to agree with the previous report that Fas treatment increased ceramide via activation of de novo ceramide synthase (39) . The immunohistochemical results using anticeramide antibody here suggest that ceramide content detected by anticeramide antibody is much higher in the nucleus after Fas treatment than at the control level.

Changes of nSMase and SM Synthase Activities in the Microsomal and Nuclear Fraction by Anti-Fas Antibody.
To investigate the possible role of the SM cycle in Fas-increased ceramide in the nucleus, we examined activities of nSMase, a ceramide-generating enzyme, and SM synthase, a ceramide-metabolizing enzyme. As shown in Fig. 4Citation , nSMase activity in the microsomal fraction was increased to 170% of the control level by treatment with 50 ng/ml of anti-Fas antibody for 4 h, whereas its activity in the nuclear fraction was increased to 150% of the control level at 4 h and additionally increased up to 200% of the control at 6 h. The control level of nuclear nSMase (100 ± 10 pmol/mg protein/h) is ~50% of microsomal nSMase (220 ± 45 pmol/mg protein/h). As shown in Fig. 4CCitation , Fas treatment increased the activity of nuclear nSMase in a dose-dependent manner.



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  		Fig. 4. Changes of microsomal and nuclear neutral sphingomyelinase (nSMase) in Fas-induced apoptosis. Jurkat T cells at an initial number of 3 x 105/ml were treated with 0 or 50 ng/ml anti-Fas antibody and harvested at the indicated times (A and B) or treated with 0, 25, and 50 ng/ml anti-Fas antibody for 6 h (C). The microsomal (A) and the nuclear (B and C) fractions then were isolated, and in each fraction, the proteins were extracted as enzyme source. The activity of nSMase was measured as described in "Materials and Methods." These results were obtained from at least three different experiments; error bars, 1 SD. Control levels of microsomal and nuclear nSMases were 220 ± 45 and 100 ± 10 pmol/mg/h, respectively.

 
In contrast to changes of nSMase, SM synthase in the microsomal fraction was not affected up to 6 h after treatment with 50 ng/ml of anti-Fas antibody (Fig. 5A)Citation . However, SM synthase in the nuclear fraction was decreased significantly to 68% and 40% of the control level after 4 h and 6 h, respectively (Fig. 5B)Citation . In addition, anti-Fas antibody decreased the activity of nuclear SM synthase to 57% and 50% of the control level at concentrations of 25 and 50 ng/ml, respectively (Fig. 5C)Citation . Control level of microsomal SM synthase (60 ± 6 pmol/mg protein/h) was similar to that of nuclear SM synthase (65 ± 5 pmol/mg protein/h). These results suggest that SMase and SM synthase are involved in regulation of nuclear ceramide in Fas-induced apoptosis.



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  		Fig. 5. Changes of microsomal and nuclear sphingomyelin (SM) synthase in Fas-induced apoptosis. Jurkat T cells at an initial number of 3 x 105/ml were treated with 0 or 50 ng/ml anti-Fas antibody and harvested at the indicated times (A and B) or treated with 0, 25, and 50 ng/ml anti-Fas antibody for 6 h (C). The microsomal (A) and the nuclear (B and C) fractions then were isolated, and after the proteins were extracted as enzyme source, the activity of SM synthase was measured as described in "Materials and Methods." These results were obtained from at least three different experiments; error bars, 1 SD. Control levels of microsomal and nuclear SM synthases were 60 ± 6 and 65 ± 5 pmol/mg/h, respectively.

 
Enhancement of Fas-Induced Apoptosis and Fas-Decreased SM Synthase Activity in the Nuclear Fraction by Addition of D-609.
To further evaluate the role of nuclear SM synthase in Fas-induced increase of ceramide and induction of apoptosis, we examined the effect of a putative inhibitor of SM synthase, D-609, on Fas-induced ceramide generation in the nuclear fraction. Although D-609 might act as a specific inhibitor of phosphatidylcholine-phospholipase C, it also inhibited the activity of SM synthase in the whole cells (40) . We found that the treatment of Jurkat cells with D-609 at concentrations from 10–20 µg/ml inhibited nuclear SM synthase (Fig. 6A)Citation . Pretreatment with 10 µg/ml of D-609 alone for 6 h inhibited the activity of SM synthase in nuclear fraction to 80% of the control level, and simultaneous treatment with 50 ng/ml of anti-Fas antibody further decreased SM synthase from 50% to 40% of the control level (Fig. 6A)Citation . In contrast, ceramide content was increased in the nuclear fraction from 200–260% of the control level (Fig. 6B)Citation . Pretreatment of 10 µg/ml D-609 with 50 ng/ml of anti-Fas antibody additionally increased apoptosis from 38–60% (Fig. 6C)Citation . Thus, the increase of nuclear ceramide through D-609-induced inhibition of SM synthase was well paralleled with the increase of Fas-induced apoptosis in the presence of D-609. Interestingly, D-609 alone did not increase nuclear ceramide and apoptosis of Jurkat cells, suggesting that inhibition of SM synthase alone may be not enough to accumulate nuclear ceramide unless some stress, such as Fas cross-linking, increases ceramide generation.



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  		Fig. 6. Enhancement by D-609 of Fas-induced inhibition of nuclear sphingomyelin (SM) synthase, and increase of nuclear ceramide and apoptosis. After preincubation for 1 h with 0, 10, or 20 µg/ml of D-609, the cells were treated with 0 or 50 ng/ml anti-Fas antibody for 4 h. The nuclear fraction then was isolated, and lipids and enzyme source were extracted. SM synthase activity (A), ceramide content (B), and apoptosis (C) were examined as described in "Materials and Methods." The results were obtained from at least three different experiments; error bars, 1 SD.

 
Activation of Nuclear Caspase-3 by Fas Cross-Linking.
Although it was already shown by Western blot analysis that cytosolic caspase-3 was cleaved to its active form and moved into the nucleus after Fas treatment, we examined whether caspase-3 was activated in the nucleus after Fas cross-linking (29) . As shown in Fig. 7Citation , the control level of caspase-3 in the nuclear fraction (15 pmol/mg protein/min) was approximately one-third of that in the postnuclear fraction (50 pmol/mg protein/min). Caspase-3 in the nuclear and postnuclear fractions was activated in a time- and dose-dependent manner (Fig. 7)Citation . Interestingly, the increasing pattern of caspase-3 activity in the nuclear fraction was slower compared with that in the postnuclear fraction (Fig. 7, A and C)Citation , suggesting the possible translocation of caspase-3 from the cytosol to the nucleus as described previously (29) .



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  		Fig. 7. Fas-induced activation of caspase-3 in the nucleus. After incubation with 0 and 50 ng/ml anti-Fas antibody for the indicated times (A and C) or with 0, 25, 50, and 100 ng/ml for 6 h (B and D), the cells were harvested, and the microsomal (A and B) and nuclear fractions (C and D) were isolated. Acetyl-Asp-Glu-Val-Asp-a-(4-methyl- coumaryl-7-amide) (DEVD-MCA) hydrolyzing activity was assessed as caspase-3 activity as described in "Materials and Methods." The results were obtained from at least three different experiments; error bars, 1 SD.

 
Caspase-3-Dependent Increase of Ceramide and Regulation of nSMase and SM Synthase in the Nucleus in Fas-Induced Apoptosis.
We also investigated whether caspase-3 activation in the nucleus was required for Fas-induced apoptosis and was involved in the increase of nuclear ceramide. We found that Fas-induced apoptosis was prevented almost completely by 20 µM of the caspase-3 inhibitor DEVD-CMK (Fig. 8C)Citation and that caspase-3 activity in the nucleus, as judged by hydrolysis of DEVD-MCA, also was inhibited, as well as in the postnuclear fraction (Fig. 8, A and B)Citation . With 20 µM DEVD-CMK, Fas-induced increase of nuclear ceramide was returned to the control level (Fig. 9A)Citation , and Fas-induced activation of nSMase and inhibition of SM synthase in the nucleus also were restored to the control levels (Fig. 9, B and C)Citation . These results suggest that increase of ceramide in the nucleus through activation of SMase and inhibition of SM synthase is mediated by caspase-3 activation, which is required for Fas-induced apoptosis.



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  		Fig. 8. Inhibition by acetyl-Asp-Glu-Val-Asp-chloromethyl ketone (DEVD-CMK) of Fas-induced increase of caspase-3 in the nucleus and apoptosis. After preincubation for 1 h with various concentrations of DEVD-CMK (A and B, 0 and 20 µM; C, 0, 10, 20, and 30 µM DEVD-CMK), the cells were treated with 0 or 50 ng/ml anti-Fas antibody for 6 h and fractionated to the microsomal and nuclear fractions. Caspase-3 activities in the microsomal (A) and nuclear (B) fractions then were determined by acetyl-Asp-Glu-Val-Asp-a-(4-methyl-coumaryl-7-amide) (DEVD-MCA) hydrolyzing ability, and apoptosis (C) was assessed by externalization of annexin V as described in "Materials and Methods." The results were obtained from at least three different experiments; error bars, 1 SD.

 


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  		Fig. 9. Restoration by acetyl-Asp-Glu-Val-Asp-chloromethyl ketone (DEVD-CMK) of Fas-induced increase of ceramide content, activation of neutral sphingomyelinase (nSMase) activity, and inhibition of sphingomyelin (SM) synthase in the nucleus. After preincubation for 1 h with 0, 10, or 20 µM of DEVD-CMK, the cells were treated with 0 or 50 ng/ml anti-Fas antibody for 6 h and fractionated to the nuclear fraction. Ceramide content in nuclei (A) then were determined by DGK assay method and nSMase (B) and SM synthase activity (C) in nuclei were measured as described in "Materials and Methods." The results were obtained from at least three different experiments; error bars, 1 SD.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The previous reports mentioned that Fas cross-linking induced early, transient ceramide elevation (in min) at the plasma membrane through Fas-associated death domain-activated acid SMase (41) , and overexpression of factor associated with nSMase activation quickly enhanced nSMase activity localized near the inner leaflet of plasma membrane in the presence of tumor necrosis factor {alpha} (42) . In contrast, others suggested that late, sustained ceramide generation (in h) by nSMase in the plasma membrane was essential for execution of apoptosis (43, 44, 45) . The biological implication of ceramide generation in the outer leaflet of plasma membrane is ambiguous because 10–20% of the SM pool, which responds to stress-induced apoptotic signals, existed even after treatment with sufficient exogenous bacterial SMase (46) . It was reported recently that ceramide generation was increased in mitochondria in Fas-sensitive cells but not in Fas-resistant cells (47) and that ceramide in the nucleus was generated through nSMase in hepatocytes after ligation of the hepatic vein (20) . Thus, the precise regulatory mechanism and topology of ceramide generation still are controversial in stress-induced apoptosis. Therefore, we investigated whether the nuclear compartment is a possible site of ceramide generation in Fas-induced apoptosis.

We showed that ceramide content in the nuclear fraction was increased in a time- and dose-dependent manner after Fas cross-linking of Jurkat T cells (Fig. 3)Citation . Ceramide content measured by diacylglycerol kinase assay in the nuclear fraction was increased to approximately twofold of the no-treatment control level after 2 h of Fas treatment (Fig. 3B)Citation ; at that time, apoptosis was hardly induced (Fig. 2)Citation . These results suggest that apoptosis is preceded by ceramide increase in the nucleus. We also tried to confirm the increase of ceramide content in the nucleus by confocal microscopy using anticeramide antibody, the specificity and sensitivity of which were reported recently (34) . The specificity of this antibody for ceramide in immunohistochemical assay seems not to be controversial because the antibody could recognize accumulation of ceramide in the lysosomes of the fibroblasts from patients with Farber’s disease because of the defect of ceramidase, but the staining corresponding to ceramide was hardly detected in the corrected fibroblasts by overexpression of ceramidase.5 As shown in Fig. 3DCitation , Fas-induced increase of ceramide (red) was evident in the nucleus as compared with the no-treatment control under the cell-permeable condition of confocal microscopy. The green of calnexin (an ER marker) turned to yellow after merging with the red of ceramide (Fig. 3D)Citation , suggesting increase of ceramide in the nucleus and, at least in part, in the ER after Fas cross-linking. The results of confocal microscopy may support the previous findings of ceramide generation in ER through de novo ceramide synthase in Fas-induced apoptosis, but these results never conflict with the increase of ceramide in the nucleus as we suggest here (48) . Additionally, confirmation of the purity and quality of nuclei (Fig. 1)Citation leaves us no doubt that ceramide content is increased in the nucleus by Fas treatment.

As a mechanism of ceramide increase in the nucleus, we found that Fas induced increase of nSMase activity in the nuclear and microsomal fractions (Fig. 4)Citation . These results agree with the reports that ceramide was increased through activation of nuclear nSMase in an in vivo model of hepatic cell apoptosis (20) and in irradiation-induced apoptosis of leukemia cells (49) . However, we interestingly found that the activity of SM synthase in the nucleus was inhibited along with activation of nSMase after Fas cross-linking (Fig. 5)Citation , suggesting the role of ceramide generation through regulation of the SM cycle in the nucleus in apoptosis. This notion also was supported by the data that the putative SM synthase inhibitor D-609 (40) , which blocks the activity of nuclear SM synthase (Fig. 6A)Citation , enhanced Fas-induced increase of ceramide and apoptosis (Fig. 6, B and C)Citation .

It is unclear how nuclear ceramide induces nuclear events in apoptosis. Among the bioactive lipids, DAG is recognized as a counterpart molecule of proapoptotic ceramide because of the similarity of its chemical structure and antiapoptotic action (50) . DAG is increased in the nucleus through activation of phosphatidylcholine-phospholipase C by treatment with insulin or tumor promoter 12-O-tetradecanoylphorbol-13-acetate (51 , 52) , and phosphoinositide hydrolysis and DAG production were increased by platelet-activating factor in isolated rat liver nuclei (53) . Thus, after hydrolysis of phosphatidylcholine or phosphoinositide in the nucleus, a generated DAG may mediate cell growth and survival by activating antiapoptotic molecules, such as protein kinase C, phosphoinositide 3 kinase/Akt kinase, and telomerase (52 , 54, 55, 56, 57) . In contrast, ceramide is known to suppress the function of these antiapoptotic molecules (57, 58, 59) . Therefore, the proapoptotic action of ceramide in the nucleus may be related closely to its inhibition against DAG-activated antiapoptotic signals. If this is the case, the role of SM synthase seems to be more critical to modulate the balance between apoptosis and growth and survival because inhibition of this enzyme simultaneously causes an increase of proapoptotic ceramide and a decrease of prosurvival DAG.

The active form of caspase-3, after cleavage of procaspase-3 in the cytosol, translocated into the nucleus and induced nuclear events in Fas-induced apoptosis (29 , 35 , 60) . Nuclear translocation of active caspase-3 also was found in apoptosis of butyric acid-induced colonic epithelial cells (31) and neuroblastoma cells (32) . We showed that enzymatic activity of caspase-3 in the nuclear fraction was time- and dose-dependently increased after Fas cross-linking (Fig. 7)Citation . Because the targets of active caspase-3 in the nucleus, several molecules, such as gelsolin, fodrin, lamin B, inhibitor of caspase-activated DNase, poly(ADP-ribose)polymerases, and DNA-dependent protein kinases, were known (29 , 60 , 61) . Although Bourteele et al. (62) reported previously that tumor necrosis factor {alpha}-induced inhibition of GC synthase and SM synthase activity in the microsomal fraction of Kym-1 rhabdomyosarcoma cells was blocked by the caspase-3 inhibitor DEVD-CMK, the relation of caspase-3 and the ceramide-generating system in the nucleus has not yet been clarified. As shown in Fig. 8Citation and Fig. 9Citation , DEVD-CMK blocked Fas-induced activation of caspase-3, generation of ceramide, and regulation of nSMase and SM synthase in the nucleus, suggesting caspase-3-dependent increase of ceramide through activation of nSMase and inhibition of SM synthase in the nucleus. Although the precise mechanism of caspase-3 to regulate ceramide-related enzymes is unknown, it is likely that caspase-3 activates nSMase by induction of Bcl-2 cleavage because Bcl-2 is reported to inhibit nSMase activity through reduction of glutathione in the nucleus (63, 64, 65, 66) .

In this work, it is suggested that the nucleus is a novel site of ceramide increase in Fas-induced apoptosis and that ceramide content in the nucleus is regulated through caspase-3-dependent activation of nSMase and inhibition of SM synthase. In the future, the mechanism by which caspase-3 regulates the SM cycle consisting of nSMase and SM synthase, and whether and how the increase of ceramide in the nucleus is related to apoptosis-executing machinery (e.g., regulation of cell cycle, DNA synthesis, and RNA translation), should be investigated.


   ACKNOWLEDGMENTS
 
We thank Hideaki Hori, Department of Medicine, Osaka Dental University, for expert technical assistance with the electron microscopy studies.


   FOOTNOTES
 
Grant support: Japanese Ministry of Education, Culture, Sports, Science, and Technology Grant 12470199 (to T. Okazaki).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Toshiro Okazaki, Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, 54 Syogoin-Kawaramachi, Sakyo-ku, Kyoto 606-8507, Japan. Phone/Fax: 81-75-751-3154; E-mail: toshiroo{at}kuhp.kyoto-u.ac.jp

5 Unpublished observations. Back

Received 5/15/03. Revised 10/13/03. Accepted 12/ 1/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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