Increase of Nuclear Ceramide Through Caspase-3-Dependent Regulation of the “Sphingomyelin Cycle” in Fas-Induced Apoptosis

[CANCER RESEARCH 64, 1000–1007, February 1, 2004] Increase of Nuclear Ceramide through Caspase-3-Dependent Regulation of the “Sphingomyelin Cycle” in Fas-Induced Apoptosis

Mitsumasa Watanabe,1 Toshiyuki Kitano,1 Tadakazu Kondo,1 Takeshi Yabu,1 Yoshimitsu Taguchi,1,3 Masaro Tashima,1 Hisanori Umehara,2 Naochika Domae,4 Takashi Uchiyama,1 and Toshiro Okazaki1 Departments of 1Hematology and Oncology and 2Rheumatology and Clinical Immunology, Graduate School of Medicine, 3Graduate School of Biostudies, Kyoto University, Kyoto, and 4Department of Medicine, Osaka Dental University, Osaka, Japan

ABSTRACT (N-myristoylamino)-1-phenyl-1-propanol, which increases ceramide by blocking the deacylation of ceramide to sphingosine, enhanced Regardless of the existence of ceramide-related molecules, such as apoptosis in human leukemia HL-60 cells (11). It also was reported sphingomyelin (SM), neutral sphingomyelinase (nSMase), and SM syn- that the activity of GC synthase, which converts ceramide to GC, was thase, in the nucleus, the regulation of ceramide in the nucleus is poorly understood in stress-induced apoptosis. In Fas-induced Jurkat T-cell apo- more increased in apoptosis-resistant cells than in apoptosis-sensitive ␬ ptosis, we found a time- and dose-dependent increase of ceramide content cells (12–14). The involvement of SM synthase in nuclear factor B in the nuclear and microsomal fractions. Fas-induced increase of ceramide activation of transformed fibroblast and basic fibroblast growth fac- content in the nucleus also was detected by confocal microscopy using tor-induced proliferation of primary astrocytes was suggested re- anticeramide antibody. Activation of nSMase and inhibition of SM syn- cently, but the implication of SM synthase has yet to be clarified in thase were evident in the nuclear fraction after Fas cross-linking, whereas Fas-induced apoptosis (15, 16). nSMase was activated, but SM synthase was not affected, in the microso- In terms of the topology of ceramide generation, SM hydrolysis in mal fraction. Pretreatment with D-609, a putative SM synthase inhibitor, plasma membrane by nSMase was first reported to be involved in enhanced Fas-induced increase of ceramide in the nucleus and induction apoptosis (17), and lysosome, endoplasmic reticulum (ER), and mi- of apoptosis along with increase of Fas-induced inhibition of nuclear SM synthase. Fas-induced activation of caspase-3 was detected in the nuclear tochondria then were proposed as possible sites of ceramide genera- 5 fraction and in whole cell lysate. A caspase-3 inhibitor, acetyl-Asp-Glu- tion (18, 19). Increase of ceramide through activation of nSMase in Val-Asp-chloromethyl ketone, blocked not only Fas-induced increases of the nucleus of hepatocytes was reported in the animal model of apoptosis and ceramide content but also Fas-induced activation of nSMase hepatic vein ligation (20), and addition of ceramide-treated cell extract and inhibition of SM synthase in the nuclear fraction. Taken together, it was shown to induce nuclear apoptotic events in the reconstituted is suggested that the nucleus is a site for ceramide increase and caspase-3 condition (21). In addition, the activities of nSMase and SM synthase activation in Fas-induced Jurkat T-cell apoptosis and that caspase-3- were found in rat nuclear membrane together with the existence of dependent regulation of the “SM cycle” consisting of nSMase and SM phosphatidylcholine and diacylglycerol (DAG; Refs. 22–25). These synthase plays a role in Fas-induced ceramide increase in the nucleus. results suggest the existence of the “SM cycle,” consisting of nSMase and SM synthase in the nucleus (1). However, ceramide regulation INTRODUCTION through the SM cycle in the nucleus still is unknown in Fas-induced apoptosis. Ceramide has been recognized as an intracellular lipid mediator Fas-induced apoptosis is known to show the typical and morpho- related to a variety of cell functions, including cell differentiation, logic changes in the nucleus, such as chromatin condensation and secretion, senescence, and apoptosis (1–3). A diverse array of stresses, DNA fragmentation. Activation of proteolytic molecules, including ␣ including Fas cross-linking, tumor necrosis factor , irradiation, heat caspases, granzyme B, cathepsin D, and histone-associated protease, shock, and anticancer drugs (4), were reported to increase intracellular is reported to play a role in execution of apoptosis (26). Among them, ceramide in induction of apoptosis (4–7). Generation of ceramide caspase-3 seems to be critical to nuclear changes in apoptosis because through many kinds of ceramide-related enzymes, such as sphingo- caspase-3-deficient breast cancer cells (MCF-7) underwent cell death myelinase (SMase), ceramidase, de novo ceramide synthase, and without chromatin condensation and DNA fragmentation (27). In glucosylceramide (GC) synthase, has been shown to play a role in Fas-induced apoptosis, active caspase-3 was shown to translocate apoptosis. For example, acid SMase-deficient human lymphoblasts Ϫ/Ϫ from the cytosol to the nucleus and to cleave nuclear proteins, such as from acid SMase mice were found to be resistant to the ionizing lamins and poly(ADP-ribose)polymerases (28–30). The translocation radiation-induced apoptosis because of a lack of ceramide generation of active caspase-3 to the nucleus also was reported in apoptosis of from sphingomyelin (SM; Ref. 6). Although the indispensable role of colonic epithelial cells (31) and neuroblastoma cells (32), and a acid SMase also was suggested in Fas-induced apoptosis (8), its role recombinant, active caspase-3 was shown to induce apoptotic changes still is controversial because Niemann-Pick disease-derived lympho- of the nucleus in a cell-free system (33). However, it remains to be cytes, which show a low activity of acid SMase because of its genetic clarified whether active caspase-3 is involved in regulation of nuclear defect, were reported to be sensitive to Fas treatment (9). Exogenous ceramide content in Fas-induced apoptosis. neutral sphingomyelinase (nSMase) and overexpression of bacterial Therefore, we investigate whether and how ceramide generation in nSMase in mammalian cells induced apoptosis with excess of ceram- the nucleus is regulated in Fas-induced apoptosis and, if this is the ide increase (10), and the ceramidase inhibitor (1S, 2R)-D-erythro-2- case, how Fas-activated caspase-3 is involved in regulation of nuclear ceramide. Our results show that ceramide content in nuclear fraction Received 5/15/03; revised 10/13/03; accepted 12/1/03. Grant support: Japanese Ministry of Education, Culture, Sports, Science, and Tech- is increased through activation of nSMase and inhibition of SM nology Grant 12470199 (to T. Okazaki). synthase in Fas-induced Jurkat T-cell apoptosis. Increase of ceramide The costs of publication of this article were defrayed in part by the payment of page in the nucleus by Fas treatment also is shown by confocal microscopy charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. using anticeramide antibody (34). Activation of nuclear caspase-3 is Requests for reprints: Toshiro Okazaki, Department of Hematology and Oncology, detected after Fas cross-linking, and the caspase-3 inhibitor acetyl- Graduate School of Medicine, Kyoto University, 54 Syogoin-Kawaramachi, Sakyo-ku, Kyoto 606-8507, Japan. Phone/Fax: 81-75-751-3154; E-mail: [email protected] u.ac.jp. 5 Unpublished observations. 1000

Asp-Glu-Val-Asp-chloromethyl ketone (DEVD-CMK) clearly blocks pepstatin A, 0.15 units/ml aprotinin, and 50 ␮g/ml leupeptin. After incubation Fas-induced apoptosis and increase of nuclear ceramide by inhibiting on ice for 15 min, the suspension in lysis buffer was loaded onto 1 M sucrose. its effects on SM synthase and nSMase in the nucleus. Therefore, we The nuclear pellet recovered from centrifugation through the sucrose cushion suggest that ceramide content in the nucleus is increased through at 1600 ϫ g for 10 min at 4°C was resuspended in 10 mM HEPES/KOH lysis ϫ caspase-3-dependent activation of nSMase and inhibition of SM syn- buffer and recentrifuged at 1600 g for 10 min at 4°C. Ј thase in the nucleus, namely regulation of the SM cycle in Fas- Marker Enzyme Assays. The 5 -nucleotidase activity was determined by using 200 ␮g of protein in the sample diluted to a total volume of 1.5 ml with induced apoptosis. assay buffer. The assay buffer contained 2 mM 5Ј-AMP, 100 mM KCL, 15 mM

MgCl2, and 80 mM glycine-NaOH (pH 9.0). The thiamine pyrophosphatase MATERIALS AND METHODS activity was determined by using 200 ␮g of protein in the sample diluted to a total volume of 1.5 ml with assay buffer. The assay buffer contained 50 mM Cell Culture and Reagents. Human leukemia Jurkat T-lymphoid leukemia sodium barbital (pH 9.0), 15 mM CaCl2, and 3.3 mM thiamine PPI (36). The cells were obtained from Riken (Saitama, Japan) and were maintained in RPMI acid phosphatase activity was determined by using 200 ␮g of protein in the 1640 medium containing heat-inactivated 10% fetal bovine serum (JRH Bio- sample diluted to a total volume of 1.5 ml with assay buffer. The assay buffer sciences, Lenexa, KS) and kanamycin sulfate (80 ng/ml) at 37°C in a humid- contained 50 mM glycerophosphate buffer (pH 5.5; Ref. 37). Addition of ified 5% CO2/95% air atmosphere. The cells in exponentially growing phase perchloric acid (5% w/v) after a 15-min incubation at 37°C stopped this ϫ 5 were resuspended in 2% serum-containing media at a concentration of 3 10 enzyme reaction. These mixtures were centrifuged for 5 min at 6000 ϫ g. For cells/ml and then treated. Anti-Fas antibody (CH-11) was purchased from phosphate assessment, 400 ␮l of the supernatants were used. Medical & Biological Laboratories (Nagoya, Japan). C -NBD ceramide and 6 Before starting the experiments, the quality of nuclei was examined using an C -NBD sphingomyelin were from Matreya, Inc. (Pleasant Gap, PA). Other 6 electron microscope (Fig. 1A), and the purity of the nuclear pellet was assessed chemicals, if manufacturer is not mentioned, were obtained from Sigma by comparison of the activities of the marker enzymes for each compartment Chemical Co. (St. Louis, MO). as follows. Activities of 5Ј-nucleotidase, thiamine pyrophosphatase, and acid Fluorescence-Activated Cell Sorter Analysis Using Annexin V Staining. phosphatase, which were recognized as marker enzymes for plasma mem- A total of 3 ϫ 105 cells were harvested and washed with PBS. To detect brane, Golgi apparatus, and lysosome, respectively, were not observed in our apoptotic changes in the position of phosphatidylserine, annexin V binding nuclear fraction before and after treatment with anti-Fas antibody (Fig. 1B). In assay was performed using the ApoAlert Annexin V-FITC kit (Clontech, Palo addition, KDEL, a specific marker protein for ER, was not detected in our Alto, CA) according to the manufacturer’s protocol. Fluorescence of 488-nm nuclear fraction (Fig. 1C). We additionally examined the difference of protein wavelength was measured with a FACScan (Becton Dickinson, Franklin and DNA levels between, before, and after Fas treatment. The results did not Lakes, NJ). Ten thousand events generally were monitored, and data analysis was performed using Cell Quest software (Becton Dickinson). show any significant difference of protein and DNA levels in whole cells or Fluorometric Assay of DEVD-MCA Hydrolyzing Activity. The cells nuclei between, before, and after Fas treatment (Fig. 1, D and E). These results were treated with anti-Fas antibody at various conditions, harvested, and clearly demonstrate that the purity and quality of nuclei in the nuclear fraction we used are enough to assess their characteristics as described previously homogenized in lysis buffer containing 10 mM HEPES/NaOH (pH 7.4), 2 mM EDTA, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic (29, 35). acid, 5 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10 ␮g/ml pepstatin A, Ceramide Measurement. Lipids in the nuclear pellet were extracted by 0.15 units/ml aprotinin, and 50 ␮g/ml leupeptin. The lysate was centrifuged at the method of Bligh and Dyer, and ceramide mass measurement using Esch- 10,000 ϫ g for 10 min at 4°C. The supernatant was collected and used as erichia coli DAG kinase, which phosphorylates ceramide to ceramide-1- enzyme source and added to the reaction mixture [10% sucrose, 10 mM phosphate, was performed as described previously (1). The solvent system to HEPES/NaOH (pH 7.4), 5 mM DTT, 0.1% 3-[(3-cholamidopropyl)dimethyl- separate ceramide-1-phosphate and phosphatidic acid on thin-layer chroma- ammonio]-1-propanesulfonic acid, and 10 ␮M acetyl-Asp-Glu-Val-Asp-a-(4- tography (TLC) plates consists of chloroform, acetone, methanol, acetic acid, methyl-coumaryl-7-amide) (DEVD-MCA), followed by incubation at 37°C for and H2O (ratio, 10:4:3:2:1). To calculate ceramide content, the positive spots 1 h. Fluorescence was measured with a microplate reader (MTP-100F; Corona on TLC plates were measured using the BASE III image analyzer system (Fuji, Electric, Ibaragi, Japan) using 360-nm excitation and 450-nm emission filters. Tokyo, Japan). Concentrations of 7-amino-4-methylcoumarin liberated as a result of hydrol- Sphingomyelin Synthase Assay. The protein of nuclear pellet, suspended ysis were calculated compared with standard 7-amino-4-methylcoumarin so- in 10 mM HEPES/KOH lysis buffer, was extracted in the presence of 0.1% lutions. Triton X-100, and protein concentration was determined by using the Protein Fractionation of Cells. Subcellular fractionation was performed by the Assay kit (Bio-Rad, Hercules, CA) Nuclei in 10 ␮l of a lysis buffer, corre- modified method described previously (29, 35). Briefly, the cells (5 ϫ 106/ml) sponding to 50 ␮g of protein, then were mixed with a reaction buffer [5 mM ␮ ␮ were lysed by passing through a 27-gauge needle in a lysis buffer containing Tris/HCl (pH 7.5), 500 M EDTA, 10 g/ml C6-NBD-ceramide, and 100 ␮ ␮ 10 mM HEPES/KOH (pH 7.4), 10 mM KCl, 3 mM MgCl2,1mM DTT, 0.2 mM g/ml phosphatidylcholine (total 100 l)] and incubated at 37°Cfor1h.The ␮ ␮ spermine, 0.5 mM spermidine, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml reaction was stopped by the addition of 900 lH2O and 2 ml chloroform/

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 (5Ј-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 ␤-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. 1001

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 ϭ 475 nm; emission ϭ 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 ␮g/ml pepstatin A, 0.15 units/ml aprotinin, and 50 ␮g/ml leupeptin. The lysate was centrifuged at 10,000 ϫ g for 10 min at 4°C. The supernatant was collected and used as nuclear extract. Fifty ␮gof protein were mixed in a reaction buffer [nSMase: 100 mM Tris/HCl (pH 7.5), ␮ 10 mM MgCl2,5mM DTT, 10 M C6-NBD-sphingomyelin, and 0.1% Triton X-100 (total 100 ␮l); acid SMase: 100 mM acetate buffer (pH 5), 10 ␮M C6-NBD-sphingosine, and 0.1% Triton X-100 (total 100 ␮l)] and incubated at ␮ 37°C for 2 h. The reaction was stopped by the addition of 900 lH2O 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 ϭ 475 nm; emission ϭ 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% paraformal- dehyde 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 for1hat37°C Fig. 2. Fas-induced apoptosis in Jurkat T cells. Jurkat T cells at an initial number of with mouse anticeramide and rabbit anticalnexin antibody (Calbiochem, San 3 ϫ 105/ml were treated with 0, 25, or 50 ng/ml of anti-Fas antibody and harvested at the Diego, CA) and then with cyanine-3-conjugated antimouse antibody (red) or indicated times. Apoptotic cells were determined by annexin V externalization method as FITC-conjugated antirabbit (green), respectively, as secondary antibody for 10 described in “Materials and Methods.” These results were obtained from at least three different experiments; error bars, 1 SD. min at 37°C (34). Statistical Examination. Statistical significance was calculated using Stu- dent’s t test. firmed by confocal microscopy using antibodies for ceramide and RESULTS 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 Increases of Ceramide Content in the Nuclear Fraction in anticeramide antibody (red in Fig. 3D) was weakly detected in the Fas-Induced Apoptosis. As judged by annexin V externalization, nuclear region under cell-permeabilizing conditions. After Fas treat- Fas cross-linking induced apoptosis of Jurkat T cells in a time- and ment for 4 h, ceramide was detected mainly in the nucleus and dose-dependent manner (Fig. 2). The percentages of annexin V- partially in the perinuclear region, probably in ER because the yellow positive cells were increased from 10% to 12%, 23%, and 33% after in Fig. 3D showing the merge image of ceramide (red) and calnexin 2,4,and6hoftreatment, respectively, with 50 ng/ml of anti-Fas (green) signal was more intensified after Fas treatment as compared antibody. with the no-treatment control. Because the perinuclear region gener- By treatment with anti-Fas antibody, ceramide content in the nu- ally corresponds to ER, increase of ceramide in ER seems to agree clear and microsomal fractions was increased in a time- and dose- with the previous report that Fas treatment increased ceramide via dependent manner. As shown in Fig. 3, the control levels of ceramide activation of de novo ceramide synthase (39). The immunohistochem- in the microsomal fraction and nuclear fraction were 5 and 6 pmol/ ical results using anticeramide antibody here suggest that ceramide nmol phosphate, respectively. Ceramide was hardly detected in the content detected by anticeramide antibody is much higher in the cytosol fraction (data not shown). These approximately equal amounts nucleus after Fas treatment than at the control level. of ceramide in the nuclear and microsomal fractions also were con- Changes of nSMase and SM Synthase Activities in the Micro- firmed in our previous data using anticeramide antibody (34). After somal and Nuclear Fraction by Anti-Fas Antibody. To investigate treatment with 50 ng/ml of anti-Fas antibody for 4 h, ceramide levels the possible role of the SM cycle in Fas-increased ceramide in the in the microsomal fraction and nuclear fraction were increased to nucleus, we examined activities of nSMase, a ceramide-generating ϳ200% and ϳ330% of the control level, respectively. Because Fas enzyme, and SM synthase, a ceramide-metabolizing enzyme. As treatment for 2 h could significantly increase ceramide level in the shown in Fig. 4, nSMase activity in the microsomal fraction was nuclear fraction (Fig. 3B; P Ͻ 0.05) but hardly induced an increase of increased to 170% of the control level by treatment with 50 ng/ml of apoptosis (Fig. 2), ceramide increase in the nucleus seemed to precede anti-Fas antibody for 4 h, whereas its activity in the nuclear fraction the execution of apoptosis. In addition, higher increasing rates of was increased to 150% of the control level at 4 h and additionally nuclear ceramide level (approximately fourfold of the control level) increased up to 200% of the control at 6 h. The control level of nuclear than those of microsomal ceramide level (approximately twofold of nSMase (100 Ϯ 10 pmol/mg protein/h) is ϳ50% of microsomal the control level) were detected, suggesting a different mechanism of nSMase (220 Ϯ 45 pmol/mg protein/h). As shown in Fig. 4C, Fas ceramide increase in nuclear fraction from that in microsomal frac- treatment increased the activity of nuclear nSMase in a dose-depen- tion. dent manner. Increase of nuclear ceramide by anti-Fas antibody also was con- In contrast to changes of nSMase, SM synthase in the microsomal 1002

Fig. 3. Fas-induced increase of ceramide content in the nucleus. Jurkat T cells at an initial number of 3 ϫ 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 for6h(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.” fraction was not affected up to 6 h after treatment with 50 ng/ml of synthase (65 Ϯ 5 pmol/mg protein/h). These results suggest that anti-Fas antibody (Fig. 5A). However, SM synthase in the nuclear SMase and SM synthase are involved in regulation of nuclear ceram- fraction was decreased significantly to 68% and 40% of the control ide in Fas-induced apoptosis. level after 4 h and 6 h, respectively (Fig. 5B). In addition, anti-Fas Enhancement of Fas-Induced Apoptosis and Fas-Decreased SM antibody decreased the activity of nuclear SM synthase to 57% and Synthase Activity in the Nuclear Fraction by Addition of D-609. 50% of the control level at concentrations of 25 and 50 ng/ml, To further evaluate the role of nuclear SM synthase in Fas-induced respectively (Fig. 5C). Control level of microsomal SM synthase increase of ceramide and induction of apoptosis, we examined the Ϯ (60 6 pmol/mg protein/h) was similar to that of nuclear SM 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 ␮g/ml inhibited nuclear SM synthase (Fig. 6A). Pretreat- ment with 10 ␮g/ml of D-609 alone for 6 h inhibited the activity of SM synthase in nuclear fraction to 80% of the control level, and simulta- neous treatment with 50 ng/ml of anti-Fas antibody further decreased SM synthase from 50% to 40% of the control level (Fig. 6A). In contrast, ceramide content was increased in the nuclear fraction from 200–260% of the control level (Fig. 6B). Pretreatment of 10 ␮g/ml D-609 with 50 ng/ml of anti-Fas antibody additionally increased apoptosis from 38–60% (Fig. 6C). 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 Fig. 4. Changes of microsomal and nuclear neutral sphingomyelinase (nSMase)in Fas-induced apoptosis. Jurkat T cells at an initial number of 3 ϫ 105/ml were treated with of D-609. Interestingly, D-609 alone did not increase nuclear ceramide 0 or 50 ng/ml anti-Fas antibody and harvested at the indicated times (A and B) or treated and apoptosis of Jurkat cells, suggesting that inhibition of SM syn- with 0, 25, and 50 ng/ml anti-Fas antibody for6h(C). The microsomal (A) and the nuclear (B and C) fractions then were isolated, and in each fraction, the proteins were extracted thase alone may be not enough to accumulate nuclear ceramide unless as enzyme source. The activity of nSMase was measured as described in “Materials and some stress, such as Fas cross-linking, increases ceramide generation. Methods.” These results were obtained from at least three different experiments; error bars, 1 SD. Control levels of microsomal and nuclear nSMases were 220 Ϯ 45 and Activation of Nuclear Caspase-3 by Fas Cross-Linking. Al- 100 Ϯ 10 pmol/mg/h, respectively. though it was already shown by Western blot analysis that cytosolic 1003

judged by hydrolysis of DEVD-MCA, also was inhibited, as well as in the postnuclear fraction (Fig. 8, A and B). With 20 ␮M DEVD- CMK, Fas-induced increase of nuclear ceramide was returned to the control level (Fig. 9A), 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). 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.

DISCUSSION The previous reports mentioned that Fas cross-linking induced early, transient ceramide elevation (in min) at the plasma membrane Fig. 5. Changes of microsomal and nuclear sphingomyelin (SM) synthase in Fas- through Fas-associated death domain-activated acid SMase (41), and induced apoptosis. Jurkat T cells at an initial number of 3 ϫ 105/ml were treated with 0 overexpression of factor associated with nSMase activation quickly or 50 ng/ml anti-Fas antibody and harvested at the indicated times (A and B) or treated enhanced nSMase activity localized near the inner leaflet of plasma with 0, 25, and 50 ng/ml anti-Fas antibody for6h(C). The microsomal (A) and the nuclear (B and C) fractions then were isolated, and after the proteins were extracted as enzyme membrane in the presence of tumor necrosis factor ␣ (42). In contrast, source, the activity of SM synthase was measured as described in “Materials and others suggested that late, sustained ceramide generation (in h) by 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 Ϯ 6 and nSMase in the plasma membrane was essential for execution of 65 Ϯ 5 pmol/mg/h, respectively. apoptosis (43–45). 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-

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 ␮g/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. 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. 7, 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). 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), suggesting the possible translocation of caspase-3 from the cytosol to the nucleus as described previously (29). Caspase-3-Dependent Increase of Ceramide and Regulation of nSMase and SM Synthase in the Nucleus in Fas-Induced Apo- ptosis. We also investigated whether caspase-3 activation in the nu- 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 cleus was required for Fas-induced apoptosis and was involved in the ng/ml for6h(B and D), the cells were harvested, and the microsomal (A and B) and increase of nuclear ceramide. We found that Fas-induced apoptosis 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 was prevented almost completely by 20 M of the caspase-3 inhibitor as described in “Materials and Methods.” The results were obtained from at least three DEVD-CMK (Fig. 8C) and that caspase-3 activity in the nucleus, as different experiments; error bars, 1 SD. 1004

It is unclear how nuclear ceramide induces nuclear events in apoptosis. Among the bioactive lipids, DAG is recognized as a counter- part 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-tetradecanoyl- phorbol-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 medi- ate cell growth and survival by activating antiapoptotic molecules, such as protein kinase C, phosphoinositide 3 kinase/Akt kinase, and telomerase (52, 54–57). In contrast, ceramide is known to suppress the function of these antiapoptotic molecules (57–59). Therefore, the proapoptotic action of ceramide in the nucleus may be related closely Fig. 8. Inhibition by acetyl-Asp-Glu-Val-Asp-chloromethyl ketone (DEVD-CMK)of to its inhibition against DAG-activated antiapoptotic signals. If this is Fas-induced increase of caspase-3 in the nucleus and apoptosis. After preincubation for the case, the role of SM synthase seems to be more critical to 1 h with various concentrations of DEVD-CMK (A and B, 0 and 20 ␮M; C, 0, 10, 20, and 30 ␮M DEVD-CMK), the cells were treated with 0 or 50 ng/ml anti-Fas antibody for 6 h modulate the balance between apoptosis and growth and survival and fractionated to the microsomal and nuclear fractions. Caspase-3 activities in the because inhibition of this enzyme simultaneously causes an increase 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) of proapoptotic ceramide and a decrease of prosurvival DAG. was assessed by externalization of annexin V as described in “Materials and Methods.” The active form of caspase-3, after cleavage of procaspase-3 in the The results were obtained from at least three different experiments; error bars, 1 SD. cytosol, translocated into the nucleus and induced nuclear events in Fas-induced apoptosis (29, 35, 60). Nuclear translocation of active linking of Jurkat T cells (Fig. 3). Ceramide content measured by caspase-3 also was found in apoptosis of butyric acid-induced colonic diacylglycerol kinase assay in the nuclear fraction was increased to epithelial cells (31) and neuroblastoma cells (32). We showed that approximately twofold of the no-treatment control level after2hof enzymatic activity of caspase-3 in the nuclear fraction was time- and Fas treatment (Fig. 3B); at that time, apoptosis was hardly induced dose-dependently increased after Fas cross-linking (Fig. 7). Because (Fig. 2). These results suggest that apoptosis is preceded by ceramide the targets of active caspase-3 in the nucleus, several molecules, such increase in the nucleus. We also tried to confirm the increase of as gelsolin, fodrin, lamin B, inhibitor of caspase-activated DNase, ceramide content in the nucleus by confocal microscopy using anti- poly(ADP-ribose)polymerases, and DNA-dependent protein kinases, ceramide antibody, the specificity and sensitivity of which were were known (29, 60, 61). Although Bourteele et al. (62) reported reported recently (34). The specificity of this antibody for ceramide in previously that tumor necrosis factor ␣-induced inhibition of GC immunohistochemical assay seems not to be controversial because the synthase and SM synthase activity in the microsomal fraction of antibody could recognize accumulation of ceramide in the lysosomes Kym-1 rhabdomyosarcoma cells was blocked by the caspase-3 inhib- of the fibroblasts from patients with Farber’s disease because of the itor DEVD-CMK, the relation of caspase-3 and the ceramide-gener- defect of ceramidase, but the staining corresponding to ceramide was ating system in the nucleus has not yet been clarified. As shown in hardly detected in the corrected fibroblasts by overexpression of Fig. 8 and Fig. 9, DEVD-CMK blocked Fas-induced activation of ceramidase.5 As shown in Fig. 3D, Fas-induced increase of ceramide caspase-3, generation of ceramide, and regulation of nSMase and SM (red) was evident in the nucleus as compared with the no-treatment synthase in the nucleus, suggesting caspase-3-dependent increase of control under the cell-permeable condition of confocal microscopy. ceramide through activation of nSMase and inhibition of SM synthase The green of calnexin (an ER marker) turned to yellow after merging with the red of ceramide (Fig. 3D), 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). Addition- ally, confirmation of the purity and quality of nuclei (Fig. 1) 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). 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 Fig. 9. Restoration by acetyl-Asp-Glu-Val-Asp-chloromethyl ketone (DEVD-CMK)of activation of nSMase after Fas cross-linking (Fig. 5), suggesting the Fas-induced increase of ceramide content, activation of neutral sphingomyelinase (nS- role of ceramide generation through regulation of the SM cycle in the Mase) activity, and inhibition of sphingomyelin (SM) synthase in the nucleus. After preincubation for 1 h with 0, 10, or 20 ␮M of DEVD-CMK, the cells were treated with 0 nucleus in apoptosis. This notion also was supported by the data that or 50 ng/ml anti-Fas antibody for 6 h and fractionated to the nuclear fraction. Ceramide the putative SM synthase inhibitor D-609 (40), which blocks the 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 activity of nuclear SM synthase (Fig. 6A), enhanced Fas-induced Methods.” The results were obtained from at least three different experiments; error bars, increase of ceramide and apoptosis (Fig. 6, B and C). 1 SD. 1005

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2004 American Association for Cancer Research. Fas-INDUCED INCREASE OF NUCLEAR CERAMIDE in the nucleus. Although the precise mechanism of caspase-3 to 20. Tsugane, K., Tamiya, K. K., Nagino, M., Nimura, Y., and Yoshida, S. A possible role regulate ceramide-related enzymes is unknown, it is likely that of nuclear ceramide and sphingosine in hepatocyte apoptosis in rat liver [see com- ments]. J. Hepatol., 31: 8–17, 1999. caspase-3 activates nSMase by induction of Bcl-2 cleavage because 21. Martin, S. J., Newmeyer, D. D., Mathias, S., Farschon, D. M., Wang, H. G., Reed, Bcl-2 is reported to inhibit nSMase activity through reduction of J. C., Kolesnick, R. N., and Green, D. R. Cell-free reconstitution of Fas-, UV radiation-, and ceramide-induced apoptosis. EMBO J., 14: 5191–5200, 1995. glutathione in the nucleus (63–66). 22. Albi, E., Cataldi, S., Rossi, G., and Magni, M. V. A possible role of cholesterol- In this work, it is suggested that the nucleus is a novel site of sphingomyelin/phosphatidylcholine in nuclear matrix during rat liver regeneration. ceramide increase in Fas-induced apoptosis and that ceramide content J. Hepatol., 38: 623–628, 2003. 23. Albi, E., and Viola Magni, M. Phosphatidylcholine-dependent phospholipase C in rat in the nucleus is regulated through caspase-3-dependent activation of liver chromatin. Biochem. Biophys. Res. Commun., 265: 640–643, 1999. nSMase and inhibition of SM synthase. In the future, the mechanism 24. Micheli, M., Albi, E., Leray, C., and Magni, M. V. Nuclear sphingomyelin protects by which caspase-3 regulates the SM cycle consisting of nSMase and RNA from RNase action. FEBS Lett., 431: 443–447, 1998. 25. Chujor, C. S., Feingold, K. R., Elias, P. M., and Holleran, W. M. 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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2004 American Association for Cancer Research. Increase of Nuclear Ceramide through Caspase-3-Dependent Regulation of the ''Sphingomyelin Cycle'' in Fas-Induced Apoptosis

Mitsumasa Watanabe, Toshiyuki Kitano, Tadakazu Kondo, et al.

Cancer Res 2004;64:1000-1007.

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