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Tumor Necrosis Factor Induces Apoptosis in Hepatoma Cells by Increasing Ca2+ Release from the Endoplasmic Reticulum and Suppressing Bcl-2 Expression

2002; Elsevier BV; Volume: 277; Issue: 35 Linguagem: Inglês

10.1074/jbc.m203465200

ISSN

1083-351X

Autores

Byung-Chul Kim, Heung‐Tae Kim, Mizuko Mamura, Indu S. Ambudkar, Kyeong Sook Choi, Seong‐Jin Kim,

Tópico(s)

Phagocytosis and Immune Regulation

Resumo

Tumor necrosis factor (TNF) plays an import role in the control of apoptosis. The most well known apoptotic pathway regulated by TNF involves the TNFR1-associated death domain protein, Fas-associated death domain protein, and caspase-8. This study examines the mechanism of TNF-induced apoptosis in FaO rat hepatoma cells. TNF treatment significantly increased the percentage of apoptotic cells. TNF did not activate caspase-8 but activated caspase-3, -10, and -12. The effect of TNF on the expression of different members of the Bcl-2 family in these cells was studied. We observed no detectable changes in the steady-state levels of Bcl-XL, Bax, and Bid, although TNF suppresses Bcl-2 expression. Dantrolene suppressed the inhibitory effect of TNF on Bcl-2 expression. TNF induced release of Ca2+ from the endoplasmic reticulum (ER) that was blocked by dantrolene. Importantly, the expression of Bcl-2 blocked TNF-induced apoptosis and decreased TNF-induced Ca2+ release. These results suggest that TNF induces apoptosis by a mechanism that involves increasing Ca2+ release from the ER and suppression of Bcl-2 expression. Tumor necrosis factor (TNF) plays an import role in the control of apoptosis. The most well known apoptotic pathway regulated by TNF involves the TNFR1-associated death domain protein, Fas-associated death domain protein, and caspase-8. This study examines the mechanism of TNF-induced apoptosis in FaO rat hepatoma cells. TNF treatment significantly increased the percentage of apoptotic cells. TNF did not activate caspase-8 but activated caspase-3, -10, and -12. The effect of TNF on the expression of different members of the Bcl-2 family in these cells was studied. We observed no detectable changes in the steady-state levels of Bcl-XL, Bax, and Bid, although TNF suppresses Bcl-2 expression. Dantrolene suppressed the inhibitory effect of TNF on Bcl-2 expression. TNF induced release of Ca2+ from the endoplasmic reticulum (ER) that was blocked by dantrolene. Importantly, the expression of Bcl-2 blocked TNF-induced apoptosis and decreased TNF-induced Ca2+ release. These results suggest that TNF induces apoptosis by a mechanism that involves increasing Ca2+ release from the ER and suppression of Bcl-2 expression. tumor necrosis factor endoplasmic reticulum transforming growth factor-β1 TNFR1-associated death domain Fas-associated death domain propidium iodide terminal dUTP nick end labeling z-Ile-Glu(OMe)-Thr-Asp(OMe)-CH2F Tumor necrosis factor (TNF)1 elicits a wide range of biological responses including inflammation, infection, injury, and apoptosis (1Aggarwal B.B. Ann. Rheum. Dis. 2000; 59: i6-i16Crossref PubMed Google Scholar, 2Baud V. Karin M. Trends Cell Biol. 2001; 11: 372-377Abstract Full Text Full Text PDF PubMed Scopus (1347) Google Scholar). TNF exerts its pleiotropic function through two distinct receptors, TNFR1 and TNFR2 (3Stanger B.Z. Leder P. Lee T.H. Kim E. Seed B. Cell. 1995; 81: 513-523Abstract Full Text PDF PubMed Scopus (853) Google Scholar). Both receptors contain several cysteine repeats in their extracellular domains, whereas their intracellular domain contains no significant homology. Both TNFR1 and TNFR2 can regulate TNF-mediated responses, but apoptosis is mainly induced through TNFR1, which has a cytoplasmic death domain that interacts with the adaptor protein TNFR1-associated death domain protein (TRADD) following ligand binding (4Nagata S. Cell. 1997; 88: 355-360Abstract Full Text Full Text PDF PubMed Scopus (4526) Google Scholar). TRADD serves as a platform to recruit at least three additional mediators, receptor-interacting protein 1, Fas-associated death domain protein (FADD) and TNF receptor-associated factor 2 (TRAF2) (3Stanger B.Z. Leder P. Lee T.H. Kim E. Seed B. Cell. 1995; 81: 513-523Abstract Full Text PDF PubMed Scopus (853) Google Scholar, 5Chinnaiyan A.M. O'Rourke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Abstract Full Text PDF PubMed Scopus (2144) Google Scholar, 6Hsu H Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1719) Google Scholar, 7Rothe M. Wong S.C. Henzel W.J. Goeddel D.V. Cell. 1994; 78: 681-692Abstract Full Text PDF PubMed Scopus (923) Google Scholar, 8Rothe M. Sarma V. Dixit V.M. Goeddel D.V. Science. 1995; 269: 1424-1427Crossref PubMed Scopus (968) Google Scholar). Caspase-8 is next recruited to this protein complex, and the active caspase-8 initiates a caspase cascade, which results in apoptosis (9Pimentel-Muinos F.X. Seed B. Immunity. 1999; 11: 783-793Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). TNFR2 lacks a death domain but synergistically enhances TNFR1-induced apoptosis. Recently, it has been shown in human T cells that TNFR2-mediated apoptosis requires RIP, which is induced during T-cell activation and promotes a change in TNFR2 signaling from the nuclear factor-κB (NF-κB) activation pathway to apoptosis (10Grell M. Zimmermann G. Gottfried E. Chen C.M. Grunwald U. Huang D.C., Wu Lee Y.H. Durkop H. Engelmann H. Scheurich P. Wajant H. Strasser A. EMBO J. 1999; 18: 3034-3043Crossref PubMed Scopus (258) Google Scholar). The endoplasmic reticulum (ER) regulates protein synthesis, protein folding and trafficking, cellular responses to stress, and intracellular calcium (Ca2+) levels (11Berridge M.J. Biochem. J. 1995; 15: 1-11Crossref Scopus (1045) Google Scholar). Intracellular Ca2+ homeostasis is very important in maintaining the normal function of the cell. Alterations in intracellular Ca2+ homeostasis are implicated in the control of apoptosis. Endogenous endonuclease activation proceeds via a Ca2+-dependent mechanism in thymocytes and certain other cell types exposed to a wide range of stimuli (12McConkey D.J. Orrenius S. Biochem. Biophys. Res. Commun. 1997; 239: 357-366Crossref PubMed Scopus (377) Google Scholar). Bcl-2 has been demonstrated to act as an ion channel in isolated lipid bilayers (13Minn A.J. Velez P. Schendel S.L. Liang H. Muchmore S.W. Fesik S.W. Fill M. Thompson C.B. Nature. 1997; 385: 353-357Crossref PubMed Scopus (717) Google Scholar, 14Schendel S.L. Xie Z. Montal M.O. Matsuyama S. Montal M. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5113-5118Crossref PubMed Scopus (545) Google Scholar) and displays a complex distribution in cells (15Lithgow T. van Driel R. Bertram J.F. Strasser A. Cell Growth Differ. 1994; 5: 411-417PubMed Google Scholar). Bcl-2 is localized to the mitochondrial membrane as well as to the ER and the nuclear membrane (16Yang E Korsmeyer S.J. 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Chen L. Nunez G. Miesfeld R.L. Distelhorst C.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6569-6573Crossref PubMed Scopus (609) Google Scholar, 21Marin M.C. Fernandez A. Bick R.J. Brisbay S. Buja L.M. Snuggs M. McConkey D.J. von Eschenbach A.C. Keating M.J. McDonnell T.J. Oncogene. 1996; 12: 2259-2266PubMed Google Scholar). Overexpression of Bcl-2 reduces the calcium concentration in the endoplasmic reticulum. However, overexpression of Bcl-2 has been shown to modulate the ER store of calcium by up-regulating calcium pump (SERCA) expression without affecting the release channel (IP3R) (22Kuo T.H. Kim H.R. Zhu L., Yu, Y. Lin H.M. Tsang W. Oncogene. 1998; 17: 1903-1910Crossref PubMed Scopus (168) Google Scholar). Other studies have shown that Bcl-2 regulates mitochondrial Ca2+ homeostasis (23Foyouzi-Youssefi R. Arnaudeau S. Borner C. Kelley W.L. Tschopp J. Lew D.P. Demaurex N. Krause K.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5723-5728Crossref PubMed Scopus (380) Google Scholar, 24Murphy A.N. Bredesen E. Cortopassi G. Wang E. Fiskum G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9893-9898Crossref PubMed Scopus (379) Google Scholar) and prevents Ca2+-induced cytochrome crelease (25Shimizu S. Eguchi Y. Kamiike W. Funahashi Y. Mignon A. Lacronique V. Matsuda H. Tsujimoto Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1455-1459Crossref PubMed Scopus (350) Google Scholar). FaO rat hepatoma cells are very sensitive to various apoptotic stimuli, including TGF-β (26Chevalier S. Roberts R.A. Carcinogenesis. 1999; 20: 1209-1213Crossref PubMed Scopus (16) Google Scholar, 27Choi K.S. Lim I.K. Brady J.N. Kim S.-J. Hepatology. 1998; 27: 415-421Crossref PubMed Scopus (46) Google Scholar, 28Choi K.S. Eom Y.W. Kang Y., Ha, M.J. Rhee H. Yoon J.-W. Kim S.-J. J. Biol. Chem. 1999; 274: 31775-31783Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 29Goll V. Viollon-Abadie C. Nicod L. Richert L. Hum. Exp. Toxicol. 2000; 19: 193-202Crossref PubMed Scopus (24) Google Scholar, 30Kim B.C. Mamura M. Choi K.S. Calabretta B. Kim S.-J. Mol. Cell. Biol. 2002; 22: 1369-1378Crossref PubMed Scopus (101) Google Scholar). Cleavage of full-length BAD is rapidly induced during TGF-β1-induced apoptosis, but steady-state levels of Bcl-2, Bcl-XL, and Bax are not changed (30Kim B.C. Mamura M. Choi K.S. Calabretta B. Kim S.-J. Mol. Cell. Biol. 2002; 22: 1369-1378Crossref PubMed Scopus (101) Google Scholar). In this study, we have examined the effect of TNF on apoptosis in FaO cells. We showed that TNF treatment induced apoptosis and activated caspase-3, caspase-10, and caspase-12. TNF specifically suppressed Bcl-2 expression and induced a rise in cytoplasmic Ca2+concentration by releasing Ca2+ from intracellular stores. Altogether the present data suggest that TNF induces apoptosis via mobilization of intracellular Ca2+ and suppression of Bcl-2. FaO rat hepatoma cells were maintained at 37 °C in DMEM (Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin, and streptomycin (100 μg/ml)). Generation of FaO cells stably expressing Bcl-2 was previously described (28Choi K.S. Eom Y.W. Kang Y., Ha, M.J. Rhee H. Yoon J.-W. Kim S.-J. J. Biol. Chem. 1999; 274: 31775-31783Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Propidium iodide, SKF96365, TMB-8, and dantrolene, a blocker of intracellular calcium release from the sarcoplasmic reticulum that acts by binding to the ryanodine receptor, were purchased from Calbiochem. EGTA and thapsigargin were purchased from Sigma. Recombinant murine TNF was purchased from R & D Systems. All other chemicals were from standard sources and were molecular grade or higher. Caspase-8 inhibitor, z-Ile-Glu(OMe)-Thr-Asp(OMe)-CH2F (IETD-fmk), was purchased from Calbiochem and was reconstituted in Me2SO to a concentration of 50 mm. It was then added to medium at a concentration of 50 μm 30 min prior to treatment with either TNF or TGF-β1. FaO cells were treated with lysis buffer (10 mm Tris-Cl, pH 7.4, 10 mm NaCl, 10 mm EDTA, and 0.5% SDS, and 0.1 mg/ml proteinase K) and were incubated at 50 °C for 2 h. The lysate was extracted with phenol, phenol/chloroform (1:1), and chloroform and precipitated with 2.5 volumes of ice-cold ethanol. The DNA was resuspended in Tris-EDTA buffer supplemented with 100 μg/ml RNase A. DNA samples were electrophoretically separated on 2% agarose gel for 2 h at 50 V. FaO cells were plated at 5 × 104cells/eight-well chamber slide (Nalge Nunc International, Rochester, NY) and incubated for 24 h. The cells were treated with TGF-β1 (5 ng/ml) or TNF (20 ng/ml) for 12 h and fixed with 4% paraformaldehyde (pH 7.4) for 10 min. Apoptotic cells were assessed by measuring DNA fragmentation in a standard TUNEL assay according to the instructions with the kit (In Situ Cell Death Detection Kit, POD; Roche Molecular Biochemicals). For the flow cytometry assay, FaO and FaO-Bcl-2 cells were grown in six-well plates and incubated for 24 h at 37 °C in the presence or absence of 20 ng/ml TNF-α. Cells were harvested and washed twice with phosphate-buffered saline (pH 7.4). After fixing in 80% ethanol for 30 min, cells were washed twice and resuspended in phosphate-buffered saline (pH 7.4) containing 0.1% Triton X-100, 5 μg/ml propidium iodide (PI) and 50 μg/ml ribonuclease A for DNA staining. Cells were then analyzed by a FACScan cytometer (CELLQUEST program; Becton Dickinson). Red fluorescence due to PI staining of DNA was expressed on a logarithmic scale simultaneously with the forward scatter of the particles. Four thousand events were counted on the scatter gate. The number of apoptotic nuclei was expressed as a percentage of the total number of events. Whole-cell extracts were obtained in a 1% Triton X-100 lysis buffer (50 mm Tris-Cl, pH 8.0, 150 mm sodium chloride, 1 mm EDTA, 1 mmEGTA, 2.5 mm sodium pyrophosphate, 1 mm sodium orthovanadate, 1 mm β-glycerophosphate, 1 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride). Western blotting was performed using anti-Bcl-2 (N-19; Santa Cruz Biotechnology, Inc.), anti-Bcl-XS/L (S-18; Santa Cruz Biotechnology), anti-Bax (P-19; Santa Cruz Biotechnology), anti-Bid (M-20; Santa Cruz Biotechnology), anti-caspase-8 (p-20; Santa Cruz Biotechnology), anti-caspase-10 (Cell Signaling Technology Inc., Beverly, MA), and anti-rynodine receptor (N-1; Santa Cruz Biotechnology) antibodies. Monoclonal rat antibody against caspase-12 was kindly provided by Dr. Junying Yuan (Harvard Medical School, Boston, MA). The antibody recognizes the full-length unprocessed form as well as the processed fragments of their respective antigen. Protein samples were heated at 95 °C for 5 min and analyzed by SDS-16% PAGE. Immunoblot signals were developed by Super Signal Ultra chemiluminescent reagent (Pierce). Fura2 fluorescence in single cells was measured as described earlier (31Liu X. Wang W. Lockwich T. Singh B.B. Wellner R. Jadloweic J. O'Connell B. Zhu X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 3403-3413Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar) by using an SLM 8000/DMX 100 spectrofluorimeter attached to an inverted Nikon Diaphot microscope with a Fluor ×40 oil immersion objective. Images were acquired using an enhanced CCD camera (CCD-72; MTI) and Image-1 software (Universal Imaging Corp., Downingtown, PA). Analog plots of the fluorescence ratio (340/380) in single cells are shown. Treatment of FaO rat hepatoma cells with TNF induced a 180–200-bp internucleosomal DNA cleavage as early as 12 h after TNF treatment (Fig.1A). We have previously shown that TGF-β1 rapidly induces apoptosis in these cells (27Choi K.S. Lim I.K. Brady J.N. Kim S.-J. Hepatology. 1998; 27: 415-421Crossref PubMed Scopus (46) Google Scholar, 28Choi K.S. Eom Y.W. Kang Y., Ha, M.J. Rhee H. Yoon J.-W. Kim S.-J. J. Biol. Chem. 1999; 274: 31775-31783Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Therefore, we used TGF-β1 treatment as a positive control for the apoptotic response. To show that the TNF-induced cell death is due to apoptosis, the TUNEL assay was also carried out (Fig. 1B). As indicated by the number of dark brown positive cells, there was a significant increase in the rate of apoptosis in a time-dependent manner following TNF treatment. To characterize the mechanism of TNF-mediated apoptosis, the steady-state levels of several Bcl-2 family proteins were measured by Western blotting analysis. We detected no changes in expression of Bcl-XL, Bax, or Bid over the TNF treatment time course; nor were any cleaved products of these proteins detected (Fig.1C). However, Bcl-2 expression decreased in a time-dependent manner (Fig. 1C). Since caspase-8 and caspase-3 have been implicated in TNF-induced apoptosis, we examined activation of caspase-8 and caspase-3 in TNF-induced apoptosis in FaO cells. TNF treatment of FaO cells induced a time-dependent processing of caspase-3 but not caspase-8 (Fig.2A). We also used TGF-β1 as a control for the caspase activation. As expected, TGF-β1 treatment caused activation of both caspase-3 and caspase-8 (Fig. 2B). To confirm this observation, we examined the effect of inhibitor of caspase-8 on TNF-mediated apoptosis. The addition of an inhibitor of caspase-8 (IETD-fmk) reduced the percentage of TUNEL-positive nuclei induced by TGF-β1, but the presence of IETD-fmk showed very little effect on TNF-mediated apoptosis (Fig. 2, C and D). We next examined activation of caspase-10 in TNF-induced apoptosis in FaO cells. It is known that in some cases caspase-10 can functionally substitute for caspase-8 in death receptor signal transduction (32Wang J. Chun H.J. Wong W. Spencer D.M. Lenardo M.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13884-13888Crossref PubMed Scopus (305) Google Scholar). The anti-caspase-10 antibody from Cell Signaling Technology does not detect the cleaved form of caspase-10. Treatment with TNF decreased the level of procaspase-10, whereas TGF-β1 treatment showed no effect on the level of procaspase-10, suggesting that TNF may induce apoptosis through the activation of caspase-10 (Fig.3A). Caspase-12, an ER-resident caspase, is specifically involved in apoptosis that results from ER stress (33Bitko V. Barik S. J. Cell. Biochem. 2001; 80: 441-454Crossref PubMed Scopus (131) Google Scholar, 34Nakagawa T. Yuan J. J. Cell Biol. 2000; 150: 887-894Crossref PubMed Scopus (1029) Google Scholar, 35Nakagawa T. Zhu H. Morishima N., Li, E., Xu, J. Yankner B.A. Yuan J. Nature. 2000; 403: 98-103Crossref PubMed Scopus (2912) Google Scholar, 36Yoneda T. Imaizumi K. Oono K. Yui D. Gomi F. Katayama T. Tohyama M. J. Biol. Chem. 2001; 276: 13935-13940Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar). Caspase-12 participates in ER stress-induced apoptosis that is blocked by benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), a general caspase inhibitor (34Nakagawa T. Yuan J. J. Cell Biol. 2000; 150: 887-894Crossref PubMed Scopus (1029) Google Scholar). Alterations in Ca2+ homeostasis and accumulation of unfolded proteins in the ER cause ER stress. Using a polyclonal serum directed against caspase-12, a single band corresponding to the p53 proform of caspase-12 was detected. After treatment with TNF, smaller fragments between 17 and 40 kDa became visible representing active caspase-12 (Fig. 3, B and C). However, cleavage of procaspase-12 was not detected during TGF-β1-induced apoptosis (Fig. 3C). These results suggest that the TNF-induced cell death is apoptosis and that the actual apoptotic pathway involves activation of caspase-3 and caspase-12.FIG. 3Activation of caspase-10 and caspase-12 after TNF and TGF-β1 treatment.A, immunoblot for caspase-10. FaO cells were treated with either TGF-β1 (5 ng/ml) or TNF (20 ng/ml) for 24 h. B, samples were taken at the indicated time points after TNF treatment and assayed by Western blotting analysis using a caspase-12-specific antibody that recognizes full-length as well as the active form. β-Actin was used as a control for protein loading. C, immunoblot for caspase-12. FaO cells were treated with either TGF-β1 (5 ng/ml) or TNF (20 ng/ml) for 24 h.View Large Image Figure ViewerDownload (PPT) Bcl-2 is known to regulate the flux of Ca2+ across the ER membrane, thereby abrogating Ca2+ signaling of apoptosis (20Lam M. Dubyak G. Chen L. Nunez G. Miesfeld R.L. Distelhorst C.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6569-6573Crossref PubMed Scopus (609) Google Scholar). To determine whether an increase in cytosolic Ca2+was required for the induction of apoptosis by TNF, the action of extracellular calcium chelators, EGTA and SKF96365, was studied. In a Ca2+-containing medium, TNF induced apoptosis in FaO cells. The low extracellular Ca2+ caused by either EGTA or SKF96365 did not alter TNF-induced apoptosis (Fig.4, A and B). Dantrolene, a drug used to treat malignant hyperthermia, inhibits Ca2+ release from ER and has been shown to inhibit cell damage induced by thapsigargin-induced ER Ca2+ depletion (37Wei H. Perry D.C. J. Neurochem. 1996; 67: 2390-2398Crossref PubMed Scopus (127) Google Scholar, 38Wei H. Leeds P. Chen R.-W. Wei W. Leng Y. Bredesen D.E. Chuang D.-M. J. Neurochem. 2000; 75: 81-90Crossref PubMed Scopus (87) Google Scholar). To assess the effects of dantrolene and TMB-8, which blocks Ca2+ release from ER, against TNF-induced apoptosis, we treated the FaO cells with TMB-8 and dantrolene. TNF-induced apoptosis was almost completely blocked by TMB-8 and dantrolene, whereas TGF-β1-induced apoptosis was unaffected by these compounds (Fig.4C). To support the view that TNF-induced apoptosis is triggered by the release of Ca2+ from intracellular Ca2+ stores, we investigated whether TNF had a direct effect on intracellular Ca2+ homeostasis. FaO cells were loaded with the Ca2+ dye, and changes in [Ca2+]i(expressed as 340/380-nm fluorescence ratio) were measured. Treatment with TNF caused a relatively fast elevation in [Ca2+]i, which peaked within 5 min and then decreased to a lower sustained level. In a Ca2+-free medium, a smaller, transient increase was seen (Fig.5, A, C, and H). However, TGF-β1 has no effect on intracellular Ca2+ homeostasis (Fig. 5G). This finding suggests that TNF induces both internal Ca2+ release and Ca2+ influx. The trace in Fig.6B represents the internal Ca2+ release component. The increases in cytosolic Ca2+ elevation after TNF treatment in both Ca2+-containing and Ca2+-free conditions, were significantly reduced in cells pretreated with dantrolene (Fig. 5, B, D, and H). This suggests that dantrolene reduces TNF-induced internal Ca2+ release. Dantrolene also inhibited internal Ca2+ release induced by a thapsigargin, a highly specific inhibitor of the ER-associated Ca2+ pump that is used to deplete internal Ca2+stores (Fig. 5, E, F, and I).FIG. 6Bcl-2 reduces the amount of releasable Ca2+ from intracellular stores. A Ca2+-containing medium (A and C) or Ca2+-free medium (B and D) containing 1 mm EGTA were used to assess intracellular Ca2+ changes in FaO cells (A and B) or FaO/Bcl-2 cells (C and D) (see the legend to Fig. 5 for details). In the bar graphs(E), the data represent the mean values of four replications, with bars indicating S.E. *, p< 0.05 compared with control.View Large Image Figure ViewerDownload (PPT) We next determined the effect of Bcl-2 overexpression on TNF-induced calcium homeostasis. We previously generated FaO cell lines expressing Bcl-2 (28Choi K.S. Eom Y.W. Kang Y., Ha, M.J. Rhee H. Yoon J.-W. Kim S.-J. J. Biol. Chem. 1999; 274: 31775-31783Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). These cell lines are individually isolated clones. In both Ca2+-containing and Ca2+-free medium, the increase of cytosolic Ca2+ after the addition of TNF was significantly reduced in FaO/Bcl-2 cells compared with FaO cells (Fig. 6). The skeletal muscle relaxant dantrolene is an inhibitor of Ca2+release through rynodine receptor. The expression of rynodine receptor protein was confirmed in FaO cells by Western blot analysis using a specific antibody raised against the ryanodine receptor (Fig.7A). We next examined the effect of dantrolene on Bcl-2 protein levels by Western blotting. As shown in Fig. 7, B and C, Bcl-2 protein levels increased significantly to 320% of the control after treatment with 25 μm dantrolene for 24 h. TNF treatment markedly decreased Bcl-2 protein level, but treatment with dantrolene blocked suppression of Bcl-2 expression by TNF, suggesting that TNF suppresses Bcl-2 expression by regulating the flux of Ca2+. To investigate whether dantrolene also blocks TNF-induced apoptosis in FaO cells, we measured nuclear incorporation of propidium iodide by fluorescence-activated cell sorting analysis. Cells were incubated in the absence or presence of TNF. TGF-β1 or thapsigargin was used as control. Dantrolene pretreatment robustly suppressed TNF-induced- or thapsigargin-induced population of apoptotic nuclei, whereas dantrolene did not block TGF-β1-induced apoptosis (Fig.8). These results further suggest that TNF activates intracellular Ca2+ release from ER, which, in turn, induces apoptosis. Overexpression of Bcl-2 has been shown to repress apoptosis by regulating ER-associated Ca2+ release. To investigate the protective effect of Bcl-2 on TNF-induced apoptosis, stable cell lines expressing Bcl-2 were generated (28Choi K.S. Eom Y.W. Kang Y., Ha, M.J. Rhee H. Yoon J.-W. Kim S.-J. J. Biol. Chem. 1999; 274: 31775-31783Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) (Fig.9A). As shown in Fig.9A, Bcl-2 protein level was significantly increased. Most of the Bcl-2-overexpressing cell lines were completely resistant to TGF-β1-induced apoptosis. We next examined the propidium iodide incorporation in the FaO cells overexpressing Bcl-2. A significant decrease in PI incorporation was seen after the addition of TNF in FaO/Bcl-2 cells compared with control cells (Fig.9B). Up to this point, our results suggest that ER-controlled calcium release is involved in TNF-induced apoptosis in FaO cells. The activation of caspase-12 is involved in a specific form of apoptosis in the ER unfolded protein response. TNF treatment increased cleaved active products of caspase-12. However, pretreatment with dantrolene or TMB-8 completely blocked activation of caspase-12 induced by TNF, whereas extracellular calcium chelators, EGTA or SKF96365, did not block the TNF-induced caspase-12 cleavage (Fig.10). Receptor-mediated apoptosis has been demonstrated for various growth factors, including TNF. The TNFs act via a large family of receptors expressed on the surface of the target cell, known as the TNF receptor superfamily (1Aggarwal B.B. Ann. Rheum. Dis. 2000; 59: i6-i16Crossref PubMed Google Scholar, 2Baud V. Karin M. Trends Cell Biol. 2001; 11: 372-377Abstract Full Text Full Text PDF PubMed Scopus (1347) Google Scholar, 3Stanger B.Z. Leder P. Lee T.H. Kim E. Seed B. Cell. 1995; 81: 513-523Abstract Full Text PDF PubMed Scopus (853) Google Scholar). TNF is responsible for a diverse range of signaling events within cells, leading to either necrosis or apoptosis. TNF exerts many of its effects by binding to either a 55-kDa cell membrane receptor termed TNFR-1 or a 75-kDa cell membrane receptor termed TNFR-2. TNF induces apoptosis by more than one pathway. The most widely accepted pathway involves TRADD, FADD, and caspase-8 (39Rath P.C. Aggarwal B.B. J. Clin. Immunol. 1999; 19: 350-364Crossref PubMed Scopus (346) Google Scholar). Indeed, FADD- and caspase-8-deficient fibroblasts are resistant to TNFR1-induced apoptosis (40Varfolomeev E.E. Schuchmann M. Luria V. Chiannilkulchai N. Beckmann J.S. Mett I.L. Rebrikov D. Brodianski V.M. Kemper O.C. Kollet O. Lapidot T. Soffer D. Sobe T. Avraham K.B. 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It has been suggested that in some cases caspase-10 can functionally substitute for caspase-8 in death receptor signal transduction (32Wang J. Chun H.J. Wong W. Spencer D.M. Lenardo M.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13884-13888Crossref PubMed Scopus (305) Google Scholar). However, the role of caspase-10 is not clear in TNF-mediated apoptosis in FaO cells. TNF induces activation of caspase-12 in FaO cells. Caspase-12 is known to be essential for cell death induced by ER stress. Procaspase-12 is enriched in ER-containing microsomal fractions from the brain and is processed by the family of cytosolic calcium-dependent cysteine proteases, calpain (34Nakagawa T. Yuan J. J. Cell Biol. 2000; 150: 887-894Crossref PubMed Scopus (1029) Google Scholar). Procaspase-12 is cleaved, and the activated forms accumulate under ER stress conditions (35Nakagawa T. Zhu H. Morishima N., Li, E., Xu, J. Yankner B.A. Yuan J. Nature. 2000; 403: 98-103Crossref PubMed Scopus (2912) Google Scholar). 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It is conceivable that cellular context dictates precisely how Bcl-2 will influence intracellular Ca2+ pools. Importantly, the connection between ER Ca2+ pool emptying and apoptosis is not disputed. TNF treatment suppressed the protein level of Bcl-2 in FaO cells. This result is somewhat unexpected, given the evidence that Bcl-2 is capable of blocking Ca2+ release from the ER, resulting in the protection of Ca2+-induced toxicity. Although the mechanism of suppression of Bcl-2 by TNF in FaO cells remains to be investigated, this study suggests another apoptotic pathway triggered by TNF. It will be interesting to find whether the residual apoptotic response observed upon TNF treatment of FADD-deficient cells (41Yeh W.C. Pompa J.L. McCurrach M.E. Shu H.B. Elia A.J. Shahinian A., Ng, M. Wakeham A. Khoo W. Mitchell K., El- Deiry W.S. Lowe S.W. Goeddel D.V. Mak T.W. Science. 1998; 279: 1954-1958Crossref PubMed Scopus (799) Google Scholar) is mediated through the mobilization of Ca2+ from the ER. Interference of Ca2+ release from the ER increases Bcl-2 mRNA and protein levels. Treatment with dantrolene, an inhibitor of Ca2+ release from the ER, prevented cell death, and this protection by dantrolene was associated with a marked increase in the protein levels of Bcl-2 in GT1 hypothalamic neurosecretory cells (38Wei H. Leeds P. Chen R.-W. Wei W. Leng Y. Bredesen D.E. Chuang D.-M. J. Neurochem. 2000; 75: 81-90Crossref PubMed Scopus (87) Google Scholar). As the ER Ca2+ level has a prominent role in regulating protein synthesis (51Brostrom C.O. Brostrom M.A. Annu. Rev. Physiol. 1990; 52: 577-590Crossref PubMed Scopus (162) Google Scholar), the effect of dantrolene on ER Ca2+could be the underlying mechanism for Bcl-2 induction. In another study, treatment with lithium, which interferes with Ca2+release from the ER (52Okamoto Y. Kagaya A. Motohashi N. 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Regardless of the detailed mechanisms underlying the cytoprotective effects of dantrolene, the ability of dantrolene to induce Bcl-2 raises the possibility that this drug may be potentially useful in the treatment of some forms of neurodegenerative diseases. We thank J. Yuan for caspase-12 antibody and S. Reffey and S. Patel for the critical reading of the manuscript.

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