Journal of Neurochemistry, 2001, 77, 1531±1541 Cleavage of Bax is mediated by caspase-dependent or -independent calpain activation in dopaminergic neuronal cells: protective role of Bcl-2 Won-Seok Choi,* Eun-Hee Lee,* Chul-Woong Chung,² Yong-Keun Jung,² Byung K. Jin,³ Seung U. Kim,³ Tae H. Oh,§ Takaomi C. Saido¶ and Young J. Oh* *Department of Biology, Yonsei University College of Science, Seoul, Korea ²Department of Life Science, Kwangju Institute of Science and Technology, Kwangju, Korea ³Brain Research Center, Ajou University School of Medicine, Suwon, Korea §Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, Maryland, USA ¶Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, Hirosawa, Wako-shi, Saitama, Japan Abstract Thus, cotreatment of cells with calpain inhibitor blocked Two cysteine protease families, caspase and calpain, are both MPP1- and STS-induced Bax cleavage. Intriguingly, known to participate in cell death. We investigated whether a overexpression of baculovirus-derived inhibiting protein of stress-speci®c protease activation pathway exists, and to caspase, p35 or cotreatment of cells with caspase inhibitor what extent Bcl-2 plays a role in preventing drug-induced blocked STS- but not MPP1-induced Bax cleavage. This protease activity and cell death in a dopaminergic neuronal appears to indicate that calpain activation may be either cell line, MN9D. Staurosporine (STS) induced caspase- dependent or independent of caspase activation within the dependent apoptosis while a dopaminergic neurotoxin, same cells. However, cotreatment with calpain inhibitor MPP1 largely induced caspase-independent necrotic cell rescued cells from MPP1-induced but not from STS-induced death as determined by morphological and biochemical neuronal cell death. In these paradigms of dopaminergic cell criteria including cytochrome c release and ¯uorogenic death, overexpression of Bcl-2 prevented both STS- and caspase cleavage assay. At the late stage of both STS- and MPP1-induced cell death and its associated cleavage of Bax. MPP1-induced cell death, Bax was cleaved into an 18-kDa Thus, our results suggest that Bcl-2 may play a protective role fragment. This 18-kDa fragment appeared only in the by primarily blocking drug-induced caspase or calpain activity mitochondria-enriched heavy membrane fraction of STS- in dopaminergic neuronal cells. treated cells, whereas it was detected exclusively in the Keywords: Bax, Bcl-2, calpain, caspase, MPP1, cytosolic fraction of MPP1-treated cells. This proteolytic staurosporine. cleavage of Bax appeared to be mediated by calpain as J. Neurochem. (2001) 77, 1531±1541. determined by incubation with [35S]methionine-labelled Bax. Apoptosis is a controlled process to remove unnecessary, aged, or damaged cells in various situations (Thompson 1995). It is well known that apoptosis is characterized by Received September 29, 2000; revised manuscript received March 12, 2001; accepted March 17, 2001. distinct morphological changes such as cellular shrinkage, Address correspondence and reprint requests to Y. J. Oh, Department blebbing and chromatin condensation. Among the cellular of Biology, Yonsei University College of Science, 134 Shinchondong molecules known to regulate apoptosis is a family of Seodaemoongu, Seoul 120±749, Korea. E-mail: [email protected] cysteine proteases ± recently termed caspases (Martin and Abbreviations used: Ac-DEVD-AMC, acetyl-Asp-Glu-Val-Asp-7- Green 1995; Cryns and Yuan 1998). Once activated through amino-4-methylcoumarin; BAF, Boc-aspartyl(OMe)-¯uoromethylketone; CCM, complete culture medium; CPT, calpeptin; MTT, 3-[4,5- a proteolytic cascade, caspases cleave endogenous cellular dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; PARP, poly- substrates and consequently amplify the death signals (ADP-ribose)polymerase; STS, staurosporine; Z-VAD, N-benzyloxy- (Thornberry and Lazebnik 1998). carbonyl-Val-Ala-Asp-¯uoromethylketone. q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 1531±1541 1531 1532 W.-S. Choi et al. In addition to caspases, activation of proteolytic pathways Materials and methods by serine proteases, aspartic proteases and proteosomes is involved in cell death. Moreover, a signi®cant focus has Cell culture and drug treatments been directed towards another cysteine protease, calpain, MN9D cells that stably overexpressed human Bcl-2 (MN9D/Bcl-2), and with a concomitant evaluation of its contribution to both or vector alone (MN9D/Neo) have been previously established, necrosis and apoptosis (Chan and Mattson 1999). Two forms characterized, and maintained (Oh et al. 1995). Both MN9D of calpains, m- and m-calpain, are expressed ubiquitously in parental cells and MN9D/Neo cells had no detectable levels of endogenous Bcl-2 as determined by immunoblot analysis (Oh et al. animal tissues, and activated by Ca21 and autolytic 1995). MN9D cells stably overexpressing baculovirus p35 (MN9D/ processing. Activated calpain induces proteolysis of speci®c P35) were established by transfecting with a pCDNA3 eukaryotic cellular substrates. These include several cytoskeletal, expression vector containing a full-length p35 cDNA and regulatory and membrane proteins of various cell types subsequently characterized by RT-PCR. Primers used included (Chan and Mattson 1999; Wang 2000). Over the past decade 50-CGACGAACGCAACGACTACT-30 (forward primer) and 50- or so, the possible implication of calpain in neuronal death CTTTTCGGATTTGCCCCAGC-3 0 (reverse primer). Cells from of the central nervous system has been suggested. For each stable cell line were plated at a density of 2 104 cells on  example, calpain expression is elevated in the brains of 25 mg/mL poly-d-lysine-coated 48-well plates (Costar, Corning, patients with multiple sclerosis, amyotrophic lateral sclero- NY, USA). Cells were maintained in Dulbecco's modi®ed Eagle's sis and Alzheimer's disease as well as in experimental medium supplemented with 10% heat-inactivated fetal bovine models of traumatic injury and ischemic neuronal death serum (Life Technologies, Rockville, MD, USA) and 250 mg/mL G418 (Life Technologies; complete culture medium, CCM) for 3 (Saito et al. 1993; Kamp¯ et al. 1997; Springer et al. 1997; days in an incubator with an atmosphere of 10% CO at 378C. Cells Lipton 1999; Shields et al. 1999; Stracher 1999; Nixon 2 were subsequently switched to serum-free N2 medium containing 2000). Recently, putative mechanisms by which calpain the various experimental reagents and further incubated for the mediates neuronal death have also been proposed in certain indicated time periods. Reagents used included STS (Sigma, St neurological disorders (Steiner et al. 1998; Zhang et al. Louis, MO, USA), MPP1 (RBI), Boc-aspartyl(OMe)-¯uoromethyl- 1999). Although calpain is known to be elevated in ketone (BAF; Enzyme Systems Products, Dublin, CA, USA), patients with Parkinson's disease (Mouatt-Prigent et al. N-benzyloxycarbonyl-Val-Ala-Asp-¯uoromethylketone (Z-VAD; 1996), the molecular mechanisms for this still remain to be Enzyme Systems Products), calpeptin (Calbiochem, San Diego, determined. CA, USA) and MDL 28170 (Calbiochem). Recent reports indicate that caspases and calpains may Transmission electron microscopy work either in concert or independently during cell death MN9D/Neo cells were plated at 2 105 cells on 25 mg/mL poly  (Wang 2000). Furthermore, caspase and calpain sequentially d-lysine-coated 6-well plates (Costar), maintained in CCM for in¯uence cell death. For example, calpain acts as a negative 3 days, switched to N2 medium and treated with 1 mm STS or or positive upstream regulator of caspase processing (Ruiz- 50 mm MPP1 for the indicated time periods. Cells were ®xed in Vela et al. 1999; Wolf et al. 1999; Chua et al. 2000). Karnovsky's ®xative overnight at 48C, and then post®xed in 1% Conversely, caspase-3-mediated degradation of an endo- osmium tetroxide/1.5% ferrocyanide solution for 30 min at room genous calpain inhibitor, calpastatin facilitates calpain temperature (218C). Following dehydration in a series of graded ethanols, cells were embedded in Epon resin, and heat-polymerized. activation that may further the proteolysis initiated by Ultrathin sections were mounted, stained with uranyl acetate and lead caspase (Porn-Ares et al. 1998; Wang et al. 1998). However, citrate, and then examined with a Zeiss EM 902 A transmission the potential cross-talk and the relative contributions of electron microscope (Zeiss, Zena, Germany). these two proteases to neuronal death are relatively unknown. In this study, we speci®cally investigated whether a stress- In vitro ¯uorogenic caspase cleavage assay Caspase activity was measured by a ¯uorometric assay. Brie¯y, speci®c calpain activation pathway exists, and examined its 1 potential role in cleaving endogenous substrate and pro- MN9D/Neo cells treated with 1 mm STS or 50 mm MPP were lysed in a buffer containing 50 mm Tris, pH 7.0/2 mm EDTA/1.0% moting cell death in a murine mesencephalon-derived Triton X-100. Cell lysates (10 mg) recovered after centrifugation at dopaminergic neuronal cell line, MN9D (Choi et al. 1991, 13 000 g were incubated with 25 mm acetyl-Asp-Glu-Val-Asp-7- 1992; Heller et al. 1996). Our results suggest that (i) calpain amino-4-methylcoumarin (Ac-DEVD-AMC, a substrate for cas- is activated and the major protease cleaving Bax protein in pase-3 as well as caspase-6, -7, -8, and -10; Calbiochem) for 1 h at 1 staurosporine (STS)-induced apoptosis and MPP -induced