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Endoplasmic Reticulum Stress–Associated Caspase 12 Mediates Cisplatin-Induced LLC-PK1 Cell Apoptosis

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Endoplasmic Reticulum Stress–Associated Caspase 12 Mediates Cisplatin-Induced LLC-PK1 Cell Apoptosis Endoplasmic Reticulum Stress–Associated Caspase 12 Mediates Cisplatin-Induced LLC-PK1 Cell Apoptosis Hua Liu and Radhakrishna Baliga Department of Pediatrics, University of Mississippi Medical Center, Jackson, Mississippi Reactive oxygen metabolites are important mediators in cisplatin-induced apoptosis in renal tubular epithelial cells (LLC-PK1). Mitochondria have been implicated to play a principal role in cisplatin-induced apoptosis. Caspase 12, an endoplasmic reticulum (ER)-specific caspase, participates in apoptosis under ER stress. Cytochrome P450 system is crucial to the generation of reactive oxygen metabolites and is present at high concentration in the ER. The direct role of caspase 12 in any model of renal injury has not previously been described. In this study, cleavage of procaspase 12 preceded that of caspases 3 and 9 after cisplatin treatment of LLC-PK1 cells. The active form of caspase 8 was not detected throughout the course of study. Preincubation of the LLC-PK1 cells with the caspase 9 inhibitor did not attenuate caspase 3 activation and provided no significant protection. Caspase 3 inhibitor provided only modest protection against cisplatin-induced apoptosis. LLC-PK1 cells that were transfected with anti–caspase 12 antibody significantly attenuated cisplatin-induced apoptosis. Taken together, these data indicate that caspase 12 plays a pivotal role in cisplatin-induced apoptosis. It is proposed that the oxidative stress that results from the interaction of cisplatin with the ER cytochrome P450 leads to activation of procaspase 12, resulting in apoptosis. J Am Soc Nephrol 16: 1985–1992, 2005. doi: 10.1681/ASN.2004090768 isplatin is a widely used chemotherapeutic agent in activated. The extrinsic, or receptor-mediated, pathway, involves the treatment of a variety of solid human tumors (1). cellular ligands such as TNF-␣ or FasL, resulting in caspase 8 C The therapeutic effectiveness of cisplatin is blunted by activation (19). The intrinsic, or mitochondrial, pathway involves the nephrotoxicity that develops primarily at the S3 segment of release of the mitochondrial protein cytochrome c, resulting in the proximal tubule (2,3). Reactive oxygen metabolites (ROM) activation of caspase 9 (20). Both caspases 8 and 9 then activate are important mediators of cisplatin-induced renal tubular cell caspase 3, the major effector caspase that is responsible for the apoptosis (4,5). The mechanism that triggers apoptosis in re- destruction of various substrates. sponse to cisplatin is not well defined. Mitochondria have been Recent studies have indicated a close link between ER stress and implicated as principal sensors and thus critical in the genera- caspase 12 activation resulting in apoptotic cell death (21–24). tion of the apoptotic cascade (6–8). The prevailing assumption Procaspase 12 is located predominantly on the cytoplasmic side of has been that all electron-transfer processes that lead to ROM the ER and is expressed at high levels in the kidney, especially the production are localized to the mitochondria (9,10). Recent renal tubular epithelial cells (25). The ER also regulates apoptosis studies, including that of ours, suggest that the cytochrome by sensitizing the mitochondria to a variety of extrinsic and in- P450 (CYP) system is crucial to the ROM-mediated cell signal- trinsic stimuli and also initiating cell death signals of its own (26). ing and that CYP is present in high concentration in the endo- We demonstrated previously that the microsomal CYP2E1 is a site plasmic reticulum (ER) of most animal cells (11–14). and a source for ROM generation in cisplatin-induced apoptosis Caspases are a family of cysteine proteases that play an impor- (5). The direct role of caspase 12 in cisplatin-induced apoptosis in tant role in the programmed cell death (15–17). Their activation renal tubular epithelial cells was not explored previously. Hence, signals a point of no return in the apoptotic pathway. The caspase our study was designed to explore the role of caspase 12 in family is broadly divided into two groups, namely the initiator cisplatin-induced apoptosis. Treatment of the LLC-PK1 cells with caspases (caspases 8, 9, and 12) and the effector caspases (caspases cisplatin resulted in cleavage of procaspase 12 and apoptosis. 3, 6, and 7). The initiator caspases undergo activation in response Preincubation of the cells with caspase 9 inhibitor did not inhibit to an apoptotic stimuli and in turn activate the effector caspases caspase 3 activation and provided no significant protection. that are responsible for the cleavage of a wide variety of physio- Caspase 3 inhibitor provided only modest protection. LLC-PK1 logic substrates (18). There are two relatively well-recognized cell cells that were transfected with anti–caspase 12 antibody (Ab) death pathways that depend on the type of procaspase that is significantly attenuated cisplatin-induced apoptosis. We also looked at the interrelation between ER stress and procaspase 12 Received September 14, 2004. Accepted April 7, 2005. cleavage. Procaspase 12 was cleaved in cisplatin-treated CYP2e1 wild-type (WT) mice, and this cleavage was significantly pro- Published online ahead of print. Publication date available at www.jasn.org. tected in the CYP2e1 knockout (KO) animals. We therefore postu- Address correspondence to: Dr. Radhakrishna Baliga, Department of Pediatrics, late that the oxidative stress that results from the interaction of Division of Nephrology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216-4505. Phone: 601-984-5970; Fax: 601-815-5902; cisplatin with ER CYP leads to the activation of procaspase 12, E-mail: [email protected] resulting in apoptosis. Copyright © 2005 by the American Society of Nephrology ISSN: 1046-6673/1607-1985 1986 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 1985–1992, 2005 Materials and Methods Transferase-Mediated dUTP Nick-End Labeling Assay Cell Culture and Treatment Transferase-mediated dUTP nick-end labeling (TUNEL) was per- LLC-PK1 cells, purchased from American Type Culture Collection formed by using ApoAlert DNA Fragmentation Assay Kit (Clontech, (CRL 1392; Manassas, VA), were maintained in Medium 199 supple- Palo Alto, CA), by which fluorescein-dUTP incorporation at the free ends of fragmented DNA is visualized by fluorescence microscopy. mented with 3% FBS in a humidified atmosphere of 95% air/5% CO2 at 37°C and fed at intervals of 3 d (27). The cells were maintained in TUNEL stain was performed following the manufacturer’s protocol. 75-cm2 tissue culture flasks, and the monolayer was subcultured using 0.05% trypsin, 0.53 mM EDTA, in HBSS. All experiments were carried Detection of DNA Fragmentation out on confluent cell monolayer between passages 203 and 215. Apo- LLC-PK1 cells were homogenized and lysed with a buffer that con- ptosis was induced in LLC-PK1 cells by incubation with 50 ␮M cispla- tained 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM NaCl, and 0.5% tin at 37°C for 12 h (28). Caspase inhibitions were performed by SDS. The lysates were incubated with proteinase K (0.2 mg/ml) at 50°C preincubation of caspase 3 inhibitor DEVD-CHO (60 ␮M; Bachem, overnight. DNA was isolated from the lysates following the procedures as Torrance, CA) and caspase 9 inhibitor LEHD-CHO (60 ␮M; Peptide described by Ramachandra and Studzinski (31). The DNA obtained was Inc., Osaka, Japan) for1hat37°C. resuspended in a buffer (10 mM Tris-HCl and 1 mM EDTA) at 1 ␮g/ml and incubated with 0.1 U/10 ␮g DNA of DNAase-free RNAase cocktail (cat. no. 2286; Ambion, Austin, TX). Electrophoresis was performed in Determination of Caspase Activity 1.6% agarose gels, and DNA was visualized with ethidium bromide. Caspases 3, 8, and 9 activities were tested by use of the Caspase Colorimetric Activity Assay Kits (Chemicon International Inc., Te- Immunocytochemistry mecula, CA) following the manufacturer’s protocols. Equal numbers of Cultured LLC-PK1 cells were fixed in B5 solution, and cell block was ϫ 6 cells (0.5 to 2 10 ) from each group were treated with cell lysis buffer made by imbedding in tissue medium. Sections were cut on a glass ϫ on ice for 10 min and centrifuged at 10,000 g for 5 min. The super- slide. After deparaffinization and antigen retrieval, the sections were natants were incubated with respective caspase substrates (DEVD-pNA immunolabeled and visualized according to an avidin-biotin complex for caspase 3, IETD-pNA for caspase 8, and LEHD-pNA for caspase 9) (ABC) method (32). at 37°C for 1 h. The activities were assayed by use of a spectrophotom- eter microtiter plate reader at 405 nm. The specificity of changes in the Transfection of Anti–Caspase 12 Ab into LLC-PK1 Cells caspase activities was confirmed by addition of a specific inhibitor to Anti–caspase 12 Ab (AB3612, Lot 22101182; Chemicon) was delivered the caspase in parallel experiments. into LLC-PK1 cells by using transfection agent Chariot (30025; Active Motif, Carlsbad, CA) following the manufacturer’s protocol. Briefly, the initial transfection mixture was prepared by adding 100 ␮l of anti–caspase Preparation of Cellular Fractions 12 Ab dilution (1.2 ␮l of stock Ab in 98.8 ␮l of PBS) to 100 ␮l of Chariot For the detection of caspases, cell fractions were prepared as de- ␮ ␮ dilution (6 l of Chariot stock solution in 96 l of distilled H2O). The scribed previously (25). In brief, LLC-PK1 cells or kidney cortex was mixture was incubated at room temperature for 30 min to allow the lysed and homogenized in buffer A (50 mM Tris-HCl [pH 8.0], 1 mM formation of Chariot-Ab complex. LLC-PK1 cells in six-well culture plate mercaptoethanol, 1 mM EDTA, 0.32 M sucrose, and 0.1 mM PMSF). (40 to 50% confluence) were overlaid with the mixture (200 ␮l/well), Nuclear fraction was collected from the pellet after first centrifugation added with 400 ␮l of serum-free culture medium (final Ab dilution 1:500), at 900 ϫ g for 10 min.
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