p53-dependent Caspase-2 Activation in Mitochondrial Release of Apoptosis-inducing Factor and Its Role in Renal Tubular Epithelial Cell Injury
2005; Elsevier BV; Volume: 280; Issue: 35 Linguagem: Inglês
10.1074/jbc.m503305200
ISSN1083-351X
AutoresRohit Seth, Cheng‐Ta Yang, Varsha Kaushal, Sudhir V. Shah, Gur P. Kaushal,
Tópico(s)Renal and related cancers
ResumoWe demonstrate the role of p53-mediated caspase-2 activation in the mitochondrial release of apoptosis-inducing factor (AIF) in cisplatin-treated renal tubular epithelial cells. Gene silencing of AIF with its small interfering RNA (siRNA) suppressed cisplatin-induced AIF expression and provided a marked protection against cell death. Subcellular fractionation and immunofluorescence studies revealed cisplatin-induced translocation of AIF from the mitochondria to the nuclei. Pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone or p53 inhibitor pifithrin-α markedly prevented mitochondrial release of AIF, suggesting that caspases and p53 are involved in this release. Caspase-2 and -3 that were predominantly activated in response to cisplatin provided a unique model to study the role of these caspases in AIF release. Cisplatin-treated caspase-3 (+/+) and caspase-3 (-/-) cells exhibited similar AIF translocation to the nuclei, suggesting that caspase-3 does not affect AIF translocation, and thus, caspase-2 may be involved in the translocation. Caspase-2 inhibitor benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethylketone or down-regulation of caspase-2 by its siRNA significantly prevented translocation of AIF. Caspase-2 activation was a critical response from p53, which was markedly induced and phosphorylated in cisplatin-treated cells. Overexpression of p53 not only resulted in caspase-2 activation but also mitochondrial release of AIF. The p53 inhibitor pifithrin-α or p53 siRNA prevented both cisplatin-induced caspase-2 activation and mitochondrial release of AIF. Caspase-2 activation was dependent on the p53-responsive gene, PIDD, a death domain-containing protein that was induced by cisplatin in a p53-dependent manner. These results suggest that caspase-2 activation mediated by p53 is an important pathway involved in the mitochondrial release of AIF in response to cisplatin injury. We demonstrate the role of p53-mediated caspase-2 activation in the mitochondrial release of apoptosis-inducing factor (AIF) in cisplatin-treated renal tubular epithelial cells. Gene silencing of AIF with its small interfering RNA (siRNA) suppressed cisplatin-induced AIF expression and provided a marked protection against cell death. Subcellular fractionation and immunofluorescence studies revealed cisplatin-induced translocation of AIF from the mitochondria to the nuclei. Pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone or p53 inhibitor pifithrin-α markedly prevented mitochondrial release of AIF, suggesting that caspases and p53 are involved in this release. Caspase-2 and -3 that were predominantly activated in response to cisplatin provided a unique model to study the role of these caspases in AIF release. Cisplatin-treated caspase-3 (+/+) and caspase-3 (-/-) cells exhibited similar AIF translocation to the nuclei, suggesting that caspase-3 does not affect AIF translocation, and thus, caspase-2 may be involved in the translocation. Caspase-2 inhibitor benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethylketone or down-regulation of caspase-2 by its siRNA significantly prevented translocation of AIF. Caspase-2 activation was a critical response from p53, which was markedly induced and phosphorylated in cisplatin-treated cells. Overexpression of p53 not only resulted in caspase-2 activation but also mitochondrial release of AIF. The p53 inhibitor pifithrin-α or p53 siRNA prevented both cisplatin-induced caspase-2 activation and mitochondrial release of AIF. Caspase-2 activation was dependent on the p53-responsive gene, PIDD, a death domain-containing protein that was induced by cisplatin in a p53-dependent manner. These results suggest that caspase-2 activation mediated by p53 is an important pathway involved in the mitochondrial release of AIF in response to cisplatin injury. Apoptosis-inducing factor (AIF) 1The abbreviations used are: AIF, apoptosis-inducing factor; RTEC, renal tubular epithelial cell(s); PIDD, p53-induced protein with a death domain; Smac, second mitochondria-derived activator of caspase; Z, benzyloxycarbonyl; fmk, fluoromethylketone; DAPI, 4′, 6′-diamidino-2-phenylindole; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Diablo, direct IAP-binding protein with low pl; HtrA2, high temperature requirement A2; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,3-diphenyltetrazolium bromide; siRNA, small interfering RNA; AMC, amino-4-methylcoumarin; IAP, hihibitor of apoptosis protein. is a highly conserved mitochondrial inter-membrane flavoprotein encoded by nuclear DNA (1Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. 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We provide evidence that cisplatin-induced caspase-2 activation but not caspase-3 activation is involved in the mitochondrial release of AIF. We have further explored the mechanism of caspase-2 activation and demonstrated the role of p53 and its responsive gene p53-induced death domain protein (PIDD) on caspase-2 activation that participates in the subsequent release of mitochondrial AIF in RTEC. LLC-PK1 cells obtained from ATCC were cultured as described in our previous studies (39Kaushal G.P. Kaushal V. Hong X. Shah S.V. Kidney Int. 2001; 60: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). The cells were grown in Gibco medium 199 supplemented with 10% heat-inactivated fetal calf serum. Cultures were maintained in a humidified incubator gassed with 5% CO2 and 95% air at 37 °C and fed with fresh medium at intervals of 48-72 h. Experiments were performed with cells grown to 80% confluence. Caspase substrates were purchased from Peptide International (Louisville, KY), and antibodies to caspase-3, AIF, histone H1A, α-tubulin, β-actin, and proteins of the Bcl-2 family were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) Antibodies to p53, Ser-15-phosphorylated p53, and active caspase-3 were obtained from Cell Signaling Technology (Beverly, MA). Antibody to cytochrome c oxidase subunit IV was from Molecular Probes, Inc. (Eugene, OR). Caspase inhibitors, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethylketone (Z-VDVAD-fmk), and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone were obtained from Enzyme System Products (Livermore, CA). The cell culture medium was replaced with fresh medium containing serum, and cells were incubated either without or with cisplatin at various concentrations (25-200 μm) for the period of time indicated (1-24 h). In initial studies, we determined the optimum exposure time and the suitable concentration of cisplatin. To determine the effect of inhibitors, cells were treated with the inhibitors for 10 min prior to the addition of cisplatin (50 μm). Primary cultures of RTEC were prepared from caspase-3 (-/-) and wild-type (+/+) mice. Caspase-3 (-/-) mice were generously provided by Richard Flavell, Ph.D. All procedures for the preparation of primary cultures were performed under sterile conditions as described (40Bergin E. Levine J.S. Koh J.S. Lieberthal W. Am. J. Physiol. 2000; 278: F758-F768Crossref PubMed Google Scholar). Briefly, renal cortices were minced and incubated with 0.5 mg/ml collagenase and 0.5 mg/ml soybean trypsin inhibitor for 30 min. After the removal of large, undigested fragments by gravity, the cell suspension was washed first with Hanks' solution containing 10% horse serum and then with Dulbecco's modified Eagle's medium by centrifugation, and the cell pellets were suspended in growth medium containing a serum-free mixture of Dulbecco's modified Eagle's medium, Ham's F-12 (1:1), 2 mm glutamine, 15 mm HEPES, 5 μg/ml transferrin, 5 μg/ml insulin, 50 mm hydrocortisone, 500 units/ml penicillin, and 50 mg/ml streptomycin. The cells previously isolated by this procedure were identified as being predominantly of proximal tubular origin (41Sheridan A.M. Schwartz J.H. Kroshian V.M. Tercyak A.M. Masino S. Lieberthal W. Am. J. Physiol. 1993; 265: F342-F350PubMed Google Scholar). The cell suspension was then plated into tissue culture flasks. After 24 h, the mono-layers were washed several times to remove unattached cells, and new growth medium was added. The cultures became confluent in 5-7 days. Isolation of Nuclear Fraction—The nuclei were prepared by a minor modification of the previously described procedure (42Wood E.R. Earnshaw W.C. J. Cell Biol. 1990; 111: 2839-2850Crossref PubMed Scopus (141) Google Scholar). LLC-PK1 cells were gently scraped using the rubber policeman, harvested by centrifugation, and washed twice with PBS. The cells were resuspended in homogenization buffer containing 210 mm mannitol, 70 mm sucrose, 1 mm EDTA, 10 mm HEPES, pH 7.5; supplemented with 1× protease inhibitor mixture (Sigma); and homogenized with 30-35 strokes of a Dounce homogenizer (Wheaton). To establish the number of stokes for cell permeabilization, the trypan blue exclusion method (0.4% trypan blue solution in PBS diluted 1:10 with cell suspension), which discriminates between permeabilized (stained) cells and intact cells (unstained), was used. The suspension was then transferred to Eppendorf centrifuge tubes and centrifuged at 500 × g for 10 min to pellet nuclei and unbroken cells. The nuclear fraction was adjusted to the final concentration of 0.25 m sucrose and 0.35% Triton X-100 and layered on top of a discontinuous sucrose density gradient prepared with 0.32, 0.8, and 1.2 m sucrose in a Beckman centrifuge. The tubes were centrifuged at 40,000 × g for 2 h. The nuclei were recovered at the interface of 0.8 m and 1.2 m sucrose. Samples were stored at -70 °C before being used for Western blot analysis. Isolation of Mitochondria—Mitochondria were isolated by sucrose density gradient centrifugation essentially as described (43Desagher S. Osen-Sand A. Nichols A. Eskes R. Montessuit S. Lauper S. Maundrell K. Antonsson B. Martinou J.-C. J. Cell Biol. 1999; 144: 891-901Crossref PubMed Scopus (1093) Google Scholar). Briefly, cells were washed with PBS containing 1 mm EDTA and resuspended in isotonic homogenization buffer supplemented with 1× protease inhibitor mixture (Sigma). Cells were broken with 30-35 strokes of a Dounce homogenizer (Wheaton), and the suspension was centrifuged at 2,000 × g in an Eppendorf centrifuge at 4 °C. The supernatant was then centrifuged at 13,000 × g at 4 °C for 10 min. The pellet was resuspended in 1 ml of homogenization buffer and layered on top of a discontinuous sucrose gradient consisting of 20 ml of 1.2 m sucrose, 10 mm HEPES, pH 7.5, 1 mm EDTA, and 0.1% bovine serum albumin on top of 17 ml of 1.6 m sucrose, 10 mm HEPES, pH 7.5, 1 mm EDTA, and 0.1% bovine serum albumin. The samples were centrifuged at 27,000 rpm for 2 h at 4 °C using a Beckman SW28 rotor. Mitochondria recovered at the 1.6-1.2 m sucrose interface were washed and resuspended in homogenization buffer. This procedure results in the mitochondrial preparation with very little contamination of other organelles. Contamination of mitochondria in the nuclear fraction was determined by immunoblotting for cytochrome oxidase subunit IV, an integral membrane protein of the mitochondria. Cells were grown on sterile glass coverslips and treated with cisplatin for various time points in the presence and absence of inhibitors. Following treatments, the cells were washed in PBS and fixed in 2% paraformaldehyde in PBS for 15 min. After washing twice in PBS, the cells were permeabilized for 1 h in blocking buffer containing 1% bovine serum albumin, 1% goat serum, 0.1% saponin, 1 mm CaCl2, 1 mm MgCl2, and 2 mm NaV2O5 in PBS. The cells were then incubated with mouse monoclonal anti-AIF antibody (1:200) and rabbit anti-caspase-3 (active) antibody (1:200) for 1 h in a 37 °C humidified incubator. After three washes in washing buffer containing 1% bovine serum albumin and 0.1% saponin in PBS, the cells were incubated at 37 °C in a humidified incubator for 1 h with a 1:500 dilution of Alexa Fluor-conjugated secondary antibody (goat anti-mouse Alexa Fluor 488 for AIF and goat anti-rabbit Alexa Fluor 594 for caspase-3) in blocking solution and again washed with washing buffer. The nuclei were stained with 0.5 μg/ml 4′,6′-diamidino-2-phenylindole (DAPI) for 5 min, and the cells were washed twice in washing buffer. Coverslips were then mounted on slides using antifade mounting medium (Molecular Probes). Localization of active caspase-3 and morphological changes of the nuclei were analyzed using a Zeiss deconvoluted microscope. Cells were harvested by centrifugation, and the pellets were washed in cold PBS twice. The washed cell pellets were lysed with 20 mm HEPES, pH 7.5, containing 10% sucrose, 0.1% CHAPS, 2 mm dithiothreitol, 0.1% Nonidet P-40, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 μg/ml pepstatin A at 4 °C. The supernatants obtained were used to determine the activities of caspase-2 and -3 by fluorometric assay using the following amino-4-methylcoumarin (AMC)-tagged substrates: VDVAD-AMC for caspase-2 and DEVD-AMC for caspase-3 (45Stennicke H.R. Salvesen G.S. Methods Enzymol. 2000; 322: 91-100Crossref PubMed Google Scholar). The enzyme extracts containing 50 μg of protein were incubated with 100 mm HEPES, pH 7.4, containing 10% sucrose, 0.1% CHAPS, 10 mm dithiothreitol, and 50 μm caspase substrate in a total reaction volume of 0.25 ml. The reaction mixture was incubated for 60 min at 30 °C. At the end of the incubation, the amount of liberated fluorescent group, AMC, was determined using a fluorescent spectrofluorometer (PerkinElmer Life Sciences) with an excitation wavelength of 380 nm and an emission wavelength of 460 nm. AMC was used as a standard. Based on the standard curve made with fluorescence reading with free AMC, the data for caspase activity are expressed as nmol of AMC liberated when 50 μg of protein extract was incubated with 50 μm of substrate for 60 min at 30 °C. The cell lysates were prepared as described above for the caspase assay, and equal amounts of protein samples were resolved by SDS-polyacrylamide gel electrophoresis using 8% polyacrylamide gels as previously described (44Kaushal G.P. Singh A.B. Shah S.V. Am. J. Physiol. 1998; 274: F587-F595PubMed Google Scholar). The proteins were electrophoretically transferred to a Transblot membrane (Bio-Rad), processed for immunoblotting with specific antibodies, and detected using the ECL system as previously described (39Kaushal G.P. Kaushal V. Hong X. Shah S.V. Kidney Int. 2001; 60: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Recombinant adenoviral vectors carrying p53, green fluorescent protein, lacZ, and AIF were generous gifts from Dr Ruth S. Slack. The amplification of packaged adenovirus was achieved by infection of HEK 293 cells. Adenoviruses were released from the cells by repeated freezing and thawing of the cells, and a titer of 1.1 × 1012 plaque-forming units/ml was produced. The adenoviral infectivity titers for LLC-PK1 cells were determined using procedures as described (46Cregan S.P. MacLaurin J. Gendron T.F. Callaghan S.M. Park D.S. Parks R.J. Graham F.L. Morley P. Slack R.S. Gene Ther. 2000; 7: 1200-1209Crossref PubMed Scopus (39) Google Scholar). LLC-PK1 cells growing at 70-80% confluence were infected with adenoviral vectors at a multiplicity of infection of 50 plaque-forming units/cell. The presence of the recombinant adenovirus was confirmed by Western blot. When the adenovirus was used for the experiment, the efficiency of the infection was confirmed by contransfection of Ad-GFP or Ad-LacZ followed by 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) staining for β-galactosidase expression. The cells were infected with adenovirus p53 or transfected with p53 small interfering RNA (siRNA) 36 h prior to the treatment of cisplatin. Total RNA from the cultured cells was obtained by using the RNeasy Mini Kit (Qiagen, Inc., Chatsworth, CA). Approximately 1 μg of total RNA was used for reverse transcription. The expression levels of various genes were determined by reverse transcription-PCR. The following primer pairs were used for amplifying gene-specific amplicons: GGAAGCTCAGGATGTTCGAG-3′ and the complementary downstream primer 5′-GTTTCTGCATCACCCAGGTT-3′ for PIDD, 5′-CAGCTCCAAGAGGTTTTTCG-3′ and 5′-CCAGCATCACTCTCCTCACA-3′ for caspase-2, and 5′-AGCCATGTACGTAGCCATCC-3′ and downstream primer 5′-TCTCAGCTGTGGTGGTGAAG-3′ for β-actin used as an internal control. To ensure that the PCR had not reached a saturation point that would skew the quantitation, aliquots were removed after certain cycle numbers and analyzed in series on an agarose gel. Then the minimum cycle that initially gives optimal product was used in the experiment. LLCPK1 cells were plated in a 6-well plate with complete medium. When the cells were 60% confluent, old medium was replaced with fresh medium without serum and antibiotics. siRNAs were designed or obtained from commercial sources to interfere with the expression of AIF, p53, and caspase-2. AIF siRNA sequence 5′-AUGCAGAACUCCAAGCACGTT-3′ (sense strand) was designed using online software (BLOCK-iT™ RNAi Designer from Invitrogen). Commercially available siRNAs for AIF, caspase 2, p53, and AIF from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) were also used in this study according to the manufacturer's instructions. Briefly, Lipofectamine (10 μl) and siRNA (10 pmol in 10 μl) in 0.5 ml of serum-free medium were mixed and incubated at room temperature for 20 min. The cells in each well were then transfected with this mixture. After 12 h, fetal bovine serum was added to a final concentration of 10%, and on day 2, the medium in the cells was replaced with fresh medium before cisplatin treatment. Cells (5,000/well) were plated in 96-well dishes and treated with 25 μm caspase inhibitors (Z-VAD-fmk or Z-VDVAD-fmk) or p53 inhibitor pifithrin-α prior to the treatment with 50 μm cisplatin. Inhibition of cell proliferation was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Roche Molecular Biochemicals, Laval, Canada) according to the manufacturer's protocol. For the detection of apoptosis, cells were grown on glass coverslips in 6-well plates. The cells were treated with caspase inhibitors or pifithrin-α for 1 h prior to treatment with 50 μm cisplatin for 12 h. Apoptosis was detected on the basis of nuclear morphology. Cells were fixed with 4% paraformaldehyde and stained with DAPI to reveal f
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