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Promotion of Caspase Activation by Caspase-9-Mediated Feedback Amplification of Mitochondrial Damage Alan D

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Promotion of Caspase Activation by Caspase-9-Mediated Feedback Amplification of Mitochondrial Damage Alan D C al & ellu ic la n r li Im C m f u Journal of Guerrero et al., J Clin Cell Immunol 2012, 3:3 o n l o a l n o r DOI: 10.4172/2155-9899.1000126 g u y o J ISSN: 2155-9899 Clinical & Cellular Immunology Research Article Article OpenOpen Access Access Promotion of Caspase Activation by Caspase-9-mediated Feedback Amplification of Mitochondrial Damage Alan D. Guerrero1, Ingo Schmitz2, Min Chen1 and Jin Wang1* 1Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas 77030, USA 2Institute for Molecular and Clinical Immunology, Otto-von-Guericke-University Magdeburg and Helmholtz Center for Infection Research, D-38124, Braunschweig, Germany Abstract Mitochondrial disruption during apoptosis results in the activation of caspase-9 and a downstream caspase cascade. Triggering this caspase cascade leads to the cleavage of anti-apoptotic Bcl-2 family proteins, resulting in feedback amplification of mitochondrial disruption. However, whether such a feedback loop plays an important role in the promotion of caspase activation and execution of apoptosis has not been well established. We observed that mutated Bcl-2 or Bcl-xL that are resistant to cleavage by caspases inhibited caspase-9-induced caspase activation in human H9 T cells. The release of Smac after the activation of caspase-9 was also inhibited by cleavage-resistant Bcl-2 or Bcl-xL. Consistently, caspase-9-deficient cells were defective in the release of Smac after induction of apoptosis. Moreover, addition of a Smac mimetic overcame the inhibitory effects of cleavage-resistant Bcl-2/Bcl- xL, and restored caspase-9-mediated cell death. Our data suggest that caspase-9-induced feedback disruption of mitochondria plays an important role in promoting the activation of caspases, while a defect in this process can be overcome by promoting Smac functions. Keywords: Caspase-9; Mitochondrial feedback; Bcl-2, Bcl-xL induced cleavage inactivates the anti-apoptosis function of Bcl-2 and Bcl-xL [24-27]. Interestingly, loss of mitochondrial membrane Abbreviations: iCasp9: Inducible Caspase-9; CID: Chemical- potential during cell death is inhibited when caspase-9 or caspases-3 Inducer of Dimerization; AIF: Apoptosis-Inducing Factor; Endo G: and -7 are deficient [28-33], suggesting an important role for caspases Endonuclease G; HtrA2: High Temperature Requirement Protein A2; in feedback disruption of mitochondria. Smac/DIABLO: Second Mitochondria-Derived Activator of Caspases/ Direct IAP-Binding Protein with Low pI; IAP: Inhibitor of Apoptosis; Nuclear fragmentation following the activation of the caspase XIAP: X-linked IAP; CAD/DFF40: Caspase-Activated DNase/DNA cascade is a hallmark of apoptosis [34]. The degradation of DNA from Fragmentation Factor 40 kDa; ICAD/DFF45: Inhibitor of CAD/DNA apoptotic cells may be important for the prevention of autoimmunity Fragmentation Factor 45 kDa; VDAC: Voltage-Dependent Anion [35,36]. Caspase-activated DNase (CAD)/DNA Fragmentation Channel; GAPDH: Glyceraldehyde 3-phosphate Dehydrogenase; HA: Factor 40 kDa (DFF40) plays a prominent role in mediating nuclear Hemagglutinin; TMRE: Tetramethylrhodamine Ethyl Ester; zVAD- fragmentation [37-40]. CAD is associated with its inhibitor and fmk: Carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylk- chaperone, inhibitor of CAD (ICAD)/DNA Fragmentation Factor etone; UV: Ultraviolet 45 kDa (DFF45) [37-39]. Processing of ICAD by caspases results in Introduction the release and activation of CAD to induce nuclear damage [37-39]. Deletion of either CAD or its inhibitor/chaperone ICAD blocks nuclear In mitochondrial apoptosis, the disruption of the mitochondrial fragmentation in apoptotic cells, suggesting the importance of CAD outer membrane allows the release of cytochrome c from the in mediating nuclear damages [41,42]. Whether caspase-9-induced mitochondria into the cytosol [1,2]. Cytochrome c, Apaf-1 and feedback disruption of mitochondria is important for promoting CAD caspase-9 form the apoptosome for the dimerization and activation activation and inducing nuclear damage is not known. of caspase-9 [3-5]. Caspase-9 then proteolytically cleaves downstream To study the signaling events downstream of caspase-9, we have effector caspases, such as caspase-3, resulting in the activation of expressed caspase-9 that can be dimerized for the direct activation of these caspases [6-8]. Other proteins released from the mitochondria, caspase-9 in intact cells [6,24]. Dimerization of caspase-9 induces the including apoptosis-inducing factor (AIF), endonuclease G (Endo activation of a downstream caspase cascade through caspase-3 [6], G), high temperature requirement protein A2 (HtrA2) and second and triggers a feedback loop for mitochondrial disruption through mitochondria-derived activator of caspases (Smac)/direct IAP-binding protein with low pI (DIABLO), also regulate cell death [9-18]. While AIF, Endo G and HtrA2 may cause cell death independent of caspases [19], Smac promotes caspase activation by releasing caspase-9 and *Corresponding author: Jin Wang, Ph.D., Department of Immunology, Baylor College of Medicine, One Baylor Plaza, Room N920, Houston, Texas 77030, USA, caspase-3 from inhibition by the inhibitor of apoptosis (IAP) proteins Tel: 713-798-6193; Fax: 713-798-3033; E-mail: [email protected] [17,18,20,21]. Received June 15, 2012; Accepted August 02, 2012; Published August 09, 2012 Anti-apoptotic Bcl-2 family proteins, such as Bcl-2 and Bcl-xL, Citation: Guerrero AD, Schmitz I, Chen M, Wang J (2012) Promotion of Caspase maintain the stability of the mitochondrial outer membrane and inhibit Activation by Caspase-9-mediated Feedback Amplification of Mitochondrial the release of mitochondrial apoptotic proteins [1,2,22,23]. However, Damage. J Clin Cell Immunol 3:126. doi:10.4172/2155-9899.1000126 Bcl-2 and Bcl-xL are cleavable by caspases in apoptotic cells [24-26]. Copyright: © 2012 Guerrero AD, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits Cleavage-resistant Bcl-2 and Bcl-xL are more effective in inhibiting unrestricted use, distribution, and reproduction in any medium, provided the apoptosis than their wild type counterparts, suggesting that caspase- original author and source are credited. J Clin Cell Immunol ISSN:2155-9899 JCCI, an open access journal Volume 3 • Issue 3 • 1000126 Citation: Guerrero AD, Schmitz I, Chen M, Wang J (2012) Promotion of Caspase Activation by Caspase-9-mediated Feedback Amplification of Mitochondrial Damage. J Clin Cell Immunol 3:126. doi:10.4172/2155-9899.1000126 Page 2 of 9 cleavage of anti-apoptotic Bcl-2 family proteins [24]. Here we show Flow cytometric analyses of sub-diploid nuclei that cleavage-resistant Bcl-2 or Bcl-xL suppresses the release of Smac and cytochrome c after dimerization of caspase-9 in H9 T cells, leading Cells were cultured for the indicated times with or without to inhibition of both endogenous caspase activation and nuclear apoptotic stimuli. Cells were then collected, washed once in PBS damage. A Smac mimetic overcame the inhibitory effects of cleavage- and re-suspended in hypotonic staining buffer (0.1% sodium citrate, resistant Bcl-2 or Bcl-xL to promote caspase activation and apoptosis, 0.1% Triton X-100 and 50 µg/mL propidium iodide) at 4° in the dark supporting the importance for using Smac mimetics in cancer therapy. overnight as described [32]. Cells were analyzed using an LSRII flow cytometer (BD Biosciences). Materials and Methods Cell death assay Cell lines Cells (20,000/well) were plated in 96-well plates and cultured 5’ HA-tagged inducible caspase-9 (iCasp9) was created by fusing with a Smac mimetic [45] or solvent control for 2 h. Cells were then a mutated FK506-binding protein with the protease domain of treated with CID for 24 h and stained with propidium iodide (PI). The caspase-9 [43]. Human H9 T cell lines that stably express iCasp9 (H9- percentage of cell loss was quantitated by flow cytometry as described iCasp9 cells) were established as described [6,24]. AP20187 (ARIAD [6,24]. Pharmaceuticals, Cambridge, MA), a dimeric FK506 analog, was used as a chemical-inducer of dimerization (CID) to activate iCasp9 [6,24]. Caspase activity assay H9-iCasp9 cells with silencing of caspase-3 or stably expressing wild type Bcl-2 or Bcl-xL, or mutant Bcl-2 or Bcl-xL resistant to proteolytic Caspase activation was determined using the Caspase-Glo 3/7 Kit cleavage by caspases (Bcl-2D/A or Bcl-xLD/A), have been reported [24]. (Promega, Madison, WI) according to the manufacturer’s instructions. JMR cells and JMR cells reconstituted with caspase-9 (JMR-C9) have Cells (10,000/well) were plated in 96-well flat bottom plates and treated been described [31,32]. with a Smac mimetic [45] or solvent control for 2 h, followed by treatment with CID for 6 h. The Caspase-Glo 3/7 reagent was added Western blotting to cells and the plate was incubated for 1 h at room temperature. Cells were lysed in lysis buffer (50 mM HEPES, pH 7.5, 150 mM Luminescence was measured using a FLUOstar OPTIMA plate reader NaCl, 1 mM EDTA, 1% Triton X-100, 1x protease inhibitor cocktail from (BMG Labtech, Offenburg, Germany). Results are shown in Relative Roche, 2 µM z-VAD-fmk) at different time points for Western blotting Light Units (RLU). as described [6,24]. The following primary antibodies were used for Measurement of mitochondrial membrane potential Western blotting: mouse monoclonal antibodies to Smac, cytochrome c and Bcl-2 (BD Biosciences, San Jose, CA), ICAD and caspase-9 Cells were cultured for the indicated times. During the last 20 minutes (Medical and Biological Laboratories, Woburn, MA), HA (Covance, of culture, cells were incubated with 25 nM of tetramethylrhodamine Princeton, NJ) and α-tubulin (Santa Cruz Biotechnology, Santa Cruz, ethyl ester (TMRE; Invitrogen). Cells were collected, washed and CA); rabbit polyclonal antibody to caspase-3 (BD Biosciences), Bcl-xL mitochondrial membrane potential was quantitated on a LSRII flow and GAPDH (Cell Signaling Technology, Danvers, MA) and VDAC1 cytometer (BD Bioscience).
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