ToxSci Advance Access originally published online on March 30, 2007
Toxicological Sciences 2007 98(1):125-136; doi:10.1093/toxsci/kfm071
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An L-Tyrosine Derivative and PPAR
Agonist, GW7845, Activates a Multifaceted Caspase Cascade in Bone Marrow B Cells
* Department of Environmental Health, Boston University School of Public Health
Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts 02118
1 To whom correspondence should be addressed at Department of Environmental Health, Boston University School of Public Health, 715 Albany Street, R-405, Boston, MA 02118. Fax: (617) 638-6463. E-mail: jschlezi{at}bu.edu.
Received January 17, 2007; accepted March 23, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Apoptosis is a critical event in the deletion of B lymphocytes prior to their migration to the periphery. Synthetic peroxisome proliferator activated receptor
(PPAR) agonists, including the drug GW7845 and the environmental contaminant mono-(2-ethylhexyl) phthalate, as well as an endogenous ligand, 15-deoxy-
12,14-prostaglandin J2, induce clonally unrestricted apoptosis in pro/pre-B cells. Considering that PPAR agonists are used clinically for the treatment of diabetes and postulated to be useful as chemotherapeutics, we used GW7845 as a model PPAR agonist to examine the mechanism of cell death that may contribute to tumor killing as well as normal bone marrow B lymphocyte toxicity. GW7845 induced rapid mitochondrial membrane depolarization and release of cytochrome c, along with nearly concurrent activation of capases-2, -3, -8, and -9 in primary pro-B cells and BU-11 cells, a nontransformed pro/pre-B cell line. GW7845-induced apoptosis was reduced significantly in Bax-deficient and Apaf-1 mutant primary pro-B cells, supporting the conclusion that GW7845-induced apoptosis is mitochondria- and apoptosome-dependent. Using benzyloxycarbonyl-VAD-fluoromethyl ketone (VAD-FMK) as a pan-caspase inhibitor, we demonstrated that an initial cytochrome c release occurred independently of caspase activation and that only caspase-9 activation was partially caspase independent. The attenuation of GW7845-induced apoptosis by multiple FMK-labeled peptide sequences suggests that multiple caspase pathways are responsible for initiating and executing apoptosis. The strong activation of Bid provides a mechanism by which caspases-2, -3, and -8 may amplify the apoptotic signal. These data support the hypothesis that pharmacologic concentrations of PPAR agonists induce an intrinsic apoptotic pathway that is driven in normal bone marrow B cells by multiple amplification loops.
Key Words: PPAR; chemotherapy; bone marrow toxicity; apoptosis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Peroxisome proliferator activated receptor
(PPAR) agonists have an established clinical use in the treatment of type II diabetes and are being developed as potential chemotherapeutics for hematologic malignancies (Konopleva and Andreeff, 2002
). Our laboratory has shown that normal, developing bone marrow B lymphocytes also are sensitive to apoptosis induced in a clonally nonrestricted fashion by environmental chemicals or by synthetic drugs, including aryl hydrocarbon receptor and PPAR agonists (Ryu et al., 2005; Schlezinger et al., 2004; Schlezinger et al., 2002). Treatment of murine bone marrow B cells with the synthetic PPAR agonists ciglitazone and GW7845 (Schlezinger et al., 2002), the endogenous PPAR agonist 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2), and the environmental PPAR
/ agonist mono-(2-ethyhexyl) phthalate (Schlezinger et al., 2004) results in an extremely potent apoptosis signal. While these structurally diverse compounds share the ability to activate PPAR, the questions remain as to whether these compounds induce comparable apoptotic pathways and whether bone marrow B cell toxicity may accompany the therapeutic benefits of PPAR agonists.
Caspase activation is a hallmark of apoptosis in many cell types. Typically, caspase cascades are assigned to one of two non-mutually exclusive pathways based on the contribution of death receptors or mitochondria to apical caspase activation. The extrinsic pathway is induced by ligation of tumor necrosis factor receptor family death receptors and requires early activation of caspase-8. The intrinsic pathway is induced by stress (e.g., cytotoxic agents, irradiation) and involves release of cytochrome c from the mitochondria, usually resulting from pore formation by proapoptotic members of the Bcl-2 family such as Bax and Bak. Assembly of an "apoptosome," a death complex composed of mitochondrial-derived cytochrome c, Apaf-1, and caspase-9 drives apoptosis (reviewed in Jin and El-Deiry, 2005). Caspase-2 also may act as the apical caspase in the intrinsic pathway, being activated upstream of the mitochondria and leading to the release of cytochrome c (Zhivotovsky and Orrenius, 2005).
Studies of cells in the immune system have begun to define the apoptotic pathways induced by PPAR agonists of various structures. Treatment of myeloid and lymphoid tumor cell lines with 15d-PGJ2, glitazone drugs, triterpenoids, or ring-substituted diindolylmethanes induces an apoptotic pathway with several common features: loss of mitochondrial membrane potential, activation of caspases-3, -8, and -9, and sensitivity to expression of antiapoptotic Bcl-2 family members (Contractor et al., 2005; Inoue et al., 2004; Konopleva et al., 2004; Nencioni et al., 2003; Piva et al., 2005; Ray et al., 2004, 2005). Cytochrome c release has been observed following treatment with 15d-PGJ2 (Nencioni et al., 2003), and this release is accompanied by changes in Bax expression following treatment with a diindolylmethane (Contractor et al., 2005). These results support the hypothesis that various PPAR agonists induce a mitochondrially-dependent apoptotic pathway; however, the actual triggers of mitochondrial disassemblage and the interaction between the caspases within the pathway remain to be defined.
Apoptosis is a critical event in the deletion of self-reactive B lymphocytes as they enter the periphery from the bone marrow and occurs at the highest rate during the pro/pre-B cell transition (Lu and Osmond, 2000). While much of the signaling pathway leading to immature B cell apoptosis has been mapped in systems in which transformed cells (e.g., WEHI-231) were induced to undergo apoptosis following surface Ig cross-linking, a model for self-antigen–induced clonal deletion, significantly less is known about mitochondrial activation and caspase cascades in nontransformed bone marrow B cells with clonally nonrestricted agents and whether these B cells may be sensitized to exogenous apoptotic agents. Results of studies of the treatment of pro/pre-B cells with the polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene suggest that apoptotic signals are amplified in these cells by activation of caspases in both the intrinsic and extrinsic pathways (Ryu et al., 2005). Considerable crossover between the intrinsic and extrinsic apoptotic pathways may occur. The extrinsic pathway can induce caspase-8–dependent cleavage of Bid (Li et al., 1998). Conversely, caspase-3 cleavage, resulting from activation of the intrinsic pathway, can activate caspase-6, which in turn activates caspase-8, also leading to Bid cleavage (Cowling and Downward, 2002; Murphy et al., 2004; Slee et al., 1999).
To delineate which components are recruited during apoptosis signaling in bone marrow B cells, apoptosis of stromal cell–dependent pro/pre-B cells (BU-11) and primary pro-B cells was induced with the PPAR agonist and putative chemotherapeutic, GW7845. Landmarks in the apoptosis signaling pathway, including cytochrome c release, apoptosome formation, and caspase-2, -3, -8, and -9 activation were mapped. The results indicate that a complex and powerful cascade of apoptosis mediators induces the rapid demise of normal bone marrow B cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Materials.
The caspase-8–specific antibody was from Axxora (San Diego, CA). The cytochrome c–specific antibody was from BD Biosciences Clonetech (Palo Alto, CA). Caspase inhibitors and cyclosporin A were from Biomol (Plymouth Meeting, PA). Antibodies specific for cleaved caspases-3 and -9 and cleaved lamin were from Cell Signaling Technology (Beverley, MA). The antibodies for
-fodrin and caspase-2 were from Chemicon International (Temecula, CA). GW7845 was the generous gift of Dr T. Willson (GlaxoSmithKline, Research Triangle Park, NC). Plasmocin was from Invivogen (San Diego, CA). JC-1 was from Molecular Probes (Eugene, OR). The Bid-specific antibody was from R&D Systems (Minneapolis, MN). Murine rIL-7 was from Research Diagnostics (Flanders, NJ). Propidium iodide (PI), Protease Inhibitor Cocktail for Mammalian Cells, DiOC6 and rhodamine 123, and the ß-actin antibody were from Sigma Chemical Co. (St Louis, MO). All other reagents were from Fisher Scientific (Suanee, GA).
Cell culture.
The stromal cell-dependent, C57BL/6-derived BU-11 cell line has been characterized previously (Ryu et al., 2005; Schlezinger et al., 2004; Schlezinger et al., 2002). BU-11 cells represent B cells at the transition between the pro- and early pre-B cell stages as they are CD43+/B220+/IgM– with rearranged Ig heavy chain genes. BMS2 is a culture dish-adherent cloned bone marrow stromal cell line that supports BU-11 cell growth. Stocks of BU-11 cells were maintained on BMS2 cell monolayers in an equal mixture of Dulbecco's modified Eagle's medium and RPMI 1640 medium with 5% fetal bovine serum (FBS), plasmocin, L-glutamine, and 2-mercaptoethanol. All cultures were maintained at 37°C in a humidified, 7.5% CO2 atmosphere. Cell cultures were determined to be mycoplasma negative using the MycoAlert Assay (Cambrex, East Rutherford, NJ).
Primary bone marrow pro-B cell cultures were prepared from wild-type C57BL/6, Bax wild-type, Bax null, Apaf-1fog/J heterozygous, and Apaf-1fog/J homozygous mice (Jackson Laboratories, Bar Harbor, ME) as described previously (Ryu et al., 2005). Bone marrow was flushed from the femurs of 4- to 6-week–old male mice. Red blood cells were lysed by incubation in 0.17M NH4Cl, 10mM KHCO3, and 1mM EDTA at 37°C for 5 min. The remaining cells were cultured for 5–7 days in primary B cell medium (RPMI containing 10% FBS, penicillin/streptomycin, L-glutamine, 2-mercaptoethanol, and 16 ng/ml murine rIL-7 [RDI, Flanders, NJ]). This procedure results in a B cell culture in which at least 95% of the cells express CD43 and B220.
For experiments, pro/pre-B cells were cultured in 24-well plates (4 x 105 cells in 1 ml medium) or in T25 flasks (6 x 106 cells in 10 ml medium) overnight in RPMI with 5% FBS and treated with vehicle (Vh) (ethanol:dimethyl sulfoxide [DMSO], 50:50, 0.1% final concentration in all experimental cultures) or GW7845 (40µM) for 15 min–2.5 h. Cultures treated with Vh alone were treated for the maximal time shown for the experiment. Results shown from experiments here, as well as in previous studies (Schlezinger et al., 2006; Schlezinger et al., 2002), demonstrated that Vh treatment alone has no significant impact on the parameters measured. Cells were pretreated with Vh (DMSO, 0.1% final concentration in all experimental cultures), cyclosporin A (1–2µM) DEVD-FMK, FA-FMK, IETD-FMK, LEHD-FMK, VAD-FMK, VDVAD-FMK, or VEID-FMK (15–30µM) for 30 min. Primary pro-B cells were cultured in 24-well plates (4 x 105 cells in 1 ml medium) or in T25 flasks (107 cells in 10 ml medium) in primary B cell medium with 7.5% FBS overnight and treated with Vh (ethanol:DMSO, 50:50, 0.2% final concentration in all experimental cultures) or GW7845 (80µM) for 1–4 h.
Analysis of apoptosis.
B cells were harvested into cold phosphate-buffered saline (PBS) containing 5% FBS and 10µM azide. Cells were resuspended in 0.25 ml hypotonic buffer containing 10 µg/ml PI, 1% sodium citrate, and 0.1% Triton X-100 and analyzed with FL-2 in the log mode on a Becton/Dickinson FACScan flow cytometer. The percentage of cells undergoing apoptosis was determined to be those having a weaker PI fluorescence than cells in the G0/G1 phase of the cell cycle (Schlezinger et al., 2002, 2006).
Analysis of mitochondrial membrane potential.
Thirty minutes prior to harvest, JC-1 (1.4µM, Molecular Probes), rhodamine 123 (1µM), or DiOC6 (2nM) were added to each well. Cells stained with rhodamine 123 were washed in PBS, centrifuged, and then resuspended in PBS prior to analysis by flow cytometry. BU-11 cells were transferred to FACS tubes without washing for JC-1 and DiOC6 and analyzed immediately by flow cytometry. Only cells in the live gate were analyzed. The percentage of cells with low mitochondrial membrane potential (
mlow) was determined to be those having an increased green fluorescence with or without a loss of red fluorescence for JC-1 or a decreased green fluorescence for rhodamine and DiOC6 (Salvioli et al., 1997).
Immunoblotting.
B cells were harvested and washed once in cold PBS. For analysis of cleavage of caspases or their substrates, cytoplasmic extracts were prepared as described previously (Schlezinger et al., 2002). For analysis of cytochrome c release and Bax and Bid translocation, cytoplasmic and mitochondrial fractions from digitonin-permeabilized cells were prepared as described previously (Waterhouse et al., 2004). Protein concentrations were determined by the Bradford method.
Proteins (5–60 µg) were resolved on 6% (-fodrin), 12% (caspases-2, -8, and -9), or 15% (Bax, Bid, caspase-2, -3, cytochrome c, and lamin) gels, transferred to a 0.2-µm nitrocellulose membrane, and incubated with primary antibody. Primary antibodies included monoclonal mouse anti-–fodrin (MAB1622, Chemicon International), polyclonal rabbit anti-Bax (sc-293, Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal rat anti-Bid (MAB860, R&D Systems), monoclonal rat anti-caspase–2 (MAB3501, Chemicon International), polyclonal rabbit anti-cleaved caspase-3 (9661, Cell Signaling Technology), polyclonal rat anti-caspase-8 (ALX-804-447, Axxora), polyclonal rabbit anti-caspase-9 (9504, Cell Signaling Technology), polyclonal rabbit anti-cytochrome c (S2050, BD Biosciences), or monoclonal mouse anti-cleaved lamin antibody (2036, Cell Signaling Technology). Immunoreactive bands were detected using horseradish peroxidase–conjugated secondary antibodies (BioRad, Hercules, CA) followed by enhanced chemiluminescence. To control for equal protein loading, blots were stripped and reprobed with a ß-actin–specific antibody (A5441, Sigma) or -tubulin–specific antibody (CP06, EMD Biosciences, San Diego, CA) and analyzed as above. To quantify changes in protein expression, band densities were determined using the UVP Bioimaging System and the Labworks 4 program (UVP, Inc., Upland, CA). The band density of the protein of interest was divided by the band density of ß-actin. To normalize for differences between experiments, the band density ratio for experimental samples then was divided by the ratio in the naive sample from that experiment.
Statistics.
Statistical analyses were performed with Statview (SAS Institute, Cary, NC). Data are presented as means ± SE. At least three experiments were performed in each BU-11 protocol. Experiments with primary pro-B cells were performed with a minimum of three pools of bone marrow cells, and each pool of bone marrow cells was prepared and maintained separately. One-factor ANOVAs were used to analyze the data. For the ANOVAs, the Dunnett's or Tukey-Kramer multiple comparisons test was used to determine significant differences.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Induction of Mitochondrial Membrane Changes in Pro/Pre-B cells by GW7845
Previous studies demonstrated that malignant B cells are susceptible to apoptosis induced by PPAR
agonists (Contractor et al., 2005; Konopleva et al., 2004; Piva et al., 2005; Ray et al., 2004). We have shown that normal bone marrow B cells also are susceptible to apoptosis induced by structurally diverse PPAR agonists, including ciglitazone, GW7845, 15d-PGJ2, and MEHP (Schlezinger et al., 2004; Schlezinger et al., 2002). The death induced by GW7845 in particular is extremely rapid, with a 40µM dose inducing apoptosis in 50% of pro/pre-B cells (BU-11) within 2 h (Schlezinger et al., 2006; Schlezinger et al., 2002). The nature of the stressor and the rapid activation of death suggested that compromise of the mitochondria is involved in initiating the death pathway. Therefore, the effect of GW7845 on the mitochondria of pro/pre-B cells was investigated. BU-11 cells were treated with Vh or GW7845 and analyzed for mitochondrial membrane potential loss by rhodamine 123, JC-1, or DiOC6 staining and for cytochrome c release by immunoblotting. Within 15 min of treatment, GW7845 induced a significant loss of mitochondrial membrane potential, and the loss of membrane potential was essentially complete by 60 min (Figs. 1A and 1B). Similar results were seen using JC-1 and DiOC6 (not shown). The GW7845-induced loss of mitochondrial membrane potential was accompanied by cytochrome c release within 15 min (Fig. 1C), supporting a role for the mitochondria and the intrinsic apoptosis pathway in GW7845-induced death.
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While the opening of the mitochondrial permeability transition pore is associated with loss of mitochondrial membrane potential and the release of small solutes from the outer membrane, the role of these events in mediating apoptotic cell death has been debated (Schinzel et al., 2005
). To begin to examine the contribution of the permeability transition pore to the GW7845-induced apoptotic process, BU-11 cells were treated with Vh or cyclosporin A (CsA, 1–2µM), an inhibitor of permeability transition pore opening through interaction with cyclophilin D (Schinzel et al., 2005), 30 min prior to treatment with Vh or GW7845. CsA (1–2 µM) modestly inhibited apoptosis (6–14%), as assayed by a reduction in the percentage of cells exhibiting a subdiploid peak following PI staining of DNA (Fig. 2A), suggesting that the mitochondrial permeability transition contributes minimally to the apoptotic process. The same concentrations of CsA had a significantly greater effect on the loss of mitochondrial membrane potential, reducing the loss by as much as 40% (Fig. 2B). Higher concentrations of CsA were increasingly toxic. Therefore, the formal possibility that permeability transition pore function was not completely inhibited by CsA cannot be ruled out. However, CsA also did not significantly affect the release of cytochrome c (Fig. 2C, fold change from naive: GW: 4.8 ± 0.3, GW + CsA 1µM: 4.9 ± 0.7, GW + CsA 2µM: 4.2 ± 0.8) suggesting that GW7845-induced release of cytochrome c and apoptosis occur largely independently of the mitochondrial permeability transition.
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Release of cytochrome c may occur downstream or upstream of caspase activation. To investigate the contribution of caspases to cytochrome c release, BU-11 cells were pretreated with Vh or the pan-caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone (VAD-FMK), then treated with Vh or GW7845, and analyzed for cytochrome c release. While almost completely blocking apoptosis (see Fig. 6), pretreatment with VAD-FMK had, at best, a small effect on cytochrome c release (Fig. 3A, fold change from naive at 30 min: GW: 2.4 ± 0.3, GW + VAD: 1.9 ± 0.2). This result suggested that cytochrome c release may be the apical event in GW7845-induced apoptosis and thus likely a result of changes in the localization of Bcl-2 family members in the mitochondria. Indeed, treatment of BU-11 cells with GW7845 (20–40µM) for 60 min resulted in Bax translocation to the mitochondria (Fig. 3B). The contribution of Bax to the apoptotic cascade was tested using Bax null primary pro-B cells. Primary bone marrow pro-B cells (> 95% B220+/CD43+) were prepared from Bax wild-type (Bax WT) and null (Bax KO) mice by culture in rIL-7 for > 5 days as we previously described, treated with Vh or GW7845 for 4 h, and analyzed for apoptosis by PI staining. GW7845-induced formation of the subdiploid population was reduced significantly in Bax null primary pro-B cells (Fig. 3C). These results strongly support the conclusion that mitochondrial activation occurs upstream of caspase activation in GW7845-induced apoptosis.
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If the caspase cascade were initiated by mitochondrial changes such as cytochrome c release, then it would be predicted that apoptosis would be inhibited in primary bone marrow B cells from Apaf-1 mutant mice, Apaf-1 being an essential component of the apoptosome and essential for maximal activation of caspase-9 (Saleh et al., 1999
). To test this prediction, primary pro-B cells were prepared from mice hetero- or homozygous for the Apaf-1fog mutation, were treated with Vh or GW7845 for 4 h, and analyzed for apoptosis by PI staining. A significant increase in apoptosis was observed with primary pro-B cells from Apaf-1fog heterozygous mice but not with primary pro-B cells from Apaf-1fog homozygous mice (Fig. 4). The results are consistent with the hypothesis that release of cytochrome c occurs upstream of caspase activation and that caspase-9 is the apical caspase in GW7845-induced early B cell apoptosis.
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Activation of Caspases Contributing to the Intrinsic Apoptotic Pathway by GW7845
Typically, it is thought that the apoptotic process is activated by initiator caspases, such as caspases-2 or -9 that drive the intrinsic pathway or caspase-8 that initiates the extrinsic pathway. The initial data showing a significant release of cytochrome c suggests that at least an intrinsic, caspase-9–driven pathway is activated by GW7845 in pro/pre-B cells. To determine the role of caspase-9 in GW7845-induced pro/pre-B cell apoptosis, BU-11 cells were treated with GW7845 and analyzed for caspase-9 activation by immunblotting for full-length and cleaved (active) caspase-9. A loss of full-length caspase-9 and the appearance of cleaved, active caspase-9 occurred within approximately the same time frame as cytochrome c release from the mitochondria, i.e., 15 min after treatment (Fig. 5A).
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To confirm that the non-transformed, stromal cell–dependent BU-11 cells accurately model caspase activation in primary bone marrow B cells, formation of active caspase fragments was analyzed in the latter population. Primary pro-B cells undergo apoptosis following exposure to GW7845 with approximately the same kinetics as BU-11 cells, although they are somewhat more resistant to this apoptosis stimulus and require exposure to a higher concentration of GW7845 (Schlezinger et al., 2004
, 2002). As seen in the BU-11 cell line, treatment of primary pro-B cells with GW7845 induced cleavage of caspase-9 (Fig. 5A). These data are consistent with activation of a mitochondria-dependent, intrinsic apoptosis pathway.
Caspase-3 is considered to be the primary apoptosis executioner of both the intrinsic and extrinsic pathways with the broadest substrate repertoire of the effector caspases. Given activation of multiple components of the intrinsic pathway, including release of cytochrome c and activation of caspase-9, it was predicted that caspase-3 would be activated with approximately the same kinetics. To test this prediction, BU-11 and primary pro-B cells were treated with GW7845 and analyzed for caspase-3 activation by immunblotting for cleaved caspase-3. As predicted, GW7845 rapidly induced the formation of the 17 kDa active caspase-3 fragment (Fig. 5B). Further confirmation of induction of caspase-3 activity was shown by the cleavage of endogenous -fodrin (Fig. 5C), a specific caspase-3 substrate.
Cooperation of the Intrinsic and Extrinsic Pathways in GW7845-Induced Apoptosis
The extremely rapid induction of apoptosis by GW7845 suggested that more than a simple intrinsic apoptotic pathway is activated by GW7845. The hypothesis that activation of multiple caspases is required to support GW7845-induced death was tested further by examining the effect of multiple caspase inhibitors. BU-11 cells were pretreated with Vh or 15µM of a series of caspase inhibitors (DEVD-FMK, FA-FMK, IETD-FMK, LEHD-FMK, VAD-FMK, VDVAD-FMK, or VEID-FMK), treated with Vh or GW7845 for 2.5 h, and analyzed for apoptosis by PI staining. The limiting inhibitor dose of 15µM, which is significantly lower than doses indicated in other publications, was used to maximize inhibitor specificity. FA-FMK, a negative control peptide, had no effect on GW785-induced apoptosis (Fig. 6). All of the peptide inhibitors significantly suppressed the apoptosis induced by GW7845 (Fig. 6), with VAD-FMK, a general caspase inhibitor, DEVD-FMK, a caspase-3-like activity inhibitor, and IETD-FMK, a caspase-8–like activity inhibitor, causing the greatest reduction in apoptosis. The participation of caspases from both the intrinsic and extrinsic pathways suggests that an amplification loop contributes to the rapid apoptosis induced by GW7845.
Caspase-3 can participate in several amplification scenarios, including supporting Bid cleavage by activating caspase-2, bridging the mitochondria-dependent intrinsic pathway and the extrinsic pathway through its ability to activate caspase-8 through caspase-6 (Cowling and Downward, 2002; Murphy et al., 2004; Slee et al., 1999), and directly stimulating the mitochondrial translocation of Bid and Bax (Lakhani et al., 2006; Slee et al., 2000). To determine if caspases-2 and -8 are activated by GW7845, BU-11 or primary pro-B cells were treated with Vh or GW7845 and analyzed by immunoblotting for full-length and cleaved (activated) caspases-2 and -8. As seen with caspase-9, activation of caspase-2 (Fig. 7A) and caspase-8 (Fig. 7B) was evident within 15 min of treatment with GW7845 in BU-11 and within 1 h of treatment in primary pro-B cells.
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Bid is a proapoptotic member of the Bcl-2 protein family. Following cleavage, the C-terminal portion (tBid) translocates to the mitochondria and integrates into the outer mitochondrial membrane instigating cytochrome c release (Slee et al., 2000
). The strong GW7845-induced apoptotic signal and the activation of multiple caspases capable of cleaving Bid suggested that Bid may play a role in amplifying the apoptotic signal. Therefore, we examined Bid cleavage following GW7845 treatment. BU-11 cells were treated with Vh or GW7845 and analyzed for formation of tBid by immunoblotting. As predicted, there was a very strong formation of tBid in the cytoplasm following treatment with GW7845 (Fig. 8A). Furthermore, tBid also was present in the mitochondria (Fig. 8B). Pretreatment of BU-11 with DEVD-FMK (a caspase-3–like activity inhibitor), IETD-FMK (a caspase-8–like activity inhibitor), or VDVAD-FMK (a caspase-2–like activity inhibitor) resulted in a reduction in the appearance of tBid in the mitochondria (Figs. 8C and 8D). The induction of mitochondrial tBid could be completely blocked by DEVD-FMK (Fig. 8C and 8D) but only partially blocked by IETD-FMK and VDVAD-FMK (Fig. 8D). This suggests that while multiple caspases may contribute to Bid cleavage, caspase-3 activation drives the amplification process. Collectively, these results indicate a nearly simultaneous activation of components of both the extrinsic and intrinsic pathways following GW7845 exposure that may support amplification of the initial mitochondrially driven apoptotic signal.
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To confirm the role of caspase-9 as the initial caspase activated in the death pathway, we used the general caspase inhibitor, VAD-FMK, to block all but the first reaction in the caspase cascade. Since this peptide inhibitor blocks the activity and not the cleavage of caspases, active fragments of only the apical caspase should form in the presence of VAD-FMK. BU-11 cells were pretreated with Vh or VAD-FMK, treated with Vh or GW7845, and analyzed for formation of active caspase fragments by immunoblotting. VAD-FMK completely blocked the GW7845-induced formation of caspase-2 and -8 fragments (Figs. 9A and 9B). However, the formation of active caspase-9 fragments induced by GW7845 was only partially blocked by VAD-FMK (Fig. 9C). These results support those above showing that caspase-9 is the most apical caspase activated by GW7845 and that activation of caspases-2 and -8 is itself caspase dependent.
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Since activation of caspase-8 was caspase dependent, we sought to determine if caspases-3 and -6 might contribute to the activation of caspase-8. BU-11 cells were pretreated with Vh, DEVD-FMK, or VEID-FMK (a caspase-6 inhibitor), treated with Vh or GW7845, and analyzed for formation of active caspase-8 fragments by immunoblotting. Pretreatment with either DEVD-FMK or VEID-FMK significantly reduced the formation of cleaved caspase-8 (Fig. 10A), supporting the hypothesis that caspase-8 is activated through a caspase-3 to caspase-6 loop. Activation of caspase-6 was confirmed by examining the cleavage of lamin A, a caspase-6–specific substrate (Ruchaud et al., 2002
). As predicted, formation of cleaved lamin A occurs following treatment with GW7845 (Fig. 10B). The apparent activation of caspase-6 following treatment with GW7845 supports the hypothesis that activation of the extrinsic apoptotic pathway through the activation of a caspase-3 to caspase-6 to caspase-8 loop significantly contributes to the execution of apoptosis induced by GW7845.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Questions remain as to whether the program of B cell development, in which apoptosis plays an essential role, is perturbed by exposure to environmental chemicals and as to whether bone marrow B cells are particularly sensitive to apoptotic agents due to prevalence of naturally occurring apoptosis in early B cell development. We have shown previously that apoptosis induced by a prototypic polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[a]anthracene, in bone marrow B cells is enhanced by the recruitment of the extrinsic apoptotic pathway following release of cytochrome c from the mitochondria (Ryu et al., 2005
), suggesting the sensitivity of early B cells to apoptotic stimuli. Understanding the mechanisms of bone marrow B cell apoptosis will aid in the evaluation of the potential for exposure to toxic chemicals in the clinical setting (e.g., exposure to antidiabetic or chemotherapeutic agents) or in the environment (e.g., exposure to organotins or phthalates) to lead to immunosuppression.
Treatment with glitazone drugs, PPAR agonists similar in structure to GW7845, can impair the metabolic function of mitochondria (Scatena et al., 2004) and induce the loss of mitochondrial membrane potential in other cell types (Perez-Ortiz et al., 2004; Ray et al., 2004), suggesting that mitochondria are a likely target for GW7845. Indeed, a prominent characteristic of GW7845-induced apoptosis in pro/pre-B cells is compromise of the mitochondrial membranes, as evidenced by the rapid decrease in mitochondrial membrane potential and the release of cytochrome c. The hallmark of the intrinsic apoptotic pathway is the release of cytochrome c and formation of the apoptosome (reviewed in Jin and El-Deiry, 2005). The fact that GW7845-induced apoptosis was abrogated in Bax null and Apaf-1fog primary pro-B cells again supports the role of the mitochondria in driving the apoptotic process. Furthermore, GW7845-induced caspase-9 cleavage was largely resistant to pretreatment with the general caspase inhibitor VAD-FMK, suggesting that its activation was the result of apical activation of the mitochondria.
While both loss of mitochondrial membrane potential and cytochrome c release occur following treatment with GW7845, it appears that these events may occur independently and that only the cytochrome c release is required to support apoptosis. Originally, it was thought that loss of mitochondrial membrane potential was an early and required event in the apoptotic process; however, more recent evidence suggests that loss of mitochondrial membrane potential is not necessarily related to the release of cytochrome c (Salvioli et al., 1997). Loss of mitochondrial membrane potential results from initiation of the mitochondrial permeability transition, and recent studies describing results with cells from cyclophilin D knockout mice suggest that the permeability transition is not required for death induced by Bcl-2 family members Bid, Bax, and Bak. Rather, opening of the mitochondrial permeability transition pore is required for reactive oxygen- and calcium-mediated necrotic cell death (Schinzel et al., 2005). The data here show that CsA significantly reduced GW7845-induced membrane depolarization while having a minimal effect on cytochrome c release and apoptosis. These results suggest that GW7845 may induce more than one death pathway, one leading to apoptosis by inducing release of cytochrome c and one leading to necrosis by inducing opening of the mitochondrial permeability transition pore, potentially resulting in necrapoptosis (Lemasters et al., 2002).
The lack of effect of VAD-FMK or CsA on GW7845-induced cytochrome c release suggests that the initial release occurred independently of caspase activation and the mitochondrial permeability transition. Results with pro/pre-B cells suggest that Bax translocates to the mitochondria where it may contribute to cytochrome c release following treatment with GW7845. To this end, it has been shown that Bcl-2 overexpressing and Bax knockout cells are relatively resistant to apoptosis induction with other PPAR agonists, such as diindolylmethanes and 15d-PGJ2 (Contractor et al., 2005). Both JNK and p38 MAPK activation are associated with apoptosis potentially through their interactions with p53 and Bax (Wada and Penninger, 2004). Our recent study with GW7845 showed that activation of MAPKs was required for release of cytochrome c and apoptosis in BU-11 and primary pro-B cells (Schlezinger et al., 2006), suggesting a mechanism by which Bax may be activated. The relationship between kinase activation and Bax activation remains to be examined.
The data presented here support the hypothesis that multiple, interacting caspases drive GW7845-induced apoptosis and that a caspase amplification loop is activated. Indeed, all inhibitor peptide sequences that were tested significantly blocked apoptosis, and activation of the typically initiator caspases-2 and -8 was caspase dependent. The apoptotic pathway may be amplified in multiple ways, and the very strong activation of Bid, a proapoptotic Bcl-2 family member, suggests that this event plays an important role in the amplification loop. Furthermore, not only was tBid formed, but it also localized in the mitochondria following treatment with GW7845. Caspase-2 can be cleaved directly by caspase-3 and may act as an executioner caspase in this system (Slee et al., 1999), and it may act as an amplifier by cleaving and activating Bid (Zhivotovsky and Orrenius, 2005). Studies in several systems suggest that the intrinsic mitochondrial pathway activates caspase-8 through caspase-3 (Cowling and Downward, 2002; Murphy et al., 2004; Slee et al., 1999). Most studies implicate caspase-6 as an intermediary, thus it is not surprising that we observed cleavage of lamin A, a caspase-6 substrate. Active caspase-8 may then cleave Bid, allowing it to translocate to the mitochondria (Li et al., 1998). Finally, caspase-3 itself may cleave and activate Bid (Slee et al., 2000). Since multiple peptide sequences inhibited both apoptosis and tBid formation, it is likely that multiple caspases participate in an apoptosis amplification loop, potentially through their ability to cleave Bid.
While compounds that have been shown to induce apoptosis in bone marrow B cells, including ciglitazone, 15d-PGJ2, and GW7845 (Schlezinger et al., 2004; Schlezinger et al., 2002), share the ability to activate PPAR, the participation of PPAR itself in the apoptotic mechanism of its agonists is controversial. PPAR antagonists reduce the magnitude of apoptosis induced by diverse PPAR agonists, including ring-substituted diindolylmethanes, triterpenoids, 15d-PGJ2, and glitazone drugs, thereby implicating this receptor in death signaling (Bodles et al., 2006; Chen et al., 2005; Contractor et al., 2005; Konopleva et al., 2004). However, troglitazone was found to be equally effective at inhibiting proliferation in both PPAR–/– and PPAR+/+ mouse embryonic stem cells (Palakurthi et al., 2001), and there is growing evidence for PPAR-independent effects of PPAR agonists, particularly at higher concentrations (Berry et al., 2005; Chintharlapalli et al., 2006). Indeed, the concentration of GW7845 required to induce apoptosis (20µM) is substantially higher than that required to activate PPAR (10–1000nM).
Evidence is mounting of alternative, extrareceptor mechanisms by which compounds classified as PPAR agonists may induce apoptosis. Production of reactive oxygen species and suppression of NF-
B–binding activity following treatment with 15d-PGJ2, but not glitazone drugs, have been suggested to initiate apoptosis (Nencioni et al., 2003). A triterpenoid PPAR agonist also can induce formation of high-molecular weight protein aggregates in the mitochondrial membrane that act as a permeability transition pore (Brookes et al., 2007). In addition, alterations in calcium distribution may be the apical event in the apoptosis induced by at least some PPAR agonists. Glitazone drugs induce endoplasmic reticulum stress (Gardner et al., 2005), and calcium chelation reduces GW7845-induced apoptosis in pro/pre-B cells (Schlezinger and Sherr, unpublished results). Investigation of the relationship between endoplasmic reticulum stress and PPAR agonist–induced apoptosis continues.
To our knowledge, no studies have investigated if the concentration of GW7845, or other PPAR agonists, that induces B cell apoptosis in vitro is attainable in vivo; therefore, caution must be used in extrapolation of the potential of these compounds to induce apoptosis in vivo. However, significantly lower concentrations of PPAR agonists may slow proliferation and induce cell cycle arrest in B cells via induction of cyclin-dependent kinase inhibitors (Schlezinger et al., 2004). In vivo studies support the hypothesis that physiologically significant concentrations of PPAR agonists that may be effective in controlling tumor growth can be reached (Grommes et al., 2004). In addition, in vivo treatment with a PPAR-activating triterpenoid significantly reduced the growth of a leukemia cell xenograft (Place et al., 2003). Interestingly, low dose, PPAR-dependent growth inhibition of a colon cancer cell line was hypothesized to act in concert with the PPAR-independent apoptosis induced by higher doses of a PPAR-active diindolylmethane, to result in suppressed tumor growth in vivo (Chintharlapalli et al., 2006).
The effects of PPAR haploinsufficiency on B cell proliferation demonstrate PPAR-dependent regulation of B cell growth as well as death, suggesting that PPAR may be a target to control B cell growth (Setoguchi et al., 2001). Exploitation of receptor-independent apoptotic pathways as well may provide synergistic pathways to control growth of lymphocyte malignancies. Further studies are required to determine how low dose and potentially receptor-dependent effects may contribute to B cell growth control in both normal and transformed B cells in vivo. Understanding both the receptor-dependent and -independent mechanisms of action of PPAR agonists is required to minimize possible immunosuppressive effects of clinical treatments (e.g., antidiabetic drugs and chemotherapeutics) and possible inadvertent immunosuppressive effects of environmental exposures (e.g., organotins and phthalates).
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ACKNOWLEDGMENTS |
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Superfund Basic Research Grant 2P42ES007381-12 and National Institutes of Health RO1-ES06086 supported this work.
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REFERENCES |
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