Bz-423 superoxide signals B cell apoptosis via Mcl-1, Bak, and Bax
Graphical abstract
Introduction
Bz-423 is a pro-apoptotic 1,4-benzodiazepine with potent therapeutic properties in murine lupus linked to specific deletion of autoreactive lymphocytes [1], [2]. Bz-423 binds to the oligomycin-sensitivity conferring protein (OSCP), a component of the mitochondrial F1F0-ATPase, and induces the formation of superoxide via a state 3 to state 4 respiratory transition [3]. The reactive oxygen species (ROS) generated by Bz-423 induces lymphocyte apoptosis both in vivo and in vitro[2]. The absence of either general toxicities or significant effects on normal immune responses in treated mice indicates that Bz-423 has selective effects on cells that are pathogenic in autoimmune disease.
We previously characterized the apoptosis induced by Bz-423 in mouse embryonic fibroblasts (MEFs) containing knockouts of key apoptotic proteins [4]. In this cell type, Bz-423-induced superoxide is followed by caspase activation, mitochondrial electrochemical gradient (ΔΨm) collapse, and the release of cytochrome c into the cytoplasm, consistent with mitochondrial outer membrane permeabilization (MOMP) and the release of cytochrome c from the mitochondrial inter-membrane space [5]. Following these events, morphological and biochemical evidence of apoptosis is detected. In isolated mitochondria, Bz-423 induces ROS, but does not cause gradient collapse or swelling. These data show that Bz-423-induced superoxide does not directly trigger opening of the permeability transition pore, and implicates extra-mitochondrial factors in the mechanism coupling Bz-423-induced ROS to apoptosis.
In MEFs, apoptosis signal-regulating kinase 1 (ASK1) was found to be a critical upstream cellular redox sensor linking mitochondrial superoxide generation to apoptosis [4]. Activation of ASK1 initiates a mitogen activated protein (MAP) kinase cascade culminating in the activation of c-Jun N-terminal kinase (JNK). Activated JNK is then necessary for activation of pro-apoptotic Bax and Bak resulting in MOMP and a commitment to apoptotic cell death, as a small molecule JNK inhibitor prevents all of these steps.
Because MAP kinases are differentially regulated across cell types [6], we sought to determine if Bz-423 activates this pathway in lymphocytes, a cell type which is more sensitive to Bz-423. In particular, we sought to identify the extra-mitochondrial factors that link Bz-423-induced superoxide to apoptosis in Ramos B cells, in order to understand the differences in the response between lymphocytes and fibroblasts. In contrast to fibroblasts, Bz-423 does not activate MAP kinases in B cells, but apoptosis nonetheless still requires activation of Bax and Bak. We identify a superoxide-dependent decrease in Mcl-1 levels, and find that Bz-423 activates multiple BH3-only proteins in order to activate Bax and Bak and induce MOMP. These results demonstrate that superoxide generated from the mitochondrial respiratory chain as a consequence of a respiratory transition can signal specific apoptotic responses that differ across cell types. Our data suggest that differences in the levels of antioxidants and expression of Bcl-2 proteins help to explain the increased sensitivity of lymphocytes to Bz-423.
Section snippets
Reagents
Bz-423 was synthesized as previously described [7]. Dihydroethidium (DHE) and 3,3′-dihexyloxacarbocyanine iodide (DIOC6(3)) were obtained from Invitrogen Corp. (Carlsbad, CA, USA). Manganese (III) tetrakis (4-benzoic acid)porphyrin (MnTBAP) was purchased from Alexis Biochemicals (Lausen, Switzerland). Unless otherwise specified, all additional reagents were obtained from Sigma–Aldrich (St. Louis, MO, USA).
Cell lines and culture
Ramos cells were obtained from the American Type Culture Collection and maintained in RPMI
Lymphocytes display increased sensitivity to Bz-423
The cell death pathway activated by Bz-423 in fibroblasts in vitro requires >10 h exposure to [Bz-423] >10 μM [4]. In contrast, apoptosis of Ramos cells begins in 3–4 h at lower [Bz-423] (Fig. 1A). As previously reported, cell death in both cell types depends on Bz-423-induced superoxide [2], [4]. Indeed, consistent with the increased sensitivity of Ramos cells to this agent, Bz-423 induces a larger superoxide response in Ramos cells at lower [Bz-423] (Fig. 1B). The apoptotic response is specific
Discussion
While modulation of the F1F0-ATPase in lymphocytes and fibroblasts results in the formation of superoxide, this signal is propagated differently in each cell type, resulting in different cell death pathways. Lymphocytes are more sensitive to Bz-423 than fibroblasts in vitro and in vivo, suggesting that the different responses result from differences in factors that limit superoxide signaling, such as cellular antioxidant defenses which include antioxidant enzymes (e.g. superoxide dismutases,
Acknowledgments
This work was supported by grants from the NIH (R01-AI 47450 to G.D.G, R01-CA 10456 to A.W.O.). N.B.B. is supported by a training grant from the NIH (T32 DK065517).
References (31)
- et al.
Identification and validation of the mitochondrial F1F0-ATPase as the molecular target of the immunomodulatory benzodiazepine Bz-423
Chem Biol
(2005) - et al.
Bz-423 superoxide signals apoptosis via selective activation of JNK, Bak, and Bax
Free Radic Biol Med
(2008) - et al.
Role of MAPKs in development and differentiation: lessons from knockout mice
Biochimie
(2006) - et al.
Superoxide dismutase and catalase conjugated to polyethylene glycol increases endothelial enzyme activity and oxidant resistance
J Biol Chem
(1988) - et al.
A novel benzodiazepine increases the sensitivity of B cells to receptor stimulation with synergistic effects on calcium signaling and apoptosis
J Biol Chem
(2004) - et al.
Free radicals and antioxidants in normal physiological functions and human disease
Int J Biochem Cell Biol
(2007) - et al.
Characterization of the antioxidant status of the heterozygous manganese superoxide dismutase knockout mouse
Arch Biochem Biophys
(1999) - et al.
Life in the balance: how BH3-only proteins induce apoptosis
Curr Opin Cell Biol
(2005) - et al.
Specific cleavage of Mcl-1 by caspase-3 in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in Jurkat leukemia T cells
J Biol Chem
(2005) - et al.
Survival factor withdrawal-induced apoptosis of TF-1 cells involves a TRB2-Mcl-1 axis-dependent pathway
J Biol Chem
(2007)
Mule/ARF-BP1, a BH3-only E3 ubiquitin ligase, catalyzes the polyubiquitination of Mcl-1 and regulates apoptosis
Cell
Glycogen synthase kinase-3 regulates mitochondrial outer membrane permeabilization and apoptosis by destabilization of MCL-1
Mol Cell
Phosphorylation-dependent scaffolding role of JSAP1/JIP3 in the ASK1-JNK signaling pathway
J Biol Chem
Attenuation of autoimmune disease in Fas-deficient mice by treatment with a cytotoxic benzodiazepine
Arthritis Rheum
Benzodiazepine-induced superoxide signals B cell apoptosis: mechanistic insight and potential therapeutic utility
J Clin Invest
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- 1
Current address: Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, United States.
- 2
Current address: Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States.