Elsevier

Biochemical Pharmacology

Volume 78, Issue 8, 15 October 2009, Pages 966-973
Biochemical Pharmacology

Bz-423 superoxide signals B cell apoptosis via Mcl-1, Bak, and Bax

https://doi.org/10.1016/j.bcp.2009.05.025Get rights and content

Abstract

Bz-423 is a pro-apoptotic 1,4-benzodiazepine with therapeutic properties in murine models of lupus demonstrating selectivity for autoreactive lymphocytes. Bz-423 modulates the F1F0-ATPase, inducing the formation of superoxide within the mitochondrial respiratory chain, which then functions as a second messenger initiating apoptosis. In order to understand some of the features that contribute to the increased sensitivity of lymphocytes, we report the signaling pathway engaged by Bz-423 in a Burkitt lymphoma cell line (Ramos). Following the generation of superoxide, Bz-423-induced apoptosis requires the activation of Bax and Bak to induce mitochondrial outer membrane permeabilization and cytochrome c release. Knockdown of the BH3-only proteins Bad, Bim, Bik, and Puma inhibits Bz-423 apoptosis, suggesting that these proteins serve as upstream sensors of the oxidant stress induced by Bz-423. Treatment with Bz-423 results in superoxide-dependent Mcl-1 degradation, implicating this protein as the link between Bz-423-induced superoxide and Bax and Bak activation. In contrast to fibroblasts, B cell death induced by Bz-423 is independent of c-Jun N-terminal kinase. These results demonstrate that superoxide generated from the mitochondrial respiratory chain as a consequence of a respiratory transition can signal a specific apoptotic response that differs across cell types.

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).

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    Current address: Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, United States.

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