Bz-423 superoxide signals apoptosis via selective activation of JNK, Bak, and Bax

doi:10.1016/j.freeradbiomed.2008.07.022

Free Radical Biology and Medicine

Volume 45, Issue 9, 1 November 2008, Pages 1232-1242

https://doi.org/10.1016/j.freeradbiomed.2008.07.022 Get rights and content

Abstract

Bz-423 is a proapoptotic 1,4-benzodiazepine with potent therapeutic properties in murine models of lupus and psoriasis. 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. Herein, we report the signaling pathway activated by Bz-423 in mouse embryonic fibroblasts containing knockouts of key apoptotic proteins. Bz-423-induced superoxide activates cytosolic ASK1 and its release from thioredoxin. A mitogen-activated protein kinase cascade follows, leading to the specific phosphorylation of JNK. JNK signals activation of Bax and Bak which then induces mitochondrial outer membrane permeabilization to cause the release of cytochrome c and a commitment to apoptosis. The response of these cells to Bz-423 is critically dependent on both superoxide and JNK activation as antioxidants and the JNK inhibitor SP600125 prevents Bax translocation, cytochrome c release, and cell death. These results demonstrate that superoxide generated from the mitochondrial respiratory chain as a consequence of a respiratory transition can signal a sequential and specific apoptotic response. Collectively, these data suggest that the selectivity of Bz-423 observed in vivo results from cell-type specific differences in redox balance and signaling by ASK1 and Bcl-2 proteins.

Introduction

Bz-423 is a proapoptotic 1,4-benzodiazepine with potent therapeutic properties against murine lupus and psoriasis [1], [2], [3]. The absence of either general toxicities or significant effects on normal immune responses in treated mice indicates that Bz-423 has selective effects on pathogenic cells. Affinity-based screening of a phage-display human cDNA expression library identified the oligomycin-sensitivity conferring protein (OSCP), a component of the mitochondrial F₁F₀-ATPase, as the molecular target of Bz-423 [4]. Binding of Bz-423 to the OSCP modulates the enzyme and induces a state 3 to state 4 respiratory transition, leading to the formation of superoxide by the mitochondrial respiratory chain (MRC).

To gain an understanding of the cellular response to Bz-423, we previously characterized the general features of apoptosis in a Burkitt lymphoma cell line (Ramos) [1]. In these cells, Bz-423-induced increase in superoxide is followed by caspase activation, mitochondrial electrochemical gradient (ΔΨ_m) collapse, and the release of cytochrome c into the cytoplasm nearly simultaneously, consistent with mitochondrial outer membrane permeabilization (MOMP) and the release of cytochrome c from the mitochondrial intermembrane space [5]. Following these events, morphological and biochemical evidence of apoptosis is detected. In rat liver isolated mitochondria, Bz-423 induces reactive oxygen species (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 extramitochondrial factors in the mechanism coupling Bz-423-induced ROS to apoptosis.

To identify factors that couple Bz-423-generated superoxide to apoptosis, the response to Bz-423 was studied in detail in mouse embryonic fibroblasts (MEFs) [6]. Although MEFs are significantly less sensitive to Bz-423-induced killing than either primary B cells or B cell lymphoma-derived cell lines (i.e., longer incubation times and higher Bz-423 concentrations are required), and in vivo data have yet to identify cytotoxic effects on nonlymphoid cells, MEFs were selected to exploit the use of well-characterized single gene knockouts [6], to identify signaling molecules that are part of the Bz-423 response, and to identify factors that potentially explain the relative resistance of fibroblasts. Using these cells, we show that cytosolic factors, including proapoptotic Bcl-2-family proteins and mitogen-activated protein (MAP) kinases, couple Bz-423-induced ROS to an apoptotic cascade that is reflected back to the mitochondria to release cytochrome c. The release of cytochrome c commits the cell to apoptosis. The events described following Bz-423 treatment of MEFs demonstrate how ROS generated by modulation of the mitochondrial F₁F₀-ATPase can induce a sequential and specific apoptotic signal transduction pathway.

Section snippets

Reagents

Bz-423 was synthesized as previously described [7]. Dihydroethidium (DHE) and 5-(and -6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H₂DCFDA) were obtained from Invitrogen Corp. (Carlsbad, CA). 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).

Cell lines and culture

SV40-transformed WT, Bad^-/-, Bax^-/-, Bak^-/-, and DKO

Isolated mitochondria respond to Bz-423

Incubating mitochondria isolated and purified from MEFs with Bz-423 under conditions supporting state 3 respiration results in increased superoxide within the mitochondria (Fig. S1A). This response is consistent with inhibition of the F₁F₀-ATPase, and demonstrates that mitochondria respond to Bz-423 independent of other components of the cell. In this cell-free system, however, Bz-423 does not cause ΔΨ_m collapse or trigger cytochrome c release (Figs. S1A and B). Together, these data show that

Discussion

Inhibition of F₁F₀-ATPase induces a state 3 to state 4 transition leading to formation of reduced intermediates in mitochondria [4], [24], [25]. These reactive intermediates (e.g., reduced ubisemiquinones) can form at both the matrix and the intermembrane sides of the inner membrane and release superoxide into both compartments [26]. Superoxide can also be formed on the matrix side at complex I most likely via the transfer of an electron from a half-reduced flavin mononucleotide to molecular

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^☆: 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).

¹: Current address: Scripps Research Institute, La Jolla, CA 92037, USA.

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Original ContributionBz-423 superoxide signals apoptosis via selective activation of JNK, Bak, and Bax☆

Abstract

Introduction

Section snippets

Reagents

Cell lines and culture

Isolated mitochondria respond to Bz-423

Discussion

Chem. Biol.

Trends Pharmacol. Sci.

J. Biol. Chem.

Chem. Biol. Interact.

Biochim. Biophys. Acta

Free Radic. Biol. Med.

Cell

FEBS Lett.

Biochem. Pharmacol.

Int. J. Biochem. Cell Biol.

Cancer Cell

Free Radic. Biol. Med.

J. Biol. Chem.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

Neuropharmacology

Biochem. Pharmacol.

Blood

J. Biol. Chem.

Eur. J. Pharmacol.

Arch. Biochem. Biophys.

Biochimie

J. Biol. Chem.

Benzodiazepine-induced superoxide signals B cell apoptosis: mechanistic insight and potential therapeutic utility

J. Clin. Invest.

Attenuation of autoimmune disease in Fas-deficient mice by treatment with a cytotoxic benzodiazepine

Arthritis Rheum.

Bz-423, a benzodiazepine, suppresses keratinocyte proliferation and has anti-psoriatic activity in the human skin-SCID mouse transplant model

J. Pharmacol. Exp. Ther.

Pharmacological manipulation of cell death: clinical applications in sight?

J. Clin. Invest.

Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death

Science

The combinatorial synthesis and chemical and biological evaluation of a 1,4-benzodiazepine library

Proc. Natl. Acad. Sci. U.S.A.

Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay

Am. J. Physiol. Cell. Physiol.

Mitochondria: pharmacological manipulation of cell death

J. Clin. Invest.

The redox regulation of thiol dependent signaling pathways in cancer

Curr. Pharm. Des.

Original Contribution
Bz-423 superoxide signals apoptosis via selective activation of JNK, Bak, and Bax☆