Abstract
7-Chloro-5-(4-hydroxyphenyl)-1-methyl-3-(napthalen-2-ylmetyl)-4,5,-dihydro-1H-benzo[b][1,4]diazepin-2(3H)-one (Bz-423) is a proapoptotic 1,4-benzodiazepine that potently suppresses disease in the murine model of lupus by selectively killing pathogenic lymphocytes. In MRL/MpJ-Faslpr (MRL-lpr) mice, Bz-423 overcomes deficient expression of the Fas death receptor and hyperactivation of antiapoptotic phosphatidylinositol 3-kinase (PI3K)-Akt signaling to specifically kill pathogenic CD4+ T cells. Bz-423 binds to the oligomycin-sensitivity-conferring protein component of the mitochondrial F0F1-ATPase, which modulates the enzyme leading to formation of superoxide by the mitochondrial respiratory chain. Scavenging this reactive oxygen species blocks all subsequent components of the apoptotic cascade. To gain insight into how apoptotic signaling activated by Bz-423-induced superoxide contributes to the selective depletion of MRL-lpr CD4+ T cells, we characterized the death mechanism in a CD4+ T cell leukemia line (Jurkat). Although Bz-423-induced superoxide indirectly inactivates Akt, this response is not required for T cell death. Apoptosis instead results from parallel increases in levels of the proapoptotic Bcl-2 proteins Noxa and Bak leading to specific activation of Bak, mitochondrial outer membrane permeabilization, and a commitment to apoptosis. By directly up-regulating proteins that trigger loss of mitochondrial outer membrane integrity, Bz-423 bypasses defective Fas function and antiapoptotic PI3K-Akt signaling in MRL-lpr CD4+ T cells. Moreover, because disease-associated abnormalities should sensitize autoreactive CD4+ T cells to transcriptional up-regulation of Noxa by redox signals and to Bak-dependent apoptosis, the apoptotic mechanism elucidated in Jurkat cells provides important clues into the cell-type- and disease-selective effects of Bz-423 in MRL-lpr mice.
- Bz-423, 7-chloro-5-(4-hydroxyphenyl)-1-methyl-3-(napthalen-2-ylmetyl)-4,5,-dihydro-1H-benzo[b][1,4]diazepin-2(3H)-one
- ASK1, apoptosis signal-regulating kinase1
- DHE, dihydroethidium
- GAPDH, glyceradehyde-3-phosphate dehydrogenase
- JNK, c-Jun N-terminal kinase
- MAP, mitogen-activated protein
- MEF, mouse embryonic fibroblast
- MnTBAP, manganese (III) tetrakis (4-benzoic acid)porphyrin
- MIS, mitochondrial intermembrane space
- MOMP, mitochondrial outer membrane permeabilization
- MRL/MpJ-Faslpr, MRL-lpr
- myrAkt, myristoylated Akt
- O2∸, superoxide
- PI, propidium iodide
- PBS, phosphate-buffered saline
- PI3K, phosphatidylinositol 3-kinase
- ROS, reactive oxygen species
- siRNA, small interfering RNA
- SP600125, anthra[1–9cd]pyrazol-6(2H)-one
- V-β, β-subunit of the F0F1-ATPase
- zVAD-fmk, benzyloxycarbonyl-valine-alanine-aspartic acid fluoromethyl ketone.
Footnotes
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This study was supported by the National Institutes of Health [Grants AI47450, CA10456].
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G.D.G. and A.W.O. acknowledge stock ownership and consulting compensation from a corporation that has licensed certain commercial rights to Bz-423.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.109.156422
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- Received May 19, 2009.
- Accepted August 24, 2009.
- © 2009 by The American Society for Pharmacology and Experimental Therapeutics
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