Research Article: A BAX/BAK and Cyclophilin D-Independent Intrinsic Apoptosis Pathway

Date Published: June 12, 2012

Publisher: Public Library of Science

Author(s): Sebastián Zamorano, Diego Rojas-Rivera, Fernanda Lisbona, Valentina Parra, Felipe A. Court, Rosario Villegas, Emily H. Cheng, Stanley J. Korsmeyer, Sergio Lavandero, Claudio Hetz, Dhyan Chandra.


Most intrinsic death signals converge into the activation of pro-apoptotic BCL-2 family members BAX and BAK at the mitochondria, resulting in the release of cytochrome c and apoptosome activation. Chronic endoplasmic reticulum (ER) stress leads to apoptosis through the upregulation of a subset of pro-apoptotic BH3-only proteins, activating BAX and BAK at the mitochondria. Here we provide evidence indicating that the full resistance of BAX and BAK double deficient (DKO) cells to ER stress is reverted by stimulation in combination with mild serum withdrawal. Cell death under these conditions was characterized by the appearance of classical apoptosis markers, caspase-9 activation, release of cytochrome c, and was inhibited by knocking down caspase-9, but insensitive to BCL-XL overexpression. Similarly, the resistance of BIM and PUMA double deficient cells to ER stress was reverted by mild serum withdrawal. Surprisingly, BAX/BAK-independent cell death did not require Cyclophilin D (CypD) expression, an important regulator of the mitochondrial permeability transition pore. Our results suggest the existence of an alternative intrinsic apoptosis pathway emerging from a cross talk between the ER and the mitochondria.

Partial Text

Apoptosis is a conserved cell death mechanism essential for normal development and tissue homeostasis in multicellular organisms. Although apoptosis presumably participates in the development of most cell lineages, alterations in the expression of apoptosis-regulatory proteins is implicated in the initiation of a variety of human diseases, including autoimmunity, immunodeficiency, cancer, and neurodegenerative diseases, among others [1], [2]. The BCL-2 family of proteins is a group of upstream regulators of the caspase cascade, comprised of both pro- and anti-apoptotic components [1], [2]. BCL-2 family members are defined by the presence of up to four α-helical conserved BCL-2 homology (BH) domains. Pro-apoptotic BCL-2 family members can be further subdivided into more highly conserved, “multidomain” members displaying homology in the BH1, BH2 and BH3 domains (i.e. BAX and BAK), and the “BH3-only” members which contain a single BH domain critical for activation of apoptosis.

In this article we have identified experimental conditions where classical intrinsic death stimuli engage the mitochondrial apoptosis machinery in the absence of BAX, BAK and CypD. BAX and BAK are fundamental components of the core apoptosis pathway upon which multiple death signals converge through activation/upregulation of specific BH3-only proteins to trigger cytochrome c release [2], [3], [53], [54]. Bax and bak double deficiency in mice is embryonic lethal due to failure of developmental programs that depend on apoptosis [4]. However, a small percentage of bax and bak DKO mice (∼10%) are viable [4], indicating that proper development can occur in the absence of these pro-apoptotic proteins. Whether developmental cell death in the absence of BAX and BAK is dependent on the CypD and mitochondrial PTP is unknown. We speculate that this may not be the case since most stimuli that trigger CypD-induced cell death rely on mitochondrial calcium uptake and production of reactive oxygen species, conditions more prompt to undergo necrotic cell death due to a rapid drop in ATP production. In fact, genetic ablation of cypD does not generate evident developmental defects, and does not alter the susceptibility to apoptosis of cells to a large number of known intrinsic death stimuli, but it modulated cell death after exposure to H2O2 (oxidative stress) in vitro and protected mice against brain and heart ischemia/reperfusion injury [16], [19], [55]. Our results, together with another report [22], suggest the existence of an alternative mitochondrial regulatory mechanism to instigate under certain circumstances the release of cytochrome c and apoptosome assembly in the absence of BAX/BAK and CypD. Similarly to Tsujimoto’s study [22], we can not exclude the possibility that Cyclosporin A-insensitive PTP (previously described in vitro[55]) operates in our experimental system to trigger cytochrome c release and apoptosis. However we did not detect any evidence for mitochondrial swelling in our EM analysis, a key feature of PRP-mediated cell death. A recent study described the activation of BAX/BAK-independent apoptosis when cell where deprived of glucose [56]. Apoptosis induction required RIPK1 and caspase-8 activation, but was independent of caspase-9 [57].