Apoptotic crosstalk between the endoplasmic reticulum and mitochondria controlled by Bcl-2

Abstract

Apoptosis involves mitochondrial steps such as the release of the apoptogenic factor cytochrome c which are e ectively blocked by Bcl-2. Although Bcl-2 may have a direct action on the mitochondrial membrane, it also resides and functions on the endoplasmic reticulum (ER), and there is increasing evidence for a role of the ER in apoptosis regulation as well. Here we uncover a hitherto unrecognized, apoptotic crosstalk between the ER and mitochondria that is controlled by Bcl-2. After triggering massive ER dilation due to an inhibition of secretion, the drug brefeldin A (BFA) induces the release of cytochrome c from mitochondria in a caspase-8-and Bid-independent manner. This is followed by caspase-3 activation and DNA/nuclear fragmentation. Surprisingly, cytochrome c release by BFA is not only blocked by wild-type Bcl-2 but also by a Bcl-2 variant that is exclusively targeted to the ER (Bcl-2/cb5). Similar ®ndings were obtained with tunicamycin, an agent interfering with N-linked glycosylations in the secretory system. Thus, apoptotic agents perturbing ER functions induce a novel crosstalk between the ER and mitochondria that can be interrupted by ER-based Bcl-2.

Endoplasmic reticulum stress-induced cell death mediated by the proteasome

“…To elicit robust cell death in response to ER stress, BFA-treated cells were coincubated with CHX so as to limit the activation of ATF6 and IRE1 translation-dependent survival pathways, as was done previously. 8 We reported that ER stress-induced apoptosis was not prevented by caspase inhibition with Z-VAD.fmk but could be blocked by Pefabloc, a general inhibitor of serine protease activity. 11 We therefore sought to identify the serine protease(s) responsible for cell death observed in our system.…”

“…CHOP/GADD153, the pro-apoptotic translational-dependent mediator of ER stress-induced cell death, 17 is not a player in our model system because we co-treated cells with CHX so as to bypass the expression of protective UPR genes, as was done previously. 8,11 Therefore, we have defined an ER stressinduced cell death pathway that is independent of CHOP/ GADD153. We also excluded calpains from playing a role in this paradigm because none of the calpain inhibitors tested had any protective effect on ER stress-induced apoptosis (data not shown).…”

“…From previous reports, several pro-apoptotic factors and signaling pathways have been shown to be involved in ER stress-induced cell death, including calpain, Cysteine aspartyl protease (caspase-4) (human), caspase-12 (murine), CHOP/GADD153, TRAF2, ASK-1, JNK, and pro-apoptotic Bcl-2 family members, among others. [7][8][9][10] In many instances, caspases have been implicated in this cell death pathway, on the grounds that broadspectrum caspase inhibitors block cell death. In some cases, however, cell death is not fully blocked by caspase inhibitors, suggesting that caspase-independent mechanisms may contribute to the execution of ER stress-induced cell death.…”

Bcl-2 complexed with Beclin-1 maintains full anti-apoptotic function

“…Cells were transfected with Flag-hBeclin-1, FlaghBeclin-1(DBcl-2BD) or hBeclin-1(DBcl-2BD)-GFP (generously provided by Beth Levine, University of Texas Southwestern Medical Center at Dallas) or mouse Beclin-1-GFP (generously provided by Zhenyu Yue, Mount Sinai School of Medicine, New York) fusion constructs alone or in combination with plasmids encoding wild-type Bcl-2 (wt-Bcl-2) or Bcl-2 variants targeted at their C-termini to the mitochondrial outer membrane (mit-Bcl-2) or the ER membrane (ER-Bcl-2) (Hacki et al, 2000;Kaufmann et al, 2003) (generously provided by Christoph Borner, IMMCR, Albert-Ludwigs-Universita¨t, Freiburg). For immunocytochemistry, immunoprecipitation, immunoblot and caspase assays, cells were cotransfected so that they expressed 0.8-or 1.7-fold molar ratios of Beclin-1 to Bcl-2.…”

A highly conserved proapoptotic gene, IKIP, located next to the APAF1 gene locus, is regulated by p53

“…11,12 Third, bcl-2 family members, including bcl-2 itself, Bax and Bak, as well as the BH3-only protein BIK, localize not only to mitochondria but also to the ER. [13][14][15] Therefore, multiple pathways exist by which the ER can contribute to pro-or antiapoptotic stimuli.…”