Abstract
Accumulation of misfolded proteins and alterations in Ca2+ homeostasis in the endoplasmic reticulum (ER) causes ER stress and leads to cell death. However, the signal-transducing events that connect ER stress to cell death pathways are incompletely understood. To discern the pathway by which ER stress-induced cell death proceeds, we performed studies on Apaf-1(-/-) (null) fibroblasts that are known to be relatively resistant to apoptotic insults that induce the intrinsic apoptotic pathway. While these cells were resistant to cell death initiated by proapoptotic stimuli such as tamoxifen, they were susceptible to apoptosis induced by thapsigargin and brefeldin-A, both of which induce ER stress. This pathway was inhibited by catalytic mutants of caspase-12 and caspase-9 and by a peptide inhibitor of caspase-9 but not by caspase-8 inhibitors. Cleavage of caspases and poly(ADP-ribose) polymerase was observed in cell-free extracts lacking cytochrome c that were isolated from thapsigargin or brefeldin-treated cells. To define the molecular requirements for this Apaf-1 and cytochrome c-independent apoptosis pathway further, we developed a cell-free system of ER stress-induced apoptosis; the addition of microsomes prepared from ER stress-induced cells to a normal cell extract lacking mitochondria or cytochrome c resulted in processing of caspases. Immunodepletion experiments suggested that caspase-12 was one of the microsomal components required to activate downstream caspases. Thus, ER stress-induced programmed cell death defines a novel, mitochondrial and Apaf-1-independent, intrinsic apoptotic pathway.
Highlights
Accumulation of misfolded proteins and alterations in Ca2؉ homeostasis in the endoplasmic reticulum (ER) causes ER stress and leads to cell death
To define the molecular requirements for this Apaf-1 and cytochrome c-independent apoptosis pathway further, we developed a cell-free system of ER stress-induced apoptosis; the addition of microsomes prepared from ER stress-induced cells to a normal cell extract lacking mitochondria or cytochrome c resulted in processing of caspases
In order to test whether these cells are susceptible to ER stress-inducing agents, cells were treated with 500 nM thapsigargin or 2 M brefeldin-A for different time periods
Summary
We developed a cell-free model of ER stressinduced cell death that involves the addition of microsomes to a 300,000 ϫ g cell-free extract that lacks Apaf-1 and cytochrome c, both of which are required for activating caspase-9 and other downstream caspases through the previously described intrinsic apoptotic pathway. This system was capable of reproducing a key element of apoptosis, namely caspase processing and activation. ER stress-induced caspase-12 activation defines a novel, ER-based intrinsic pathway for apoptosome-independent effector caspase activation and cell death
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