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Coupling endoplasmic reticulum stress to the cell death program. An Apaf-1-independent intrinsic pathway
Rammohan V Rao1, Susana Castro-Obregon, Harald Frankowski
1Buck Institute for Age Research, Novato, California 94945 and the Department of Medicine III, Johannes Gutenberg University, Mainz D-55101, Germany.
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.
Insights
Endoplasmic reticulum (ER) stress triggers cell death through a novel pathway independent of Apaf-1 and mitochondria. This discovery reveals a new intrinsic apoptotic mechanism involving caspase-12, crucial for understanding cell death signaling.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Endoplasmic reticulum (ER) stress, caused by misfolded proteins and calcium imbalance, can lead to cell death.
- The precise signaling pathways connecting ER stress to cell death remain unclear.
- Apaf-1-deficient cells are generally resistant to apoptosis via the intrinsic pathway.
Purpose of the Study:
- To elucidate the specific signaling cascade involved in ER stress-induced cell death.
- To investigate whether ER stress-induced apoptosis utilizes the canonical Apaf-1 and cytochrome c-dependent pathway.
- To identify key molecular players in this cell death pathway.
Main Methods:
- Utilized Apaf-1(-/-) fibroblasts to assess apoptosis susceptibility to ER stressors (thapsigargin, brefeldin-A) versus other apoptotic stimuli.
- Employed caspase inhibitors (caspase-12, -9, -8) to dissect the apoptotic pathway.
- Developed a cell-free system using microsomes from ER-stressed cells and cell extracts lacking mitochondria to study caspase activation.
- Conducted immunodepletion experiments to identify essential microsomal components.
Main Results:
- Apaf-1(-/-) fibroblasts underwent apoptosis upon ER stress induction, despite resistance to other apoptotic stimuli.
- ER stress-induced apoptosis was sensitive to caspase-12 and caspase-9 inhibition, but not caspase-8.
- Cell-free extracts from ER-stressed cells induced caspase processing independently of cytochrome c.
- Caspase-12 was identified as a critical microsomal component required for downstream caspase activation.
Conclusions:
- ER stress-induced cell death represents a novel intrinsic apoptotic pathway.
- This pathway operates independently of Apaf-1 and mitochondria.
- Caspase-12 plays a crucial role in initiating ER stress-mediated apoptosis.