Abstract
The endoplasmic reticulum (ER) is the cellular site of polypeptide folding and modification. When these processes are hampered, an unfolded protein response (UPR) is activated. If the damage is too broad, the mammalian UPR launches the apoptotic program. As a consequence, mobilization of ER calcium stores sensitizes mitochondria to direct proapoptotic stimuli. We make use of a mouse Apaf1-deficient cell system of proneural origin to understand the roles played in this context by the apoptosome, the most studied apoptotic machinery along the mitochondrial pathway of death. We show here that in the absence of the apoptosome ER stress induces cytochrome c release from the mitochondria but that apoptosis cannot occur. Under these circumstances, Grp78/BiP and GADD153/CHOP, both hallmarks of UPR, are canonically up-regulated, and calcium is properly released from ER stores. We also demonstrate that caspase 12, a protease until now believed to play a central role in the initiation of ER stress-induced cell death in the mouse system, is dispensable for the mitochondrial pathway of death to take place.
Highlights
Stress conditions interfering with the homeostasis of the ER4 initiate diverse signaling responses, resulting in a decreased rate of protein translation so as to prevent further accumulation of unfolded proteins [1]
To cause endoplasmic reticulum (ER) stress, in the series of experiments we present here we applied three pharmacological agents to the ETNA cells, widely used death inducers that inhibit N-linked glycosywith brefeldin A (BFA) at the indicated concentrations for 48 h, stained with propidium iodide (PI), and analyzed by flow cytometry. d, cells were treated with thapsigargin (TG) at the indicated concentrations for 48 h, stained with PI, and analyzed by flow cytometry. b– d, results are means Ϯ S.D. of three independent determinations
When compared with cells transfected with wt caspase 9, the C9DN mutant hampers apoptosis in ETNAϩ/ϩ cells, as shown by reduction of caspase 3 and poly(ADP-ribose) polymerase (PARP) processing (Fig. 2d) and lack of TUNEL positivity, analogously to what we have shown within ETNAϪ/Ϫ cells with the same stimuli
Summary
Plasmid Construction—Apaf expression plasmid was created by cloning of Apaf cDNA under control of the strong promoter CAGGs (CMV/-actin, a kind gift of Dr Miyazaki, Osaka, Japan).
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