Chemical Biology Investigation of Cell Death Pathways Activated by Endoplasmic Reticulum Stress Reveals Cytoprotective Modulators of ASK1

Abstract

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) is caused by many disease-relevant conditions, inducing conserved signaling events collectively known as the unfolded protein response. When ER stress is excessive or prolonged, cell death (usually occurring by apoptosis) is triggered. We undertook a chemical biology approach for investigating mechanisms of ER stress-induced cell death. Using a cell-based high throughput screening assay to identify compounds that rescued a neuronal cell line from thapsigargin-induced cell death, we identified benzodiazepinones that selectively inhibit cell death caused by inducers of ER stress (thapsigargin and tunicamycin) but not by inducers of extrinsic (tumor necrosis factor) or intrinsic (mitochondrial) cell death pathways. The compounds displayed activity in several cell lines and primary cultured neurons. Mechanism of action studies revealed that these compounds inhibit ER stress-induced activation of p38 MAPK and kinases responsible for c-Jun phosphorylation. Active benzodiazepinones suppressed cell death at the level of apoptotic signal kinase-1 (ASK1) within the IRE1 pathway but without directly inhibiting the kinase activity of ASK1 or >400 other kinases tested. Rather, active compounds enhanced phosphorylation of serine 967 of ASK1, promoting ASK1 binding to 14-3-3, an event associated with suppression of ASK1 function. Reducing ASK1 protein expression using small interfering RNA also protected cells from ER stress-induced apoptosis, confirming the importance of this protein kinase. Taken together, these findings demonstrate an essential role for ASK1 in cell death induced by ER stress. The compounds identified may prove useful for revealing endogenous mechanisms that regulate inhibitory phosphorylation of ASK1.

The unfolded protein response at the crossroads of cellular life and death during endoplasmic reticulum stress

“…This interaction was reported to promote a cascade of phosphorylation events involving the MAP3K apoptosis signal-regulated kinase 1 (ASK1) and culminating in c-JUN N-terminal kinase (JNK) activation (Nishitoh et al, 2002;Urano et al, 2000). ASK1-deficient cells or cells treated with small molecules interfering with ASK1 function have been shown to be protected against ER stressinduced apoptosis, highlighting the importance of the IRE1-ASK1-JNK axis in these cells (Kim et al, 2009;Nishitoh et al, 2002). JNK is a key regulator of many cellular events and has pro-or anti-apoptotic functions depending on the cell type, the nature of the death stimulus and on the duration of its activation.…”

p28GANK inhibits endoplasmic reticulum stress-induced cell death via enhancement of the endoplasmic reticulum adaptive capacity

“…The mechanism of ER stress-induced p38 MAPK activation is obscure, and a most recent study reported that an IRE1-TRAF2-ASK1 module mediates the p38 MAPK pathway activation [37]. This raises the question whether p28…”

Cyclosporine A and bromocriptine attenuate cell death mediated by intracellular calcium mobilization

“…Salubrinal, the first identified chemical inhibitor of ER stress-induced cell death (12), and benzodiazepinone derivatives (BZDs) (14, 16) suppresses cell death induced by thapsigargin, tunicamycin, and other inducers of ER stress (15), indicating that they act on common cell death pathways activated during ER stress. Interestingly, salubrinal has been shown to arrest UPR signaling in the PERK pathway by modulating phosphorylation of eIF2-α, whereas BZD compounds operate downstream of IRE1, thus modulating phosphorylation of ASK1 (14,16 ]i) by blocking calcium pumping into ER, resulting in the induction of ER stress by exerting effects on Ca 2+ -dependent chaperones, which reside in the ER. In addition to triggering ER stress-induced UPR, however, it is likely that thapsigargin also activates other Ca Our results indicate that cyclosporine A and bromocriptine are selective inhibitors of thapsigargin-triggered cell death, thus demonstrating their cytoprotective activity.…”

“…As a positive control for each test plate, salubrinal, a known inhibitor of ER stress-induced cell death was employed (12). The robustness of the assay was assessed by calculating the Z' factor of each test plate using a positive control (salubrinal in DMSO followed by thapsigargin), and negative control (DMSO followed by thapsigargin) (13,14). Two specific chemical libraries (LOPAC TM from Sigma, and Spectrum collection TM from Microsource Discovery) enriched with pharmacologically active small molecules (Fig.…”