TRIM13 regulates caspase-8 ubiquitination,translocation to autophagosomes and activation during ER stress induced cell death

The emerging evidences suggest that endoplasmic (ER) stress is involved in onset of many pathological conditions like cancer and neurodegeneration. The persistent ER stress results in misfolded protein aggregates, which are degraded through the process of autophagy or lead to cell death through activation of caspases. The regulation of crosstalk of autophagy and cell death during ER stress is emerging. Ubiquitination plays regulatory role in crosstalk of autophagy and cell death. In the current study, we describe the role of TRIM13, RING E3 ubiquitin ligase, in regulation of ER stress induced cell death. The expression of TRIM13 sensitizes cells to ER stress induced death. TRIM13 induced autophagy is essential for ER stress induced caspase activation and cell death. TRIM13 induces K63 linked poly-ubiquitination of caspase-8, which results in its stabilization and activation during ER stress. TRIM13 regulates translocation of caspase-8 to autophagosome and its fusion with lysosome during ER stress. This study first time demonstrated the role of TRIM13 as novel regulator of caspase-8 activation and cell death during ER stress.     

ER stress induces caspase-8 activation,stimulating cytochrome c release and caspase-9 activation

Excess ER stress induces caspase-12 activation and/or cytochrome c release, causing caspase-9 activation. Little is known about their relationship during ER stress-mediated cell death. Upon ER stress, P19 embryonal carcinoma (EC) cells showed activation of various caspases, including caspase-3, caspase-8, caspase-9, and caspase-12, and extensive DNA fragmentation. We examined the relationship between ER stress-mediated cytochrome c/caspase-9 and caspase-12 activation by using caspase-9- and caspase-8-deficient mouse embryonic fibroblasts and a P19 EC cell clone [P19-36/12 (-) cells] lacking expression of caspase-12. Caspase-9 and caspase-8 deficiency inhibited and delayed the onset of DNA fragmentation but did not inhibit caspase-12 processing induced by ER stress. P19-36/12 (-) cells underwent apoptosis upon ER stress, with cytochrome c release and caspase-8 and caspase-9 activation. The dominant negative form of FADD and z-VAD-fmk inhibited caspase-8, caspase-9, Bid processing, cytochrome c release, and DNA fragmentation induced by ER stress, suggesting that caspase-8 and caspase-9 are the main caspases involved in ER stress-mediated apoptosis of P19-36/12 (-) cells. Caspase-8 deficiency also inhibited the cytochrome c release induced by ER stress. Thus, in parallel with the caspase-12 activation, ER stress triggers caspase-8 activation, resulting in cytochrome c/caspase-9 activation via Bid processing.     

cFLIP downregulation is an early event required for endoplasmic reticulum stress-induced apoptosis in tumor cells

Protein misfolding or unfolding and the resulting endoplasmic reticulum (ER) stress frequently occur in highly proliferative tumors. How tumor cells escape cell death by apoptosis after chronic ER stress remains poorly understood. We have investigated in both two-dimensional (2D) cultures and multicellular tumor spheroids (MCTSs) the role of caspase-8 inhibitor cFLIP as a regulator of the balance between apoptosis and survival in colon cancer cells undergoing ER stress. We report that downregulation of cFLIP proteins levels is an early event upon treatment of 2D cultures of colon cancer cells with ER stress inducers, preceding TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) upregulation, caspase-8 activation, and apoptosis. Maintaining high cFLIP levels during ER stress by ectopic expression of cFLIP markedly inhibits ER stress-induced caspase-8 activation and apoptosis. Conversely, cFLIP knockdown by RNA interference significantly accelerates caspase-8 activation and apoptosis upon ER stress. Despite activation of the proapoptotic PERK branch of the unfolded protein response (UPR) and upregulation of TRAIL-R2, MCTSs are markedly more resistant to ER stress than 2D cultures of tumor cells. Resistance of MCTSs to ER stress-induced apoptosis correlates with sustained cFLIP_L expression. Interestingly, resistance to ER stress-induced apoptosis is abolished in MCTSs generated from cFLIP_L knockdown tumor cells. Overall, our results suggest that controlling cFLIP levels in tumors is an adaptive strategy to prevent tumor cell’s demise in the unfavorable conditions of the tumor microenvironment.Subject terms: Cancer microenvironment, Apoptosis     

Coupling endoplasmic reticulum stress to the cell death program: role of the ER chaperone GRP78

Alterations in Ca(2+) homeostasis and accumulation of unfolded proteins in the endoplasmic reticulum (ER) lead to an ER stress response. Prolonged ER stress may lead to cell death. Glucose-regulated protein (GRP) 78 (Bip) is an ER lumen protein whose expression is induced during ER stress. GRP78 is involved in polypeptide translocation across the ER membrane, and also acts as an apoptotic regulator by protecting the host cell against ER stress-induced cell death, although the mechanism by which GRP78 exerts its cytoprotective effect is not understood. The present study was carried out to determine whether one of the mechanisms of cell death inhibition by GRP78 involves inhibition of caspase activation. Our studies indicate that treatment of cells with ER stress inducers causes GRP78 to redistribute from the ER lumen with subpopulations existing in the cytosol and as an ER transmembrane protein. GRP78 inhibits cytochrome c-mediated caspase activation in a cell-free system, and expression of GRP78 blocks both caspase activation and caspase-mediated cell death. GRP78 forms a complex with caspase-7 and -12 and prevents release of caspase-12 from the ER. Addition of (d)ATP dissociates this complex and may facilitate movement of caspase-12 into the cytoplasm to set in motion the cytosolic component of the ER stress-induced apoptotic cascade. These results define a novel protective role for GRP78 in preventing ER stress-induced cell death.     

TRIM8 regulated autophagy modulates the level of cleaved Caspase-3 subunit to inhibit genotoxic stress induced cell death

In cancer patients, treatment modalities like chemotherapy and radiation exert their anticancer effects by inducing DNA damage. The cancer cells can survive under genotoxic stress by inducing DNA damage response (DDR) or can undergo cell death. The process of autophagy is emerging as crucial regulator of cell survival during different stress conditions. Post translational modification through ubiquitin plays an essential role in DDR during genotoxic stress conditions. Ubiquitin ligases regulate autophagy and cell death pathways however their role during genotoxic stress conditions is not understood. In the current study we identified TRIM8, RING E3 Ligase, as a novel regulator of autophagy during DDR. TRIM8 regulates lysosomal biogenesis and autophagy flux. The turnover of TRIM8 is high and is stabilized during genotoxic stress conditions. TRIM8 regulated autophagy is essential for its cytoprotective role during genotoxic stress induced cell death. TRIM8 stabilizes the turnover of XIAP during genotoxic stress and forms complex with XIAP and caspase-3 to inhibit its activation in presence of etoposide. TRIM8 mediated autophagy promotes degradation of cleaved caspase-3 subunits. This study described TRIM8, as a novel regulator of DDR-autophagy crosstalk, which may play role in survival of cancer cells in presence of genotoxic agents.     

Neuropeptide Y mitigates ER stress–induced neuronal cell death by activating the PI3K–XBP1 pathway

The unfolded protein response (UPR) is an evolutionarily conserved adaptive reaction that increases cell survival under endoplasmic reticulum (ER) stress conditions. ER stress–associated neuronal cell death pathways play roles in the pathogenesis of neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and Huntington’s disease. Neuropeptide Y (NPY) has an important role in neuroprotection against neurodegenerative diseases. In this study, we investigated whether NPY has a protective role in ER stress–induced neuronal cell death in SK-N-SH human neuroblastoma cells. An ER stress–inducing chemical, tunicamycin, increased the activities of caspase-3 and -4, whereas pretreatment with NPY decreased caspase-3 and -4 activities during the ER stress response. In addition, NPY suppressed the activation of three major ER stress sensors during the tunicamycin-induced ER stress response. NPY-mediated activation of PI3K increased nuclear translocation of XBP1s, which in turn induced expression of Grp78/BiP. Taken together, our data indicated that NPY plays a protective role in ER stress–induced neuronal cell death through activation of the PI3K–XBP1 pathway, and that NPY signaling can serve as therapeutic target for ER stress–mediated neurodegenerative diseases.     

Enhanced lysosomal activity is involved in Bax inhibitor-1-induced regulation of the endoplasmic reticulum (ER) stress response and cell death against ER stress: involvement of vacuolar H+-ATPase (V-ATPase)

Bax inhibitor-1 (BI-1) is an evolutionarily conserved protein that protects cells against endoplasmic reticulum (ER) stress while also affecting the ER stress response. In this study, we examined BI-1-induced regulation of the ER stress response as well as the control of the protein over cell death under ER stress. In BI-1-overexpressing cells (BI-1 cells), proteasome activity was similar to that of control cells; however, the lysosomal fraction of BI-1 cells showed sensitivity to degradation of BSA. In addition, areas and polygonal lengths of lysosomes were greater in BI-1 cells than in control cells, as assessed by fluorescence and electron microscopy. In BI-1 cells, lysosomal pH was lower than in control cells and lysosomal vacuolar H(+)-ATPase(V-ATPase), a proton pump, was activated, suggesting high H(+) uptake into lysosomes. Even when exposed to ER stress, BI-1 cells maintained high levels of lysosomal activities, including V-ATPase activity. Bafilomycin, a V-ATPase inhibitor, leads to the reversal of BI-1-induced regulation of ER stress response and cell death due to ER stress. In BI-1 knock-out mouse embryo fibroblasts, lysosomal activity and number per cell were relatively lower than in BI-1 wild-type cells. This study suggests that highly maintained lysosomal activity may be one of the mechanisms by which BI-1 exerts its regulatory effects on the ER stress response and cell death.     

Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions

Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells.     

Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions

Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells.     

An endoplasmic reticulum-specific stress-activated caspase (caspase-12) is implicated in the apoptosis of A549 epithelial cells by respiratory syncytial virus

Respiratory syncytial virus (RSV) infection induced programmed cell death or apoptosis in the cultured lung epithelial cell line, A549. The apoptotic cells underwent multiple changes, including fragmentation and degradation of genomic DNA, consistent with the activation of the DNA fragmentation factor or caspase-activated DNase (DFF or CAD). The infection led to activation of FasL; however, a transdominant mutant of FAS-downstream death domain protein, FADD, did not inhibit apoptosis. Similarly, modest activation of cytoplasmic apoptotic caspases, caspase-3 and -8, were observed; however, only a specific inhibitor of caspases-3 inhibited apoptosis, while an inhibitor of caspase-8 had little effect. No activation of caspase-9 and -10, indicators of the mitochondrial apoptotic pathway, was observed. In contrast, RSV infection strongly activated caspase-12, an endoplasmic reticulum (ER) stress response caspase. Activation of the ER stress response was further evidenced by upregulation of ER chaperones BiP and calnexin. Antisense-mediated inhibition of caspase-12 inhibited apoptosis. Inhibitors of NF-kappa B had no effect on apoptosis. Thus, RSV-induced apoptosis appears to occur through an ER stress response that activates caspase-12, and is uncoupled from NF-kappa B activation.     

ω-Hydroxyundec-9-enoic acid induces apoptosis through ROS-mediated endoplasmic reticulum stress in non-small cell lung cancer cells

ω-Hydroxyundec-9-enoic acid (ω-HUA), a hydroxyl unsaturated fatty acid derivative, is involved in the antifungal activity of wild rice (Oryza officinalis). Here, we investigated the anti-cancer activity of ω-HUA on a non-small cell lung cancer (NSCLC) cell line. ω-HUA increased apoptosis and induced cleavages of caspase-6, caspase-9, and poly (ADP-ribose) polymerase (PARP). ω-HUA treatment significantly induced endoplasmic reticulum (ER) stress response. Suppression of CHOP expression and inhibiting ER stress by 4-phenylbutyrate (4-PBA) significantly attenuated the ω-HUA treatment-induced activation of caspase-6, caspase-9, and PARP, and subsequent apoptotic cell death, indicating a role for ER stress in ω-HUA-induced apoptosis. In addition, cells subjected to ω-HUA exhibited significantly increased quantity of reactive oxygen species (ROS), and the ROS scavenger N-acetyl-l-cysteine (NAC) inhibited ω-HUA-induced apoptotic cell death and ER stress signals, indicating a role for ROS in ER stress-mediated apoptosis in ω-HUA-treated cells. Taken together, these results suggest that sequential ROS generation and ER stress activation are critical in ω-HUA treatment-induced apoptosis and that ω-HUA represents a promising candidate for NSCLC treatment.     

Coupling endoplasmic reticulum stress to the cell death program. An Apaf-1-independent intrinsic pathway

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.     

Apoptosome-independent activation of the lysosomal cell death pathway by caspase-9

The apoptosome, a heptameric complex of Apaf-1, cytochrome c, and caspase-9, has been considered indispensable for the activation of caspase-9 during apoptosis. By using a large panel of genetically modified murine embryonic fibroblasts, we show here that, in response to tumor necrosis factor (TNF), caspase-8 cleaves and activates caspase-9 in an apoptosome-independent manner. Interestingly, caspase-8-cleaved caspase-9 induced lysosomal membrane permeabilization but failed to activate the effector caspases whereas apoptosome-dependent activation of caspase-9 could trigger both events. Consistent with the ability of TNF to activate the intrinsic apoptosis pathway and the caspase-9-dependent lysosomal cell death pathway in parallel, their individual inhibition conferred only a modest delay in TNF-induced cell death whereas simultaneous inhibition of both pathways was required to achieve protection comparable to that observed in caspase-9-deficient cells. Taken together, the findings indicate that caspase-9 plays a dual role in cell death signaling, as an activator of effector caspases and lysosomal membrane permeabilization.     

Ursolic acid induces ER stress response to activate ASK1–JNK signaling and induce apoptosis in human bladder cancer T24 cells

Here we studied the cellular mechanisms of ursolic acid's anti-bladder cancer ability by focusing on endoplasmic reticulum stress (ER stress) signaling. We show that ursolic acid induces a significant ER stress response in cultured human bladder cancer T24 cells. ER stress inhibitor salubrinal, or PERK silencing, diminishes ursolic acid-induced anti-T24 cell effects. Salubrinal inhibits ursolic acid-induced CHOP expression, Bim ER accumulation and caspase-3 activation in T24 cells. Ursolic acid induces IRE1–TRAF2–ASK1 signaling complex formation to activate pro-apoptotic ASK1–JNK signaling. We suggest that ER stress contributes to ursolic acid's effects against bladder cancer cells.     

Licochalcone A induces apoptosis through endoplasmic reticulum stress via a phospholipase Cγ1-, Ca2+-, and reactive oxygen species-dependent pathway in HepG2 human hepatocellular carcinoma cells

Licochalcone A (LicA), an estrogenic flavonoid, induces apoptosis in multiple types of cancer cells. In this study, the molecular mechanisms underlying the anti-cancer effects of LicA were investigated in HepG2 human hepatocellular carcinoma cells. LicA induced apoptotic cell death, activation of caspase-4, -9, and -3, and expression of endoplasmic reticulum (ER) stress-associated proteins, including C/EBP homologous protein (CHOP). Inhibition of ER stress by CHOP knockdown or treatment with the ER stress inhibitors, salubrinal and 4-phenylbutyric acid, reduced LicA-induced cell death. LicA also induced reactive oxygen species (ROS) accumulation and the anti-oxidant N-acetylcysteine reduced LicA-induced cell death and CHOP expression. In addition, LicA increased the levels of cytosolic Ca²⁺, which was blocked by 2-aminoethoxydiphenyl borate (an antagonist of inositol 1,4,5-trisphosphate receptor) and BAPTA-AM (an intracellular Ca²⁺ chelator). 2-Aminoethoxydiphenyl borate and BAPTA-AM inhibited LicA-induced cell death. Interestingly, LicA induced phosphorylation of phospholipase Cγ1 (PLCγ1) and inhibition of PLCγ1 reduced cell death and ER stress. Moreover, the multi-targeted receptor tyrosine kinase inhibitors, sorafenib and sunitinib, reduced LicA-induced cell death, ER stress, and cytosolic Ca²⁺ and ROS accumulation. Finally, LicA induced phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) and c-Met receptor and inhibition of both receptors by co-transfection with VEGFR2 and c-Met siRNAs reversed LicA-induced cell death, Ca²⁺ increase, and CHOP expression. Taken together, these findings suggest that induction of ER stress via a PLCγ1-, Ca²⁺-, and ROS-dependent pathway may be an important mechanism by which LicA induces apoptosis in HepG2 hepatocellular carcinoma cells.     

The stressosome,a caspase-8-activating signalling complex assembled in response to cell stress in an ATG5-mediated manner

Stress-induced apoptosis is mediated primarily through the intrinsic pathway that involves caspase-9. We previously reported that in caspase-9-deficient cells, a protein complex containing ATG5 and Fas-associated death domain (FADD) facilitated caspase-8 activation and cell death in response to endoplasmic reticulum (ER) stress. Here, we investigated whether this complex could be activated by other forms of cell stress. We show that diverse stress stimuli, including etoposide, brefeldin A and paclitaxel, as well as heat stress and gamma-irradiation, caused formation of a complex containing ATG5-ATG12, FADD and caspase-8 leading to activation of downstream caspases in caspase-9-deficient cells. We termed this complex the ‘stressosome’. However, in these cells, only ER stress and heat shock led to stressosome-dependent cell death. Using in silico molecular modelling, we propose the structure of the stressosome complex, with FADD acting as an adaptor protein, interacting with pro-caspase-8 through their respective death effector domains (DEDs) and interacting with ATG5-ATG12 through its death domain (DD). This suggests that the complex could be regulated by cellular FADD-like interleukin-1β-converting enzyme–inhibitory protein (cFLIP_L), which was confirmed experimentally. This study provides strong evidence for an alternative mechanism of caspase-8 activation involving the stressosome complex.     

Autophagy Protects against Oxaliplatin-Induced Cell Death via ER Stress and ROS in Caco-2 Cells

Oxaliplatin is included in a number of effective combination regimens used as first and subsequent lines of therapy for metastatic colorectal cancer. Accumulating evidence indicates that autophagy plays a significant role in response to cancer therapy. However, the role of autophagy in oxaliplatin-induced cell death remains to be clarified. In this study, we showed that oxaliplatin induced cell death and autophagy in Caco-2 colorectal cancer cells. The suppression of autophagy using either pharmacologic inhibitors (3-methyladenine, bafilomycin A1) or RNA interference in essential autophagy genes (ATG5 or Beclin1) enhanced the cell death and reactive oxygen species (ROS) production induced by oxaliplatin in Caco-2 cells. Blocking oxaliplatin-induced ROS production by using ROS scavengers (NAC or Tiron) decreased autophagy. Furthermore, numerous dilated endoplasmic reticula (ER) were present in oxaliplatin-treated Caco-2 cells, and blocking ER stress by RNA interference against candidate of metastasis-1 (P8) and C/EBP-homologous protein (CHOP) decreased autophagy and ROS production. Taken together, these data indicate that oxaliplatin activates autophagy as a cytoprotective response via ER stress and ROS in human colorectal cancer cells.     

Caspases and their role in inflammation and ischemic neuronal death. Focus on caspase-12

Caspases are cysteine proteases, which play important roles in different processes including, apoptosis and inflammation. Caspase-12, expressed in mouse and human, is classified as an inflammatory caspase. However, in humans caspase-12 gene has acquired different mutations that result in the expression of different variants. Caspase-12 is generally recognized as a negative regulator of the inflammatory response induced by infections, because it inhibits the activation of caspase-1 in inflammasome complexes, the production of the pro-inflammatory cytokines IL-1β and IL-18 and the overall response to sepsis. In contrast, caspase-4, the human paralog of caspase-12, exerts a positive modulatory action of the inflammatory response to infectious agents. The role of caspase-12 and caspase-4 in inflammation associated with cerebral ischemia, a condition that results from a transient or permanent reduction of cerebral blood flow, is still unknown. Among the mechanisms involved in ischemic brain injury, apoptosis and inflammation have important roles. Under these conditions, disturbances in the homeostasis of the endoplasmic reticulum (ER) take place, leading to ER stress, caspase activation and apoptosis. Caspase-12 up-regulation and processing has been observed after the ischemic episode but its role in apoptosis is controversial. Cleavage of caspase-4 also occurs during ER stress but its role in ischemic brain injury is unknown. Throughout this review evidence supporting a role of caspase-12 and caspase-4 on the modulation of the inflammatory response to infection and their potential contribution to ER stress-induced apoptosis, is discussed. Understanding the actions of rodent caspase-12 and human caspase-4 will help us to elucidate their role in different pathological conditions, which to date is not well understood.     

Involvement of ERK MAP kinase in endoplasmic reticulum stress in SH-SY5Y human neuroblastoma cells

Endoplasmic reticulum (ER) stress has increasingly come into focus as a factor contributing to neuronal injury. Although caspase-dependent mechanisms have been implicated in ER stress, the signaling pathways involved remain unclear. In this study, we examined the role of the extracellular signal-regulated kinase (ERK), a mitogen-activated protein (MAP) kinase pathway that is highly conserved in many systems for balancing cell survival and death. Prolonged treatment of the human neuroblastoma cell line SH-SY5Y with thapsigargin, an inducer of ER stress, increased cell death over 24-48 h, as measured by LDH release. Caspases were involved; increased levels of active caspase-3 and cleaved caspase substrate PARP were detected, and treatment with Z-VAD-FMK reduced thapsigargin-induced cytotoxicity. In contrast, inhibition of calpain was not protective, although calpain was activated following thapsigargin treatment. An early and transient phosphorylation of ERK1/2 occurred after thapsigargin-induced ER stress, and targeting this pathway with the MEK inhibitors U0126 or PD98059 significantly reduced cell death. Similar cytoprotection was obtained against brefeldin A, another ER stress agent. However, protection against ER stress via ERK inhibition was not accompanied by amelioration of caspase-3 activation, PARP cleavage, or DNA laddering. These data indicate that ERK may contribute to non-caspase-dependent pathways of injury after ER stress.