Bifunctional apoptosis regulator (BAR), an endoplasmic reticulum (ER)-associated E3 ubiquitin ligase, modulates BI-1 protein stability and function in ER Stress

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates inositol-requiring protein-1 (IRE1), among other ER-associated signaling proteins of the unfolded protein response (UPR) in mammalian cells. IRE1 signaling becomes attenuated under prolonged ER stress. The mechanisms by which this occurs are not well understood. An ER resident protein, Bax inhibitor-1 (BI-1), interacts with IRE1 and directly inhibits IRE1 activity. However, little is known about regulation of the BI-1 protein. We show here that bifunctional apoptosis regulator (BAR) functions as an ER-associated RING-type E3 ligase, interacts with BI-1, and promotes proteasomal degradation of BI-1. Overexpression of BAR reduced BI-1 protein levels in a RING-dependent manner. Conversely, knockdown of endogenous BAR increased BI-1 protein levels and enhanced inhibition of IRE1 signaling during ER stress. We also found that the levels of endogenous BAR were reduced under prolonged ER stress. Our findings suggest that post-translational regulation of the BI-1 protein by E3 ligase BAR contributes to the dynamic control of IRE1 signaling during ER stress.     

CHOP is involved in endoplasmic reticulum stress-induced apoptosis by enhancing DR5 expression in human carcinoma cells

It has been shown that excess stress to the endoplasmic reticulum (ER) triggers apoptosis, but the mechanisms underlying these processes remain unclear. We and others have reported previously that DR5 expression is up-regulated in thapsigargin (THG)-treated human cancer cells. Here, we provide evidence that CHOP is involved in THG up-regulation of DR5, which is a critical step for ER stress-induced apoptosis in human cancer cells. In human colon cancer HCT116 cells, knockdown of DR5 by siRNA blocked THG-induced Bax conformational change along with caspase-3 activation and cell death. Moreover, inhibition of CHOP expression attenuated DR5 up-regulation and apoptosis induced by THG, whereas ectopic expression of DR5 restored the sensitivity of CHOP siRNA-transfected cells to THG-induced apoptosis. In addition to HCT116 cells, inhibition of CHOP or DR5 induction also attenuated THG-induced cell death in other cancer cell lines including LNCaP, A2780S, and DU145, indicating that CHOP and DR5 are critical for ER stress-mediated apoptosis in human carcinoma cells. Furthermore, we identified a potential CHOP-binding site in the 5'-flanking region of the DR5 gene. Mutation of this site abrogated the enhanced reporter activity in response to THG treatment. Together, our findings suggest that CHOP regulates ER stress-induced apoptosis, at least in part, through enhancing DR5 expression in some types of human cancer cells.     

Inhibition of endoplasmic reticulum stress counteracts neuronal cell death and protein aggregation caused by N-terminal mutant huntingtin proteins

Accumulation of abnormal proteins occurs in many neurodegenerative diseases including Huntington's disease (HD). However, the precise role of protein aggregation in neuronal cell death remains unclear. We show here that the expression of N-terminal huntingtin proteins with expanded polyglutamine (polyQ) repeats causes cell death in neuronal PC6.3 cell that involves endoplasmic reticulum (ER) stress. These mutant huntingtin fragment proteins elevated Bip, an ER chaperone, and increased Chop and the phosphorylation of c-Jun-N-terminal kinase (JNK) that are involved in cell death regulation. Caspase-12, residing in the ER, was cleaved in mutant huntingtin expressing cells, as was caspase-3 mediating cell death. In contrast, cytochrome-c or apoptosis inducing factor (AIF) was not released from mitochondria after the expression of these proteins. Treatment with salubrinal that inhibits ER stress counteracted cell death and reduced protein aggregations in the PC6.3 cells caused by the mutant huntingtin fragment proteins. Salubrinal upregulated Bip, reduced cleavage of caspase-12 and increased the phosphorylation of eukaryotic translation initiation factor-2 subunit-alpha (eIF2alpha) that are neuroprotective. These results show that N-terminal mutant huntingtin proteins activate cellular pathways linked to ER stress, and that inhibition of ER stress by salubrinal increases cell survival. The data suggests that compounds targeting ER stress may be considered in designing novel approaches for treatment of HD and possibly other polyQ diseases.     

Tunicamycin-induced ER stress upregulates the expression of mitochondrial HtrA2 and promotes apoptosis through the cytosolic release of HtrA2

Recent studies provide some evidence that the HtrA2 protein is intimately associated with the pathogenesis of neurodegenerative disorders and that endoplasmic reticulum (ER) quality control and ER stress-associated cell death play critical roles in neuronal cell death. However, little is known about the intimate relationship between HtrA2 and ER stress-associated cellular responses. In the present study, we have demonstrated that the HtrA2 protein level was gradually and significantly increased by up to 10-fold in the mitochondria under tunicamycin (Tm)-induced ER stress, which eventually promoted cell death through the release of HtrA2 into the cytoplasm. Using an ecdysoneinducible mammalian expression system, we demonstrate that the extent of cell death in 293-HtrA2 cells was approximately 20 times higher under Tm-induced ER stress, indicating that the increase in the HtrA2 protein level in the mitochondria itself is necessary but not sufficient for the promotion of cell death. Taken together, these results suggest that HtrA2 may serve as a mediator of ER stress-induced apoptosis and ER-mitochondrial cross-talk in some cellular processes.     

Transient expression of wild-type or mitochondrially targeted Bcl-2 induces apoptosis, whereas transient expression of endoplasmic reticulum-targeted Bcl-2 is protective against Bax-induced cell death

Bcl-2 protein family members function either to promote or inhibit programmed cell death. Bcl-2, typically an inhibitor of apoptosis, has also been demonstrated to have pro-apoptotic activity (Cheng, E. H., Kirsch, D. G., Clem, R. J., et al. (1997) Science 278, 1966-1968). The pro-apoptotic activity has been attributed to the cleavage of Bcl-2 by caspase-3, which converts Bcl-2 to a pro-apoptotic molecule. Bcl-2 is a membrane protein that is localized in the endoplasmic reticulum (ER) membrane, the outer mitochondrial membrane, and the nuclear envelope. Here, we demonstrate that transient expression of Bcl-2 at levels comparable to those found in stably transfected cells induces apoptosis in human embryonic kidney 293 cells and in the human breast cell line MDA-MB-468 cells. Furthermore, we have targeted Bcl-2 specifically to either the ER or the outer mitochondrial membrane to test whether induction of apoptosis by Bcl-2 is dependent upon its localization within either of these membranes. Our findings indicate that Bcl-2 specifically targeted to the mitochondria induces cell death, whereas Bcl-2 that is targeted to the ER does not. The expression of Bcl-2 does result in its cleavage to a 20-kDa protein; however, mutation of the caspase-3 cleavage site (D34A) does not inhibit its ability to induce cell death. Additionally, we find that transiently expressed ER-targeted Bcl-2 inhibits cell death induced by Bax overexpression. In conclusion, the ability of Bcl-2 to promote apoptosis is associated with its localization at the mitochondria. Furthermore, the ability of ER-targeted Bcl-2 to protect against Bax-induced apoptosis suggests that the ER localization of Bcl-2 may play an important role in its protective function.     

Involvement of caspase-4 in endoplasmic reticulum stress-induced apoptosis and Abeta-induced cell death

Recent studies have suggested that neuronal death in Alzheimer's disease or ischemia could arise from dysfunction of the endoplasmic reticulum (ER). Although caspase-12 has been implicated in ER stress-induced apoptosis and amyloid-beta (Abeta)-induced apoptosis in rodents, it is controversial whether similar mechanisms operate in humans. We found that human caspase-4, a member of caspase-1 subfamily that includes caspase-12, is localized to the ER membrane, and is cleaved when cells are treated with ER stress-inducing reagents, but not with other apoptotic reagents. Cleavage of caspase-4 is not affected by overexpression of Bcl-2, which prevents signal transduction on the mitochondria, suggesting that caspase-4 is primarily activated in ER stress-induced apoptosis. Furthermore, a reduction of caspase-4 expression by small interfering RNA decreases ER stress-induced apoptosis in some cell lines, but not other ER stress-independent apoptosis. Caspase-4 is also cleaved by administration of Abeta, and Abeta-induced apoptosis is reduced by small interfering RNAs to caspase-4. Thus, caspase-4 can function as an ER stress-specific caspase in humans, and may be involved in pathogenesis of Alzheimer's disease.     

Inhibition of PKR protects against tunicamycin-induced apoptosis in neuroblastoma cells

Endoplasmic reticulum (ER) dysfunction is thought to play a significant role in several neurological disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, cerebral ischemia, and the prion diseases. ER dysfunction can be mimicked by cellular stress signals such as disruption of calcium homeostasis, inhibition of protein glycosylation, and reduction of disulfide bonds, which results in accumulation of misfolded proteins in the ER and leads to cell death by apoptosis. Tunicamycin, which is an inhibitor of protein glycosylation, induces ER stress and apoptosis. In this study, we examined the involvement of double stranded (ds) RNA-activated protein kinase PKR in tunicamycin-induced apoptosis. We used overexpression of the trans-dominant negative, catalytically inactive mutant K296R to inhibit PKR activity in neuroblastoma cells. We demonstrate that inhibition of PKR activation in response to tunicamycin protects neuronal cells from undergoing apoptosis. Furthermore, K296R overexpressing cells show defective PKR activation, delayed eIF2α phosphorylation, dramatically delayed ATF4 expression. In addition, both caspase-3 activation and C/EBP homologous protein (CHOP, also known as GADD153) induction, which are markers of apoptotic cells, are absent from K296R overexpression cells in response to tunicamycin. These results establish that PKR activation plays a major regulatory role in induction of apoptosis in response to ER stress and indicates the potential of PKR as possible target for neuroprotective therapeutics.     

Characterization of a p75(NTR) apoptotic signaling pathway using a novel cellular model.

The p75 neurotrophin receptor (p75(NTR)) belongs to the tumor necrosis factor receptor/nerve growth factor receptor superfamily. In some cells derived from neuronal tissues it causes cell death through a poorly characterized pathway. We developed a neuronal system using conditionally immortalized striatal neurons, in which the expression of p75(NTR) is inducibly controlled by the ecdysone receptor. In these cells p75(NTR) induces apoptosis through its death domain in a nerve growth factor-independent manner. Caspases 9, 6, and 3 are activated by receptor expression indicating the activation of the common effector pathway of apoptosis. Cell death is blocked by a dominant negative form of caspase 9 and Bcl-X(L) consistent with a pathway that involves mitochondria. Significantly, the viral flice inhibitory protein E8 protects from p75(NTR)-induced cell death indicating that death effector domains are involved. A p75(NTR) construct with a deleted death domain dominantly interferes with p75(NTR) signaling, implying that receptor multimerization is required. However, in contrast to the other receptors of the family, p75(NTR)-mediated apoptosis does not involve the adaptor proteins Fas-associated death domain protein or tumor necrosis factor-associated death domain protein, and the apical caspase 8 is not activated. We conclude that p75(NTR) signals apoptosis by similar mechanisms as other death receptors but uses different adaptors and apical caspases.     

Estrogen‐regulated developmental neuronal apoptosis is determined by estrogen receptor subtype and the Fas/Fas ligand system

Adult sexual dimorphism in neuronal cell number is controlled by estrogen exposure during a tightly defined period of rat brain development. The mechanisms of estrogen's effect are unknown; one possibility is regulation of programmed cell death (apoptosis). In this study we have shown that estradiol can function as a neuroprotective agent or an inducer of apoptosis, depending on the estrogen receptor‐subtype present in the cell. Thus, ERα has a neuroprotective effect, while ERβ mediates the induction of apoptosis in neuronal cells. Moreover, we show that estrogen‐induced apoptosis through ER‐β requires the expression of Fas‐ and Fas ligand (FasL) proteins, since the absence of FasL in neurons prevents this effect. Furthermore, we demonstrate that microglia‐secreted products induce the expression of FasL necessary to mediate estradiol–ERβ apoptotic effect. These findings may explain the dichotomous effect of fetal estradiol on the adult neuronal number. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 64–78, 2000     

Saturated fatty acids induce endoplasmic reticulum stress and apoptosis independently of ceramide in liver cells

Accumulation of lipids in nonadipose tissues can lead to cell dysfunction and cell death, a phenomenon known as lipotoxicity. However, the signaling pathways and mechanisms linking lipid accumulation to cell death are poorly understood. The present study examined the hypothesis that saturated fatty acids disrupt endoplasmic reticulum (ER) homeostasis and promote apoptosis in liver cells via accumulation of ceramide. H4IIE liver cells were exposed to varying concentrations of saturated (palmitate or stearate) or unsaturated (oleate or linoleate) fatty acids. ER homeostasis was monitored using markers of the ER stress response pathway, including phosphorylation of IRE1alpha and eIF2alpha, splicing of XBP1 mRNA, and expression of molecular chaperone (e.g., GRP78) and proapoptotic (CCAAT/enhancer-binding protein homologous protein) genes. Apoptosis was monitored using caspase activity and DNA laddering. Palmitate and stearate induced ER stress, caspase activity, and DNA laddering. Inhibition of caspase activation prevented DNA laddering. Unsaturated fatty acids did not induce ER stress or apoptosis. Saturated fatty acids increased ceramide concentration; however, inhibition of de novo ceramide synthesis did not prevent saturated fatty acid-induced ER stress and apoptosis. Unsaturated fatty acids rescued palmitate-induced ER stress and apoptosis. These data demonstrate that saturated fatty acids disrupt ER homeostasis and induce apoptosis in liver cells via mechanisms that do not involve ceramide accumulation.     

Caspase-12 and caspase-4 are not required for caspase-dependent endoplasmic reticulum stress-induced apoptosis

Alterations in cellular homeostasis that affect protein folding in the endoplasmic reticulum (ER) trigger a signaling pathway known as the unfolded protein response (UPR). The initially cytoprotective UPR will trigger an apoptotic cascade if the cellular insult is not corrected; however, the proteins required to initiate this cell death pathway are poorly understood. In this study, we show that UPR gene expression is induced in cells treated with ER stress agents in the presence or absence of murine caspase-12 or human caspase-4 expression and in cells that overexpress Bcl-x(L) or a dominant negative caspase-9. We further demonstrate that ER stress-induced apoptosis is a caspase-dependent process that does not require the expression of caspase-12 or caspase-4 but can be inhibited by overexpression of Bcl-x(L) or a dominant negative caspase-9. Additionally, treatment of human and murine cells with ER stress agents led to the cleavage of the caspase-4 fluorogenic substrate, LEVD-7-amino-4-trifluoromethylcoumarin, in the presence or absence of caspase-12 or caspase-4 expression, whereas Bcl-x(L) or a dominant negative caspase-9 overexpression inhibited LEVD-7-amino-4-trifluoromethylcoumarin cleavage. These data suggest that caspase-12 and caspase-4 are not required for the induction of ER stress-induced apoptosis and that caspase-4-like activity is not always associated with an initiating event.