Smac mimetic suppresses tunicamycin-induced apoptosis via resolution of ER stress

Abstract Since Inhibitor of Apoptosis (IAP) proteins have been implicated in cellular adaptation to endoplasmic reticulum (ER) stress, we investigated the regulation of ER stress-induced apoptosis by small-molecule second mitochondria-derived activator of caspase (Smac) mimetics that antagonize IAP proteins. Here, we discover that Smac mimetic suppresses tunicamycin (TM)-induced apoptosis via resolution of the unfolded protein response (UPR) and ER stress. Smac mimetics such as BV6 selectively inhibit apoptosis triggered by pharmacological or genetic inhibition of protein N-glycosylation using TM or knockdown of DPAGT1, the enzyme that catalyzes the first step of protein N-glycosylation. In contrast, BV6 does not rescue cell death induced by other typical ER stressors (i.e., thapsigargin (TG), dithiothreitol, brefeldin A, bortezomib, or 2-deoxyglucose). The protection from TM-triggered apoptosis is found for structurally different Smac mimetics and for genetic knockdown of cellular IAP (cIAP) proteins in several cancer types, underlining the broader relevance. Interestingly, lectin microarray profiling reveals that BV6 counteracts TM-imposed inhibition of protein glycosylation. BV6 consistently abolishes TM-stimulated accumulation of ER stress markers such as glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) and reduces protein kinase RNA-like ER kinase (PERK) phosphorylation and X box-binding protein 1 (XBP1) splicing upon TM treatment. BV6-stimulated activation of nuclear factor-κB (NF-κB) contributes to the resolution of ER stress, since NF-κB inhibition by overexpression of dominant-negative IκBα superrepressor counteracts the suppression of TM-stimulated transcriptional activation of CHOP and GRP78 by BV6. Thus, our study is the first to show that Smac mimetic protects from TM-triggered apoptosis by resolving the UPR and ER stress. This provides new insights into the regulation of cellular stress responses by Smac mimetics.

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Activation of Hepatitis B Virus S Promoter by a Cell Type-Restricted IRE1-Dependent Pathway Induced by Endoplasmic Reticulum Stress

Molecular and Cellular Biology ◽

10.1128/mcb.25.17.7522-7533.2005 ◽

2005 ◽

Vol 25 (17) ◽

pp. 7522-7533 ◽

Cited By ~ 22

Author(s):

Zhi-Ming Huang ◽

Thomas Tan ◽

Hiderou Yoshida ◽

Kazutoshi Mori ◽

Yanjun Ma ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Er Stress ◽

Transcriptional Activation ◽

Active Form ◽

Dominant Negative ◽

Dependent Manner ◽

Cell Type ◽

B Virus

ABSTRACT IRE1-alpha is an integral membrane protein of the endoplasmic reticulum (ER) that is a key sensor in the cellular transcriptional response to stress in the ER. Upon induction of ER stress, IRE1-alpha is activated, resulting in the synthesis of the active form of the transcription factor XBP1 via IRE1-mediated splicing of its mRNA. In this report, we have examined the role of IRE1-alpha and XBP1 in activation of the hepatitis B virus S promoter by ER stress. Cotransfection experiments revealed that overexpression of either IRE1-alpha or XBP1 activated this promoter. Conversely, cotransfected dominant-negative IRE1-alpha or small interfering RNA directed against XBP1 decreased the activation of the S promoter by ER stress, confirming an important role for the IRE1-alpha/XBP1 signaling pathway in activation of the S promoter. However, XBP1 does not bind directly to the S promoter; rather, a novel S promoter-binding complex that does not contain XBP1 is induced in cells undergoing ER stress in an XBP1-dependent manner. This complex, as well as transcriptional activation of the S promoter, is induced by ER stress in hepatocytes but not in fibroblasts, despite the presence of active XBP1 in the latter. Thus, the hepatitis B virus S promoter responds to a novel, cell type-restricted transcriptional pathway downstream of IRE1-alpha and XBP1.

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Polyphenol-rich extract induces apoptosis with immunogenic markers in melanoma cells through the ER stress-associated kinase PERK

Cell Death Discovery ◽

10.1038/s41420-019-0214-2 ◽

2019 ◽

Vol 5 (1) ◽

Cited By ~ 7

Author(s):

Karol Prieto ◽

Yu Cao ◽

Eslam Mohamed ◽

Jimena Trillo-Tinoco ◽

Rosa A. Sierra ◽

...

Keyword(s):

Er Stress ◽

Stress Responses ◽

Calcium Release ◽

Melanoma Cells ◽

Melanoma Tumor ◽

Cancer Cell Death ◽

Extracellular Release ◽

Induced Apoptosis ◽

Stress Mediators

Abstract Polyphenols elicit antitumor activities, in part, through the induction of anti- or pro-oxidant effects in cancer cells which promote priming of protective anti-tumor immunity. We recently characterized a polyphenol-rich extract from Caesalpinia spinosa (P2Et) that stimulates in vivo antitumor responses against breast and melanoma tumor models via the promotion of immunogenic cancer cell death (ICD). However, the primary mediators whereby P2Et promotes ICD remained unknown. Here, we sought to elucidate the role that severe endoplasmic reticulum (ER) stress plays in mediating P2Et-induced apoptosis and ICD in murine melanoma cells. Our findings demonstrate a substantial selective induction of specific ER-stress mediators in B16-F10 melanoma cells treated with P2Et. While knockout of the ER stress-associated PKR-like ER kinase (PERK) prevented induction of apoptosis and expression of ICD markers in P2Et-treated cells, deletion of X-box binding protein 1 (Xbp1) did not. P2Et-driven activation of PERK in melanoma cells was found to promote ER-calcium release, disrupt mitochondrial membrane potential, and trigger upregulation of ICD drivers, surface calreticulin expression, and extracellular release of ATP and HMGB1. Notably, calcium release inhibition, but not targeting of PERK-driven integrated stress responses, prevented P2Et-induced apoptosis. Collectively, these results underline the central role of PERK-directed calcium release in mediating the antitumor and immunogenic actions of P2Et in melanoma cells.

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IKKβ-mediated nuclear factor-κB activation attenuates smac mimetic–induced apoptosis in cancer cells

Molecular Cancer Therapeutics ◽

10.1158/1535-7163.mct-09-0068 ◽

2009 ◽

Vol 8 (6) ◽

pp. 1636-1645 ◽

Cited By ~ 16

Author(s):

Lang Bai ◽

Wenshu Chen ◽

Wenjie Chen ◽

Xia Wang ◽

Hong Tang ◽

...

Keyword(s):

Cancer Cells ◽

Nuclear Factor Κb ◽

Nuclear Factor ◽

Smac Mimetic ◽

Induced Apoptosis ◽

Apoptosis In Cancer Cells

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Abnormal disulfide-linked oligomerization results in ER retention and altered signaling by TNFR1 mutants in TNFR1-associated periodic fever syndrome (TRAPS)

Blood ◽

10.1182/blood-2005-11-006783 ◽

2006 ◽

Vol 108 (4) ◽

pp. 1320-1327 ◽

Cited By ~ 165

Author(s):

Adrian A. Lobito ◽

Fiona C. Kimberley ◽

Jagan R. Muppidi ◽

Hirsh Komarow ◽

Adrianna J. Jackson ◽

...

Keyword(s):

Protein Misfolding ◽

Periodic Fever ◽

Nuclear Factor Κb ◽

Dominant Negative ◽

Tnf Receptor ◽

Wild Type ◽

Er Retention ◽

Self Association ◽

Induced Apoptosis ◽

Mutant Receptors

AbstractTumor necrosis factor (TNF) receptor–associated periodic syndrome (TRAPS) is an autosomal dominant systemic autoinflammatory disease associated with heterozygous mutations in TNF receptor 1 (TNFR1). Here we examined the structural and functional alterations caused by 9 distinct TRAPS-associated TNFR1 mutations in transfected cells and a mouse “knock-in” model of TRAPS. We found that these TNFR1 mutants did not generate soluble versions of the receptor, either through membrane cleavage or in exosomes. Mutant receptors did not bind TNF and failed to function as dominant-negative inhibitors of TNFR1-induced apoptosis. Instead, TRAPS mutant TNFR1 formed abnormal disulfide-linked oligomers that failed to interact with wild-type TNFR1 molecules through the preligand assembly domain (PLAD) that normally governs receptor self-association. TRAPS mutant TNFR1 molecules were retained intracellularly and colocalized with endoplasmic reticulum (ER) markers. The capacity of mutant receptors to spontaneously induce both apoptosis and nuclear factor κB (NF-κB) activity was reduced. In contrast, the R92Q variant of TNFR1 behaved like the wild-type receptor in all of these assays. The inflammatory phenotype of TRAPS may be due to consequences of mutant TNFR1 protein misfolding and ER retention.

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Nuclear factor-κB activation by the photochemotherapeutic agent verteporfin

Blood ◽

10.1182/blood.v95.1.256.001k29_256_262 ◽

2000 ◽

Vol 95 (1) ◽

pp. 256-262 ◽

Cited By ~ 1

Author(s):

D. J. Granville ◽

C. M. Carthy ◽

H. Jiang ◽

J. G. Levy ◽

B. M. McManus ◽

...

Keyword(s):

Stress Responses ◽

Nuclear Factor Κb ◽

Light Irradiation ◽

Luciferase Reporter ◽

Nuclear Factor ◽

Luciferase Reporter Construct ◽

Nuclear Extracts ◽

The Status ◽

General Caspase Inhibitor ◽

Induced Apoptosis

The nuclear factor-kappa B (NF-κB) gene transactivator serves in the formation of immune, inflammatory, and stress responses. In quiescent cells, NF-κB principally resides within the cytoplasm in association with inhibitory κ (IκB) proteins. The status of IκB and NF-κB proteins was evaluated for promyelocytic leukemia HL-60 cells treated at different intensities of photodynamic therapy (PDT). The action of the potent photosensitizer, benzoporphyrin derivative monoacid ring A (verteporfin), and visible light irradiation were assessed. At a verteporfin concentration that produced the death of a high proportion of cells after light irradiation, evidence of caspase-3 and caspase-9 processing and of poly(ADP-ribose) polymerase cleavage was present within whole cell lysates. The general caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) effectively blocked these apoptosis-related changes. Recent studies indicate that IκB proteins may be caspase substrates during apoptosis. However, the level of IκBβ was unchanged for HL-60 cells undergoing PDT-induced apoptosis. IκB levels decreased during PDT-induced apoptosis, though ZVAD.fmk did not affect this change. At a less intensive level of photosensitization, cellular IκB levels were transiently depressed after PDT. At these times, p50 and RelA NF-κB species were increased within nuclear extracts, as revealed by electrophoretic mobility supershift assays. HL-60 cells transiently transfected with a κB-luciferase reporter construct exhibited elevated luciferase activity after PDT or treatment with tumor necrosis factor-, a well-characterized NF-κB activator. Productive NF-κB activation and associated gene transcription may influence the phenotype and behavior of cells exposed to less intensive PDT regimens. However, IκB is not subject to caspase-mediated degradation as a component of PDT-induced apoptosis. (Blood. 2000;95:256-262)

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Protective role of Gipie, a Girdin family protein, in endoplasmic reticulum stress responses in endothelial cells

Molecular Biology of the Cell ◽

10.1091/mbc.e10-08-0724 ◽

2011 ◽

Vol 22 (6) ◽

pp. 736-747 ◽

Cited By ~ 18

Author(s):

Etsushi Matsushita ◽

Naoya Asai ◽

Atsushi Enomoto ◽

Yoshiyuki Kawamoto ◽

Takuya Kato ◽

...

Keyword(s):

Endothelial Cells ◽

Endoplasmic Reticulum ◽

Er Stress ◽

Stress Responses ◽

Protective Role ◽

Jnk Pathway ◽

Interacting Protein ◽

Family Protein ◽

Induced Apoptosis ◽

The Unfolded Protein Response

Continued exposure of endothelial cells to mechanical/shear stress elicits the unfolded protein response (UPR), which enhances intracellular homeostasis and protect cells against the accumulation of improperly folded proteins. Cells commit to apoptosis when subjected to continuous and high endoplasmic reticulum (ER) stress unless homeostasis is maintained. It is unknown how endothelial cells differentially regulate the UPR. Here we show that a novel Girdin family protein, Gipie (78 kDa glucose-regulated protein [GRP78]-interacting protein induced by ER stress), is expressed in endothelial cells, where it interacts with GRP78, a master regulator of the UPR. Gipie stabilizes the interaction between GRP78 and the ER stress sensor inositol-requiring protein 1 (IRE1) at the ER, leading to the attenuation of IRE1-induced c-Jun N-terminal kinase (JNK) activation. Gipie expression is induced upon ER stress and suppresses the IRE1-JNK pathway and ER stress-induced apoptosis. Furthermore we found that Gipie expression is up-regulated in the neointima of carotid arteries after balloon injury in a rat model that is known to result in the induction of the UPR. Thus our data indicate that Gipie/GRP78 interaction controls the IRE1-JNK signaling pathway. That interaction appears to protect endothelial cells against ER stress-induced apoptosis in pathological contexts such as atherosclerosis and vascular endothelial dysfunction.

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Gene expression during ER stress–induced apoptosis in neurons

The Journal of Cell Biology ◽

10.1083/jcb.200305149 ◽

2003 ◽

Vol 162 (4) ◽

pp. 587-597 ◽

Cited By ~ 253

Author(s):

Claus Reimertz ◽

Donat Kögel ◽

Abdelhaq Rami ◽

Thomas Chittenden ◽

Jochen H.M. Prehn

Keyword(s):

Gene Expression ◽

Er Stress ◽

Hippocampal Neurons ◽

Target Genes ◽

Neuroblastoma Cells ◽

Human Cells ◽

Protein Glycosylation ◽

Prolonged Treatment ◽

Domain 3 ◽

Induced Apoptosis

Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of ischemic and neurodegenerative disorders. Treatment of human SH-SY5Y neuroblastoma cells with tunicamycin, an inhibitor of protein glycosylation, rapidly induced the expression of target genes of the unfolded protein response. However, prolonged treatment also triggered a delayed, caspase-dependent cell death. Microarray analysis of gene expression changes during tunicamycin-induced apoptosis revealed that the Bcl-2 homology domain 3-only family member, Bcl-2 binding component 3/p53 upregulated modulator of apoptosis (Bbc3/PUMA), was the most strongly induced pro-apoptotic gene. Expression of Bbc3/PUMA correlated with a Bcl-xL–sensitive release of cytochrome c and the activation of caspase-9 and -3. Increased expression of Bbc3/PUMA was also observed in p53-deficient human cells, in response to the ER stressor thapsigargin, and in rat hippocampal neurons after transient forebrain ischemia. Overexpression of Bbc3/PUMA was sufficient to trigger apoptosis in SH-SY5Y neuroblastoma cells, and human cells deficient in Bbc3/PUMA showed dramatically reduced apoptosis in response to ER stress. Our data suggest that the transcriptional induction of Bbc3/PUMA may be sufficient and necessary for ER stress–induced apoptosis.

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Molecular Symbiosis of CHOP and C/EBPβ Isoform LIP Contributes to Endoplasmic Reticulum Stress-Induced Apoptosis

Molecular and Cellular Biology ◽

10.1128/mcb.01507-09 ◽

2010 ◽

Vol 30 (14) ◽

pp. 3722-3731 ◽

Cited By ~ 73

Author(s):

Calin-Bogdan Chiribau ◽

Francesca Gaccioli ◽

Charlie C. Huang ◽

Celvie L. Yuan ◽

Maria Hatzoglou

Keyword(s):

Endoplasmic Reticulum ◽

Transcription Factor ◽

Er Stress ◽

Transcriptional Activation ◽

Nuclear Import ◽

Cellular Response ◽

Transcriptional Control ◽

Nuclear Translocation ◽

Basic Leucine Zipper ◽

Induced Apoptosis

ABSTRACT Induction of the transcription factor CHOP (CCAAT-binding homologous protein; GADD 153) is a critical cellular response for the transcriptional control of endoplasmic reticulum (ER) stress-induced apoptosis. Upon nuclear translocation, CHOP upregulates the transcription of proapoptotic factors and downregulates antiapoptotic genes. Transcriptional activation by CHOP involves heterodimerization with other members of the basic leucine zipper transcription factor (bZIP) family. We show that the bZIP protein C/EBPβ isoform LIP is required for nuclear translocation of CHOP during ER stress. In early ER stress, LIP undergoes proteasomal degradation in the cytoplasmic compartment. During later ER stress, LIP binds CHOP in both cytoplasmic and nuclear compartments and contributes to its nuclear import. By using CHOP-deficient cells and transfections of LIP-expressing vectors in C/EBPβ−/− mouse embryonic fibroblasts (MEFs), we show that the LIP-CHOP interaction has a stabilizing role for LIP. At the same time, CHOP uses LIP as a vehicle for nuclear import. LIP-expressing C/EBPβ−/− MEFs showed enhanced ER stress-induced apoptosis compared to C/EBPβ-null cells, a finding in agreement with the decreased levels of Bcl-2, a known transcriptional control target of CHOP. In view of the positive effect of CHOP-LIP interaction in mediating their proapoptotic functions, we propose this functional cooperativity as molecular symbiosis between proteins.

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Conditions of endoplasmic reticulum stress stimulate lipid droplet formation in Saccharomyces cerevisiae

Biochemical Journal ◽

10.1042/bj20090785 ◽

2009 ◽

Vol 424 (1) ◽

pp. 61-67 ◽

Cited By ~ 100

Author(s):

Weihua Fei ◽

Han Wang ◽

Xin Fu ◽

Christopher Bielby ◽

Hongyuan Yang

Keyword(s):

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Endoplasmic Reticulum Stress ◽

Er Stress ◽

Cell Biology ◽

Neutral Lipids ◽

Brefeldin A ◽

Protein Glycosylation ◽

Yeast Mutants ◽

Cellular Organelles

LDs (lipid droplets) are cellular organelles which can be found in nearly all eukaryotic cells. Despite their importance in cell biology, the mechanism underlying LD biogenesis remains largely unknown. In the present study we report that conditions of ER (endoplasmic reticulum) stress stimulate LD formation in Saccharomyces cerevisiae. We found that LDs accumulated in yeast mutants with compromised protein glycosylation or ER-associated protein degradation. Moreover, tunicamycin and Brefeldin A, agents which induce ER stress, were found to stimulate LD formation. In contrast, the restoration of protein glycosylation reduced LD accumulation. Interestingly, enhanced neutral lipids synthesis and LD formation under conditions of ER stress was not dependent on Ire1p. Lastly, we demonstrated that the absence of LDs did not compromise cell viability under ER stress. Our results suggest that although more LDs are produced, LDs are not essential to cell survival under ER stress.

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Chronic Carbon Tetrachloride Applications Induced Hepatocyte Apoptosis in Lipocalin 2 Null Mice through Endoplasmic Reticulum Stress and Unfolded Protein Response

International Journal of Molecular Sciences ◽

10.3390/ijms21155230 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5230

Author(s):

Erawan Borkham-Kamphorst ◽

Ute Haas ◽

Eddy Van de Leur ◽

Anothai Trevanich ◽

Ralf Weiskirchen

Keyword(s):

Endoplasmic Reticulum ◽

Carbon Tetrachloride ◽

Liver Injury ◽

Er Stress ◽

Stress Responses ◽

Protein Glycosylation ◽

Initiation Factor ◽

Wild Type ◽

Hepatocyte Apoptosis ◽

Unfolded Protein

The lack of Lipocalin (LCN2) provokes overwhelming endoplasmic reticulum (ER) stress responses in vitro and in acute toxic liver injury models, resulting in hepatocyte apoptosis. LCN2 is an acute phase protein produced in hepatocytes in response to acute liver injuries. In line with these findings we investigated ER stress responses of Lcn2−/− mice in chronic ER stress using a long-term repetitive carbon tetrachloride (CCl4) injection model. We found chronic CCl4 application to enhance ER stress and unfolded protein responses (UPR), including phosphorylation of eukaryotic initiation factor 2α (eIF2α), increased expression of binding immunoglobulin protein (BiP) and glucose-regulated protein 94 (GRP94). IRE1α/TRAF2/JNK signaling enhanced mitochondrial apoptotic pathways, and showed slightly higher in Lcn2−/− mice compared to the wild type counterparts, leading to increased hepatocyte apoptosis well evidenced by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Hepatocyte injuries were confirmed by significant high serum alanine transaminase (ALT) levels in CCl4-treated Lcn2−/− mice. Lcn2−/− mice furthermore developed mild hepatic steatosis, supporting our finding that ER stress promotes lipogenesis. In a previous report we demonstrated that the pharmacological agent tunicamycin (TM) induced ER stress through altered protein glycosylation and induced high amounts of C/EBP-homologous protein (CHOP), resulting in hepatocyte apoptosis. We compared TM-induced ER stress in wild type, Lcn2−/−, and Chop null (Chop−/−) primary hepatocytes and found Chop−/− hepatocytes to attenuate ER stress responses and resist ER stress-induced hepatocyte apoptosis through canonical eIF2α/GADD34 signaling, inhibiting protein synthesis. Unexpectedly, in later stages of TM incubation, Chop−/− hepatocytes resumed activation of IRE1α/JNK/c-Jun and p38/ATF2 signaling, leading to late hepatocyte apoptosis. This interesting observation indicates Chop−/− mice to be unable to absolutely prevent all types of liver injury, while LCN2 protects the hepatocytes by maintaining homeostasis under ER stress conditions.

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