ER Stress-induced Apoptosis Research Articles

Enhanced Antitumor Effects of Adenoviral-Mediated siRNA against GRP78 Gene on Adenosine-Induced Apoptosis in Human Hepatoma HepG2 Cells

Our previous studies show that adenosine-induced apoptosis is involved in endoplasmic reticulum stress in HepG2 cells. In this study, we have investigated whether knockdown of GRP78 by short hairpin RNA (shRNA) increases the cytotoxic effects of adenosine in HepG2 cells. The adenovirus vector-delivered shRNA targeting GRP78 (Ad-shGRP78) was constructed and transfected into HepG2 cells. RT-PCR assay was used to determine RNA interference efficiency. Effects of knockdown of GRP78 on adenosine-induced cell viabilities, cell-cycle distribution and apoptosis, as well as relative protein expressions were determined by flow cytometry and/or Western blot analysis. The intracellular Ca2+ concentration was detected by laser scanning confocal microscope. Mitochondrial membrane potential (ΔΨm) was measured by a fluorospectrophotometer. The results revealed that GRP78 mRNA was significantly downregulated by Ad-shGRP78 transfection. Knockdown of GRP78 enhanced HepG2 cell sensitivity to adenosine by modulating G0/G1 arrest and stimulating Bax, Bak, m-calpain, caspase-4 and CHOP protein levels. Knockdown of GRP78 worsened cytosolic Ca2+ overload and ΔΨm loss. Knockdown of caspase-4 by shRNA decreased caspase-3 mRNA expression and cell apoptosis. These findings indicate that GRP 78 plays a protective role in ER stress-induced apoptosis and show that the combination of chemotherapy drug and RNA interference adenoviruses provides a new treatment strategy against malignant tumors.

HDAC4 protects cells from ER stress induced apoptosis through interaction with ATF4

Histone deacetylase 4 (HDAC4) is involved in the regulation of many fundamental cell processes such as proliferation, differentiation, and survival via the modification of their substrates or protein–protein interactions. In this study, we found that HDAC4 could be upregulated under ER stress. There exists a direct interaction between HDAC4 and activating transcription factor 4 (ATF4). In vitro, overexpression of HDAC4 caused the retention of ATF4 in cytoplasm and inhibition of ATF4 transcriptional activity. ER stress could promote cell apoptosis through the upregulation of ATF4 levels and its target genes such as CHOP and TRB3. This effect was exacerbated by downregulation of HDAC4 levels. These results demonstrated that HDAC4 played an important role in the regulation of ER stress-induced apoptosis through interacting with ATF4 and inhibiting its transcriptional activity.

Discrepancy of uterine leiomyoma and myometrium to hypoxia-induced endoplasmic reticulum stress after uterine occlusion therapy accounts for therapeutic effect

Uterine artery occlusion (UAO) is a promising method for the treatment of leiomyoma. This study is intended to demonstrate the discrepancy of ER stress-induced apoptosis in leiomyoma and myometrium as a result of UAO therapy. Primary cultured leiomyoma and myometrial cells were incubated in low oxygen supply (1 % O2). Then, real time RT-PCR and Western blotting were performed to analyze the mRNA and protein levels of ER stress-related molecules including GRP78, CHOP, JNK, Bax, Bcl-2 and Caspase4. Furthermore, the activity of Caspase4 was detected. Tissues of leiomyoma and myometria were also collected before and 30 min after UAO during surgery and evaluated. The leiomyoma cells and tissues expressed higher ER stress-related molecules compared to myometrial cells or tissues, while the levels of Bcl-2, an anti-apoptotic protein, declined. In myometrial cells, an elevated level of Caspase4 activation as well as its expression was not significant during the first 12 h, suggesting that hypoxia might not intensely affect the myometrium compared with leiomyoma. ER stress-related apoptosis partly accounts for the effects of UAO therapy on uterine leiomyoma, which leads to the death of leiomyoma while maintaining the survival of the uterus itself.

Phosphorylation at Tyrosine 262 Promotes GADD34 Protein Turnover

In mammalian cells, metabolic and environmental stress increases the phosphorylation of the eukaryotic translational initiation factor, eIF2α, and attenuates global protein synthesis. Subsequent transcriptional activation of GADD34 assembles an eIF2α phosphatase that feeds back to restore mRNA translation. Active proteasomal degradation of GADD34 protein then reestablishes the sensitivity of cells to subsequent bouts of stress. Mass spectrometry established GADD34 phosphorylation on multiple serines, threonines, and tyrosines. Phosphorylation at tyrosine 262 enhanced the rate of the GADD34 protein turnover. Substrate-trapping studies identified TC-PTP (PTPN2) as a potential GADD34 phosphatase, recognizing phosphotyrosine 262. Reduced GADD34 protein levels in TC-PTP-null MEFs following ER stress emphasized the importance of TC-PTP in determining the cellular levels of GADD34 protein. The susceptibility of TC-PTP-null MEFs to ER stress-induced apoptosis was significantly ameliorated by ectopic expression of GADD34. The data suggested that GADD34 phosphorylation on tyrosine 262 modulates endoplasmic reticulum stress signaling and cell fate.

Characterization of FKGK18 as Inhibitor of Group VIA Ca2+-Independent Phospholipase A2 (iPLA2β): Candidate Drug for Preventing Beta-Cell Apoptosis and Diabetes

Ongoing studies suggest an important role for iPLA2β in a multitude of biological processes and it has been implicated in neurodegenerative, skeletal and vascular smooth muscle disorders, bone formation, and cardiac arrhythmias. Thus, identifying an iPLA2βinhibitor that can be reliably and safely used in vivo is warranted. Currently, the mechanism-based inhibitor bromoenol lactone (BEL) is the most widely used to discern the role of iPLA2β in biological processes. While BEL is recognized as a more potent inhibitor of iPLA2 than of cPLA2 or sPLA2, leading to its designation as a “specific” inhibitor of iPLA2, it has been shown to also inhibit non-PLA2 enzymes. A potential complication of its use is that while the S and R enantiomers of BEL exhibit preference for cytosol-associated iPLA2β and membrane-associated iPLA2γ, respectively, the selectivity is only 10-fold for both. In addition, BEL is unstable in solution, promotes irreversible inhibition, and may be cytotoxic, making BEL not amenable for in vivo use. Recently, a fluoroketone (FK)-based compound (FKGK18) was described as a potent inhibitor of iPLA2β. Here we characterized its inhibitory profile in beta-cells and find that FKGK18: (a) inhibits iPLA2β with a greater potency (100-fold) than iPLA2γ, (b) inhibition of iPLA2β is reversible, (c) is an ineffective inhibitor of α-chymotrypsin, and (d) inhibits previously described outcomes of iPLA2β activation including (i) glucose-stimulated insulin secretion, (ii) arachidonic acid hydrolysis; as reflected by PGE2 release from human islets, (iii) ER stress-induced neutral sphingomyelinase 2 expression, and (iv) ER stress-induced beta-cell apoptosis. These findings suggest that FKGK18 is similar to BEL in its ability to inhibit iPLA2β. Because, in contrast to BEL, it is reversible and not a non-specific inhibitor of proteases, it is suggested that FKGK18 is more ideal for ex vivo and in vivo assessments of iPLA2β role in biological functions.

Blocking TLR2 activity diminishes and stabilizes advanced atherosclerotic lesions in apolipoprotein E-deficient mice

Toll-like receptor 2 (TLR2) signaling plays a critical role in the initiation of atherosclerosis. The aim of this study was to investigate whether blocking TLR2 activity could produce therapeutic effects on advanced atherosclerosis. Forty-week old apolipoprotein E-deficient (ApoE(-/-)) mice fed on a normal diet were intravenously injected with a TLR2-neutralizing antibody or with an isotype-matched IgG for 18 weeks. Double-knockout ApoE(-/-)Tlr2(-/-) mice were taken as a positive control. At the end of the treatments, the plasma lipid levels were measured, and the plaque morphology, pro-inflammatory cytokines expression and apoptosis in arteries were analyzed. In the second part of this study, 6-week old ApoE(-/-) and ApoE(-/-)Tlr2(-/-) mice fed on a high-cholesterol diet for 12 to 24 weeks, the expression levels of TLR2 and apoptotic markers in arteries were examined. Blockade of TLR2 activity with TLR2-neutralizing antibody or knockout of Tlr2 gene did not alter the plasma lipid levels in ApoE(-/-) mice. However, the pharmacologic and genetic manipulations significantly reduced the plaque size and vessel stenosis, and increased plaque stability in the brachiocephalic arteries. The protective effects of TLR2 antagonism were associated with the suppressed expression of pro-inflammatory cytokines IL-6 and TNF-α and the inactivation of transcription factors NF-κB and Stat3. In addition, blocking TLR2 activity attenuated ER stress-induced macrophage apoptosis in the brachiocephalic arteries, which could promote the resolution of necrotic cores in advanced atherosclerosis. Moreover, high-cholesterol diet more prominently accelerated atherosclerotic formation and increased the expression of pro-apoptotic protein CHOP and apoptosis in ApoE(-/-) mice than in ApoE(-/-)Tlr2(-/-) mice. The pharmacologic or genetic blockade of TLR2 activity diminishes and stabilizes advanced atherosclerotic lesions in ApoE(-/-) mice. Thus, targeting TLR2 signaling may be a promising therapeutic strategy against advanced atherosclerosis.

Major Vault Protein Regulates Class A Scavenger Receptor-mediated Tumor Necrosis Factor-α Synthesis and Apoptosis in Macrophages

Atherosclerosis is considered a disease of chronic inflammation largely initiated and perpetuated by macrophage-dependent synthesis and release of pro-inflammatory mediators. Class A scavenger receptor (SR-A) expressed on macrophages plays a key role in this process. However, how SR-A-mediated pro-inflammatory response is modulated in macrophages remains ill defined. Here through immunoprecipitation coupled with mass spectrometry, we reported major vault protein (MVP) as a novel binding partner for SR-A. The interaction between SR-A and MVP was confirmed by immunofluorescence staining and chemical cross-linking assay. Treatment of macrophages with fucoidan, a SR-A ligand, led to a marked increase in TNF-α production, which was attenuated by MVP depletion. Further analysis revealed that SR-A stimulated TNF-α synthesis in macrophages via the caveolin- instead of clathrin-mediated endocytic pathway linked to p38 and JNK, but not ERK, signaling pathways. Importantly, fucoidan invoked an enrichment of MVP in lipid raft, a caveolin-reliant membrane structure, and enhanced the interaction among SR-A, caveolin, and MVP. Finally, we demonstrated that MVP elimination ameliorated SR-A-mediated apoptosis in macrophages. As such, MVP may fine-tune SR-A activity in macrophages which contributes to the development of atherosclerosis.

Pinocembrin protects brain against ischemia/reperfusion injury by attenuating endoplasmic reticulum stress induced apoptosis

Endoplasmic reticulum stress (ER stress) is known to play a vital role in mediating ischemic reperfusion damage in brain. Our previous studies showed that pinocembrin alleviated cerebral ischemic injury in ischemia/reperfusion and vascular dementia animal models, but whether attenuation of ER stress-induced apoptosis contributes to the mechanisms remains to be elucidated. In this study, an attempt was therefore made to investigate the modulation effect of pinocembrin on ischemia/reperfusion-induced ER stress in brain. Focal cerebral ischemia/reperfusion rats were induced by middle cerebral artery occlusion (MCAO) for 2h followed by 6h reperfusion. Pinocembrin was administered in different doses (1mg/kg, 3mg/kg, and 10mg/kg, respectively) at the same time of onset of reperfusion. Neurological function and brain infarction were evaluated. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method, and flow cytometer (FCM) were used to investigate cell apoptosis in penumbra cortex. DNA fragmentation assay was also performed using electrophoresis. The expression of ER stress proteins of GRP78, CHOP/GADD153, ATF4, eIF2α phosphorylation was detected by western blot, and caspase-12 was evaluated by immunohistochemical analysis. Our results demonstrate that pinocembrin-treatment (3mg/kg and 10mg/kg) significantly reduced neurological deficit scores, infarct volume, and neuron apoptosis in the ischemia/reperfusion rats. It can also significantly modulate the protein levels by increasing GRP78 (10mg/kg) and attenuating CHOP/GADD153 expression along with caspase-12 activation (3mg/kg and 10mg/kg). At the same time, eIF2α phosphorylation was restrained and the expression of ATF4 was reduced (3mg/kg and 10mg/kg). These results suggest that the attenuation of ER stress induced apoptosis may be involved in the mechanisms of pinocembrin.

Abstract B10: ER stress protein GRP78 as a therapeutic target combined with antiangiogenic therapy in renal cell carcinoma

Abstract Background: Antiangiogenic therapy deprives oxygen and nutrition from the tumor. These stress conditions cause unfolded or misfolded proteins in tumor cells. Glucose regulated protein 78 (GRP78) binds to unfolded proteins and dissociates from the membrane-bound ER stress sensors. Dissociation of GRP78 from these stress sensors allows their subsequent activation which suppresses global mRNA translation to protect cells from excessive unfolded proteins. We investigated a potential role of GRP78 as a combined therapeutic target in renal cell carcinoma treated with antiangiogenic therapy. Methods: Renal cell carcinoma cells (Caki-1, Caki-2, UMRC-3 and UMRC-6) were used to investigate the effect of GRP78 knockdown, which was performed by small interfering RNA. Caki-1 xenografts were developed and treated with sunitinib 40mg/kg/day to evaluate in vivo expression of GRP78 during antiangiogenic therapy. Caki-1 cells stably overexpressing GRP78 were developed to investigate the role of GRP78 in cancer cell. Hypoxic stress was induced by 1% hypoxia chamber and hypoglycaemic stress was induced by glucose-free media. Downstream signalling pathways of GRP78 were evaluated by Western blots. Results: In vitro hypoxia and/or glucose deprivation induced GRP78 upregulation in Caki-1 cells. GRP78 was also induced in Caki-1 xenografts treated by sunitinib. Overexpression of GRP78 increased tumor proliferation in hypoxic and/or hypoglycemic stresses by activating PERK/eIF2α pathway and protected tumor cells from stress-induced apoptosis. Knockdown of GRP78 using small interference RNA inhibited cancer cell survival and induced apoptosis in renal cell carcinoma cells in vitro. GRP78 knockdown also sensitized renal cell carcinoma cells to ER stress-induced apoptosis and hypoxic and hypoplycemic stress-induces apoptosis. Conclusions: Antiangiogenic therapy induces ER stress by depriving oxygen and glucose from renal cell carcinoma. ER protein GRP78 has a critical role in protecting renal cell carcinoma cells from hypoxic and hypoglycemic stress induced by antiangiogenic therapy. Knockdown of GRP78 sensitizes RCC cells to apoptotic cell death from anti-angiogenic stresses. Our results suggest that GRP78 is a novel therapeutic target in renal cell carcinoma management. Citation Format: Kyung Seok Han, Na Li, Estelle Li, Fazli Ladan, Gleave Martin, Alan So. ER stress protein GRP78 as a therapeutic target combined with antiangiogenic therapy in renal cell carcinoma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B10.

Activation of α7 nicotinic acetylcholine receptor protects M2 macrophages from ER stress‐induced apoptosis

The α7 nicotinic acetylcholine receptor (α7nAChR) is expressed in many non‐neuronal cell types including immune cells, and is now recognized to be involved in a variety of inflammatory events. Atherosclerosis is a chronic inflammatory disease of the vascular wall, and macrophages (MΦ) are essential contributors to the development of atherosclerotic lesions. MΦ apoptosis and inefficient efferocytosis are associated with necrotic core formation and lesion destabilization. MΦs express α7nAChR but the potential role of α7nAChR in MΦ survival and apoptosis has yet to be explored. In bone marrow‐derived MΦs (BMDMs) which were polarized to pro‐inflammatory “M1” or anti‐inflammatory “M2” phenotypes, nicotine exerted a protective action against ER stress‐induced apoptosis in M2 but not M1 MΦs; this effect was lost in α7KO M2 MΦs. Concomitantly, nicotine treatment resulted in increased STAT3 phosphorylation which was reduced in the α7KO MΦs, and abrogated upon inhibition of the upstream regulator JAK2. Our results suggest that activation of the α7nAChR protects M2 MΦs from apoptosis, presumably through regulation of survival genes through activation of the JAK2/STAT3 pathway. Supported by R01HL111877–01.

Bcl-2 associated athanogene 5 (Bag5) is overexpressed in prostate cancer and inhibits ER-stress induced apoptosis

BackgroundThe Bag (Bcl-2 associated athanogene) family of proteins consists of 6 members sharing a common, single-copied Bag domain through which they interact with the molecular chaperone Hsp70. Bag5 represents an exception in the Bag family since it consists of 5 Bag domains covering the whole protein. Bag proteins like Bag1 and Bag3 have been implicated in tumor growth and survival but it is not known whether Bag5 also exhibits this function.MethodsBag5 mRNA and protein expression levels were investigated in prostate cancer patient samples using real-time PCR and immunoblot analyses. In addition immunohistological studies were carried out to determine the expression of Bag5 in tissue arrays. Analysis of Bag5 gene expression was carried out using one-way ANOVA and Bonferroni’s Multiple Comparison test. The mean values of the Bag5 stained cells in the tissue array was analyzed by Mann-Whitney test. Functional studies of the role of Bag5 in prostate cancer cell lines was performed using overexpression and RNA interference analyses.ResultsOur results show that Bag5 is overexpressed in malignant prostate tissue compared to benign samples. In addition we could show that Bag5 levels are increased following endoplasmic reticulum (ER)-stress induction, and Bag5 relocates from the cytoplasm to the ER during this process. We also demonstrate that Bag5 interacts with the ER-resident chaperone GRP78/BiP and enhances its ATPase activity. Bag5 overexpression in 22Rv.1 prostate cancer cells inhibited ER-stress induced apoptosis in the unfolded protein response by suppressing PERK-eIF2-ATF4 activity while enhancing the IRE1-Xbp1 axis of this pathway. Cells expressing high levels of Bag5 showed reduced sensitivity to apoptosis induced by different agents while Bag5 downregulation resulted in increased stress-induced cell death.ConclusionsWe have therefore shown that Bag5 is overexpressed in prostate cancer and plays a role in ER-stress induced apoptosis. Furthermore we have identified GRP78/BiP as a novel interaction partner of Bag5.

ER stress protein GRP78 as a therapeutic target combined with antiangiogenic therapy in renal cell carcinoma.

428 Background: Antiangiogenic therapy deprives oxygen and nutrition from the tumor. These stresses cause unfolded proteins in tumor cells. Glucose regulated protein 78 (GRP78) binds to unfolded proteins and its subsequent activation suppresses global mRNA translation to protect cells from excessive unfolded proteins. We investigated a potential role of GRP78 as a combined therapeutic target in renal cell carcinoma treated with antiangiogenic therapy. Methods: Renal cell carcinoma cells (Caki-1, Caki-2, UMRC-3, and UMRC-6) were used to investigate the effect of GRP78 knockdown, which was performed by small interfering RNA. Caki-1 xenografts were developed and treated with sunitinib 40mg/kg/day to evaluate in vivo expression of GRP78 during antiangiogenic therapy. Caki-1 cells stably overexpressing GRP78 were developed to investigate the role of GRP78 in cancer cell. Hypoxic stress was induced by 1% hypoxia chamber and hypoglycaemic stress was induced by glucose-free media. Downstream signalling pathways of GRP78 were evaluated by Western blots. Results: In vitro hypoxia and/or glucose deprivation induced GRP78 upregulation in Caki-1 cells. GRP78 was also induced in Caki-1 xenografts treated by sunitinib. Overexpression of GRP78 increased tumor proliferation in hypoxic and/or hypoglycemic stresses by activating PERK/eIF2α pathway and protected tumor cells from stress-induced apoptosis. Knockdown of GRP78 using small interference RNA inhibited cancer cell survival and induced apoptosis in renal cell carcinoma cells in vitro. GRP78 knockdown also sensitized renal cell carcinoma cells to ER stress-induced apoptosis and hypoxic and hypoplycemic stress-induces apoptosis. Conclusions: Antiangiogenic therapy induces ER stress by depriving oxygen and glucose from renal cell carcinoma. ER protein GRP78 has a critical role in protecting renal cell carcinoma cells from hypoxic and hypoglycemic stress induced by antiangiogenic therapy. Knockdown of GRP78 sensitizes RCC cells to apoptotic cell death from anti-angiogenic stresses. Our results suggest that GRP78 is a novel therapeutic target in renal cell carcinoma management.

Clusterin Protects Hepatocellular Carcinoma Cells from Endoplasmic Reticulum Stress Induced Apoptosis through GRP78

Clusterin (CLU) is a stress-activated chaperone, which plays an important role in cancer development and progression through promoting cell survival. However, the exact mechanism of how CLU exerts its cell protective role under ER stress condition is still unclear. Therefore, in order to explore the molecular mechanisms by which CLU inhibited ER stress-induced apoptosis, HCC cell lines were treated with tunicamycin (TN), an ER stress inducer. We found that the expressions of both CLU and GRP78 were increased after TN treatment. Knockdown of CLU expression in SMMC7721 and HCCLM3 cells inhibited GRP78 expression after TN treatment and enhanced ER stress-induced apoptosis, whereas over-expression of CLU in HepG2 cells increased GRP78 expression after TN induction and abolished the effect of TN on cell apoptosis. Furthermore, knockdown of GRP78 expression in CLU-HepG2 cells abrogated the protective role of CLU under ER stress condition. Co-immunoprecipitation (co-IP) and confocal microscopy experiments confirmed the direct interaction between CLU and GRP78 under ER stress condition. The effect of CLU knockdown on GRP78 expression and cell apoptosis in HCC tumors were further determined in orthotopic xenograft tumor model. Knockdown of CLU expression in HCCLM3 cells inhibited GRP78 expression in tumor tissues, accompanied with increased number of apoptotic cancer cells. Moreover, the correlation between CLU and GRP78 expression was further determined in clinical HCC specimens. Taken together, these findings reveal that CLU protects HCC cells from ER stress induced apoptosis at least partially through interacting with GRP78.

Genetic modulation of islet β-cell iPLA2β expression provides evidence for its impact on β-cell apoptosis and autophagy

β-cell apoptosis is a significant contributor to β-cell dysfunction in diabetes and ER stress is among the factors that contributes to β-cell death. We previously identified that the Ca2+-independent phospholipase A2β (iPLA2β), which in islets is localized in β-cells, participates in ER stress-induced β-cell apoptosis. Here, direct assessment of iPLA2β role was made using β-cell-specific iPLA2β overexpressing (RIP-iPLA2β-Tg) and globally iPLA2β-deficient (iPLA2β-KO) mice. Islets from Tg, but not KO, express higher islet iPLA2β and neutral sphingomyelinase, decrease in sphingomyelins, and increase in ceramides, relative to WT group. ER stress induces iPLA2β, ER stress factors, loss of mitochondrial membrane potential (∆Ψ), caspase-3 activation, and β-cell apoptosis in the WT and these are all amplified in the Tg group. Surprisingly, β-cells apoptosis while reduced in the KO is higher than in the WT group. This, however, was not accompanied by greater caspase-3 activation but with larger loss of ∆Ψ, suggesting that iPLA2β deficiency impacts mitochondrial membrane integrity and causes apoptosis by a caspase-independent manner. Further, autophagy, as reflected by LC3-II accumulation, is increased in Tg and decreased in KO, relative to WT. Our findings suggest that (1) iPLA2β impacts upstream (UPR) and downstream (ceramide generation and mitochondrial) pathways in β-cells and (2) both over- or under-expression of iPLA2β is deleterious to the β-cells. Further, we present for the first time evidence for potential regulation of autophagy by iPLA2β in islet β-cells. These findings support the hypothesis that iPLA2β induction under stress, as in diabetes, is a key component to amplifying β-cell death processes.

NOXA contributes to the sensitivity of PERK-deficient cells to ER stress

PKR-like ER kinase (PERK) deficient mouse embryonic fibroblasts (MEFs) are hypersensitive to ER stress-induced apoptosis. However, the molecular determinants of increased sensitivity of PERK−/− MEFs are not clearly understood. Here we show that induction of several Unfolded Protein Response (UPR) target genes is attenuated in PERK−/− MEFs. We also report elevated expression of the BH3-only protein, NOXA in PERK−/− MEFs. Further, shRNA-mediated knockdown of NOXA rescued the hypersensitivity of PERK−/− MEFs to ER stress-induced apoptosis. Taken together our results suggest that compromised induction of UPR and increased NOXA expression contributes to hypersensitivity of PERK−/− MEFs to ER stress-induced apoptosis.

Stress-Inducible Caspase Substrate TRB3 Promotes Nuclear Translocation of Procaspase-3

Pseudokinase TRB3 is a stress-inducible nuclear protein, which has recently been shown to be involved in ER stress-induced apoptosis. However, it remains unclear how TRB3 contributes to the process. We recently demonstrated that TRB3 was cleaved by caspase-3 (CASP3) in vitro and also in apoptosis-induced cells. Thus, we investigate the role of TRB3 cleavage in the apoptotic process to address the above question. Overexpression studies revealed that the cleavage of TRB3 promoted CASP3/7 activation and apoptosis. In contrast, the anti-apoptotic effects were found under TRB3 non-cleavable conditions, such as ER stress, and also when the CASP3/7 activation was enhanced by knockdown of endogenous TRB3 expression. Interestingly, nuclear translocation of procaspase-3 (proCASP3) was observed in cells either overexpressing TRB3 or under tunicamycin-induced ER stress. Although forced cytoplasmic expression of proCASP3 enhanced apoptosis significantly, its nuclear expression did not produce any pro-apoptotic effect, suggesting that nuclear distribution of proCASP3 is not critical for the execution of apoptosis. Thus, TRB3 might prevent cytoplasmic activation of CASP3 by promoting proCASP3 entry into the nucleus, and thereby inhibit apoptosis. Taken together, our results suggest that TRB3, through its own cleavage, functions as a molecular switch between the cell survival and apoptotic pathways under stressful conditions.

Deficiency of αB crystallin augments ER stress-induced apoptosis by enhancing mitochondrial dysfunction

AbstractEndoplasmic reticulum (ER) stress is linked to several pathological conditions including age-related macular degeneration. Excessive ER stress initiates cell death cascades which are mediated, in part, through mitochondrial dysfunction. Here, we identify αB crystallin as an important regulator of ER stress-induced cell death. Retinal pigment epithelial (RPE) cells from αB crystallin (–/–) mice, and human RPE cells transfected with αB crystallin siRNA, are more vulnerable to ER stress induced by tunicamycin. ER stress-mediated cell death is associated with increased levels of reactive oxygen species, depletion of glutathione in mitochondria, decreased superoxide dismutase activity, increased release of cytochrome c, and activation of caspases 3 and 4. The ER stress signaling inhibitors, salubrinal and 4-(2-aminoethyl) benzenesulfonyl fluoride, decrease mitochondrial damage and reduce RPE apoptosis induced by ER stress. Prolonged ER stress decreases levels of αB crystallin, thus exacerbating mitochondrial dysfunction. Overexpression of αB crystallin protects RPE cells from ER stress-induced apoptosis by attenuating increases in Bax, CHOP, mitochondrial permeability transition, and cleaved caspase 3. Thus, these data collectively demonstrate that αB crystallin provides critical protection of mitochondrial function during ER stress-induced RPE apoptosis.

4-O-Carboxymethyl Ascochlorin Causes ER Stress and Induced Autophagy in Human Hepatocellular Carcinoma Cells

The synthetic derivative of ascochlorin, 4-O-carboxymethyl ascochlorin (AS-6) is an agonist of the nuclear hormone receptor PPARγ and has been shown to induce differentiation in mouse pre-adipocytes and to ameliorate type II diabetes in a murine model. AS-6 was cytotoxic when added at micromolar concentrations to cultures of three different human cancer cell lines. We used gel electrophoresis and mass spectrometry to identify proteins with altered expression in human hepatocarcinoma cells (HepG2) cells after 12 h in the presence of AS-6 and found 58 proteins that were differentially expressed. Many of the proteins showing increased expression in cells treated with AS-6 are involved in protein quality control, including glucose-regulated protein 78 (GRP78/BiP), a regulator of ER stress responses, and the transcriptional regulator CHOP, which mediates ER stress-induced apoptosis. Cells treated with AS-6 undergo an autophagic response accompanied by increased expression of beclin1, ATG5, and LC3-II and autophagosome formation marked by the appearance of large vesicles containing LC3-II. Grp78 induction was inhibited when the PPARγ antagonist, GW9662, was added together with AS-6, and autophagy and cell death were partially blocked. 3-methyl-adenine (3-MA), an inhibitor of phosphatidyl inositol 3-kinase (PI3-kinase) prevented induction of ATG5 and activation of LC3-II and blocked autophagosome formation. 3-MA also blocked induction of GRP78 and CHOP, suggesting that PI3-kinase, which is known to mediate ER stress-induced autophagy, also plays a role in initiating apoptosis in response to ER stress. Together these data establish that the cytotoxicity of AS-6 operates by a mechanism dependent on ER stress-induced autophagy and apoptosis.

Abstract 4990: PP2A signaling is overridden by MEK/ERK activity leading to suppression of Bim and protection of melanoma cells from ER stress-induced apoptosis

Abstract Endoplasmic reticulum (ER) stress triggers apoptosis by activating Bim in diverse types of cells. This involves dephosphorylation of Bim by protein phosphatase 2A (PP2A). However, melanoma cells are largely resistant to ER stress-induced apoptosis, indicating that Bim activation is suppressed in melanoma cells undergoing ER stress. We show here that this is due to increased activation of MEK/ERK signaling that overrides the PP2A activity leading to enhanced Bim phosphorylation and subsequent proteosomal degradation. Upon pharmacological ER stress, the Bim protein expression was rapidly downregulated after an initial increase, despite its sustained upregulation at the transcriptional level. Although PP2A exerted the dephosphorylating effect on BimEL, its activity appeared to be superseded by increased activation of MEK/ERK signaling that led to enchanced phosphorylation and accelerated turnover of the protein. Moreover, in contrast to its upregulation by ER stress in MCF-7 and HEK293 cells, PP2A was downregulated in melanoma cells undergoing ER stress. Pharmacological activation or enforced expression of PP2A decreased ERK and Bim phosphorylation, up-regulated Bim expression, and rendered melanoma cells sensitive to ER stress-induced apoptosis. Notably, instead of direct interaction with Bim, PP2A reduced Bim phosphorylation indirectly by dephosphorylating ERK in melanoma cells. Together, these results identify a key resistance mechanism of melanoma cells against apoptosis induced by ER stress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4990. doi:1538-7445.AM2012-4990

Molecular Intricacies and the Role of ER Stress in Diabetes

Molecular Intricacies and the Role of ER Stress in Diabetes