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At least 127 records · Page 7Linked to original sources
PubMed2005

Vesicular stomatitis viruses expressing wild-type or mutant M proteins activate apoptosis through distinct pathways.

Vesicular stomatitis virus (VSV) induces apoptosis by at least two mechanisms. The viral matrix (M) protein induces apoptosis via the mitochondrial pathway due to the inhibition of host gene expression. However, in some cell types, the inhibition of host gene expression by VSV expressing wild-type (wt) M protein delays VSV-induced apoptosis, indicating that another mechanism is involved. In support of this, the recombinant M51R-M (rM51R-M) virus, expressing a mutant M protein that is defective in its ability to inhibit host gene expression, induces apoptosis much more rapidly in L929 cells than do viruses expressing wt M protein. Here, we determine the caspase pathways by which the rM51R-M virus induces apoptosis. An analysis of caspase activity, using fluorometric caspase assays and Western blots, indicated that each of the main initiator caspases, caspase-8, caspase-9, and caspase-12, were activated during infection with the rM51R-M virus. The overexpression of Bcl-2, an inhibitor of the mitochondrial pathway, or MAGE-3, an inhibitor of caspase-12 activation, did not delay apoptosis induction in rM51R-M virus-infected L929 cells. However, an inhibitor of caspase-8 activity significantly delayed apoptosis induction. Furthermore, the inhibition of caspase-8 activity prevented the activation of caspase-9, suggesting that caspase-9 is activated by cross talk with caspase-8. These data indicate that VSV expressing the mutant M protein induces apoptosis via the death receptor apoptotic pathway, a mechanism distinct from that induced by VSV expressing the wt M protein.

Amino Acid Substitution↗
PubMed2006

Activation of caspase-8 and caspase-12 pathways by 7-ketocholesterol in human retinal pigment epithelial cells.

PURPOSE: To determine whether caspase or cathepsin pathways are activated in human retinal pigment epithelial cells (ARPE-19) after exposure to 7-ketocholesterol (7kCh). METHODS: ARPE-19 cells were exposed to 7kCh with or without z-VAD-fmk, a pan-caspase inhibitor. Caspase-3, -8, and -9 activities were measured by a fluorochrome inhibitor of caspase (FLICA) assay. Caspase-12 activity was detected by Western blotting. RT-PCR was performed for 18s, mortalin-2, cathepsins B, D, and L/V2. RESULTS: At 24 hours, 7kCh-treated cultures had increased caspase-8 (P < 0.001) and caspase-3 (P < 0.001) activities compared with vehicle-treated cultures. 7kCh-induced caspase-3 activation was blocked by z-VAD-fmk (P < 0.001). Caspase-9 was not activated by 7kCh treatment (P > 0.05). Procaspase-12 was cleaved into its active form after treatment with 7kCh for 24 hours. At 6 hours, the RNA level for mortalin-2, a pro-survival gene, was upregulated. ARPE-19 cells did not express RNA for cathepsins B, D, or L/V2 under any conditions. CONCLUSIONS: In ARPE-19 cells, 7kCh-induced apoptosis uses the receptor-mediated caspase-8 pathway and the endoplasmic reticulum stress-induced caspase-12 pathway but not the mitochondrial caspase-9 pathway. The cathepsin pathways are not involved in 7kCh-induced cell death. These data demonstrate that 7kCh causes a loss of cell viability through caspase-dependent apoptosis and can act as an oxidative stressor leading to retinal pigment epithelial cell atrophy. Elucidating the specific apoptotic pathways involved may have therapeutic potential for AMD and other retinal diseases.

Amino Acid Chloromethyl Ketones↗
PubMed2006

Flt-1 intraceptor induces the unfolded protein response, apoptotic factors, and regression of murine injury-induced corneal neovascularization.

PURPOSE: To determine whether Flt24K, a recombinant construct of domains 2 to 4 of VEGFR-1 (Flt) coupled with an endoplasmic reticulum retention signal (KDEL) can bind VEGFR-2 and induce apoptosis, unfolded protein response (UPR), and regression of injury-induced corneal neovascularization. METHODS: Human microvascular endothelial cells were transfected with pCMV.Flt24K and subjected to hypoxia. Cell lysates underwent Western blot analysis with anti-XBP-1 antibody and RT-PCR for CHOP. Human malignant melanoma cells (which express VEGFR-2 but not Flt), were transfected with pCMV.Flt24K, and lysates underwent immunoprecipitation with anti-FLT antibody, and Western blot analysis for VEGF and VEGFR-2. Mouse corneas sustained injury induced by topical NaOH and mechanical scraping and were injected with pCMV.Flt24K 2 weeks later. Corneas were harvested 2 days later for Western blot analysis for XBP-1 and caspase-3 or 1 week later for quantification of neovascularization and TUNEL staining. Saline and empty pCMV vector were used in control experiments. RESULTS: The mean percentage area of corneal neovascularization in mice 3 weeks after corneal injury and 1 week after intrastromal injection of empty pCMV vector or pCMV.Flt24K was 55.4% +/- 2.7% vs. 19.3% +/- 6.1%, respectively (P < 0.001). Flt24K was found to bind VEGFR-2 and upregulate activated XBP-1 and CHOP in vitro. In vivo, pCMV.Flt24K upregulated activated XBP-1 and caspase-3. Apoptosis was observed in corneal neovascular endothelium in corneas treated with pCMV.Flt24K but not in the control. CONCLUSIONS: The Flt24K intraceptor can bind VEGFR-2 within cells, induce the unfolded protein response in vitro and in vivo, elicit apoptosis of vascular endothelial cells in vivo, and induce regression of corneal neovascularization in vivo.

Animals↗
PubMed2004

Caspase-12: a developmental link between G-protein-coupled receptors and integrin alphaIIbbeta3 activation.

Fibrinogen binding by integrin alphaIIbbeta3 is promoted by platelet agonists that increase the affinity and avidity of alphaIIbbeta3 for fibrinogen through a process called "inside-out" signaling. Having previously demonstrated that inside-out activation of alphaIIbbeta3 is defective in murine megakaryocytes that lack the transcription factor NF-E2, we screened for NF-E2-regulated genes that affect alphaIIbbeta3 activation. Caspase-12 is the most down-regulated gene we identified in NF-E2(-/-) megakaryocytes. Therefore, the role of this protein in alphaIIbbeta3 activation was determined using platelets from caspase-12(-/-) mice. Despite wild-type levels of alphaIIbbeta3, caspase-12(-/-) platelets exhibit reduced fibrinogen binding to alphaIIbbeta3 following stimulation by adenosine diphosphate (ADP) or protease-activated receptor 4 (PAR4) receptor-activating peptide. The defect in alphaIIbbeta3 activation is associated with decreased cytosolic free calcium and inositol triphosphate levels, and with reduced aggregation, despite wild-type phospholipase Cbeta expression levels. In contrast, agonist-induced surface expression of P-selectin, suppression of cAMP levels following ADP stimulation, and spreading on immobilized fibrinogen are unimpaired. Moreover, although caspase-12 is highly expressed in mature megakaryocytes, it is undetectable in platelets. Taken together, these studies establish that caspase-12 expression in murine megakaryocytes is regulated, directly or indirectly, by NF-E2, and suggest that caspase-12 participates in the development of fully functional signaling pathways linking some G-protein-coupled receptors to alphaIIbbeta3 activation.

Alprostadil↗
PubMed2003

Caspase-12 and ER-stress-mediated apoptosis: the story so far.

The labyrinth of the endoplasmic reticulum (ER) interweaves the cytosol and connects to the nucleus, mitochondria, and the plasma membrane. In the lumen of the ER, the essential function of lipid synthesis, Ca(2+) storage, folding, and maturation of proteins take place. Therefore, the tight regulation and maintenance of ER homeostasis is vital. Disturbance of the Ca(2+) homeostasis during hypoxia, or imbalance between the demand and capacity of the protein-folding apparatus, initiates an adaptive response of the cell, termed the unfolded protein response (UPR, ER stress response). As a result, ER-localized chaperones are induced, protein synthesis is slowed down, and a protein degrading system is initiated. However, if the ER stress cannot be alleviated, it culminates in apoptosis. This paper reviews the newly outlined signaling pathways of the unfolded protein response and describes the central role of caspase-12 in the initiation of cell death. The complex role of the ER and its signaling pathways provides a novel angle on apoptosis research and may offer a key to apoptosis-associated diseases.

Animals↗
PubMed2006

The unfolded protein response: a stress signaling pathway critical for health and disease.

The endoplasmic reticulum (ER) is an intracellular organelle consisting of a membranous labyrinth network that extends throughout the cytoplasm of the cell and is contiguous with the nuclear envelope. In all eukaryotic cells, the ER is the site where folding and assembly occurs for proteins destined to the extracellular space, plasma membrane, and the exo/endocytic compartments. The ER is exquisitely sensitive to alterations in homeostasis, and provides stringent quality control systems to ensure that only correctly folded proteins transit to the Golgi and unfolded or misfolded proteins are retained and ultimately degraded. A number of biochemical and physiologic stimuli, such as perturbation in calcium homeostasis or redox status, elevated secretory protein synthesis, expression of misfolded proteins, sugar/glucose deprivation, altered glycosylation, and overloading of cholesterol can disrupt ER homeostasis, impose stress to the ER, and subsequently lead to accumulation of unfolded or misfolded proteins in the ER lumen. The ER has evolved highly specific signaling pathways called the unfolded protein response (UPR) to cope with the accumulation of unfolded or misfolded proteins. Recent discoveries of the mechanisms of ER stress signaling have led to major new insights into the diverse cellular and physiologic processes that are regulated by the UPR. This review summarizes the complex regulation of UPR signaling and its relevance to human physiology and disease.

Apoptosis↗
PubMed2006

ER stress-induced apoptosis and caspase-12 activation occurs downstream of mitochondrial apoptosis involving Apaf-1.

Accumulation of unfolded proteins induces endoplasmic reticulum (ER) stress. Excessive and prolonged stresses lead cells to apoptosis. However, the precise molecular mechanisms of ER stress-induced apoptosis have not been fully elucidated. We investigated the involvement of the apoptosome in ER stress-induced cell death pathway using mouse embryonic fibroblasts (MEFs) and mice deficient for Apaf-1. Apaf-1-deficient MEFs showed more resistance to ER stress-inducing reagents as compared with wild type cells. Despite comparable induction of ER stress in both wild type and Apaf-1-deficient cells, activation of caspase-3 was only observed in wild type, but not Apaf-1-deficient, MEFs. Under ER stress conditions, BAX translocated to mitochondria and cytochrome c was released from mitochondria. We also demonstrated that caspase-12 was processed downstream of Apaf-1 and caspase-3, and neither overexpression nor knockdown of caspase-12 affected susceptibility of the cells to ER stress-induced cell death. Furthermore, in the kidneys of Apaf-1-deficient mice, apoptosis induced by in vivo administration of tunicamycin was remarkably suppressed as compared with wild type mice. These data collectively demonstrated that Apaf-1 and the mitochondrial pathway of apoptosis play significant roles in ER stress-induced apoptosis.

Animals↗
PubMed2001

Wild-type, mitochondrial and ER-restricted Bcl-2 inhibit DNA damage-induced apoptosis but do not affect death receptor-induced apoptosis.

The proto-oncogene Bcl-2 is expressed in membranes of mitochondria and endoplasmic reticulum and mediates resistance against a broad range of apoptotic stimuli. Although several mechanisms of Bcl-2 action have been proposed, its role in different cellular organelles remains elusive. Here, we analyzed the function of Bcl-2 targeted specifically to certain subcellular compartments in Jurkat cells. Bcl-2 expression was restricted to the outer mitochondrial membrane by replacing its membrane anchor with the mitochondrial insertion sequence of ActA (Bcl-2/MT) or the ER-specific sequence of cytochrome b5 (Bcl-2/ER). Additionally, cells expressing wild-type Bcl-2 (Bcl-2/WT) or a transmembrane domain-lacking mutant (Bcl-2/DeltaTM) were employed. Apoptosis induced by ionizing radiation or by the death receptors for CD95L or TRAIL was analyzed by determination of the mitochondrial membrane potential (DeltaPsi(m)) and activation of different caspases. Bcl-2/WT and Bcl-2/MT strongly inhibited radiation-induced apoptosis and caspase activation, whereas Bcl-2/DeltaTM had completely lost its anti-apoptotic effect. Interestingly, Bcl-2/ER conferred protection against radiation-induced mitochondrial damage and apoptosis similarly to Bcl-2/MT. The finding that ER-targeted Bcl-2 interfered with mitochondrial DeltaPsi(m) breakdown and caspase-9 activation indicates the presence of a crosstalk between both organelles in radiation-induced apoptosis. By contrast, Bcl-2 in either subcellular position did not influence CD95- or TRAIL-mediated apoptosis.

Apoptosis↗
PubMed2003

Calcium as a mediator of apoptosis in bovine oocytes and preimplantation embryos.

Cell death pathways and their regulation during development of the preimplantation embryo are poorly understood. Our previous studies indicate that Ca2+ is a key mediator of apoptosis in different cell models. We hypothesized that Ca2+ signaling mediates apoptotic cell death during early embryonic development via activation of Ca2+-dependent proteases, micro-calpain, and caspase-12. To test this hypothesis, established procedures for in vitro production of bovine embryos in concert with fluorescence, high-resolution, digital Ca2+ imaging, detection of Ca2+-dependent apoptotic mediators, and measurement of apoptotic cell death were used in the present studies. We found that an increase in intracellular free Ca2+ concentration ([Ca2+]i) in the individual embryo cells (2.6-fold) and mature oocytes (1.6-fold) was associated with activation of micro-calpain, induction of pan-caspase activity (5-10-fold), and expression of the Ca2+-dependent caspase-12. Inhibition of calpain or caspase activity significantly (1.5-2-fold) reduced apoptotic indices in embryos treated with the mobilizer of intracellular Ca2+ stores, thapsigargin, or the Ca2+ ionophore, ionomycin. Taken together, these results support our hypothesis that Ca2+ is involved in apoptosis of the preimplantation bovine embryo and that Ca2+-dependent apoptotic proteases are micro-calpain and caspase-12.

Animals↗
PubMed2002

Na/Ca exchanger overexpression induces endoplasmic reticulum-related apoptosis and caspase-12 activation in insulin-releasing BRIN-BD11 cells.

Ca(2+) may trigger programmed cell death (apoptosis) and regulate death-specific enzymes. Therefore, the development of strategies to control Ca(2+) homeostasis may represent a potential approach to prevent or enhance cell apoptosis. To test this hypothesis, the plasma membrane Na/Ca exchanger (NCX1.7 isoform) was stably overexpressed in insulin-secreting tumoral cells. NCX1.7 overexpression increased apoptosis induced by endoplasmic reticulum (ER) Ca(2+)-ATPase inhibitors, but not by agents increasing intracellular calcium concentration ([Ca(2+)](i)), through the opening of plasma membrane Ca(2+)-channels. NCX1.7 overexpression reduced the rise in [Ca(2+)](i) induced by all agents, depleted ER Ca(2+) stores, sensitized the cells to Ca(2+)-independent proapoptotic signaling pathways, and reduced cell proliferation by approximately 40%. ER Ca(2+) stores depletion was accompanied by the activation of the ER-specific caspase (caspase-12), and the activation was enhanced by ER Ca(2+)-ATPase inhibitors. Hence, Na/Ca exchanger overexpression, by depleting ER Ca(2+) stores, triggers the activation of caspase-12 and increases apoptotic cell death. By increasing apoptosis and decreasing cell proliferation, overexpression of Na/Ca exchanger may represent a new potential approach in cancer gene therapy. On the other hand, our results open the way to the development of new strategies to control cellular Ca(2+) homeostasis that could, on the contrary, prevent the process of apoptosis that mediates, in part, beta-cell autoimmune destruction in type 1 diabetes.

Animals↗
PubMed2005

The effect of C-peptide on cognitive dysfunction and hippocampal apoptosis in type 1 diabetic rats.

Primary diabetic encephalopathy is a recently recognized late complication of diabetes resulting in a progressive decline in cognitive faculties. In the spontaneously type 1 diabetic BB/Wor rat, we recently demonstrated that cognitive impairment was associated with hippocampal apoptotic neuronal loss. Here, we demonstrate that replacement of proinsulin C-peptide in this insulinopenic model significantly prevented spatial learning and memory deficits and hippocampal neuronal loss. C-peptide replacement prevented oxidative stress-, endoplasmic reticulum-, nerve growth factor receptor p75-, and poly(ADP-ribose) polymerase-related apoptotic activities. It partially ameliorated apoptotic stresses mediated via impaired insulin and IGF activities. These findings were associated with the prevention of increased expression of Bax and active caspase 3 and the frequency of caspase 3-positive neurons. The results show that several partially interrelated apoptotic mechanisms are involved in primary encephalopathy and suggest that impaired insulinomimetic action by C-peptide plays a prominent role in cognitive dysfunction and hippocampal apoptosis in type 1 diabetes. Although these abnormalities were not fully prevented by C-peptide replacement, the findings suggest that this regime will substantially prevent cognitive decline in the type 1 diabetic population.

Animals↗
PubMed2007

Aggregated myocilin induces russell bodies and causes apoptosis: implications for the pathogenesis of myocilin-caused primary open-angle glaucoma.

Primary open-angle glaucoma with elevated intraocular pressure is a leading cause of blindness worldwide. Mutations of myocilin are known to play a critical role in the manifestation of the disease. Misfolded mutant myocilin forms secretion-incompetent intracellular aggregates. The block of myocilin secretion was proposed to alter the extracellular matrix environment of the trabecular meshwork, with subsequent impediment of aqueous humor outflow leading to elevated intraocular pressure. However, the molecular pathogenesis of myocilin-caused glaucoma is poorly defined. In this study, we show that heteromeric complexes composed of wild-type and mutant myocilin were retained in the rough endoplasmic reticulum, aggregating to form inclusion bodies typical of Russell bodies. The presence of myocilin aggregates induced the unfolded protein response proteins BiP and phosphorylated endoplasmic reticulum-localized eukaryotic initiation factor-2alpha kinase (PERK) with the subsequent activation of caspases 12 and 3 and expression of C/EBP homologous protein (CHOP)/GADD153, leading to apoptosis. Our findings identify endoplasmic reticulum stress-induced apoptosis as a pathway to explain the reduction of trabecular meshwork cells in patients with myocilin-caused glaucoma. As a consequence, the phagocytotic capacity of the remaining trabecular meshwork cell population would be insufficient for effective cleaning of aqueous humor, constituting a major pathogenetic factor for the development of increased intraocular pressure in primary open-angle glaucoma.

Animals↗
PubMed2006

Co-involvement of the mitochondria and endoplasmic reticulum in cell death induced by the novel ER-targeted protein HAP.

HAP (a homologue of the ASY/Nogo-B protein), a novel human apoptosis-inducing protein, was found to be identical to RTN3. In an earlier study, we demonstrated that HAP localized exclusively to the endoplasmic reticulum (ER) and that its overexpression could induce cell apoptosis via a depletion of endoplasmic reticulum (ER) Ca(2+) stores. In this study, we show that overexpression of HAP causes the activation of caspase-12 and caspase-3. We still detected the collapse of mitochondrial membrane potential (Deltaomegam) and the release of cytochrome c in HAP-overexpressing HeLa cells. All the results indicate that both the mitochondria and the ER are involved in apoptosis caused by HAP overexpression, and suggest that HAP overexpression may initiate an ER overload response (EOR) and bring about the downstream apoptotic events.

Animals↗
PubMed2005

Preparation of anti-mouse caspase-12 mRNA hammerhead ribozyme and identification of its activity in vitro.

AIM: To prepare and identify specific anti-mouse caspase-12 hammerhead ribozymes in vitro, in order to select a more effective ribozyme against mouse caspase-12 as a potential tool to rescue cells from endoplasmic reticulum stress induced apoptosis. METHODS: Two hammerhead ribozymes directed separately against 138 and 218 site of nucleotide of mouse caspase-12 mRNA were designed by computer software, and their DNA sequences were synthesized. The synthesized ribozymes were cloned into an eukaryotic expression vector-neo(r)pBSKU6 and embedded in U6 SnRNA context for further study. Mouse caspase-12 gene segment was cloned into PGEM-T vector under the control of T7 RNA polymerase promoter (containing gene sequence from positions nt 41 to nt 894) as target. In vitro transcription both the ribozymes and target utilize T7 promoter. The target was labeled with (alpha- (32)P)UTP, while ribozymes were not labeled. After gel purification the RNAs were dissolved in RNase free water. Ribozyme and target were incubated for 90 min at 37 degrees in reaction buffer (40 mmol/L Tris-HCL, pH 7.5, 10 mmol/L Mg(2+)). Molar ratio of ribozyme vs target was 30:1. Samples were analyzed on 6% PAGE(containing 8 mol/L urea). RESULTS: Both caspase-12 and ribozyme gene sequences were successfully cloned into expression vector confirmed by sequencing. Ribozymes and caspase-12 mRNA were obtained by in vitro transcription. Cleavage experiment showed that in a physiological similar condition (37 degrees, pH 7.5), Rz138 and Rz218 both cleaved targets at predicted sites, for Rz138 the cleavage efficiency was about 100%, for Rz218 the value was 36.66%. CONCLUSION: Rz138 prepared in vitro can site specific cleave mouse caspase-12 mRNA with an excellent efficiency. It shows a potential to suppress the expression of caspase-12 in vivo, thus provided a new way to protect cells from ER stress induced apoptosis.

Animals↗
PubMed2004

Induction of endoplasmic reticulum stress by ellipticine plant alkaloids.

Anticancer drugs often show complex mechanisms of action, including effects on multiple cellular targets. Detailed understanding of these intricate effects is important for the understanding of cytotoxicity. In this study, we examined apoptosis induction by ellipticines, a class of cytotoxic plant alkaloids known to inhibit topoisomerase II. The potent ellipticine derivative 6-propanamine ellipticine (6-PA-ELL) induced rapid apoptosis in MDA-MB-231 breast cancer cells, preceded by a conformational change in Bak and cytochrome c release. Experiments using knock-out mouse embryo fibroblasts established that Bak was of particular importance for cytotoxicity. 6-PA-ELL increased the expression of the endoplasmic reticulum chaperones GRP78/BiP and GRP94, suggesting induction of endoplasmic reticulum stress. Induction of GRP78 expression was dependent on the endoplasmic reticulum stress response element (ERSE) of the GRP78 promoter. Examination of different ellipticine derivatives revealed a correlation between pro-apoptotic activity and the ability to induce GRP78 expression. Furthermore, 6-PA-ELL was found to induce splicing of the mRNA encoding the XBP1 transcription factor, characteristic of endoplasmic reticulum stress, and to induce activation of the endoplasmic reticulum-specific caspase-12 in mouse colon cancer cells. We finally demonstrate that 6-PA-ELL induces apoptotic signaling also in enucleated cells, consistent with the existence of a cytoplasmic target for this compound. Our data suggest that induction of endoplasmic reticulum stress may contribute to the cytotoxicity of ellipticines.

Animals↗