Role of proteolytic activation of protein kinase Cdelta in oxidative stress-induced apoptosis

Protein kinase Cdelta (PKCdelta), a member of the novel PKC family, is emerging as a redox-sensitive kinase in various cell types. Oxidative stress activates the PKCdelta kinase by translocation, tyrosine phosphorylation, or proteolysis. During proteolysis, caspase-3 cleaves the native PKCdelta (72-74 kDa) into 41-kDa catalytically active and 38-kDa regulatory fragments to persistently activate the kinase. The proteolytic activation of PKCdelta plays a key role in promoting apoptotic cell death in various cell types, including neuronal cells. Attenuation of PKCdelta proteolytic activation by antioxidants suggests that the cellular redox status can influence activation of the proapoptotic kinase. PKCdelta may also amplify apoptotic signaling via positive feedback activation of the caspase cascade. Thus, the dual role of PKCdelta as a mediator and amplifier of apoptosis may be important in the pathogenesis of major neurodegenerative disorders, such as Parkinson's disease, Alzheimer's disease, and Huntington disease.     

Infected cell protein No. 22 is subject to proteolytic cleavage by caspases activated by a mutant that induces apoptosis

Earlier reports have shown that the d120 mutant of herpes simplex virus 1 lacking both copies of the gene encoding the infected cells protein No. 4 (ICP4) induces apoptosis in a variety of cell lines. The programmed cell death induced by this mutant is blocked by overexpression of Bcl-2 or by transduction of infected cells with the gene encoding the viral U(S)3 protein kinase. HEp-2 cells infected with the d120 mutant express predominantly alpha proteins. Studies on these proteins revealed the accumulation of a M(r) 37,500 protein that reacted with antibody directed against the carboxyl-terminal domain of ICP22. We report that the M(r) 37,500 protein is a product of the proteolytic cleavage of ICP22 by a caspase activated by the d120 mutant. Thus the accumulation of the M(r) 37,500 protein was blocked in cells transduced with the U(S)3 protein kinase, in cells overexpressing Bcl-2, or in infected cells treated with the general caspase inhibitor zVAD-fmk. Exposure of ICP22 made in wild-type virus-infected cells to caspase 3 yielded two polypeptides, of which one could not be differentiated from the M(r) 37,500 protein with respect to electrophoretic mobility. We conclude that the cellular apoptotic response targets at least one viral protein for destruction.     

Mannheimia haemolytica leukotoxin induces apoptosis of bovine lymphoblastoid cells (BL-3) via a caspase-9-dependent mitochondrial pathway

Mannheimia haemolytica is a key pathogen in the bovine respiratory disease complex. It produces a leukotoxin (LKT) that is an important virulence factor, causing cell death in bovine leukocytes. The LKT binds to the beta(2) integrin CD11a/CD18, which usually activates signaling pathways that facilitate cell survival. In this study, we investigated mechanisms by which LKT induces death in bovine lymphoblastoid cells (BL-3). Incubation of BL-3 cells with a low concentration of LKT results in the activation of caspase-3 and caspase-9 but not caspase-8. Similarly, the proapoptotic proteins Bax and BAD were significantly elevated, while the antiapoptotic proteins Bcl-2, Bcl(XL) and Akt-1 were downregulated. Following exposure to LKT, we also observed a reduction in mitochondrial cytochrome c and corresponding elevation of cytosolic cytochrome c, suggesting translocation from the mitochondrial compartment to the cytosol. Consistent with this observation, tetramethylrhodamine ethyl ester perchlorate staining revealed that mitochondrial membrane potential was significantly reduced. These data suggest that LKT induces apoptosis of BL-3 cells via a caspase-9-dependent mitochondrial pathway. Furthermore, scanning electron micrographs of mitochondria from LKT-treated BL-3 cells revealed lesions in the outer mitochondrial membrane, which are larger than previous reports of the permeability transition pore through which cytochrome c is usually released.     

Hepatitis B Virus X Protein Sensitizes Primary Mouse Hepatocytes to Ethanol-and TNF-α-Induced Apoptosis by a Caspase-3-Dependent Mechanism

It is well-documented that alcohol drinking together with hepatitis viral infection accelerates liver injury;howeverthe underlying mechanisms remain unknown.In this paper,we demonstrated that primary hepatocytes fromtransgenic mice overexpressing hepatitis B virus X protein(HBX)were more susceptible to ethanol- and TNF-α-induced apoptotic killing.Compared to normal control mouse hepatocytes,ethanol and/or TNF-α treatment led toa significant increase in reactive oxygen species,mitochondrial permeability transition,cytochrome C release,caspase-3 activity,and poly(ADP-ribose)polymerase degradation in hepatocytes from HBX transgenic mice.Blocking caspase-3 activity antagonized ethanol-and TNF-α-induced apoptosis in primary hepatocytes from HBXtransgenic mice.Taken together,our findings suggest that HBX sensitizes primary mouse hepatocytes to ethanol-and TNF-α-induced apoptosis by a caspase-3-dependent mechanism,which may partly explain the synergisticeffects of alcohol consumption and hepatitis B virus infection on liver injury.Cellular & Molecular Immunology.2005;2(1):40-48.     

Nodosin通过Apaf-1和caspase-3诱导HepG2细胞凋亡

目的:研究传统中药提取物nodosin对人肝细胞癌Hep G2细胞凋亡的影响,并探讨其作用机制。方法:将nodosin设置为1. 25μmol/L、2. 5μmol/L、5μmol/L、10μmol/L和20μmol/L不同浓度组,作用于Hep G2细胞24 h后,用Hoechst 33258染色和电镜观察不同浓度的药物对细胞形态学的影响,用流式细胞术检测细胞凋亡率,用RT-qPCR检测凋亡蛋白酶激活因子1 (Apaf-1) mRNA的表达,用Western blot检测caspase-3及其前体和活化体的蛋白水平。结果:形态学结果显示,随着用药剂量的增加,细胞皱缩和细胞核偏移越明显,凋亡小体在5μmol/L、10μmol/L和20μmol/L剂量组明显增多。Apaf-1 mRNA的表达增加,caspase-3及其前体的表达和cleaved caspase-3的蛋白水平随着用药剂量的增加逐渐增加(P 0. 01)。结论:Nodosin能够诱导Hep G2细胞凋亡。该作用可能是通过增加Apaf-1 mRNA的表达继之激活caspase-3实现的。     

Vibrio vulnificus cytolysin induces apoptosis in HUVEC,SGC-7901 and SMMC-7721 cells via caspase-9/3-dependent pathway

Vibrio vulnificus cytolysin (VVC) is known to be a pore-forming toxin which shows cytotoxicity for mammalian cells in culture and induces apoptosis in endothelial cells. In order to determine whether VVC induces apoptosis in vascular endothelial cells and tumor cells, the cytotoxicity induced by recombinant VVC (rVVC) and its potential mechanism in HUVEC, SGC-7901 and SMMC-7721 cells were investigated. Our study demonstrated that rVVC induced the release of intracellular K⁺ from all the target cells, yet lactate dehydrogenase was not released by rVVC. It indicates that osmotic lysis might not contribute to the cytolysin-induced cytotoxicity. The study also demonstrated that rVVC induced apoptosis in HUVEC, SGC-7901 and SMMC-7721 cells in time- and dosage-dependent manners, which was associated with the activation of caspase-9 and -3, but not caspase-8. During the apoptotic process of the target cells, rVVC labeled with FITC was monitored to attach initially to the surface of the cells and entered the cytoplasma subsequently. These findings suggest that VVC may be not only a pore-forming toxin, but also a transmembrane toxin with powerful ability to induce apoptosis in human vascular endothelial cells and tumor cells.     

Virus infection induces proteolytic processing of IL-18 in human macrophages via caspase-1 and caspase-3 activation

There is increasing evidence that IL-18 is a key pro-inflammatory cytokine and an important mediator of Th1 immune response. The main source of IL-18 is macrophage-like cells. In the present study we have investigated IL-18 protein expression in primary human macrophages in response to influenza A and Sendai virus infections. Macrophages constitutively expressed proIL-18 but produced biologically active IL-18 only after virus infection. The IL-18 release was due to virus infection-induced proteolytic processing of 24-kDa proIL-18 into its mature 18-kDa form. ProIL-18 processing required active caspase-1 enzyme and the release of mature IL-18 was blocked with a caspase-1-specific inhibitor. Caspase-3 inhibitor also reduced IL-18 production in response to virus infection. Inactive proforms of caspase-1 and caspase-3 were basally expressed in macrophages, and virus infection induced the cleavage of procaspases into their mature forms. Besides increasing the expression of caspase proteins, virus infection enhanced caspase mRNA expression in macrophages. The enhancement of caspase gene expression was abrogated by anti-IFN-alpha antibody. Furthermore, IFN-alpha and IFN-gamma could induce caspase gene expression. These results imply that interferons are involved in virus-induced caspase activation that leads to proIL-18 processing and subsequent release of mature IL-18.     

Involvement of proinflammatory factors, apoptosis, caspase-3 activation and Ca2+ disturbance in microglia activation-mediated dopaminergic cell degeneration

Increasing evidences suggest that activated microglia may contribute to neurodegeneration in Parkinson's disease (PD). In the present study, primary ventral mesencephalic (VM) cultures from E14 rats and PC12 cells were utilized as in vitro models to examine the mechanism underlying microglia activation mediated dopaminergic neurodegeneration. Using lipopolysaccharide (LPS) (1-100 ng/ml) as a tool, we observed that microglia activation-mediated a selective dopaminergic neurodegeneration in VM neuron-glia cultures, which was supported by the further study showing that conditioned medium (CM) from microglia-enriched cultures treated with LPS (10-100 ng/ml) decreased PC12 cell viability. The results from antibody neutralization, NO inhibition and superoxide neutralization suggested that the dopaminergic cell death was due to the production of microglia-derived proinflammatory factors (TNF-alpha, NO and superoxide), among which reactive oxygen species (ROS) might outweigh proinflammatory cytokines. Apoptosis assay on PC12 cells and primary dopaminergic neurons showed that apoptosis was a mechanism for both microglia activation-mediated dopaminergic cell death. Through Western blot and immunocytochemistry, we found that caspase-3 activation was involved in both dopaminergic cell injuries. Finally, the results from laser scanning confocal microscope demonstrated that PC12 cell intracellular free Ca(2+) ([Ca(2+)](i)) increased early after CM treatment. [Ca(2+)](i) increase involved influx of calcium from the extracellular milieu and release from intracellular stores and participated in the CM-induced PC12 cell apoptosis. Further investigations indicated that TNF-alpha, IL-1beta, NO and superoxide contributed at different degrees to CM-induced [Ca(2+)](i) increase and apoptosis in PC12 cells. Using primary VM cultures and PC12 cells, our study shows the roles of proinflammatory factors, apoptosis, caspase-3 activation and Ca(2+) disturbance in microglia activation-mediated dopaminergic cell degeneration. Understanding the mechanism for microglia activation-mediated dopaminergic neurodegeneration may contribute to the development of new neuroprotective strategies against PD.     

Individual expression of poliovirus 2Apro and 3Cpro induces activation of caspase-3 and PARP cleavage in HeLa cells

The expression of individual viral genes enables the study of their effects on cellular functions. Our group previously generated stable HeLa cell lines that efficiently express poliovirus proteases 2A (clone 2A7d) and 3C (clone 3C7) under the control of tetracycline [Virology 266 (2000a) 352; J. Virol. 74 (2000b) 2383]. Upon induction of these proteases, the cells undergo drastic morphological alterations and eventually die. The present paper characterizes, in detail, the cellular and molecular events that lead to cell death in these lines. Several signs of apoptosis were observed in both 2A7d- and 3C7-induced cells, such as nuclear fragmentation, DNA breakdown (as determined by TUNEL), and phosphatidylserine translocation. Protease 2A induces the cleavage of poly-ADP-ribose-polymerase (PARP). This is blocked by the caspase-3 inhibitor DEVD in both 2A7d-On and 3C7-On cells suggesting that this enzyme might account for PARP cleavage in both cell lines. The results indicate that both poliovirus proteases induce apoptosis by mechanisms involving caspase activation, although the kinetics of apoptosis differs.     

Phagocytosis of crocidolite asbestos induces oxidative stress, DNA damage, and apoptosis in mesothelial cells

Phagocytosis of asbestos fibers may be a necessary step for asbestos-induced injury to mesothelial cells, but this has not been established because quantification of fiber uptake is difficult and ways to increase fiber phagocytosis without also increasing total dose were not available. We quantified phagocytosis by counting intracellular fibers after removing adherent fibers with trypsin; we selectively increased fiber phagocytosis by coating crocidolite asbestos fibers with the adhesive serum protein vitronectin (VN), which we have shown increases fiber uptake via integrins. We measured various aspects of asbestos-induced cytotoxicity: intracellular oxidation by the shift of fluorescence of cells loaded with an oxidative probe, DNA strand breakage by the alkaline unwinding ethidium bromide fluorometric assay, apoptosis by annexin V binding and by nuclear morphology, and cell-cycle progression. We found that, compared with control fibers or particles, asbestos increased intracellular oxidation, DNA strand breakage, and apoptosis. Selective increases in fiber uptake by VN-coating of the fibers further increased the oxidation, DNA strand breakage, and apoptosis, and induced a cell-cycle arrest in G2/M. Selective decreases in fiber uptake by cytochalasin or by integrin blockade with RGD peptides inhibited several of these measures of injury. We conclude that phagocytosis is important and perhaps necessary for asbestos-induced injury to mesothelial cells.     

二硫苏糖醇通过激活calpain-2/caspase-12信号通路诱导BRL-3A细胞凋亡

目的:探讨二硫苏糖醇(DTT)诱导大鼠正常肝细胞株BRL-3A发生内质网应激时细胞内葡萄糖调节蛋白78(GRP78)、钙蛋白酶2 (calpain-2)、caspase-12及caspase-3的表达变化及对细胞凋亡的影响。方法:采用2. 5 mmol/L DTT分别处理BRL-3A细胞12 h和24 h,应用real-time PCR检测细胞内GRP78、calpain-2、caspase-12及caspase-3的mRNA水平;采用细胞免疫荧光检测细胞内GRP78、calpain-2、caspase-12及caspase-3的蛋白表达;应用Western blot检测cleaved caspase-12及cleaved caspase-3的表达变化;采用流式细胞术检测细胞凋亡情况。结果:BRL-3A细胞经DTT处理12 h及24 h后,GRP78、calpain-2及caspase-12的mRNA表达较正常对照组显著升高(P 0. 01),而caspase-3的mRNA水平与正常对照组比较无显著变化;细胞免疫荧光及Western blot检测发现,DTT处理细胞12 h及24 h后,BRL-3A细胞内GRP78、calpain-2、caspase-12及caspase-3的蛋白表达均较正常对照组显著增高,同时,cleaved caspase-12及cleaved caspase-3的表达也较正常对照组明显增多(P 0. 05);流式细胞术检测发现,经DTT处理后BRL-3A细胞的凋亡率较正常对照组显著增加(P 0. 05)。结论:二硫苏糖醇诱导BRL-3A细胞凋亡可能与calpain-2/caspase-12信号通路激活有关。     

Deregulated miR-155 promotes Fas-mediated apoptosis in human intervertebral disc degeneration by targeting FADD and caspase-3

The role of apoptosis in the pathogenesis of intervertebral disc degeneration (IDD) remains enigmatic. Accumulating evidence has shown that the apoptotic machinery is regulated by miRNAs. We hypothesized that miRNAs might contribute to apoptosis in IDD. We have found that 29 miRNAs were differentially expressed and miR-155 was down-regulated in degenerative nucleus pulposus (NP). The deregulation of miR-155 was further verified using real-time PCR (0.56 fold, p < 0.05). Bioinformatics target prediction identified FADD and caspase-3 as putative targets of miR-155. Furthermore, miR-155 inhibited FADD and caspase-3 expression by directly targeting their 3'-UTRs, which was abolished by mutation of the miR-155 binding sites. In vitro up-regulation of miR-155 in human NP cells by transfection with lentiviral pre-miR-155 resulted in repression of FADD and caspase-3; whereas knockdown of miR-155 with lentiviral antigomiR-155 led to over-expression of FADD and caspase-3. Also, Fas-mediated apoptosis was increased when antagonizing miR-155 and decreased when using pre-miR-155 in human NP cells. In addition, we presented direct evidence of NP cells undergoing apoptosis in IDD tissues using transmission electron microscopy analysis. Moreover, a combination of in situ hybridization (ISH) and immunohistochemistry (IHC) revealed that miR-155 expressed in the cytoplasm of human NP cells with reverse correlation with FADD and caspase-3. In summary, this is the first study addressing the underlying mechanisms of IDD in terms of apoptosis and miRNAs. Furthermore, caspase-3 is identified as a novel target of miR-155. Our results suggest that deregulated miR-155 promotes Fas-mediated apoptosis in human IDD by targeting FADD and caspase-3, implicating an aetiological and therapeutic role of miR-155 in IDD.     

Induction of apoptosis signal-regulating kinase 1 and oxidative stress mediate age-dependent vulnerability to 3-nitropropionic acid in the mouse striatum

The mitochondrial toxin, 3-nitropropionic acid (3-NP), produces age-dependent oxidative stress and selective striatal damage, which may simulate Huntington's disease starting in middle age. Recent reports showed that apoptosis signal-regulating kinase 1 (Ask1) activated by oxidative stress triggers a cell death signaling pathway. 3-NP was injected to the striatum in C57BL/6J mice. We have confirmed that striatal lesion volume and DNA fragmentation were age-dependent after 3-NP treatment. In the non-injured striatum of the middle-aged group, the protein levels of Ask1 and its active form, phosphorylated Ask1 (pAsk1), were significantly higher than in the young group. Ask1 increased more in the 3-NP injured striatum of the middle-aged group than in the non-injured striatum, and subsequently the activity of pAsk1 was significantly higher than in the young group. However, middle-aged SOD1Tg mice showed significant reductions of Ask1 and pAsk1 in the injured and the non-injured striatum compared to the middle-aged group. In particular, apoptosis signal transduction and cell death were significantly inhibited by the reduction of Ask1 expression using siRNA. Present results suggest that age-related upregulation of Ask1 and oxidative stress may mediate age-dependent striatal vulnerability to 3-NP.     

Strain-dependent induction of enterocyte apoptosis by Giardia lamblia disrupts epithelial barrier function in a caspase-3-dependent manner

We recently demonstrated that Giardia lamblia rearranges cytoskeletal proteins and reduces transepithelial electrical resistance. The effect of G. lamblia on enterocyte apoptosis is unknown, and a possible link between microbially induced enterocyte apoptosis and altered epithelial permeability has yet to be established. The aim of this study was to assess whether G. lamblia induces enterocyte apoptosis in duodenal epithelial monolayers and whether this effect increases epithelial permeability. Monolayers of nontransformed human duodenal epithelial cells were incubated with sonicated or live G. lamblia trophozoites (NF, S2, WB, or PB strains) for 8, 24, and 48 h. Cell cultures were assessed for apoptosis by Hoechst fluorescence staining, enzyme-linked immunosorbent assay for apoptotic nucleosomes, and electron microscopy. In separate experiments, monolayers were pretreated with or without 120 microM caspase-3 inhibitor (Z-DEVD-FMK) for 1 h and were assessed for production of apoptotic nucleosomes, tight junctional integrity (with fluorescent ZO-1 staining followed by confocal laser microscopy), and transepithelial permeability for fluorescein isothiocyanate-dextran. G. lamblia strains NF and S2, but not strains WB or PB, induced enterocyte apoptosis within the monolayers, and this effect was inhibited by Z-DEVD-FMK pretreatment. Using the G. lamblia NF isolate, additional experiments investigated the possible link between enterocyte apoptosis and altered epithelial permeability. G. lamblia NF disrupted tight junctional ZO-1 and increased epithelial permeability, but these effects were also prevented by pretreatment with the caspase-3 inhibitor. These findings indicate that strain-dependent induction of enterocyte apoptosis may contribute to the pathogenesis of giardiasis. This effect is responsible for a loss of epithelial barrier function by disrupting tight junctional ZO-1 and increasing permeability in a caspase-3-dependent manner.     

Induction of apoptosis by Hypericin through activation of caspase-3 in human carcinoma cells

Potent photosensitizer Hypericin (HY), is a lipid soluble perylquinone derivative of the genus Hypericum and has a strong photodynamic effect on tumors and viruses. However, the mechanisms of tumor cell death induced by this compound is still unclear. Furthermore, there are no reports on mechanisms in cell apoptosis induced by perylquinones in human nasopharyngeal carcinoma (NPC) and other mucosal cells. We studied the photodynamic effects of HY compound in poorly differentiated (CNE2) and moderately differentiated (TW0-1) human NPC cells as well as human mucosal colon (CCL-220.1) and bladder (SD) cells. Using these cell lines we investigated few hall marks of apoptotic commitments in a drug and light dose dependent manner. Tumor cells photoactivated with HY showed cell size shrinkage and an increase in the sub-diploid DNA content. A loss of membrane phospholipid asymmetry associated with apoptosis was induced in all tumor cell lines as evidenced by the externalization of phosphatidylserine. Under apoptotic conditions, Western blot analysis of poly (ADP-ribose) polymerase, a caspase substrate, showed the classical cleavage pattern (116-85 kDa) associated with apoptosis in PDT-treated cell lysates. In addition, 85 kDa cleaved product was blocked by using tetrapeptide caspase inhibitors such as DEVD-CHO or z-VAD-fmk. These results demonstrate that tumor cell death induced by photoactivated HY is mediated by caspase proteases. This study also identifies that CNE2, CCL-220.1 (colon) and SD (bladder) cell lines are more sensitive than TW0-1 cell line to PDT using perylquinone HY.     

Molecular mechanism of satratoxin-induced apoptosis in HL-60 cells: activation of caspase-8 and caspase-9 is involved in activation of caspase-3

Satratoxins have been recognized as potential immunomodulatory agents in outbreaks of building-related illness. Here we report that satratoxin G-treated human leukemia HL-60 cells underwent apoptosis through the action of caspase-3 which was activated by both caspase-8 and caspase-9. Western blot analysis of caspase-3 in the satratoxin G-treated cells apparently indicated the appearance of a catalytically active fragment of 17 kDa. Increased caspase-3 activity was also detected by using a fluorogenic substrate, DEVD-AMC. Next, exposure to satratoxin G led to cleavage of PARP from its native 116 kDa form to a 85 kDa product. Moreover, DFF-45/ICAD were cleaved into a 12.5 kDa fragment via satratoxin G treatment. Enzymic assay on IETD-AMC revealed that caspase-8 is strongly activated by exposure to satratoxin G while T-2 toxin (T-2) could not activate caspase-8 at an early stage of apoptosis. Furthermore, satratoxin G caused a release of cytochrome c from mitochondria into the cytosol and increased the activity of caspase-9 against LEHD-AMC. These findings indicate that satratoxin G-induced apoptosis involves activation of caspase-3 and DFF-40/CAD through both activation of caspase-8 and cytosolic accumulation of cytochrome c along with activation of caspase-9.     

Ca2(+)-dependent proteolytic modification of the cAMP-dependent protein kinase in Drosophila wild-type and dunce memory mutants

Two cAMP-dependent protein kinases with different activation constants were separated from Drosophila melanogaster head extracts. Both are only found in nervous tissue. The first cAMP-dependent kinase, with Mr = 190,000, has been already characterized as tetrameric Drosophila type II cAMP-dependent protein kinase R2C2. The second purified cAMP-dependent protein kinase, with Mr = 80,000, is dimeric in structure RPC, and the cAMP-concentration required for half maximal activation is 4 fold lower than for the type II kinase. The generation of RP can be stimulated in vitro by addition of exogenous calcium and is due to an endogenous Ca2(+)-dependent protease that selectively degrades the regulatory subunit. Extraction in the presence of various protease inhibitors does not affect the amounts of RP, suggesting that the observed quantitative change in RP occurs in vivo. The amounts of RP in the nervous tissue of the memory mutants dunce1 and dunce2, which have increased cAMP levels, are different from the amount of RP in wild-type flies. Also treatments of wild-type flies with drugs affecting cAMP-metabolism and acetylcholine levels led to amounts of RP different from untreated flies.