Calcium-independent phospholipase A2 (iPLA2 beta)-mediated ceramide generation plays a key role in the cross-talk between the endoplasmic reticulum (ER) and mitochondria during ER stress-induced insulin-secreting cell apoptosis

Endoplasmic reticulum (ER) stress induces INS-1 cell apoptosis by a pathway involving Ca(2+)-independent phospholipase A(2) (iPLA(2)beta)-mediated ceramide generation, but the mechanism by which iPLA(2)beta and ceramides contribute to apoptosis is not well understood. We report here that both caspase-12 and caspase-3 are activated in INS-1 cells following induction of ER stress with thapsigargin, but only caspase-3 cleavage is amplified in iPLA(2)beta overexpressing INS-1 cells (OE), relative to empty vector-transfected cells, and is suppressed by iPLA(2)beta inhibition. ER stress also led to the release of cytochrome c and Smac and, unexpectedly, their accumulation in the cytosol is amplified in OE cells. These findings raise the likelihood that iPLA(2)beta participates in ER stress-induced apoptosis by activating the intrinsic apoptotic pathway. Consistent with this possibility, we find that ER stress promotes iPLA(2)beta accumulation in the mitochondria, opening of mitochondrial permeability transition pore, and loss in mitochondrial membrane potential (Delta Psi) in INS-1 cells and that these changes are amplified in OE cells. ER stress also led to greater ceramide generation in ER and mitochondria fractions of OE cells. Exposure to ceramide alone induces loss in Delta Psi and apoptosis and these are suppressed by forskolin. ER stress-induced mitochondrial dysfunction and apoptosis are also inhibited by forskolin, as well as by inactivation of iPLA(2)beta or NSMase, suggesting that iPLA(2)beta-mediated generation of ceramides via sphingomyelin hydrolysis during ER stress affect the mitochondria. In support, inhibition of iPLA(2)beta or NSMase prevents cytochrome c release. Collectively, our findings indicate that the iPLA(2)beta-ceramide axis plays a critical role in activating the mitochondrial apoptotic pathway in insulin-secreting cells during ER stress.     

Cytosolic and mitochondrial ROS in staurosporine-induced retinal cell apoptosis

In this study, we investigated the involvement of reactive oxygen species (ROS) and calcium in staurosporine (STS)-induced apoptosis in cultured retinal neurons, under conditions of maintained membrane integrity. The antioxidants idebenone (IDB), glutathione-ethylester (GSH/EE), trolox, and Mn(III)tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) significantly reduced STS-induced caspase-3-like activity and intracellular ROS generation. Endogenous sources of ROS production were investigated by testing the effect of the following inhibitors: 7-nitroindazole (7-NI), a specific inhibitor of the neuronal isoform of nitric oxide synthase (nNOS); arachidonyl trifluoromethyl ketone (AACOCF(3)), a phospholipase A(2) (PLA(2)) inhibitor; allopurinol, a xanthine oxidase inhibitor; and the mitochondrial inhibitors rotenone and oligomycin. All these compounds decreased caspase-3-like activity and ROS generation, showing that both mitochondrial and cytosolic sources of ROS are implicated in this mechanism. STS induced a significant increase in intracellular calcium concentration ([Ca(2+)](i)), which was partially prevented in the presence of IDB and GSH/EE, indicating its dependence on ROS generation. These two antioxidants and the inhibitors allopurinol and 7-NI also reduced the number of TdT-mediated dUTP nick-end labeling-positive cells. Thus, endogenous ROS generation and the rise in intracellular calcium are important inter-players in STS-triggered apoptosis. Furthermore, the antioxidants may help to prolong retinal cell survival upon apoptotic cell death.     

Are mitochondrial reactive oxygen species required for autophagy?

Reactive oxygen species (ROS) are said to participate in the autophagy signaling. Supporting evidence is obscured by interference of autophagy and apoptosis, whereby the latter heavily relies on ROS signaling. To dissect autophagy from apoptosis we knocked down expression of cytochrome c, the key component of mitochondria-dependent apoptosis, in HeLa cells using shRNA. In cytochrome c deficient HeLa1.2 cells, electron transport was compromised due to the lack of electron shuttle between mitochondrial respiratory complexes III and IV. A rapid and robust LC3-I/II conversion and mitochondria degradation were observed in HeLa1.2 cells treated with staurosporine (STS). Neither generation of superoxide nor accumulation of H₂O₂ was detected in STS-treated HeLa1.2 cells. A membrane permeable antioxidant, PEG-SOD, plus catalase exerted no effect on STS-induced LC3-I/II conversion and mitochondria degradation. Further, STS caused autophagy in mitochondria DNA-deficient ρ° HeLa1.2 cells in which both electron transport and ROS generation were completely disrupted. Counter to the widespread view, we conclude that mitochondrial ROS are not required for the induction of autophagy.     

The group VIA calcium-independent phospholipase A2 participates in ER stress-induced INS-1 insulinoma cell apoptosis by promoting ceramide generation via hydrolysis of sphingomyelins by neutral sphingomyelinase

Beta-cell mass is regulated by a balance between beta-cell growth and beta-cell death, due to apoptosis. We previously reported that apoptosis of INS-1 insulinoma cells due to thapsigargin-induced ER stress was suppressed by inhibition of the group VIA Ca2+-independent phospholipase A2 (iPLA2beta), associated with an increased level of ceramide generation, and that the effects of ER stress were amplified in INS-1 cells in which iPLA2beta was overexpressed (OE INS-1 cells). These findings suggested that iPLA2beta and ceramides participate in ER stress-induced INS-1 cell apoptosis. Here, we address this possibility and also the source of the ceramides by examining the effects of ER stress in empty vector (V)-transfected and iPLA2beta-OE INS-1 cells using apoptosis assays and immunoblotting, quantitative PCR, and mass spectrometry analyses. ER stress induced expression of ER stress factors GRP78 and CHOP, cleavage of apoptotic factor PARP, and apoptosis in V and OE INS-1 cells. Accumulation of ceramide during ER stress was not associated with changes in mRNA levels of serine palmitoyltransferase (SPT), the rate-limiting enzyme in de novo synthesis of ceramides, but both message and protein levels of neutral sphingomyelinase (NSMase), which hydrolyzes sphingomyelins to generate ceramides, were temporally increased in the INS-1 cells. The increases in the level of NSMase expression in the ER-stressed INS-1 cells were associated with corresponding temporal elevations in ER-associated iPLA2beta protein and catalytic activity. Pretreatment with BEL inactivated iPLA2beta and prevented induction of NSMase message and protein in ER-stressed INS-1 cells. Relative to that in V INS-1 cells, the effects of ER stress were accelerated and/or amplified in the OE INS-1 cells. However, inhibition of iPLA2beta or NSMase (chemically or with siRNA) suppressed induction of NSMase message, ceramide generation, sphingomyelin hydrolysis, and apoptosis in both V and OE INS-1 cells during ER stress. In contrast, inhibition of SPT did not suppress ceramide generation or apoptosis in either V or OE INS-1 cells. These findings indicate that iPLA2beta activation participates in ER stress-induced INS-1 cell apoptosis by promoting ceramide generation via NSMase-catalyzed hydrolysis of sphingomyelins, raising the possibility that this pathway contributes to beta-cell apoptosis due to ER stress.     

Inhibition of apoptosis in pulmonary endothelial cells by altered pH,mitochondrial function,and ATP supply

We investigated the effect of altered extracellular pH, mitochondrial function, and ATP content on development of apoptosis in human pulmonary artery endothelial cells after treatment with staurosporine (STS). STS produced a concentration- and time-dependent increase in caspase-3 activity in pH 7.4 medium that reached a peak at 6 h. The increase in caspase activity was associated with significant DNA fragmentation. Fluorescent imaging of treated monolayers in pH 7.4 medium with Hoechst-33342-propidium iodide demonstrated a large percentage of apoptotic cells ( approximately 40%) with no evidence of necrosis. Caspase activity, DNA fragmentation, and percentage of apoptotic cells were reduced after STS treatment in acidic media (pH 7.0 and 6.6). The Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM inhibited STS-induced apoptosis, whereas the rise in intracellular Ca2+concentration in STS-treated cells in pH 7.4 medium was reduced in pH 7.0 medium. These results suggest that one mechanism for inhibitory effects of acidosis may be a pH-induced alteration in Ca2+ signaling. Treatment with STS in the presence of oligomycin (10 microM), an inhibitor of the mitochondrial F(0)F(1)-ATPase, in glucose-free media abolished caspase activation and DNA fragmentation in association with severe ATP depletion ( approximately 2% of control cells). Imaging demonstrated a change in the mode of cell death from apoptosis to necrosis under these conditions. This change was linked to the level of ATP depletion, because STS treatment in the absence of glucose or the presence of oligomycin in media with glucose still leads to apoptosis in the presence of only moderate ATP depletion. These results demonstrate that pH, mitochondrial function, and ATP supply are important variables that regulate STS-induced apoptosis in human pulmonary artery endothelial cells.     

Axin expression reduces staurosporine-induced mitochondria-mediated cell death in HeLa cells

Cytoplasmic axin expression frequently produces punctuate structures in cells, but the nature of axin puncta has not been fully elucidated. In an effort to analyze cytoplasmic axin puncta, we established HeLa cells expressing axin in a doxycycline-inducible manner (HeLa-Axin). We observed that axin accumulated in an aggregate-like pattern in perinuclear areas and appeared to be associated with mitochondria, Golgi apparatus, and endoplasmic reticulum (ER), but not lysosomes. Further biochemical analysis suggested that some part of the cytoplasmic axin pool was associated with mitochondria. In addition, mitochondrial proteins [i.e., cytochrome oxidase IV (CoxIV) and cytochrome c] were slightly higher in HeLa-Axin cells than in HeLa-EV cells, suggesting altered mitochondrial degradation. HeLa-Axin cells were then treated with staurosporine (STS) to determine if the mitochondria-induced apoptosis pathway was altered. Compared to STS-treated control cells (HeLa-EV), HeLa-Axin cells had less STS-induced cytotoxicity and reduced caspase-3 activation and PARP cleavage. Given that mitochondria outer membrane potential was unchanged, HeLa-Axin cells might be relatively resistant to STS-mediated mitochondrial damage. Mitochondria associated with axin aggregates were resistant to detergent-mediated permeabilization. These results suggest that axin forms aggregate-like structures in association with mitochondria, which render mitochondria resistant to STS-induced membrane damage and cytotoxicity.     

Sphingosine 1-phosphate promotes antigen processing and presentation to CD4+ T cells in Mycobacterium tuberculosis-infected monocytes

It was previously shown that cells die with increased cytosolic ATP after stimulation with apoptotic inducers including staurosporine (STS). To identify the source of apoptotic ATP elevation, we monitored, in real time, the cytosolic ATP level in luciferase-expressing HeLa cells. A mitochondrial uncoupler or a respiration chain inhibitor was found to decrease cytosolic ATP by about 50%. However, even when mitochondrial ATP synthesis was suppressed, STS induced a profound elevation of intracellular ATP. In contrast, the STS-induced ATP increase was prevented by any of three inhibitors of the glycolytic pathway: 2-deoxyglucose, iodoacetamide, and NaF. The STS effect strongly depended on intracellular calcium and was mimicked by a calcium ionophore. We conclude that Ca(2+)-dependent activation of anaerobic glycolysis, but not aerobic mitochondrial oxidative phosphorylation, is responsible for the STS-induced elevation of ATP in apoptotic HeLa cells.     

Akt is activated in response to an apoptotic signal

Akt is a serine-threonine kinase known to exert antiapoptotic effects through several downstream targets. Akt is cleaved during mitochondrial-mediated apoptosis in a caspase-dependent manner. The reason for this is not clear, however, because Akt has not been demonstrated to be activated in response to mitochondrial apoptotic stimuli. Accordingly, we explored whether the well described mitochondrial apoptotic stimuli staurosporine (STS) and etoposide activate Akt and whether such activation impacts apoptosis. Both STS and etoposide activated Akt in NIH 3T3 cells, maximally at 8 and 2 h, respectively, preceding the onset of apoptosis and poly(ADP-ribose) polymerase cleavage. The overexpression of Akt delayed STS-induced apoptosis with an even more pronounced delay observed with overexpression of constitutively active Akt. Akt activation by proapoptotic stimuli lay upstream of mitochondria, because neither caspase inhibitors nor overexpression of Bcl-2 or Bcl-x(L) could prevent it. Activation depended on phosphatidylinositol 3-kinase activity, however. Conversely, inhibition of phosphatidylinositol 3-kinase with wortmannin sensitized cells to apoptosis initiated by STS. These data demonstrate that mitochondrial apoptotic stimuli also activate Akt and such activation modulates apoptosis in this setting.     

Antiproliferative effects of goniothalamin on Ca9-22 oral cancer cells through apoptosis, DNA damage and ROS induction

Goniothalamin (GTN), a plant bioactive styryl-lactone, is a natural product with potent anti-tumorigenesis effects for several types of cancer. Nonetheless, the anticancer effect of GTN has not been examined in oral cancer. The present study was designed to evaluate its potential anticancer effects in an oral squamous cell carcinoma (OSCC) model and to determine the possible mechanisms with respect to apoptosis, DNA damage, reactive oxygen species (ROS) induction, and mitochondrial membrane potential. Our data demonstrated that cell proliferation was significantly inhibited by GTN in Ca9-22 OSCC cancer cells in concentration- and time-dependent manners (p<0.05). For cell cycle and apoptotic effects of GTN-treated Ca9-22 cancer cells, the sub-G1 population and annexin V-intensity significantly increased in a concentration-dependent manner (p<0.001). For the analysis of DNA double strand breaks, γH2AX intensity significantly increased in GTN-treated Ca9-22 cancer cells in concentration-response relationship (p<0.05). Moreover, GTN significantly induced intracellular ROS levels in Ca9-22 cancer cells in a concentration- and time-dependent manner (p<0.05). For membrane depolarization of mitochondria, the DiOC(2)(3) (3,3'-diethyloxacarbocyanine iodide) intensity of GTN-treated Ca9-22 cancer cells was significantly decreased in concentration- and time-dependent relationships (p<0.001). Taken together, these results suggest that the anticancer effect of GTN against oral cancer cells is valid and GTN-induced growth inhibition and apoptosis influence the downstream cascade including ROS induction, DNA damage, and mitochondria membrane depolarization. Therefore, GTN has potential as a chemotherapeutic agent against oral cancer.     

Differential effects of bcl-2 on cell death triggered under ATP-depleting conditions

The intracellular ATP concentration decides on the onset of either apoptosis or necrosis in Jurkat cells exposed to death stimuli. Bcl-2 can block apoptotic demise, which occurs preferably under conditions of high cellular ATP levels. Here, we investigated the effects of Bcl-2 on the necrotic type of cell demise that prevails under conditions of energy loss. ATP levels were modulated by using mitochondrial inhibitors, such as rotenone or S-nitrosoglutathione, in medium either lacking glucose or supplemented with glucose to stimulate glycolytic ATP generation. Under conditions of ATP depletion, staurosporine (STS) induced >90% necrosis in vector control-transfected cells, whereas bcl-2-transfected cells were protected. Thus, the antiapoptotic protein Bcl-2 can reduce the overall amount of cell death in ATP-depleted cells regardless whether it occurs by apoptosis or necrosis. Cytochrome c release, normally preceding STS-induced necrosis, was also inhibited by Bcl-2. However, Bcl-2 did not prevent an initial STS-induced drop of the mitochondrial membrane potential (DeltaPsi(m)). Therefore, the mechanisms whereby Bcl-2 prevents cell death and favors retention of cytochrome c in the mitochondria require neither the maintenance of mitochondrial DeltaPsi nor the maintenance of normal ATP levels.     

Excessive reactive oxygen species induces apoptosis in fibroblasts: role of mitochondrially accumulated hyaluronic acid binding protein 1 (HABP1/p32/gC1qR)

Constitutively expressed HABP1 in normal murine fibroblast cell line induces growth perturbation, morphological abnormalities along with initiation of apoptosis. Here, we demonstrate that though HABP1 accumulation started in mitochondria from 48 hr of growth, induction of apoptosis with the release of cytochrome c and apoptosome complex formation occurred only after 60 hr. This mitochondrial dysfunction was due to gradual increase in ROS generation in HABP1 overexpressing cells. Along with ROS generation, increased Ca 2+ influx in mitochondria leading to drop in membrane potential was evident. Interestingly, upon expression of HABP1, the respiratory chain complex I was shown to be significantly inhibited. Electronmicrograph confirmed defective mitochondrial ultrastructure. The reduction in oxidant generation and drop in apoptotic cell population accomplished by disruption of HABP1 expression, corroborating the fact that excess ROS generation in HABP1 overexpressing cells leading to apoptosis was due to mitochondrial HABP1 accumulation.     

Involvement of mitochondrial dysfunction in human islet amyloid polypeptide-induced apoptosis in INS-1E pancreatic beta cells: An effect attenuated by phycocyanin

Misfolded human islet amyloid polypeptide (hIAPP) in pancreatic islets is associated with the loss of insulin-secreting beta cells in type 2 diabetes. Insulin secretion impairment and cell apoptosis can be due to mitochondrial dysfunction in pancreatic beta cells. Currently, there is little information about the effect of hIAPP on mitochondrial function. In this study, we used INS-1E rat insulinoma beta cells as a model to investigate the role of mitochondria in hIAPP-induced apoptosis and the protective effects of phycocyanin (PC). We demonstrated that hIAPP induced apoptosis in INS-1E cells was associated with the disruption of mitochondrial function, as evidenced by ATP depletion, mitochondrial mass reduction, mitochondrial fragmentation and loss of mitochondrial membrane potential (ΔΨ(m)). Further molecular analysis showed that hIAPP induced changes in the expression of Bcl-2 family members, release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria into cytosol, activation of caspases and cleavage of poly (ADP-ribose) polymerase. Interestingly, the hIAPP-induced mitochondrial dysfunction in INS-1E cells was effectively restored by co-treatment of PC. Moreover, there was crosstalk between the extrinsic and intrinsic apoptotic pathways as demonstrated by cleavage of Bid by caspase-8 in the apoptotic process triggered by hIAPP. Taken together, we demonstrated for the first time the involvement of mitochondrial dysfunction in hIAPP-induced INS-1E cell apoptosis. Attenuation of mitochondrial dysfunction provides a mechanism for the protective effects of PC.     

Lysosomal enzymes promote mitochondrial oxidant production, cytochrome c release and apoptosis.

Exposure of mammalian cells to oxidant stress causes early (iron catalysed) lysosomal rupture followed by apoptosis or necrosis. Enhanced intracellular production of reactive oxygen species (ROS), presumably of mitochondrial origin, is also observed when cells are exposed to nonoxidant pro-apoptotic agonists of cell death. We hypothesized that ROS generation in this latter case might promote the apoptotic cascade and could arise from effects of released lysosomal materials on mitochondria. Indeed, in intact cells (J774 macrophages, HeLa cells and AG1518 fibroblasts) the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH) causes lysosomal rupture, enhanced intracellular ROS production, and apoptosis. Furthermore, in mixtures of rat liver lysosomes and mitochondria, selective rupture of lysosomes by MSDH promotes mitochondrial ROS production and cytochrome c release, whereas MSDH has no direct effect on ROS generation by purifed mitochondria. Intracellular lysosomal rupture is associated with the release of (among other constituents) cathepsins and activation of phospholipase A2 (PLA2). We find that addition of purified cathepsins B or D, or of PLA2, causes substantial increases in ROS generation by purified mitochondria. Furthermore, PLA2 - but not cathepsins B or D - causes rupture of semipurified lysosomes, suggesting an amplification mechanism. Thus, initiation of the apoptotic cascade by nonoxidant agonists may involve early release of lysosomal constituents (such as cathepsins B and D) and activation of PLA2, leading to enhanced mitochondrial oxidant production, further lysosomal rupture and, finally, mitochondrial cytochrome c release. Nonoxidant agonists of apoptosis may, thus, act through oxidant mechanisms.     

Mechanism of A23187-induced apoptosis in HL-60 cells: dependency on mitochondrial permeability transition but not on NADPH oxidase

Calcium ions (Ca(2+)) are involved in a number of physiological cellular functions including apoptosis. An elevation in intracellular levels of Ca(2+) in A23187-treated HL-60 cells was associated with the generation of both intracellular and extracellular reactive oxygen species (ROS) and induction of apoptotic cell death. A23187-induced apoptosis was prevented by cyclosporin A, a potent inhibitor of mitochondrial permeability transition (MPT). The generation of extracellular ROS was suppressed by the NADPH oxidase inhibitor diphenylene iodonium, and by superoxide dismutase, but these agents had no effect on A23187-induced apoptosis. In contrast, the blocking of intracellular ROS by a cell-permeant antioxidant diminished completely the induction of MPT and apoptosis. In isolated mitochondria, the addition of Ca(2+) induced a typical MPT concomitant with the generation of ROS, which leads to augmentation of intracellular ROS levels. These results indicate that intracellular not extracellular ROS generated by A23187 is associated with the opening of MPT pores that leads to apoptotic cell death.     

Stimulation of membrane permeability transition by alpha-lipoic acid and its biochemical characteristics

Mitochondria play an important role in apoptosis by generating reactive oxygen species (ROS) and inducing membrane permeability transition (MPT). Recent studies on alpha-lipoic acid (LA) and its reduced form, dihydrolipoic acid, suggest that these agents (LAs) inhibit apoptosis of cells by means of their antioxidant activity. On the other hand, LAs also stimulate Ca2+-dependent mitochondrial MPT and induce apoptosis of certain cells. Thus, the role of LAs in apoptotic cell death remains obscure. We investigated the mechanism of LA-induced MPT of mitochondria. Biochemical analysis revealed, in the presence of Ca2+, inorganic phosphate and succinate, LA induced uncoupling of oxidative phosphorylation, stimulated oxidation of pyridine nucleotides and enhanced Ca2+-induced MPT, as characterized by decrease in Ca2+ loading, ROS generation, oxidation of thiol groups of adenine nucleotide translocator, membrane depolarization, swelling, and cytochrome c release in an incubation time and concentration dependent manner. LA also stimulated hydroxyl radical-induced MPT in a alpha-tocopherol-inhibitable manner. Cyclosporine A, a potent inhibitor of mitochondrial MPT, inhibited all these events induced by LA. These results indicate that, under certain conditions, LA stimulates Ca2+-induced MPT through the decrease in loading capacity of Ca2+ and that MPT is involved in LA-induced apoptotic cell death. Since fairly high doses of LA have been used as a dietary supplement, the possible occurrence of such side effects, including mitochondrial dysfunction and induction of apoptosis in normal tissues, should be studied.     

Studies of insulin secretory responses and of arachidonic acid incorporation into phospholipids of stably transfected insulinoma cells that overexpress group VIA phospholipase A2 (iPLA2beta ) indicate a signaling rather than a housekeeping role for iPLA2beta

A cytosolic 84-kDa group VIA phospholipase A(2) (iPLA(2)beta) that does not require Ca(2+) for catalysis has been cloned from several sources, including rat and human pancreatic islet beta-cells and murine P388D1 cells. Many potential iPLA(2)beta functions have been proposed, including a signaling role in beta-cell insulin secretion and a role in generating lysophosphatidylcholine acceptors for arachidonic acid incorporation into P388D1 cell phosphatidylcholine (PC). Proposals for iPLA(2)beta function rest in part on effects of inhibiting iPLA(2)beta activity with a bromoenol lactone (BEL) suicide substrate, but BEL also inhibits phosphatidate phosphohydrolase-1 and a group VIB phospholipase A(2). Manipulation of iPLA(2)beta expression by molecular biologic means is an alternative approach to study iPLA(2)beta functions, and we have used a retroviral construct containing iPLA(2)beta cDNA to prepare two INS-1 insulinoma cell clonal lines that stably overexpress iPLA(2)beta. Compared with parental INS-1 cells or cells transfected with empty vector, both iPLA(2)beta-overexpressing lines exhibit amplified insulin secretory responses to glucose and cAMP-elevating agents, and BEL substantially attenuates stimulated secretion. Electrospray ionization mass spectrometric analyses of arachidonic acid incorporation into INS-1 cell PC indicate that neither overexpression nor inhibition of iPLA(2)beta affects the rate or extent of this process in INS-1 cells. Immunocytofluorescence studies with antibodies directed against iPLA(2)beta indicate that cAMP-elevating agents increase perinuclear fluorescence in INS-1 cells, suggesting that iPLA(2)beta associates with nuclei. These studies are more consistent with a signaling than with a housekeeping role for iPLA(2)beta in insulin-secreting beta-cells.     

PTEN: A crucial mediator of mitochondria-dependent apoptosis

The highly frequent mutation of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in various cancers has attracted much attention to study its role in tumorigenesis. As an important tumor suppressor, the pro-apoptotic function of PTEN has been linked to its capacity antagonizing the PI3K/Akt signaling pathway. However, less data are available concerning its role in neurodegeneration in which apoptotic processes are also involved. In the present study, we attempted to study the role and the underlying mechanism of PTEN in neuronal apoptosis. Using primary rat hippocampal cultures, staurosporine (STS, 100 nM) induced a time-dependent apoptosis, accompanied by a marked production of reactive oxygen species (ROS), release of cytochrome c and activation of caspase 9 and 3. However, the expression of PTEN, and the levels of phospho-PTEN and phospho-Akt were not changed at all time points tested (0.5–24 h) after STS stimulation, suggesting that the protein level as well as the phosphorylation status of PTEN were not related to the procession of apoptosis. Interestingly, immunostaining revealed a punctate intracellular distribution of PTEN from 2 to 8 h after adding STS. Double labeling and Western blotting of mitochondrial fraction demonstrated a mitochondrial location and accumulation of PTEN, respectively, after challenging with STS. Furthermore, we provide evidence for the first time that PTEN was associated with Bax in the absence and the presence of STS. Of note, the STS-induced marked increase in the cellular ROS level, release of cytochrome c and activation of caspase 3 were inhibited in cultured hippocampal cells when PTEN was knocked down by a specific antisense. Moreover, knockdown of PTEN significantly protected hippocampal cells from apoptotic damage. These findings demonstrated that PTEN is a crucial mediator of mitochondria-dependent apoptosis, and thus could become a molecular target for interfering with neurodegenerative diseases.     

Drp-1-dependent division of the mitochondrial network blocks intraorganellar Ca2+ waves and protects against Ca2+-mediated apoptosis

By transiently or stably overexpressing the mitochondrial fission factor dynamin-related protein-1 (Drp-1), we evaluated the role of mitochondrial division in organelle Ca2+ homeostasis and apoptotic signaling. Quantitative 3D digital microscopy revealed a split mitochondrial network in Drp-1-overexpressing cells without changes in cell viability. High-speed mitochondrial [Ca2+] ([Ca2+]m) imaging revealed propagating intramitochondrial Ca2+ waves in intact cells, which were blocked in the Drp-1-fragmented network, leaving a fraction of individual mitochondria without substantial [Ca2+]m elevation. Consequently, in Drp-1-expressing cells the apoptotic efficacy of ceramide, which causes a Ca2+-dependent perturbation of mitochondrial structure and function, was drastically reduced. Conversely, the sensitivity to staurosporine-induced apoptosis, previously shown to be directly triggered by Drp-1-dependent recruitment of proapoptotic proteins to mitochondria, was enhanced. These results demonstrate that the regulated process of mitochondrial fusion and fission controls the spatiotemporal properties of mitochondrial Ca2+ responses and, thus, physiological and pathological consequences of cellular Ca2+ signals.     

Activation of mitochondrial calcium-independent phospholipase A2γ (iPLA2γ) by divalent cations mediating arachidonate release and production of downstream eicosanoids

Calcium-independent phospholipase A(2)γ (iPLA(2)γ) (PNPLA8) is the predominant phospholipase activity in mammalian mitochondria. However, the chemical mechanisms that regulate its activity are unknown. Here, we utilize iPLA(2)γ gain of function and loss of function genetic models to demonstrate the robust activation of iPLA(2)γ in murine myocardial mitochondria by Ca(2+) or Mg(2+) ions. Calcium ion stimulated the production of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) from 1-palmitoyl-2-[(14)C]arachidonoyl-sn-glycero-3-phosphocholine during incubations with wild-type heart mitochondrial homogenates. Furthermore, incubation of mitochondrial homogenates from transgenic myocardium expressing iPLA(2)γ resulted in 13- and 25-fold increases in the initial rate of radiolabeled 2-AA-LPC and arachidonic acid (AA) production, respectively, in the presence of calcium ion. Mass spectrometric analysis of the products of calcium-activated hydrolysis of endogenous mitochondrial phospholipids in transgenic iPLA(2)γ mitochondria revealed the robust production of AA, 2-AA-LPC, and 2-docosahexaenoyl-LPC that was over 10-fold greater than wild-type mitochondria. The mechanism-based inhibitor (R)-(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL) (iPLA(2)γ selective), but not its enantiomer, (S)-BEL (iPLA(2)β selective) or pyrrolidine (cytosolic PLA(2)α selective), markedly attenuated Ca(2+)-dependent fatty acid release and polyunsaturated LPC production. Moreover, Ca(2+)-induced iPLA(2)γ activation was accompanied by the production of downstream eicosanoid metabolites that were nearly completely ablated by (R)-BEL or by genetic ablation of iPLA(2)γ. Intriguingly, Ca(2+)-induced iPLA(2)γ activation was completely inhibited by long-chain acyl-CoA (IC(50) ～20 μm) as well as by a nonhydrolyzable acyl-CoA thioether analog. Collectively, these results demonstrate that mitochondrial iPLA(2)γ is activated by divalent cations and inhibited by acyl-CoA modulating the generation of biologically active metabolites that regulate mitochondrial bioenergetic and signaling functions.