Glutathione and modulation of cell apoptosis

Apoptosis is a highly organized form of cell death that is important for tissue homeostasis, organ development and senescence. To date, the extrinsic (death receptor mediated) and intrinsic (mitochondria derived) apoptotic pathways have been characterized in mammalian cells. Reduced glutathione, is the most prevalent cellular thiol that plays an essential role in preserving a reduced intracellular environment. glutathione protection of cellular macromolecules like deoxyribose nucleic acid proteins and lipids against oxidizing, environmental and cytotoxic agents, underscores its central anti-apoptotic function. Reactive oxygen and nitrogen species can oxidize cellular glutathione or induce its extracellular export leading to the loss of intracellular redox homeostasis and activation of the apoptotic signaling cascade. Recent evidence uncovered a novel role for glutathione involvement in apoptotic signaling pathways wherein post-translational S-glutathiolation of protein redox active cysteines is implicated in the potentiation of apoptosis. In the present review we focus on the key aspects of glutathione redox mechanisms associated with apoptotic signaling that includes: (a) changes in cellular glutathione redox homeostasis through glutathione oxidation or GSH transport in relation to the initiation or propagation of the apoptotic cascade, and (b) evidence for S-glutathiolation in protein modulation and apoptotic initiation.     

Modulation of NMDA-mediated excitotoxicity by protein kinase C

Excessive activation of N-methyl-D-aspartate (NMDA) receptors leads to cell death in human embryonic kidney-293 (HEK) cells which have been transfected with recombinant NMDA receptors. To evaluate the role of protein kinase C (PKC) activation in NMDA-mediated toxicity, we have analyzed the survival of transfected HEK cells using trypan blue exclusion. We found that NMDA-mediated death of HEK cells transfected with NR1/NR2A subunits was increased by exposure to phorbol esters and reduced by inhibitors of PKC activation, or PKC down-regulation. The region of NR2A that provides the PKC-induced enhancement of cell death was localized to a discrete segment of the C-terminus. Use of isoform-specific PKC inhibitors showed that Ca(2+)- and lipid-dependent PKC isoforms (cPKCs), specifically PKCbeta1, was responsible for the increase in cell death when phorbol esters were applied prior to NMDA in these cells. PKC activity measured by an in vitro kinase assay was also increased in NR1A/NR2A-transfected HEK cells following NMDA stimulation. These results suggest that PKC acts on the C-terminus of NR2A to accentuate cell death in NR1/NR2A-transfected cells and demonstrate that this effect is mediated by cPKC isoforms. These data indicate that elevation of cellular PKC activity can increase neurotoxicity mediated by NMDA receptor activation.     

Activation of PKC is sufficient to induce an apoptotic program in salivary gland acinar cells

Accumulating evidence suggests that specific isoforms of PKC may function to promote apoptosis. We show here that activation of the conventional and novel isoforms of PKC with 12-O-tetradecanoyl phorbol-13- ester (TPA) induces apoptosis in salivary acinar cells as indicated by DNA fragmentation and activation of caspase-3. TPA-induced DNA fragmentation, caspase-3 activation, and morphologic indicators of apoptosis, can be enhanced by pretreatment of cells with the calpain inhibitor, calpeptin, prior to the addition of TPA. Analysis of PKC isoform expression by immunoblot shows that TPA-induced downregulation of PKC alpha and PKC delta is delayed in cells pre-treated with calpeptin, and that this correlates with an increase of these isoforms in the membrane fraction of cells. TPA-induced apoptosis is accompanied by biphasic activation of the c-jun-N-terminal kinase (JNK) pathway and inactivation of the extracellular regulated kinase (ERK) pathway. Expression of constitutively activated PKC alpha or PKC delta, but not kinase negative mutants of these isoforms, or constitutively activated PKC epsilon, induces apoptosis in salivary acinar cells, suggesting a role for these isoforms in TPA-induced apoptosis. These studies demonstrate that activation of PKC is sufficient for initiation of an apoptotic program in salivary acinar cells. Cell Death and Differentiation (2000) 7, 1200 - 1209.     

Overexpression of protein kinase C isoforms protects RAW 264.7 macrophages from nitric oxide-induced apoptosis: involvement of c-Jun N-terminal kinase/stress-activated protein kinase, p38 kinase, and CPP-32 protease pathways

Nitric oxide (NO) induces apoptotic cell death in murine RAW 264.7 macrophages. To elucidate the inhibitory effects of protein kinase C (PKC) on NO-induced apoptosis, we generated clones of RAW 264.7 cells that overexpress one of the PKC isoforms and explored the possible interactions between PKC and three structurally related mitogen-activated protein (MAP) kinases in NO actions. Treatment of RAW 264.7 cells with sodium nitroprusside (SNP), a NO-generating agent, activated both c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 kinase, but did not activate extracellular signal-regulated kinase (ERK)-1 and ERK-2. In addition, SNP-induced apoptosis was slightly blocked by the selective p38 kinase inhibitor (SB203580) but not by the MAP/ERK1 kinase inhibitor (PD098059). PKC transfectants (PKC-beta II, -delta, and -eta) showed substantial protection from cell death induced by the exposure to NO donors such as SNP and S-nitrosoglutathione (GSNO). In contrast, in RAW 264.7 parent or in empty vector-transformed cells, these NO donors induced internucleosomal DNA cleavage. Moreover, overexpression of PKC isoforms significantly suppressed SNP-induced JNK/SAPK and p38 kinase activation, but did not affect ERK-1 and -2. We also explored the involvement of CPP32-like protease in the NO-induced apoptosis. Inhibition of CPP32-like protease prevented apoptosis in RAW 264.7 parent cells. In addition, SNP dramatically activated CPP32 in the parent or in empty vector-transformed cells, while slightly activated CPP32 in PKC transfectants. Therefore, we conclude that PKC protects NO-induced apoptotic cell death, presumably nullifying the NO-mediated activation of JNK/SAPK, p38 kinase, and CPP32-like protease in RAW 264.7 macrophages.     

PKC epsilon -mediated ERK1/2 activation involved in radiation-induced cell death in NIH3T3 cells

Protein kinase C (PKC) isoforms play distinct roles in cellular functions. We have previously shown that ionizing radiation activates PKC isoforms (alpha, delta, epsilon, and zeta), however, isoform-specific sensitivities to radiation and its exact mechanisms in radiation mediated signal transduction are not fully understood. In this study, we showed that overexpression of PKC isoforms (alpha, delta, epsilon, and zeta) increased radiation-induced cell death in NIH3T3 cells and PKC epsilon overexpression was predominantly responsible. In addition, PKC epsilon overexpression increased ERK1/2 activation without altering other MAP-kinases such as p38 MAPK or JNK. Co-transfection of dominant negative PKC epsilon (PKC epsilon -KR) blocked both PKC epsilon -mediated ERK1/2 activation and radiation-induced cell death, while catalytically active PKC epsilon construction augmented these phenomena. When the PKC epsilon overexpressed cells were pretreated with PD98059, MEK inhibitor, radiation-induced cell death was inhibited. Co-transfection of the cells with a mutant of ERK1 or -2 (ERK1-KR or ERK2-KR) also blocked these phenomena, and co-transfection with dominant negative Ras or Raf cDNA revealed that PKC epsilon -mediated ERK1/2 activation was Ras-Raf-dependent. In conclusion, PKC epsilon -mediated ERK1/2 activation was responsible for the radiation-induced cell death.     

Molecular mechanisms of protein kinase C-induced apoptosis in prostate cancer cells

Protein kinase C (PKC) isozymes, a family of serine-threonine kinases, are important regulators of cell proliferation and malignant transformation. Phorbol esters, the prototype PKC activators, cause PKC translocation to the plasma membrane in prostate cancer cells, and trigger an apoptotic response. Studies in recent years have determined that each member of the PKC family exerts different effects on apoptotic or survival pathways. PKCdelta, one of the novel PKCs, is a key player of the apoptotic response via the activation of the p38 MAPK pathway. Studies using RNAi revealed that depletion of PKCdelta totally abolishes the apoptotic effect of the phorbol ester PMA. Activation of the classical PKCalpha promotes the dephosphorylation and inactivation of the survival kinase Akt. Studies have assigned a pro-survival role to PKCepsilon, but the function of this PKC isozyme remains controversial. Recently, it has been determined that the PKC apoptotic effect in androgen-dependent prostate cancer cells is mediated by the autocrine secretion of death factors. PKCdelta stimulates the release of TNFalpha from the plasma membrane, and blockade of TNFalpha secretion or TNFalpha receptors abrogates the apoptotic response of PMA. Molecular analysis indicates the requirement of the extrinsic apoptotic cascade via the activation of death receptors and caspase-8. Dissecting the pathways downstream of PKC isozymes represents a major challenge to understanding the molecular basis of phorbol ester-induced apoptosis.     

Apoptosis signalling by 4-hydroxynonenal: a role for JNK-c-Jun/AP-1 pathway

4-Hydroxynonenal (HNE) is one of the most abundant aldehyde components of ox-LDL and it exerts various effects on intracellular and extracellular signaling cascades. In this mini-review, a brief synopsis of HNE-modulated signaling pathways will be presented mainly focused on cell death, including recent studies from our laboratory. The results of a number of studies demonstrate the ability of HNE to induce apoptosis and ROS formation in a dose-dependent manner. Several signaling pathways have been shown to be modulated by HNE, including MAP kinases, PKC isoforms, cell-cycle regulators, receptor tyrosine kinases and caspases. In order to get insight into the mechanisms of apoptotic response by HNE, MAP kinase and caspase activation pathways have been studied in 3T3 fibroblasts; HNE induced early activation of JNK and p38 proteins but down-regulated the basal activity of ERK-1/2. We have shown that HNE-induced release of cytochrome c from mitochondria, caspase-9 and caspase-3 activation. Activation of AP-1 along with increased c-Jun and phospho-c-Jun levels could be inhibited by pretreatment of cells with certain molecules such as resveratrol. Additionally, overexpression of dominant negative c-Jun and JNK1 in 3T3 fibroblasts prevented HNE-induced apoptosis, which indicated a role for JNK-c-Jun/AP-1 pathway. JNK-dependent induction of c-Jun/AP-1 activation data in the literature indicates a critical potential role for JNK in the cellular response against toxic products of lipid peroxidation.     

Beneficial effect of (-)schisandrin B against 3-nitropropionic acid-induced cell death in PC12 cells

Huntington's disease (HD) is characterized by the dysfunction of mitochondrial energy metabolism, which is associated with the functional impairment of succinate dehydrogenase (mitochondrial complex II), and pyruvate dehydrogenase (PDH). Treatment with 3-nitropropionic acid (3-NP), a potent irreversible inhibitor of succinate dehydrogenase, replicates most of the pathophysiological features of HD. In the present study, we investigated the effect of (-)schisandrin B [(-)Sch B, a potent enantiomer of schisandrin B] on 3-NP-induced cell injury in rat differentiated neuronal PC12 cells. The 3-NP caused cell necrosis, as assessed by lactate dehydrogenase (LDH) leakage, and mitochondrion-dependent cell apoptosis, as assessed by caspase-3 and caspase-9 activation, in differentiated PC12 cells. The cytotoxicity induced by 3-NP was associated with a depletion of cellular reduced glutathione (GSH) as well as the activation of redox-sensitive c-Jun N-terminal kinase (JNK) pathway and the inhibition of PDH. (-)Sch B pretreatment (5 and 15 μM) significantly reduced the extent of necrotic and apoptotic cell death in 3-NP-challenged cells. The cytoprotection afforded by (-)Sch B pretreatment was associated with the attenuation of 3-NP-induced GSH depletion as well as JNK activation and PDH inhibition. (-)Sch B pretreatment enhanced cellular glutathione redox status and ameliorated the 3-NP-induced cellular energy crisis, presumably by suppressing the activated JNK-mediated PDH inhibition, thereby protecting against necrotic and apoptotic cell death in differentiated PC12 cells.     

Phorbol ester-induced apoptosis in prostate cancer cells via autocrine activation of the extrinsic apoptotic cascade: a key role for protein kinase C delta

It is well established that activation of protein kinase C (PKC) by phorbol esters promotes apoptosis in androgen-dependent prostate cancer cells. However, there is limited information regarding the cellular mechanisms involved in this effect. In this report we identified a novel autocrine pro-apoptotic loop triggered by PKCdelta activation in prostate cancer cells that is mediated by death receptor ligands. The apoptotic effect of phorbol 12-myristate 13-acetate in LNCaP cells was impaired by inhibition or depletion of tumor necrosis factor alpha-converting enzyme, the enzyme responsible for tumor necrosis factor alpha (TNFalpha) shedding. Moreover, the apoptogenic effect of conditioned medium collected after phorbol 12-myristate 13-acetate treatment could be inhibited by blocking antibodies against TNFalpha and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), but not FasL, as well as by RNA interference depletion of TNFalpha and TRAIL receptors. Moreover, depletion or inhibition of death receptor downstream effectors, including caspase-8, FADD, p38 MAPK, and JNK, significantly reduced the apoptogenic effect of the conditioned medium. PKCdelta played a major role in this autocrine loop, both in the secretion of autocrine factors as well as a downstream effector. Taken together, our results demonstrate that activation of PKCdelta in prostate cancer cells causes apoptosis via the release of death receptor ligands and the activation of the extrinsic apoptotic cascade.     

Hepatocyte resistance to oxidative stress is dependent on protein kinase C-mediated down-regulation of c-Jun/AP-1

The prevention of injury from reactive oxygen species is critical for cellular resistance to many death stimuli. Resistance to death from the superoxide generator menadione in the hepatocyte cell line RALA255-10G is dependent on down-regulation of the c-Jun N-terminal kinase (JNK)/AP-1 signaling pathway by extracellular signal-regulated kinase 1/2 (ERK1/2). Because protein kinase C (PKC) regulates both oxidant stress and JNK signaling, the ability of PKC to modulate hepatocyte death from menadione through effects on AP-1 was examined. PKC inhibition with Ro-31-8425 or bisindolylmaleimide I sensitized this cell line to death from menadione. Menadione treatment led to activation of PKCmicro, or protein kinase D (PKD), but not PKCalpha/beta, PKCzeta/lambda, or PKCdelta/. Menadione induced phosphorylation of PKD at Ser-744/748, but not Ser-916, and translocation of PKD to the nucleus. PKC inhibition blocked menadione-induced phosphorylation of PKD, and expression of a constitutively active PKD prevented death from Ro-31-8425/menadione. PKC inhibition led to a sustained overactivation of JNK and c-Jun in response to menadione as determined by in vitro kinase assay and immunoblotting for the phosphorylated forms of both proteins. Cell death from PKC inhibition and menadione treatment resulted from c-Jun activation, since death was blocked by adenoviral expression of the c-Jun dominant negative TAM67. PKC and ERK1/2 independently down-regulated JNK/c-Jun, since inhibition of either kinase failed to affect activation of the other kinase, and simultaneous inhibition of both pathways caused additive JNK/c-Jun activation and cell death. Resistance to death from superoxide therefore requires both PKC/PKD and ERK1/2 activation in order to down-regulate proapoptotic JNK/c-Jun signaling.     

Activation of protein kinase C beta II by the stereo-specific phosphatidylserine receptor is required for phagocytosis of apoptotic thymocytes by resident murine tissue macrophages

We showed previously that protein kinase C (PKC) is required for phagocytosis of apoptotic leukocytes by murine alveolar (AM?) and peritoneal macrophages (PM?) and that such phagocytosis is markedly lower in AM? compared with PM?. In this study, we examined the roles of individual PKC isoforms in phagocytosis of apoptotic thymocytes by these two M? populations. By immunoblotting, AM? expressed equivalent PKC eta but lower amounts of other isoforms (alpha, betaI, betaII, delta, epsilon, mu, and zeta), with the greatest difference in betaII expression. A requirement for PKC betaII for phagocytosis was demonstrated collectively by phorbol 12-myristate 13-acetate-induced depletion of PKC betaII, by dose-response to PKC inhibitor Ro-32-0432, and by use of PKC betaII myristoylated peptide as a blocker. Exposure of PM? to phosphatidylserine (PS) liposomes specifically induced translocation of PKC betaII and other isoforms to membranes and cytoskeleton. Both AM? and PM? expressed functional PS receptor, blockade of which inhibited PKC betaII translocation. Our results indicate that murine tissue M? require PKC betaII for phagocytosis of apoptotic cells, which differs from the PKC isoform requirement previously described in M? phagocytosis of other particles, and imply that a crucial action of the PS receptor in this process is PKC betaII activation.     

Inhibition of thioredoxin reductase induces apoptosis in neuronal cell lines: role of glutathione and the MKK4/JNK pathway

The Thioredoxin (Trx)/Thioredoxin reductase (TrxR)-system has emerged as a crucial component of many cellular functions particularly antioxidant defence. We investigated the effect of the selective TrxR inhibitor 1-chloro-2,4-dinitrobenzene (CDNB) on survival and redox status in neuronal cell lines. CDNB was found to cause apoptosis without depletion of glutathione or loss of mitochondrial complex I-activity. Cells treated with CDNB displayed an early increase of reactive oxygen species and rapid activation of stress inducible protein kinases c-Jun N-terminal kinase (JNK) and mitogen activated protein kinase kinase 4 (MKK4). Thus TrxR inhibition by CDNB results in generation of reactive oxygen species and subsequent activation of stress-inducible kinases without impairment of the cellular antioxidant status or mitochondrial function. Inhibition of the specific kinases involved in cell death triggered by Trx/TrxR dysfunction could represent a novel and selective therapeutic approach in neurodegenerative disorders.     

15-Deoxy-Delta12,14-prostaglandin J(2) protects against nitrosative PC12 cell death through up-regulation of intracellular glutathione synthesis

Nitrosative stress with subsequent inflammatory cell death has been associated with many neurodegenerative disorders. Expression of inducible nitric-oxide synthase and production of nitric oxide (NO) have been frequently elevated in many inflammatory disorders. NO can rapidly react with superoxide anion, producing more reactive peroxynitrite. In the present study, exposure of rat pheochromocytoma (PC12) cells to the peroxynitrite donor 3-morpholinosydnonimine hydrochloride (SIN-1) induced apoptosis, which accompanied depletion of intracellular glutathione (GSH), c-Jun N-terminal kinase activation, mitochondrial membrane depolarization, the cleavage of poly(ADP-ribose)polymerase, and DNA fragmentation. During SIN-1-induced apoptotic cell death, expression of inducible cyclooxygenase (COX-2), and peroxisome proliferator-activated receptor-gamma (PPARgamma) was elevated. SIN-1 treatment resulted in elevated production of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), an endogenous PPARgamma activator. Preincubation with 15d-PGJ(2) rendered PC12 cells resistant to nitrosative stress induced by SIN-1. 15d-PGJ(2) fortified an intracellular GSH pool through up-regulation of glutamylcysteine ligase, thereby preventing cells from SIN-1-induced GSH depletion. The above findings suggest that 15d-PGJ(2) may act as a survival mediator capable of augmenting cellular thiol antioxidant capacity through up-regulation of the intracellular GSH synthesis in response to the nitrosative insult.