Hepatocytes sensitized to tumor necrosis factor-alpha cytotoxicity undergo apoptosis through caspase-dependent and caspase-independent pathways

Hepatocytes can be sensitized to tumor necrosis factor (TNF)-alpha toxicity by repression of NF-kappaB activation or inhibition of RNA synthesis. To determine whether both forms of sensitization lead to TNF-alpha cytotoxicity by similar mechanisms, TNF-alpha-induced cell death in RALA255-10G hepatocytes was examined following infection with an adenovirus, Ad5IkappaB, that blocks NF-kappaB activation or following cotreatment with actinomycin D (ActD). TNF-alpha treatment of Ad5IkappaB-infected cells resulted in 44% cell death within 6 h. ActD/TNF-alpha induced no death within 6 h but did lead to 37% cell death by 24 h. In both instances, cell death occurred by apoptosis and was associated with caspase activation, although caspase activation in ActD-sensitized cells was delayed. CrmA and chemical caspase inhibitors blocked Ad5IkappaB/TNF-alpha-induced cell death but did not inhibit ActD/TNF-alpha-induced apoptosis. A Fas-associated protein with death domain (FADD) dominant negative decreased Ad5IkappaB/TNF-alpha- and ActD/TNF-alpha-induced cell death by 81 and 47%, respectively. However, downstream events differed, since Ad5IkappaB/TNF-alpha but not ActD/TNF-alpha treatment caused mitochondrial cytochrome c release. These results suggest that NF-kappaB inactivation and inhibition of RNA synthesis sensitize RALA255-10G hepatocytes to TNF-alpha toxicity through distinct cell death pathways that diverge below the level of FADD. ActD-induced hepatocyte sensitization to TNF-alpha cytotoxicity occurs through a FADD-dependent, caspase-independent pathway of apoptosis.     

Distinct functions of JNK and c-Jun in oxidant-induced hepatocyte death

Overactivation of c‐Jun N‐terminal kinase (JNK)/c‐Jun signaling is a central mechanism of hepatocyte injury and death including that from oxidative stress. However, the functions of JNK and c‐Jun are still unclear, and this pathway also inhibits hepatocyte death. Previous studies of menadione‐induced oxidant stress demonstrated that toxicity resulted from sustained JNK/c‐Jun activation as death was blocked by the c‐Jun dominant negative TAM67. To further delineate the function of JNK/c‐Jun signaling in hepatocyte injury from oxidant stress, the effects of direct JNK inhibition on menadione‐induced death were examined. In contrast to the inhibitory effect of TAM67, pharmacological JNK inhibition by SP600125 sensitized the rat hepatocyte cell line RALA255‐10G to death from menadione. SP600125 similarly sensitized mouse primary hepatocytes to menadione toxicity. Death from SP600125/menadione was c‐Jun dependent as it was blocked by TAM67, but independent of c‐Jun phosphorylation. Death occurred by apoptosis and necrosis and activation of the mitochondrial death pathway. Short hairpin RNA knockdowns of total JNK or JNK2 sensitized to death from menadione, whereas a jnk1 knockdown was protective. Jnk2 null mouse primary hepatocytes were also sensitized to menadione death. JNK inhibition magnified decreases in cellular ATP content and β‐oxidation induced by menadione. This effect mediated cell death as chemical inhibition of β‐oxidation also sensitized cells to death from menadione, and supplementation with the β‐oxidation substrate oleate blocked death. Components of the JNK/c‐Jun signaling pathway have opposing functions in hepatocyte oxidant stress with JNK2 mediating resistance to cell death and c‐Jun promoting death. J. Cell. Biochem. 113: 3254–3265, 2012. © 2012 Wiley Periodicals, Inc.     

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.     

Caspase 3 inhibition attenuates hydrogen peroxide-induced DNA fragmentation but not cell death in neuronal PC12 cells

Exposure of neurons to H(2)O(2) results in both necrosis and apoptosis. Caspases play a pivotal role in apoptosis, but exactly how they are involved in H(2)O(2)-mediated cell death is unknown. We examined H(2)O(2)-induced toxicity in neuronal PC12 cells and the effects of inducible overexpression of the H(2)O(2)-scavenging enzyme catalase on this process. H(2)O(2) caused cell death in a time- and concentration-dependent manner. Cell death induced by H(2)O(2) was found to be mediated in part through an apoptotic pathway as H(2)O(2)-treated cells exhibited cell shrinkage, nuclear condensation and marked DNA fragmentation. H(2)O(2) also triggered activation of caspase 3. Genetic up-regulation of catalase not only significantly reduced cell death but also suppressed caspase 3 activity and DNA fragmentation. While the caspase 3 inhibitor DEVD inhibited both caspase 3 activity and DNA fragmentation induced by H(2)O(2) it did not prevent cell death. Treatment with the general caspase inhibitor ZVAD, however, resulted in complete attenuation of H(2)O(2)-mediated cellular toxicity. These results suggest that DNA fragmentation induced by H(2)O(2) is attributable to caspase 3 activation and that H(2)O(2) may be critical for signaling leading to apoptosis. However, unlike inducibly increased catalase expression and general caspase inhibition both of which protect cells from cytotoxicity, caspase 3 inhibition alone did not improve cell survival suggesting that prevention of DNA fragmentation is insufficient to prevent H(2)O(2)-mediated cell death.     

Cell cycle effects and caspase-dependent and independent death of HL-60 and Jurkat cells treated with the inhibitor of NF-kappaB parthenolide

The sesquiterpene parthenolide (PRT) is an active component of Mexican-Indian medicinal plants and also of the common herb of European origin feverfew. PRT is considered to be a specific inhibitor of NF-kappaB. Human leukemic HL-60, Jurkat, and Jurkat IkappaBalphaM cells, the latter expressing a dominant-negative IkappaBalpha and thus having non-functional NF-kappaB, were treated with PRT and activation of caspases, plasma membrane integrity, DNA fragmentation, chromatin condensation (probed by DNA susceptibility to denaturation), and changes in cell morphology were determined. As a positive control for apoptosis cells were treated with topotecan (TPT) and H2O2. At 2-8 microM concentration PRT induced transient cell arrest in G2 and M followed by apoptosis. A narrow range of PRT concentration (2-10 microM) spanned its cytostatic effect, induction of apoptosis and induction of necrosis. In fact, necrotic cells were often seen concurrently with apoptotic cells at the same PRT concentration. Atypical apoptosis was characterized by loss of plasma membrane integrity very shortly after caspases activation. In contrast, a prolonged phase of caspase activation with preserved integrity of plasma membrane was seen during apoptosis induced by TPT or H2O2. Necrosis induced by PRT was also atypical, characterized by rapid rupture of plasma membrane and no increase in DNA susceptibility to denaturation. Using Jurkat cells with inactive NF-kappaB we demonstrate that cell cycle arrest and the mode of cell death induced by PRT were not caused by inhibition of NF-kappaB. The data suggest that regardless of caspase activation PRT targets plasma membrane causing its destruction. A caution, therefore, should be exercised in interpreting data of the experiments in which PRT is used with the intention to specifically prevent activation of NF-kappaB.     

Reactive oxygen species promote TNFalpha-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases

TNFalpha is a pleiotropic cytokine that induces either cell proliferation or cell death. Inhibition of NF-kappaB activation increases susceptibility to TNFalpha-induced death, concurrent with sustained JNK activation, an important contributor to the death response. Sustained JNK activation in NF-kappaB-deficient cells was suggested to depend on reactive oxygen species (ROS), but how ROS affect JNK activation was unclear. We now show that TNFalpha-induced ROS, whose accumulation is suppressed by mitochondrial superoxide dismutase, cause oxidation and inhibition of JNK-inactivating phosphatases by converting their catalytic cysteine to sulfenic acid. This results in sustained JNK activation, which is required for cytochrome c release and caspase 3 cleavage, as well as necrotic cell death. Treatment of cells or experimental animals with an antioxidant prevents H(2)O(2) accumulation, JNK phosphatase oxidation, sustained JNK activity, and both forms of cell death. Antioxidant treatment also prevents TNFalpha-mediated fulminant liver failure without affecting liver regeneration.     

Novel synergistic mechanism for sst2 somatostatin and TNFalpha receptors to induce apoptosis: crosstalk between NF-kappaB and JNK pathways

Somatostatin is a multifunctional hormone that modulates cell proliferation, differentiation and apoptosis. Mechanisms for somatostatin-induced apoptosis are at present mostly unsolved. Therefore, we investigated whether somatostatin receptor subtype 2 (sst2) induces apoptosis in the nontransformed murine fibroblastic NIH3T3 cells. Somatostatin receptor subtype 2 expression induced an executioner caspase-mediated apoptosis through a tyrosine phosphatase SHP-1 (Src homology domain phosphatase-1)-dependent stimulation of nuclear factor kappa B (NF-kappaB) activity and subsequent inhibition of the mitogen-activated protein kinase JNK. Tumor necrosis factor alpha (TNFalpha) stimulated both NF-kappaB and c-Jun NH2-terminal kinase (JNK) activities, which had opposite action on cell survival. Importantly, sst2 sensitized NIH3T3 cells to TNFalpha-induced apoptosis by (1) upregulating TNFalpha receptor protein expression, and sensitizing to TNFalpha-induced caspase-8 activation; (2) enhancing TNFalpha-mediated activation of NF-kappaB, resulting in JNK inhibition and subsequent executioner caspase activation and cell death. We have here unraveled a novel signaling mechanism for a G protein-coupled receptor, which directly triggers apoptosis and crosstalks with a death receptor to enhance death ligand-induced apoptosis.     

Death receptor-induced signaling pathways are differentially regulated by gamma interferon upstream of caspase 8 processing

FasL and gamma interferon (IFN-gamma) are produced by activated T cells and NK cells and synergistically induce apoptosis. Although both cytokines can also elicit proinflammatory responses, a possible cross talk of these ligands with respect to nonapoptotic signaling has been poorly addressed. Here, we show that IFN-gamma sensitizes KB cells for apoptosis induction by facilitating death-inducing signaling complex (DISC)-mediated caspase 8 processing. Moreover, after protection against death receptor-induced apoptosis by caspase inhibition or Bcl2 overexpression, IFN-gamma also sensitized for Fas- and TRAIL death receptor-mediated NF-kappaB activation leading to synergistic upregulation of a variety of proinflammatory genes. In contrast, Fas-mediated activation of JNK, p38, and p42/44 occurred essentially independent from IFN-gamma sensitization, indicating that the apoptosis- and NF-kappaB-related FasL-IFN-gamma cross talk was not due to a simple global enhancement of Fas signaling. Overexpression of FLIP(L) and FLIP(S) inhibited Fas- as well as TRAIL-mediated NF-kappaB activation and apoptosis induction in IFN-gamma-primed cells suggesting that both responses are coregulated at the level of the DISC.     

Modulation of tumor necrosis factor apoptosis-inducing ligand- induced NF-kappa B activation by inhibition of apical caspases.

Tumor necrosis factor (TNF) apoptosis-inducing ligand (TRAIL), a member of the TNF family, induces apoptosis in many transformed cells. We report TRAIL-induced NF-kappaB activation, concomitant with production of the pro-inflammatory cytokine Interleukin-8 in the relatively TRAIL-insensitive cell line, HEK293. In contrast, TRAIL-induced NF-kappaB activation occurred in HeLa cells only upon pretreatment with the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (z-VAD.fmk), indicating that this was due to a caspase-sensitive component of TRAIL-induced NF-kappaB activation. NF-kappaB activation was mediated by the death receptors, TRAIL-R1 and -R2, but not by TRAIL-R3 or -R4 and was only observed in HeLa cells in the presence of z-VAD.fmk. Receptor-interacting protein, an obligatory component of TNF-alpha-induced NF-kappaB activation, was cleaved during TRAIL-induced apoptosis. We show that receptor-interacting protein is recruited to the native TRAIL death-inducing signaling complex (DISC) and that recruitment is enhanced in the presence of z-VAD.fmk, thus providing an explanation for the potentiation of TRAIL-induced NF-kappaB activation by z-VAD.fmk in TRAIL-sensitive cell lines. Examination of the TRAIL DISC in sensitive and resistant cells suggests that a high ratio of c-FLIP to caspase-8 may partially explain cellular resistance to TRAIL-induced apoptosis. Sensitivity to TRAIL-induced apoptosis was also modulated by inhibition or activation of NF-kappaB. Thus, in some contexts, modulation of NF-kappaB activation possibly at the level of apical caspase activation at the DISC may be a key determinant of sensitivity to TRAIL-induced apoptosis.     

Aspirin prevents apoptosis and NF-kappaB activation induced by H2O2 in hela cells

The classical pathway of nuclear factor-kappa B (NF-kappaB) activation by several inducers mainly involves the phosphorylation of IkappaBalpha by a signalsome complex composed of IkappaBalpha kinases (IKKalpha and IKKbeta). However, in some cell types hydrogen peroxide (H2O2) has been shown to activate an alternative pathway that does not involve the classical signalsome activation process. In this study, we demonstrate that H2O2 induced NF-kappaB activation in HeLa cells through phosphorylation and degradation of IkappaB proteins as shown by immunblot analysis. Our studies reveal that a commonly used non-steroid anti-inflammatory drug, acetylsalicylic acid (aspirin) prevents H2O2-induced NF-kappaB activation in a dose-dependent manner through inhibition of phosphorylation and degradation of IkappaBalpha and IkappaBbeta. Differential staining and DNA fragmentation analysis also show that aspirin preloading of HeLa cells also prevents H2O2-induced apoptosis in a dose-dependent manner with maximum efficiency at 10 mM concentration. Additionally, aspirin effectively prevents caspase-3 and caspase-9 (cysteinyl aspartate-specific proteases) activation by H2O2. These results suggest that NF-kappaB activation is involved in H2O2-induced apoptosis and aspirin may inhibit both processes simultaneously.     

Vitamin E prevents cell death induced by mild oxidative stress in chicken skeletal muscle cells

Apoptosis and necrosis are two forms of cell death that can occur in response to various agents and oxidative damage. In addition to necrosis, apoptosis contributes to muscle fiber loss in various muscular dystrophies as well participates in the exudative diathesis in chicken, pathology caused by dietary deficiency of vitamin E and selenium, which affects muscle tissue. We have used chicken skeletal muscle cells and bovine fibroblasts to study molecular events involved in the cell death induced by oxidative stress and apoptotic agents. The effect of vitamin E on cell death induced by oxidants was also investigated. Treatment of cells with anti-Fas antibody (50 to 400 ng/mL), staurosporine (0.1 to 100 microM) and TNF-alpha (10 and 50 ng/mL) resulted in a little loss of Trypan blue exclusion ability. Those stimuli conducted cells to apoptosis detected by an enhancement in caspase activity upon fluorogenic substrates but this activity was not fully blocked by the caspase inhibitor Z-VAD-fmk. Oxidative stress induced by menadione (10, 100 and 250 muM) promoted a significant reduction in cell viability (10%, 20% and 35% for fibroblasts; 20%, 30% and 75% for muscle cells, respectively) and caused an increase in caspase activity and DNA fragmentation. H2O2 also promoted apoptosis verified by caspase activation and DNA fragmentation, but in higher doses induced necrosis. Vitamin E protected cells from death induced by low doses of oxidants. Although it was ineffective in reducing caspase activity in fibroblasts, this vitamin diminished the enzyme activity in muscle cells. These data suggested that oxidative stress could activate apoptotic mechanisms; however the mode of cell death will depend on the intensity and duration of the stimulus, and on the antioxidant status of the cells.     

Macrophages require constitutive NF-kappaB activation to maintain A1 expression and mitochondrial homeostasis

NF-kappaB is a critical mediator of macrophage inflammatory responses, but its role in regulating macrophage survival has yet to be elucidated. Here, we demonstrate that constitutive NF-kappaB activation is essential for macrophage survival. Blocking the constitutive activation of NF-kappaB with pyrrolidine dithiocarbamate or expression of IkappaBalpha induced apoptosis in macrophagelike RAW 264.7 cells and primary human macrophages. This apoptosis was independent of additional death-inducing stimuli, including Fas ligation. Suppression of NF-kappaB activation induced a time-dependent loss of mitochondrial transmembrane potential (DeltaPsi(m)) and DNA fragmentation. Examination of initiator caspases revealed the cleavage of caspase 9 but not caspase 8 or the effector caspase 3. Addition of a general caspase inhibitor, z-VAD. fmk, or a specific caspase 9 inhibitor reduced DNA fragmentation but had no effect on DeltaPsi(m) collapse, indicating this event was caspase independent. To determine the pathway leading to mitochondrial dysfunction, analysis of Bcl-2 family members established that only A1 mRNA levels were reduced prior to DeltaPsi(m) loss and that ectopic expression of A1 protected against cell death following inactivation of NF-kappaB. These data suggest that inhibition of NF-kappaB in macrophages initiates caspase 3-independent apoptosis through reduced A1 expression and mitochondrial dysfunction. Thus, constitutive NF-kappaB activation preserves macrophage viability by maintaining A1 expression and mitochondrial homeostasis.     

Lysosomal and mitochondrial pathways in H2O2-induced apoptosis of alveolar type II cells

Increasing evidence suggests a role for apoptosis in the maintenance of the alveolar epithelium under normal and pathological conditions. However, the signaling pathways modulating alveolar type II (AT II) cell apoptosis remain poorly defined. Here we investigated the role of lysosomes as modulators of oxidant-mediated AT II cell apoptosis using an in vitro model of H(2)O(2)-stress. H(2)O(2) stress led to time-dependent increases in intracellular oxidants, mitochondrial membrane polarization, cytochrome c release, lysosomal rupture, and AT II cells apoptosis. Increased apoptosis was prevented by specific inhibition of the caspase cascade using the broad-spectrum caspase inhibitor z-VAD-fmk or a caspase 3 inhibitor, or by using functional inhibitors for cathepsin D (pepstatin A) or cathepsin B. Inhibition of cathepsin D also prevented mitochondrial permeabilization and cythocrome c release suggesting that lysosomal rupture precedes and is necessary for the activation of the mitochondrial pathway of cell death.     

Interferon gamma (IFNgamma ) and tumor necrosis factor alpha synergism in ME-180 cervical cancer cell apoptosis and necrosis. IFNgamma inhibits cytoprotective NF-kappa B through STAT1/IRF-1 pathways

We investigated the molecular mechanism of the synergism between interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) documented in a variety of biological occasions such as tumor cell death and inflammatory responses. IFNgamma/TNFalpha synergistically induced apoptosis of ME-180 cervical cancer cells. IFNgamma induced STAT1 phosphorylation and interferon regulatory factor 1 (IRF-1) expression. Transfection of phosphorylation-defective STAT1 inhibited IFNgamma/TNFalpha-induced apoptosis, whereas IRF-1 transfection induced susceptibility to TNFalpha. Dominant-negative IkappaBalpha transfection sensitized ME-180 cells to TNFalpha. IFNgamma pretreatment attenuated TNFalpha- or p65-induced NF-kappaB reporter activity, whereas it did not inhibit p65 translocation or DNA binding of NF-kappaB. IRF-1 transfection alone inhibited TNFalpha-induced NF-kappaB activity, which was reversed by coactivator p300 overexpression. Caspases were activated by IFNgamma/TNFalpha combination; however, caspase inhibition did not abrogate IFNgamma/TNFalpha-induced cell death. Instead, caspase inhibitors directed IFNgamma/TNFalpha-treated ME-180 cells to undergo necrosis, as demonstrated by Hoechst 33258/propidium iodide staining and electron microscopy. Taken together, our results indicate that IFNgamma and TNFalpha synergistically act to destroy ME-180 tumor cells by either apoptosis or necrosis, depending on caspase activation, and STAT1/IRF-1 pathways initiated by IFNgamma play a critical role in IFNgamma/TNFalpha synergism by inhibiting cytoprotective NF-kappaB. IFNgamma/TNFalpha synergism appears to activate cell death machinery independently of caspase activation, and caspase activation seems to merely determine the mode of cell death.     

Role of caspase 2 in apoptotic signaling in primate and murine germ cells

This study investigates the role of caspase 2 in apoptotic signaling of nonhuman primate male germ cells triggered by mild testicular hyperthermia, testosterone (T(e)) implants, or by combined interventions. Mean incidence of germ cell apoptosis increased significantly by Day 3 in the heat (H(e)) alone group and by Day 8 in the Te alone group but peaked at Day 3 in H(e) + T(e) group. We found activation of caspase 2 in both germ cells and Sertoli cells after induction of apoptosis. Most notably, active caspase 2 immunoreactivity was detected only in those germ cells susceptible to apoptosis compared with controls, where little or no such staining is detected. To further explore the role of caspase 2 in regulating male germ cell death, we next evaluated the efficacy of caspase 2 inhibition in preventing or attenuating heat-induced germ cell apoptosis in rats. Caspase 2 inhibition significantly (P < 0.05) prevented such heat-induced germ cell apoptosis. The protection offered by the caspase 2 inhibitor occurred upstream of mitochondria, involving suppression of mitogen-activated protein kinase (MAPK) 14 activation and inducible nitric oxide synthase (NOS2) induction and, in turn, suppression of cytochrome c-mediated death pathway. Together, our results show that caspase 2 is activated in male germ cells undergoing apoptosis in nonhuman primates after heat stress, hormonal deprivation, or after combined interventions. Blockade of caspase 2 activation prevents heat-induced germ cell apoptosis in rats by suppressing the MAPK14- and NO-mediated intrinsic pathway signaling.