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.     

Simvastatin potentiates TNF-alpha-induced apoptosis through the down-regulation of NF-kappaB-dependent antiapoptotic gene products: role of IkappaBalpha kinase and TGF-beta-activated kinase-1

Numerous recent reports suggest that statins (hydroxy-3-methylglutaryl-CoA reductase inhibitors) exhibit potential to suppress tumorigenesis through a mechanism that is not fully understood. Therefore, in this article, we investigated the effects of simvastatin on TNF-alpha-induced cell signaling. We found that simvastatin potentiated the apoptosis induced by TNF-alpha as indicated by intracellular esterase activity, caspase activation, TUNEL, and annexin V staining. This effect of simvastatin correlated with down-regulation of various gene products that mediate cell proliferation (cyclin D1 and cyclooxygenase-2), cell survival (Bcl-2, Bcl-x(L), cellular FLIP, inhibitor of apoptosis protein 1, inhibitor of apoptosis protein 2, and survivin), invasion (matrix mellatoproteinase-9 and ICAM-1), and angiogenesis (vascular endothelial growth factor); all known to be regulated by the NF-kappaB. We found that simvastatin inhibited TNF-alpha-induced NF-kappaB activation, and l-mevalonate reversed the suppressive effect, indicating the role of hydroxy-3-methylglutaryl-CoA reductase. Simvastatin suppressed not only the inducible but also the constitutive NF-kappaB activation. Simvastatin inhibited TNF-alpha-induced IkappaBalpha kinase activation, which led to inhibition of IkappaBalpha phosphorylation and degradation, suppression of p65 phosphorylation, and translocation to the nucleus. NF-kappaB-dependent reporter gene expression induced by TNF-alpha, TNFR1, TNFR-associated death domain protein, TNFR-associated factor 2, TGF-beta-activated kinase 1, receptor-interacting protein, NF-kappaB-inducing kinase, and IkappaB kinase beta was abolished by simvastatin. Overall, our results provide novel insight into the role of simvastatin in potentially preventing and treating cancer through modulation of IkappaB kinase and NF-kappaB-regulated gene products.     

Fas ligand induces cell-autonomous NF-kappaB activation and interleukin-8 production by a mechanism distinct from that of tumor necrosis factor-alpha

Fas ligand (FasL) has been well characterized as a death factor. However, recent studies revealed that FasL possesses inflammatory activity. Here we found that FasL induces production of the inflammatory chemokine IL-8 without inducing apoptosis in HEK293 cells. Reporter gene assays involving wild-type and mutated IL-8 promoters and NF-kappaB- and AP-1 reporter constructs indicated that an FasL-induced NF-kappaB and AP-1 activity are required for maximal promoter activity. FasL induced NF-kappaB activation with slower kinetics than did TNF-alpha, yet this response was cell autonomous and not mediated by secondary paracrine factors. The death domain of Fas, FADD, and caspase-8 were required for NF-kappaB activation by FasL. A dominant-negative mutant of IKKgamma inhibited the FasL-induced NF-kappaB activation. However, TRADD and RIP, which are essential for the TNF-alpha-induced NF-kappaB activation, were not involved in the FasL-induced NF-kappaB activation. Moreover, CLARP/FLIP inhibited the FasL- but not the TNF-alpha-induced NF-kappaB activation. These results show that FasL induces NF-kappaB activation and IL-8 production by a novel mechanism, distinct from that of TNF-alpha. In addition, we found that mouse FADD had a dominant-negative effect on the FasL-induced NF-kappaB activation in HEK293 cells, which may indicate a species difference between human and mouse in the FasL-induced NF-kappaB activation.     

E1A sensitizes cells to tumor necrosis factor alpha by downregulating c-FLIP S

Tumor necrosis factor alpha (TNF-alpha) activates both apoptosis and NF-kappaB-dependent survival pathways, the former of which requires inhibition of gene expression to be manifested. c-FLIP is a TNF-alpha-induced gene that inhibits caspase-8 activation during TNF-alpha signaling. Adenovirus infection and E1A expression sensitize cells to TNF-alpha by allowing apoptosis in the absence of inhibitors of gene expression, suggesting that it may be disabling a survival signaling pathway. E1A promoted TNF-alpha-mediated activation of caspase-8, suggesting that sensitivity was occurring at the level of the death-inducing signaling complex. Furthermore, E1A expression downregulated c-FLIP(S) expression and prevented its induction by TNF-alpha. c-FLIP(S) and viral FLIP expression rescued E1A-mediated sensitization to TNF-alpha by restoring the resistance of caspase-8 to activation, thereby preventing cell death. E1A inhibited TNF-alpha-dependent induction of c-FLIP(S) mRNA and stimulated ubiquitination- and proteasome-dependent degradation of c-FLIP(S) protein. Since elevated c-FLIP levels confer resistance to apoptosis and promote tumorigenicity, interference with its induction by NF-kappaB and stimulation of its destruction in the proteasome may provide novel therapeutic approaches for facilitating the elimination of apoptosis-refractory tumor cells.     

Downregulation of Bcl-2, FLIP or IAPs (XIAP and survivin) by siRNAs sensitizes resistant melanoma cells to Apo2L/TRAIL-induced apoptosis

Melanoma cells are relatively resistant to Apo2L/TRAIL (TNF-related apoptosis-inducing ligand). We postulated that resistance might result from higher expression of inhibitors of apoptosis including Bcl-2, FLIP (FLICE-like inhibitory protein) or IAPs such as XIAP (X-linked inhibitor of apoptosis) or survivin. Compared to scrambled or mismatch controls, targeting individual inhibitors with siRNA (si-Bcl-2, si-XIAP, si-FLIP or si-Surv), followed by Apo2L/TRAIL resulted in marked increase in apoptosis in melanoma cells. Compared to Bcl-2 or FLIP, siRNAs against XIAP and survivin were most potent in sensitizing melanoma cells. A similar substantial increase in apoptosis was seen in renal carcinoma cells (SKRC-45, Caki-2), following the inhibition of either XIAP or survivin by siRNAs. Apo2L/TRAIL treatment in IAP-targeted cells resulted in cleavage of Bid, activation of caspase-9 and cleavage of PARP (poly ADP-ribose polymerase). Thus, Apo2L/TRAIL resistance can be overcome by interfering with expression of inhibitors of apoptosis regulating both extrinsic (death receptor) or intrinsic (mitochondrial) pathways of apoptosis in melanoma cells.     

Suppression of endoplasmic reticulum stress-induced caspase activation and cell death by the overexpression of Bcl-xL or Bcl-2

Continuous endoplasmic reticulum (ER) stress, such as the accumulation of unfolded proteins, results in cell death and relates to the pathogenesis of some neurodegenerative diseases. Treatment of brefeldin A, an inhibitor of transport between the ER and Golgi complex, induced cell death during 24 h, which accompanied activation of caspase-2, caspase-3 and caspase-9, starting at 12 h and increasing time-dependently up to 28 h. Caspase-2 was expressed and activated in not only mitochondria and cytosol, but also in the microsomal fraction containing ER and Golgi. Of note is that overexpression of Bcl-x(L) or Bcl-2 in PC12 cells markedly suppressed brefeldin A-induced activation of caspases and resulting cell death. Delivery of anti-Bcl-2 antibody into the Bcl-2-overexpressed cells again recovered apoptosis. While the brefeldin A-treatment induced the phosphorylation of both c-Jun N-terminal kinase (JNK) and p38 MAPK, overexpression of Bcl-x(L) or Bcl-2 reduced the prolonged phosphorylation of JNK, but not of p38 MAPK. Pretreatment with a JNK inhibitor, SP600125, suppressed the brefeldin A-induced caspase-2 activation and cell death significantly. Thus, our results suggest that protective effects of Bcl-x(L) and Bcl-2 against brefeldin A-induced cell death appear to be dependent on the regulation of JNK activation.     

Bcl-2 and Bcl-xL inhibit CD95-mediated apoptosis by preventing mitochondrial release of Smac/DIABLO and subsequent inactivation of X-linked inhibitor-of-apoptosis protein.

Bcl-2 and Bcl-x(L) are reported to inhibit CD95-mediated apoptosis in "type II" but not in "type I" cells. In the present studies, we found that stimulation of CD95 receptors, with either agonistic antibody or CD95 ligand, resulted in the activation of caspase-8, which in turn processed caspase-3 between its large and small subunits. However, in contrast to control cells, those overexpressing either Bcl-2 or Bcl-x(L) displayed a distinctive pattern of caspase-3 processing. Indeed, the resulting p20/p12 caspase-3 was not active and did not undergo normal autocatalytic processing to form p17/p12 caspase-3, because it was bound to and inhibited by endogenous X-linked inhibitor-of-apoptosis protein (XIAP). Importantly, Bcl-2 and Bcl-x(L) inhibited the release of both cytochrome c and Smac from mitochondria. However, since Smac alone was sufficient to promote caspase-3 activity in vitro by inactivating XIAP, we proposed the existence of a death receptor-induced, Smac-dependent and apoptosome-independent pathway. This type II pathway was subsequently reconstituted in vitro using purified recombinant proteins at endogenous concentrations. Thus, mitochondria and associated Bcl-2 and Bcl-x(L) proteins may play a functional role in death receptor-induced apoptosis by modulating the release of Smac. Our data strongly suggest that the relative ratios of XIAP (and other inhibitor-of-apoptosis proteins) to active caspase-3 and Smac may dictate, in part, whether a cell exhibits a type I or type II phenotype.     

Activation of intrinsic and extrinsic proapoptotic signaling pathways in interleukin-18-mediated human cardiac endothelial cell death

Endothelial cells are the primary targets of circulating immune and inflammatory mediators. We hypothesize that interleukin-18, a proinflammatory cytokine, induces endothelial cell apoptosis. Human cardiac microvascular endothelial cells (HCMEC) were treated with interleukin (IL) 18. mRNA expression was analyzed by ribonuclease protection assay, protein levels by immunoblotting, and cell death by enzyme-linked immunosorbent assay and fluorescence-activated cell sorter analysis. We also investigated the signal transduction pathways involved in IL-18-mediated cell death. Treatment of HCMEC with IL-18 increases 1) NF-kappaB DNA binding activity; 2) induces kappaB-driven luciferase activity; 3) induces IL-1beta and TNF-alpha expression via NF-kappaB activation; 4) inhibits antiapoptotic Bcl-2 and Bcl-X(L); 5) up-regulates proapoptotic Fas, Fas-L, and Bcl-X(S) expression; 6) induces fas and Fas-L promoter activities via NF-kappaB activation; 7) activates caspases-8, -3, -9, and BID; 8) induces cytochrome c release into the cytoplasm; 9) inhibits FLIP; and 10) induces HCME cell death by apoptosis as seen by increased annexin V staining and increased levels of mono- and oligonucleosomal fragmented DNA. Whereas overexpression of Bcl-2 significantly attenuated IL-18-induced endothelial cell apoptosis, Bcl-2/Bcl-X(L) chimeric phosphorothioated 2'-MOE-modified antisense oligonucleotides potentiated the proapoptotic effects of IL-18. Furthermore, caspase-8, IKK-alpha, and NF-kappaB p65 knockdown or dominant negative IkappaB-alpha and dominant negative IkappaB-beta or kinase dead IKK-beta significantly attenuated IL-18-induced HCME cell death. Effects of IL-18 on cell death are direct and are not mediated by intermediaries such as IL-1beta, tumor necrosis factor-alpha, or interferon-gamma. Taken together, our results indicate that IL-18 activates both intrinsic and extrinsic proapoptotic signaling pathways, induces endothelial cell death, and thereby may play a role in myocardial inflammation and injury.     

Bcl-x(L) prevents the initial decrease in mitochondrial membrane potential and subsequent reactive oxygen species production during tumor necrosis factor alpha-induced apoptosis

The Bcl-2 family of proteins are involved in regulating the redox state of cells. However, the mode of action of Bcl-2 proteins remains unclear. This work analyzed the effects of Bcl-x(L) on the cellular redox state after treatment with tumor necrosis factor alpha (TNF-alpha) or exogenous oxidants. We show that in cells that undergo TNF-alpha-induced apoptosis, TNF-alpha induces a partial decrease in mitochondrial membrane potential (DeltaPsi(m)) followed by high levels of reactive oxygen species (ROS). ROS scavengers delay the progression of mitochondrial depolarization and apoptotic cell death. This indicates that ROS are important mediators of mitochondrial depolarization. However, ROS scavengers fail to prevent the initial TNF-alpha-induced decrease in DeltaPsi(m). In contrast, expression of Bcl-x(L) prevents both the initial decrease in DeltaPsi(m) following TNF-alpha treatment and the subsequent induction of ROS. Bcl-x(L) itself does not act as a ROS scavenger. In addition, Bcl-x(L) does not block the initial decrease in DeltaPsi(m) following treatment with the oxidant hydrogen peroxide. However, unlike control-transfected cells, Bcl-x(L)-expressing cells can recover their mitochondrial membrane potential following the initial drop in DeltaPsi(m) induced by hydrogen peroxide. These data suggest that Bcl-x(L) plays a regulatory role in controlling the membrane potential of and ROS production by mitochondria rather than acting as a direct antioxidant.     

Bcl-rambo, a novel Bcl-2 homologue that induces apoptosis via its unique C-terminal extension

The Bcl-2 family of proteins plays a central regulatory role in apoptosis. We have identified a novel, widely expressed Bcl-2 member which we have named Bcl-rambo. Bcl-rambo shows overall structural homology to the anti-apoptotic Bcl-2 members containing conserved Bcl-2 homology (BH) motifs 1, 2, 3, and 4. Unlike Bcl-2, however, the C-terminal membrane anchor region is preceded by a unique 250 amino acid insertion containing two tandem repeats. No interaction of Bcl-rambo with either anti-apoptotic (Bcl-2, Bcl-x(L), Bcl-w, A1, MCL-1, E1B-19K, and BHRF1) or pro-apoptotic (Bax, Bak, Bik, Bid, Bim, and Bad) members of the Bcl-2 family was observed. In mammalian cells, Bcl-rambo was localized to mitochondria, and its overexpression induces apoptosis that is specifically blocked by the caspase inhibitors, IAPs, whereas inhibitors controlling upstream events of either the 'death receptor' (FLIP, FADD-DN) or the 'mitochondrial' pro-apoptotic pathway (Bcl-x(L)) had no effect. Surprisingly, the Bcl-rambo cell death activity was induced by its membrane-anchored C-terminal domain and not by the Bcl-2 homology region. Thus, Bcl-rambo constitutes a novel type of pro-apoptotic Bcl-2 member that triggers cell death independently of its BH motifs.     

Osmotic stress sensitizes naturally resistant cells to TNF-alpha-induced apoptosis

Most cells are naturally resistant to TNF-alpha-induced cell death and become sensitized when NF-kappaB transactivation is blocked or in the presence of protein synthesis inhibitors that prevent the expression of anti-apoptotic genes. In this report we analyzed the role of osmotic stress on TNF-alpha-induced cell death. We found that it sensitizes the naturally resistant HeLa cells to TNF-alpha-induced apoptosis, with the involvement of an increase in the activity of several kinases, the inhibition of Bcl-2 expression, and a late increase on NF-kappaB activation. Cell death occurs regardless of the enhanced NF-kappaB activity, whose inhibition produces an increase in apoptosis. The inhibition of p38 kinase, also involved in NF-kappaB activation, significantly increases the effect of osmotic stress on TNF-alpha-induced cell death.     

Sequencing, chromosomal mapping, and functional characterization of bovine FLICE-like inhibitory protein (FLIP)

FLICE-like inhibitory protein (FLIP) has been shown in both humans and mice to inhibit apoptosis and NF-kappaB activation induced by pro-inflammatory mediators. The activation of NF-kappaB and the induction of apoptosis are critical events in the pathogenesis of a variety of disease states in cattle, including mastitis. Since FLIP is known to moderate these events in other species, we mapped the bovine FLIP gene, sequenced bovine FLIP cDNA, and characterized its expression in cultured primary bovine endothelial cells. Sequencing of bovine FLIP revealed approximately 83, 74, and 68% amino acid sequence identity to its porcine, human, and murine orthologs, respectively. Bovine FLIP was mapped to chromosome 2 by radiation hybrid mapping. Interestingly the region to which bovine FLIP maps contains a putative quantitative trait locus for functional herd life which is an indicator of a cow's ability to survive involuntary culling due primarily to mastitis and infertility. In addition to sequencing and mapping, the function of bovine FLIP was studied. Over-expression of bovine FLIP protected against bacterial lipopolysaccharide (LPS)- and TNF-alpha-induced apoptosis in bovine endothelial cells consistent with previous studies of human FLIP. In addition, elevated expression of bovine FLIP blocked LPS- and TNF-alpha-induced upregulation of NF-kappaB-dependent gene products as assayed by E-selectin expression. Only the full-length bovine FLIP protein could inhibit NF-kappaB activation induced by LPS, whereas the death effector domain region alone was able to inhibit TNF-alpha-induced NF-kappaB activation. Together, these data demonstrate the conservation of FLIP's ability to inhibit apoptosis and to downregulate NF-kappaB activation across species.     

Mycobacterium tuberculosis promotes apoptosis in human neutrophils by activating caspase-3 and altering expression of Bax/Bcl-xL via an oxygen-dependent pathway

In addition to direct bactericidal activities, such as phagocytosis and generation of reactive oxygen species (ROS), neutrophils can regulate the inflammatory response by undergoing apoptosis. We found that infection of human neutrophils with Mycobacterium tuberculosis (Mtb) induced rapid cell death displaying the characteristic features of apoptosis such as morphologic changes, phosphatidylserine exposure, and DNA fragmentation. Both a virulent (H37Rv) and an attenuated (H37Ra) strain of Mtb were equally effective in inducing apoptosis. Pretreatment of neutrophils with antioxidants or an inhibitor of NADPH oxidase markedly blocked Mtb-induced apoptosis but did not affect spontaneous apoptosis. Activation of caspase-3 was evident in neutrophils undergoing spontaneous apoptosis, but it was markedly augmented and accelerated during Mtb-induced apoptosis. The Mtb-induced apoptosis was associated with a speedy and transient increase in expression of Bax protein, a proapoptotic member of the Bcl-2 family, and a more prominent reduction in expression of the antiapoptotic protein Bcl-x(L). Pretreatment with an inhibitor of NADPH oxidase distinctly suppressed the Mtb-stimulated activation of caspase-3 and alteration of Bax/Bcl-x(L) expression in neutrophils. These results indicate that infection with Mtb causes ROS-dependent alteration of Bax/Bcl-x(L) expression and activation of caspase-3, and thereby induces apoptosis in human neutrophils. Moreover, we found that phagocytosis of Mtb-induced apoptotic neutrophils markedly increased the production of proinflammatory cytokine TNF-alpha by human macrophages. Therefore, the ROS-dependent apoptosis in Mtb-stimulated neutrophils may represent an important host defense mechanism aimed at selective removal of infected cells at the inflamed site, which in turn aids the functional activities of local macrophages.     

Caspase-9 is activated in a cytochrome c-independent manner early during TNFalpha-induced apoptosis in murine cells

FL5.12 pro-B lymphoma cells utilize the mitochondrial pathway to apoptosis in response to tumor necrosis factor (TNF) receptor occupation, yet high levels of the Bcl-2 family antiapoptotic protein, Bcl-x(L), fail to protect these cells against TNF-receptor-activated death. Bcl-x(L) expression delays, but does not totally block, the release of mitochondrial cytochrome c (cyt c) in these cells in response to TNFalpha-induced apoptosis and caspase-9 is processed prior to mitochondrial cyt c release under these circumstances. Early processing of caspase-9 also occurred in Apaf-1 knockout murine fibroblasts in response to TNF-receptor occupation. A caspase-9-specific inhibitor was more effective in delaying the progression of apoptosis in the FL5.12 Bcl-x(L) cells than was an inhibitor specific to caspase-3. Furthermore, downregulation of caspase-9 levels by RNA interference resulted in partial protection of these cells against TNF-receptor-activated apoptosis, indicating that caspase-9 activation contributed to early amplification of the caspase cascade. Consistent with this, proteolytic processing of caspase-9 was observed prior to processing by caspase-3, suggesting that caspase-3 was not responsible for early caspase-9 activation. We show that murine caspase-9 is efficiently processed by active caspase-8 at SEPD, the motif at which caspase-9 autoprocesses following its recruitment to the apoptosome. Our results suggest that, in addition to processing procaspase-3 and the BH3 protein Bid, active caspase-8 can cleave and activate procaspase-9 in response to TNF receptor crosslinking in murine cells.     

In mouse embryonic fibroblasts, neither caspase-8 nor cellular FLICE-inhibitory protein (FLIP) is necessary for TNF to activate NF-κB, but caspase-8 is required for TNF to cause cell death, and induction of FLIP by NF-κB is required to prevent it

Binding of TNF to TNF receptor-1 can give a pro-survival signal through activation of p65/RelA NF-κB, but also signals cell death. To determine the roles of FLICE-inhibitory protein (FLIP) and caspase-8 in TNF-induced activation of NF-κB and apoptosis, we used mouse embryonic fibroblasts derived from FLIP and caspase-8 gene-deleted mice, and treated them with TNF and a smac-mimetic compound that causes degradation of cellular inhibitor of apoptosis proteins (cIAPs). In cells treated with smac mimetic, TNF and Fas Ligand caused wild-type and FLIP(-/-) MEFs to die, whereas caspase-8(-/-) MEFs survived, indicating that caspase-8 is necessary for death of MEFs triggered by these ligands when IAPs are degraded. By contrast, neither caspase-8 nor FLIP was required for TNF to activate p65/RelA NF-κB, because IκB was degraded, p65 translocated to the nucleus, and an NF-κB reporter gene activated normally in caspase-8(-/-) or FLIP(-/-) MEFs. Reconstitution of FLIP(-/-) MEFs with the FLIP isoforms FLIP-L, FLIP-R, or FLIP-p43 protected these cells from dying when treated with TNF or FasL, whether or not cIAPs were depleted. These results show that in MEFs, caspase-8 is necessary for TNF- and FasL-induced death, and FLIP is needed to prevent it, but neither caspase-8 nor FLIP is required for TNF to activate NF-κB.