Deficiency in caspase-9 or caspase-3 induces compensatory caspase activation

Dysregulation of apoptosis contributes to the pathogenesis of many human diseases. As effectors of the apoptotic machinery, caspases are considered potential therapeutic targets. Using an established in vivo model of Fas-mediated apoptosis, we demonstrate here that elimination of certain caspases was compensated in vivo by the activation of other caspases. Hepatocyte apoptosis and mouse death induced by the Fas agonistic antibody Jo2 required proapoptotic Bcl-2 family member Bid and used a Bid-mediated mitochondrial pathway of caspase activation; deficiency in caspases essential for this pathway, caspase-9 or caspase-3, unexpectedly resulted in rapid activation of alternate caspases after injection of Jo2, and therefore failed to protect mice against Jo2 toxicity. Moreover, both ultraviolet and gamma irradiation, two established inducers of the mitochondrial caspase-activation pathway, also elicited compensatory activation of caspases in cultured caspase-3(-/-) hepatocytes, indicating that the compensatory caspase activation was mediated through the mitochondria. Our findings provide direct experimental evidence for compensatory pathways of caspase activation. This issue should therefore be considered in developing caspase inhibitors for therapeutic applications.     

Soluble Fas gene therapy protects against Fas-mediated apoptosis of hepatocytes but not the lethal effects of Fas-induced TNF-alpha production by Kupffer cells

The elevation of soluble Fas (sFas) in the sera of patients with liver disease suggests a role for sFas in the disease process; whether it is protective or not is controversial. To determine the effects of sFas on Fas-induced liver apoptosis, we manipulated mice to produce sFas by transfecting them in vivo with different amounts of an adenovirus that produces mouse sFas driven by the CMV promoter (AdsFas). Fas-mediated apoptosis was induced by administration of anti-mouse Fas (Jo2; 10 microg/mouse) one week later. The administration of AdsFas (10(3), 10(7), or 10(9) pfu/mouse), which was associated with only minimal side-effects, resulted in a significant reduction in the liver transaminase levels and mortality of the mice on challenge with Jo2, as compared to control mice treated with AdLacZ. However, the protective effect of AdsFas was not complete. The possibility that Jo2-induction of TNF-alpha in the Kupffer cells of the liver contributes to the pathology was therefore tested. Although administration of soluble TNF receptor (sTNFRI) alone did not protect the mice from the lethal effects of Jo2, administration of sTNFRI (200 microg/mouse) after infection with AdsFas (10(9) pfu/mouse) resulted in 100% survival of the mice on challenge with Jo2. To confirm that the production of TNF-alpha by Kupffer cells produce the lethal effects of Jo2 that remained after treatment with AdsFas, these cells were selectively ablated by treatment of the mice with gadolinium chloride prior to challenge with Jo2. This treatment greatly reduced early mortality and hepatocellular damage as well as TNF-alpha production 6 h after injection of Jo2. These results indicate that: (1) AdsFas prevents Jo2-induced apoptosis of hepatocytes; (2) In addition to mediating Fas-mediated apoptosis of hepatocytes, Jo2 can separately induce TNF-alpha production by Kupffer cells resulting in early mortality, and (3) Optimal protection from Jo2-induced mortality can be achieved by protection of liver cells by pretreatment with both AdsFas and sTNFRI.     

The mitochondrial permeability transition augments Fas-induced apoptosis in mouse hepatocytes

Tumor necrosis factor-alpha receptor 1 and Fas recruit overlapping signaling pathways. To clarify the differences between tumor necrosis factor alpha (TNFalpha) and Fas pathways in hepatocyte apoptosis, primary mouse hepatocytes were treated with TNFalpha or an agonist anti-Fas antibody after infection with an adenovirus expressing an IkappaB superrepressor (Ad5IkappaB). Treatment with TNFalpha induced apoptosis in Ad5IkappaB-infected mouse hepatocytes, as we previously reported for rat hepatocytes. Ad5IkappaB plus anti-Fas antibody or actinomycin D plus anti-Fas antibody rapidly induced apoptosis, whereas anti-Fas antibody alone produced little cytotoxicity. The proteasome inhibitor (MG-132) and a dominant-negative mutant of nuclear factor-kappaB-inducing kinase also promoted TNFalpha- and Fas-mediated apoptosis. Expression of either crmA or a dominant-negative mutant of the Fas-associated death domain protein prevented TNFalpha- and Fas-mediated apoptosis. In addition, the caspase inhibitors, DEVD-cho and IETD-fmk, inhibited TNFalpha- and Fas-mediated apoptosis. In Ad5IkappaB-infected hepatocytes, caspases-3 and -8 were activated within 2 h after treatment with anti-Fas antibody or within 6 h after TNFalpha treatment. Confocal microscopy demonstrated onset of the mitochondrial permeability transition (MPT) and mitochondrial depolarization by 2-3 h after anti-Fas antibody treatment and 8-10 h after TNFalpha treatment, followed by cytochrome c release. The combination of the MPT inhibitors, cyclosporin A, and trifluoperazine, protected Ad5IkappaB-infected hepatocytes from TNFalpha-mediated apoptosis. After anti-Fas antibody, cyclosporin A and trifluoperazine decreased cytochrome c release but did not prevent caspase-3 activation and cell-death. In conclusion, nuclear factor-kappaB activation protects mouse hepatocytes against both TNFalpha- and Fas-mediated apoptosis. TNFalpha and Fas recruit similar but nonidentical, pathways signaling apoptosis. The MPT is obligatory for TNFalpha-induced apoptosis. In Fas-mediated apoptosis, the MPT accelerates the apoptogenic events but is not obligatory for them.     

Actin cytoskeleton is required for early apoptosis signaling induced by anti-Fas antibody but not Fas ligand in murine B lymphoma A20 cells.

Murine B lymphoma A20 cells are highly sensitive to Fas-mediated death signals induced by anti-Fas antibody Jo2 or cross-linked Fas ligand (FasL). We have found that the microfilament poison cytochalasin D blocks Fas-mediated apoptosis induced by Jo2 but not FasL in A20 cells. The induction of Fas-mediated apoptosis by Jo2 was antagonized by anti-Fcgamma RII/RIII receptor (FcgammaR) antibody, and defective in FcgammaR-negative A20 cells. Since the induction of Jo2-mediated apoptosis in FcgammaR-negative A20 cells was reversed by the addition of wild type A20 cells or the cross-linking agent protein A, Fas-expressing bystander A20 cells seem to be killed by other A20 cells that capture and cross-link monomeric Jo2 via FcgammaR. Although cytochalasin D affected FcgammaR-mediated cross-linking of Jo2 molecules, the drug markedly inhibited the intracellular signaling pathway induced by Jo2. The blockade of Jo2-induced apoptosis by cytochalasin D occurred upstream of caspase-8 activation. Thus, these observations suggest that actin cytoskeleton is required for early apoptosis signaling induced by Jo2, but not physiological FasL.     

Expression of apoptosis on rat liver by hepatic vagus hyperactivity after ventromedial hypothalamic lesioning

We examined whether the Fas (APO-1/CD95)/Fas ligand system mediates apoptosis in rats with ventromedial hypothalamus (VMH) lesions. Northern and Western blotting indicated that VMH lesions lead to a significant increase in Fas mRNA and protein expression from day 1 to day 7 and in Fas ligand mRNA and protein expression from day 2 to day 7. Immunohistochemistry indicated that the region of strongest Fas expression shifted from acinar zone 1 to zones 2 and 3 by day 7 after VMH lesioning and that at days 2-7 Fas-ligand-positive hepatocyte cell membranes and cytoplasm were randomly distributed in acinar zones 1-3. We also analyzed activation of caspase 3-like proteases in hepatocytes, Kupffer cells, and sinusoidal endothelial cells. Spectrofluorometric assay demonstrated that caspase 3-like activity significantly increased only in hepatocytes after VMH lesioning. Moreover, electron microscopy and TUNEL assay showed that VMH lesions induced apoptosis. All of these effects were completely inhibited by hepatic vagotomy and administration of atropine. Vagal firing after VMH lesioning may stimulate Fas/Fas ligand system-mediated apoptosis through the cholinergic system in the rat liver.     

Fas-Mediated Apoptosis in Primary Cultured Mouse Hepatocytes

The Fas antigen is a member of the tumor necrosis factor/nerve growth factor receptor family and is expressed in tissues such as the thymus, liver, and ovary. Agonistic anti-Fas antibodies have cytolytic activity against cell lines expressing the Fas antigen. In this study, we show that anti-Fas antibody can induce the death of mouse hepatocytes in primary culture. Cell death via apoptosis was evidenced by the fact that the dying cells displayed DNA fragmentation, extensive surface bleb formation, and chromatin condensation. Anti-Fas antibody alone induced apoptosis in less than 20% of the cultured hepatocytes, whereas all cells were killed within 24 h by anti-Fas antibody in the presence of actinomycin D, cycloheximide, or protein kinase C (PKC) inhibitors such as H-7 and HA1004. These results indicate that the Fas antigen expressed in mouse hepatocytes functionally transduces the apoptotic signal and suggest that cultured mouse hepatocytes express protective proteins against apoptosis and that phosphorylation by PKC is also involved in protection of the hepatocytes from Fas-mediated apoptosis.     

Simple epithelium keratins 8 and 18 provide resistance to Fas-mediated apoptosis. The protection occurs through a receptor-targeting modulation

Keratins 8 and 18 belong to the keratin family of intermediate filament (IF) proteins and constitute a hallmark for all simple epithelia, including the liver. Hepatocyte IFs are made solely of keratins 8 and 18 (K8/K18). In these cells, the loss of one partner via a targeted null mutation in the germline results in hepatocytes lacking K8/K18 IFs, thus providing a model of choice for examining the function(s) of simple epithelium keratins. Here, we report that K8-null mouse hepatocytes in primary culture and in vivo are three- to fourfold more sensitive than wild-type (WT) mouse hepatocytes to Fas-mediated apoptosis after stimulation with Jo2, an agonistic antibody of Fas ligand. This increased sensitivity is associated with a higher and more rapid caspase-3 activation and DNA fragmentation. In contrast, no difference in apoptosis is observed between cultured K8-null and WT hepatocytes after addition of the Fas-related death-factors tumor necrosis factor (TNF) alpha or TNF-related apoptosis-inducing ligand. Analyses of the Fas distribution in K8-null and WT hepatocytes in culture and in situ demonstrate a more prominent targeting of the receptor to the surface membrane of K8-null hepatocytes. Moreover, altering Fas trafficking by disrupting microtubules with colchicine reduces by twofold the protection generated against Jo2-induced lethal action in K8-null versus WT hepatocytes. Together, the results strongly suggest that simple epithelium K8/K18 provide resistance to Fas-mediated apoptosis and that this protection occurs through a modulation of Fas targeting to the cell surface.     

Fas-mediated autophagy requires JNK activation in HeLa cells

Fas has been reported to play an important role in apoptosis; however, Fas-mediated autophagy and its mechanism are still unclear. Here, we found that Fas agonistic antibody CH11-induced autophagy in HeLa cells, and inhibition of autophagy by 3-MA increased CH11-induced apoptosis. A Fas antagonistic antibody (UB2) suppressed both CH11-induced autophagy and apoptosis. In addition, the CH11-induced autophagy was blocked by JNK inhibitor (SP600125), but it was not affected by caspase 8 inhibitor (Z-IETD); whereas the CH11-induced apoptosis was increased by SP600125, and it was suppressed by Z-IETD. Further experiments confirmed that JNK was activated by CH11 dose-dependently, and the activation was suppressed when autophagy was blocked by 3-MA. Together, our results suggest that JNK, but not caspase 8, involves in Fas-mediated CH11-induced autophagy in HeLa cells, and this autophagy plays a protective role in CH11-induced cell death.     

A Bcl-2 transgene expressed in hepatocytes does not protect mice from fulminant liver destruction induced by Fas ligand

We compared the biological mechanism of cell death during hepatotoxicity induced by ligation of the Fas receptor in wild-type and liver-specific bcl-2 transgenic mice. Transgenic overexpression of Bcl-2 in mouse hepatocytes can prevent lethal hepatitis induced by agonistic anti-Fas antibodies. In contrast, Fas ligand (FasL)-induced death cannot be overcome in bcl-2 transgenic mice, indicating that anti-Fas antibodies do not reliably mimic the more physiological ligand. Different apoptotic parameters, viz. caspase activation, cytochrome c release and nuclear DNA degradation were analysed. No differences, however, could be observed between wild-type and bcl-2 transgenic mice after injection with a lethal dose of soluble FasL, indicating that apoptosis by FasL-dependent ligation is not modulated by Bcl-2 in vivo. These results demonstrate that the stimulus determines the outcome between type I mitochondria-independent apoptosis, in the case of FasL, or type II mitochondria-dependent and Bcl-2-inhibitable apoptosis, in the case of anti-Fas antibodies.     

IL-1 beta protects human chondrocytes from CD95-induced apoptosis

This study addresses the effects of IL-1 beta on apoptosis induced by agonistic anti-CD95 (Fas) Ab. IL-1 beta inhibited anti-CD95 Ab-induced apoptosis in all preparations of normal human articular chondrocytes tested. Inhibitors of nitric oxide synthase or cyclooxygenase did not influence the protective effect of IL-1 beta, indicating that nitric oxide and PGs were not involved in the modulation of CD95-induced apoptosis. However, when the IL-1 beta-dependent induction of NF-kappa B was inhibited, the antiapoptotic effect of IL-1 beta was partially reversed, suggesting that NF-kappa B-mediated gene activation is part of the protective mechanism. In addition, IL-1 beta significantly increased the expression of Bcl-2. The protein tyrosine kinase inhibitor herbimycin A completely eliminated the protective effect of IL-1 beta on CD95-induced apoptosis. These findings suggest that IL-1 beta modulates the CD95 death cascade in chondrocytes by mechanisms that involve tyrosine phosphorylation events and NF-kappa B-dependent gene activation.     

Secoisolariciresinol and isotaxiresinol inhibit tumor necrosis factor-alpha-dependent hepatic apoptosis in mice

The effects of secoisolariciresinol (1) and isotaxiresinol (2), two major lignans isolated from the wood of Taxus yunnanensis, on tumor necrosis factor-alpha (TNF-alpha)-dependent hepatic apoptosis induced by D-galactosamine (d-GalN)/lipopolysaccharide (LPS) were investigated in mice. Co-administration of d-GalN (700 mg/kg) and LPS (10 microg/kg) resulted in a typical hepatic apoptosis characterized by DNA fragmentation and the formation of apoptotic bodies. Serum glutamic pyruvic transaminase (sGPT) and glutamic oxaloacetic transaminase (sGOT) levels were also raised at 8 h after d-GalN/LPS intoxication due to a severe necrosis of hepatocytes. Pre-administration of 1 or 2 (50, 10 mg/kg, i.p.) 12 and 1 h before d-GalN/LPS significantly reduced DNA fragmentation and prevented chromatin condensation, apoptotic body formation and hepatitis. Pro-inflammatory cytokines such as TNF-alpha and interferon-gamma (IFN-gamma) secreted from LPS-activated macrophages are important mediators of hepatocyte apoptosis in this model. Pre-treatment with 1 or 2 significantly inhibited the elevation of serum TNF-alpha and IFN-gamma levels. In a separate experiment, both lignans had a significant dose-dependent protective effect on d-GalN/TNF-alpha-induced cell death in primary cultured mouse hepatocytes and TNF-alpha-mediated cell death in murine L929 fibrosarcoma cells. These results indicated that 1 and 2 prevent d-GalN/LPS-induced hepatic injury by inhibiting hepatocyte apoptosis through the blocking of TNF-alpha and IFN-gamma production by activated macrophages and direct inhibition of the apoptosis induced by TNF-alpha.     

Unique sequence of a high molecular weight myosin light chain kinase is involved in interaction with actin cytoskeleton

Resolution of neutrophil mediated inflammation is achieved, in part, through induction of neutrophil apoptosis. This constitutively expressed programme can be delayed by inflammatory mediators and induced by ligation of the Fas receptor. However, functional activation of the neutrophil results in resistance to Fas signalled death. We evaluated the effects of Fas antibody engagement on caspase activation and mitochondrial permeability, and the impact of co-stimulation by lipopolysaccharide (LPS) or granulocyte macrophage-colony stimulating factor (GM-CSF) on these events. Fas engagement by an agonistic anti-Fas antibody resulted in enhanced caspase 3 and 8 activity and increased mitochondrial permeability. Studies with pharmacological inhibitors of caspase activity showed that activation of caspase 8 occurred before, and activation of caspase 3 occurred after mitochondrial disruption. The mitochondrial stabilising agent bongkrekic acid also inhibited caspase activation and apoptosis. LPS, GM-CSF and increased glutathione stabilised the mitochondria and inhibited caspase 3. Caspase 8 activity was also inhibited by co-stimulation through a mechanism independent of mitochondrial stabilisation. Glutathione directly inhibited caspase 3 and 8 activity. We conclude inhibition of Fas antibody induced apoptosis by inflammatory proteins is associated with augmented mitochondrial stability and reduced caspase 3 activity that may be glutathione mediated.     

Fc gamma Rs modulate cytotoxicity of anti-Fas antibodies: implications for agonistic antibody-based therapeutics

Development of anti-Fas Abs to treat diseases with insufficient Fas-mediated apoptosis has been limited by concern about hepatotoxicity. We report here that hepatotoxicity elicited by anti-Fas Ab Jo2 is dependent on FcgammaRIIB. Thus, following Jo2 treatment, all FcgammaRIIB(-/-) mice survived while 80% of wild-type and all FcR-gamma(-/-) mice died from acute liver failure. Microscopic examination suggests that FcgammaRIIB deficiency protects the hepatic sinusoidal endothelium, a cell type that normally coexpresses Fas and FcgammaRIIB. In vitro studies showed that FcgammaRIIB, but not FcgammaRI and FcgammaRIII, on neighboring macrophages substantially enhanced Jo2 mediated apoptosis of Fas expressing target cells. However, FcgammaRI and FcgammaRIII appeared essential for apoptosis-inducing activity of a non-hepatotoxic anti-Fas mAb HFE7A. These findings imply that by interacting with the Fc region of agonistic Abs, FcgammaRs can modulate both the desired and undesired consequences of Ab-based therapy. Recognizing this fact should facilitate development of safer and more efficacious agonistic Abs.     

New therapeutic strategy of hinokitiol in haemorrhagic shock‐induced liver injury

Haemorrhagic shock and resuscitation (HS/R) may cause global ischaemia‐reperfusion injury, which can result in systemic inflammation, multiorgan failure (particularly liver failure) and high mortality. Hinokitiol, a bioactive tropolone‐related compound, exhibits antiplatelet and anti‐inflammatory activities. Targeting inflammatory responses is a potential strategy for ameliorating hepatic injury during HS/R. Whether hinokitiol prevents hepatic injury during HS/R remains unclear. In the present study, we determined the role of hinokitiol following HS/R. The in vivo assays revealed that hinokitiol markedly attenuated HS/R‐induced hepatic injury. Hinokitiol could inhibited NF‐κB activation and IL‐6 and TNF‐α upregulation in liver tissues. Moreover, hinokitiol reduced caspase‐3 activation, upregulated Bax and downregulated Bcl‐2. These findings suggest that hinokitiol can ameliorate liver injury following HS/R, partly through suppression of inflammation and apoptosis. Furthermore, the in vitro data revealed that hinokitiol significantly reversed hypoxia/reoxygenation (H/R)‐induced cell death and apoptosis in the primary hepatocytes. Hinokitiol prevented H/R‐induced caspase‐3 activation, PPAR cleavage, Bax overexpression and Bcl‐2 downregulation. Moreover, hinokitiol attenuated H/R‐stimulated NF‐κB activation and reduced the levels of IL‐6 and TNF‐α mRNAs, suggesting that hinokitiol can protect hepatocytes from H/R injury. Collectively, our data suggest that hinokitiol attenuates liver injury following HS/R, partly through the inhibition of NF‐κB activation.     

Activation of cAMP-guanine exchange factor confers PKA-independent protection from hepatocyte apoptosis

cAMP has previously been shown to promote cell survival in a variety of cell types, but the downstream signaling pathway(s) of this antiapoptotic effect is unclear. Thus the role of cAMP signaling through PKA and cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs) in cAMP's antiapoptotic action was investigated in the present study. cAMP's protective effect against bile acid-, Fas ligand-, and TNF-alpha-induced apoptosis in rat hepatocytes was largely unaffected by the selective PKA inhibitor, Rp-8-(4-chlorophenylthio)-cAMP (Rp-cAMP). In contrast, a novel cAMP analog, 8-(4-chlorophenylthio)-2'-O-methyl (CPT-2-Me)-cAMP, which activated cAMP-GEFs in hepatocytes without activating PKA, protected hepatocytes against apoptosis induced by bile acids, Fas ligand, and TNF-alpha. The role of cAMP-GEF and PKA on activation of Akt, a kinase implicated in cAMP survival signaling, was investigated. Inhibition of PKA with RP-cAMP had no effect on cAMP-mediated Akt phosphorylation, whereas CPT-2-Me-cAMP, which did not activate PKA, induced phosphatidylinositol 3-kinase (PI3-kinase)-dependent activation of Akt. Pretreatment of hepatocytes with the PI3-kinase inhibitor, Ly-294002, prevented CPT-2-Me-cAMP's protective effect against bile acid and Fas ligand, but not TNF-alpha-mediated apoptosis. Glucagon, CPT-cAMP, and CPT-2-Me-cAMP all activated Rap 1, a downstream effector of cAMP-GEF. These results suggest that a PKA-independent cAMP/cAMP-GEF/Rap pathway exists in hepatocytes and that activation of cAMP-GEFs promotes Akt phosphorylation and hepatocyte survival. Thus a cAMP/cAMP-GEF/Rap/PI3-kinase/Akt signaling pathway may confer protection against bile acid- and Fas-induced apoptosis in hepatocytes.     

Activation of caspases in lethal experimental hepatitis and prevention by acute phase proteins.

Lethal hepatitis can be induced by an agonistic anti-Fas Ab in normal mice or by TNF in mice sensitized to d -(+)-galactosamine or actinomycin D. In all three models, we found that apoptosis of hepatocytes is an early and necessary step to cause lethality. In the three models, we observed activation of the major executioner caspases-3 and -7. Two acute-phase proteins, alpha1-acid glycoprotein and alpha1-antitrypsin, differentially prevent lethality: alpha1-acid glycoprotein protects in both TNF models and not in the anti-Fas model, while alpha1-antitrypsin confers protection in the TNF/d -(+)-galactosamine model only. The protection is inversely correlated with activation of caspase-3 and caspase-7. The data suggest that activation of caspase-3 and -7 is essential in the in vivo induction of apoptosis leading to lethal hepatitis and that acute phase proteins are powerful inhibitors of apoptosis and caspase activation. Furthermore, Bcl-2 transgenic mice, expressing Bcl-2 specifically in hepatocytes, are protected against a lethal challenge with anti-Fas or with TNF/d -(+)-galactosamine, but not against TNF/actinomycin D. The acute-phase proteins might constitute an inducible anti-apoptotic protective system, which in pathology or disturbed homeostasis prevents excessive apoptosis.