Recruitment Of Fas-associated Death Domain Research Articles

CTRP3 promotes TNF-α-induced apoptosis and barrier dysfunction in salivary epithelial cells

BackgroundC1q/tumour necrosis factor-related protein 3 (CTRP3) plays important roles in metabolism and inflammatory responses in various cells and tissues. However, the expression and function of CTRP3 in salivary glands have not been explored. MethodsThe expression and distribution of CTRP3 were detected by western blot, polymerase chain reaction, immunohistochemical and immunofluorescence staining. The effects of CTRP3 on tumour necrosis factor (TNF)-α-induced apoptosis and barrier dysfunction were detected by flow cytometry, western blot, co-immunoprecipitation, and measurement of transepithelial resistance and paracellular tracer flux. ResultsCTRP3 was distributed in both acinar and ductal cells of human submandibular gland (SMG) and was primarily located in the ducts of rat and mouse SMGs. TNF-α increased the apoptotic rate, elevated expression of cleaved caspase 3 and cytochrome C, and reduced B cell lymphoma-2 (Bcl-2) levels in cultured human SMG tissue and SMG-C6 cells, and CTRP3 further enhanced TNF-α-induced apoptosis response. Additionally, CTRP3 aggravated TNF-α-increased paracellular permeability. Mechanistically, CTRP3 promoted TNF-α-enhanced TNF type I receptor (TNFR1) expression, inhibited the expression of cellular Fas-associated death domain (FADD)-like interleukin-1β converting enzyme inhibitory protein (c-FLIP), and increased the recruitment of FADD with receptor-interacting protein kinase 1 and caspase 8. Moreover, CTRP3 was significantly increased in the labial gland of Sjögren's syndrome patients and in the serum and SMG of nonobese diabetic mice. ConclusionsThese findings suggest that the salivary glands are a novel source of CTRP3 synthesis and secretion. CTRP3 might promote TNF-α-induced cell apoptosis through the TNFR1-mediated complex II pathway.

Hepatitis B Virus X Protein Sensitizes TRAIL-Induced Hepatocyte Apoptosis by Inhibiting the E3 Ubiquitin Ligase A20.

Hepatitis B virus (HBV) infection causes hepatocyte death and liver damage, which may eventually lead to cirrhosis and liver cancer. Hepatitis B virus X protein (HBx) is a key antigen that is critically involved in HBV-associated liver diseases. However, the molecular basis for its pathogenesis, particularly in liver damage, has not been well defined. Herein, we report that HBx was able to enhance the susceptibility of hepatocytes to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Increased sensitivity to TRAIL was associated with HBx-induced upregulation of miR-125a, which, in turn, suppressed the expression of its putative target gene, A20 E3 ligase. Importantly, we demonstrate that the defective expression of A20 impaired the K63-linked polyubiquitination of caspase-8, which reciprocally enhanced the activation of caspase-8, the recruitment of Fas-associated death domain (FADD), and the formation of death-inducing signaling complex (DISC), thereby promoting HBx-mediated apoptotic signaling. Accordingly, antagonizing miR-125a or ectopically expressing A20 in hepatocytes abolished the pro-apoptotic effect of HBx. Conversely, the overexpression of miR-125a or knockdown of A20 mimicked HBx to enhance TRAIL susceptibility in hepatocytes. Thus, we establish, for the first time, a miR-125a/A20-initiated and caspase-8-targeted mechanism by which HBx modulates apoptotic signaling and increases hepatic susceptibility to the damaging agent, which might provide novel insight into HBV-related liver pathology.

Identification of a novel pro-apoptotic role of NF-κB in the regulation of TRAIL- and CD95-mediated apoptosis of glioblastoma cells

We recently reported that nuclear factor-kappa B (NF-κB) promotes DNA damage-triggered apoptosis in glioblastoma, the most common brain tumor. In the present study, we investigated the role of NF-κB in death receptor-mediated apoptosis. Here, we identify a novel pro-apopotic function of NF-κB in TRAIL- and CD95-induced apoptosis. Inhibition of NF-κB by overexpression of the dominant-negative IκBα-superrepressor (IκBα-SR) significantly decreases tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)- or CD95-induced apoptosis. Vice versa, activation of NF-κB via overexpression of constitutively active IκB kinase complex (IKK)β (IKK-EE) significantly increases TRAIL-mediated apoptosis. Intriguingly, NF-κB inhibition reduces the recruitment of Fas-associated death domain and caspase-8 and formation of the death-inducing signaling complex (DISC) upon stimulation of TRAIL receptors or CD95. This results in reduced TRAIL-mediated activation of caspases, loss of mitochondrial potential and cytochrome c release in IκBα-SR-expressing cells. In comparison, NF-κB inhibition strongly enhances TNF-α-mediated apoptosis. Comparative studies revealed that TNF-α rapidly stimulates transcriptional activation and upregulation of anti-apoptotic proteins, whereas TRAIL causes apoptosis before transcriptional activation. Thus, this study demonstrates for the first time that NF-κB exerts a pro-apoptotic role in TRAIL- and CD95-induced apoptosis in glioblastoma cells by facilitating DISC formation.

Trifluoperazine regulation of calmodulin binding to Fas: a computational study.

Death-inducing signaling complex (DISC) formation is a critical step in Fas-mediated signaling for apoptosis. Previous experiments have demonstrated that the calmodulin (CaM) antagonist, trifluoperazine (TFP) regulates CaM-Fas binding and affects Fas-mediated DISC formation. In this study, we investigated the anti-cooperative characteristics of TFP binding to CaM and the effect of TFP on the CaM-Fas interaction from both structural and thermodynamic perspectives using combined molecular dynamics simulations and binding free energy analyses. We studied the interactions of different numbers of TFP molecules with CaM and explored the effects of the resulting conformational changes in CaM on CaM-Fas binding. Results from these analyses showed that the number of TFP molecules bound to CaM directly influenced α-helix formation and hydrogen bond occupancy within the α-helices of CaM, contributing to the conformational and motion changes in CaM. These changes affected CaM binding to Fas, resulting in secondary structural changes in Fas and conformational and motion changes of Fas in CaM-Fas complexes, potentially perturbing the recruitment of Fas-associated death domain for DISC formation. The computational results from this study reveal the structural and molecular mechanisms that underlie the role of the CaM antagonist, TFP, in regulation of CaM-Fas binding and Fas-mediated DISC formation in a concentration-dependent manner.

TNF-α-Induced Mitochondrial Alterations in Human T Cells Requires FADD and Caspase-8 Activation but Not RIP and Caspase-3 Activation

Although much is known about how TNF-alpha induces apoptosis in the presence of inhibitors of protein synthesis, little is known about how it induces apoptosis without these inhibitors. In this report we investigated temporal sequence of events induced by TNF-alpha in the absence of protein synthesis. Regardless of whether we measured the effects by plasma membrane phosphotidylserine accumulation, by DNA strand breaks, or activation of caspases, significant changes were observed only between 12-24 h of TNF-alpha treatment. One of the earliest changes observed after TNF-alpha treatment was mitochondrial swelling at 10 min; followed by cytochrome c and Smac release at 10-30 min, and then heterochromatin clumping occurred at 60 min. While genetic deletion of receptor-interaction protein (RIP) had no effect on TNF-alpha-induced mitochondrial damage, deletion of Fas-associated death domain (FADD) abolished the TNF-induced mitochondrial swelling. Since pan-caspase inhibitor z-VAD-fmk abolished the TNF-alpha-induced mitochondrial changes, z-DEVD-fmk, an inhibitor of caspase-3 had no effect, suggesting that TNF-alpha-induced mitochondrial changes or cytochrome c and Smac release requires caspase-8 but not caspase-3 activation. Overall, our results indicated that mitochondrial changes are early events in TNF-alpha-induced apoptosis and that these mitochondrial changes require recruitment of FADD and caspase-8 activation, but not caspase-3 activation or RIP recruitment.

Inhibition of methionine adenosyltransferase II induces FasL expression, Fas-DISC formation and caspase-8-dependent apoptotic death in T leukemic cells

Methionine adenosyltransferase II (MAT II) is a key enzyme in cellular metabolism and catalyzes the formation of S-adenosylmethionine (SAMe) from L-methionine and ATP. Normal resting T lymphocytes have minimal MAT II activity, whereas activated proliferating T lymphocytes and transformed T leukemic cells show significantly enhanced MAT II activity. This work was carried out to examine the role of MAT II activity and SAMe biosynthesis in the survival of leukemic T cells. Inhibition of MAT II and the resultant decrease in SAMe levels enhanced expression of FasL mRNA and protein, and induced DISC (Death Inducing Signaling Complex) formation with FADD (Fas-associated Death Domain) and procaspase-8 recruitment, as well as concomitant increase in caspase-8 activation and decrease in c-FLIP(s) levels. Fas-initiated signaling induced by MAT II inhibition was observed to link to the mitochondrial pathway via Bid cleavage and to ultimately lead to increased caspase-3 activation and DNA fragmentation in these cells. Furthermore, blocking MAT 2A mRNA expression, which encodes the catalytic subunits of MAT II, using a small-interfering RNA approach enhanced FasL expression and cell death, validating the essential nature of MAT II activity in the survival of T leukemic cells.

Receptor-mediated Endocytosis Is Not Required for Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-induced Apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is selectively toxic to tumor compared with normal cells. Other members of the TNF family of death ligands (TNF, CD95L) engage their respective receptors (TNF-R1 and CD95), resulting in internalization of receptor and ligand and recruitment of adaptor proteins to the caspase activation platform known as the death-inducing signaling complex (DISC). Recently, TNF-R1 and CD95 have been shown to induce apoptosis with an absolute requirement for internalization of their corresponding receptors in the formation of a DISC. We show that TRAIL and its receptors are rapidly endocytosed in a time- and concentration-dependent manner. Blockade of receptor internalization with hyperosmotic sucrose did not inhibit TRAIL-induced apoptosis but, rather, amplified the apoptotic signaling of TRAIL. Plate-bound and soluble TRAIL induced similar levels of apoptosis. Together these results suggest that neither ligand nor receptor internalization is required for TRAIL-induced apoptosis. Internalization of TRAIL is mediated primarily by clathrin-dependent endocytosis and also by clathrin-independent pathways. Inhibition of clathrin-dependent internalization by overexpression of dominant negative forms of dynamin or AP180 did not inhibit TRAIL-induced apoptosis. Consistent with the finding that neither internalization of TRAIL nor its receptors is required for transmission of its apoptotic signal, recruitment of FADD (Fas-associated death domain) and procaspase-8 to form the TRAIL-associated DISC occurred at 4 degrees C, independent of endocytosis. Our findings demonstrate that TRAIL and TRAIL receptor 1/2, unlike TNF-TNF-R1 or CD95L-CD95, do not require internalization for formation of the DISC, activation of caspase-8, or transmission of an apoptotic signal in BJAB type I cells.

Peltophorum dubium and soybean Kunitz-type trypsin inhibitors induce human Jurkat cell apoptosis

Plants constitute an important source of compounds which can induce apoptosis in a variety of cells. Previously, we reported the isolation of a trypsin inhibitor from Peltophorum dubium seeds (PDTI). This inhibitor, as well as soybean trypsin inhibitor (SBTI), both belonging to the Kunitz family, have lectin-like properties and trigger rat lymphoma cell apoptosis. In the present study, we demonstrate for the first time that PDTI and SBTI induce human leukemia Jurkat cell death. Induction of apoptosis was confirmed by flow cytometry after propidium iodide labeling of apoptotic nuclei, showing a considerable increase of the sub G 0/ G 1 fraction, with no cell cycle arrest. With the purpose of gaining insight into the signaling pathways involved, we investigated the activation of caspases and the effect of caspase inhibitors, and showed caspases-3 and -8-like activation by PDTI or SBTI-treatment. Consistent with these results, pan caspase inhibitor and caspase-8 inhibitor protected Jurkat cells from apoptosis. However, there was no caspase-9 activation, confirmed by the failure of caspase-9 inhibitor to prevent cell death. No significant release of cytochrome c from mitochondria was detected suggesting that the intrinsic mitochondrial pathway is not predominant in the apoptotic process. On the other hand, recruitment of Fas-associated death domain (FADD) to the cell membrane indicates the involvement of this adaptor protein in PDTI- and SBTI-induced apoptosis in Jurkat cells. Furthermore, human peripheral lymphocytes, either stimulated with phytohemagglutinin or not, are also susceptible to viability decrease induced by these inhibitors.

Fas Signaling in Thyroid Carcinomas Is Diverted from Apoptosis to Proliferation

The death receptor Fas is present in thyroid carcinomas, yet fails to trigger apoptosis. Interestingly, Fas has been reported to be actually overexpressed in papillary thyroid carcinomas, suggesting that it may confer a survival advantage. We investigated the expression and activation status of Fas pathway mediators in thyroid carcinoma cell lines and tumor specimens. All cell lines tested express Fas-associated death domain, procaspase-8, procaspase-9, and procaspase-3; resistance to Fas-mediated apoptosis could not be attributed to lack of any of these apoptosis mediators. Moreover, Fas death domain mutations were not found in our study. The proteasome inhibitors MG132 and PS-341 (bortezomib, Velcade), which lead to accumulation of the nuclear factor kappaB (NF-kappaB) inhibitor IkappaB, did not sensitize SW579 cells to Fas-mediated apoptosis, suggesting that resistance to Fas-mediated apoptosis is not due to proteasome or NF-kappaB activity. Cross-linking of Fas in vitro induced recruitment of Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein (FLIP) instead of procaspase-8. Inhibition of FLIP expression with a FLIP antisense oligonucleotide resulted in significant sensitization to Fas-mediated apoptosis. Fas cross-linking promoted BrdUrd incorporation; activated the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase, NF-kappaB, and activator protein-1 pathways in thyroid carcinoma cells in vitro; and protected cells from tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. We also found that good prognosis papillary thyroid carcinoma specimens exhibited higher immunoreactivity for cleaved (activated) caspase-8 than poor prognosis tumors. In thyroid carcinomas, the proteolytic cleavage and activation of caspase-8 depends on the balance between expression levels for procaspase-8 and FLIP and correlates with favorable clinical prognosis. Fas may actually stimulate proliferation and confer a survival advantage to thyroid cancer cells.

Doxazosin Induces Apoptosis of Benign and Malignant Prostate Cells via a Death Receptor–Mediated Pathway

Quinazoline-based alpha1-adrenoceptor antagonists such as doxazosin and terazosin have been previously shown to induce apoptosis in prostate cancer cells via an alpha1-adrenoceptor-independent pathway, involving activation of transforming growth factor-beta1 (TGF-beta1) signaling. In this study, the molecular events initiating this apoptotic effect were further investigated in vitro using the human androgen-independent prostate cancer cells PC-3 and the human benign prostate epithelial cells BPH-1. Quantitative microarray assays were done in PC-3 and BPH-1 cells after treatment with doxazosin (25 micromol/L, 6 and 24 hours) to identify the early gene changes. Transient changes in the expression of several apoptosis regulators were identified, including up-regulation of Bax and Fas/CD95 and down-regulation of Bcl-xL and TRAMP/Apo3. Moreover, there were significant changes in the expression pattern of signaling components of the extracellular matrix such as integrins alpha2, alphaV, beta1, and beta8. Western blot analysis revealed activation of caspase-8 and caspase-3 within the first 6 to 12 hours of treatment with doxazosin in both PC-3 and BPH-1 cells. Doxazosin-induced apoptosis was blocked by specific caspase-8 inhibitors, supporting the functional involvement of caspase-8 in doxazosin-induced apoptosis. The effect of doxazosin on recruitment of Fas-associated death domain (FADD) and procaspase-8 to the Fas receptor was examined via analysis of death-inducing signaling complex formation. Doxazosin increased FADD recruitment and subsequent caspase-8 activation, implicating Fas-mediated apoptosis as the underlying mechanism of the effect of doxazosin in prostate cells. These results show that doxazosin exerts its apoptotic effects against benign and malignant prostate cells via a death receptor-mediated mechanism with a potential integrin contribution towards cell survival outcomes.

Involvement of NADPH Oxidase Isoforms and Src Family Kinases in CD95-dependent Hepatocyte Apoptosis

CD95 ligand (CD95L) triggers a rapid formation of reactive oxygen species (ROS) as an upstream event of CD95 activation and apoptosis induction in rat hepatocytes. This ROS response was sensitive to inhibition by diphenyleneiodonium, apocynin, and neopterin, suggestive of an involvement of NADPH oxidases. In line with this, hepatocytes expressed mRNAs not only of the phagocyte gp91phox (Nox 2), but also of the homologs Nox 1 and 4 and Duox 1 and 2, as well as the regulatory subunit p47phox. gp91phox (Nox 2) and p47phox were also identified at the protein level in rat hepatocytes. CD95L induced within 1 min ceramide formation and serine phosphorylation of p47phox, which was sensitive to inhibitors of sphingomyelinase and protein kinase Czeta (PKCzeta). These inhibitors and p47phox protein knockdown inhibited the early CD95L-induced ROS response, suggesting that ceramide and PKCzeta are upstream events of the CD95L-induced Nox/Duox activation. CD95L also induced rapid activation of the Src family kinase Yes, being followed by activation of c-Src, Fyn, and c-Jun-N-terminal kinases (JNK). Only Yes and JNK activation were sensitive to N-acetylcysteine, inhibitors of NADPH oxidase, PKCzeta, or sphingomyelinase, indicating that the CD95L-induced ROS response is upstream of Yes and JNK but not of Fyn and c-Src activation. Activated Yes rapidly associated with the epidermal growth factor receptor (EGFR), which became phosphorylated at Tyr845 and Tyr1173 but not at Tyr1045. Activated EGFR then triggered an AG1478-sensitive CD95-tyrosine phosphorylation, which was a signal for membrane targeting of the EGFR/CD95 complex, subsequent recruitment of Fas-associated death domain and caspase 8, and apoptosis induction. All of these events were significantly blunted by inhibitors of sphingomyelinase, PKCzeta, NADPH oxidases, Yes, or EGFR-tyrosine kinase activity and after protein knockdown of either p47phox, Yes, or EGFR. The data suggest that CD95L-induced apoptosis involves a sphingomyelinase- and PKCzeta-dependent activation of NADPH oxidase isoforms, which is required for Yes/EGFR/CD95 interactions as upstream events of CD95 activation.

NADPH oxidase-dependent reactive oxygen species are important to the early stage of CD95 engagement in hepatocytes

CD95 ligand (CD95L) triggers a rapid formation of reactive oxygen species (ROS) as an upstream event of CD95 activation and apoptosis induction in rat hepatocytes. This ROS response was sensitive to inhibition by diphenyleneiodonium, apocynin, and neopterin, suggestive of an involvement of NADPH oxidases. In line with this, hepatocytes expressed mRNAs not only of the phagocyte gp91phox (Nox 2), but also of the homologs Nox 1 and 4 and Duox 1 and 2, as well as the regulatory subunit p47phox. gp91phox (Nox 2) and p47phox were also identified at the protein level in rat hepatocytes. CD95L induced within 1 min ceramide formation and serine phosphorylation of p47phox, which was sensitive to inhibitors of sphingomyelinase and protein kinase Cζ (PKCζ). These inhibitors and p47phox protein knockdown inhibited the early CD95L-induced ROS response, suggesting that ceramide and PKCζ are upstream events of the CD95Linduced Nox/Duox activation. CD95L also induced rapid activation of the Src family kinase Yes, being followed by activation of c-Src, Fyn, and c-Jun-N-terminal kinases (JNK). Only Yes and JNK activation were sensitive to N-acetylcysteine, inhibitors of NADPH oxidase, PKCζ, or sphingomyelinase, indicating that the CD95L-induced ROS response is upstream of Yes and JNK but not of Fyn and c-Src activation. Activated Yes rapidly associated with the epidermal growth factor receptor (EGFR), which became phosphorylated at Tyr845 and Tyr1173 but not at Tyr1045. Activated EGFR then triggered an AG1478-sensitive CD95-tyrosine phosphorylation, which was a signal for membrane targeting of the EGFR/CD95 complex, subsequent recruitment of Fas-associated death domain and caspase 8, and apoptosis induction. All of these events were significantly blunted by inhibitors of sphingomyelinase, PKCζ, NADPH oxidases, Yes, or EGFR-tyrosine kinase activity and after protein knockdown of either p47phox, Yes, or EGFR. The data suggest that CD95L-induced apoptosis involves a sphingomyelinase- and PKCζ-dependent activation of NADPH oxidase isoforms, which is required for Yes/EGFR/CD95 interactions as upstream events of CD95 activation. (HEPATOLOGY 2005;42:956–958.)

The C-terminal Tails of Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) and Fas Receptors Have Opposing Functions in Fas-associated Death Domain (FADD) Recruitment and Can Regulate Agonist-specific Mechanisms of Receptor Activation

Members of the tumor necrosis factor (TNF) superfamily of receptors such as Fas/CD95 and the TNF-related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5 induce apoptosis by recruiting adaptor molecules and caspases. The central adaptor molecule for these receptors is a death domain-containing protein, FADD, which binds to the activated receptor via death domain-death domain interactions. Here, we show that in addition to the death domain, the C-terminal tails of DR4 and DR5 positively regulate FADD binding, caspase activation and apoptosis. In contrast, the corresponding region in the Fas receptor has the opposite effect and inhibits binding to the receptor death domain. Replacement of wild-type or mutant DR5 molecules into DR5-deficient BJAB cells indicates that some agonistic antibodies display an absolute requirement for the C-terminal tail for FADD binding and signaling while other antibodies can function in the absence of this mechanism. These data demonstrate that regions outside the death domains of DR4 and DR5 have opposite effects to that of Fas in regulating FADD recruitment and show that different death receptor agonists can use distinct molecular mechanisms to activate signaling from the same receptor.

Casein Kinase I Attenuates Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Induced Apoptosis by Regulating the Recruitment of Fas-Associated Death Domain and Procaspase-8 to the Death-Inducing Signaling Complex

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a wide variety of malignant cell lines, in contrast to normal cells, but with considerable heterogeneity in response. Death receptor-mediated apoptosis may be attenuated by a variety of different mechanisms, including phosphorylation-based signaling pathways. We have demonstrated that casein kinase I can attenuate TRAIL-induced apoptosis in human cell lines derived from colon adenocarcinoma (HT29 and HCT8) and pediatric rhabdomyosarcoma (JR1). Inhibition of casein kinase I (CKI) phosphorylation events in HT29, HCT8, and JR1 cells by CKI-7 dramatically increased apoptosis after exposure to TRAIL, in the absence of apoptosis induced by TRAIL treatment alone. CKI inhibition enhanced the recruitment of Fas-associated death domain and procaspase-8 to the death-inducing signaling complex after TRAIL treatment and enhanced cleavage of procaspase-8 at the death-inducing signaling complex. In HT29 cells studied further, rapid cleavage of caspase-8, caspase-3, Bid, and the caspase substrate poly(ADP-ribose) polymerase occurred when CKI-7 and TRAIL were combined. Overexpression of Bcl-2, Bcl-xL, or mutant DN-Fas-associated death domain protected HT29 cells from TRAIL-induced apoptosis in the presence of the CKI inhibitor. In addition, TRAIL combined with CKI-7 promoted the release of cytochrome c, Smac/DIABLO, HtrA2/Omi, and AIF from the mitochondria and down-regulated the expression of XIAP and c-IAP1. Small hairpin RNAs directed against CKI revealed that the CKIalpha isoform contributed significantly to the inhibition of TRAIL-induced apoptosis. These findings suggest that CKIalpha plays an antiapoptotic role through the generation of phosphorylated sites at the level of the death-inducing signaling complex, thereby conferring resistance to caspase cleavage mediated by TRAIL.

PLEIOTROPIC EFFECT IN LYMPHOCYTE ACTIVATION CAUSED BY CASPASE-8 MUTATIONS LEAD TO HUMAN IMMUNODEFICIENCY

Chun HJ, Zheng L, Ahmad M, et al. Nature. 2002;419:395–399Defects in genes involved in the apoptosis (programmed cell death) of lymphocytes have been shown to cause disorders characterized by significant adenopathy and autoimmunity. This study defines a new defect in that category and characterizes the clinical presentation. Mutations in the caspase-8 gene not only affect apoptosis but also affect host defense. This article describes a heretofore unrecognized function of caspase-8 in lymphocyte activation.The study relies on a single kindred with 2 affected children and the heterozygous parents and sibling. The carriers of the mutation were asymptomatic.The authors carefully characterize the apoptosis of cells from the affected children as well as the activation of their lymphocytes. Traditional killing studies were performed to define the defect in apoptosis as well as flow cytometric determinations of T cell activation. Cytokine production and proliferation were also measured to delineate the defect in T cell function.The 2 patients with defects in caspase-8 had significant adenopathy and splenomegaly. These 2 features have been observed in all patients with defects in genes involved in lymphocyte apoptosis. In addition, these patients had several features that appear to be specific for caspase-8 defects. The patients had diminished CD4 counts, elevated CD8 T cell counts, poor responses to immunization, failure to thrive, recurrent herpes infections, and other recurrent infections. Mechanistic studies revealed defective apoptosis attributable to Fas-mediated signals but preserved apoptosis in response to mitochondrial-mediated signals. Western blots suggested that the Fas signal was intact up to recruitment of Fas-associated death domain, but that subsequent recruitment of caspase-8 was defective. Sequencing revealed a homozygous caspase-8 mutation. In addition to the defect in apoptosis, the authors describe a very significant defect in lymphocyte activation that probably accounts for the immunodeficiency phenotype. B cells, T cells, and NK cells are all defective in this disorder.Caspase-8 mutations lead to a disorder characterized by expansion of lymphocytes in secondary lymphoid organs and abnormal lymphocyte activation.This article is significant for introducing us to a new immunodeficiency and for the demonstration that caspase-8 is involved in lymphocyte activation. The 3 known defects of lymphocyte apoptosis, Fas deficiency, FasL deficiency, and caspase 10 deficiency all have a similar phenotype: the early presentation of significant adenopathy, splenomegaly, and autoimmune disease. Caspase-8 is now known to have an undefined but integral function in lymphocyte activation and the phenotype of patients with caspase-8 deficiency reflects this, with both lymphoid expansion and immunodeficiency.

Chemotherapy enhances TNF-related apoptosis-inducing ligand DISC assembly in HT29 human colon cancer cells.

Cytokines such as Fas-ligand (Fas-L) and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) can induce human colon cancer cell apoptosis through engagement of their death domain receptors. All the cancer cells are not sensitive to these cytokines. We have shown recently that low doses of cytotoxic drugs could restore TRAIL-induced cell death in resistant colon cancer cell lines. The present work further explores the death pathway triggered by the cytotoxic drug/TRAIL combination in HT-29 colon cancer cells (www.alexis-corp.com). Clinically relevant concentrations of cisplatin, doxorubicin and 5-fluorouracil synergize with TRAIL to trigger HT-29 cell death. Activation of this pathway leads to apoptosis that involves both caspases and the mitochondria. An increased recruitment of Fas-associated death domain (FADD) and procaspase-8 to the TRAIL-induced death-inducing signaling complex (DISC) was shown in cells exposed to anticancer drugs. Following caspase-8 activation at the DISC level, the mitochondria-dependent death pathway is activated, as demonstrated by the cleavage of Bid, the dissipation of DeltaPsi(m), the release of mitochondrial proteins in the cytosol and the inhibitory effect of Bcl-2 expression. Importantly, besides mitochondrial potentiation, we show here that cytotoxic drugs sensitize HT-29 colon cancer cells to TRAIL-induced cell death by enhancing FADD and procaspase-8 recruitment to the DISC, a novel mechanism whose efficacy could depend partly on Bcl-2 expression level.

Expression of Dominant-negative Fas-associated Death Domain Blocks Human Keratinocyte Apoptosis and Vesication Induced by Sulfur Mustard

DNA damaging agents up-regulate levels of the Fas receptor or its ligand, resulting in recruitment of Fas-associated death domain (FADD) and autocatalytic activation of caspase-8, consequently activating the executioner caspases-3, -6, and -7. We found that human epidermal keratinocytes exposed to a vesicating dose (300 microm) of sulfur mustard (SM) exhibit a dose-dependent increase in the levels of Fas receptor and Fas ligand. Immunoblot analysis revealed that the upstream caspases-8 and -9 are both activated in a time-dependent fashion, and caspase-8 is cleaved prior to caspase-9. These results are consistent with the activation of both death receptor (caspase-8) and mitochondrial (caspase-9) pathways by SM. Pretreatment of keratinocytes with a peptide inhibitor of caspase-3 (Ac-DEVD-CHO) suppressed SM-induced downstream markers of apoptosis. To further analyze the importance of the death receptor pathway in SM toxicity, we utilized Fas- or tumor necrosis factor receptor-neutralizing antibodies or constructs expressing a dominant-negative FADD (FADD-DN) to inhibit the recruitment of FADD to the death receptor complex and block the Fas/tumor necrosis factor receptor pathway following SM exposure. Keratinocytes pretreated with Fas-blocking antibody or stably expressing FADD-DN and exhibiting reduced levels of FADD signaling demonstrated markedly decreased caspase-3 activity when treated with SM. In addition, the processing of procaspases-3, -7, and -8 into their active forms was observed in SM-treated control keratinocytes, but not in FADD-DN cells. Blocking the death receptor complex by expression of FADD-DN additionally inhibited SM-induced internucleosomal DNA cleavage and caspase-6-mediated nuclear lamin cleavage. Significantly, we further found that altering the death receptor pathway by expressing FADD-DN in human skin grafted onto nude mice reduces vesication and tissue injury in response to SM. These results indicate that the death receptor pathway plays a pivotal role in SM-induced apoptosis and is therefore a target for therapeutic intervention to reduce SM injury.

The Mycotoxin Penicillic Acid Inhibits Fas Ligand-induced Apoptosis by Blocking Self-processing of Caspase-8 in Death-inducing Signaling Complex

Upon engagement with Fas ligand (FasL), Fas rapidly induces recruitment and self-processing of caspase-8 via the adaptor protein Fas-associated death domain (FADD), and activated caspase-8 cleaves downstream substrates such as caspase-3. We have found that penicillic acid (PCA) inhibits FasL-induced apoptosis and concomitant loss of cell viability in Burkitt's lymphoma Raji cells. PCA prevented activation of caspase-8 and caspase-3 upon treatment with FasL. However, PCA did not affect active caspase-3 in FasL-treated cells, suggesting that PCA primarily blocks early signaling events upstream of caspase-8 activation. FasL-induced processing of caspase-8 was severely impaired in the death-inducing signaling complex, although FasL-induced recruitment of FADD and caspase-8 occurred normally in PCA-treated cells. Although PCA inhibited the enzymatic activities of active recombinant caspase-3, caspase-8, and caspase-9 at similar concentrations, PCA exerted weak inhibitory effects on activation of caspase-9 and caspase-3 in staurosporine-treated cells but strongly inhibited caspase-8 activation in FasL-treated cells. Glutathione and cysteine neutralized an inhibitory effect of PCA on caspase-8, and PCA bound directly to the active center cysteine in the large subunit of caspase-8. Thus, our present results demonstrate that PCA inhibits FasL-induced apoptosis by targeting self-processing of caspase-8.

Prevention of bile acid–induced apoptosis by betaine in rat liver

Bile acid–induced apoptosis plays an important role in the pathogenesis of cholestatic liver disease, and its prevention is of therapeutic interest. The effects of betaine were studied on taurolithocholate 3-sulfate (TLCS) and glycochenodeoxycholate (GCDC)-induced apoptosis in rat hepatocytes in vitro and in vivo. Hepatocyte apoptosis, caspase activation, and poly (ADP-ribose) polymerase (PARP) cleavage, which are normally observed in response to both bile acids, were largely prevented after preincubation of hepatocytes with betaine. Betaine uptake was required for this protective effect, which was already observed at betaine concentrations of 1 mmol/L. Betaine did not affect the TLCS-induced membrane trafficking of CD95 and tumor necrosis factor–related apoptosis inducing ligand (TRAIL) receptor 2 to the plasma membrane or the TLCS-induced recruitment of Fas-associated death domain (FADD) and caspase 8 to the CD95 receptor. However, betaine largely prevented cytochrome c release and oxidative stress exerted otherwise by TLCS. Inhibition of caspase 9 strongly blunted TLCS-induced caspase-8 activation. Further betaine did not prevent the TLCS-induced c-Jun N-terminal kinase (JNK), extracellular signal–regulated kinase (Erk), and p38 mitogen-activated protein kinase (p38 MAPK) activation or TLCS-induced protein kinase B (PKB) dephosphorylation. The protective betaine effect was insensitive to inhibition of Erks by PD089059, of p38 MAPK by SB203580, or of phosphatidylinositol 3-kinase (PI3-kinase) by LY294002. Betaine supplementation in the drinking water significantly ameliorated in vivo hepatocyte apoptosis following bile duct ligation. In conclusion, this study identifies betaine as a potent protectant against bile acid–induced apoptosis in vivo and in vitro, and its antiapoptotic action largely resides on an inhibition of the proapoptotic mitochondrial pathway. (H EPATOLOGY 2002;36:829-839.)

Death Domain Mutagenesis of KILLER/DR5 Reveals Residues Critical for Apoptotic Signaling

The Fas/tumor necrosis factor (TNF)/TRAIL receptors signal death through a cytoplasmic death domain (DD) containing six alpha-helices with positively charged helix 2 interacting with negatively charged helix 3 of another DD. DD mutation occurs in head/neck and lung cancer (TRAIL receptor KILLER/DR5) and in lpr mice (Fas). We examined the apoptotic potential of known KILLER/DR5 lung tumor-derived mutants (n = 6) and DD mutants (n = 18) generated based on conservation with DR4, Fas, Fas-associated death domain (FADD), and tumor necrosis factor receptor 1 (TNFR1). With the exception of Arg-330 required in Fas or FADD for aggregation or for TNFR1 cytotoxicity, surprisingly major loss-of-function KILLER/DR5 alleles (W325A, L334A (lpr-like), I339A, and W360A) contained hydrophobic residues. Loss-of-function of I339A (highly conserved) has not been reported in DDs. Charged residue mutagenesis revealed the following points. 1) E326A, conserved in DR4, is dispensable for death; the homologous residue is positively charged in Fas, TNFR1, and FADD and is critical for DD interactions. 2) K331A, D336A, E338A, K340A, K343A, and D351A have partial loss-of-function suggesting multiple charges stabilize receptor-adapter interactions. Analysis of the tumor-derived KILLER/DR5 mutants revealed the following. 1) L334F has partial loss-of-function versus L334A, whereas E338K has major loss-of-function versus E338A, examples where alanine and tumor-specific substitutions have divergent phenotypes. 2) Unexpectedly, S324F, E326K, K386N, and D407Y have no loss-of-function with tumor-specific or alanine substitutions. Loss-of-function KILLER/DR5 mutants were deficient in recruitment of FADD and caspase 8 to TRAIL death-inducing signaling complexes. The results reveal determinants within KILLER/DR5 for death signaling and drug design.