IL-10 protects mouse intestinal epithelial cells from Fas-induced apoptosis via modulating Fas expression and altering caspase-8 and FLIP expression

We have previously shown that the absence of Fas/Fas ligand significantly reduced tissue damage and intestinal epithelial cell (IEC) apoptosis in an in vivo model of T cell-mediated enteropathy. This enteropathy was more severe in IL-10-deficient mice, and this was associated with increased serum levels of IFN-gamma and TNF-alpha and an increase in Fas expression on IECs. In this study, we investigated the potential of IL-10 to directly influence Fas expression and Fas-induced IEC apoptosis. Mouse intestinal epithelial cell lines MODE-K and IEC4.1 were cultured with IFN-gamma, TNF-alpha, or anti-Fas monoclonal antibody (mAb) in the presence or absence of IL-10. Fas expression and apoptosis were determined by FACScan analysis of phycoerythrin-anti-Fas mAb staining and annexin V staining, respectively. Treatment with a combination of IFN-gamma and TNF-alpha induced significant apoptosis. Anti-Fas mAb alone did not induce much apoptosis unless cells were pretreated with IFN-gamma and TNF-alpha. These IECs constitutively expressed low levels of Fas, which significantly increased by preincubation of the cells with IFN-gamma and TNF-alpha. Treatment with cytokine or cytokine plus anti-Fas mAb increased apoptosis, which correlated with a decreased Fas-associated death domain IL-1-converting enzyme-like inhibitory protein (FLIP) level, increased caspase-8 activity, and subsequently increased caspase-3 activity. IL-10 diminished both cytokine- and anti-Fas mAb-induced apoptosis, and this was correlated with decreased cytokine-induced Fas expression, increased FLIP, and decreased caspase-8 and caspase-3 activity. In conclusion, IL-10 modulated cytokine induction of Fas expression on IEC cell lines and regulated IEC susceptibility to TNF-alpha, IFN-gamma, and Fas-mediated apoptosis. These findings suggest that IL-10 directly modulates IEC responses to T cell-mediated apoptotic signals.     

Enhanced expression of Fas-associated death domain-like IL-1-converting enzyme (FLICE)-inhibitory protein induces resistance to Fas-mediated apoptosis in activated mast cells

Mast cells play a critical role in host immune responses and are implicated in the pathogenesis of allergic inflammation. Though mouse mast cell line MC/9 expresses cell surface Fas Ag and is sensitive to Fas-induced apoptosis, activated MC/9 cells are resistant to Fas-induced cell death by cross-linking of FcepsilonRI or FcgammaR. Fas-associated death domain-like IL-1-converting enzyme (FLICE)-inhibitory protein (FLIP), a caspase-8 inhibitor that lacks the cysteine domain, is one of the negative regulators of receptor-mediated apoptosis. In this report, we show that activation of mast cells by cross-linking of FcepsilonRI or FcgammaR can induce enhanced expression of FLIP and consequently a resistance to Fas-induced apoptosis, although the expression level of Fas Ag is not changed. Addition of antisense oligonucleotide for FLIP prevents resistance to Fas-induced apoptosis of activated mast cells, suggesting that endogenous FLIP inhibits Fas-mediated apoptosis in activated mast cells. Thus, the enhanced expression of FLIP in activated mast cells contributes to the resistance to Fas-induced apoptosis, which may result in the development and prolongation of allergic inflammation.     

Nitric oxide negatively regulates Fas CD95-induced apoptosis through inhibition of ubiquitin-proteasome-mediated degradation of FLICE inhibitory protein

Stimulation of cell surface Fas (CD95) results in recruitment of cytoplasmic proteins and activation of caspase-8, which in turn activates downstream effector caspases leading to programmed cell death. Nitric oxide (NO) plays a key role in the regulation of apoptosis, but its role in Fas-induced cell death and the underlying mechanism are largely unknown. Here we show that stimulation of the Fas receptor by its ligand (FasL) results in rapid generation of NO and concomitant decrease in cellular FLICE inhibitory protein (FLIP) expression without significant effect on Fas and Fas-associated death domain (FADD) adapter protein levels. FLIP down-regulation as well as caspase-8 activation and apoptosis induced by FasL were all inhibited by the NO-liberating agent sodium nitroprusside and dipropylenetriamine NONOate, whereas the NO synthase inhibitor aminoguanidine and NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO) had opposite effects, indicating an anti-apoptotic role of NO in the Fas signaling process. FasL-induced down-regulation of FLIP is mediated by a ubiquitin-proteasome pathway that is negatively regulated by NO. S-nitrosylation of FLIP is an important mechanism rendering FLIP resistant to ubiquitination and proteasomal degradation by FasL. Deletion analysis shows that the caspase-like domain of FLIP is a key target for S-nitrosylation by NO, and mutations of its cysteine 254 and cysteine 259 residues completely inhibit S-nitrosylation, leading to increased ubiquitination and proteasomal degradation of FLIP. These findings indicate a novel pathway for NO regulation of FLIP that provides a key mechanism for apoptosis regulation and a potential new target for intervention in death receptor-associated diseases.     

Calmodulin binding to cellular FLICE-like inhibitory protein modulates Fas-induced signalling

We and others have demonstrated that Fas-mediated apoptosis is a potential therapeutic target for cholangiocarcinoma. Previously, we reported that CaM (calmodulin) antagonists induced apoptosis in cholangiocarcinoma cells through Fas-related mechanisms. Further, we identified a direct interaction between CaM and Fas with recruitment of CaM into the Fas-mediated DISC (death-inducing signalling complex), suggesting a novel role for CaM in Fas signalling. Therefore we characterized the interaction of CaM with proteins recruited into the Fas-mediated DISC, including FADD (Fas-associated death domain)-containing protein, caspase 8 and c-FLIP {cellular FLICE [FADD (Fas-associated death domain)-like interleukin 1beta-converting enzyme]-like inhibitory protein}. A Ca(2+)-dependent direct interaction between CaM and FLIP(L), but not FADD or caspase 8, was demonstrated. Furthermore, a 37.3+/-5.7% increase (n=6, P=0.001) in CaM-FLIP binding was observed at 30 min after Fas stimulation, which returned to the baseline after 60 min and correlated with a Fas-induced increase in intracellular Ca(2+) that reached a peak at 30 min and decreased gradually over 60 min in cholangiocarcinoma cells. A CaM antagonist, TFP (trifluoperazine), inhibited the Fas-induced increase in CaM-FLIP binding concurrent with inhibition of ERK (extracellular-signal-regulated kinase) phosphorylation, a downstream signal of FLIP. Direct binding between CaM and FLIP(L) was demonstrated using recombinant proteins, and a CaM-binding region was identified in amino acids 197-213 of FLIP(L). Compared with overexpression of wild-type FLIP(L) that resulted in decreased spontaneous as well as Fas-induced apoptosis, mutant FLIP(L) with deletion of the CaM-binding region resulted in increased spontaneous and Fas-induced apoptosis in cholangiocarcinoma cells. Understanding the biology of CaM-FLIP binding may provide new therapeutic targets for cholangiocarcinoma and possibly other cancers.     

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.     

Mechanisms of spontaneous resolution versus fibrosis in granulomatous experimental autoimmune thyroiditis

When granulomatous experimental autoimmune thyroiditis (G-EAT) was induced in CBA/J or DBA/1 mice, thyroid lesions resolved in less severe (3+) G-EAT in wild-type mice or severe (5+) G-EAT in IFN-gamma(-/-) mice, but progressed to fibrosis in 5+ G-EAT in wild-type mice. To define the mechanisms leading to these distinct outcomes, the expression of inflammatory and apoptotic molecules and infiltrating cells was evaluated using immunohistochemistry, RT-PCR, and confocal microscopy. The ratio of CD4(+)/CD8(+) T cells in thyroid infiltrates was one factor that predicted G-EAT outcome. CD4(+) T cells outnumbered CD8(+) T cells when lesions progressed to fibrosis, while CD8(+) T cells outnumbered CD4(+) T cells in thyroids that resolved. Fas, Fas ligand, FLIP, TNF-alpha, inducible NO synthase, TGF-beta, and IFN-gamma were highly expressed by infiltrating cells when G-EAT progressed to fibrosis. The expression of active caspase-3 was low, possibly contributing to the persistence of CD4(+) T cells in fibrosis. In contrast, FLIP was mainly expressed by thyrocytes in resolving G-EAT, the expression of active caspase-3 was high, and resolution correlated with apoptosis of infiltrating cells. There was also relatively less expression of TGF-beta, IFN-gamma, TNF-alpha, and inducible NO synthase and higher expression of IL-10 in resolving G-EAT than in G-EAT that progressed to fibrosis. These differences were particularly striking when comparing IFN-gamma(-/-) vs wild-type mice. These results suggest that several opposing biological mechanisms contribute to the outcome of an ongoing autoimmune response. These include differential expression of pro- and antiapoptotic molecules, cytokines, and the ratio of CD4(+) vs CD8(+) T cells.     

NFkappaB activation by Fas is mediated through FADD, caspase-8, and RIP and is inhibited by FLIP

Fas (APO-1/CD95) is the prototypic death receptor, and the molecular mechanisms of Fas-induced apoptosis are comparably well understood. Here, we show that Fas activates NFkappaB via a pathway involving RIP, FADD, and caspase-8. Remarkably, the enzymatic activity of the latter was dispensable for Fas-induced NFkappaB signaling pointing to a scaffolding-related function of caspase-8 in nonapoptotic Fas signaling. NFkappaB was activated by overexpressed FLIPL and FLIPS in a cell type-specific manner. However, in the context of Fas signaling both isoforms blocked FasL-induced NFkappaB activation. Moreover, down-regulation of both endogenous FLIP isoforms or of endogenous FLIPL alone was sufficient to enhance FasL-induced expression of the NFkappaB target gene IL8. As NFkappaB signaling is inhibited during apoptosis, FasL-induced NFkappaB activation was most prominent in cells that were protected by Bcl2 expression or caspase inhibitors and expressed no or minute amounts of FLIP. Thus, protection against Fas-induced apoptosis in a FLIP-independent manner converted a proapoptotic Fas signal into an inflammatory NFkappaB-related response.     

Fas receptor clustering and involvement of the death receptor pathway in rituximab-mediated apoptosis with concomitant sensitization of lymphoma B cells to fas-induced apoptosis

Ab binding to CD20 has been shown to induce apoptosis in B cells. In this study, we demonstrate that rituximab sensitizes lymphoma B cells to Fas-induced apoptosis in a caspase-8-dependent manner. To elucidate the mechanism by which Rituximab affects Fas-mediated cell death, we investigated rituximab-induced signaling and apoptosis pathways. Rituximab-induced apoptosis involved the death receptor pathway and proceeded in a caspase-8-dependent manner. Ectopic overexpression of FLIP (the physiological inhibitor of the death receptor pathway) or application of zIETD-fmk (specific inhibitor of caspase-8, the initiator-caspase of the death receptor pathway) both specifically reduced rituximab-induced apoptosis in Ramos B cells. Blocking the death receptor ligands Fas ligand or TRAIL, using neutralizing Abs, did not inhibit apoptosis, implying that a direct death receptor/ligand interaction is not involved in CD20-mediated cell death. Instead, we hypothesized that rituximab-induced apoptosis involves membrane clustering of Fas molecules that leads to formation of the death-inducing signaling complex (DISC) and downstream activation of the death receptor pathway. Indeed, Fas coimmune precipitation experiments showed that, upon CD20-cross-linking, Fas-associated death domain protein (FADD) and caspase-8 were recruited into the DISC. Additionally, rituximab induced CD20 and Fas translocation to raft-like domains on the cell surface. Further analysis revealed that, upon stimulation with rituximab, Fas, caspase-8, and FADD were found in sucrose-gradient raft fractions together with CD20. In conclusion, in this study, we present evidence for the involvement of the death receptor pathway in rituximab-induced apoptosis of Ramos B cells with concomitant sensitization of these cells to Fas-mediated apoptosis via Fas multimerization and recruitment of caspase-8 and FADD to the DISC.     

Inhibition of histone deacetylase activity enhances Fas receptor-mediated apoptosis in leukemic lymphoblasts

We recently reported that butyrate, an inhibitor of histone deacetylases, is capable of inducing Fas-independent apoptosis in the acute lymphoblastic leukemia cell line CCRF-CEM. Here we demonstrate that butyrate enhances Fas-induced apoptosis in this cell line. The application of different histone deacetylase inhibitors revealed that tetra-acetylated histone H4 is associated with the amplifying effect of butyrate on Fas-induced cell death. FasL, Fas, FADD, RIP, caspase-8, caspase-3, Bid, FLIP(S+L), FLASH and FAP-1, proteins known to act within the Fas-apoptosis cascade, showed no changes in their expression levels in cells treated with butyrate compared with untreated cells. Analyses of Fas-oligomerization and Western blotting as well as enzyme activity assays of caspase-2, caspase-3 and caspase-8 suggest that butyrate enhances Fas-induced apoptosis downstream of Fas but upstream of caspase-8 activation. In immunoprecipitation experiments a 37 kD butyrate-regulated protein was detected which specifically interacts with caspase-8.     

Cycloheximide-induced T-cell death is mediated by a Fas-associated death domain-dependent mechanism

Cycloheximide (CHX) can contribute to apoptotic processes, either in conjunction with another agent (e.g. tumor necrosis factor-alpha) or on its own. However, the basis of this CHX-induced apoptosis has not been clearly established. In this study, the molecular mechanisms of CHX-induced cell death were examined in two different human T-cell lines. In T-cells undergoing CHX-induced apoptosis (Jurkat), but not in T-cells resistant to the effects of CHX (CEM C7), caspase-8 and caspase-3 were activated. However, the Fas ligand was not expressed in Jurkat cells either before or after treatment with CHX, suggesting that the activation of these caspases does not involve the Fas receptor. To determine whether CHX-induced apoptosis was mediated by a Fas-associated death domain (FADD)-dependent mechanism, a FADD-DN protein was expressed in cells prior to CHX treatment. Its expression effectively inhibited CHX-induced cell death, suggesting that CHX-mediated apoptosis primarily involves a FADD-dependent mechanism. Since CHX treatment did not result in the induction of Fas or FasL, and neutralizing anti-Fas and anti-tumor necrosis factor receptor-1 antibodies did not block CHX-mediated apoptosis, these results may also indicate that FADD functions in a receptor-independent manner. Surprisingly, death effector filaments containing FADD and caspase-8 were observed during CHX treatment of Jurkat, Jurkat-FADD-DN, and CEM C7 cells, suggesting that their formation may be necessary, but not sufficient, for cell death.     

The long form of FLIP is an activator of caspase-8 at the Fas death-inducing signaling complex

Death receptors, such as Fas and tumor necrosis factor-related apoptosis-inducing ligand receptors, recruit Fas-associated death domain and pro-caspase-8 homodimers, which are then autoproteolytically activated. Active caspase-8 is released into the cytoplasm, where it cleaves various proteins including pro-caspase-3, resulting in apoptosis. The cellular Fas-associated death domain-like interleukin-1-beta-converting enzyme-inhibitory protein long form (FLIP(L)), a structural homologue of caspase-8 lacking caspase activity because of several mutations in the active site, is a potent inhibitor of death receptor-induced apoptosis. FLIP(L) is proposed to block caspase-8 activity by forming a proteolytically inactive heterodimer with caspase-8. In contrast, we propose that FLIP(L)-bound caspase-8 is an active protease. Upon heterocomplex formation, a limited caspase-8 autoprocessing occurs resulting in the generation of the p43/41 and the p12 subunits. This partially processed form but also the non-cleaved FLIP(L)-caspase-8 heterocomplex are proteolytically active because they both bind synthetic substrates efficiently. Moreover, FLIP(L) expression favors receptor-interacting kinase (RIP) processing within the Fas-signaling complex. We propose that FLIP(L) inhibits caspase-8 release-dependent pro-apoptotic signals, whereas the single, membrane-restricted active site of the FLIP(L)-caspase-8 heterocomplex is proteolytically active and acts on local substrates such as RIP.     

Tocotrienol-induced caspase-8 activation is unrelated to death receptor apoptotic signaling in neoplastic mammary epithelial cells

Tocotrienols, a subclass in the vitamin E family of compounds, have been shown to induce apoptosis by activating caspase-8 and caspase-3 in neoplastic mammary epithelial cells. Since caspase-8 activation is associated with death receptor apoptotic signaling, studies were conducted to determine the exact death receptor/ligand involved in tocotrienol-induced apoptosis. Highly malignant +SA mouse mammary epithelial cells were grown in culture and maintained in serum-free media. Treatment with 20 microM gamma-tocotrienol decreased+SA cell viability by inducing apoptosis, as determined by positive terminal dUTP nick end labeling (TUNEL) immunocytochemical staining. Western blot analysis showed that gamma-tocotrienol treatment increased the levels of cleaved (active) caspase-8 and caspase-3. Combined treatment with caspase inhibitors completely blocked tocotrienol-induced apoptosis. Additional studies showed that treatment with 100 ng/ml tumor necrosis factor-alpha (TNF-alpha), 100 ng/ml FasL, 100 ng/ml TNF-related apoptosis-inducing ligand (TRAIL), or 1 microg/ml apoptosis-inducing Fas antibody failed to induce death in +SA cells, indicating that this mammary tumor cell line is resistant to death receptor-induced apoptosis. Furthermore, treatment with 20 microM gamma-tocotrienol had no effect on total, membrane, or cytosolic levels of Fas, Fas ligand (FasL), or Fas-associated via death domain (FADD) and did not induce translocation of Fas, FasL, or FADD from the cytosolic to the membrane fraction, providing additional evidence that tocotrienol-induced caspase-8 activation is not associated with death receptor apoptotic signaling. Other studies showed that treatment with 20 microM gamma-tocotrienol induced a large decrease in the relative intracellular levels of phospho-phosphatidylinositol 3-kinase (PI3K)-dependent kinase 1 (phospho-PDK-1 active), phospho-Akt (active), and phospho-glycogen synthase kinase3, as well as decreasing intracellular levels of FLICE-inhibitory protein (FLIP), an antiapoptotic protein that inhibits caspase-8 activation, in these cells. Since stimulation of the PI3K/PDK/Akt mitogenic pathway is associated with increased FLIP expression, enhanced cellular proliferation, and survival, these results indicate that tocotrienol-induced caspase-8 activation and apoptosis in malignant +SA mammary epithelial cells is associated with a suppression in PI3K/PDK-1/Akt mitogenic signaling and subsequent reduction in intracellular FLIP levels.     

Potentiation of Fas- and TRAIL-mediated apoptosis by IFN-gamma in A549 lung epithelial cells: enhancement of caspase-8 expression through IFN-response element

Epithelial cell apoptosis triggered cooperatively by multiple cytokines contributes to the injury induced by inflammatory responses in the lung and elsewhere. Here we show that interferon-gamma (IFN-gamma) sensitizes A549 cells, human lung epithelial cells, to cytokine-mediated apoptosis by upregulating caspase-8 expression. Pretreating the cells with IFN-gamma potentiated Fas- and TNF-related apoptosis inducing ligand (TRAIL)-induced cell death, but other forms of apoptosis, not mediated via receptors, were unaffected. Western blotting and inhibitor assays showed that IFN-gamma selectively increased expression of caspases-7 and -8, but not caspases-2, -3, -9, or -10, as a necessary step leading to apoptosis. Assaying promoter activity using a luciferase reporter gene indicated that an IFN-gamma response element was located in the 5'-flanking region of the caspase-8 gene, spanning positions -227 to -219. Taken together, these findings suggest that IFN-gamma potentiates Fas- and TRAIL-mediated apoptosis by increasing caspase-8 expression via an IFN-gamma response element in A549 cells.     

Rho family proteins modulate rapid apoptosis induced by cytotoxic T lymphocytes and Fas

Little is known about the role of Rho proteins in apoptosis produced by stimuli evolved specifically to produce apoptosis, such as granzymes from cytotoxic T lymphocytes (CTLs) and Fas. Here we demonstrate that all three Rho family members are involved in CTL- and Fas-induced killing. Dominant-negative mutants of each Rho family member and Clostridium difficile toxin B, an inhibitor of all family members, strongly inhibited the susceptibility of cells to CTL- and Fas-induced apoptosis. Fas-induced caspase-3 activation was inhibited by C. difficile toxin. Activated mutants of each GTPase increased susceptibility to apoptosis, and activation of Cdc42 increased within 5 min of Fas stimulation. In contrast, during the time required for CTL and Fas killing, no apoptosis was produced by dominant-negative or activated mutants or by C. difficile toxin alone. Inhibition of actin polymerization using latrunculin A reduced the ability of constitutively active GTPase mutants to stimulate apoptosis and blocked Fas-induced activation of caspase-3. Furthermore, the ability of Rac to enhance apoptosis was decreased by point mutations reported to block Rac induction of actin polymerization. Rho family proteins may regulate apoptosis through their effects on the actin cytoskeleton.     

Downregulation of FLIP by cycloheximide sensitizes human fat cells to CD95-induced apoptosis

Adipocyte apoptosis is an important regulator of adipocyte number in fat depots. We have previously shown that an inhibition of protein synthesis sensitizes human adipocytes for apoptosis. In vivo, dramatic changes in the fat cell's protein expression should be anticipated under special conditions such as calorie restriction. Here, we studied the underlying mechanism by which human preadipocytes and adipocytes are sensitized for death receptor induced apoptosis in vitro.The protein synthesis blocker cycloheximide (CHX) sensitized human fat cells for CD95-induced apoptosis in a caspase-dependent manner. Treatment with CHX differentially changed expression of pro- and anti-apoptotic proteins. Most noticeably, FLICE-like inhibitory protein (FLIP) expression rapidly decreased during CHX treatment. Reduction of FLIP levels resulted in undetectable amounts of FLIP at the CD95 death-inducing signaling complex (DISC) upon CD95 stimulation, thereby enhancing recruitment and activation at caspase-8. Down-regulation of FLIP by shRNA sensitized preadipocytes for CD95-induced apoptosis. In mice, adipose tissue mRNA levels of Flip were down-regulated upon fasting.In conclusion, we identify FLIP as an important regulator of apoptosis sensitivity in fat cells. Modulating adipocyte homeostasis by apoptosis might provide a new therapeutic concept to get rid of excess adipose tissue, and FLIP might be a possible target molecule.     

The TRAF3-binding site of human molluscipox virus FLIP molecule MC159 is critical for its capacity to inhibit Fas-induced apoptosis

Members of the viral Flice/caspase-8 inhibitory protein (v-FLIP) family prevent induction of apoptosis by death receptors through inhibition of the processing and activation of procaspase-8 and -10 at the level of the receptor-associated death-inducing signaling complex (DISC). Here, we have addressed the molecular function of the v-FLIP member MC159 of the human molluscum contagiosum virus. MC159 FLIP powerfully inhibited both caspase-dependent and caspase-independent cell death induced by Fas. The C-terminal region of MC159 bound TNF receptor-associated factor (TRAF)3, was necessary for optimal TRAF2 binding, and mediated the recruitment of both TRAFs into the Fas DISC. TRAF-binding-deficient mutants of MC159 showed impaired inhibition of FasL-induced caspase-8 processing and Fas internalization, and had reduced antiapoptotic activity. Our findings provide evidence that a MC159/TRAF2/TRAF3 complex regulates a new aspect of Fas signaling, and identify MC159 FLIP as a molecule that targets multiple features of Fas-induced cell death.     

Matrix attachment regulates Fas-induced apoptosis in endothelial cells: a role for c-flip and implications for anoikis

Survival of endothelial cells is critical for cellular processes such as angiogenesis. Cell attachment to extracellular matrix inhibits apoptosis in endothelial cells both in vitro and in vivo, but the molecular mechanisms underlying matrix-induced survival signals or detachment-induced apoptotic signals are unknown. We demonstrate here that matrix attachment is an efficient regulator of Fas-mediated apoptosis in endothelial cells. Thus, matrix attachment protects cells from Fas-induced apoptosis, whereas matrix detachment results in susceptibility to Fas-mediated cell death. Matrix attachment modulates Fas-mediated apoptosis at two different levels: by regulating the expression level of Fas, and by regulating the expression level of c-Flip, an endogenous antagonist of caspase-8. The extracellular signal-regulated kinase (Erk) cascade functions as a survival pathway in adherent cells by regulating c-Flip expression. We further show that detachment-induced cell death, or anoikis, itself results from activation of the Fas pathway by its ligand, Fas-L. Fas-L/Fas interaction, Fas-FADD complex formation, and caspase-8 activation precede the bulk of anoikis in endothelial cells, and inhibition of any of these events blocks anoikis. These studies identify matrix attachment as a survival factor against death receptor-mediated apoptosis and provide a molecular mechanism for anoikis and previously observed Fas resistance in endothelial cells.     

Regulation of the Fas death pathway by FLICE-inhibitory protein in primary human B cells

The Fas/Fas ligand (L) system plays an important role in the maintenance of peripheral B cell tolerance and the prevention of misguided T cell help. CD40-derived signals are required to induce Fas expression on virgin B cells and to promote their susceptibility to Fas-mediated apoptosis. In the current study, we have analyzed the early biochemical events occurring upon Fas ligation in CD40L-activated primary human tonsillar B cells with respect to Fas-associated death domain protein (FADD), caspase-8/FADD-like IL-1beta-converting enzyme (FLICE), and c-FLICE inhibitory protein (FLIP). We report here that Fas-induced apoptosis in B cells does not require integrity of the mitochondrial Apaf-1 pathway and that caspase-8 is activated by association with the death-inducing signaling complex (DISC), i.e., upstream of the mitochondria. We show that both FADD and the zymogen form of caspase-8 are constitutively expressed at high levels in virgin B cells, whereas c-FLIP expression is marginal. In contrast, c-FLIP, but neither FADD nor procaspase-8, is strongly up-regulated upon ligation of CD40 or the B cell receptor on virgin B cells. Finally, we have found that c-FLIP is also recruited and cleaved at the level of the DISC in CD40L-activated virgin B cells. We propose that c-FLIP expression delays the onset of apoptosis in Fas-sensitive B cells. The transient protection afforded by c-FLIP could offer an ultimate safeguard mechanism against inappropriate cell death or allow recruitment of phagocytes to ensure efficient removal of apoptotic cells.