Focal adhesion kinase determines the fate of death or survival of cells in response to TNFα in the presence of actinomycin D

We speculated that focal adhesion kinase (FAK) might play a critical role in the TNFα-induced cell death. In this study, we found that FAK−/− cells are more sensitive to TNFα-induced apoptosis in the presence of actinomycin D (Act D) compared to FAK+/− cells. Prosurvival pathways are activated by the rapid recruitment of complex I, comprising TNFR1, TRADD, RIP and TRAF2, which leads to the activation of the NF-κB pathway. On the other hand, proapoptotic pathways are activated by complex II, the death-inducing signaling complex (DISC), which contains TNFR1, TRADD, RIP, and FADD, and procaspase-8 proteins. As TNFR1, TRADD, and RIP are included in both Complex I and DISC, we speculated that RIP might be a key protein. Coimmunoprecipitation assays revealed that RIP is included in complex I in FAK+/− cells, and FAK was associated with RIP. On the other hand, RIP is included in DISC in FAK−/− cells. FAK might be a key protein in the formation of complex I and the activation of NF-κB. Furthermore, Akt was activated in FAK+/− cells, but not FAK−/− cells. In conclusion, we first demonstrated that FAK determines the pathway leading to death or survival in TNFα/ActD-stimulated fibroblasts.     

Focal adhesion kinase determines the fate of death or survival of cells in response to TNFalpha in the presence of actinomycin D

We speculated that focal adhesion kinase (FAK) might play a critical role in the TNFalpha-induced cell death. In this study, we found that FAK-/- cells are more sensitive to TNFalpha-induced apoptosis in the presence of actinomycin D (Act D) compared to FAK+/- cells. Prosurvival pathways are activated by the rapid recruitment of complex I, comprising TNFR1, TRADD, RIP and TRAF2, which leads to the activation of the NF-kappaB pathway. On the other hand, proapoptotic pathways are activated by complex II, the death-inducing signaling complex (DISC), which contains TNFR1, TRADD, RIP, and FADD, and procaspase-8 proteins. As TNFR1, TRADD, and RIP are included in both Complex I and DISC, we speculated that RIP might be a key protein. Coimmunoprecipitation assays revealed that RIP is included in complex I in FAK+/- cells, and FAK was associated with RIP. On the other hand, RIP is included in DISC in FAK-/- cells. FAK might be a key protein in the formation of complex I and the activation of NF-kappaB. Furthermore, Akt was activated in FAK+/- cells, but not FAK-/- cells. In conclusion, we first demonstrated that FAK determines the pathway leading to death or survival in TNFalpha/ActD-stimulated fibroblasts.     

Structure-activity relationship of the p55 TNF receptor death domain and its lymphoproliferation mutants.

Upon stimulation with tumor necrosis factor (TNF), the TNF receptor (TNFR55) mediates a multitude of effects both in normal and in tumor cells. Clustering of the intracellular domain of the receptor, the so-called death domain (DD), is responsible for both the initiation of cell killing and the activation of gene expression. To characterize this domain further, TNFR55 DD was expressed and purified as a thioredoxin fusion protein in Escherichia coli. Circular dichroism, steady-state and time-resolved fluorescence spectroscopy were used to compare TNFR55 DD with DDs of the Fas antigen (Fas), the Fas-associating protein with DD (FADD) and p75 nerve growth factor receptor, for which the 3-dimensional structure are already known. The structural information derived from the measurements strongly suggests that TNFR55 DD adopts a similar fold in solution. This prompted a homology modeling of the TNFR DD 3-D structure using FADD as a template. In vivo studies revealed a difference between the two lymphoproliferation (lpr) mutations. Biophysical techniques were used to analyze the effect of changing Leu351 to Ala and Leu351 to Asn on the global structure and its impact on the overall stability of TNFR55 DD. The results obtained from these experiments in combination with the modeled structure offer an explanation for the in vivo observed difference.     

Formation of the death domain complex between FADD and RIP1 proteins in vitro

Fas-associated death domain (FADD) protein is an adapter molecule that bridges the interactions between membrane death receptors and initiator caspases. The death receptors contain an intracellular death domain (DD) which is essential to the transduction of the apoptotic signal. The kinase receptor-interacting protein 1 (RIP1) is crucial to programmed necrosis. The cell type interplay between FADD and RIP1, which mediates both necrosis and NF-κB activation, has been evaluated in other studies, but the mechanism of the interaction of the FADD and RIP1 proteins remain poorly understood. Here, we provided evidence indicating that the DD of human FADD binds to the DD of RIP1 in vitro. We developed a molecular docking model using homology modeling based on the structures of FADD and RIP1. In addition, we found that two structure-based mutants (G109A and R114A) of the FADD DD were able to bind to the RIP1 DD, and two mutations (Q169A and N171A) of FADD DD and four mutations (G595, K596, E620, and D622) of RIP1 DD disrupted the FADD–RIP1 interaction. Six mutations (Q169A, N171A, G595, K596, E620, and D622) lowered the stability of the FADD–RIP1 complex and induced aggregation that structurally destabilized the complex, thus disrupting the interaction.     

FADD deficiency sensitises Jurkat T cells to TNF-alpha-dependent necrosis during activation-induced cell death

Activation-induced cell death (AICD) in activated T lymphocytes is largely mediated by Fas/Fas ligand (FasL) interaction. The cytoplasmic adaptor molecule Fas-associated death domain protein (FADD) plays an essential role in the apoptotic signalling of the Fas death pathway. In the present study, we observed that FADD deficient (FADD(-/-)) Jurkat T cells undergo AICD to a similar extent as wild-type cells. AICD in wild-type Jurkat T cells is via apoptosis, whereas it is non-apoptotic in FADD(-/-) cells. The latter took up propidium iodide, exhibit a loss in mitochondrial membrane potential and have no detectable cleavage products of caspase-8 or -3 activation, suggesting that these cells die by necrosis. Wild-type Jurkat T cells undergo apoptosis when incubated with recombinant FasL and Trail but not with TNF-alpha. In contrast, FADD(-/-) Jurkat T cells are resistant to FasL and Trail but die of necrosis when incubated with TNF-alpha. We showed that neutralising anti-TNF-alpha blocked AICD as well as TNF-alpha-induced necrosis in FADD(-/-) Jurkat T cells. Furthermore, down regulating the receptor interacting protein, RIP, with geldanamycin treatment, which is essential for TNF-alpha signalling, markedly inhibited AICD in FADD(-/-) Jurkat T cells. In addition, caspase-8-deficient Jurkat T cells are resistant to Fas- and TNF-alpha-induced cell death. Taken together, our results suggest that a deficiency in FADD and not caspase-8 or the inhibition of the Fas signalling pathway sensitises Jurkat T cells to TNF-alpha-dependent necrosis during AICD.     

Kinase-independent function of RIP1, critical for mature T-cell survival and proliferation

The death receptor, Fas, triggers apoptotic death and is essential for maintaining homeostasis in the peripheral lymphoid organs. RIP1 was originally cloned when searching for Fas-binding proteins and was later shown to associate also with the signaling complex of TNFR1. Although Fas exclusively induces apoptosis, TNFR1 primarily activates the pro-survival/pro-inflammatory NF-κB pathway. Mutations in Fas lead to lymphoproliferative (lpr) diseases, and deletion of TNFR1 results in defective innate immune responses. However, the function of RIP1 in the adult lymphoid system has not been well understood, primarily owing to perinatal lethality in mice lacking the entire RIP1 protein in germ cells. This current study investigated the requirement for RIP1 in the T lineage using viable RIP1 mutant mice containing a conditional and kinase-dead RIP1 allele. Disabling the kinase activity of RIP1 had no obvious impact on the T-cell compartment. However, T-cell-specific deletion of RIP1 led to a severe T-lymphopenic condition, owing to a dramatically reduced mature T-cell pool in the periphery. Interestingly, the immature T-cell compartment in the thymus appeared intact. Further analysis showed that mature RIP1^−/− T cells were severely defective in antigen receptor-induced proliferative responses. Moreover, the RIP1^−/− T cells displayed greatly increased death and contained elevated caspase activities, an indication of apoptosis. In total, these results revealed a novel, kinase-independent function of RIP1, which is essential for not only promoting TCR-induced proliferative responses but also in blocking apoptosis in mature T cells.The pro-survival signaling pathways provide protection against cell death responses at various stages during T lymphopoiesis as well as maintenance of the mature population.^{1, 2} Apoptosis is a major programmed cell death pathway, which can be induced through either intrinsic or extrinsic signals.³ Under normal circumstances, the pro-survival and apoptosis signaling pathways are tightly regulated, which ensures generation of diverse T-cell repertoires, while avoiding autoimmunity. For instance, the Bcl-2 and Bcl-X_L genes, which inhibit the intrinsic apoptotic pathway, are essential for both T-cell development and peripheral maintenance.^{4, 5} However, lack of cell death, as in the case of inactivation of Bim, a pro-apoptotic protein of the Bcl-2 family, results in lymphoproliferative and autoimmune diseases.⁶ The extrinsic pathway of apoptosis is triggered through cell receptors, including Fas/Apo-1 and tumor necrosis factor receptor 1 (TNFR1). Whereas Fas is a professional death receptor, TNFR1 primarily signals the pro-survival pathway by activating NF-κB, which also promotes inflammation.^{7, 8}Receptor-interacting protein (RIP or RIP1) was originally cloned as a potential Fas-interacting protein.⁹ However, later studies found that lack of RIP1 has no effect on Fas-induced apoptosis.^{10, 11} Subsequently, RIP1 was also found to associate with the signaling complex of TNFR1.¹² It was shown that RIP1 deficiency disrupts NF-κB activation induced by TNFR1 in primary mouse embryonic fibroblast cells or human Jurkat T lymphoma cells.^{10, 11} Several functional domains of RIP1 have been defined. In particular, RIP1 contains a serine/threonine kinase domain (KD) at the amino-terminus and a death domain (DD) at the carboxy-terminus. The intermediate domain, but not the protein serine/threonine KD of RIP1, is required for the activation of NF-κB.¹³ The DD of RIP1 interacts with the DD of TNFR1-associated death domain (TRADD) protein, a signaling adaptor, leading to both apoptosis and NF-κB activation.¹² Therefore, RIP1 may serve as a scaffold protein in addition to being a protein serine/threonine kinase.The function of the KD of RIP1 remained unknown until the landmark work by Holler et al.,¹⁴ implicating a novel function for RIP1 in a caspase-independent cell death process with certain characteristics of necrosis, namely necroptosis. Importantly, mutations targeting the kinase activity of RIP1 abolish necroptotic cell death induced by TNFR1. The in vivo role of RIP1-mediated necroptosis was first revealed by analysis of the embryonic defect displayed by mice lacking the Fas-associated death domain (FADD) protein.¹⁵ The FADD adaptor protein relays exclusively apoptotic signals in the pathways triggered by Fas, TNFR1, and TNF-related apoptosis-inducing ligand receptors (TRAIL-Rs or DR4/5).^{16, 17, 18} Whereas none of the DRs are essential for mouse development, FADD deficiency resulted in midgestation death of mouse embryos.^{19, 20} Interestingly, when RIP1 is absent, normal embryonic development is restored in FADD^−/− mice,¹⁵ indicating that FADD^−/− embryonic lethality is caused by RIP1-dependent necroptosis.Although normal during embryogenesis, RIP1^−/− FADD^−/− double knockout (DKO) mice display perinatal lethality,¹⁵ similar to the phenotype of RIP1^−/− single knockout mice.¹⁰ In contrast, deletion of a RIP1-related protein kinase, RIP3, fully restores normal embryonic as well as postnatal development in FADD^−/− mice.²¹ Recent studies demonstrated that RIP1^−/− mice can only reach adulthood when both FADD and RIP3 are absent, indicating that RIP1 protects neonatal cells from FADD-mediated apoptosis and RIP3-dependent necroptosis.^{22, 23, 24, 25} Importantly, FADD^−/− RIP3^−/− DKO mice and RIP1^−/− FADD^−/− RIP3^−/− triple knockout mice develop age-dependent lymphadenopathy and splenomegaly, reminiscent of the lymphoproliferative (lpr) disease displayed by Fas^−/− mice. Therefore, both apoptosis and necroptosis are essential for homeostasis in the peripheral lymphoid organs.Previous studies have indicated that RIP1 is essential for T-cell development, because RIP1-deficient fetal liver cells fail to reconstitute the T-cell compartment in immunodeficient recipient mice.^{15, 26} A recent study showed that lack of RIP1 in hematopoietic stem cells and progenitors (HSCs/Ps) leads to a severe defect in hematopoiesis.²⁷ However, the temporal requirement for RIP1, particularly at postlineage commitment stages, remains unclear. In the current study, T lineage-specific deletion of RIP1 revealed a novel stage-specific requirement for RIP1 to protect T cells from apoptosis as well as to allow normal proliferative responses.     

The adaptor protein FADD and the initiator caspase-8 mediate activation of NF-��B by TRAIL

Besides inducing apoptosis, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) activates NF-κB. The apoptosis signaling pathway of TRAIL is well characterized involving TRAIL receptors, Fas-associated protein with death domain (FADD) and caspase-8. In contrast, the molecular mechanism of TRAIL signaling to NF-κB remains controversial. Here, we characterized the receptor–proximal mediators of NF-κB activation by TRAIL. Deletion of the DD of TRAIL receptors 1 and 2 revealed that it is essential in NF-κB signaling. Because FADD interacts with the TRAIL receptor DD, FADD was tested. RNAi-mediated knockdown of FADD or FADD deficiency in JURKAT T-cell leukemia cells decreased or disabled NF-κB signaling by TRAIL. In contrast, TRAIL-induced activation of NF-κB was maintained upon loss of receptor interacting protein 1 (RIP1) or knockdown of FLICE-like inhibitory protein (FLIP). Exogenous expression of FADD rescued TRAIL-induced NF-κB signaling. Loss-of-function mutations of FADD within the RHDLL motif of the death effector domain, which is required for TRAIL-induced apoptosis, abrogated FADD''s ability to recruit caspase-8 and mediate NF-κB activation. Accordingly, deficiency of caspase-8 inhibited TRAIL-induced activation of NF-κB, which was rescued by wild-type caspase-8, but not by a catalytically inactive caspase-8 mutant. These data establish the mechanism of TRAIL-induced NF-κB activation involving the TRAIL receptor DD, FADD and caspase-8, but not RIP1 or FLIP. Our results show that signaling of TRAIL-induced apoptosis and NF-κB bifurcates downstream of caspase-8.     

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.     

Novel pandemic influenza A (H1N1) virus infection modulates apoptotic pathways that impact its replication in A549 cells

It is not well-known whether apoptosis signaling affects influenza virus infection and reproduction in human lung epithelial cells. Using A549 cell line, we studied the relationship of some apoptosis-associated molecules with novel pandemic influenza A (H1N1) virus, A/California/04/2009. Infected cells displayed upregulated Fas ligand, activated FADD and caspase-8, and downregulated FLIP in the extrinsic apoptotic pathway. p53 expression increased and Bcl-X_L expression decreased in the intrinsic pathway. Expression of pre-apoptotic molecules (FasL, FADD, and p53) increased virus replication, while inhibition of activity of FADD, caspase-8 and caspase-3, and expression of anti-apoptotic proteins (FLIP and Bcl-X_L) decreased virus replication. p38, ERK and JNK from MAPK pathways were activated in infected cells, and inhibition with their inhibitors diminished virus replication. In the p38 superfamily, p38α expression increased viral RNA production, while expression of p38β and p38γ decreased. These data indicated that influenza virus induces apoptotic signaling pathways, which benefit virus replication.     

Structural insight for the roles of fas death domain binding to fadd and oligomerization degree of the fas–fadd complex in the death‐inducing signaling complex formation: A computational study

Fas binding to Fas‐associated death domain (FADD) activates FADD–caspase‐8 binding to form death‐inducing signaling complex (DISC) that triggers apoptosis. The Fas–Fas association exists primarily as dimer in the Fas–FADD complex, and the Fas–FADD tetramer complexes have the tendency to form higher order oligomer. The importance of the oligomerized Fas–FADD complex in DISC formation has been confirmed. This study sought to provide structural insight for the roles of Fas death domain (Fas DD) binding to FADD and the oligomerization of Fas DD–FADD complex in activating FADD–procaspase‐8 binding. Results show Fas DD binding to FADD stabilized the FADD conformation, including the increased stability of the critical residues in FADD death effector domain (FADD DED) for FADD–procaspase‐8 binding. Fas DD binding to FADD resulted in the decreased degree of both correlated and anticorrelated motion of the residues in FADD and caused the reversed correlated motion between FADD DED and FADD death domain (FADD DD). The exposure of procaspase‐8 binding residues in FADD that allows FADD to interact with procaspase‐8 was observed with Fas DD binding to FADD. We also observed different degrees of conformational and motion changes of FADD in the Fas DD–FADD complex with different degrees of oligomerization. The increased conformational stability and the decreased degree of correlated motion of the residues in FADD in Fas DD–FADD tetramer complex were observed compared to those in Fas DD–FADD dimer complex. This study provides structural evidence for the roles of Fas DD binding to FADD and the oligomerization degree of Fas DD–FADD complex in DISC formation to signal apoptosis. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Establishment of lal-/- Myeloid Lineage Cell Line That Resembles Myeloid-Derived Suppressive Cells

Myeloid-derived suppressor cells (MDSCs) in mouse are inflammatory cells that play critical roles in promoting cancer growth and metastasis by directly stimulating cancer cell proliferation and suppressing immune surveillance. In order to facilitate characterization of biochemical and cellular mechanisms of MDSCs, it is urgent to establish an “MDSC-like” cell line. By cross breeding of immortomouse (simian virus 40 large T antigen transgenic mice) with wild type and lysosomal acid lipase (LAL) knock-out (lal-/-) mice, we have established a wild type (HD1A) and a lal-/- (HD1B) myeloid cell lines. Compared with HD1A cells, HD1B cells demonstrated many characteristics similar to lal-/- MDSCs. HD1B cells exhibited increased lysosomes around perinuclear areas, dysfunction of mitochondria skewing toward fission structure, damaged membrane potential, and increased ROS production. HD1B cells showed increased glycolytic metabolism during blockage of fatty acid metabolism to fuel the energy need. Similar to lal-/- MDSCs, the mTOR signal pathway in HD1B cells is overly activated. Rapamycin treatment of HD1B cells reduced ROS production and restored the mitochondrial membrane potential. HD1B cells showed much stronger immunosuppression on CD4⁺ T cell proliferation and function in vitro, and enhanced cancer cells proliferation. Knockdown of mTOR with siRNA reduced the HD1B cell ability to immunosuppress T cells and stimulate cancer cell proliferation. Therefore, the HD1B myeloid cell line is an “MDSC-like” cell line that can be used as an alternative in vitro system to study how LAL controls various myeloid cell functions.     

Distinct signaling pathways in TRAIL- versus tumor necrosis factor-induced apoptosis

Trimeric tumor necrosis factor (TNF) binding leads to recruitment of TRADD to TNFR1. In current models, TRADD recruits RIP, TRAF2, and FADD to activate NF-kappaB, Jun N-terminal protein kinase (JNK), and apoptosis. Using stable short-hairpin RNA (shRNA) knockdown (KD) cells targeting these adaptors, TNF death-inducing signaling complex immunoprecipitation demonstrates competitive binding of TRADD and RIP to TNFR1, whereas TRAF2 recruitment requires TRADD. Analysis of KD cells indicates that FADD is necessary for Fas-L- or TRAIL- but not TNF-induced apoptosis. Interestingly, TRADD is dispensable, while RIP is required for TNF-induced apoptosis in human tumor cells. TRADD is required for c-Jun phosphorylation upon TNF exposure. RIP KD abrogates formation of complex II following TNF exposure, whereas TRADD KD allows efficient RIP-caspase 8 association. Treatment with TRAIL also induces formation of a complex II containing FADD, RIP, IKKalpha, and caspase 8 and 10, leading to activation of caspase 8. Our data suggest that TNF triggers apoptosis in a manner distinct from that of Fas-L or TRAIL.     

Deletion of FADD in Macrophages and Granulocytes Results in RIP3- and MyD88-Dependent Systemic Inflammation

Myeloid cells, which include monocytes, macrophages, and granulocytes, are important innate immune cells, but the mechanism and downstream effect of their cell death on the immune system is not completely clear. Necroptosis is an alternate form of cell death that can be triggered when death receptor-mediated apoptosis is blocked, for example, in stimulated Fas-associated Death Domain (FADD) deficient cells. We report here that mice deficient for FADD in myeloid cells (mFADD^-/-) exhibit systemic inflammation with elevated inflammatory cytokines and increased levels of myeloid and B cell populations while their dendritic and T cell numbers are normal. These phenotypes were abolished when RIP3 deficiency was introduced, suggesting that systemic inflammation is caused by RIP3-dependent necroptotic and/or inflammatory activity. We further found that loss of MyD88 can rescue the systemic inflammation observed in these mice. These phenotypes are surprisingly similar to that of dendritic cell (DC)-specific FADD deficient mice with the exception that DC numbers are normal in mFADD^-/- mice. Together these data support the notion that innate immune cells are constantly being stimulated through the MyD88-dependent pathway and aberrations in their cell death machinery can result in systemic effects on the immune system.     

RIP death domain structural interactions implicated in TNF-mediated proliferation and survival

Death domain (DD)-containing proteins are involved in both apoptosis and survival/proliferation signaling induced by activated death receptors. Here, a phylogenetic and structural analysis was performed to highlight differences in DD domains and their key regulatory interaction sites. The phylogenetic analysis shows that receptor DDs are more conserved than DDs in adaptors. Adaptor DDs can be subdivided into those that activate or inhibit apoptosis. Modeling of six homotypic DD interactions involved in the TNF signaling pathway implicates that the DD of RIP (Receptor interacting protein kinase 1) is capable of interacting with the DD of TRADD (TNFR1-associated death domain protein) in two different, exclusive ways: one that subsequently recruits CRADD (apoptosis/inflammation) and another that recruits NFkappaB (survival/proliferation).     

Pigment Epithelial-derived Factor (PEDF)-triggered Lung Cancer Cell Apoptosis Relies on p53 Protein-driven Fas Ligand (Fas-L) Up-regulation and Fas Protein Cell Surface Translocation

Pigment epithelium-derived factor (PEDF), a potent antiangiogenesis agent, has recently attracted attention for targeting tumor cells in several types of tumors. However, less is known about the apoptosis-inducing effect of PEDF on human lung cancer cells and the underlying molecular events. Here we report that PEDF has a growth-suppressive and proapoptotic effect on lung cancer xenografts. Accordingly, in vitro, PEDF apparently induced apoptosis in A549 and Calu-3 cells, predominantly via the Fas-L/Fas death signaling pathway. Interestingly, A549 and Calu-3 cells are insensitive to the Fas-L/Fas apoptosis pathway because of the low level of cell surface Fas. Our results revealed that, in addition to the enhancement of Fas-L expression, PEDF increased the sensitivity of A549 and Calu-3 cells to Fas-L-mediated apoptosis by triggering the translocation of Fas protein to the plasma membrane in a p53- and FAP-1-dependent manner. Similarly, the up-regulation of Fas-L by PEDF was also mediated by p53. Furthermore, peroxisome proliferator-activated receptor γ was determined to be the upstream regulator of p53. Together, these findings uncover a novel mechanism of tumor cell apoptosis induced by PEDF and provide a potential therapeutic strategy for tumors that are insensitive to Fas-L/Fas-dependent apoptosis because of a low level of cell surface Fas.     

Fas ligand-independent, FADD-mediated activation of the Fas death pathway by anticancer drugs

Trimerization of the Fas receptor (CD95, APO-1), a membrane bound protein, triggers cell death by apoptosis. The main death pathway activated by Fas receptor involves the adaptor protein FADD (for Fas-associated death domain) that connects Fas receptor to the caspase cascade. Anticancer drugs have been shown to enhance both Fas receptor and Fas ligand expression on tumor cells. The contribution of Fas ligand-Fas receptor interactions to the cytotoxic activity of these drugs remains controversial. Here, we show that neither the antagonistic anti-Fas antibody ZB4 nor the Fas-IgG molecule inhibit drug-induced apoptosis in three different cell lines. The expression of Fas ligand on the plasma membrane, which is identified in untreated U937 human leukemic cells but remains undetectable in untreated HT29 and HCT116 human colon cancer cell lines, is not modified by exposure to various cytotoxic agents. These drugs induce the clustering of Fas receptor, as observed by confocal laser scanning microscopy, and its interaction with FADD, as demonstrated by co-immunoprecipitation. Overexpression of FADD by stable transfection sensitizes tumor cells to drug-induced cell death and cytotoxicity, whereas down-regulation of FADD by transient transfection of an antisense construct decreases tumor cell sensitivity to drug-induced apoptosis. These results were confirmed by transient transfection of constructs encoding either a FADD dominant negative mutant or MC159 or E8 viral proteins that inhibit the FADD/caspase-8 pathway. These results suggest that drug-induced cell death involves the Fas/FADD pathway in a Fas ligand-independent fashion.