Two CD95 (APO-1/Fas) signaling pathways.

We have identified two cell types, each using almost exclusively one of two different CD95 (APO-1/Fas) signaling pathways. In type I cells, caspase-8 was activated within seconds and caspase-3 within 30 min of receptor engagement, whereas in type II cells cleavage of both caspases was delayed for approximately 60 min. However, both type I and type II cells showed similar kinetics of CD95-mediated apoptosis and loss of mitochondrial transmembrane potential (DeltaPsim). Upon CD95 triggering, all mitochondrial apoptogenic activities were blocked by Bcl-2 or Bcl-xL overexpression in both cell types. However, in type II but not type I cells, overexpression of Bcl-2 or Bcl-xL blocked caspase-8 and caspase-3 activation as well as apoptosis. In type I cells, induction of apoptosis was accompanied by activation of large amounts of caspase-8 by the death-inducing signaling complex (DISC), whereas in type II cells DISC formation was strongly reduced and activation of caspase-8 and caspase-3 occurred following the loss of DeltaPsim. Overexpression of caspase-3 in the caspase-3-negative cell line MCF7-Fas, normally resistant to CD95-mediated apoptosis by overexpression of Bcl-xL, converted these cells into true type I cells in which apoptosis was no longer inhibited by Bcl-xL. In summary, in the presence of caspase-3 the amount of active caspase-8 generated at the DISC determines whether a mitochondria-independent apoptosis pathway is used (type I cells) or not (type II cells).     

The CD95 (APO-1/Fas) and the TRAIL (APO-2L) apoptosis systems

Heat shock protein 70 (hsp70) is a stress-inducible protein that prevents apoptosis induced by a wide range of cytotoxic agents by an as yet undefined mechanism. The caspase family of cysteine proteases have been attributed a central role in the execution of apoptosis. However, several cases of caspase-independent apoptosis have been recently reported, suggesting that caspases may not be necessary for apoptosis in all cells. This study examines the protective role of hsp70 in both caspase-dependent and -independent apoptosis. Hydrogen peroxide (H2O2) used at low and high concentrations in Jurkat T cells induces caspase-dependent and -independent apoptosis, respectively. A hsp70-transfected Jurkat clone was used to observe the protection mediated by hsp70 during these two forms of apoptosis. Results reveal that hsp70 inhibits both caspase-dependent and -independent apoptosis. Furthermore, measurement of caspase-3 activity during caspase-dependent apoptosis revealed that caspase activation was inhibited in hsp70 transfectants. Early apoptotic events, such as mitochondrial depolarization, cytochrome c release, and increased intracellular calcium, were demonstrated to be common to both caspase-dependent and -independent H2O2-induced apoptosis. The inhibition of these events by hsp70 suggests that hsp70 may be an important anti-apoptotic regulator, functioning at a very early stage in the apoptotic pathway.     

Dendritic cells are resistant to apoptosis through the Fas (CD95/APO-1) pathway.

Immunoregulation of lymphocytes and macrophages in the peripheral immune system is achieved in part by activation-induced cell death. Members of the TNF receptor family including Fas (CD95) are involved in the regulation of activation-induced cell death. To determine whether activation-induced cell death plays a role in regulation of dendritic cells (DCs), we examined interactions between Ag-presenting murine DCs and Ag-specific Th1 CD4+ T cells. Whereas mature bone marrow- or spleen-derived DCs expressed high levels of Fas, these DCs were relatively insensitive to Fas-mediated killing by the agonist mAb, Jo-2, as well as authentic Fas ligand expressed on the CD4+ T cell line, A.E7. The insensitivity to Fas-mediated apoptosis was not affected by priming with IFN-gamma and/or TNF-alpha or by blocking the DC survival signals TNF-related activation-induced cytokine and CD40L. However, apoptosis could be induced with C2-ceramide, suggesting that signals proximal to the generation of ceramide might mediate resistance to Fas. Analysis of protein expression of several anti-apoptotic mediators revealed that expression of the intracellular inhibitor of apoptosis Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein was significantly higher in Fas-resistant DCs than in Fas-sensitive macrophages, suggesting a possible role for Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein in DC resistance to Fas-mediated apoptosis. Our results demonstrate that murine DCs differ significantly from other APC populations in susceptibility to Fas-mediated apoptosis during cognate presentation of Ag. Because DCs are most notable for initiation of an immune response, resistance to apoptosis may contribute to this function.     

人FasL基因转染成熟树突状细胞对T淋巴细胞增殖和凋亡的影响

探讨转染人FasL基因的成熟树突状细胞(DC)对异体T淋巴细胞增殖和凋亡的影响,为实现临床器官移植免疫耐受提供初步实验依据.从健康成年人外周静脉血中获得成熟树突状细胞.将人FasL基因成功转染成熟树突状细胞,检测其表面分子的表达和自身凋亡情况,并对其抗原递呈功能进行分析.从异体健康成人外周血中获取T淋巴细胞,将转染成功的树突状细胞与T淋巴细胞混合培养,检测其对T淋巴细胞增殖和凋亡的影响.结果表明:人FasL基因转染没有明显影响成熟树突状细胞表面分子CD40、CD80、CD86和HLA-DR的表达;没有诱导树突状细胞自身发生凋亡;没有影响DC的抗原递呈功能.转染FasL基因后的树突状细胞使异体T淋巴细胞刺激指数明显下降,凋亡增加.因此认为,人FasL基因转染对成熟树突状细胞的表面分子表达、自身凋亡、抗原递呈等生物学性状无影响;转染FasL基因的树突状细胞使异体T淋巴细胞的增殖能力减弱,并能明显诱导T淋巴细胞凋亡.     

A novel adenoviral vector expressing human Fas/CD95/APO-1 enhances p53-mediated apoptosis.

Recent evidence suggests an intriguing link between p53 and the Fas pathway. To evaluate this association further, we utilized a recombinant adenoviral vector (AdWTp53) to overexpress wild-type p53 in lung cancer (A549, H23, EKVX and HOP92) and breast cancer (MDA-MB-231 and MCF-7) cell lines and observed an increase in the Fas/CD95/APO-1 protein levels. Furthermore, this increase correlated with the sensitivity of the cell lines to p53-mediated cytotoxicity. To examine the effects of Fas over-expression in cells resistant to p53 over-expression, we constructed AdFas, an adenoviral vector capable of transferring functional human Fas to cancer cells. Interestingly, infection of p53-resistant MCF-7 cells with AdFas sensitized them to p53-mediated apoptosis. These studies indicate that combined over-expression of Fas and wild-type p53 may be an effective cancer gene therapy approach, especially in cells relatively resistant to p53 over-expression.     

Scythe cleavage during Fas (APO-1)-and staurosporine-mediated apoptosis

Scythe is a nuclear protein that has been implicated in the apoptotic process in Drosophila melanogaster; however, its role in apoptosis of mammalian cells is not fully elucidated. Here we show that cleavage of Scythe by caspase-3 occurs after activation of both the extrinsic (i.e. Fas/APO-1-mediated) and the intrinsic (i.e. staurosporine-induced) apoptosis pathway. Moreover, this caspase-dependent cleavage correlates with Scythe translocation from the nucleus to the cytosol. We also show that cytosolic re-localization of Scythe is required for Fas/APO-1-triggered phosphatidylserine (PS) exposure, a signal for macrophage clearance of apoptotic cells. Our data suggest that Scythe cleavage may represent a marker for caspase-3 activation and implicate cytosolic re-localization of Scythe in the pathway of PS exposure.     

The Fas/APO-1 receptor and its deadly ligand

The cell surface receptor Fas/APO-1 and its ligand have recently been identified as important mediators of apoptosis. Both molecules are crucial for the maintenance of a sound immune system, and when defective they give rise to severe autoimmune disorders. Understanding the mechanism and regulation of Fas/APO-1-triggered cell death promises important insights for the pathogenesis of AIDS, cancer and autoimmune diseases.     

RIP mediates tumor necrosis factor receptor 1 activation of NF-kappaB but not Fas/APO-1-initiated apoptosis.

The CD95 (Fas/APO-1) and tumor necrosis factor (TNF) receptor pathways share many similarities, including a common reliance on proteins containing 'death domains' for elements of the membrane-proximal signal relay. We have created mutant cell lines that are unable to activate NF-kappaB in response to TNF. One of the mutant lines lacks RIP, a 74 kDa Ser/Thr kinase originally identified by its ability to associate with Fas/APO-1 and induce cell death. Reconstitution of the line with RIP restores responsiveness to TNF. The RIP-deficient cell line is susceptible to apoptosis initiated by anti-CD95 antibodies. An analysis of cells reconstituted with mutant forms of RIP reveals similarities between the action of RIP and FADD/MORT-1, a Fas-associated death domain protein.     

Activation of CD95 (APO-1/Fas) signaling by ceramide mediates cancer therapy-induced apoptosis.

We report here that anticancer drugs such as doxorubicin lead to induction of the CD95 (APO-1/Fas) system of apoptosis and the cellular stress pathway which includes JNK/SAPKs. Ceramide, which accumulates in response to different types of cellular stress such as chemo- and radiotherapy, strongly induced expression of CD95-L, cleavage of caspases and apoptosis. Antisense CD95-L as well as dominant-negative FADD inhibited ceramide- and cellular stress-induced apoptosis. Fibroblasts from type A Niemann-Pick patients (NPA), genetically deficient in ceramide synthesis, failed to up-regulate CD95-L expression and to undergo apoptosis after gamma-irradiation or doxorubicin treatment. In contrast, JNK/SAPK activity was still inducible by doxorubicin in the NPA cells, suggesting that activation of JNK/SAPKs alone is not sufficient for induction of the CD95 system and apoptosis. CD95-L expression and apoptosis in NPA fibroblasts were restorable by exogenously added ceramide. In addition, NPA fibroblasts undergo apoptosis after triggering of CD95 with an agonistic antibody. These data demonstrate that ceramide links cellular stress responses induced by gamma-irradiation or anticancer drugs to the CD95 pathway of apoptosis.     

Two distinct signaling pathways regulate peroxynitrite-induced apoptosis in PC12 cells

The mechanisms of peroxynitrite-induced apoptosis are not fully understood. We report here that peroxynitrite-induced apoptosis of PC12 cells requires the simultaneous activation of p38 and JNK MAP kinase, which in turn activates the intrinsic apoptotic pathway, as evidenced by Bax translocation to the mitochondria, cytochrome c release to the cytoplasm and activation of caspases, leading to cell death. Peroxynitrite induces inactivation of the Akt pathway. Furthermore, overexpression of constitutively active Akt inhibits both peroxynitrite-induced Bax translocation and cell death. Peroxynitrite-induced death was prevented by overexpression of Bcl-2 and by cyclosporin A, implicating the involvement of the intrinsic apoptotic pathway. Selective inhibition of mixed lineage kinase (MLK), p38 or JNK does not attenuate the decrease in Akt phosphorylation showing that inactivation of the Akt pathway occurs independently of the MLK/MAPK pathway. Together, these results reveal that peroxynitrite-induced activation of the intrinsic apoptotic pathway involves interactions with the MLK/MAPK and Akt signaling pathways.     

How the Bcl-2 family of proteins interact to regulate apoptosis

Commitment of cells to apoptosis is governed largely by protein-protein interactions between members of the Bcl-2 protein family. Its three sub-families have distinct roles： the BH3-only proteins trigger apoptosis by binding via their BH3 domain to pro-survival relatives, while the pro-apoptotic Bax and Bak have an essential downstream role involving disruption of organellar membranes and induction of caspase activation. The BH3-only proteins act as damage sensors, held inert until their activation by stress signals. Once activated, they were thought to bind promiscuously to pro-survival protein targets but unexpected selectivity has recently emerged from analysis of their interactions. Some BH3-only proteins also bind to Bax and Bak. Whether Bax and Bak are activated directly by these BH3-only proteins, or indirectly as a consequence of BH3-only proteins neutralizing their pro-survival targets is the subject of intense debate. Regardless of this, a detailed understanding of the interactions between family members, which are often selective, has notable implications for designing anti-cancer drugs to target the Bcl-2 family.     

Bcl-2 mutants with restricted subcellular location reveal spatially distinct pathways for apoptosis in different cell types.

Human Bcl-2 is located in multiple intracellular membranes when expressed in MDCK and Rat-1/myc cells. We restricted expression to the endoplasmic reticulum or mitochondria by exchanging the Bcl-2 carboxy-terminal insertion sequence for an equivalent sequence from cytochrome b5 or ActA, respectively. MDCK cells are protected from serum deprivation-induced apoptosis by both wild-type Bcl-2 and the mutant targeted to mitochondria but not by the mutant targeted to endoplasmic reticulum. In contrast, when expressed in Rat-1/myc cells, the Bcl-2 mutant located at the endoplasmic reticulum is more effective than that targeted to mitochondria. In MDCK cells both mutants bind Bax as effectively as wild-type, demonstrating that Bax binding is not sufficient to prevent apoptosis.     

CD95 (APO-1/Fas)-associating signalling proteins

The CD95 (APO-1/Fas) receptor has attracted great interest in recent years because it transduces an apoptotic signal in a variety of different tissues. CD95 belongs to the NGF/TNF-receptor superfamily, members of which need to be trimerized by specific protein ligands in order to generate a signal. This review focuses on recent advances in the understanding of the proximal signal transduction mechanism of CD95. The cloning of numerous proteins that interact with CD95 and other members of this receptor family and the in vivo identification of several proteins that associate with CD95 in a ligand-dependent fashion opens the way to delineate the death pathway and to explain crosstalk among these receptors on a molecular basis.     

The CD95(APO-1/Fas) DISC and beyond

CD95 (APO-1/Fas) is a prototype death receptor characterized by the presence of an 80 amino acid death domain in its cytoplasmic tail. This domain is essential for the recruitment of a number of signaling components upon activation by either agonistic anti-CD95 antibodies or cognate CD95 ligand that initiate apoptosis. The complex of proteins that forms upon triggering of CD95 is called the death-inducting signaling complex (DISC). The DISC consists of an adaptor protein and initiator caspases and is essential for induction of apoptosis. A number of proteins have been reported to regulate formation or activity of the DISC. This review discusses recent developments in this area of death receptor research.     

Underphosphorylated BAD interacts with diverse antiapoptotic Bcl-2 family proteins to regulate apoptosis

Survival factors activate kinases which, in turn, phosphorylate the proapoptotic Bcl-xl/Bcl-2-associated death promoter homolog (BAD) protein at key serine residues. Phosphorylated BAD interacts with 14-3-3 proteins, and overexpression of 14-3-3 attenuates BAD-mediated apoptosis. Although BAD is known to interact with Bcl-2, Bcl-w, and Bcl-xL, the exact relationship between BAD and anti- or proapoptotic Bcl-2 proteins has not been analyzed systematically. Using the yeast two-hybrid protein interaction assay, we found that BAD interacted negligibly with proapoptotic Bcl-2 proteins. Even though wild type BAD only interacted with selected numbers of antiapoptotic proteins, underphosphorylated mutant BAD interacted with all antiapoptotic Bcl-2 proteins tested (Bcl-2, Bcl-w, Bcl-xL, Bfl-1/A1, Mcl-1, Ced-9, and BHRF-1). Using nonphosphorylated recombinant BAD expressed in bacteria, direct interactions between BAD and diverse antiapoptotic Bcl-2 members were also observed. Furthermore, apoptosis induced by BAD was blocked by coexpression with Bcl-2, Bcl-w, and Bfl-1. Comparison of BAD orthologs from zebrafish to human indicated the conservation of a 14-3-3 binding site and the BH3 domain during evolution. Thus, highly conserved BAD interacts with diverse antiapoptotic Bcl-2 members to regulate apoptosis.     

CD95(Fas/APO-1) signals ceramide generation independent of the effector stage of apoptosis

Although numerous studies document caspase-independent ceramide generation preceding apoptosis upon environmental stress, the molecular ordering of ceramide generation during cytokine-induced apoptosis remains uncertain. Here, we show that CD95-induced ceramide elevation occurs during the initiation phase of apoptosis. We titrated down the amount of FADD transfected into HeLa and 293T cells until it was insufficient for apoptosis, although cycloheximide (CHX) still triggered the effector phase. Even in the absence of CHX, ceramide levels increased rapidly, peaking at 2.7 +/- 0.2-fold of control 8 h post-transfection. Dominant negative FADD failed to confer ceramide generation or CHX-mediated apoptosis. Ceramide generation induced by FADD was initiator caspase-dependent, being blocked by crmA. Limited pro-caspase 8 overexpression also increased ceramide levels 2.7 +/- 0.2-fold, yet failed, without CHX, to initiate apoptosis. Expression of membrane-targeted oligomerized CD-8 caspase 8 induced apoptosis without CHX, yet elevated ceramide only to a level equivalent to limited pro-caspase 8 transfection. Ceramide elevations were detected concurrently by diacylglycerol kinase and electrospray tandem mass spectrometry. These investigations provide evidence that ceramide generation is initiator caspase-dependent and occurs prior to commitment to the effector phase of apoptosis, definitively ordering ceramide as proximal in CD95 signaling.     

Specificity of anti-human CD95 (APO-1/Fas) antibodies

The CD95 (APO-1/Fas) death receptor plays an important role in many physiological and pathophysiological systems. Thus, the CD95 system contributes to activation-induced cell death. Therefore, reliable antibodies recognizing human CD95 are of great interest. Detection of CD95 expression often relies on antibodies, e.g., suitable for Western blotting. To detect CD95, we compared the specificity of nine different anti-human CD95 antibodies recognizing different epitopes by using postnuclear supernatants of four different cell lines. Only two of the antibodies tested, both directed against intracellular epitopes of human CD95, detected endogenous human CD95 by Western blotting. Therefore, we conclude that results obtained with other anti-CD95 antibodies should be treated with caution.