Michael Hahne

Inhibition of death receptor signals by cellular FLIP

The widely expressed protein Fas is a member of the tumour necrosis factor receptor family which can trigger apoptosis. However, Fas surface expression does not necessarily render cells susceptible to Fas ligand-induced death signals,, indicating that inhibitors of the apoptosis-signalling pathway must exist. Here we report the characterization of an inhibitor of apoptosis, designated FLIP (for FLICE-inhibitory protein), which is predominantly expressed in muscle and lymphoid tissues. The short form, FLIPS, contains two death effector domains and is structurally related to the viral FLIP inhibitors of apoptosis, whereas the long form, FLIPL, contains in addition a caspase-like domain in which the active-centre cysteine residue is substituted by a tyrosine residue. FLIPS and FLIPL interact with the adaptor protein FADD, and the protease FLICE,, and potently inhibit apoptosis induced by all known human death receptors. FLIPL is expressed during the early stage of T-cell activation, but disappears when T cells become susceptible to Fas ligand-mediated apoptosis. High levels of FLIPL protein are also detectable in melanoma cell lines and malignant melanoma tumours. Thus FLIP may be implicated in tissue homeostasis as an important regulator of apoptosis.

Melanoma cell expression of Fas(Apo-1/CD95) ligand : Implications for tumor Immune escape

Malignant melanoma accounts for most of the increasing mortality from skin cancer. Melanoma cells were found to express Fas (also called Apo-1 or CD95) ligand (FasL). In metastatic lesions, Fas-expressing T cell infiltrates were proximal to FasL+ tumor cells. In vitro, apoptosis of Fas-sensitive target cells occurred upon incubation with melanoma tumor cells; and in vivo, injection of FasL+ mouse melanoma cells in mice led to rapid tumor formation. In contrast, tumorigenesis was delayed in Fas-deficient lpr mutant mice in which immune effector cells cannot be killed by FasL. Thus, FasL may contribute to the immune privilege of tumors.

The caspase-8 inhibitor FLIP promotes activation of NF-κB and Erk signaling pathways

BACKGROUND Activation of Fas (CD95) by its ligand (FasL) rapidly induces cell death through recruitment and activation of caspase-8 via the adaptor protein Fas-associated death domain protein (FADD). However, Fas signals do not always result in apoptosis but can also trigger a pathway that leads to proliferation. We investigated the level at which the two conflicting Fas signals diverge and the protein(s) that are implicated in switching the response. RESULTS Under conditions in which proliferation of CD3-activated human T lymphocytes is increased by recombinant FasL, there was activation of the transcription factors NF-kappaB and AP-1 and recruitment of the caspase-8 inhibitor and FADD-interacting protein FLIP (FLICE-like inhibitory protein). Fas-recruited FLIP interacts with TNF-receptor associated factors 1 and 2, as well as with the kinases RIP and Raf-1, resulting in the activation of the NF-kappaB and extracellular signal regulated kinase (Erk) signaling pathways. In T cells these two signal pathways are critical for interleukin-2 production. Increased expression of FLIP in T cells resulted in increased production of interleukin-2. CONCLUSIONS We provide evidence that FLIP is not simply an inhibitor of death-receptor-induced apoptosis but that it also mediates the activation of NF-kappaB and Erk by virtue of its capacity to recruit adaptor proteins involved in these signaling pathways.

TRAMP, a Novel Apoptosis-Mediating Receptor with Sequence Homology to Tumor Necrosis Factor Receptor 1 and Fas(Apo-1/CD95)

A novel member of the tumor necrosis factor (TNF) receptor family, designated TRAMP, has been identified. The structural organization of the 393 amino acid long human TRAMP is most homologous to TNF receptor 1. TRAMP is abundantly expressed on thymocytes and lymphocytes. Its extracellular domain is composed of four cysteine-rich domains, and the cytoplasmic region contains a death domain known to signal apoptosis. Overexpression of TRAMP leads to two major responses, NF-kappaB activation and apoptosis. TRAMP-induced cell death is inhibited by an inhibitor of ICE-like proteases, but not by Bcl-2. In addition, TRAMP does not appear to interact with any of the known apoptosis-inducing ligands of the TNF family.

APRIL modulates B and T cell immunity

The TNF-like ligands APRIL and BLyS are close relatives and share the capacity to bind the receptors TACI and BCMA. BLyS has been shown to play an important role in B cell homeostasis and autoimmunity, but the biological role of APRIL remains less well defined. Analysis of T cells revealed an activation-dependent increase in APRIL mRNA expression. We therefore generated mice expressing APRIL as a transgene in T cells. These mice appeared normal and showed no signs of B cell hyperplasia. Transgenic T cells revealed a greatly enhanced survival in vitro as well as enhanced survival of staphylococcal enterotoxin B-reactive CD4+ T cells in vivo, which both directly correlate with elevated Bcl-2 levels. Analysis of humoral responses to T cell-dependent antigens in the transgenic mice indicated that APRIL affects only IgM but not IgG responses. In contrast, T cell-independent type 2 (TI-2) humoral response was enhanced in APRIL transgenic mice. As TACI was previously reported to be indispensable for TI-2 antibody formation, these results suggest a role for APRIL/TACI interactions in the generation of this response. Taken together, our data indicate that APRIL is involved in the induction and/or maintenance of T and B cell responses.

Therapeutic neutralization of CD95-ligand and TNF attenuates brain damage in stroke.

Stroke is the third most common cause of death in the Western world. The mechanisms of brain damage in the affected areas are largely unknown. Hence, rational treatment strategies are limited. Previous experimental evidence suggested that cerebral lesions were less prominent in CD95 (APO-1/Fas)-deficient (lpr) than in wild-type mice. Additional results strongly suggested that the CD95-ligand (CD95L) was a major cause of neuronal autocrine suicide in the penumbra. These data and the assumption that death-receptor systems might determine stroke-related damage in the brain prompted us to examine these systems in in vitro and in vivo models of ischemia. We showed that hybrids of TNF-deficient and gld mice were strongly resistant towards stroke-induced damage. To determine the mechanism of action of TNF and CD95L, we separately investigated their influence on primary ischemic death and secondary inflammatory injury. Inhibition of both TNF and CD95L in vitro prevented death of primary neurons induced by oxygen-glucose deprivation and reperfusion. The recruitment of inflammatory cells to the ischemic hemisphere was abrogated in the absence of both TNF and CD95L. Significantly, mice injected with a mixture of neutralizing anti-TNF and anti-CD95L antibodies 30 min after induction of stroke showed a marked decrease in both infarct volumes and mortality. Accordingly, the locomotor performance of these animals was not significantly impaired in comparison to sham-operated animals. These data reveal that inhibition of TNF and CD95L blocks stroke-related damage at two levels, the primary ischemic and the secondary inflammatory injury. These results offer new approaches in stroke treatment. Cell Death and Differentiation (2001) 8, 679–686

Biologically active APRIL is secreted following intracellular processing in the Golgi apparatus by furin convertase.

Tumor necrosis factor (TNF) ligand family members are synthesized as transmembrane proteins, and cleavage of the membrane‐anchored proteins from the cell surface is frequently observed. The TNF‐related ligands APRIL and BLyS and their cognate receptors BCMA/TACI form a two ligand/two receptor system that has been shown to participate in B‐ and T‐cell stimulation. In contrast to BLyS, which is known to be cleaved from the cell surface, we found that APRIL is processed intracellularly by furin convertase. Blockage of protein transport from the endoplasmic reticulum to the Golgi apparatus by Brefeldin A treatment abrogated APRIL processing, whereas monensin, an inhibitor of post‐Golgi transport, did not interfere with cleavage of APRIL, but blocked secretion of processed APRIL. Thus, APRIL shows a unique maturation pathway among the TNF ligand family members, as it not detectable as a membrane‐anchored protein at the cell surface, but is processed in the Golgi apparatus prior to its secretion.

Oncogenic Ras inhibits Fas ligand-mediated apoptosis by downregulating the expression of Fas

Tumor growth is the result of deregulated tissue homeostasis which is maintained through the delicate balance of cell growth and apoptosis. One of the most efficient inducers of apoptosis is the death receptor Fas. We report here that oncogenic Ras (H‐Ras) downregulates Fas expression and renders cells of fibroblastic and epitheloid origin resistant to Fas ligand‐induced apoptosis. In Ras‐transformed cells, Fas mRNA is absent. Inhibition of DNA methylation restores Fas expression. H‐Ras signals via the PI 3‐kinase pathway to downregulate Fas, suggesting that the known anti‐apoptotic effect of the downstream PKB/Akt kinase may be mediated, at least in part, by the repression of Fas expression. Thus, the oncogenic potential of H‐ras may reside on its capacity not only to promote cellular proliferation, but also to simultaneously inhibit Fas‐triggered apoptosis.

An endogenous hybrid mRNA encodes TWE‐PRIL, a functional cell surface TWEAK–APRIL fusion protein

TWEAK and APRIL are two recently identified tumour necrosis factor (TNF) ligand family members, implicated in angiogenesis and immune regulation, respectively. TWEAK is a transmembrane protein expressed on the cell surface, whereas APRIL acts solely as a secreted factor. In this report, using RACE, RT–PCR, cDNA library screening and an RNase protection assay, we characterize a hybrid transcript between TWEAK and APRIL mRNAs. The encoded TWE‐PRIL protein is composed of TWEAK cytoplasmic and transmembrane domains fused to the APRIL C‐terminal domain. TWE‐PRIL mRNA is expressed and translated in human primary T cells and monocytes, and endogenous TWE‐PRIL protein was detected in primary human T lymphocytes and monocytic cell lines. TWE‐PRIL is membrane anchored and presents the APRIL receptor‐binding domain at the cell surface. It is a biologically active ligand, as it stimulates cycling in T‐ and B‐lymphoma cell lines. Much like membrane‐bound and secreted TNF‐α, the different cellular localizations of TWE‐PRIL and APRIL suggest that they exert distinct biological roles.

Functional expression of Fas and Fas ligand on human gut lamina propria T lymphocytes. A potential role for the acidic sphingomyelinase pathway in normal immunoregulation.

The expression and function of Fas (CD95/APO-1), a cell surface receptor directly responsible for triggering cell death by apoptosis, was investigated on human T lymphocytes resident within the intestinal lamina propria, a major site of antigen challenge and persistent lymphocyte activation. Three color immunofluorescence and FACS analysis indicated that virtually all freshly isolated human gut lamina propria T lymphocytes (T-LPL) express Fas, together with the marker of progress activation CD45R0. A discrete fraction of freshly isolated T-LPL also constitutively expressed Fas ligand (FasL), perhaps as a result of recent in vivo activation. Importantly, whereas Fas cross-linking did not result in apoptosis induction in peripheral blood T lymphocytes (T-PBL), Fas was found to be fully effective in generating the apoptotic signal in T-LPL. This was associated with the activation of an acidic sphingomyelinase and with ceramide generation, early events known to be involved in Fas-mediated apoptotic signaling. By contrast, acidic sphingomyelinase activation and ceramide production were not detectable in T-PBL after Fas cross-linking. However C2-ceramide, a cell permeant synthetic analog of ceramide, could efficiently induce apoptosis in T-LPL and T-PBL when added exogenously. These data indicate that T-LPL constitutively express both Fas and FasL and that Fas cross-linking generates signals resulting in sphingomyelin hydrolysis and apoptosis, outlining a potential mechanism involved in intestinal tolerance. Moreover, they provide the first evidence of a role for ceramide-mediated pathways in normal immunoregulation.