Takao Kataoka

TRAIL Receptors 1 (DR4) and 2 (DR5) Signal FADD-Dependent Apoptosis and Activate NF-κB

TRAIL induces apoptosis through two closely related receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Here we show that TRAIL-R1 can associate with TRAIL-R2, suggesting that TRAIL may signal through heteroreceptor signaling complexes. Both TRAIL receptors bind the adaptor molecules FADD and TRADD, and both death signals are interrupted by a dominant negative form of FADD and by the FLICE-inhibitory protein FLIP. The recruitment of TRADD may explain the potent activation of NF-kappaB observed by TRAIL receptors. Thus, TRAIL receptors can signal both death and gene transcription, functions reminiscent of those of TNFR1 and TRAMP, two other members of the death receptor family.

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.

N-Terminal Fragment of c-FLIP(L) Processed by Caspase 8 Specifically Interacts with TRAF2 and Induces Activation of the NF-κB Signaling Pathway

ABSTRACT Caspase 8 is required not only for death receptor-mediated apoptosis but also for lymphocyte activation in the immune system. FLIP(L), the long-splice form of c-FLIP, is one of the specific substrates for caspase 8, and increased expression of FLIP(L) promotes activation of the NF-κB signaling pathway. The synthetic caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk) markedly blocked NF-κB activation induced by overexpression of FLIP(L). FLIP(L) is specifically processed by caspase 8 into N-terminal FLIP(p43) and C-terminal FLIP(p12). Only FLIP(p43) was able to induce NF-κB activation as efficiently as FLIP(L), and FLIP(p43)-induced NF-κB activation became insensitive to zVAD-fmk. In caspase 8-deficient cells, FLIP(p43) provoked NF-κB activation only when procaspase 8 or caspase 8(p43) was complemented. FLIP(p43)-induced NF-κB activation was profoundly blocked by the dominant-negative TRAF2. Moreover, endogenous TRAF2 interacted specifically with FLIP(p43), and the formation of the FLIP(p43)-caspase 8-TRAF2 tertiary complex was a prerequisite to induction of NF-κB activation. zVAD-fmk prevented the recruitment of TRAF2 into the death-inducing signaling complex. Thus, our present results demonstrate that FLIP(p43) processed by caspase 8 specifically interacts with TRAF2 and subsequently induces activation of the NF-κB signaling pathway.

The Caspase 8 Inhibitor c-FLIP L Modulates T-Cell Receptor-Induced Proliferation but Not Activation-Induced Cell Death of Lymphocytes

ABSTRACT The caspase 8 inhibitor c-FLIPL can act in vitro as a molecular switch between cell death and growth signals transmitted by the death receptor Fas (CD95). To elucidate its function in vivo, transgenic mice were generated that overexpress c-FLIPL in the T-cell compartment (c-FLIPL Tg mice). As anticipated, FasL-induced apoptosis was inhibited in T cells from the c-FLIPL Tg mice. In contrast, activation-induced cell death of T cells in c-FLIPL Tg mice was unaffected, suggesting that this deletion process can proceed in the absence of active caspase 8. Accordingly, c-FLIPL Tg mice differed from Fas-deficient mice by showing no accumulation of B220+ CD4− CD8− T cells. However, stimulation of T lymphocytes with suboptimal doses of anti-CD3 or antigen revealed increased proliferative responses in T cells from c-FLIPL Tg mice. Thus, a major role of c-FLIPL in vivo is the modulation of T-cell proliferation by decreasing the T-cell receptor signaling threshold.

Prodigiosins as a New Group of H+/Cl−Symporters That Uncouple Proton Translocators

We reported previously (Kataoka, T., Muroi, M., Ohkuma, S., Waritani, T., Magae, J., Takatsuki, A., Kondo, S., Yamasaki, M., and Nagai, K. (1995) FEBS Lett. 359, 53–59) that prodigiosin 25-C uncoupled vacuolar H+-ATPase, inhibited vacuolar acidification, and affected glycoprotein processing. In the present study we show that prodigiosins (prodigiosin, metacycloprodigiosin, and prodigiosin 25-C) inhibit the acidification activity of H+-ATPase chloride dependently, but not membrane potential formation or ATP hydrolysis activity, and suggest that they promote H+/Cl− symport (or OH−/Cl− exchange, in its equivalence) across vesicular membranes. In fact, prodigiosins displayed H+/Cl− symport activity on liposomal membranes. First of all, they decreased the internal pH of liposomes depending on the external chloride, and raised it depending on the internal chloride when external buffer was free from chloride. Second, their effect was electroneutral and not seriously affected by the application of an inside positive membrane potential generated by K+ and valinomycin. Finally, they promoted the uptake of [36Cl] from external buffers with concomitant intraliposomal acidification when external pH was acidic relative to liposome interior. As prodigiosins hardly inhibit the catalytic activity (ATP hydrolysis) unlike well known OH−/Cl− exchangers (for example, tributyltin chloride), they should provide powerful tools for the study of molecular machinery and cellular activities involving transport of protons and/or chloride.

Cellular FLIP (Long Form) Regulates CD8+ T Cell Activation through Caspase-8-Dependent NF-κB Activation

Cellular FLIP long form (c-FLIPL) was originally identified as an inhibitor of Fas (CD95/Apo-1). Subsequently, additional functions of c-FLIPL were identified through its association with receptor-interacting protein (RIP)1 and TNFR-associated factor 2 to activate NF-κB, as well as by its association with and activation of caspase-8. T cells from c-FLIPL-transgenic (Tg) mice manifest hyperproliferation upon activation, although it was not clear which of the various functions of c-FLIPL was involved. We have further explored the effect of c-FLIPL on CD8+ effector T cell function and its mechanism of action. c-FLIPL-Tg CD8+ T cells have increased proliferation and IL-2 responsiveness to cognate Ags as well as to low-affinity Ag variants, due to increased CD25 expression. They also have a T cytotoxic 2 cytokine phenotype. c-FLIPL-Tg CD8+ T cells manifest greater caspase activity and NF-κB activity upon activation. Both augmented proliferation and CD25 expression are blocked by caspase inhibition. c-FLIPL itself is a substrate of the caspase activity in effector T cells, being cleaved to a p43FLIP form. p43FLIP more efficiently recruits RIP1 than full-length c-FLIPL to activate NF-κB. c-FLIPL and RIP1 also coimmunoprecipitate with active caspase-8 in effector CD8+ T cells. Thus, one mechanism by which c-FLIPL influences effector T cell function is through its activation of caspase-8, which in turn cleaves c-FLIPL to allow RIP1 recruitment and NF-κB activation. This provides a partial explanation of why caspase activity is required to initiate proliferation of resting T cells.

Safflower polysaccharides activate the transcription factor NF-κB via Toll-like receptor 4 and induce cytokine production by macrophages

Two active polysaccharide fractions (SF1 and SF2) purified from dried safflower petals (Carthamus tinctorius L.) stimulated the synthesis of various cytokines by peritoneal macrophages. In a number of cell types, SF1 and SF2 induced a rapid degradation of IkappaB alpha essential for the activation of the transcription factor NF-kappaB. Toll-like receptor 4 (TLR4), but not TLR2, was expressed in all cell lines that responded to SF1 and SF2. Enforced expression of TLR4 and MD-2 rendered responsiveness to SF1 and SF2. Moreover, these safflower polysaccharides failed to induce the production of TNF-alpha and NO by peritoneal macrophages prepared from C3H/HeJ mice that have a point mutation in the Tlr4 gene. Thus, these observations clearly indicate that safflower polysaccharides activate the NF-kappaB signaling pathway via TLR4.

The fungal metabolite gliotoxin: immunosuppressive activity on CTL-mediated cytotoxicity.

Gliotoxin, a potential etiologic agent which is synthesized by Aspergillus fumigatus and other pathogenic fungi, exhibits a variety of immunosuppressive activities. We have found that gliotoxin markedly inhibits both perforin-dependent and Fas ligand-dependent cytotoxic T-lymphocyte (CTL)-mediated cytotoxicity. Gliotoxin blocked granule exocytosis and the production of inositol phosphates in response to anti-CD3 stimulation. Apparently, activation signals were not efficiently received by the gliotoxin-treated CTL clone, perhaps because gliotoxin profoundly disturbed CTL cell attachment to immobilized anti-CD3. Although the expression of surface molecules of the CTL clone such as CD3 was unaffected by gliotoxin, the effector/target conjugate formation was inhibited dose-dependently by gliotoxin treatment of the effector CTL clone. These results suggest that gliotoxin prevents CTL from interacting with target cells.

Development of improved soluble inhibitors of FasL and CD40L based on oligomerized receptors.

TNF receptor family members fused to the constant domain of immunoglobulin G have been widely used as immunoadhesins in basic in vitro and in vivo research and in some clinical applications. In this study, we assemble soluble, high avidity chimeric receptors on a pentameric scaffold derived from the coiled-coil domain of cartilage oligomeric matrix protein (COMP). The affinity of Fas and CD40 (but not TNFR-1 and TRAIL-R2) to their ligands is increased by fusion to COMP, when compared to the respective Fc chimeras. In functional assays, Fas:COMP was at least 20-fold more active than Fas:Fc at inhibiting the action of sFasL, and CD40:COMP could block CD40L-mediated proliferation of B cells, whereas CD40:Fc could not. In conclusion, members of the TNF receptor family can display high specificity and excellent avidity for their ligands if they are adequately multimerized.

Prodigiosin 25-C uncouples vacuolar type H+-ATPase, inhibits vacuolar acidification and affects glycoprotein processing

Prodigiosin 25‐C inhibited the accumulation of 3‐(2,4‐dinitroanilino)‐3′‐amino‐N‐methyldipropylamine and acridine orange in the acidic compartments of baby hamster kidney cells with little perturbation of cellular ATP levels. In rat liver lysosomes, prodigiosin 25‐C inhibited the proton pump activity with an IC50 of approximately 30 nM, but did not affect ATPase activity up to 1 μM. It also delayed the transport of vesicular stomatitis virus G protein and induced a drastic swelling of Golgi apparatus and mitochondria. These results indicate that prodigiosin 25‐C raises the pH of acidic compartments through inhibition of the proton pump activity of vacuolar type H+‐ATPase, thereby causing the functional and morphological changes to the Golgi apparatus.