Apoptosis induced by TGF-beta 1 in Burkitt's lymphoma cells is caspase 8 dependent but is death receptor independent.
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TGF-beta is a potent inducer of apoptosis in many Burkitt's lymphoma (BL) cell lines. In this study, we characterize this apoptotic process in the EBV-negative BL41 cell line. Induction of apoptosis was detected as early as 8 h after TGF-beta treatment, as assayed by TUNEL and poly(ADP-ribose) polymerase cleavage. FACS analysis demonstrates that this proceeds predominately from the G1, but also from the G2/M phases of the cell cycle. We observed no early detectable changes in the steady-state levels of Bcl-2 and several of its family members after TGF-beta treatment. We detected cleavage of caspases 2, 3, 7, 8, and 9 into their active subunits. Consistent with the involvement of these enzymes in TGF-beta-mediated apoptosis, the broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(Ome)-flouromethylketone (ZVAD-fmk) blocked TGF-beta-induced apoptosis and revealed a G1 arrest in treated cells. Use of specific caspase inhibitors revealed that the induction of apoptosis is caspase 8 dependent, but caspase 3 independent. Activation of caspase 8 has been shown to be a critical event in death receptor-mediated apoptosis. However, TGF-beta treatment of BL41 cells was found not to affect the cell surface expression of Fas, TNF-R1, DR3, DR4, or DR5, or the steady-state expression levels of Fas ligand, TNF-R1, DR3, DR4, and DR5. Furthermore, blocking experiments indicated that TGF-beta-mediated apoptosis is not dependent on Fas ligand, TNF-alpha, tumor necrosis-like apoptosis-inducing ligand, or TNF-like weak inducer of apoptosis signaling. Therefore, it appears that TGF-beta induces apoptosis in BL cell lines via caspase 8 in a death receptor-independent fashion. (+info)
Studies on the interaction between TWEAK and the death receptor WSL-1/TRAMP (DR3).
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WSL-1/TRAMP (DR3) is a member of the tumour necrosis factor (TNF) receptor superfamily which exhibits effects on NF-kappaB activation and apoptosis. TWEAK, a novel TNF-related molecule, has been proposed as the ligand for this receptor. Utilising both human and murine TWEAK ligand, it is shown that TWEAK and WSL-1/TRAMP do not interact in an in vitro binding assay and that TWEAK binds strongly to cells that do not express WSL-1/TRAMP on the cell surface. Biological activity of TWEAK is also observed in these cells. Finally, cells isolated from WSL-1/TRAMP knockout mice are shown to retain their ability to interact with TWEAK. These results suggest that WSL-1/TRAMP is not the major receptor for TWEAK (+info)
DR3 regulates negative selection during thymocyte development.
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DR3 (Ws1, Apo3, LARD, TRAMP, TNFSFR12) is a member of the death domain-containing tumor necrosis factor receptor (TNFR) superfamily, members of which mediate a variety of developmental events including the regulation of cell proliferation, differentiation, and apoptosis. We have investigated the in vivo role(s) of DR3 by generating mice congenitally deficient in the expression of the DR3 gene. We show that negative selection and anti-CD3-induced apoptosis are significantly impaired in DR3-null mice. In contrast, both superantigen-induced negative selection and positive selection are normal. The pre-T-cell receptor-mediated checkpoint, which is dependent on TNFR signaling, is also unaffected in DR3-deficient mice. These data reveal a nonredundant in vivo role for this TNF receptor family member in the removal of self-reactive T cells in the thymus. (+info)
The cleavage of Akt/protein kinase B by death receptor signaling is an important event in detachment-induced apoptosis.
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Epithelial cells undergo death receptor-dependent apoptosis when detached from matrix, a process termed anoikis. Activation of Akt/protein kinase B (PKB) by matrix attachment protects cells from anoikis. In this study, we establish a link between anoikis and Akt/PKB-mediated survival by demonstrating that Akt/PKB is cleaved by caspases in matrix-detached epithelial cells by a mechanism that involves death receptors. Reduced levels of Akt/PKB protein were observed in detached Madin-Darby canine kidney cells relative to cells attached to collagen. Equivalent levels of Akt/PKB, however, were detected in matrix-adherent and detached cells after inhibition of caspase activity or expression of an Akt/PKB mutant (D108+119A) that is resistant to caspase cleavage. The contribution of death domain-containing proteins to Akt/PKB cleavage was evidenced by the ability of dominant negative Fas-associated death domain to restore normal levels of Akt/PKB in matrix-detached cells. Importantly, expression of a cleavage-resistant Akt/PKB mutant protected matrix-detached cells from apoptosis. These studies suggest that members of the death receptor family promote the caspase-mediated cleavage of Akt/PKB and that this event contributes to anoikis. (+info)
Multiple pathways of TWEAK-induced cell death.
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TWEAK, a recently identified member of the TNF family, is expressed on IFN-gamma-stimulated monocytes and induces cell death in certain tumor cell lines. In this study, we characterized the TWEAK-induced cell death in several tumor cell lines that exhibited distinct features. Although the TWEAK-induced cell death in Kym-1 cells was indirectly mediated by TNF-alpha and was inhibited by cycloheximide, the TWEAK-induced cell death in HSC3 cells or IFN-gamma-treated HT-29 cells was not inhibited by anti-TNF-alpha mAb or cycloheximide, suggesting a direct triggering of cell death via TWEAK receptor in the latter cell lines. The TWEAK-induced apoptosis in HSC3 cells and IFN-gamma-treated HT-29 cells was associated with caspase-8 and caspase-3 activation. Although a pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, inhibited the TWEAK-induced cell death in HSC3 cells, it rather sensitized HT-29 cells to TWEAK-induced cell death by necrosis. This necrosis was abrogated by lysosomal proteinase inhibitors, particularly a cathepsin B inhibitor, [L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl]-L-isoleucyl-L-proline methyl ester. During the process of TWEAK-induced necrosis, cathepsin B was released from lysosome to cytosol. Although DR3 has been reported to be a receptor for TWEAK, all TWEAK-sensitive tumor cell lines used in this study did not express DR3 at either protein or mRNA level, but did bind CD8-TWEAK specifically. These results indicated that TWEAK could induce multiple pathways of cell death, including both caspase-dependent apoptosis and cathepsin B-dependent necrosis, in a cell type-specific manner via TWEAK receptor(s) distinct from DR3. (+info)
TL1A is a TNF-like ligand for DR3 and TR6/DcR3 and functions as a T cell costimulator.
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DR3 is a death domain-containing receptor that is upregulated during T cell activation and whose overexpression induces apoptosis and NF-kappaB activation in cell lines. Here we show that an endothelial cell-derived TNF-like factor, TL1A, is a ligand for DR3 and decoy receptor TR6/DcR3 and that its expression is inducible by TNF and IL-1alpha. TL1A induces NF-kappaB activation and apoptosis in DR3-expressing cell lines, while TR6-Fc protein antagonizes these signaling events. Interestingly, in T cells, TL1A acts as a costimulator that increases IL-2 responsiveness and secretion of proinflammatory cytokines both in vitro and in vivo. Our data suggest that interaction of TL1A with DR3 promotes T cell expansion during an immune response, whereas TR6 has an opposing effect. (+info)
Legionella pneumophila induces apoptosis via the mitochondrial death pathway.
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Legionella pneumophila has been shown to induce apoptosis within macrophages, monocytic cell lines and alveolar epithelial cells. The mechanisms and significance of L. pneumophila-associated apoptosis are not well understood. It has been speculated that L. pneumophila may induce apoptosis through ligation of death receptors by bacterial surface components or by secreted bacterial factors. Translocation of apoptotic factor(s) through the Dot/Icm secretion machinery followed by direct activation of caspases within the cytosol is discussed as another possible mechanism of apoptosis induction by L. pneumophila. Here, it is shown that L. pneumophila induced the mitochondrial release of cytochrome c in CD95 (Fas/Apo-1)-negative monocytic Mono Mac 6 cells, indicating that Legionella-induced apoptosis is mediated via the mitochondrial signalling pathway. In addition, blocking of the death receptor pathway at distinct stages using CD95-, FADD- or caspase-8-deficient Jurkat cells did not affect induction of apoptosis by L. pneumophila. Conversely, inhibition of the mitochondrial death pathway by overexpression of the anti-apoptotic protein Bcl-2 potently inhibited the processing of caspases and the induction of apoptosis. Therefore, these findings support a model in which the induction of apoptosis by L. pneumophila is mediated by activation of the intrinsic mitochondrial death pathway in the absence of external death receptor signalling. (+info)
Expression of silencer of death domains and death-receptor-3 in normal human kidney and in rejecting renal transplants.
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We have previously reported the pattern of cellular expression of tumor necrosis factor receptors (TNFR) in human kidney and their altered expression in transplant rejection. We have extended our studies to examine the expression of Silencer of Death Domains (SODD), a protein that binds to the cytoplasmic portion of TNFR1 to inhibit signaling in the absence of ligand. In normal human kidney SODD is expressed in glomerular endothelial cells where it colocalizes with TNFR1. During acute rejection both SODD and TNFR1 are lost from glomeruli, but we found strong expression of SODD on the luminal surface of tubular epithelial cells. This occurs in the absence of detectable TNFR1 expression, suggesting that SODD could interact with other proteins at these sites. Several other members of the TNF superfamily, including Fas and death receptors (DR)-3, -4, and -5, also contain intracellular death domains, but SODD only interacts with the death domain of DR3. We therefore studied the expression of DR3 in human kidney, and report that this death receptor is up-regulated in renal tubular epithelial cells and endothelial cells of some interlobular arteries, in parallel with SODD, during acute transplant rejection. In less severe rejection episodes, DR3 and SODD were more focally induced, generally at sites of mononuclear cell infiltrates. In ischemic allografts, eg, with acute tubular necrosis but no cellular rejection, DR3 was induced on tubular epithelial cells and on glomerular endothelial cells. These data confirm that TNF receptor family members are expressed in a regulated manner during renal transplant rejection, and identify DR3 as a potential inducible mediator of tubular inflammation and injury. (+info)