Bcl-2 transgene expression fails to prevent fatal hepatocyte apoptosis induced by endogenous TNFalpha in mice lacking RelA.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
L A O'Reilly
Publications and source records attributed to L A O'Reilly.
Modifications and intracellular trafficking of FADD/MORT1 and caspase-8 after stimulation of T lymphocytes.
The adaptor protein FADD/MORT1 is essential for apoptosis induced by 'death receptors', such as Fas (APO-1/CD95), mediating aggregation and autocatalytic activation of caspase-8. Perhaps surprisingly, FADD and caspase-8 are also critical for mitogen-induced proliferation of T lymphocytes. We generated novel monoclonal antibodies specific for mouse FADD and caspase-8 to investigate whether cellular responses, apoptosis or proliferation, might be explained by differences in post-translational modification and subcellular localisation of these proteins. During both apoptosis signalling and mitogenic activation, FADD and caspase-8 aggregated in multiprotein complexes and formed caps at the plasma membrane but they did not colocalise with lipid rafts. Interestingly, mitogenic stimulation, but not Fas ligation, induced a unique post-translational modification of FADD. These different modifications may determine whether FADD and caspase-8 induce cell death or proliferation.
Adaptor Proteins, Signal Transducing↗
Caspase-2 is not required for thymocyte or neuronal apoptosis even though cleavage of caspase-2 is dependent on both Apaf-1 and caspase-9.
We have generated rat monoclonal antibodies that specifically recognise caspase-2 from many species, including mouse, rat and humans. Using these antibodies, we have investigated caspase-2 expression, subcellular localisation and processing. We demonstrate that caspase-2 is expressed in most tissues and cell types. Cell fractionation and immunohistochemistry experiments show that caspase-2 is found in the nuclear and cytosolic fractions, including a significant portion present in the Golgi complex. We found that caspase-2 is processed in response to many apoptotic stimuli but experiments with caspase-2 deficient mice demonstrated that it is not required for apoptosis of thymocytes or dorsal root ganglia (DRG) neurons in response to a variety of cytotoxic stimuli. Caspase-2 processing does not occur in thymocytes lacking Apaf-1 or caspase-9, suggesting that in this cell type, activation of caspase-2 occurs downstream of apoptosome formation.
Animals↗
Bmf: a proapoptotic BH3-only protein regulated by interaction with the myosin V actin motor complex, activated by anoikis.
Bcl-2 family members bearing only the BH3 domain are essential inducers of apoptosis. We identified a BH3-only protein, Bmf, and show that its BH3 domain is required both for binding to prosurvival Bcl-2 proteins and for triggering apoptosis. In healthy cells, Bmf is sequestered to myosin V motors by association with dynein light chain 2. Certain damage signals, such as loss of cell attachment (anoikis), unleash Bmf, allowing it to translocate and bind prosurvival Bcl-2 proteins. Thus, at least two mammalian BH3-only proteins, Bmf and Bim, function to sense intracellular damage by their localization to distinct cytoskeletal structures.
Adaptor Proteins, Signal Transducing↗
Tissue expression and subcellular localization of the pro-survival molecule Bcl-w.
Anti-apoptotic members of the Bcl-2 family, such as Bcl-w, maintain cell viability by preventing the activation of the cell death effectors, the caspases. Gene targeting experiments in mice have demonstrated that Bcl-w is required for spermatogenesis and for survival of damaged epithelial cells in the gut. Bcl-w is, however, dispensable for physiological cell death in other tissues. Here we report on the analysis of Bcl-w protein expression using a panel of novel monoclonal antibodies. Bcl-w is found in a diverse range of tissues including colon, brain and testes. A survey of transformed cell lines and purified hematopoietic cells demonstrated that Bcl-w is expressed in cells of myeloid, lymphoid and epithelial origin. Subcellular fractionation and confocal laser scanning microscopy demonstrated that Bcl-w protein is associated with intracellular membranes. The implications of these results are discussed in the context of the phenotype of Bcl-w-null mice and recent data that suggest that Bcl-w may play a role in colon carcinogenesis.
Animals↗
The anti-apoptotic activities of Rel and RelA required during B-cell maturation involve the regulation of Bcl-2 expression.
Rel and RelA, individually dispensable for lymphopoiesis, serve unique functions in activated B and T cells. Here their combined roles in lymphocyte development were examined in chimeric mice repopulated with c-rel(-/-) rela(-/-) fetal liver hemopoietic stem cells. Mice engrafted with double-mutant cells lacked mature IgM(lo)IgD(hi) B cells, and numbers of peripheral CD4(+) and CD8(+) T cells were markedly reduced. The absence of mature B cells was associated with impaired survival that coincided with reduced expression of bcl-2 and A1. bcl-2 transgene expression not only prevented apoptosis and increased peripheral B-cell numbers, but also induced further maturation to an IgM(lo)IgD(hi) phenotype. In contrast, the survival of double-mutant T cells was normal and the bcl-2 transgene could not rectify the peripheral T-cell deficit. These findings indicate that Rel and RelA serve essential, albeit redundant, functions during the later antigen-independent stages of B- and T-cell maturation, with these transcription factors promoting the survival of peripheral B cells in part by upregulating Bcl-2.
Animals↗
Fas ligand, Bcl-2, granulocyte colony-stimulating factor, and p38 mitogen-activated protein kinase: Regulators of distinct cell death and survival pathways in granulocytes.
The short life span of granulocytes, which limits many inflammatory responses, is thought to be influenced by the Bcl-2 protein family, death receptors such as CD95 (Fas/APO-1), stress-activated protein kinases such as p38 mitogen-activated protein kinase (MAPK), and proinflammatory cytokines like granulocyte colony-stimulating factor (G-CSF). To clarify the roles of these various regulators in granulocyte survival, we have investigated the spontaneous apoptosis of granulocytes in culture and that induced by Fas ligand or chemotherapeutic drugs, using cells from normal, CD95-deficient lpr, or vav-bcl-2 transgenic mice. CD95-induced apoptosis, which required receptor aggregation by recombinant Fas ligand or the membrane-bound ligand, was unaffected by G-CSF treatment or Bcl-2 overexpression. Conversely, spontaneous and drug-induced apoptosis occurred normally in lpr granulocytes but were suppressed by G-CSF treatment or Bcl-2 overexpression. Although activation of p38 MAPK has been implicated in granulocyte death, their apoptosis actually was markedly accelerated by specific inhibitors of this kinase. These results suggest that G-CSF promotes granulocyte survival largely through the Bcl-2-controlled pathway, whereas CD95 regulates a distinct pathway to apoptosis that is not required for either their spontaneous or drug-induced death. Moreover, p38 MAPK signaling contributes to granulocyte survival rather than their apoptosis.
Animals↗
Pro-apoptotic apoptosis protease-activating factor 1 (Apaf-1) has a cytoplasmic localization distinct from Bcl-2 or Bcl-x(L).
How Bcl-2 and its pro-survival relatives prevent activation of the caspases that mediate apoptosis is unknown, but they appear to act through the caspase activator apoptosis protease-activating factor 1 (Apaf-1). According to the apoptosome model, the Bcl-2-like proteins preclude Apaf-1 activity by sequestering the protein. To explore Apaf-1 function and to test this model, we generated monoclonal antibodies to Apaf-1 and used them to determine its localization within diverse cells by subcellular fractionation and confocal laser scanning microscopy. Whereas Bcl-2 and Bcl-x(L) were prominent on organelle membranes, endogenous Apaf-1 was cytosolic and did not colocalize with them, even when these pro-survival proteins were overexpressed or after apoptosis was induced. Immunogold electron microscopy confirmed that Apaf-1 was dispersed in the cytoplasm and not on mitochondria or other organelles. After the death stimuli, Bcl-2 and Bcl-x(L) precluded the release of the Apaf-1 cofactor cytochrome c from mitochondria and the formation of larger Apaf-1 complexes, which are steps that presage apoptosis. However, neither Bcl-2 nor Bcl-x(L) could prevent the in vitro activation of Apaf-1 induced by the addition of exogenous cytochrome c. Hence, rather than sequestering Apaf-1 as proposed by the apoptosome model, Bcl-2-like proteins probably regulate Apaf-1 indirectly by controlling upstream events critical for its activation.
Animals↗
bcl-2 transgene expression inhibits apoptosis in the germinal center and reveals differences in the selection of memory B cells and bone marrow antibody-forming cells.
Immunization with T cell-dependent antigens generates long-lived memory B cells and antibody-forming cells (AFCs). Both populations originate in germinal centers and, predominantly, produce antibodies with high affinity for antigen. The means by which germinal center B cells are recruited into these populations remains unclear. We have examined affinity maturation of antigen-specific B cells in mice expressing the cell death inhibitor bcl-2 as a transgene. Such mice had reduced apoptosis in germinal centers and an excessive number of memory B cells with a low frequency of V gene somatic mutation, including those mutations encoding amino acid exchanges known to enhance affinity. Despite the frequency of AFCs being increased in bcl-2-transgenic mice, the fraction secreting high-affinity antibody in the bone marrow at day 42 remained unchanged compared with controls. The inability of BCL-2 to alter selection of bone marrow AFCs is consistent with these cells being selected within the germinal center on the basis of their affinity being above some threshold rather than their survival being due to a selective competition for an antigen-based signal. Continuous competition for antigen does, however, explain formation of the memory compartment.
ADP-ribosyl Cyclase↗
The proapoptotic BH3-only protein bim is expressed in hematopoietic, epithelial, neuronal, and germ cells.
Proapoptotic Bcl-2 family members activate cell death by neutralizing their anti-apoptotic relatives, which in turn maintain cell viability by regulating the activation of the cell death effectors, the caspases. Bim belongs to a distinct subgroup of proapoptotic proteins that only resemble other Bcl-2 family members within the short BH3 domain. Gene targeting experiments in mice have shown that Bim is essential for the execution of some but not all apoptotic stimuli, for hematopoietic cell homeostasis, and as a barrier against autoimmunity. There are three Bim isoforms, Bim(S), Bim(L), and Bim(EL), which have different proapoptotic potencies due at least in part to differences in interaction with the dynein motor complex. The expression pattern of Bim was investigated by immunohistochemical staining, immunoprecipitation followed by Western blotting, and in situ hybridization. Bim was found in hematopoietic, epithelial, neuronal, and germ cells. Bim(L) and Bim(EL) were coexpressed at similar levels in many cell types, but Bim(S) was not detected. Microscopic examination revealed a punctate pattern of Bim(L) and Bim(EL) immunostaining, indicating association with cytoplasmic structures. These results are discussed in the context of the phenotype of Bim-deficient mice and the post-translational regulation of Bim's pro-apoptotic activity.
3T3 Cells↗
The role of bim, a proapoptotic BH3-only member of the Bcl-2 family in cell-death control.
Apoptosis is an evolutionarily conserved process for killing unwanted cells. Genetic and biochemical experiments have indicated that three groups of proteins are necessary for activation of the cell-death effector machinery: cysteine proteases, their adaptors, and proapoptotic Bcl-2 family members. Antiapoptotic Bcl-2 family members are needed for cell survival. We have cloned Bim, a proapoptotic Bcl-2 family member that shares with the family only a 9-16 aa region of homology [Bcl-3 homology region(BH3)], but is otherwise unique. Bim requires its BH3 region for binding to Bcl-2 and activation of apoptosis. Analysis of Bim-deficient mice has shown that Bim is essential for the execution of some but not all apoptotic stimuli that can be antagonized by Bcl-2. Bim-deficient mice have increased numbers of lymphocytes, plasma cells, and myeloid cells, and most develop fatal autoimmune glomerulonephritis. In healthy cells, Bim is bound to the microtubule-associated dynein motor complex, and is thereby sequestered from Bcl-2. Certain apoptotic signals unleash Bim and allow it to translocate to intracellular membranes, where it interacts with Bcl-2 or its homologues. These results indicate that BH3-only proteins are essential inducers of apoptosis that can be unleashed by certain death signals. Unleashed BH3-only proteins neutralize the prosurvival function of Bcl-2-like molecules, and this is thought to liberate Apaf-l-like adapters to activate caspase zymogens, which then initiate cell degradation.
Animals↗
The role of the pro-apoptotic Bcl-2 family member bim in physiological cell death.
Apoptosis, an evolutionarily conserved process for killing unwanted cells in multicellular organisms, is essential for normal development, tissue homeostasis and as a defense against pathogens. The control of apoptosis is of considerable importance for clinical medicine, as its deregulation can lead to cancer, autoimmunity or degenerative diseases. We have disrupted the Bim gene in the mouse and demonstrated that it plays a major and non-redundant role in embryogenesis, in the control of hematopoietic cell death, and as a barrier against autoimmunity.
Animals↗
Apoptosis and autoimmune disease.
The process of programmed cell death or apoptosis was already noted in 1842 by Vogt [1], but it was not until the more recent studies of Kerr et al. 1972 [2] that an explosion of interest in apoptosis research occurred. Genetic, biochemical and cellular analysis in certain mammals, in the nematode Caenorhabditis elegans and in the fruitfly Drosophila melanogaster have identified several apoptosis regulating genes. This indicates that programmed cell death is an active, genetically controlled process. Many of the known cell death regulators are homologous in mammals, nematodes and insects, indicating that apoptosis is an evolutionarily conserved process. Apoptosis can be induced via multiple independent signalling pathways which converge upon a common final effector machinery. This stimulates activation of latent cysteine proteases (caspases), which cleave vital cellular substrates and thereby lead to the death of cells. The regulatory pathways of apoptosis are becoming clear with the discovery of specific signalling molecules. It has become evident that many disease processes including autoimmunity and cancer can be caused by deregulation of the apoptotic process. With the discovery of novel cell surface-bound death receptors, their ligands and further insight into the apoptotic machinery within the cell, research may ultimately lead to the design of therapies that allow intervention in the apoptotic process. The aims of such strategies would be to turn on apoptosis in neoplastic cells or in lymphocytes that are causing autoimmune disease or to prevent cell death in degenerative disorders. This review describes current understanding of the molecular regulation of apoptosis, and focuses on issues relating to possible roles of defective cell death control in autoimmunity.
Animals↗
The proapoptotic activity of the Bcl-2 family member Bim is regulated by interaction with the dynein motor complex.
Bcl-2 family members that have only a single Bcl-2 homology domain, BH3, are potent inducers of apoptosis, and some appear to play a critical role in developmentally programmed cell death. We examined the regulation of the proapoptotic activity of the BH3-only protein Bim. In healthy cells, most Bim molecules were bound to LC8 cytoplasmic dynein light chain and thereby sequestered to the microtubule-associated dynein motor complex. Certain apoptotic stimuli disrupted the interaction between LC8 and the dynein motor complex. This freed Bim to translocate together with LC8 to Bcl-2 and to neutralize its antiapoptotic activity. This process did not require caspase activity and therefore constitutes an initiating event in apoptosis signaling.
Amino Acid Sequence↗
B lymphocytes differentially use the Rel and nuclear factor kappaB1 (NF-kappaB1) transcription factors to regulate cell cycle progression and apoptosis in quiescent and mitogen-activated cells.
Rel and nuclear factor (NF)-kappaB1, two members of the Rel/NF-kappaB transcription factor family, are essential for mitogen-induced B cell proliferation. Using mice with inactivated Rel or NF-kappaB1 genes, we show that these transcription factors differentially regulate cell cycle progression and apoptosis in B lymphocytes. Consistent with an increased rate of mature B cell turnover in naive nfkb1-/- mice, the level of apoptosis in cultures of quiescent nfkb1-/-, but not c-rel-/-, B cells is higher. The failure of c-rel-/- or nfkb1-/- B cells to proliferate in response to particular mitogens coincides with a cell cycle block early in G1 and elevated cell death. Expression of a bcl-2 transgene prevents apoptosis in resting and activated c-rel-/- and nfkb1-/- B cells, but does not overcome the block in cell cycle progression, suggesting that the impaired proliferation is not simply a consequence of apoptosis and that Rel/NF-kappaB proteins regulate cell survival and cell cycle control through independent mechanisms. In contrast to certain B lymphoma cell lines in which mitogen-induced cell death can result from Rel/NF-kappaB-dependent downregulation of c-myc, expression of c-myc is normal in resting and stimulated c-rel-/- B cells, indicating that target gene(s) regulated by Rel that are important for preventing apoptosis may differ in normal and immortalized B cells. Collectively, these results are the first to demonstrate that in normal B cells, NF-kappaB1 regulates survival of cells in G0, whereas mitogenic activation induced by distinct stimuli requires different Rel/NF-kappaB factors to control cell cycle progression and prevent apoptosis.
Animals↗
Bim: a novel member of the Bcl-2 family that promotes apoptosis.
Certain members of the Bcl-2 family inhibit apoptosis while others facilitate this physiological process of cell death. An expression screen for proteins that bind to Bcl-2 yielded a small novel protein, denoted Bim, whose only similarity to any known protein is the short (nine amino acid) BH3 motif shared by most Bcl-2 homologues. Bim provokes apoptosis, and the BH3 region is required for Bcl-2 binding and for most of its cytotoxicity. Like Bcl-2, Bim possesses a hydrophobic C-terminus and localizes to intracytoplasmic membranes. Three Bim isoforms, probably generated by alternative splicing, all induce apoptosis, the shortest being the most potent. Wild-type Bcl-2 associates with Bim in vivo and modulates its death function, whereas Bcl-2 mutants that lack survival function do neither. Significantly, Bcl-xL and Bcl-w, the two closest homologues of Bcl-2, also bind to Bim and inhibit its activity, but more distant viral homologues, adenovirus E1B19K and Epstein-Barr virus BHRF-1, can do neither. Hence, Bim appears to act as a 'death ligand' which can only neutralize certain members of the pro-survival Bcl-2 sub-family.
Amino Acid Sequence↗
Rapid hybridoma screening method for the identification of monoclonal antibodies to low-abundance cytoplasmic proteins.
Screening assays are the most time-consuming and labor-intensive part of generating monoclonal antibodies (MAbs). Antibodies identified by enzyme-linked immunosorbent assay (ELISA) screening often are not suitable for their intended application such as immunofluorescence staining. We describe here a rapid and efficient flow cytometric screening procedure for the identification of MAbs directed against low-abundance cytoplasmic proteins, in our case, the pro-apoptotic molecule Bim. Cells from an equal mixture of a parental cell line and a subline expressing Bim were fixed, permeabilized and incubated with hybridoma supernatants. The supernatants were derived from a fusion of Sp2/0 plasmacytoma cells and spleen cells from a rat immunized with recombinant glutathione-S-transferase (GST)-BimL fusion protein. Secondary staining with fluorochrome-labeled anti-rat Ig antibodies allowed detection of clones expressing Bim-specific antibodies. The screening procedure was rapid and efficient, and most monoclonal antibodies identified were proven to be useful for immunofluorescence staining and other applications.
Animals↗