Role of membrane microdomain rafts in TNFR-mediated signal transduction.
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Animals↗
Biomedical subjects
A O Hueber
Publications and source records attributed to A O Hueber.
Expression of Mad1 in T cells leads to reduced thymic cellularity and impaired mitogen-induced proliferation.
To investigate Mad1 function in vivo, transgenic mice were generated that express a Mad1 transgene in T lineage cells under the control of the proximal lck promoter. Thymus size in lck-Mad1 transgenic mice is drastically reduced although representation of the various thymocyte sub populations appears normal. To investigate more closely any effects of Mad1 expression on thymocytes, we examined thymic selection using MHC class I-restricted H-Y-TCR transgenic mice. Mad1 expression in vivo reduces the efficiency of positive selection. Furthermore, thymocytes and splenic T cells from lck-Mad1 transgenic mice display a profound proliferative defect in response to activation with either PMA/Ionomycin or immobilized anti-CD3/CD28 antibody. This proliferative defect is not reversed by addition of exogenous IL-2 and is p53-independent. The growth inhibition caused by Mad1 is overcome by expression of active c-Myc.
Animals↗
A dominant negative Fas-associated death domain protein mutant inhibits proliferation and leads to impaired calcium mobilization in both T-cells and fibroblasts.
Death domain-containing members of the tumor necrosis factor (TNF) receptor family ("death receptors") can induce apoptosis upon stimulation by their natural ligands or by agonistic antibodies. Activated death receptors recruit death domain adapter proteins like Fas-associated death domain protein (FADD), and this ultimately leads to proteolytic activation of the caspase cascade and cell death. Recently, FADD has also been implicated in the regulation of proliferation; functional inhibition of FADD results in p53-dependent impairment of proliferation in activated T-cells. In this study we have further analyzed T-cells derived from transgenic mice expressing a dominant negative FADD mutant (FADD DN) under control of the lck promoter in vitro so as to identify the signaling pathways that become engaged upon T-cell receptor stimulation and that are regulated by death receptors. FADD DN expression inhibits T-cell proliferation, both at the G(0) --> S transition and in the G(1) phase of continuously proliferating cells. We observe a decrease in the release of calcium from intracellular stores after T-cell receptor stimulation, whereas influx of extracellular calcium seems to be unaffected. FADD DN-expressing fibroblasts show a similarly inhibited cell growth and impaired calcium mobilization indicating that the modulation of proliferation and calcium response by death receptors is not cell type-specific.
3T3 Cells↗
Reversible activation of c-Myc in thymocytes enhances positive selection and induces proliferation and apoptosis in vitro.
In order to study the effect of c-Myc activation in T lymphocytes in vivo, we generated transgenic mice that express a 4-hydroxytamoxifen (4-OHT)-dependent switchable c-myc oncoprotein under the control of the proximal lck promoter. Activation of c-MycER causes no obvious alteration in T cell ontogeny. However, using MHC class I restricted H-Y-TCR transgenic mice, we found that c-Myc activation in vivo enhances the efficiency of positive selection. Moreover, splenic T cells derived from lck-c-mycER transgenic mice in which c-Myc had been activated exhibited increased proliferation in vitro in response to activation with anti-CD3/CD28 antibody. Activation of c-MycER also promotes apoptosis in thymocytes in vitro.
Animals↗
CD95: more than just a death factor?
The CD95 protein delivers crucial signals for lymphocyte death, and may also negatively regulate T-lymphocyte activation by preventing the influx of calcium ions from the cell's exterior. The block in calcium-ion influx occurs through the activation of acidic sphingomyelinase and the release of ceramide, a metabolite that can also induce cell death.
Calcium Signaling↗
c-Myc-induced sensitization to apoptosis is mediated through cytochrome c release.
Expression of c-Myc sensitizes cells to a wide range of pro-apoptotic stimuli. We here show that this pro-apoptotic effect is mediated through release of mitochondrial holocytochrome c into the cytosol. First, activation of c-Myc triggers release of cytochrome c from mitochondria. This release is caspase-independent and blocked by the survival factor IGF-1. Second, c-Myc-induced apoptosis is blocked by microinjection of anticytochrome c antibody. In addition, we show that microinjection of holocytochrome c mimics the effect of c-Myc activation, sensitizing cells to DNA damage and to the CD95 pathway. Both p53 and CD95/Fas signaling have been implicated in c-Myc-induced apoptosis but neither was required for c-Myc-induced cytochrome c release. Nonetheless, inhibition of CD95 signaling in fibroblasts did prevent c-Myc-induced apoptosis, apparently by obstructing the ability of cytosolic cytochrome c to activate caspases. We conclude that c-Myc promotes apoptosis by causing the release of cytochrome c, but the ability of cytochrome c to activate apoptosis is critically dependent upon other signals.
Adaptor Proteins, Signal Transducing↗
c-Myc and E1A induced cellular sensitivity to activated NK cells involves cytotoxic granules as death effectors.
The contact of natural killer (NK) cells with foreign cells and with certain virus-infected or tumor cells triggers the cytolytic machinery of NK cells. This triggering leads to exocytosis of the cytotoxic NK cell granules. The oncoproteins c-Myc and E1A render cells vulnerable to NK cell mediated cytolysis yet the mechanisms of sensitization are not well understood. In a model where foreign cells (rat fibroblasts) were cocultured with human IL-2 activated NK cells, we observed that NK cells were capable of efficiently killing their targets only if the cells overexpressed the oncogene c-Myc or E1A. Both the parental and the oncogene expressing fibroblasts similarly triggered phosphoinositide hydrolysis in the bound NK cells, demonstrating that NK cells were cytolytically activated in contact with both resistant parental and oncogene expressing sensitive target fibroblasts. The cell death was independent of wild-type p53 and was not inhibited by an anti-apoptotic protein EIB19K. These results provided evidence that c-Myc and E1A activated the NK cell induced cytolysis at a post-triggering stage of NK cell-target cell interaction. In consistence, the c-Myc and E1A overexpressing fibroblasts were more sensitive to the cytolytic effects of isolated NK cell-derived granules than parental cells. The data indicate that oncogenes activate the cytotoxicity of NK cell granules. This mechanism can have a role in directing the cytolytic action of NK cells towards the virus-infected and cancer cells.
Actins↗
The opposing roles of the Akt and c-Myc signalling pathways in survival from CD95-mediated apoptosis.
Expression of the proto-oncogene c-myc stimulates cell proliferation in the presence of the appropriate survival factors and triggers apoptosis in their absence; this dual capacity ensures that cell growth is restricted to the correct paracrine environment and is thereby strictly controlled. Recently our laboratory demonstrated that c-Myc-induced apoptosis requires the CD95 death receptor pathway and that insulin-like growth factor (IGF-1) signalling suppresses this killing. To investigate further the links between c-Myc and IGF-1 pathways in CD95-induced apoptosis, we examined the effects of c-Myc and a downstream IGF-1 survival kinase, Akt, on killing mediated by CD95 and its recruited effector proteins (FADD and caspase-8). Here, we show that c-Myc activation does not exacerbate killing induced by FADD or pro-caspase-8, which narrows the point at which c-Myc exerts its action downstream of the interaction of CD95 with its ligand and upstream of FADD. We show further that activated Akt suppresses CD95-induced apoptosis and that Akt exerts its activity at a point downstream of FADD but upstream of caspase-8. These results restrict the possible mechanisms by which CD95-induced apoptosis is modulated by death signals and survival factors.
3T3 Cells↗
Impaired cutaneous immune responses in Thy-1-deficient mice.
Thy-1 is a cell surface glycoprotein expressed mainly on brain and lymphoid tissue. Although the functions of Thy-1 are incompletely understood, evidence exists that Thy-1 participates in T cell activation. To examine the functional role of Thy-1 in cutaneous immune responses in vivo, Thy-1 gene-targeted mice (Thy-1-/-) and wild-type mice (Thy-1+/+) were immunized with the hapten oxazolone. After challenge with oxazolone, contact hypersensitivity responses in Thy-1-/- mice were reduced by 25% compared with Thy-1+/+ mice. Likewise, irritant dermatitis induced by croton oil was also decreased. In addition, Thy-1-/- mice showed a significantly reduced delayed-type hypersensitivity response after injection of allogeneic spleen cells into the hind footpads of allosensitized animals when compared with Thy-1+/+ mice. Moreover, proliferative responses to immobilized anti-CD3 were decreased in peripheral Thy-1-/- lymphocytes; this decrease was associated with a significantly reduced intracellular Ca2+ influx and protein tyrosine phosphorylation, indicating impairment of early lymphocyte activation. In contrast, the T cell proliferation induced by mitogens was normal, suggesting that Thy-1 expression weakly contributes to TCR-mediated T cell activation. Epidermal Langerhans cells and bone marrow-derived dendritic cells from Thy-1-/- mice exhibited a normal expression of costimulatory surface molecules as well as an unaltered ability to stimulate allogeneic T cells. Taken together, these findings demonstrate that a lack of Thy-1 expression does not generally compromise the immune system; however, Thy-1 expression may be involved in the fine-tuning of T cell-mediated immune responses.
Animals↗
p53-dependent impairment of T-cell proliferation in FADD dominant-negative transgenic mice.
Members of the tumour necrosis factor (TNF) receptor family exert pleiotropic effects and can trigger both apoptosis and proliferation [1]. In their cytoplasmic region, some of these receptors share a conserved sequence motif - the 'death domain' - which is required for transduction of the apoptotic signal by recruiting other death-domain-containing adaptor molecules like the Fas-associated protein FADD/MORT1 or the TNF receptor-associated protein TRADD [2-4]. FADD links the receptor signal to the activation of the caspase family of cysteine proteases [5,6]. Functional inactivation of individual receptor family members often fails to exhibit a distinctive phenotype, probably because of redundancy [7-9]. To circumvent this problem, we used a dominant-negative mutant of FADD (FADD-DN) which should block all TNF receptor family members that use FADD as an adaptor. We established transgenic mice expressing FADD-DN under the influence of the lck promoter and investigated the consequences of its expression in T cells. As expected, FADD-DN thymocytes were protected from death induced by CD95 (Fas/Apo1), whereas apoptosis induced by ultraviolet (UV) irradiation, anti-CD3 antibody treatment or dexamethasone was unaffected, as was spontaneous cell death. Surprisingly, however, we also observed profound inhibition of thymocyte proliferation in vivo and of activation-induced proliferation of thymocytes and mature T cells in vitro. This inhibition of proliferation was not due to increased cell death and appeared to be p53 dependent.
Adaptor Proteins, Signal Transducing↗
Traps to catch unwary oncogenes.
The MYC proto-oncogene has long been implicated in the control of normal cell growth and its deregulation is associated with the development of neoplasia. The MYC protein has a well-established role as a component of signal-transduction pathways promoting both proliferation and apoptosis. Because signalling pathways that drive cell death and cell proliferation are so tightly coupled, a synergy between genetic lesions leading to suppression of cell death and those promoting cell proliferation is observed during carcinogenesis. We discuss such synergy with respect to the cooperating oncogenes MYC, RAS and BCL2.
Animals↗
A dominant-negative mutant of the Rab5 GTPase enhances T cell signaling by interfering with TCR down-modulation in transgenic mice.
TCR triggering results in the down-modulation of engaged receptors by endocytosis. As a result of this process, Ag-binding sites are depleted from the surface and signaling responses should be attenuated. To test the importance of TCR down-regulation on T cell signaling, we generated mice expressing a dominant-negative form of Rab5 (Rab5N133I) in T cells. Rab5, a monomeric GTPase of the Ras superfamily, has been implicated in the regulation of early steps in the endocytic pathway. In Rab5N133I mice, mature thymocytes developed, but the absolute number of CD4+CD8+ double positive thymocytes was reduced. Fluid phase endocytosis was severely impaired in the transgenic thymocytes. In peripheral T cells, the kinetics and rate of ligand-induced TCR down-modulation were delayed and reduced. These effects were correlated with enhanced early and late signaling responses. Analysis of thymocyte development in doubly transgenic mice for Rab5N133I and a lymphocytic choriomeningitis virus (LCMV) peptide-specific TCR demonstrated that TCR signaling was enhanced by dominant inhibition of Rab5 function, resulting in altered thymic selection. These findings suggest that TCR endocytosis is an important regulatory component of TCR signaling and that defects in this regulation can result in prolonged signaling and alter thymic development.
Animals↗
Requirement for the CD95 receptor-ligand pathway in c-Myc-induced apoptosis.
Induction of apoptosis by oncogenes like c-myc may be important in restraining the emergence of neoplasia. However, the mechanism by which c-myc induces apoptosis is unknown. CD95 (also termed Fas or APO-1) is a cell surface transmembrane receptor of the tumor necrosis factor receptor family that activates an intrinsic apoptotic suicide program in cells upon binding either its ligand CD95L or antibody. c-myc-induced apoptosis was shown to require interaction on the cell surface between CD95 and its ligand. c-Myc acts downstream of the CD95 receptor by sensitizing cells to the CD95 death signal. Moreover, IGF-I signaling and Bcl-2 suppress c-myc-induced apoptosis by also acting downstream of CD95. These findings link two apoptotic pathways previously thought to be independent and establish the dependency of Myc on CD95 signaling for its killing activity.
3T3 Cells↗
Gene structure, cDNA sequence, and expression of murine Bak, a proapoptotic Bcl-2 family member.
To facilitate the creation of Bak knockout mice and the further analysis of this Bcl-2 family member, we have isolated and sequenced the complete mouse Bak cDNA. The cDNA is 2 kb long and shares an overall nucleotide identity to the human Bak cDNA of 62%. The mouse Bak protein is 208 amino acids long with a predicted molecular weight of 23 kDa. The mouse Bak mRNA could be detected in all mouse tissues examined. In addition we mapped the murine bak gene. It consists of six exons spanning about 10 kb on chromosome 17B. The 5' region of the murine bak gene is unmethylated on the dinucleotide CpG in the area around exon 1. Furthermore, it contains potential binding sites for transcription factors such as Sp1 and c-Myb.
Amino Acid Sequence↗
Thymocytes in Thy-1-/- mice show augmented TCR signaling and impaired differentiation.
Thy-1, a single variable-like immunoglobulin superfamily domain anchored in the plasma membrane by a glycosyl phosphaditylinositol tail [1], is a major surface glycoprotein in adult mammalian neurons and rodent thymocytes [2]; the function of Thy-1 has remained enigmatic since its discovery [3]. Studies in vitro have implicated Thy-1 in homotypic and heterotypic cell-cell interactions [2,4]. Ligation of Thy-1 initiates transmembrane signaling pathways that lead to diverse physiological outcomes in different cells [2,5-7]. In rodents, Thy-1 is highly expressed on the surface of CD4+CD8+ double-positive immature thymocytes and downregulated in mature T cells. Here, we report that thymocytes from Thy-1-/- mice [8] had altered cell-cell contacts, and hyperresponsiveness to T-cell receptor (TCR) triggering as demonstrated by the heightened activation of p56lck, phosphorylation of TCR subunits, Ca2+ fluxes and cell proliferation. Thy-1-/- thymocytes exhibited impaired maturation from the double positive to single positive stage of thymocyte development, possibly due to inappropriate negative selection, and were prone to T lymphomas in aged mice. These observations indicate that Thy-1 negatively regulates TCR-mediated signaling and controls activation thresholds during thymocyte differentiation.
Animals↗
Thy-1 triggers mouse thymocyte apoptosis through a bcl-2-resistant mechanism.
Programmed cell death plays an important role during thymocyte development, since a vast majority (97%) of mouse cortical thymocytes die in thymus, whereas only 3% of these cells are rescued from cell death and positively selected. Although it seems well established that thymocyte fate depends upon appropriate surface-expressed T cell receptor, little is known about the molecular mechanism(s) responsible for the massive thymocyte elimination that occurs in the thymus. We report here that Thy-1 is capable of triggering mouse thymocyte death in vitro through a bcl-2-resistant mechanism. We have previously shown that Thy-1 is involved in mouse thymocyte adhesion to thymic stroma through interaction with an epithelial cell ligand. To examine the Thy-1 signaling function in thymocytes, we have mimicked its interaction with stromal cells by culturing mouse thymocytes onto tissue culture plates coated with monoclonal antibodies (mAb) directed at distinct Thy-1 epitope regions. mAb recognizing determinants in a defined Thy-1 structural domain, but not others, were found to induce marked thymocyte apoptosis as evidenced by morphological and biochemical data. Use of a quantitative DNA dot blot assay indicated that Thy-1-mediated thymocyte apoptosis was not blocked by RNA or protein synthesis inhibitors, EGTA, or by cyclosporin A, and differed, therefore, from "activation-driven cell death". Moreover, Thy-1(+)-transfected, but not wild-type AKR1 (Thy-1-d) thymoma cells underwent apoptosis after ligation with apoptosis-inducing, Thy-1-specific mAb. In contrast to thymocytes, the latter event was inhibitable by RNA and protein synthesis inhibitors, an indication that thymocytes, but not thymoma cells, contain the molecular components necessary for Thy-1-driven apoptosis. We further showed that Thy-1-triggered thymocyte death is a developmentally regulated process operative in fetal thymocytes from day 17 of gestation, but not in peripheral T cells. Indeed, the target of apoptosis by anti-Thy-1 was found to reside mainly within the CD4+8+3- and CD4+8+3lo double positive immature thymocyte subsets. Finally, it is of major interest that Thy-1-mediated apoptosis, which was found to be readily detectable in thymocytes from bcl-2-transgenic mice, represents a thus far unique experimental system for studying bcl-2-resistant thymocyte death mechanism(s).
Animals↗
Quantitating apoptosis by a nonradioactive DNA dot blot assay.
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Animals↗
Thy-1 functions as a recognition/signaling molecule during mouse T lymphocyte development.
Thy-1 is a prototype of mammalian glycosyl phosphatidylinositol (GPI)-anchored molecules and belongs to the Ig superfamily. This cell surface glycoprotein is expressed on mouse T lymphocytes, neurons and hematopoietic stem cells. Despite detailed structural studies, little is known about the physiological role(s) of Thy-1. We discuss here our results on the role of Thy-1 in immature T lymphocytes during intrathymic maturation. It was observed that Thy-1-mediated adhesion of mouse thymocytes to thymic stromal cells occurs through interaction with an unknown ligand. The interaction occurs by a Ca(2+)-independent mechanism and does not require TCR/CD3 surface expression. To evaluate the signal transduction upon Thy-1 ligation in immature thymocytes, we cultured mouse thymocytes with monoclonal antibodies specific for Thy-1, immobilized onto the tissue culture plates. Monoclonal antibodies directed at determinants located in a defined epitope domain, but not others, triggered marked physiological cell death (apoptosis) of immature thymocytes, as evidenced by morphological and biochemical data. This apoptosis is independent of the cell surface expression of TCR/CD3. It is a developmentally regulated process since the period in which thymocytes are sensitive to Thy-1-dependent apoptosis corresponds to the developmental "window" during which massive death of immature thymocytes takes place within the thymus. Therefore, we propose that Thy-1 could function as a cell survival/death regulator in mouse T lymphocyte development.
Animals↗