Astar Winoto

Fas-mediated apoptosis and activation-induced T-cell proliferation are defective in mice lacking FADD/Mort1

Programmed cell death, or apoptosis, is important in homeostasis of the immune system: for example, non-functional or auto-reactive lymphocytes are eliminated through apoptosis. One member of the tumour necrosis factor receptor (TNFR) family, Fas (also known as CD95 or Apo-1), can trigger cell death and is essential for lymphocyte homeostasis,. FADD/Mort1 (refs 3–6) is a Fas-associated protein that is thought to mediate apoptosis by recruiting the protease caspase-8 (refs 7, 8). A dominant-negative mutant of FADD inhibits apoptosis initiated by Fas and other TNFR family members,. Other proteins, notably Daxx, also bind Fas and presumably mediate a FADD-independent apoptotic pathway. Here we investigate the role of FADD in vivo by generating FADD-deficient mice. As homozygous mice die in utero, we generated FADD−/− embryonic stem cells and FADD−/− chimaeras in a background devoid of the recombination activating gene RAG-1, which activates rearrangement of the immunoglobulin and T-cell receptor genes. We found that thymocyte subpopulations were apparently normal in newborn chimaeras. Fas-induced apoptosis was completely blocked, indicating that there are no redundant Fas apoptotic pathways. As these mice age, their thymocytes decrease to an undetectable level, although peripheral T cells are present in all older FADD−/− chimaeras. Unexpectedly, activation-induced proliferation is impaired in these FADD−/− T cells, despite production of the cytokine interleukin (IL)-2. These results and the similarities between FADD−/− mice and mice lacking the β-subunit of the IL-2 receptor suggest that there is an unexpected connection between cell proliferation and apoptosis.

Cloning of GT box-binding proteins: a novel Sp1 multigene family regulating T-cell receptor gene expression.

Analysis of a T-cell antigen receptor (TCR) alpha promoter from a variable gene segment (V) revealed a critical GT box element which is also found in upstream regions of several V alpha genes, TCR enhancer, and regulatory elements of other genes. This element is necessary for TCR gene expression and binds several proteins. These GT box-binding proteins were identified as members of a novel Sp1 multigene family. Two of them, which we term Sp2 and Sp3, were cloned. Sp2 and Sp3 contain zinc fingers and transactivation domains similar to those of Sp1. Like Sp1, Sp2 and Sp3 are expressed ubiquitously, and their in vitro-translated products bind to the GT box in TCR V alpha promoters. Sp3, in particular, also binds to the Sp1 consensus sequence GC box and has binding activity similar to that of Sp1. As the GT box has also previously been shown to play a role in gene regulation of other genes, these newly isolated Sp2 and Sp3 proteins might regulate expression not only of the TCR gene but of other genes as well.

IDENTIFICATION OF HUMAN AND MOUSE P19, A NOVEL CDK4 AND CDK6 INHIBITOR WITH HOMOLOGY TO P16INK4

The cell cycle in mammalian cells is regulated by a series of cyclins and cyclin-dependent kinases (CDKs). The G1/S checkpoint is mainly dictated by the kinase activities of the cyclin D-CDK4 and/or cyclin D-CDK6 complex and the cyclin E-CDK2 complex. These G1 kinases can in turn be regulated by cell cycle inhibitors, which may cause the cells to arrest at the G1 phase. In T-cell hybridomas, addition of anti-T-cell receptor antibody results not only in G1 arrest but also in apoptosis. In searching for a protein(s) which might interact with Nur77, an orphan steroid receptor required for activation-induced apoptosis of T-cell hybridomas, we have cloned a novel human and mouse CDK inhibitor, p19. The deduced p19 amino acid sequence consists of four ankyrin repeats with 48% identity to p16. The human p19 gene is located on chromosome 19p13, distinct from the positions of p18, p16, and p15. Its mRNA is expressed in all cell types examined. The p19 fusion protein can associate in vitro with CDK4 but not with CDK2, CDC2, or cyclin A, B, E, or D1 to D3. Addition of p19 protein can lead to inhibition of the in vitro kinase activity of cyclin D-CDK4 but not that of cyclin E-CDK2. In T-cell hybridoma DO11.10, p19 was found in association with CDK4 and CDK6 in vivo, although its association with Nur77 is not clear at this point. Thus, p19 is a novel CDK inhibitor which may play a role in the cell cycle regulation of T cells.

FUNCTIONAL REDUNDANCY OF THE NUR77 AND NOR-1 ORPHAN STEROID RECEPTORS IN T-CELL APOPTOSIS

The transcription factor Nur77 (NGFI‐B), a member of the steroid nuclear receptor superfamily, is induced to a high level during T‐cell receptor (TCR)‐mediated apoptosis. A transgenic dominant‐negative Nur77 protein can inhibit the apoptotic process accompanying negative selection in thymocytes, while constitutive expression of Nur77 leads to massive cell death. Nur77‐deficient mice, however, have no phenotype, suggesting the possible existence of a protein with redundant function to Nur77. To explore this possibility, we have characterized the role of two Nur77 family members, Nurr1 and Nor‐1, in TCR‐induced apoptosis. We found that Nor‐1 and Nurr1 can transactivate through the same DNA element as Nur77, and that their transactivation activities can be blocked by a Nur77 dominant‐negative protein. In thymocytes, Nor‐1 protein is induced to a very high level upon TCR stimulation and has similar kinetics to Nur77. In contrast, Nurr1 is undetectable in stimulated thymocytes. Furthermore, constitutive expression of Nor‐1 in thymocytes leads to massive apoptosis and up‐regulation of CD25, suggesting a functional redundancy between Nur77 and Nor‐1 gene products. As in the case of our Nur77‐FL mice, FasL is not detectable in the thymocytes of Nor‐1 transgenic mice. Constitutive expression of Nur77 in gld/gld mice rescues the lymphoproliferative phenotype of the FasL mutant mice. Thus, Nor‐1 and Nur77 demonstrate functional redundancy in an apparently Fas‐independent apoptosis.

FADD Is Required for DR4- and DR5-mediated Apoptosis LACK OF TRAIL-INDUCED APOPTOSIS IN FADD-DEFICIENT MOUSE EMBRYONIC FIBROBLASTS

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a member of the tumor necrosis factor family that can kill a wide variety of tumor cells but not normal cells. TRAIL-induced apoptosis in humans is mediated by its receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2). What constitutes the signaling molecules downstream of these receptors, however, remains highly controversial. Using the FADD dominant negative molecule, several groups have reached different conclusions with respect to the role of FADD in TRAIL-induced apoptosis. More recently, usingFADD-deficient (−/−) mouse embryonic fibroblasts, Yehet al. (Yeh, W.-C., Pompa, J. L., McCurrach, M. E., Shu, H.-B., Elia, A. J., Shahinian, A., Ng, M., Wakeham, A., Khoo, W., Mitchell, K., El-Deiry, W. S., Lowe, S. W., Goeddel, D. V., and Mak, T. W. (1998) Science279, 1954–1958) concluded that DR4 utilizes a FADD-independent apoptotic pathway. The latter experiment, however, involved transient overexpression, which often leads to nonspecific aggregation of death domain-containing receptors. To address this issue in a more physiological setting, we stably transfected mouse DR4/5, human DR4, or human DR5 intoFADD −/− mouse embryonic fibroblast cells. We showed that FADD −/− MEF cells stably transfected with TRAIL receptors are resistant to TRAIL-mediated cell death. In contrast, TRAIL receptors stably transfected into heterozygous FADD +/− cells orFADD −/− cells reconstituted with a FADD retroviral construct are sensitive to the TRAIL cytotoxic effect. We conclude that FADD is required for DR4- and DR5-mediated apoptosis.

TRAIL-R as a Negative Regulator of Innate Immune Cell Responses

TRAIL receptor (TRAIL-R) signaling has been implicated in inducing apoptosis in tumor cells, but little is understood about its physiological function. Here, we report the generation and characterization of TRAIL-R(-/-) mice, which develop normal lymphocyte populations but possess enhanced innate immune responses. TRAIL-R(-/-) mice exhibited increased clearance of murine cytomegalovirus that correlated with increased levels of IL-12, IFN-alpha, and IFN-gamma. Stimulation of macrophages with Mycobacterium and Toll-like receptor (TLR)-2, -3, and -4, but not TLR9, ligands resulted in high levels of TRAIL upregulation and enhanced cytokine production in TRAIL-R(-/-) cells. The immediate-early TLR signaling events in TRAIL-R(-/-) macrophages and dendritic cells are normal, but I kappa B-alpha homeostatic regulation and NF-kappa B activity at later time points is perturbed. These data suggest that TRAIL-R negatively regulates innate immune responses.

Nuclear Hormone Receptors in T Lymphocytes

Among the numerous steroid and orphan nuclear receptors encoded within mammalian genomes, several are involved in regulating immune system functions. We review here recent studies on the glucocorticoid receptor and the orphan receptors Nur77 and RORgamma. These molecules play key roles in the development and the effector functions of T lymphocytes.

ERK5 is a novel type of mitogen-activated protein kinase containing a transcriptional activation domain.

ABSTRACT Previous studies have shown that upregulation of the orphan steroid receptor Nur77 is required for the apoptosis of immature T cells in response to antigen receptor signals. Transcriptional upregulation of Nur77 in response to antigen receptor signaling involves two binding sites for the MEF2 family of transcription factors located in the Nur77 promoter. Calcium signals greatly increase the activity of MEF2D in T cells via a posttranslational mechanism. The mitogen-activated protein (MAP) kinase ERK5 was isolated in a yeast two-hybrid screen using the MADS-MEF2 domain of MEF2D as bait. ERK5 resembles the other MAP kinase family members in its N-terminal half, but it also contains a 400-amino-acid C-terminal domain of previously uncharacterized function. We report here that the C-terminal region of ERK5 contains a MEF2-interacting domain and, surprisingly, also a potent transcriptional activation domain. These domains are both required for coactivation of MEF2D by ERK5. The MEF2-ERK5 interaction was found to be activation dependent in vivo and inhibitable in vitro by the calcium-sensitive MEF2 repressor Cabin 1. The transcriptional activation domain of ERK5 is required for maximal MEF2 activity in response to calcium flux in T cells, and it can activate the endogenous Nur77 gene when constitutively recruited to the Nur77 promoter via MEF2 sites. These studies provide insights into a mechanism whereby MEF2 activity can respond to calcium signaling and suggest a novel, unexpected mechanism of MAP kinase function.

Inhibition of Fas-mediated apoptosis by the B cell antigen receptor through c-FLIP

Cross‐linking of the B cell antigen receptor (BCR) induces resistance to Fas (APO‐1 / CD95)‐dependent apoptosis and thereby regulates one mechanism of B cell selection during antigen stimulation. To investigate the molecular mechanism by which BCR signaling regulates the Fas pathway, we examined the expression of constituents of the death‐inducing signaling complex (DISC), including Fas, FADD, caspase‐8 and cellular FLICE‐inhibitory protein (c‐FLIP). No significant changes in the cellular levels of Fas, FADD or caspase‐8 were observed after BCR cross‐linking. By contrast, the long isoform of c‐FLIP (c‐FLIPL) was significantly up‐regulated by BCR cross‐linking in primary B cells and in two B cell lines, A20 and WEHI‐279. Moreover, transfection of c‐FLIPL into A20 cells inhibited Fas‐dependent apoptosis and suppressed recruitment of caspase‐8 to the DISC. BCR cross‐linking or FLIP overexpression also protects B cells from TRAIL‐induced apoptosis. Thus, BCR signaling up‐regulates c‐FLIPL and suppresses the Fas‐ and TRAIL‐receptor apoptosis pathways which could be important for tolerance and selection of antigen‐specific B cells.

Essential Role of Survivin, an Inhibitor of Apoptosis Protein, in T Cell Development, Maturation, and Homeostasis

Survivin is an inhibitor of apoptosis protein that also functions during mitosis. It is expressed in all common tumors and tissues with proliferating cells, including thymus. To examine its role in apoptosis and proliferation, we generated two T cell–specific survivin-deficient mouse lines with deletion occurring at different developmental stages. Analysis of early deleting survivin mice showed arrest at the pre–T cell receptor proliferating checkpoint. Loss of survivin at a later stage resulted in normal thymic development, but peripheral T cells were immature and significantly reduced in number. In contrast to in vitro studies, loss of survivin does not lead to increased apoptosis. However, newborn thymocyte homeostatic and mitogen-induced proliferation of survivin-deficient T cells were greatly impaired. These data suggest that survivin is not essential for T cell apoptosis but is crucial for T cell maturation and proliferation, and survivin-mediated homeostatic expansion is an important physiological process of T cell development.