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Biomedical subjects

Astar Winoto

Publications and source records attributed to Astar Winoto.

13 recordsLinked to original sources
PubMed2005

Phosphorylation of FADD at serine 194 by CKIalpha regulates its nonapoptotic activities.

FADD is essential for death receptor (DR)-induced apoptosis. However, it is also critical for cell cycle progression and proliferation, activities that are regulated by phosphorylation of its C-terminal Ser194, which has also been implicated in sensitizing cancer cells to chemotherapeutic drugs and in regulating FADD's intracellular localization. We now demonstrate that casein kinase Ialpha (CKIalpha) phosphorylates FADD at Ser194 both in vitro and in vivo. FADD-CKIalpha association regulates the subcellular localization of FADD, and phosphorylated FADD was found to colocalize with CKIalpha on the spindle poles in metaphase. Inhibition of CKIalpha diminished FADD phosphorylation, prevented the ability of Taxol to arrest cells in mitosis, and blocked mitogen-induced proliferation of mouse splenocytes. In contrast, a low level of cycling splenocytes from mice expressing FADD with a mutated phosphorylation site was insensitive to CKI inhibition. These data suggest that phosphorylation of FADD by CKI is a crucial event during mitosis.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Reduction of the incidence and severity of collagen-induced arthritis by constitutive Nur77 expression in the T cell lineage.

OBJECTIVE: To test the hypothesis that constitutive expression of Nur77 in the T cell lineage will suppress the development and pathogenesis of collagen-induced arthritis (CIA) and to understand the mechanisms by which Nur77 overexpression influences the arthritogenic response to type II collagen (CII). METHODS: Nur77-transgenic and wild-type C57BL/6 mice were immunized with CII and were monitored for the development and severity of arthritis. Pathologic changes were examined by histology and radiography. The effects of Nur77 overexpression on immune responses were evaluated by cytokine production in vitro and serum levels of CII-specific antibodies. Sensitivity of T cells to apoptosis induction was analyzed in vitro following stimulation with anti-CD3 monoclonal antibody or glucocorticoid. RESULTS: The incidence and severity of CIA was significantly decreased in Nur77-transgenic mice compared with wild-type controls. Attenuation of the disease was associated with increased apoptosis induction in transgenic T cells and decreased production of CII-specific IgG2a antibodies in transgenic mice. Overexpression of Nur77 in the T cell compartment did not affect Th1/Th2 cytokine production or balance. CONCLUSION: Nur77 overexpression in the T cell lineage attenuates the development and progression of CIA, probably by promoting activation-induced T cell apoptosis and by inhibiting CII-specific antibody production.

Animals↗
PubMed2005

Transcriptional regulation of tissue-specific genes by the ERK5 mitogen-activated protein kinase.

The ERK5 mitogen-activated protein kinase (MAPK) differs from other MAPKs in possessing a potent transcriptional activation domain. ERK5-/- embryos die from angiogenic defects, but the precise physiological role of ERK5 remains poorly understood. To elucidate molecular functions of ERK5 in the development of vasculature and other tissues, we performed gene profile analyses of erk5-/- mouse embryos and erk5-/- fibroblast cells reconstituted with ERK5 or ERK5(1-740), which lacks the transactivation domain. These experiments revealed several potential ERK5 target genes, including a proapoptotic gene bnip3, known angiogenic genes flt1 and lklf (lung Krüppel-like factor), and genes that regulate cardiovascular development. Among these, LKLF, known for its roles in angiogenesis, T-cell quiescence, and survival, was found to be absolutely dependent on ERK5 for expression in endothelial and T cells. We show that ERK5 drives lklf transcription by activating MEF2 transcription factors. Expression of erk5 short hairpin or a dominant-negative form of the ERK5 upstream activator, MEK5, in T cells led to downregulation of LKLF, increased cell size and upregulation of activation markers. Thus, through its kinase and transcriptional activation domains, ERK5 regulates transcriptional responses of cell survival and quiescence critical for angiogenesis and T-cell function.

Amino Acid Motifs↗
PubMed2004

Distinct transcriptional programs in thymocytes responding to T cell receptor, Notch, and positive selection signals.

T cell antigen receptor (TCR) signaling is necessary but not sufficient to promote the positive selection of CD4+CD8+ thymocytes into CD4+ or CD8+ mature T cells. Notch signaling has also been implicated as a potential regulator of both CD4/CD8 T cell development and TCR signaling. However, the relationship between positive selection, TCR signaling, and Notch remains unclear. Here we use DNA microarray analysis to compare gene expression changes in CD4+CD8+ double-positive thymocytes undergoing positive selection, TCR stimulation, and Notch activation. We find that the genes induced during positive selection can be resolved into two distinct sets. One set, which we term "TCR-induced," is also induced by in vitro TCR stimulation and contains a large proportion of transcription factors. A second set, which we term "positive-selection-induced," is not induced by in vitro TCR simulation and contains a large proportion of genes involved in signal transduction pathways. Genes induced by Notch activity overlap substantially with genes induced during positive selection. We also find that Notch activity potentiates the effects of TCR stimulation on gene expression. These results help to identify TCR- and positive-selection-specific transcriptional events and help to clarify the relationship between positive selection and Notch.

Animals↗
PubMed2004

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.

Amino Acid Sequence↗
PubMed2003

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.

Animals↗
PubMed2003

Transcriptional activation of known and novel apoptotic pathways by Nur77 orphan steroid receptor.

Nur77 is a nuclear orphan steroid receptor that has been implicated in negative selection. Expression of Nur77 in thymocytes and cell lines leads to apoptosis through a mechanism that remains unclear. In some cell lines, Nur77 was reported to act through a transcription-independent mechanism involving translocation to mitochondria, leading to cytochrome c release. However, we show here that Nur77-mediated apoptosis in thymocytes does not involve cytoplasmic cytochrome c release and cannot be rescued by Bcl-2. Microarray analysis shows that Nur77 induces many genes, including two novel genes (NDG1, NDG2) and known apoptotic genes FasL and TRAIL. Characterization of NDG1 and NDG2 indicates that NDG1 initiates a novel apoptotic pathway in a Bcl-2-independent manner. Thus Nur77-mediated apoptosis in T cells involves Bcl-2 independent transcriptional activation of several known and novel apoptotic pathways.

Amino Acid Sequence↗
PubMed2003

The orphan steroid receptor Nur77 family member Nor-1 is essential for early mouse embryogenesis.

Nur77 and its family members, Nor-1 and Nurr1, are orphan steroid receptors implicated in a wide variety of biological processes, including apoptosis and dopamine neuron agenesis. Expression of these family members can be detected at low levels in many tissues but they are expressed at very high levels when cells are stimulated by outside signals, including serum, nerve growth factor, and receptor engagement. Introduction of a dominant negative Nur77 protein that blocks the activities of all family members led to inhibition of apoptosis in T cells. Nur77-deficient mice, however, exhibit no phenotype, and a line of Nor-1 mutant mice was reported to exhibit a mild ear development phenotype but no other gross abnormalities. Here, we report the generation of Nor-1-deficient mice with a block in early embryonic development. Nor-1 is expressed early during embryogenesis, and its loss leads to embryonic lethality around embryonic day 8.5 of gestation. The mutant embryos fail to complete gastrulation and display distinct morphological abnormalities, including a decrease in overall size, developmental delay and an accumulation of mesoderm in the primitive streak during gastrulation. Abnormal expression of a number of early developmental markers and defects in growth or distribution of emerging mesoderm cells were also detected. These data suggest that Nor-1 plays a crucial role in gastrulation.

Animals↗
PubMed2003

Apoptosis during lymphoid development.

Recent investigations have provided important insights into how signaling through the antigen receptors determines whether a cell survives or dies. In T cells, Grb2 and MAP kinases play essential roles in differentiating between apoptotic and survival signals. The PTEN phosphatase and Bim, a pro-apoptotic Bcl-2 family member, regulate apoptosis in both T and B cells. In B cells, antigen receptor-mediated death can be rescued by co-stimulation, in which the roles of protein kinase C and BAFF, a TNF family member, have been recently elucidated. In a recently identified mechanism of regulating inflammation, receptors such as c-mer and glycoproteins such as MFG-E8 were found to participate in the clearance of apoptotic cells.

Animals↗
PubMed2003

A function of Fas-associated death domain protein in cell cycle progression localized to a single amino acid at its C-terminal region.

FADD is an adaptor known to transmit apoptotic signals from members of the tumor necrosis factor receptor family. We show here that FADD has a domain implicated in cell proliferation. Mice bearing the Asp mutation in the serine 191 phosphorylation site are runted and anemic and display splenomegaly. Apoptosis is unimpaired in these mice, but they exhibit many immune developmental problems indicative of proliferative defects. Mutant FADD T cells are defective in cell cycle progression, suggesting that regulation of phosphorylation at serine 191 is essential for growth/proliferation. Remarkably, serine 191 is conserved among mammalian FADD proteins, but this C-terminal region is absent in lower organisms, suggesting that FADD acquired a domain during evolution, rendering it a "proliferation-apoptosis coupler" that balances cell proliferation and apoptosis.

Adaptor Proteins, Signal Transducing↗
PubMed2002

ERK5 MAPK regulates embryonic angiogenesis and acts as a hypoxia-sensitive repressor of vascular endothelial growth factor expression.

During angiogenesis, endothelial cells undergo proliferation, reorganization, and stabilization to establish a mature vascular network. This process is critical for establishing a functional circulatory system during development and contributes to the pathological process of tumor growth. Here we report that embryos deficient for the ERK5 MAPK die between embryonic days 10.5 and 11.5 with angiogenic failure and cardiovascular defects. We show that ERK5 deficiency leads to an increased expression of the vascular endothelial growth factor (VEGF), dysregulation of which has been shown to impede angiogenic remodeling and vascular stabilization. Our data also reveal that ERK5 negatively regulates transcription from the vegf locus during hypoxic responses. Importantly, ERK5 is required at an earlier developmental stage than p38alpha, and p38alpha does not compensate for ERK5 deficiency. These results demonstrate that ERK5 plays a specific role in the regulation of early angiogenesis.

Animals↗
PubMed2002

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.

Animals↗