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K D Fischer

Publications and source records attributed to K D Fischer.

At least 19 recordsLinked to original sources
PubMed2001

Vav-1 regulates NK T cell development and NK cell cytotoxicity.

The hematopoietic-specific Rho-family GTP exchange factor Vav-1 is a regulator of lymphocyte antigen receptor signaling and mediates normal maturation and activation of B and T cells. Recent findings suggest that Vav-1 also forms part of signaling pathways required for natural and antibody dependent cellular cytotoxicity (ADCC) of human NK cells. In this study, we show that Vav-1 is also expressed in murine NK cells. Vav-1(-/-) mice had normal numbers of splenic NK cells, and these displayed a similar expression profile of NK cell receptors as wild-type mice. Unexpectedly, IL-2-activated Vav-1(-/-) NK cells retained normal ADCC. Fc-receptor mediated activation of ERK, JNK, and p38 was also normal. In contrast, Vav-1(-/-) NK cells exhibited reduced natural cytotoxicity against EL4, C4.4.25, RMA and RMA/S. Together, the results demonstrate that Vav-1 is dispensable for mainstream NK cell development, but is required for NK natural cytotoxicity. Unlike the findings for NK cells, NK T cells were dramatically diminished in Vav-1(-/-) mice and splenocytes from Vav-1 mutant mice failed to produce IL-4 in response to in vivo CD3 stimulation. These data highlight the important role of Vav-1 in NK T cell development and NK cell function.

Animals↗
PubMed2001

Compensation between Vav-1 and Vav-2 in B cell development and antigen receptor signaling.

Vav-1 and Vav-2 are closely related Dbl-homology GTP exchange factors (GEFs) for Rho GTPases. Mutation of Vav-1 disrupts T cell development and T cell antigen receptor-induced activation, but has comparatively little effect on B cells. We found that combined deletion of both Vav-1 and Vav-2 in mice resulted in a marked reduction in mature B lymphocyte numbers. Vav-1(-/-)Vav-2(-/-) B cells were unresponsive to B cell antigen receptor (BCR)-driven proliferation in vitro and to thymus-independent antigen in vivo. BCR-stimulated intracellular calcium mobilization was greatly impaired in Vav-1(-/-)Vav-2(-/-) B cells. These findings establish a role for Vav-2 in BCR calcium signaling and reveal that the Vav family of GEFs is critical to B cell development and function.

Animals↗
PubMed2001

Lsc is required for marginal zone B cells, regulation of lymphocyte motility and immune responses.

Lsc (the murine homolog of human p115 Rho GEF) is a member of the Dbl-homology family of GTP exchange factors and is a specific activator of Rho. Lsc is activated by the G alpha(13) subunit of heterotrimeric G proteins and contains a regulator of G protein signaling domain that downmodulates G alpha(12) and G alpha(13). Lsc is expressed primarily in the hematopoietic system and links the activation of G alpha(12) and G alpha(13)-coupled receptors to actin polymerization in B and T cells. Lsc is essential for marginal zone B (MZB) cell homeostasis and for the generation of immune responses. Although Lsc-deficient lymphocytes show reduced basal motility, MZB cells show enhanced migration after serum activation. Thus, Lsc is a critical regulator of MZB cells and immune functions.

Animals↗
PubMed2001

The B lymphocyte-specific coactivator BOB.1/OBF.1 is required at multiple stages of B-cell development.

The transcriptional coactivator BOB.1/OBF.1 confers B-cell specificity on the transcription factors Oct1 and Oct2 at octamer site-containing promoters. A hallmark of the BOB.1/OBF.1 mutation in the mouse is the absence of germinal center development in secondary lymphoid organs, demonstrating the requirement for BOB.1/OBF.1 in antigen-dependent stages of B-cell differentiation. Here we analyzed earlier stages of B lymphopoiesis in BOB.1/OBF.1-deficient mice. Examination of B-cell development in the bone marrow revealed that the numbers of transitional immature (B220(+) IgM(hi)) B cells were reduced and that B-cell apoptosis was increased. When in competition with wild-type cells, BOB.1/OBF.1(-/-) bone marrow cells exhibited defects in repopulating the bone marrow B-cell compartment and were unable to establish a presence in the periphery of host mice. The defective bone marrow populations in BOB.1/OBF.1(-/-) mice were rescued by conditional expression of a BOB.1/OBF.1 transgene controlled by the tetracycline gene expression system. However, the restored populations did not restore the numbers of IgD(hi) B cells in the periphery, where the BOB.1/OBF.1 transgene was not expressed. These results show that BOB.1/OBF.1(-/-) B cells exhibit multistage defects in B-cell development, including impaired production of transitional B cells and defective maturation of recirculating B cells.

Animals↗
PubMed2000

Site-specific photocrosslinking to probe interactions of Arf1 with proteins involved in budding of COPI vesicles.

ADP-ribosylation factor 1 (Arf1) plays an important role in early and intra-Golgi protein trafficking. During this process, Arf1 interacts with many different proteins and other molecules that regulate its state of activation or are involved in its intracellular function. To determine which of these proteins interact directly with Arf1 during coat protein type I (COPI) vesicle biogenesis, we probed the molecular environment of Arf1 by use of site-specific photocrosslinking. This method was first used successfully in the field of protein trafficking to study the mechanisms involved in protein translocation across the endoplasmic reticulum during protein synthesis. In such a hydrophobic environment, crosslink yields of up to 30% have been observed. We have now applied this method to study the mechanism of vesicle budding from the cytosolic face of the Golgi apparatus, an aqueous environment. Although the crosslink yield is significantly lower under these conditions, due to predominant reaction of the photolabile probes with water, a specific interaction of Arf1 with subunits of coatomer, the major coat protein of COPI vesicles, could readily be identified.

ADP-Ribosylation Factor 1↗
PubMed1999

The guanine-nucleotide exchange factor Vav is a crucial regulator of B cell receptor activation and B cell responses to nonrepetitive antigens.

The proto-oncogene product Vav is required for receptor clustering, proliferation, and differentiation of T cells, and Vav was identified as a substrate in the TCR and B cell receptor signaling pathway. The role of Vav in B cell responses to Ag challenge in vivo is not known. In this study, we show that Vav regulates B cell proliferation following in vitro activation of Ag receptors, but Vav has no apparent role in CD40-, IL-4-, or LPS-induced B cell activation. Increased degrees of Ag receptor cross-linking can partially reverse the proliferative defect in the anti-IgM response of vav-/- B cells. In vivo, vav-/- mice mounted protective antiviral IgM and IgG responses to infections with vesicular stomatitis virus and recombinant vaccinia virus expressing the vesicular stomatitis virus glycoprotein, which harbor repetitive surface epitopes that directly cross-link the Ag receptor and activate B cells in the absence of T cell help. vav-/- B cells also responded normally to the polyvalent, repetitive hapten Ag trinitrophenyl (TNP)-Ficoll that effectively cross-links B cell receptors. However, vav-/- mice failed to mount immune responses to the nonrepetitive, T cell-dependent hapten Ag (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP)-OVA. These results provide the first genetic evidence on the role of the guanine exchange factor Vav in immune responses to viral infections and antigenic challenge in vivo, and suggest that Vav adjusts the threshold for Ag receptor-mediated B cell activation depending on the nature of the Ag.

Animals↗
PubMed1999

The oncogene product Vav is a crucial regulator of primary cytotoxic T cell responses but has no apparent role in CD28-mediated co-stimulation.

The guanine nucleotide-exchange factor Vav is a regulator of antigen-mediated cytoskeletal reorganization required for receptor clustering, proliferation and thymic selection. Moreover, Vav has been identified as a major substrate in the CD28 signal transduction pathway and overexpression of Vav enhances TCR-mediated IL-2 secretion in T cells. Here we show that CD3- plus CD28-mediated proliferation and IL-2 production were reduced in vav gene-deficient T cells. However, Vav had no apparent role in phorbol 12-myristate 13-acetate-plus CD28-mediated proliferation and IL-2 production, suggesting that Vav acts downstream of the TCR/CD3 complex. In vivo, Vav expression was crucial to generate primary vesicular stomatitis virus (VSV)-specific cytotoxic T cell responses. In contrast, vav-/- mice exhibited a reduced but significant footpad swelling after lymphocytic choriomeningitis virus (LCMV) infections and mounted a measurable primary cytotoxic T cell response to LCMV. Upon in vitro restimulation, cytotoxic T cell responses of both VSV- and LCMV-infected mice reached near normal levels. Our data provide the first genetic evidence that Vav is an important effector molecule that relays antigen receptor signaling to IL-2 production and activation of cytotoxic T cells.

Animals↗
PubMed1999

Defective liver formation and liver cell apoptosis in mice lacking the stress signaling kinase SEK1/MKK4.

The stress signaling kinase SEK1/MKK4 is a direct activator of stress-activated protein kinases (SAPKs; also called Jun-N-terminal kinases, JNKs) in response to a variety of cellular stresses, such as changes in osmolarity, metabolic poisons, DNA damage, heat shock or inflammatory cytokines. We have disrupted the sek1 gene in mice using homologous recombination. Sek1(-/- )embryos display severe anemia and die between embryonic day 10.5 (E10.5) and E12.5. Haematopoiesis from yolk sac precursors and vasculogenesis are normal in sek1(-/- )embryos. However, hepatogenesis and liver formation were severely impaired in the mutant embryos and E11.5 and E12.5 sek1(-/- )embryos had greatly reduced numbers of parenchymal hepatocytes. Whereas formation of the primordial liver from the visceral endoderm appeared normal, sek1(-/-) liver cells underwent massive apoptosis. These results provide the first genetic link between stress-responsive kinases and organogenesis in mammals and indicate that SEK1 provides a crucial and specific survival signal for hepatocytes.

Animals↗
PubMed1998

Vav regulates peptide-specific apoptosis in thymocytes.

The protooncogene Vav functions as a GDP/GTP exchange factor (GEF) for Rho-like small GTPases involved in cytoskeletal reorganization and cytokine production in T cells. Gene-targeted mice lacking Vav have a severe defect in positive and negative selection of T cell antigen receptor transgenic thymocytes in vivo, and vav-/- thymocytes are completely resistant to peptide-specific and anti-CD3/anti-CD28-mediated apoptosis. Vav acts upstream of mitochondrial pore opening and caspase activation. Biochemically, Vav regulates peptide-specific Ca2+ mobilization and actin polymerization. Peptide-specific cell death was blocked both by cytochalasin D inhibition of actin polymerization and by inhibition of protein kinase C (PKC). Activation of PKC with phorbol ester restored peptide-specific apoptosis in vav-/- thymocytes. Vav was found to bind constitutively to PKC-theta in thymocytes. Our results indicate that peptide-triggered thymocyte apoptosis is mediated via Vav activation, changes in the actin cytoskeleton, and subsequent activation of a PKC isoform.

Animals↗
PubMed1998

Vav is a regulator of cytoskeletal reorganization mediated by the T-cell receptor.

BACKGROUND: Vav is a guanine-nucleotide exchange factor for the Rho-like small GTPases RhoA, Rac1 and Cdc42, which regulate cytoskeletal reorganization and activation of stress-activated protein kinases (SAPK/JNKs). Vav is expressed in hematopoietic cells and is phosphorylated in T and B cells following activation of various growth factor or antigen receptors. Vav interacts with several signaling molecules in T cells, but the functional relevance of these interactions is established only for Slp76: they cooperate to induce activity of the transcription factor NF-AT and interleukin-2 expression. We have investigated the role of Vav in T cells by generating vav-/- mice. RESULTS: Mice deficient for vav were viable and healthy, but had impaired T-cell development. In vav-/- T cells, in response to activation of the T-cell receptor (TCR), cell cycle progression, induction of NF-ATc1 activity, downregulation of the cell-cycle inhibitor p27Kip1, interleukin-2 production, actin polymerization and the clustering of TCRs into patches and caps--a cytoskeletal reorganization process--were defective. TCR-mediated activation of mitogen-activated protein kinase and SAPK/JNK was unaffected. Ca2+ mobilization was impaired in vav-/- thymocytes and T cells. In wild-type cells, Vav constitutively associated with the cytoskeletal membrane anchors talin and vinculin. In the absence of Vav, phosphorylation of Slp76, Slp76-talin interactions, and recruitment of the actin cytoskeleton to the CD3 zeta chain of the TCR co-receptor were impaired. CONCLUSIONS: Vav is a crucial regulator of TCR-mediated Ca2+ flux, cytoskeletal reorganization and TCR clustering, and these are required for T-cell maturation, interleukin-2 production and cell cycle progression.

Actins↗
PubMed1998

Vav links antigen-receptor signaling to the actin cytoskeleton.

The haematopoietic-specific Rho-family guanine-nucleotide exchange factor Vav is a regulator of lymphocyte antigen receptor signaling leading to proliferation of B and T cells, generation of the B1 cell lineage and IL-2 production and maturation in T cells. The specific role it plays in these events, however, has not yet been resolved. Recent findings suggest that Vav is recruited to activated antigen receptors and requires both tyrosine phosphorylation and the presence of activating phospholipids for catalytic activity towards Rho-family GTPases. Studies form vav-deficient mice show that in response to antigen receptor activation, Vav is not essential for activation of JNK kinase pathways, but is required for actin polymerisation and T cell capping. We discuss Vav function in the light of these new findings.

Actins↗
PubMed1997

Impaired CD28-mediated interleukin 2 production and proliferation in stress kinase SAPK/ERK1 kinase (SEK1)/mitogen-activated protein kinase kinase 4 (MKK4)-deficient T lymphocytes.

The dual specific kinase SAPK/ERK1 kinase (SEK1; mitogen-activated protein kinase kinase 4/Jun NH2 terminal kinase [ JNK] kinase) is a direct activator of stress-activated protein kinases ([SAPKs]/JNKs) in response to CD28 costimulation, CD40 signaling, or activation of the germinal center kinase. Here we show that SEK1(-/-) recombination-activating gene (RAG)2(-/-) chimeric mice have a partial block in B cell maturation. However, peripheral B cells displayed normal responses to IL-4, IgM, and CD40 cross-linking. SEK1(-/-) peripheral T cells showed decreased proliferation and IL-2 production after CD28 costimulation and PMA/Ca2+ ionophore activation. Although CD28 expression was absolutely crucial to generate vesicular stomatitis virus (VSV)-specific germinal centers, SEK1(-/-)RAG2(-/-) chimeras mounted a protective antiviral B cell response, exhibited normal IgG class switching, and made germinal centers in response to VSV. Interestingly, PMA/Ca2+ ionophore stimulation, which mimics TCR-CD3 and CD28-mediated signal transduction, induced SAPK/JNK activation in peripheral T cells, but not in thymocytes, from SEK1(-/-) mice. These results show that signaling pathways for SAPK activation are developmentally regulated in T cells. Although SEK1(-/-) thymocytes failed to induce SAPK/JNK in response to PMA/Ca2+ ionophore, SEK1(-/-)RAG2(-/-) thymocytes proliferated and made IL-2 after PMA/Ca2+ ionophore and CD3/CD28 stimulation, albeit at significantly lower levels compared to SEK1(+/+)RAG2(-/-) thymocytes, implying that CD28 costimulation and PMA/Ca2+ ionophore-triggered signaling pathways exist that can mediate proliferation and IL-2 production independently of SAPK activation. Our data provide the first genetic evidence that SEK1 is an important effector molecule that relays CD28 signaling to IL-2 production and T cell proliferation.

Animals↗
PubMed1997

A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis.

Mouse embryos lacking the receptor tyrosine kinase, Flk1, die without mature endothelial and hematopoietic cells. To investigate the role of Flk1 during vasculogenesis and hematopoiesis, we examined the developmental potential of Flk1-/- embryonic stem cells in chimeras. We show that Flk1 is required cell autonomously for endothelial development. Furthermore, Flk1-/- cells do not contribute to primitive hematopoiesis in chimeric yolk sacs or definitive hematopoiesis in adult chimeras and chimeric fetal livers. We also demonstrate that cells lacking Flk1 are unable to reach the correct location to form blood islands, suggesting that Flk1 is involved in the movement of cells from the posterior primitive streak to the yolk sac and, possibly, to the intraembryonic sites of early hematopoiesis.

Amnion↗
PubMed1997

Stress-signalling kinase Sek1 protects thymocytes from apoptosis mediated by CD95 and CD3.

Distinct and evolutionarily conserved signal transduction cascades mediate survival or death in response to developmental and environmental cues. The stress-activated protein kinases, or Jun N-terminal kinases (SAPKs/JNKs), are activated in response to a variety of cellular stresses such as changes in osmolarity and metabolism, DNA damage, heat shock, ischaemia, or inflammatory cytokines. Sek1 (JNKK/MKK4) is a direct activator of SAPKs/JNKs in response to environmental stresses or mitogenic factors. Here we investigate the role of Sek1 in development and apoptosis by deleting sek1 in embryonic stem (ES) cells by homologous recombination. We provide genetic evidence that different stresses utilize distinct signalling pathways for SAPK/JNK activation. sek1(-/-) rag2(-/-) chimaeric mice have normal numbers of mature T cells but fewer immature CD4+CD8+ thymocytes. The sek1 mutation did not affect the induction of apoptosis in response to environmental stresses in ES and T cells: instead, sek1 protected thymocytes from CD95 (Fas)- and CD3-mediated apoptosis. These data indicate that SEK1 mediates survival signals in T-cell development.

Animals↗
PubMed1997

Intestinal growth is associated with elevated levels of glucagon-like peptide 2 in diabetic rats.

Glucagon-like peptide 2 (GLP-2) has recently been identified as a novel intestinal growth factor. Because experimental diabetes is associated with bowel growth, we examined the relationship between GLP-2 and intestinal growth in rats made diabetic by streptozotocin (STZ) injection and treated with or without insulin for 3 wk. Ileal concentrations of the intestinal proglucagon-derived peptides, i.e., glicentin + oxyntomodulin, and GLPs 1 and 2, were increased by 57 +/- 20% above those of controls in untreated STZ diabetes (P < 0.05-0.001). Similar increases in plasma concentrations of glicentin + oxyntomodulin (77 +/- 15% above controls, P < 0.01) and GLP-2 (91 +/- 32% above controls, P < 0.05) were seen in untreated STZ diabetes. Both wet and dry small intestinal weight increased by 74 +/- 20% above controls (P < 0.01) in STZ diabetes, and macromolecular analysis indicated parallel increases in both protein (P < 0.001) and lipid (P < 0.05) content. Villus height (P < 0.001) and crypt depth (P < 0.01) were also increased in untreated diabetic rat intestine. Insulin therapy prevented the changes in plasma GLP-2 and intestinal mass seen in untreated STZ diabetes. Thus STZ diabetes is associated with both increased production of GLP-2 and enhanced bowel weight, thereby suggesting a role for GLP-2 in diabetes-associated bowel growth.

Animals↗