Background Phosphatidylinositol 3′-hydroxyl kinase (PI 3-kinase) is activated by many growth factor receptors and is thought to exert its cellular functions through the elevation of ...phosphatidylinositol (3,4,5)-triphosphate levels in the cell. PI 3-kinase is required for growth-factor induced changes of the actin cytoskeleton which are mediated by the GTPases Rac and Rho. Recently, a role for Rac and Rho in regulating gene transcription has become evident.
Results Here, we show that membrane targeting of the p110 catalytic subunit, but not the p85 regulatory subunit, of PI 3-kinase generates a constitutively active enzyme that allows us to assess the relative contribution of PI 3-kinase activation to a particular cellular response. Expression of this active PI 3-kinase induced actin reorganization in the form of Rac-mediated lamellipodia and focal complexes, and Rho-mediated stress fibres and focal adhesions. However, expression of active PI 3-kinase did not induce the Ras/Rac/Rho signalling pathways that regulate gene transcription controlled by the c-fos promoter, the c-fos serum response element or the transcription factors Elk-1 and AP-1.
Conclusions Our results demonstrate that PI 3-kinase induces a selective subset of cellular responses, but is not sufficient to stimulate the full repertoire of Rac- or Rho-mediated responses.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Cell cycle progression initiated by interleukin-2 (IL-2) in T cells is critical for lymphoproliferation and an immune response. Phosphatidyl inositol 3-kinase (PI3K) is activated by IL-2. However, ...nuclear targets for PI3K are not known. Here we identify the cell cycle regulator E2F as an IL-2 target in T lymphocytes and PI3K as the critical signaling pathway. We eliminate both Stat5 and Raf/MEK pathways from E2F regulation. Protein kinase B (PKB) is activated by IL-2 via PI3K. The expression of an active PKB is sufficient to induce E2F activity. Inhibition of PI3K inhibits phosphorylation of Rb, induction of cyclin D3, and degradation of p27
kip1
. These results establish a crucial PI3K/PKB–mediated link between the IL-2 receptor and the cell cycle machinery.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Eosinophilic inflammation and Th2 cytokine production are central to the pathogenesis of asthma. Agents that target either eosinophils or single Th2 cytokines have shown benefits in subsets of ...biomarker-positive patients. More broadly effective treatment or disease-modifying effects may be achieved by eliminating more than one inflammatory stimulator. Here we present a strategy to concomitantly deplete Th2 T cells, eosinophils, basophils, and type-2 innate lymphoid cells (ILC2s) by generating monoclonal antibodies with enhanced effector function (19A2) that target CRTh2 present on all 4 cell types. Using human CRTh2 (hCRTh2) transgenic mice that mimic the expression pattern of hCRTh2 on innate immune cells but not Th2 cells, we demonstrate that anti-hCRTh2 antibodies specifically eliminate hCRTh2
basophils, eosinophils, and ILC2s from lung and lymphoid organs in models of asthma and
infection. Innate cell depletion was accompanied by a decrease of several Th2 cytokines and chemokines. hCRTh2-specific antibodies were also active on human Th2 cells in vivo in a human Th2-PBMC-SCID mouse model. We developed humanized hCRTh2-specific antibodies that potently induce antibody-dependent cell cytotoxicity (ADCC) of primary human eosinophils and basophils and replicated the in vivo depletion capacity of their murine parent. Therefore, depletion of hCRTh2
basophils, eosinophils, ILC2, and Th2 cells with h19A2 hCRTh2-specific antibodies may be a novel and more efficacious treatment for asthma.
In an effort to identify potent and isoform selective inhibitors of PI3KI', GNE-293 (34) was identified. Inhibitor 2 was found to induce micronuclei formation in both the MNT and HCA in vitro assays. ...Compounds testing negative for genotoxicity were successfully identified through modifications of the 2-benzimidazole substituent and the methylene moiety to disrupt planarity. A variety of heteroatom linkers were explored to examine their effect on potency and isoform selectivity by restricting torsional angles to favor ligand interactions with PI3KI'as Trp760. These modifications also resulted in an improved in vivo pharmacokinetic profile.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Abstract
The p110δ isoform of PI3K is a therapeutic target for rheumatoid arthritis, but the cellular and molecular mechanisms by which PI3K mediates inflammation are poorly understood. Here we ...describe a novel highly selective p110δ small molecule inhibitor, G-286, that allowed dissecting the contribution specifically of p110δ to cellular pathways that are implicated in arthritis disease pathogenesis in human and mice. Using G-286, we demonstrate that p110δ regulates B cell- but not myeloid-cell dependent inflammatory arthritis in mice. G-286 blocks B cell receptor dependent proliferation and reduces autoantibody levels in collagen-induced arthritis abrogating disease. However, G-286 does not significantly inhibit FcγR-mediated inflammatory cytokine production in murine macrophages. Accordingly, selective p110δ inhibition did not significantly affect disease progression in FcγR- and myeloid cell-dependent autoantibody-induced arthritis. Importantly, we provide evidence that human and mice diverge in their relative dependency on p110δ to regulate FcγR responses. In addition to B cells and myeloid cells, T cells and osteoclasts contribute to rheumatoid arthritis pathogenesis. Utilizing G-286, we dissect the relative impact of p110δ inhibition on human and mouse T cell and osteoclast function. These results provide new insight in the role of p110δ in arthritis disease processes and offer a compelling rationale for targeting p110δ for the treatment of human rheumatoid arthritis.
Abstract
Activating Fc receptors and inflammatory cytokine production by macrophages have been implicated in the development and pathology of arthritis. However, the molecular components that mediate ...these events are not well characterized. Here we identify Bruton’s tyrosine kinase (BTK) as an essential player not only in B-cell dependent but also in myeloid-cell dependent inflammatory arthritis using a novel highly specific small molecule inhibitor of BTK (BTK SMI). In rodent models of collagen-induced arthritis, treatment with BTK SMI inhibits disease by at least two mechanisms: preventing autoantibody production and suppressing inflammatory cytokine production. Specifically, BTK SMI treatment prevents signaling and inflammatory cytokine production in response to immune complex activation in macrophages. Pathways found in rodents translate to human as BTK SMI prevents Fc receptor induced cytokine production in human monocytes. These data indicate that BTK SMI may be beneficial for the treatment of arthritis and that BTK contributes substantially to myeloid cell-dependent disease pathogenesis.
A potent inhibitor of PI3KI' that is aCO34200 fold selective for the remaining three Class I PI3K isoforms and additional kinases is described. The hypothesis for selectivity is illustrated through ...structure activity relationships and crystal structures of compounds bound to a K802T mutant of PI3KI3. Pharmacokinetic data in rats and mice support the use of 3 as a useful tool compound to use for in vivo studies.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Rho family members include RhoA, RhoB, and RhoC, Rac1 and Rac2, Cdc42Hs, RhoG, and TC10. These proteins function as binary switches by cycling between the active GTP-bound state and the inactive ...GDP-bound state. Three classes of regulatory proteins control the guanine nucleotide binding cycle of Rho family GTPase: guanine nucleotide exchange factors (GEFs) promote the transition from the inactive GDP-bound state to the active GTP-bound conformation; GTPase-activating proteins (GAPs) stimulate the inactivation; and guanine nucleotide dissociation inhibitors (GDIs) act to lock the GTPase in either the active or inactive state. Activated GTPases are consequently able to regulate diverse cellular processes by coupling to multiple biochemical effector signaling pathways. Immunological interest in Rho GTPases stems from genetic evidence demonstrating that Rho GTPases play a critical role in regulating cell survival, proliferation, and differentiation during lymphocyte development. Moreover, in biochemical studies, regulators of Rho GTPases have emerged as important components of signal transduction pathways used by antigen receptors, costimulatory, cytokine, and chemokine receptors to regulate the immune response. The present review will summarize data regarding the role of the Rho family GTPases Cdc42Hs, Rac1, and RhoA in cells of the immune system and will discuss current models for the signaling networks regulating the activity of these GTPases in lymphocytes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Apoptosis may be triggered, in a variety of tissues, by interaction of the cell surface molecule CD95 with its specific ligand, CD95L. CD95 plays a physiological role in the regulation of the immune ...response; furthermore, alterations in CD95/CD95L function may contribute to the pathogenesis of a number of human diseases, including cancer, autoimmune diseases and viral infections. Many cells that express CD95, however, are not susceptible to CD95‐mediated apoptosis. It is therefore important to identify the mechanisms that counteract the CD95 apoptotic process that are still poorly understood. Growth factors and lymphokines such as interleukin (IL)‐4 that counteract CD95‐mediated apoptosis may activate phosphatidylinositide 3‐kinase (PI 3‐kinase). We therefore used two different approaches to investigate the role of PI 3‐kinase on CD95‐mediated apoptosis. First we tested the effect of two pharmacological PI 3‐kinase inhibitors, wortmannin and LY294002, on CD95 agonistic antibody‐induced apoptosis in three different cell lines. Second, we co‐expressed in COS7 cells CD95 with constitutively active PI 3‐kinase. Results of both approaches indicate that active PI 3‐kinase effectively protects against CD95‐mediated apoptosis. Furthermore we extended our studies on the CD95 downstream mediator, FADD, and on the PI 3‐kinase downstream mediator, the serine/threonine protein kinase PKB, using the co‐expression approach in COS7 cells. We provide evidence that apoptosis induced by triggering the CD95 cell death receptor is counteracted by PI 3‐kinase activation; moreover, PKB but not p70S6K represents the relevant downstream target of PI 3‐kinase signaling.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Background: A delicate balance between positive and negative regulatory mechanisms during T-cell activation determines the specificity and magnitude of an immune response. Phosphoinositide 3-kinase ...(PI 3-kinase) is activated by a diverse set of receptors that determine T-cell function, including the T-cell antigen receptor (TCR), the costimulatory receptor CD28, and negative regulators of T-cell activation such as CTLA-4. PI 3-kinase is also regulated by the haematopoietic cytokines that determine T-cell differentiation and lymphocyte proliferation. PI 3-kinase can thus dynamically influence the outcome of the immune reactions at various stages. In this study, we investigated the importance of PI 3-kinase in TCR-directed T-cell activation using activated or inhibitory versions of PI 3-kinase.
Results: Certain aspects of TCR responses such as the induction of transcriptional activity of AP1 and serum response factor were not affected by expression of the mutant forms of PI 3-kinase. We found, however, that PI 3-kinase profoundly influenced the transactivation capacity of ‘nuclear factor of activated T cells’ (NF-AT) elicited by the TCR: expression of an activated form of PI 3-kinase inhibited TCR-mediated NF-AT responses, whereas expression of a dominant negative mutant of PI 3-kinase potently enhanced TCR-controlled NF-AT induction. These effects of PI 3-kinase were not mediated by previously identified PI 3-kinase effectors, such as protein kinase B, a positive regulator of PI 3-kinase, or the GTPase Rac, and are therefore likely to involve a novel, as yet unknown, effector molecule.
Conclusions: Our results establish that PI 3-kinase can both positively and negatively regulate T-cell function, and uncover a previously unrecognized function for PI 3-kinase in T cells as a selective negative regulator of TCR-signalling events and therefore as a determinant of T-cell homeostasis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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