Phosphatidylinositol 3-kinase and extracellular signal-regulated kinase are recruited for Fc receptor-mediated phagocytosis during monocyte-to-macrophage differentiation

Shiga toxin-producing enterohemorrhagic E. coli infections cause bloody diarrhea, which may progress to life-threatening complications such as the hemolytic-uremic syndrome (HUS). HUS patients frequently have elevated levels of the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) detectable in urine. Thus, sequelae may develop following the localized production of proinflammatory cytokines within the kidneys. A possible source of these cytokines are macrophages, which respond to the toxins by producing TNF-alpha. We have shown previously that THP-1 cells produce soluble TNF-alpha in response to the toxins, whose production requires host-cell tyrosine-kinase activity and toxin-enzymatic activity. To further examine signaling pathways involved in TNF-alpha expression, we determined that JNK1 and -2 and p38, but not ERK1 or -2, were phosphorylated following toxin exposure. Blockade of p38 activation reduced TNF-alpha production following Shiga toxin 1 treatment. Finally, we present a model of the ribotoxic stress response triggered in human macrophages by Shiga toxins.     

Kinase suppressor of Ras 1 is required for full ERK activation in thymocytes but not for thymocyte selection

The scaffold protein kinase suppressor of Ras 1 (KSR1) is critical for efficient activation of ERK in a number of cell types. Consistent with this, we observed a defect in ERK activation in thymocytes that lack KSR1. Interestingly, we found that the defect was much greater after PMA stimulation than by CD3 activation. Since ERK activation is believed to be important for thymocyte development, we analyzed thymocyte selection in KSR1‐deficient (KSR1^?/?) mice. We found that positive selection in two different TCR transgenic models, HY and AND, was normal. On the other hand, negative selection in the HY model was slightly impaired in KSR1^?/? mice. However, a defect in negative selection was not apparent in the AND TCR model system or in an endogenous superantigen‐mediated model of negative selection. These results suggest that, despite a requirement for KSR1 for full ERK activation in thymocytes, full and efficient ERK activation is not essential for the majority of thymocyte selection events.     

Phosphatidylinositol 3-kinase and ERK1/2 are not involved in adenosine A1, A2A or A3 receptor-mediated preconditioning in rat ventricle strips

Mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase 1 and 2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3-kinase)/protein kinase B (PKB; also known as Akt) are important antiapoptotic signalling pathways which have recently been implicated in cardioprotection. However, at present the involvement of ERK1/2 and PI3-kinase/PKB in adenosine receptor-mediated cardioprotection is poorly understood. In this study we used isolated rat right ventricular strips, contracted by electrical-field stimulation, in order to investigate the role of ERK1/2 and PI3-kinase/PKB in adenosine receptor-induced cardioprotection. Ventricle strips were pretreated for 2 min with the agonists adenosine (non-selective), CPA (A1 selective), CGS 21680 (A2A selective) and Cl-IB-MECA (A3 selective) before 30 min hypoxia followed by 30 min reoxygenation. Each agonist significantly improved posthypoxic percentage contraction recovery compared to control strips. Similarly hypoxic preconditioning (10 min hypoxia followed by 20 min reoxygenation) significantly improved posthypoxic percentage contraction recovery compared to non-preconditioned strips. The selective adenosine receptor antagonists DPCPX (A1), ZM 241385 (A2A) and MRS 1220 (A3) attenuated cardioprotection induced by CPA, CGS 21680 and Cl-IB-MECA, respectively. Pre-incubation (30 min) of ventricle strips with the MEK1 inhibitor PD 98059 (50 microM) or the PI3-kinase inhibitor wortmannin (100 nM) significantly reduced posthypoxic percentage contraction recovery induced by hypoxic preconditioning. In contrast, PD 98059 and wortmannin had no significant effect on cardioprotection induced by CPA, Cl-IB-MECA or CGS 21680. Overall these data indicate that although selective A1, A2A and A3 adenosine receptor agonists induce preconditioning in rat right ventricular strips the effects are independent of ERK1/2- and PI3-kinase-dependent pathways. In contrast ERK1/2 and PI3-kinase-dependent pathways do appear to be involved in early hypoxic preconditioning.     

Sustained ERK1/2 but not STAT1 or 3 activation is required for thanatophoric dysplasia phenotypes in PC12 cells

Mutations in fibroblast growth factor receptor 3 (FGFR3) cause the most common genetic form of short-limbed dwarfism, achondroplasia (ACH), as well as neonatal lethal forms, thanatophoric dysplasia (TD) I and II. The causative mutations induce graded levels of constitutive activation of the receptor that correspond to the severity of the disorder, resulting in premature entry into hypertrophic differentiation and reduced proliferation of chondrocytes in developing cartilage. Although FGFR3 promotes growth in most tissues, it is a negative regulator of endochondral bone growth. Several signaling pathways have been implicated in these skeletal disorders including the Ras/MEK/ERK pathway and the JAK/STAT, the latter in the most severe phenotypes, however their functional relevance remains incompletely understood. Using PC12 cell lines stably expressing inducible mutant receptors containing the TDII mutation, K650E, sustained activation of ERK1/2 and activation of STAT1 and STAT3, but not STAT5, is observed in the absence of ligand. This activation leads to neurite outgrowth, a phenotypic readout of constitutive receptor activity, and sustained ERK1/2 activity is required for this ligand-independent differentiation. To assess the functional relevance of STAT activation induced by the mutant receptor, STATs were specifically downregulated using RNA-interference. Silencing of STAT1 or 3 independently or in combination had no significant effect on ligand-independent neurite outgrowth, ERK1/2 activation or p21(WAF1/CIP1) protein levels. These results support a model in which sustained activation of ERK1/2 is a key regulator of the increased transition to hypertrophic differentiation of the growth plate, whereas activation of STATs 1 and 3 is not required.     

Caspase-3-like proteases are activated by infection but are not required for replication of vesicular stomatitis virus

Infection with vesicular stomatitis virus (VSV), the prototype rhabdovirus, causes apoptotic DNA fragmentation, but the role of apoptosis in the VSV-host interaction remains unclear. Apoptosis is the gene-regulated mechanism triggered by a wide variety of stimuli that lead to cell death in a choreographed manner. In the present study, infection of the Jurkat T cell line with VSV led to activation of caspase-3 and caspase-7, with subsequent apoptotic events involving poly (ADP ribose) polymerase (PARP) cleavage, DNA fragmentation, and membrane damage. Caspase activation was correlated with viral protein expression suggesting a link between viral replication and apoptosis. We hypothesized that VSV replication might depend on apoptosis and that the inhibition of apoptosis would lead to significant decreases in viral titers. When various inhibitors of apoptosis in VSV-infected cells were used, PARP cleavage and DNA fragmentation were inhibited but the production of infectious progeny was not affected. In addition, we demonstrated that the activation of caspase-3-like proteases is required for VSV-induced apoptosis but not in vitro viral replication. Apoptosis following VSV infection is likely to be either a host-cell attempt to control viral replication or may be a ploy used by the virus to facilitate its in vivo replication and spread.     

Phosphatidylinositol 3-kinase inhibition enhances human sperm motility

BACKGROUND: The number of spermatozoa with forward motility after capacitation procedures represents the limiting factor for application of IVF versus intracytoplasmatic sperm injection (ICSI) procedure in cases of oligoasthenozoospermia. The possibility of increasing this number may thus be of help to the patient. A complex array of signalling pathways is involved in the regulation of sperm motility and recent data pointed out an important role for kinase/phosphatase-regulated phosphorylation of proteins. Here, we investigated the role of phosphatidylinositol 3-kinase (PI3K), a lipid and protein kinase involved in the regulation of several biological aspects of somatic cells, on human sperm motility by using the specific PI3K inhibitor LY294002. METHODS AND RESULTS: We demonstrated that in-vitro incubation of swim-up selected or unselected human spermatozoa with LY294002 determined an increase of percentage forward motility in all the treated samples. The effect was dose-dependent with an EC(50) of 1.09 +/- 0.54 micromol/l. LY294002 also increased sperm movement characteristics and hyperactivation as evaluated by computer-assisted motion analyser. The compound was also able to overcome the detrimental effect of hydrogen peroxide and lithium chloride on sperm motility. CONCLUSIONS: Our results suggest a negative role for PI3K in the development and maintenance of sperm motility and suggest a possible use of PI3K inhibitors to enhance motility in cases of asthenozoospermia.     

ERK1 and ERK2 are required for radial glial maintenance and cortical lamination

ERK1/2 is involved in a variety of cellular processes during development, but the functions of these isoforms in brain development remain to be determined. Here, we generated double knockout (DKO) mice to study the individual and combined roles of ERK1 and ERK2 during cortical development. Mice deficient in Erk2, and more dramatically in the DKOs, displayed proliferation defects in late radial glial progenitors within the ventricular zone, and a severe disruption of lamination in the cerebral cortex. Immunohistochemical analyses revealed that late‐generated cortical neurons were misplaced and failed to migrate the upper cortical layers in DKO mice. Moreover, these mice displayed fewer radial glial fibers, which provide architectural guides for radially migrating neurons. These results suggest that extracellular signal‐regulated kinase signaling is essential for the expansion of the radial glial population and for the maintenance of radial glial scaffolding. Tangential migration of interneurons and oligodendrocytes from the ganglionic eminences (GE) to the dorsal cortex was more severely impaired in DKO mice than in mice deficient for Erk2 alone, because of reduced progenitor proliferation in the GE of the ventral telencephalon. These data demonstrate functional overlaps between ERK1 and ERK2 and indicate that extracellular signal‐regulated kinase signaling plays a crucial role in cortical development.     

Parvalbumin-positive CA1 interneurons are required for spatial working but not for reference memory

Parvalbumin-positive GABAergic interneurons in cortical circuits are hypothesized to control cognitive function. To test this idea directly, we functionally removed parvalbumin-positive interneurons selectively from hippocampal CA1 in mice. We found that parvalbumin-positive interneurons are dispensable for spatial reference, but are essential for spatial working memory.     

Phosphatidylinositol 3-phosphate 5-kinase is required for the cellular response to nutritional starvation and mating pheromone signals in Schizosaccharomyces pombe

BACKGROUND: Phosphatidylinositol (3,5) bisphosphate, which is converted from phosphatidylinositol 3-phosphate by phosphatidylinositol 3-phosphate 5-kinase, is implicated in vacuolar functions and the sorting of cell surface proteins within endosomes in the endocytic pathway of budding yeast. A homologous protein, SpFab1p, has been found in the fission yeast Schizosaccharomyces pombe, but its role is not known. RESULTS: Here we report that SpFab1p is encoded by ste12+ known as a fertility gene in S. pombe. The ste12 mutant grew normally under stress-free conditions, but was highly vacuolated and swelled at high temperatures and under starvation conditions. In nitrogen-free medium, ste12 cells were arrested in G1 phase, but partially defective in the expression of genes responsible for mating and meiosis. The ste12 mutant was defective both in the production of, and in the response to, mating pheromones. The amount of the pheromone receptor protein Map3p, was substantially decreased in ste12 cells. Map3p was transported to the cell surface, then internalized and eventually transported to the vacuolar lumen, even in the ste12 mutant. CONCLUSION: The results indicate that phosphatidylinositol(3,5)bisphosphate is essential for cellular responses to various stresses and for the mating pheromone signalling under starvation conditions.     

Phosphatidylinositol 3-kinase improves the efficiency of positive selection

We have generated transgenic mice expressing the amino-terminal fragment of the phosphatidylinositol 3-kinase (PI3K) catalytic subunit (p110ABD) in thymocytes. Expression of p110ABD results in constitutive activation of PI3K and in significant increases in the numbers of mature, single-positive thymocytes. We previously reported that the increase in mature cells was in part due to a defect in thymic emigration. In this study we identify another component to this phenotype. Expression of p110ABD results in an enhancement of positive selection, without alterations in thymocyte lifespan or negative selection. Since PI3K can affect activation of Btk, which in turn potentiates calcium fluxes, during B cell development, our results suggest that PI3K could play a role in the regulation of Itk kinases in T cells, and that both cell types share a common signaling network to modulate calcium responses downstream of their antigen receptor.     

Reactive oxygen species and not lipoxygenase products are required for mouse B-lymphocyte activation and differentiation

A potential role for lipoxygenase (LO) products and reactive oxygen species (ROS) in mouse B-lymphocyte activation and differentiation was investigated. Previously published investigations with the nonspecific 5-LO (EC 1.13.11.34) and 12-LO (EC 1.13.11.31) inhibitors such as nordihydroguaiaretic acid (NDGA) and 6,7-dihydroxycoumarin (Esculetin), are misleading in that they suggest lymphocyte LO activity is required for activation and differentiation of these cells. In initial support of this concept, we report that NDGA and Esculetin completely inhibited B-lymphocyte activation mediated by either membrane immunoglobulin (mlg), or the lipopolysaccharide (LPS) receptor. NDGA and Esculetin completely inhibited cell enlargement and proliferation, exhibiting half maximal inhibitory concentrations (IC₅₀S) of approximately 1 × 10⁻⁶ M. In contrast, the highly specific 5-LO inhibitors BAY X 1005, MK-886 and Wy 50,295 did not inhibit cell enlargement or proliferation. Moreover, 5,8,11-eicosatriynoic acid (ETI) which inhibits 5- and 12-LO, and 5, 8, 11, 14-eicosatetraynoic acid (ETYA) which inhibits all known LOs did not affect B-lymphocyte proliferation. Interestingly, NDGA and Esculetin are antioxidants, unlike BAY X 1005, MK-886, Wy 50,295, ETI and ETYA. Our hypothesis was that the antioxidant activities of NDGA and Esculetin were responsible for inhibiting B-lymphocyte activation and proliferation and we speculated that ROS and not LO activity was required for both processes. Additional antioxidants such as butylated hydroxy toluene, o-phenanthroline, thiourea, and α-tocopherol (vitamin E), also inhibited B-lymphocyte proliferation induced by either the LPS or mIg receptors. These agents exhibited IC₅₀s of 1 × 10⁻⁸ M, 5 × 10⁻¹⁰ M, 6 × 10⁻³ M and 5 × 10⁻⁵ M, respectively. When resting B-lymphocytes were treated with a source of ROS (1 × 10⁻⁵ M H₂O₂), cells enlarged in a temperature-sensitive manner, which is similar to LPS-induced enlargement. Both NDGA and Esculetin completely inhibited H₂O₂-induced enlargement. These results further indicate that ROS are required for B-lymphocyte activation and proliferation. Similar results were obtained for B-lymphocyte differentiation. NDGA and Esculetin completely inhibited the development of plasma cells and displayed IC₅₀S of 5 × 10⁻⁶ M. Conversely, BAY X 1005, MK-886, Wy 50,295, ETI, and ETYA did not block the formation of plasma cells. Therefore, ROS are also crucial for differentiation into plasma cells. These experiments are the first to directly illustrate that intracellular ROS mediate B-lymphocyte activation, proliferation and differentiation and that LO products are not required for these processes. Moreover, this investigation ilustrates that NDGA and Esculetin are not specific LO inhibitors and that their use cannot implicate a role for LO activity.     

Phosphatidylinositol-3-kinase and Akt are required for RIG-I-mediated anti-viral signalling through cross-talk with IPS-1

Retinoic acid-inducible gene I (RIG-I) is a cytosolic pattern-recognition receptor that recognizes viruses and triggers anti-viral immune responses. Activation of intracellular RIG-I signalling is mediated through interferon-β (IFN-β) promoter stimulator-1 (IPS-1), an adaptor of RIG-I, which induces IFN regulatory factor (IRF) 3 activation and type I IFN expression. The phosphatidylinositol-3-kinase (PI3K) and Akt pathway is activated in host immune cells upon viral infection. However, the mechanism as to how they work in RIG-I signalling has not been fully elucidated. Therefore, we investigated the role of PI3K and Akt in the regulation of RIG-I-mediated IRF3 activation and type I IFN expression in macrophages. Our results show that Sendai virus infection, which is recognized by RIG-I, led to IRF3 activation and IFN-β expression and these responses were attenuated by the PI3K inhibitor (LY294002) and an Akt dominant-negative mutant in the macrophage cell line(RAW264.7). IRF3 phosphorylation and dimerization as well as IFN-β expression induced by a synthetic RIG-I agonist, short poly(I:C), were suppressed by LY294002 or siRNA-Akt in bone marrow-derived macrophages. Suppression of PI3K and Akt using a dominant-negative mutant and siRNA knockdown resulted in attenuation of IRF3 activation and IFN-β expression induced by RIG-I itself or its adaptor, IPS-1. Association of Akt with IPS-1 increased with short poly(I:C) stimulation and required the pleckstrin homology domain of Akt and caspase-recruitment domain in IPS-1. Collectively, our results show that PI3K and Akt are required downstream of IPS-1 for RIG-I-mediated anti-viral immune responses. The results describe a novel, interactive relationship between RIG-I downstream signalling molecules resulting in efficient anti-viral immunity.     

Raf-1 and ERK2 kinases are required for phorbol 12,13-dibutyrate-stimulated proliferation of rat lymphoblasts. ERK2 activation precedes Raf-1 hyperphosphorylation

Rat lymphoblasts are arrested in the G₁ phase of the cell cycle and can be promoted to proceed up to the S phase, when they are stimulated by phorbol ester. In this work, we have studied some details of the phorbol 12,13-dibutyrate (PBu₂)-stimulated proliferation. We show that in response to PBu₂ at least four different protein kinase C (PKC) isoforms translocate to the membrane. A specific PKC ζ antibody recognizes two bands of 75 and 82 kDa. These two activities are separated using a Mono Q chromatography and we show that p75 is the classical PKC ζ isoform, while p82 might be a related isoform which is PBu₂ sensitive. Our data show that there is a correlation between the ability of PBu₂ to promote mitogenesis and to activate ERK2 kinase, suggesting that ERK2 kinase might be the limiting step of the process. We also show that ERK kinase activation precedes Raf-1 kinase hyperphosphorylation, suggesting that Raf-1 kinase activation is not required for ERK kinase activation. This idea was checked using a Raf-1 kinase antisense (AS) oligonucleotide. The results obtained with the Raf-1 AS oligonucleotide indicate that this serine/threonine kinase is dispensable for ERK kinase activation, but needed for the PBu₂ mitogenic signaling even as late as 7 h after the delivery of the signal.     

Dendritic cells in lupus are not required for activation of T and B cells but promote their expansion, resulting in tissue damage

Dendritic cells (DCs) initiate and control the adaptive immune response against infections. However, their contributions to the anti-self adaptive immune response in autoimmune disorders like systemic lupus erythematosus are uncertain. By constitutively deleting DCs in MRL.Fas(lpr) mice, we show that they have complex roles in murine lupus. The net effect of DC deletion was to ameliorate disease. DCs were crucial for the expansion and differentiation of T?cells but, surprisingly, not required for their initial activation. Correspondingly, kidney interstitial infiltrates developed in the absence of DCs, but failed to progress. DC deletion concomitantly decreased inflammatory and regulatory T?cell numbers. Unexpectedly, plasmablast numbers and autoantibody concentrations depended on DCs, in contrast to total serum immunoglobulin concentrations, suggesting an effect of DCs on extrafollicular humoral responses. These findings reveal that DCs operate in unanticipated ways in murine lupus and validate them as a potential therapeutic target in autoimmunity.     

Extracellular but not intracellular calcium mobilization is required for Epstein-Barr virus-containing supernatant-induced B cell activation

Changes in intracytosolic free calcium concentration ([Ca2+]i) are though to be an important trigger for the initiation of the cellular events culminating in B cell activation. After exposure of human B lymphocytes loaded with the fluorescent indicator quin-2 to the Epstein-Barr virus (EBV)-containing supernatant of B95-8 cell line, a rise in [Ca2+]i is observed. To determine the respective contribution of the intra- and extracellular Ca2+ pools in EBV-induced B cell activation, the pharmacologic modulation of these processes was investigated using an intracellular calcium chelator, 1,2-bis(o-aminophenoxy)ethane- N,N,N',N'-tetraacetic (BAPTA) or the calcium channel blocking drugs. When the extracellular Ca2+ contribution was minimized in the presence of calcium channel-blocking drugs or incubation of the cells in Ca2+-free medium, the EBV-induced human B cell activation was fully inhibited. When the calcium channel-blocking drugs, either verapamil or diltiazem, were withdrawn or when exogenous Ca2+ was added to the Ca2+-free medium, EBV-induced B cell activation was noted, demonstrating the reversibility of the inhibition. On the contrary, when the intracellular Ca2+ contribution was reduced after loading the cell with BAPTA, no alteration of the EBV-induced B cell activation was observed. Thus, the EBV-induced rise of [Ca2+]i required in the activation of human B cells appears to be essentially related to the entry of extracellular Ca2+ and not to the release of Ca2+ from internal stores.     

FlaA proteins in Leptospira interrogans are essential for motility and virulence but are not required for formation of the flagellum sheath

Spirochetes have periplasmic flagella composed of a core surrounded by a sheath. The pathogen Leptospira interrogans has four flaB (proposed core subunit) and two flaA (proposed sheath subunit) genes. The flaA genes are organized in a locus with flaA2 immediately upstream of flaA1. In this study, flaA1 and flaA2 mutants were constructed by transposon mutagenesis. Both mutants still produced periplasmic flagella. The flaA1 mutant did not produce FlaA1 but continued to produce FlaA2 and retained normal morphology and virulence in a hamster model of infection but had reduced motility. The flaA2 mutant did not produce either the FlaA1 or the FlaA2 protein. Cells of the flaA2 mutant lacked the distinctive hook-shaped ends associated with L. interrogans and lacked translational motility in liquid and semisolid media. These observations were confirmed with a second, independent flaA2 mutant. The flaA2 mutant failed to cause disease in animal models of acute infection. Despite lacking FlaA proteins, the flagella of the flaA2 mutant were of the same thickness as wild-type flagella, as measured by electron microscopy, and exhibited a normal flagellum sheath, indicating that FlaA proteins are not essential for the synthesis of the flagellum sheath, as observed for other spirochetes. This study shows that FlaA subunits contribute to leptospiral translational motility, cellular shape, and virulence.     

Phosphatidylinositol 3-kinase inhibitors prevent mouse cytotoxic T-cell development in vitro

To become competent killer cells, CD8(+) T cells require stimulation through signal transduction pathways associated with the T-cell receptor, costimulatory molecules such as CD28, and cytokine receptors such as the interleukin (IL)-2 receptor. We used wortmannin and LY294002, two inhibitors of phosphatidylinositol 3-kinase (PI3-K), to study the role of PI3-K in mouse cytotoxic T-lymphocyte (CTL) induction in response to mitogenic anti-CD3 antibody. Anti-CD3-induced CD8(+) T-cell proliferation and CTL development were inhibited dose dependently by both PI3-K inhibitors. IL-2 synthesis by anti-CD3-activated CD8(+) T cells was also diminished by PI3-K inhibition. PI3-K inhibition resulted in a modest decrease in anti-CD3-induced CD4(+) T-cell proliferation but failed to affect IL-2 expression by anti-CD3-activated CD4(+) T cells. PI3-K inhibition during CTL induction resulted in decreased levels of mRNAs coding for granzyme B, perforin, and Fas ligand. In addition, CTL induced in the presence of PI3-K inhibitors failed to conjugate normally with P815 target cells. Exogenous IL-2 did not reverse the effects of PI3-K inhibition on CD8(+) T-cell proliferation and CTL induction. These results support the conclusion that PI3-K activation is involved in T-cell receptor, CD28, and IL-2 receptor signaling of CD8(+) T cells. PI3-K is, therefore, an important component of multiple signal transduction pathways involved in CTL generation.