Trevor R. Walker

Phosphatidylinositol 3-kinase mediates mitogen-induced human airway smooth muscle cell proliferation.

Hypertrophy and hyperplasia of airway smooth muscle (ASM) are important pathological features that contribute to airflow obstruction in chronic severe asthma. Despite considerable research effort, the cellular mechanisms that modulate ASM growth remain unknown. Recent evidence suggests that mitogen-induced activation of phosphoinositide (PI)-specific phospholipase C (PLC) and PI-dependent calcium mobilization are neither sufficient nor necessary to stimulate human ASM proliferation. In this study, we identify phosphatidylinositol (PtdIns) 3-kinase as a key regulator of human ASM proliferation. Pretreatment of human ASM with the PtdIns 3-kinase inhibitors wortmannin and LY-294002 significantly reduced thrombin- and epidermal growth factor (EGF)-induced DNA synthesis (IC(50) approximately 10 nM and approximately 3 microM, respectively). In separate experiments, wortmannin and LY-294002 markedly inhibited PtdIns 3-kinase and 70-kDa S6 protein kinase (pp70(S6k)) activation induced by stimulation of human ASM cells with EGF and thrombin but had no effect on EGF- and thrombin-induced p42/p44 mitogen-activated protein kinase (MAPK) activation. The specificity of wortmannin and LY-294002 was further suggested by the demonstrated inability of these compounds to alter thrombin-induced calcium transients, total PI hydrolysis, or basal cAMP levels. Transient expression of constitutively active PtdIns 3-kinase (p110*) activated pp70(S6k), whereas a dominant-negative PtdIns 3-kinase (Deltap85) blocked EGF- and thrombin-stimulated pp70(S6k) activity. Collectively, these data suggest that activation of PtdIns 3-kinase is required for the mitogenic effect of EGF and thrombin in human ASM cells. Further investigation of the role of PtdIns 3-kinase may offer new therapeutic approaches in the treatment of diseases characterized by smooth muscle cell hyperplasia such as asthma and chronic bronchitis.Hypertrophy and hyperplasia of airway smooth muscle (ASM) are important pathological features that contribute to airflow obstruction in chronic severe asthma. Despite considerable research effort, the cellular mechanisms that modulate ASM growth remain unknown. Recent evidence suggests that mitogen-induced activation of phosphoinositide (PI)-specific phospholipase C (PLC) and PI-dependent calcium mobilization are neither sufficient nor necessary to stimulate human ASM proliferation. In this study, we identify phosphatidylinositol (PtdIns) 3-kinase as a key regulator of human ASM proliferation. Pretreatment of human ASM with the PtdIns 3-kinase inhibitors wortmannin and LY-294002 significantly reduced thrombin- and epidermal growth factor (EGF)-induced DNA synthesis (IC50 ∼10 nM and ∼3 μM, respectively). In separate experiments, wortmannin and LY-294002 markedly inhibited PtdIns 3-kinase and 70-kDa S6 protein kinase (pp70S6k) activation induced by stimulation of human ASM cells with EGF and thrombin but had no effect on EGF- and thrombin-induced p42/p44 mitogen-activated protein kinase (MAPK) activation. The specificity of wortmannin and LY-294002 was further suggested by the demonstrated inability of these compounds to alter thrombin-induced calcium transients, total PI hydrolysis, or basal cAMP levels. Transient expression of constitutively active PtdIns 3-kinase (p110*) activated pp70S6k, whereas a dominant-negative PtdIns 3-kinase (Δp85) blocked EGF- and thrombin-stimulated pp70S6kactivity. Collectively, these data suggest that activation of PtdIns 3-kinase is required for the mitogenic effect of EGF and thrombin in human ASM cells. Further investigation of the role of PtdIns 3-kinase may offer new therapeutic approaches in the treatment of diseases characterized by smooth muscle cell hyperplasia such as asthma and chronic bronchitis.

Regulation of Phosphatidylinositol 3-Kinase Activity and Phosphatidylinositol 3,4,5-Trisphosphate Accumulation by Neutrophil Priming Agents

Neutrophil priming by agents such as TNF-α and GM-CSF causes a dramatic increase in the response of these cells to secretagogue agonists and affects the capacity of neutrophils to induce tissue injury. In view of the central role of phosphatidylinositol 3-kinase (PI3-kinase) in regulating NADPH oxidase activity we examined the influence of priming agents on agonist-stimulated phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) accumulation in human neutrophils. Pretreatment of neutrophils with TNF-α or GM-CSF, while not influencing fMLP-stimulated PtdIns(3,4,5)P3 accumulation at 5 s, caused a major increase in PtdIns(3,4,5)P3 at later times (10–60 s), which paralleled the augmented superoxide anion (O2−) response. The intimate relationship between PtdIns(3,4,5)P3 accumulation and O2− release was confirmed using platelet-activating factor, which caused full but transient priming of both responses. Likewise, LY294002, a PI3-kinase inhibitor, and genistein, a tyrosine kinase inhibitor, caused parallel inhibition of O2− generation and PtdIns(3,4,5)P3 accumulation; in contrast, radicicol, which inhibits receptor-mediated activation of p85 PI3-kinase, had no effect on either response. Despite major increases in PI3-kinase activity observed in p85 and anti-phosphotyrosine immunoprecipitates in growth factor-stimulated smooth muscle cells, no such increase was observed in primed/stimulated neutrophils. In contrast, both fMLP and TNF-α alone caused a 3-fold increase in PI3-kinase activity in p110γ PI3-kinase immunoprecipitates. p21ras activation (an upstream regulator of PI3-kinase) was unaffected by priming. These data demonstrate that timing and magnitude of PtdIns(3,4,5)P3 accumulation in neutrophils correlate closely with O2− generation, that PI3-kinase-γ is responsible for the enhanced PtdIns(3,4,5)P3 production seen in primed cells, and that factors other than activation of p21ras underlie this response.

Soluble E-selectin acts in synergy with platelet-activating factor to activate neutrophil beta 2-integrins. Role of tyrosine kinases and Ca2+ mobilization.

Selectins play a critical role in neutrophil recruitment to sites of inflammation, in tethering and rolling of neutrophils on vascular endothelium, as well as triggering β2-integrin-mediated adhesion. We have previously demonstrated potential pro-inflammatory effects of soluble E-selectin upon neutrophil effector functions, using a soluble recombinant molecule (E-zz), which increased β2-integrin-mediated adhesion, decreased β2-integrin-dependent migration, and triggered reactive oxygen species generation and release. In this study, we have examined the intracellular signals following neutrophil activation by soluble E-selectin. We show that exposure of neutrophils to E-selectin and platelet-activating factor (PAF) in combination induced a synergistic effect upon β2-integrin-mediated adhesion. Although soluble E-selectin did not induce Ca2+ mobilization in neutrophils by itself, elevation of intracellular Ca2+ was specifically prolonged in response to PAF but not leukotriene B4 orN-formyl-Met-Leu-Phe. The prolonged Ca2+mobilization observed in the presence of E-selectin was dependent on Ca2+ influx from intracellular stores rather than influx of extracellular Ca2+ through SKF 96365-sensitive channels. The specific alteration of Ca2+ mobilization reported here appears not to have a role in the synergistic effects of E-selectin and PAF upon neutrophil O⨪2 release but may be involved in augmentation of β2-integrin-mediated adhesion.

Phosphoinositide 3-kinase: a critical signalling event in pulmonary cells

Phosphoinositide 3-kinases (PI-3Ks) are enzymes that generate lipid second messenger molecules, resulting in the activation of multiple intracellular signalling cascades. These events regulate a broad array of cellular responses including survival, activation, differentiation and proliferation and are now recognised to have a key role in a number of physiological and pathophysiological processes in the lung. PI-3Ks contribute to the pathogenesis of asthma by influencing the proliferation of airways smooth muscle and the recruitment of eosinophils, and affect the balance between the harmful and protective responses in pulmonary inflammation and infection by the modulation of granulocyte recruitment, activation and apoptosis. In addition they also seem to exert a critical influence on the malignant phenotype of small cell lung cancer. PI-3K isoforms and their downstream targets thus provide novel therapeutic targets for intervention in a broad spectrum of respiratory diseases.

The diacylglycerol kinase inhibitor, R59949, potentiates secretion but not increased phosphorylation of a 47 kDalton protein in human platelets

We have investigated the action of a novel inhibitor of DG-kinase, R59949. This agent was found to produce partial inhibition of formation of phosphatidic acid in human platelets challenged with thrombin, DC8 or OAG. However, this effect was not associated with enhanced phosphorylation of a 47 kDa protein, a known substrate for protein kinase C. We therefore believe that this compound does not represent a major advance on its earlier prototype, R59022.

The Use of Inhibitors of Protein Kinases and Protein Phosphatases to Investigate the Role of Protein Phоsphorylation in Platelet Activation

A diverse range of agents have been shown to activate human platelets through cell surface proteins, including thrombin, collagen, ADP, thromboxane A2, vasopressin, adrenaline, platelet activating factor and various monoclonal antibodies. In the majority of cases, activation is associated with stimulated formation of the second messengers inositol 1,4,5 trisphosphate (IP3) and 1,2-diacylglycerol (DG), produced by hydrolysis of the minor membrane phospholipid, phosphatidylinositol 4,5-bisphosphate; receptors for ADP and adrenaline are notable exceptions. IP3 releases Ca2+ from intracellular stores and DG activates protein kinase C. These two messengers interact synergistically in a way that is poorly understood to induce platelet shape change, aggregation and secretion.

A critical ‘threshold’ of β2-integrin engagement regulates augmentation of cytokine-mediated superoxide anion release

Neutrophil adhesion regulates a number of processes involved in the pathogenesis of inflammatory diseases including rheumatoid arthritis. Neutrophil destructive potential can be modulated by adhesion, allowing alteration of inflammatory cell behaviour while preserving antimicrobial defences. β2‐Integrin‐mediated neutrophil adhesion to albumin‐coated latex beads (ACLB) allows modulation of integrin clustering and ligation and analysis of the effects of adhesion on neutrophil responses. Tumour necrosis factor‐α (TNFα) enhanced neutrophil binding of different diameter ACLB equally, by almost four‐fold, and independently of bead size. Adhesion of neutrophils to ACLB caused a size‐dependent generation and release of O2− and also potentiated TNFα‐induced O2− release. Binding of ACLB was not affected by disruption of cytoskeletal integrity with nocodazole or cytochalasin D or following blockade of tyrosine kinase activity. In contrast, tyrosine phosphorylation and an intact cytoskeleton were essential for adhesion‐ and cytokine‐induced O2− release from neutrophils. Inhibition of adhesion‐ and cytokine‐induced O2− release by 4‐amino‐5‐(4‐chlorophenyl)‐7‐(t‐butyl)pyrazol[3,4‐d]pyrimidine (PP2) indicated that a Src‐family tyrosine kinase was the principal regulatory pathway mediating this response in neutrophils, a distal role for p38 MAPK was revealed by use of SB203580. Tyrosine phosphorylation of c‐Fgr, a Src‐family tyrosine kinase, occurred following ACLB adhesion and exposure to TNFα, and was susceptible to inhibition by PP2. We suggest that activation of the key regulatory enzyme c‐Fgr is achieved following ligation of a critical threshold of integrins following binding of large (>3 μm) ACLB.