Signal transduction mediated by Bid,a pro—death Bcl—2 family proteins,connects the death receptor and mitochondria apoptosis pathways

Two major apoptosis pathways have been defined in mammalian cells,the Fas/TNF-R1 death receptor pathway and the mitochondria pathway.The Bcl-2 family proteins consist of both anti-apoptosis and pro-apoptosis members that regulate apoptosis,mainly by controlling the release of cytochrome c and other mitochondrial apoptotic events.However,death signals mediated by Fas/TNF-R1 receptors can usually activate caspases directly,bypassing the need for mitochondria and escaping the regulation by Bcl-2 family proteins.Bid is a novel pro-apoptosis Bcl-2 family protein that is activated by caspase 8 in response to Fas/TNF-R1 death receptor signals.Activated Bid is translocated to mitochondria and induces cytochrome c release,which in turn activates downstream caspases.Such a connection between the two apoptosis pathways could be important for induction of apoptosis in certain types of cells and responsible for the pathogenesis of a number of human diseases.     

Structural basis for stem cell factor-KIT signaling and activation of class III receptor tyrosine kinases

Stem cell factor (SCF) binds to and activates the KIT receptor, a class III receptor tyrosine kinase (RTK), to stimulate diverse processes including melanogenesis, gametogenesis and hematopoeisis. Dysregulation of KIT activation is associated with many cancers. We report a 2.5 A crystal structure of the functional core of SCF bound to the extracellular ligand-binding domains of KIT. The structure reveals a 'wrapping' SCF-recognition mode by KIT, in which KIT adopts a bent conformation to facilitate each of its first three immunoglobulin (Ig)-like domains to interact with SCF. Three surface epitopes on SCF, an extended loop, the B and C helices, and the N-terminal segment, contact distinct KIT domains, with two of the epitopes undergoing large conformational changes upon receptor binding. The SCF/KIT complex reveals a unique RTK dimerization assembly, and a novel recognition mode between four-helix bundle cytokines and Ig-family receptors. It serves as a framework for understanding the activation mechanisms of class III RTKs.     

P2Y(6) nucleotide receptor mediates monocyte interleukin-8 production in response to UDP or lipopolysaccharide

Extracellular nucleotides are autocrine and paracrine cellular mediators that signal through P2 nucleotide receptors. Monocytic cells express several P2Y receptors but the role of these G protein-coupled receptors in monocytes is not known. Here, we present evidence that P2Y(6) regulates chemokine production and release in monocytes. We find that UDP, a selective P2Y(6) agonist, stimulates interleukin (IL)-8 release in human THP-1 monocytic cells whereas other nucleotides are relatively inactive. P2 receptor antagonists or P2Y(6) antisense oligonucleotides inhibit IL-8 release induced by UDP. Furthermore, UDP specifically activated IL-8 production in astrocytoma 1321N1 cells transfected with human P2Y(6). Since lipopolysaccharide has been suggested to activate P2 receptors via nucleotide release, we tested whether IL-8 production stimulated by lipopolysaccharide might result from P2Y(6) activation. P2 antagonists or apyrase, an enzyme which hydrolyzes nucleotides including UDP, inhibit IL-8 production induced by lipopolysaccharide but not by other stimuli. Furthermore, IL-8 gene expression activated by lipopolysaccharide is enhanced by P2Y(6) overexpression and inhibited by P2Y(6) antisense oligonucleotides. Thus, UDP activates IL-8 production via P2Y(6) in monocytic cells. Furthermore, lipopolysaccharide mediates IL-8 production at least in part by autocrine P2Y(6) activation. These findings indicate a novel role for P2Y(6) in innate immune defenses.     

Regulation of insulin exocytosis by calcium-dependent protein kinase C in beta cells

The control of insulin release from pancreatic beta cells helps ensure proper blood glucose level, which is critical for human health. Protein kinase C has been shown to be one key control mechanism for this process. After glucose stimulation, calcium influx into beta cells triggers exocytosis of insulin-containing dense-core granules and activates protein kinase C via calcium-dependent phospholipase C-mediated generation of diacylglycerol. Activated protein kinase C potentiates insulin release by enhancing the calcium sensitivity of exocytosis, likely by affecting two main pathways that could be linked: (1) the reorganization of the cortical actin network, and (2) the direct phosphorylation of critical exocytotic proteins such as munc18, SNAP25, and synaptotagmin. Here, we review what is currently known about the molecular mechanisms of protein kinase C action on each of these pathways and how these effects relate to the control of insulin release by exocytosis. We identify remaining challenges in the field and suggest how these challenges might be addressed to advance our understanding of the regulation of insulin release in health and disease.     

Excitatory amino acid signal transduction in the hippocampus: role of noradrenergic afferents and nitric oxide in cGMP increases in vivo.

Previous studies have demonstrated that excitatory amino acid (EAA)-dependent increases in cerebellar cGMP are dependent upon the prior activation of nitric oxide (NO) synthase. Additionally, the actions of NMDA, but not kainate or quisqualate, in elevating cerebellar cGMP have been shown to be dependent upon intact noradrenergic innervation of the cerebellum. In the current study we extended these observations to the hippocampus and again found that EAA-dependent increases in hippocampal cGMP also involve prior formation of NO. And as in the case of the cerebellum, NMDA-dependent increases in hippocampal cGMP involve prior release of norepinephrine which in turn apparently activates an alpha 1-adrenergic receptor to elicit cGMP increases. In toto, these data suggest that a key role of NMDA receptors in these brain regions is to presynaptically regulate the release of norepinephrine, thereby modulating the tone of this monoaminergic system. This may be a general principle which needs experimentation in other terminal fields of noradrenergic pathways.     

Modulation of receptor-mediated signal transduction by diacylglycerol mimetics in astrocytes

1. When rat astrocytes in primary culture were incubated with bradykinin, inositol phosphate formation and arachidonic acid release were stimulated. 2. By themselves, phorbol esters inhibited inositol phosphate formation, but phorbol esters and other cell-permeant diacylglycerol analogues stimulated arachidonic acid release. Preincubation of the cells with phorbol esters or diacylglycerol analogues blocked bradykinin-stimulated inositol phosphate formation but augmented bradykinin-stimulated arachidonic acid release. 3. The present results suggest that, in astrocytes, bradykinin activates at least two signal transduction pathways bradykinin stimulates a phosphatidylinositol-specific phospholipase C leading to enhanced inositol phosphate formation, and bradykinin stimulates a second phospholipase to enhance arachidonic acid release. The pathways may be distinguished using phorbol esters and other diacylglycerol mimetics. 4. The possibility is raised that diacylglycerol, formed in response to bradykinin, may serve as a transducer of receptor-receptor interactions by altering the ability of receptors to stimulate phospholipase activity.     

Purinergic P2Y2 receptors induce increased MCP-1/CCL2 synthesis and release from rat alveolar and peritoneal macrophages

Macrophages play a key role in inflammation by synthesis and release of proinflammatory cytokines and chemokines. Extracellular nucleotides released at sites of tissue damage may be an early danger signal for immune cells, and ATP-gated P2X(7) receptors are well known to mediate the rapid release of proinflammatory IL-18 and IL-1beta. However, there is little direct evidence for the involvement of other purine receptor subtypes in the release of other cytokines or chemokines. We initially used protein arrays to address whether extracellular ATP can release cytokines and/or chemokines from rat NR8383 alveolar macrophage, which lack the P2X(7) receptor. ATPgammaS increased the release of the proinflammatory chemokine, MCP-1 (MCP-1/CCL2). Pharmacological profiling identified the receptor responsible as the P2Y(2) receptor. Brief activation (10 min) of P2Y(2) receptors increased MCP-1 mRNA levels within 30 min and increased its release at 60 min. Similar results were obtained from rat peritoneal macrophages. We investigated likely downstream signaling cascades that may be involved, specifically the canonical G(q)-mediated phospholipase C (PLC) and subsequent MAP kinase pathways, and G(i)/G(o)-mediated signaling. We could find no evidence for these pathways being involved in the P2Y(2)R-induced increase in mRNA levels although inhibition of PLC blocked the UTP-induced increased release of MCP-1. Thus, the PLC-activated pathway can account for the increased release of MCP-1, but a novel signaling pathway may be involved in the increase in MCP-1 mRNA by activation of P2Y(2) receptors in alveolar and peritoneal macrophage.     

Cannabinoid pharmacology in the cardiovascular system: potential protective mechanisms through lipid signalling

Cannabinoids include not only plant-derived compounds (of which delta9-tetrahydrocannabinol is the primary psychoactive ingredient of cannabis), but also synthetic agents and endogenous substances termed endocannabinoids which include anandamide (2-arachidonoylethanolamide) and 2-arachidonoylglycerol. Cannabinoids act on specific, G-protein-coupled, receptors which are currently divided into two types, CB1 and CB2. Relatively selective agonists and antagonists for these receptors have been developed, although one agent (SR141716A) widely used as an antagonist at CB1 receptors has non-cannabinoid receptor-mediated effects at concentrations which are often used to define the presence of the CB1 receptor. Both cannabinoid receptors are primarily coupled to Gi/o proteins and act to inhibit adenylyl cyclase. Stimulation of CB1 receptors also modulates the activity of K+ and Ca2+ channels and of protein kinase pathways including protein kinase B (Akt) which might mediate effects on apoptosis. CB, receptors may activate the extracellular signal-regulated kinase cascade through ceramide signalling. Cannabinoid actions on the cardiovascular system have been widely interpreted as being mediated by CB1 receptors although there are a growing number of observations, particularly in isolated heart and blood vessel preparations, that suggest that other cannabinoid receptors may exist. Interestingly, the currently identified cannabinoid receptors appear to be related to a wider family of lipid receptor, those for the lysophospholipids, which are also linked to Gi/o protein signalling. Anandamide also activates vanilloid VR1 receptors on sensory nerves and releases the vasoactive peptide, calcitonin gene-related peptide (CGRP), which brings about vasodilatation through its action on CGRP receptors. Current evidence suggests that endocannabinoids have important protective roles in pathophysiological conditions such as shock and myocardial infarction. Therefore, their cardiovascular effects and the receptors mediating them are the subject of increasing investigative interest.     

Cholinergic signal transduction cascades in rat pinealocytes: functional and ontogenetic aspects.

In adult rat pinealocytes, acetylcholine activates nicotinic receptors whose stimulation causes a depolarization of the cells, opening of voltage-gated cation channels of the L-type and subsequent increase in the intracellular calcium ion concentration. These events trigger a release of glutamate that, by its action on metabotropic glutamate type 3 receptors, activates an inhibitory cyclic AMP cascade and suppresses norepinephrine-induced melatonin biosynthesis. The nicotinic response is fully developed in the third postnatal week. Prior to this timepoint, rat pinealocytes possess functional muscarinic receptors whose activation causes a rise in the intracellular calcium ion concentration through a calcium release from thapsigargin-sensitive intracellular calcium stores and an opening of store-operated calcium channels. This cascade may influence tissue differentiation and maturation of the melatonin pathway. The demonstration of functional cholinoreceptors and the ontogenetic switch from muscarinic to nicotinic signalling in rat pinealocytes supports the concept that pineal functions in mammals are influenced by neuronal inputs other than the sympathetic innervation which serves as the major regulatory system.     

Activation of protein kinase C by presynaptic FLRFamide receptors facilitates transmitter release at an aplysia cholinergic synapse

Modulation of evoked quantal transmitter release by protein kinase C (PKC) was investigated at an identified cholinergic neuro-neuronal synapse of the Aplysia buccal ganglion. Evoked acetylcholine release was increased by a diacylglycerol analog that activates PKC and was decreased by H-7, a blocker of PKC. FLRFamide facilitated evoked quantal release by increasing presynaptic Ca2+ influx. The inhibition of PKC by H-7 prevented both the increase of presynaptic Ca2+ influx and the facilitation of evoked acetylcholine release induced by the activation of presynaptic FLRFamide receptors. These results provide evidence that the activation of PKC could be a step in the intracellular pathway by which FLRFamide receptors increase evoked quantal acetylcholine release.     

Intracellular signaling events at the leading edge of migrating cells

Cell migration is an important facet of the life cycle of immune and other cell types. A complex set of events must take place at the leading edge of motile cells before these cells can migrate. Chemokines induce the motility of various cell types by activating multiple intracellular signaling pathways. These include the activation of chemokine receptors, which are coupled to the heterotrimeric G proteins. The release of G beta gamma subunits from chemokine receptors results in the recruitment to the plasma membrane, with subsequent activation of various down-stream signaling molecules. Among these molecules are the pleckstrin homology domain-containing proteins and the phosphoinositide 3-kinase gamma which phosphorylates phospholipids and activates members of the GTP exchange factors (GEFs). These GEFs facilitate the exchange of GTP for GDP in members of GTPases. The latter are important for reorganizing the cell cytoskeleton, and in inducing chemotaxis. Chemokines also induce the mobilization of intracellular calcium from intracellular stores. Second messengers such as inositol 1,4,5 trisphosphate, and cyclic adenosine diphosphate ribose are among those induced by chemokines. In addition, the G beta gamma subunits recruit members of the G protein-coupled receptor kinases, which phosphorylate chemokine receptors, resulting in desensitization and termination of the motility signals. This review will discuss the intracellular signaling pathways induced by chemokines, particularly those activated at the leading edge of migrating cells which lead to cell polarization, cytoskeleton reorganization and motility.     

Autocrine signaling involved in cell volume regulation: the role of released transmitters and plasma membrane receptors

Cell volume regulation is a basic homeostatic mechanism transcendental for the normal physiology and function of cells. It is mediated principally by the activation of osmolyte transport pathways that result in net changes in solute concentration that counteract cell volume challenges in its constancy. This process has been described to be regulated by a complex assortment of intracellular signal transduction cascades. Recently, several studies have demonstrated that alterations in cell volume induce the release of a wide variety of transmitters including hormones, ATP and neurotransmitters, which have been proposed to act as extracellular signals that regulate the activation of cell volume regulatory mechanisms. In addition, changes in cell volume have also been reported to activate plasma membrane receptors (including tyrosine kinase receptors, G-protein coupled receptors and integrins) that have been demonstrated to participate in the regulatory process of cell volume. In this review, we summarize recent studies about the role of changes in cell volume in the regulation of transmitter release as well as in the activation of plasma membrane receptors and their further implications in the regulation of the signaling machinery that regulates the activation of osmolyte flux pathways. We propose that the autocrine regulation of Ca2+-dependent and tyrosine phosphorylation-dependent signaling pathways by the activation of plasma membrane receptors and swelling-induced transmitter release is necessary for the activation/regulation of osmolyte efflux pathways and cell volume recovery. Furthermore, we emphasize the importance of studying these extrinsic signals because of their significance in the understanding of the physiology of cell volume regulation and its role in cell biology in vivo, where the constraint of the extracellular space might enhance the autocrine or even paracrine signaling induced by these released transmitters.     

Dual Signalling by the Adenosine A2a Receptor Involves Activation of Both N- and P-Type Calcium Channels by Different G Proteins and Protein Kinases in the Same Striatal Nerve Terminals

Abstract: Many G_s-linked receptors have been reported to use multiple signalling pathways in transfected cells but few in their normal cell environment. We show that the adenosine A_2a receptor uses two signalling pathways to increase the release of acetylcholine from striatal nerve terminals. One pathway involves activation of G_s, adenylyl cyclase, protein kinase A, and P-type calcium channels; the other is mediated by a cholera toxin-insensitive G protein, protein kinase C, and N-type calcium channels. The effects of these two pathways are not additive, the second pathway being inhibited by the first; but they are equally sensitive to the A_2a receptor antagonist KF17837. This demonstrates that the A_2a receptor activates two signalling systems in striatal cholinergic neurons.     

Purinergic-independent calcium signaling mediates recovery from hepatocellular swelling: implications for volume regulation.

Swelling of hepatocytes and other epithelia activates volume-sensitive ion channels that facilitate fluid and electrolyte efflux to restore cell volume, but the responsible signaling pathways are incompletely defined. Previous work in model HTC rat hepatoma cells has indicated that swelling elicits ATP release, which stimulates P2 receptors and activates Cl(-) channels, and that this mechanism is essential for hepatocellular volume recovery. Since P2 receptors are generally coupled to Ca(2+) signaling pathways, we determined whether hepatocellular swelling affected cytosolic [Ca(2+)], and if this involved a purinergic mechanism. Exposure of HTC cells to hypotonic media evoked an increase in cytosolic [Ca(2+)], which was followed by activation of K(+) and Cl(-) currents. Maneuvers that interfered with swelling-induced increases in cytosolic [Ca(2+)], including extracellular Ca(2+) removal and intracellular Ca(2+) store depletion with thapsigargin, inhibited activation of membrane currents and volume recovery. However, the swelling-induced increases in cytosolic [Ca(2+)] were unaffected by either extracellular ATP depletion with apyrase or blockade of P2 receptors with suramin. These findings indicate that swelling elicits an increase in hepatocellular Ca(2+), which is essential for ion channel activation and volume recovery, but that this increase does not stem from activation of volume-sensitive P2 receptors. Collectively, these observations imply that regulatory responses to hepatocellular swelling involve a dual requirement for a purinergic-independent Ca(2+) signaling cascade and a Ca(2+)-independent purinergic signaling pathway.     

Imbalance of central nitric oxide and reactive oxygen species in the regulation of sympathetic activity and neural mechanisms of hypertension

Nitric oxide (NO) and reactive oxygen species (ROS) play important roles in blood pressure regulation via the modulation of the autonomic nervous system, particularly in the central nervous system (CNS). In general, accumulating evidence suggests that NO inhibits, but ROS activates, the sympathetic nervous system. NO and ROS, however, interact with each other. Our consecutive studies and those of others strongly indicate that an imbalance between NO bioavailability and ROS generation in the CNS, including the brain stem, activates the sympathetic nervous system, and this mechanism is involved in the pathogenesis of neurogenic aspects of hypertension. In this review, we focus on the role of NO and ROS in the regulation of the sympathetic nervous system within the brain stem and subsequent cardiovascular control. Multiple mechanisms are proposed, including modulation of neurotransmitter release, inhibition of receptors, and alterations of intracellular signaling pathways. Together, the evidence indicates that an imbalance of NO and ROS in the CNS plays a pivotal role in the pathogenesis of hypertension.     

FcepsilonRI- and Fcgamma receptor-mediated production of reactive oxygen species by mast cells is lipoxygenase- and cyclooxygenase-dependent and NADPH oxidase-independent

We investigated the enzymes responsible for FcepsilonRI-dependent production of reactive oxygen species (ROS) and the influence of ROS on mast cell secretory responses. 5-Lipoxygenase (5-LO) was the primary enzyme involved in ROS production by human mast cells (huMC) and mouse bone marrow-derived mast cells (mBMMC) following FcepsilonRI aggregation because incubation with 5-LO inhibitors (AA861, nordihydroguaiaretic acid, zileuton) but not a flavoenzyme inhibitor (diphenyleneiodonium) completely abrogated Ag-induced dichlorodihydrofluorescein (DCF) fluorescence. Furthermore, 5-LO-deficient mBMMC had greatly reduced FcepsilonRI-dependent DCF fluorescence compared with wild type mBMMC or those lacking a functional NADPH oxidase (i.e., gp91(phox)- or p47(phox)-deficient cells). A minor role for cyclooxygenase (COX)-1 in FcepsilonRI-dependent ROS production was demonstrated by inhibition of Ag-mediated DCF fluorescence by a COX-1 inhibitor (FR122047) and reduced DCF fluorescence in COX-1-deficient mBMMC. Complete abrogation of FcepsilonRI-dependent ROS production in mast cells had no effect on degranulation or cytokine secretion. In response to the NADPH oxidase-stimulating agents including PMA, mBMMC and huMC produced negligible ROS. IgG-coated latex beads did stimulate ROS production in huMC, and in this experiment 5-LO and COX again appeared to be the enzymatic sources of ROS. In contrast, IgG-coated latex bead-induced ROS production in human polymorphonuclear leukocytes occurred by the NADPH oxidase pathway. Thus mBMMC and huMC generate ROS by 5-LO and COX-1 in response to FcepsilonRI aggregation; huMC generate ROS upon exposure to IgG-coated latex beads by 5-LO and COX; and ROS appear to have no significant role in FcepsilonRI-dependent degranulation and cytokine production.     

Host Responses to Group A Streptococcus: Cell Death and Inflammation

Infections caused by group A Streptococcus (GAS) are characterized by robust inflammatory responses and can rapidly lead to life-threatening disease manifestations. However, host mechanisms that respond to GAS, which may influence disease pathology, are understudied. Recent works indicate that GAS infection is recognized by multiple extracellular and intracellular receptors and activates cell signalling via discrete pathways. Host leukocyte receptor binding to GAS-derived products mediates release of inflammatory mediators associated with severe GAS disease. GAS induces divergent phagocyte programmed cell death responses and has inflammatory implications. Epithelial cell apoptotic and autophagic components are mobilized by GAS infection, but can be subverted to ensure bacterial survival. Examination of host interactions with GAS and consequences of GAS infection in the context of cellular receptors responsible for GAS recognition, inflammatory mediator responses, and cell death mechanisms, highlights potential avenues for diagnostic and therapeutic intervention. Understanding the molecular and cellular basis of host symptoms during severe GAS disease will assist the development of improved treatment regimens for this formidable pathogen.