Neuropeptide signalling systems in flatworms

Two distinct families of neuropeptides are known to endow platyhelminth nervous systems-the FMRFamide-like peptides (FLPs) and the neuropepticle Fs (NPFs). Flatworm FLPs are strusturally simple, each 4-6 amino acids in length with a carboxy terminal aromatic-hydropliobic-Arg-Phe-amide motif. Thus far, four distinct flatworm FLPs have been characterized, with only one of these from a parasite. They have a widespread distribution within the central and peripheral nervous system of every flatworm examined, including neurones serving the attachment organs, the somatic Musculature and the reproductive system. The only physiological role that has been identified for flatworm FLPs is myoexcitation. Flatworm NPFs are believed to be invertebrate homologues of the vertebrate neuropeptide Y (NPY) family of peptides. Flatworm NPFs are 36-39 amino acids in length and are characterized by a caboxy terminal GRPRFarnide signature and conserved tyrosine residues at positions 10 and 17 from the carboxy terminal. Like FLPs, NPF occurs throughout flatworm nervous systems, although less is known about its biological role. While there is some evidence for a myoexcitatory action in cestodes and flukes, more compelling physiological data indicate that flatworm NPF inhibits cAMP levels in a manner that is characteristic of NPY action in vertebrates. The widespread expression of these neuropeptides in flanworm parasites highlights the potential of these signalling systems to yield new targets for novel anthelmintics. Although platyhelminth FLP and NPF receptors await identification, other molecules that play pivotal roles in neuropeptide signalling have been uncovered. These enzymes, involved in the biosynthesis and processing of flatworm neuropeptides, have recently been described and offer other distinct and attractive targets for therapeutic interference.

A novel small-molecule inhibitor of IL-6 signalling.

A small library of pyrrolidinesulphonylaryl molecules has been synthesized via an efficient 4-step route, and members evaluated for their ability to inhibit IL-6 signalling. One molecule (6a) was found to have promising activity against IL-6/STAT3 signalling at the low micromolar level, and to selectively inhibit phosphorylation of STAT3 (but not STAT1) in IL-6 stimulated MDA-MB-231 breast cancer and HeLa cell lines. It was also selectively cytostatic in MDA-MB-231 (STAT3-dependent) versus A4 (STAT3-null) cells suggesting STAT3-specific inhibitory properties.

A molecular photoionic AND gate based on fluorescent signalling

MOLECULES that perform logic operations are prerequisites for molecular information processing and computation. We and others have previously reported receptor molecules that can be considered to perform simple logic operations by coupling ionic bonding or more complex molecular-recognition processes with photonic (fluorescence) signals: in these systems, chemical binding (the 'input') results in a change in fluorescence intensity (the 'output') from the receptor. Here we describe a receptor (molecule (1) in Fig. 1) that operates as a logic device with two input channels: the fluorescence signal depends on whether the molecule binds hydrogen ions, sodium ions or both. The input/output characteristics of this molecular device correspond to those of an AND gate.

Ectodysplasin A (EDA) - EDA receptor signalling and its pharmacological modulation.

The TNF family ligand ectodysplasin A (EDA) regulates the induction, morphogenesis and/or maintenance of skin-derived structures such as teeth, hair, sweat glands and several other glands. Deficiencies in the EDA - EDA receptor (EDAR) signalling pathway cause hypohidrotic ectodermal dysplasia (HED). This syndrome is characterized by the absence or malformation of several skin-derived appendages resulting in hypotrychosis, hypodontia, heat-intolerance, dry skin and dry eyes, susceptibility to airways infections and crusting of various secretions. The EDA-EDAR system is an important effector of canonical Wnt signalling in developing skin appendages. It functions by stimulating NF-κB-mediated transcription of effectors or inhibitors of the Wnt, Sonic hedgehog (SHH), fibroblast growth factor (FGF) and transforming growth factor beta (TGFβ) pathways that regulate interactions within or between epithelial and mesenchymal cells and tissues. In animal models of Eda-deficiency, soluble EDAR agonists can precisely correct clinically relevant symptoms with low side effects even at high agonist doses, indicating that efficient negative feedback signals occur in treated tissues. Hijacking of the placental antibody transport system can help deliver active molecules to developing foetuses in a timely manner. EDAR agonists may serve to treat certain forms of ectodermal dysplasia.

The role of Ken&Barbie in JAK/STAT signalling during development of Drosophila melanogaster

Cell-cell interactions during embryonic development are crucial in the co-ordination of growth, differentiation and maintenance of many different cell types. To achieve this co-ordination each cell must properly translate signals received from neighbouring cells, into spatially and temporally appropriate developmental responses. A surprisingly limited number of signal pathways are responsible for the differentiation of enormous variety of cell types. As a result, pathways are frequently 'reused' during development. Thus, in mammals the JAK/STAT pathway is required during early embryogenesis, mammary gland formation, hematopoiesis and, finally, plays a pivotal role in immune response. In the canonical way, the JAK/STAT pathway is represented by a transmembrane receptor associated with a Janus kinase (JAK), which upon stimulation by an extra-cellular ligand, phosphorylates itself, the receptor and, finally, the signal transducer and activator of transcription (STAT) molecules. Phosphorylated STATs dimerise and translocate to the nucleus where they activate transcription of target genes. The JAK/STAT pathway has been conserved throughout evolution, and all known components are present in the genome of Drosophila melanogaster. Besides hematopoietic and immunity functions, the pathway is also required during development for processes including embryonic segmentation, tracheal morphogenesis, posterior spiracle formation etc. This study describes Drosophila Ken&Barbie (Ken) as a selective regulator of JAK/STAT signalling. ken mutations identified in a screen for modulators of an eye overgrowth phenotype, caused by over-expression of the pathway ligand unpaired, also interact genetically with the pathway receptor domeless (dome) and the transcription factor stat92E. Over-expression of Ken can phenocopy developmental defects known to be caused by the loss of JAK/STAT signalling. These genetic interactions suggest that Ken may function as a negative regulator of the pathway. Ken has C-terminal Zn-finger domain, presumably for DNA binding, and N-terminal BTB/POZ domain, often found in transcriptional repressors. Using EGFP-fused construct expressed in vivo revealed nuclear accumulation of Ken. Therefore, it is proposed that Ken may act as a suppresser of STAT92E target genes. An in vitro assay, termed SELEX, determined that Ken specifically binds to a DNA sequence, with the essential for DNA recognition core overlapping that of STAT92E. This interesting observation suggests that not all STAT92E sites may also allow Ken binding. Strikingly, when effects of ectopic Ken on the expression of putative JAK/STAT pathway target genes were examined, only a subset of the genes tested, namely vvl, trh and kni, were down-regulated by Ken, whereas some others, such as eve and fj, appeared to be unresponsive. Further analysis of vvl, one of the genes susceptible to ectopic Ken, was undertaken. In the developing hindgut, expression of vvl is JAK/STAT pathway dependent, but remains repressed in the posterior spiracles, despite the stimulation of STAT92E by Upd in their primordia. Importantly, ken is also expressed in the developing posterior spiracles. Strikingly, up-regulation of vvl is observed in these tissues in ken mutant embryos. These imply that while ectopic Ken is sufficient to repress the expression of vvl in the hindgut, endogenous Ken is also necessary to prevent its activation in the posterior spiracles. It is therefore conceivable that ectopic vvl expression in the posterior spiracles of the ken mutants may be the result of de-repression of endogenous STAT92E activity. Another consequence of these observations is a fine balance that must exist between STAT92E and Ken activities. Apparently, endogenous level of Ken is sufficient to repress vvl, but not other, as yet unidentified, JAK/STAT pathway targets, whose presumable activation by STAT92E is required for posterior spiracle development as the embryos mutant for dome, the receptor of the pathway, show severe spiracle defects. These defects are also observed in the embryos mis-expressing Ken. Though it is possible that the posterior spiracle phenotype caused by higher levels of Ken results from a JAK/STAT pathway independent activity, it seems to be more likely that Ken acts in a dosage dependent manner, and extra Ken is able to further antagonise JAK/STAT pathway target genes. While STAT92E binding sites required for target gene expression have been poorly characterised, the existence of genome data allows the prediction of candidate STAT92E sites present in target genes promoters to be attempted. When a 6kb region containing the putative regulatory domains flanking the vvl locus are examined, only a single potential STAT92E binding site located 825bp upstream of the translational start can be detected. Strikingly, this site also includes a perfect Ken binding sequence. Such an in silico observation, though consistent with both Ken DNA binding assay in vitro and regulation of STAT92E target genes in vivo, however, requires further analysis. The JAK/STAT pathway is implicated in a variety of processes during embryonic and larval development as well as in imago. In each case, stimulation of the same transcription factor results in different developmental outcomes. While many potential mechanisms have been proposed and demonstrated to explain such pleiotropy, the present study indicates that Ken may represent another mechanism, with which signal transduction pathways are controlled. Ken selectively down-regulates a subset of potential target genes and so modifies the transcriptional profile generated by activated STAT92E - a mechanism, which may be partially responsible for differences in the morphogenetic processes elicited by JAK/STAT signalling during development.

The interactions of flavonoids within neuronal signalling pathways

Emerging evidence suggests that dietary phytochemicals, in particular flavonoids, may exert beneficial effects in the central nervous system by protecting neurons against stress-induced injury, by suppressing neuroinflammation and by promoting neurocognitive performance, through changes in synaptic plasticity. It is likely that flavonoids exert such effects in neurons, through selective actions on different components within a number of protein kinase and lipid kinase signalling cascades, such as phosphatidylinositol-3 kinase (PI3K)/Akt, protein kinase C and mitogen-activated protein kinase. This review details the potential inhibitory or stimulatory actions of flavonoids within these pathways, and describes how such interactions are likely to affect cellular function through changes in the activation state of target molecules and/or by modulating gene expression. Although, precise sites of action are presently unknown, their abilities to: (1) bind to ATP binding sites on enzymes and receptors; (2) modulate the activity of kinases directly; (3) affect the function of important phosphatases; (4) preserve neuronal Ca2+ homeostasis; and (5) modulate signalling cascades lying downstream of kinases, are explored. Future research directions are outlined in relation to their precise site(s) of action within the signalling pathways and the sequence of events that allow them to regulate neuronal function in the central nervous system.

Targeting phosphoinositide 3-kinase signalling in lung cancer

Lung cancer is the leading cause of cancer-related mortality worldwide and more than 1 million people annually die in consequence of lung cancer. Although an improvement in lung cancer treatment could be achieved, especially in the last decade, the development of additional therapeutic strategies is urgently required in order to provide improved survival benefit for patients. Lung cancer formation is caused by genetic modifications commonly caused by tobacco smoking. Numerous studies have demonstrated the role of extracellular growth factors in lung cancer cell proliferation, metastasis, and chemoresistance. Mutations and amplifications in molecules related to receptor tyrosine signalling, such as EGFR, ErbB2, c-Met, c-Kit, VEGFR, PI3K, and PTEN are only some of the alterations known to contribute to the development of lung cancer. The phosphoinositide 3-kinase (PI3K) pathway, fundamental for cell development, growth, and survival, is known to be frequently altered in neoplasia, including carcinomas of the lung. Based on the high frequency of alterations, which include mutations and amplifications, leading to over-activation of certain upstream/downstream mediators, targeting components of the PI3K signalling pathway is considered to be a promising therapeutic approach in cancer treatment. In this article we will summarize the current knowledge about the involvement of PI3K signalling in lung cancer and discuss the development of targeted therapies involving PI3K pathway inhibitors.

The expression levels of three raft-associated molecules in cultivated vascular cells are dependent on culture conditions

Relaying a signal across the plasma membrane requires functional connections between the partner molecules. Membrane microdomains or lipid rafts provide an environment in which such specific interactions can take place. The integrity of these sites is often taken for granted when signalling pathways are investigated in cell culture. However, it is well known that smooth muscle and endothelial cells undergo cytoskeletal rearrangements during monolayer culturing. Likewise affected--and with potentially important consequences for signalling events--is the organization of the plasma membrane. The expression levels of three raft markers were massively upregulated, and raft-associated 5'-nucleotidase activity increased in conventional monolayer cultures as compared with a spheroidal coculture model, shown to promote the differentiation of endothelial cells. Our data point to a shift of raft components in monolayer cultures and demonstrate potential advantages of the spheroid coculture system for investigation of raft-mediated signalling events in endothelial cells.

Development of Drugs Targeting the PI3K Signalling Pathway in Leukaemias and Lymphomas

The phosphoinositide 3-kinase (PI3K) family of signalling enzymes play a key role in the transduction of signals from activated cell surface receptors controlling cell growth and proliferation, survival, metabolism, and migration. The intracellular signalling pathway from activated receptors to PI3K and its downstream targets v-akt murine thymoma viral oncogene homolog (Akt) and mechanistic target of rapamycin (mTOR) is very frequently deregulated by genetic and epigenetic mechanisms in human cancer, including leukaemia and lymphoma. In the past decade, an arsenal of small molecule inhibitors of key enzymes in this pathway has been developed and evaluated in pre-clinical studies and clinical trials in cancer patients. These include pharmacological inhibitors of Akt, mTOR, and PI3K, some of which are approved for the treatment of leukaemia and lymphoma. The PI3K family comprises eight different catalytic isoforms in humans, which have been subdivided into three classes. Class I PI3K isoforms have been extensively studied in the context of human cancer, and the isoforms p110α and p110δ are validated drug targets. The recent approval of a p110δ-specific PI3K inhibitor (idelalisib/Zydelig®) for the treatment of selected B cell malignancies represents the first success in developing these molecules into anti-cancer drugs. In addition to PI3K inhibitors, mTOR inhibitors are intensively studied in leukaemia and lymphoma, and temsirolimus (Torisel®) is approved for the treatment of a type of lymphoma. Based on these promising results it is hoped that additional novel PI3K pathway inhibitors will in the near future be further developed into new drugs for leukaemia and lymphoma.

Classical cadherin adhesion molecules: coordinating cell adhesion, signaling and the cytoskeleton

Classical cadherin adhesion molecules are fundamental determinants of tissue organization in both health and disease. Recent advances in understanding the molecular and cellular basis of cadherin function have revealed that these adhesion molecules serve as molecular couplers, linking cell surface adhesion and recognition to both the actin cytoskeleton and cell signalling pathways. We will review some of these developments. to provide an overview of progress in this rapidly-developing area of cell and developmental biology.

The challenges of abundance: epithelial junctions and small GTPase signalling

Small GTPases of the Ras superfamily play critical roles in epithelial biogenesis. Many key morphogenetic functions occur when small GTPases act at epithelial junctions, where they mediate an increasingly complex interplay between cell-cell adhesion molecules and fundamental cellular processes, such as cytoskeletal activity, polarity and trafficking. Important recent advances in this field include the role of additional members of the Ras superfamily in cell-cell contact stability and the capacity for polarity determinants to regulate small GTPase signalling. Interestingly, small GTPases may participate in the cross-talk between different adhesive receptors: in tissues classical cadherins can selectively regulate other junctions through cell signalling rather than through a global influence on cell-cell cohesion.

Orexin-stimulated MAP kinase cascades are activated through multiple G-protein signalling pathways in human H295R adrenocortical cells: diverse roles for orexins A and B

Orexins A and B (ORA and ORB) are neuropeptide hormones found throughout the central nervous system and periphery. They are required for a host of physiological processes including mitogen-activated protein kinase (MAPK) regulation, steroidogenesis, appetite control and energy regulation. While some signalling mechanisms have been proposed for individual recombinant orexin receptors in generic mammalian cell types, it is clear that the peripheral effects of orexin are spatially and temporally complex. This study dissects the different G-protein signalling and MAPK pathways activated in a pluripotent human adrenal H295R cell line capable of all the physiological steps involved in steroidogenesis. Both extracellular receptor kinase 1/2 (ERK1/2) and p38 were phosphorylated rapidly with a subsequent decline, in a time- and dose-dependent manner, in response to both ORA and ORB. Conversely, there was little or no direct activation of the ERK5 or JNK pathway. Analysis using signalling and MAPK inhibitors as well as receptor-specific antagonists determined the precise mediators of the orexin response in these cells. Both ERK1/2 and p38 activation were predominantly Gq- and to a lesser extent Gs-mediated; p38 activation even had a small Gi-component. Effects were broadly comparable for both orexin sub-types ORA and ORB and although most of the effects were transmitted through the orexin receptor-1 subtype, we did observe a role for orexin receptor-2-mediated activation of both ERK1/2 and p38. Cortisol secretion also differed in response to ORA and ORB. These data suggest multiple roles for orexin-mediated MAPK activation in an adrenal cell-line, this complexity may help to explain the diverse biological actions of orexins with wide-ranging consequences for our understanding of the mechanisms initiated by these steroidogenic molecules.

Ceramide and reactive oxygen species (ROS) as signal transduction molecules in inflammation

Reactive oxygen species (ROS) and the sphingolipid ceramide are each partly responsible for the intracellular signal transduction of a variety of physiological, pharmacological or environmental agents. Furthermore, the enhanced production of many of these agents, that utilise ROS and ceramide as signalling intermediates, is associated with the aetiologies of several vascular diseases (e.g. atherosclerosis) or disorders of inflammatory origin (e.g. rheumatoid arthritis; RA). Excessive monocyte recruitment and uncontrolled T cell activation are both strongly implicated in the chronic inflammatory responses that are associated with these pathologies. Therefore the aims of this thesis are (1) to further elucidate the cellular responses to modulations in intracellular ceramide/ROS levels in monocytes and T cells, in order to help resolve the mechanisms of progression of these diseases and (2) to examine both existing agents (methotrexate) and novel targets for possible therapeutic manipulation. Utilising synthetic, short chain ceramide to mimic the cellular responses to fluctuations in natural endogenous ceramide or, stimulation of CD95 to induce ceramide formation, it is described here that ceramide targets and manipulates two discrete sites responsible for ROS generation, preceding the cellular responses of growth arrest in U937 monocytes and apoptosis in Jurkat T-cells. In both cell types, transient elevations in mitochondrial ROS generation were observed. However, the prominent redox altering effects appear to be the ceramide-mediated reduction in cytosolic peroxide, the magnitude of which dictates in part the cellular response in U937 monocytes, Jurkat T-cells and primary human peripheral blood resting or PHA-activated T-cells in vitro. The application of synthetic ceramides to U937 monocytes for short (2 hours) or long (16 hours) treatment periods reduced the membrane expression of proteins associated with cell-cell interaction. Furthermore, ceramide treated U937 monocytes demonstrated reduced adhesion to 5 or 24 hour LPS activated human umbilical vein endothelial cells (HUVEC) but not resting HUVEC. Consequently it is hypothesised that the targeted treatment of monocytes from patients with cardiovascular diseases with short chain synthetic ceramide may reduce disease progression. Herein, the anti-inflammatory and immunosuppressant drug, methotrexate, is described to require ROS production for the induction of cytostasis or cytotoxicity in U937 monocytes and Jurkat T-cells respectively. Further, ROS are critical for methotrexate to abrogate monocyte interaction with activated HUVEC in vitro. The histological feature of RA of enhanced infiltration, survivability and hyporesponsiveness of T-cells within the diseased synovium has been suggested to arise from aberrant signalling. No difference in the concentrations of endogenous T-cell ceramide, the related lipid diacylglycerol (DAG) and cytosolic peroxide ex vivo was observed. TCR activation following PHA exposure in vitro for 72 hours did not induced maintained perturbations in DAG or ceramide in T-cells from RA patients or healthy individuals. However, T-cells from RA patients failed to upregulate cytosolic peroxide in response to PHA, unlike those from normals, despite expressing identical levels of the activation marker CD25. This inability to upregulate cytosolic peroxide may contribute to the T-cell pathology associated with RA by affecting the signalling capacity of redox sensitive biomolecules. These data highlight the importance of two distinctive cellular pools of ROS in mediating complex biological events associated with inflammatory disease and suggest that modulation of cellular ceramides represents a novel therapeutic strategy to minimise monocyte recruitment.

Oxidised phospholipid regulation of toll-like receptor signalling

Toll-like receptors (TLRs) serve to initiate inflammatory signalling in response to the detection of conserved microbial molecules or products of host tissue damage. Recent evidence suggests that TLR-signalling plays a considerable role in a number of inflammatory diseases, including atherosclerosis and arthritis. Agents which modulate TLR-signalling are, therefore, receiving interest in terms of their potential to modify inflammatory disease processes. One such family of molecules, the oxidised phospholipids (OxPLs), which are formed as a result of inflammatory events and accumulate at sites of chronic inflammation, have been shown to modulate TLR-signalling in both in vitro and in vivo systems. As the interaction between OxPLs and TLRs may play a significant role in chronic inflammatory disease processes, consideration is given in this review to the potential role of OxPLs in the regulation of TLR-signalling.

Reduced epithelial suppressor of cytokine signalling 1 in severe eosinophilic asthma

Severe asthma represents a major unmet clinical need. Eosinophilic inflammation persists in the airways of many patients with uncontrolled asthma, despite high-dose inhaled corticosteroid therapy. Suppressors of cytokine signalling (SOCS) are a family of molecules involved in the regulation of cytokine signalling via inhibition of the Janus kinase-signal transducers and activators of transcription pathway. We examined SOCS expression in the airways of asthma patients and investigated whether this is associated with persistent eosinophilia.

Healthy controls, mild/moderate asthmatics and severe asthmatics were studied. Whole genome expression profiling, quantitative PCR and immunohistochemical analysis were used to examine expression of SOCS1, SOCS2 and SOCS3 in bronchial biopsies. Bronchial epithelial cells were utilised to examine the role of SOCS1 in regulating interleukin (IL)-13 signalling in vitro.

SOCS1 gene expression was significantly lower in the airways of severe asthmatics compared with mild/moderate asthmatics, and was inversely associated with airway eosinophilia and other measures of T-helper type 2 (Th2) inflammation. Immunohistochemistry demonstrated SOCS1 was predominantly localised to the bronchial epithelium. SOCS1 overexpression inhibited IL-13-mediated chemokine ligand (CCL) 26 (eotaxin-3) mRNA expression in bronchial epithelial cells.

Severe asthma patients with persistent airway eosinophilia and Th2 inflammation have reduced airway epithelial SOCS1 expression. SOCS1 inhibits epithelial IL-13 signalling, supporting its key role in regulating Th2-driven eosinophilia in severe asthma.