Home About us Contact
|
Home About us Contact | ||
|
|
||
Signaling Cascade (signaling + cascade)
Kinds of Signaling Cascade Selected AbstractsMorphogenesis of the node and notochord: The cellular basis for the establishment and maintenance of left,right asymmetry in the mouseDEVELOPMENTAL DYNAMICS, Issue 12 2008Jeffrey D. Lee Abstract Establishment of left,right asymmetry in the mouse embryo depends on leftward laminar fluid flow in the node, which initiates a signaling cascade that is confined to the left side of the embryo. Leftward fluid flow depends on two cellular processes: motility of the cilia that generate the flow and morphogenesis of the node, the structure where the cilia reside. Here, we provide an overview of the current understanding and unresolved questions about the regulation of ciliary motility and node structure. Analysis of mouse mutants has shown that the motile cilia must have a specific structure and length, and that they must point posteriorly to generate the necessary leftward fluid flow. However, the precise structure of the motile cilia is not clear and the mechanisms that position cilia on node cells have not been defined. The mouse node is a teardrop-shaped pit at the distal tip of the early embryo, but the morphogenetic events that create the mature node from cells derived from the primitive streak are only beginning to be characterized. Recent live imaging experiments support earlier scanning electron microscopy (SEM) studies and show that node assembly is a multi-step process in which clusters of node precursors appear on the embryo surface as overlying endoderm cells are removed. We present additional SEM and confocal microscopy studies that help define the transition stages during node morphogenesis. After the initiation of left-sided signaling, the notochordal plate, which is contiguous with the node, generates a barrier at the embryonic midline that restricts the cascade of gene expression to the left side of the embryo. The field is now poised to dissect the genetic and cellular mechanisms that create and organize the specialized cells of the node and midline that are essential for left,right asymmetry. Developmental Dynamics 237:3464,3476, 2008. © 2008 Wiley-Liss, Inc. [source] Left-asymmetric expression of Galanin in the linear heart tube of the mouse embryo is independent of the nodal co-receptor gene crypticDEVELOPMENTAL DYNAMICS, Issue 12 2008Axel Schweickert Abstract Only very few left/right asymmetrically expressed genes are known in the mammalian embryo. In a screen for novel factors we identified the gene encoding the neuropeptide Galanin in mouse. At embryonic day (E) 8.5 asymmetric mRNA transcription was found in the left half of the linear heart tube. During heart looping and morphogenesis expression became restricted to the atrio-ventricular (AV) canal, followed by specific staining of the AV-node and AV-rings in the four-chambered heart. Expression was inverted in inv/inv and randomized in homozygous iv mutant embryos. Left-sided heart-specific transcription of mouse Gal thus should be controlled by the left-right pathway. The asymmetric pattern was retained in cryptic mutant embryos, in which the Nodal signaling cascade is disrupted. Surprisingly, Pitx2c was found to be expressed in 50% of cryptic mutant hearts as well, suggesting that some aspects of asymmetric gene expression in the heart are independent of cryptic. Developmental Dynamics 237:3557,3564, 2008. © 2008 Wiley-Liss, Inc. [source] Mice with mutations in Mahogunin ring finger-1 (Mgrn1) exhibit abnormal patterning of the left,right axisDEVELOPMENTAL DYNAMICS, Issue 12 2006Christina D. Cota Abstract Mahogunin Ring Finger 1 (Mgrn1) encodes a RING-containing protein with ubiquitin ligase activity that has been implicated in pigment-type switching. In addition to having dark fur, mice lacking MGRN1 develop adult-onset spongy degeneration of the central nervous system and have reduced embryonic viability. Observation of complete situs inversus in a small proportion of adult Mgrn1 mutant mice suggested that embryonic lethality resulted from congenital heart defects due to defective establishment and/or maintenance of the left,right (LR) axis. Here we report that Mgrn1 is expressed in a pattern consistent with a role in LR patterning during early development and that many Mgrn1 mutant embryos show abnormal expression of asymmetrically expressed genes involved in LR patterning. A range of complex heart defects was observed in 20,25% of mid-to-late gestation Mgrn1 mutant embryos and another 20% were dead. This finding was consistent with 46,60% mortality of mutants by weaning age. Our results indicate that Mgrn1 acts early in the LR signaling cascade and is likely to provide new insight into this developmental process as Nodal expression was uncoupled from expression of other Nodal-responsive genes in Mgrn1 mutant embryos. Our work identifies a novel role for MGRN1 in embryonic patterning and suggests that the ubiquitination of MGRN1 target genes is essential for the proper establishment and/or maintenance of the LR axis. Developmental Dynamics 235:3438,3447, 2006. © 2006 Wiley-Liss, Inc. [source] A role for acetylcholine receptors in their own aggregation on muscle cellsDEVELOPMENTAL NEUROBIOLOGY, Issue 8 2007Rebecca B.R. Milholland Abstract Both neurotrophic factors and activity regulate synaptogenesis. At neuromuscular synapses, the neural factor agrin released from motor neuron terminals stimulates postsynaptic specialization by way of the muscle specific kinase MuSK. In addition, activity through acetylcholine receptors (AChRs) has been implicated in the stabilization of pre- and postsynaptic contacts on muscle at various stages of development. We show here that activation of AChRs with specific concentrations of nicotine is sufficient to induce AChR aggregation and that this induction requires the function of L-type calcium channels (L-CaChs). Furthermore, AChR function is required for agrin induced AChR aggregation in C2 muscle cells. The same concentrations of nicotine did not induce observable tyrosine phosphorylation on either MuSK or the AChR , subunit, suggesting significant differences between the mechanisms of agrin and activity induced aggregation. The AChR/L-CaCh pathway provides a mechanism by which neuromuscular signal transmission can act in concert with the agrin-MuSK signaling cascade to regulate NMJ formation. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007. [source] Signaling events leading to the curative effect of cystatin on experimental visceral leishmaniasis: Involvement of ERK1/2, NF-,B and JAK/STAT pathwaysEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 3 2009Susanta Kar Abstract Curative effect of cystatin, a natural cystein protease inhibitor, on experimental visceral leishmaniasis was associated with strong upregulation of iNOS. The transductional mechanisms underlying this cellular response was investigated in the murine macrophage cell line RAW 264.7 and in the BALB/c mouse model of visceral leishmaniasis. Cystatin synergizes with IFN-, in inducing ERK1/2 phosphorylation and NF-,B DNA-binding activity. Pretreatment of cells with specific inhibitors of NF-,B or ERK1/2 pathway blocked the cystatin plus IFN-,-inducible NF-,B activity and markedly reduced the expression of iNOS at both mRNA and protein levels. Silencing of mitogen- and stress-activated protein kinase 1 significantly reduced cystatin-mediated NF-,B-dependent iNOS gene transcription suggesting the involvement of mitogen- and stress-activated protein kinase 1 activation in ERK1/2 signaling. DNA binding as well as silencing experiments revealed the requirement of IFN-,-mediated JAK-STAT activation even though cystatin did not modulate this signaling cascade by itself. In the in vivo situation, key steps in the activation cascade of NF-,B, including nuclear translocation of NF-,B subunits, I,B phosphorylation and I,B kinase, are all remarkably enhanced in Leishmania -infected mice by cystatin. Understanding the molecular mechanisms through which cystatin modulates macrophage effector responses will contribute to better define its potential for macrophage-associated diseases, in general. [source] Notch signaling is activated by TLR stimulation and regulates macrophage functionsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 1 2008Tanapat Palaga Dr. Abstract Notch signaling is a well-conserved pathway involved in cell fate decisions, proliferation and apoptosis. We report on the involvement of Notch signaling in regulating gene expression in activated macrophages. Toll-like receptors (TLR) agonists such as bacterial lipopeptide, polyI:C, lipopolysaccharide and unmethylated CpG DNA all induced up-regulation of Notch1 in primary and macrophage-like cell lines. Notch1 up-regulation was dependent on the MyD88 pathway when stimulated through TLR2, but not TLR4. Activated Notch1 and expression of the Notch target genes, Hes1 and Deltex, were detected in activated macrophages, suggesting that Notch signaling was activated upon stimulation. Inhibiting processing of Notch receptor by ,-secretase using a ,-secretase inhibitor (GSI), the expression of Notch1 was down-regulated to basal levels. This treatment significantly modulated expression of TNF-,, IL-6, and IL-10. In addition, the amount of nitric oxide produced was significantly lower and the expression of MHC class II was up-regulated in GSI-treated cells. Treatment with GSI or silencing Notch1 resulted in decreased translocation of NF-,Bp50 into nucleus upon stimulation. Taken together, stimulation of macrophages through the TLR signaling cascade triggered activation of Notch signaling, which in turn regulated gene expression patterns involved in pro-inflammatory responses, through activation of NF-,B. [source] Nod1 and Nod2 induce CCL5/RANTES through the NF-,B pathwayEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 9 2007Catherine Werts Abstract The Nod-like receptor proteins Nod1 and Nod2 participate in innate immune responses against bacteria through intracellular detection of peptidoglycan, a component of bacterial cell wall. Recent evidence has demonstrated that Nod1 stimulates the release of chemokines that attract neutrophils at the site of infection, such as CXCL8/IL-8 in humans, and CXCL1/keratinocyte-derived chemokine and CXCL2/MIP-2 in mice. We aimed to determine whether Nod proteins could trigger the release of CCL5/RANTES, a chemokine known to attract a number of immune cells, but not neutrophils. Our results demonstrate that activation of both Nod1 and Nod2 results in substantial secretion of CCL5 by murine macrophages. Moreover, in vivo, the intraperitoneal injection of murine Nod1 or Nod2 agonists resulted in a rapid secretion of CCL5 into the bloodstream. We also observed that Nod-dependent secretion of CCL5 did not correlate with the induction of the interferon-, pathway, a major signaling cascade for the activation of CCL5 by viruses. In contrast, we identified a key role of the NF-,B pathway in Nod-dependent stimulation of the CCL5 promoter. Together, these results identify a novel target downstream of Nod1 and Nod2, which is likely to play a key role in orchestrating the global Nod-dependent immune defense during bacterial infections. [source] IL-12 and IL-18 down-regulate B cell migration in an Ly49D-dependent mannerEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 7 2007Gili Hart Abstract In order to complete their maturation and participate in the humoral immune response, immature B cells that leave the bone marrow are targeted to specific areas in the spleen, where they differentiate into mature cells. Previously, we showed that immature B cells actively down-regulate their integrin-mediated migration to LN or to sites of inflammation, enabling their targeting to the spleen. This inhibition is mediated by IFN-,, which is transcribed and secreted at low levels by these immature B cells; its expression is subsequently down-regulated following B cell maturation. The activating and inhibitory MHC class,I receptors, Ly49D and Ly49G2, regulate IFN-, secretion in B cells, preventing their migration to antigen-enriched sites and their premature encounter with an antigen, while enabling their entry into the LN when mature. In the present study, we elucidate the pathways by which the Ly49 receptors regulate IFN-, levels. We show that Ly49D stimulation triggers a signaling cascade that increases transcription of both IL-12B and IL-18; these, in turn, can interact with their specific receptors, which are expressed at elevated levels on immature B cells. Ligation of the IL-12B and IL-18 receptors induces the secretion of IFN-,, thereby regulating their cytoskeleton rearrangement and migration. [source] Selective cross-talk among natural cytotoxicity receptors in human natural killer cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2003Raffaella Augugliaro Abstract The cytolytic activity of human natural killer cells is induced by several triggering cell surface receptors upon interaction with specific cellular ligands. These receptors include NKp46, NKp30 and NKp44, collectively termed natural cytotoxicity receptors (NCR). Co-operation among NCR has been shown to occur for optimal recognition and killing of most tumor target cells. In this study, we show that the mAb-mediated engagement and clustering of one or another NCR results in the activation of an identical set of tyrosine kinases. These kinases are included in the signaling cascade leading to tyrosine phosphorylation of different receptor-associated signal transducing molecules i.e. CD3, (associated with NKp46 and NKp30) and KARAP/DAP12 (associated with NKp44). In line with the notion that the engagement of inhibitory receptors prevents NCR-mediated responses, we show that the engagement of CD94/NKG2A virtually abrogates the tyrosine phosphorylation of the NCR-associated signaling molecules, i.e. it acts at the very early steps of the signaling cascade. Importantly, the engagement of a single NCR resulted in the activation of the signaling cascades associated with the other NCR. This "cross-talk" is confined to NKp46, NKp30 and NKp44 since neither CD16-nor KIR2DS4-associated signaling polypeptides were phosphorylated following the NCR engagement. These results suggest that a functional cross-talk specifically occurs among different NCR, possibly resulting in the amplification of the activating signals. [source] Distinct contributions of different CD40 TRAF binding sites to CD154-induced dendritic cell maturation and IL-12 secretionEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 3 2003Matthew Abstract The mechanisms by which CD40 controls the maturation and antigen presentation functions of dendritic cells (DC) remains largely undefined in this critical cell type. To examine this question, we have employed retroviral transduction of primary bone marrow-derived mouse DC. Mutation of the distinct binding sites for TNF receptor-associated factor 6 (TRAF6) and for TRAF 2, 3, and 5 in the CD40 cytoplasmic domain revealed their independent contributions to DC maturation and activation of NF-,B. In contrast, disruption of the TRAF6 but not the TRAF 2,3,5 binding site markedly decreased IL-12 p40 secretion along with p38 and JNK activation in response to CD154 stimulation. These data document a clear bifurcation of the CD40 signaling cascade in primary DC at the level of thereceptor's two distinct and autonomous TRAF binding sites, and reveal the predominant role of the TRAF6 binding site in CD40-induced pro-inflammatory cytokine production by these cells. [source] T cell costimulation by the hepatitis C virus envelope protein E2 binding to CD81 is mediated by LckEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 2 2003Elisabetta Soldaini Abstract Binding of the hepatitis C virus (HCV) envelope protein E2 to CD81 provides a costimulatory signal for human T cells. This phenomenon may play a role in liver damage and autoimmune manifestations associated with HCV infection. Here we show that cross-linking of CD81 by HCV E2 induced a calcium flux in T cells that depends on Lck since it was blocked by PP1 and absent in Lck-deficient Jurkat T cells. In wild-type Jurkat cells, Lck was activated by CD81 cross-linking, and CD81, like Lck, was found in lipid rafts. Indeed, the integrity of the raft compartment was required for the induction of a calcium flux by E2, since methyl-,-cyclodextrin abolished this response. A requirement for TCR/CD3 expression was indicated by the absence of a calcium flux following E2 stimulation of TCR/CD3-deficient Jurkat cells. CD81 cross-linking increased and prolonged the anti-CD3-induced tyrosine phosphorylation of TCR, and of other proteins, indicating that the CD81-mediated signal converges with the TCR/CD3 signaling cascade at its most upstream step. In conclusion, we propose that the costimulatory effects of HCV E2 on T cells depend on CD81 cross-linking that activates Lck through raft aggregation and thus leads to enhanced TCR signaling. [source] Extracellular signal-regulated kinase activation is required for consolidation and reconsolidation of memory at an early stage of ontogenesisEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2009Solčne Languille Abstract The ability to form long-term memories exists very early during ontogeny; however, the properties of early memory processes, brain structures involved and underlying cellular mechanisms are poorly defined. Here, we examine the role of extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase/ERK signaling cascade, which is crucial for adult memory, in the consolidation and reconsolidation of an early memory using a conditioned taste aversion paradigm in 3-day-old rat pups. We show that intraperitoneal injection of SL327, the upstream mitogen-activated protein kinase kinase inhibitor, impairs both consolidation and reconsolidation of early memory, leaving short-term memory after acquisition and after reactivation intact. The amnesic effect of SL327 diminishes with increasing delays after acquisition and reactivation. Biochemical analyses revealed ERK hyperphosphorylation in the amygdala but not the hippocampus following acquisition, suggesting functional activation of the amygdala as early as post-natal day 3, although there was no clear evidence for amygdalar ERK activation after reactivation. These results indicate that, despite an immature brain, the basic properties of memory and at least some of the molecular mechanisms and brain structures implicated in aversion memory share a number of similarities with the adult and emerge very early during ontogeny. [source] Translating nociceptor sensitivity: the role of axonal protein synthesis in nociceptor physiologyEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2009Theodore J. Price Abstract The increased sensitivity of peripheral pain-sensing neurons, or nociceptors, is a major cause of the sensation of pain that follows injury. This plasticity is thought to contribute to the maintenance of chronic pain states. Although we have a broad knowledge of the factors that stimulate changes in nociceptor sensitivity, the cellular mechanisms that underlie this plasticity are still poorly understood; however, they are likely to involve changes in gene expression required for the phenotypic and functional changes seen in nociceptive neurons after injury. While the regulation of gene expression at the transcriptional level has been studied extensively, the regulation of protein synthesis, which is also a tightly controlled process, has only recently received more attention. Despite the established role of protein synthesis in the plasticity of neuronal cell bodies and dendrites, little attention has been paid to the role of translation control in mature undamaged axons. In this regard, several recent studies have demonstrated that the control of protein synthesis within the axonal compartment is crucial for the normal function and regulation of sensitivity of nociceptors. Pathways and proteins regulating this process, such as the mammalian target of rapamycin signaling cascade and the fragile X mental retardation protein, have recently been identified. We review here recent evidence for the regulation of protein synthesis within a nociceptor's axonal compartment and its contribution to this neuron's plasticity. We believe that an increased understanding of this process will lead to the identification of novel targets for the treatment of chronic pain. [source] Basolateral amygdala inactivation by muscimol, but not ERK/MAPK inhibition, impairs the use of reward expectancies during working memoryEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2007Lisa M. Savage Abstract Rats were trained on a delayed matching to position (DMTP) task that embedded either a differential outcomes procedure (DOP) or a non-differential outcomes procedure (NOP). The DOP, via Pavlovian conditioning (stimulus,outcome associations), results in the use of unique reward expectancies that facilitate learning and memory performance above subjects trained with a NOP that requires subjects to retain cue information for accurate choice behavior (stimulus,response associations). This enhancement in learning and/or memory produced by the DOP is called the differential outcomes effect (DOE). After being trained on the DMTP task, rats were implanted with two cannulae aimed at the basolateral amygdala (BLA) nuclei. Rats trained with the DOP, relative to those trained with the NOP, displayed enhanced short-term memory (STM) performance under vehicle conditions (i.e. the DOE). However, injections of the ,-aminobutyric acid (GABA)A agonist muscimol into the BLA dose-dependently (0.0625 and 0.125 µg) impaired STM performance only in DOP-trained rats. These results support the role of the BLA in the use of established reward expectancies during a short-term working memory task. Despite the fact that extracellular signal-regulated kinase/mitogen-activated protein kinases (ERK/MAPK) have been shown to be necessary for amygdala-dependent long-term potentiation and some forms of long-term and STM, inhibition of the ERK/MAPK signaling cascade by U0126 (2.0 or 4.0 µg) in the BLA was not critical for updating the STM of either spatial information or reward expectation. [source] CXCL10-induced cell death in neurons: role of calcium dysregulationEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2006Yongjun Sui Abstract Chemokines play a key role in the regulation of central nervous system disease. CXCL10 over-expression has been observed in several neurodegenerative diseases, including multiple sclerosis, Alzheimer's disease and HIV-associated dementia. More recent studies by others and us have shown that CXCL10 elicits apoptosis in fetal neurons. The mechanism of CXCL10-mediated neurotoxicity, however, remains unclear. In this study, we provide evidence for the direct role of Ca2+ dysregulation in CXCL10-mediated apoptosis. We demonstrate that treatment of fetal neuronal cultures with exogenous CXCL10 produced elevations in intracellular Ca2+ and that this effect was modulated via the binding of CXCL10 to its cognate receptor, CXCR3. We further explored the association of intracellular Ca2+ elevations with the caspases that are involved in CXC10-induced neuronal apoptosis. Our data showed that increased Ca2+, which is available for uptake by the mitochondria, is associated with membrane permeabilization and cytochrome c release from this compartment. The released cytochrome c then activates the initiator active caspase-9. This initiator caspase sequentially activates the effector caspase-3, ultimately leading to apoptosis. This study identifies the temporal signaling cascade involved in CXCL10-mediated neuronal apoptosis and provides putative targets for pharmaceutical intervention of neurological disorders associated with CXCL10 up-regulation. [source] Coupling of endothelin receptors to the ERK/MAP kinase pathway,FEBS JOURNAL, Issue 20 2001Roles of palmitoylation Endothelins are potent mitogens that stimulate extracellular signal-regulated kinases (ERK/MAP kinases) through their cognate G-protein-coupled receptors, ETA and ETB. To address the role of post-translational ET receptor modifications such as acylation on ERK activation and to identify relevant downstream effectors coupling the ET receptor to the ERK signaling cascades we have constructed a panel of palmitoylation-deficient ET receptor mutants with differential G, protein binding capacity. Endothelin-1 stimulation of wild-type ETA or ETB induced a fivefold to sixfold increase in ERK in COS-7 and CHO cells whereas full-length nonpalmitoylated ETA and ETB mutants failed to stimulate ERK. A truncated ETB lacking the C-terminal tail domain including putative phosphorylation and arrestin binding site(s) but retaining the critical palmitoylation site(s) was still able to fully stimulate ERK activation. Using mutated ET receptors with selective G-protein-coupling we found that endothelin-induced stimulation of G,q, but not of G,i or G,s, is essential for endothelin-mediated ERK activation. Inhibition of protein kinases A and C or epidermal growth factor receptor kinase failed to prevent ETA - and ETB -mediated ERK activation whereas blockage of phospholipase C-, completely abrogated endothelin-promoted ERK activation through ETA and ETB in recombinant COS-7 and native C6 cells. Complex formation of Ca2+ or inhibition of Src family tyrosine kinases prevented ET-1-induced ERK-2 activation in C6-cells. Our results indicate that endothelin-promoted ERK/MAPK activation criticially depends on palmitoylation but not on phosphorylation of ET receptors, and that the G,q/phospholipase C-,/Ca2+/Src signaling cascade is necessary for efficient coupling of ET receptors to the ERK/MAPK pathway. [source] Three-dimensional structure of the histidine-containing phosphocarrier protein (HPr) from Enterococcus faecalis in solutionFEBS JOURNAL, Issue 3 2001Till Maurer The histidine-containing phosphocarrier protein (HPr) transfers a phosphate group between components of the prokaryotic phosphoenolpyruvate-dependent phosphotransferase system (PTS), which is finally used to phosphorylate the carbohydrate transported by the PTS through the cell membrane. Recently it has also been found to act as an intermediate in the signaling cascade that regulates transcription of genes related to the carbohydrate-response system. Both functions involve phosphorylation/dephosphorylation reactions, but at different sites. Using multidimensional 1H-NMR spectroscopy and angular space simulated annealing calculations, we determined the structure of HPr from Enterococcus faecalis in aqueous solution using 1469 distance and 44 angle constraints derived from homonuclear NMR data. It has a similar overall fold to that found in HPrs from other organisms. Four , strands, A, B, C, D, encompassing residues 2,7, 32,37, 40,42 and 60,66, form an antiparallel , sheet lying opposite the two antiparallel , helices, a and c (residues 16,26 and 70,83). A short , helix, b, from residues 47,53 is also observed. The pairwise root mean square displacement for the backbone heavy atoms of the mean of the 16 NMR structures to the crystal structure is 0.164 nm. In contrast with the crystalline state, in which a torsion angle strain in the active-center loop has been described [Jia, Z., Vandonselaar, M., Quail, J.W. & Delbaere, L.T.J. (1993) Nature (London) 361, 94,97], in the solution structure, the active-site His15 rests on top of helix a, and the phosphorylation site N,1 of the histidine ring is oriented towards the surface, making it easily accessible to the solvent. Back calculation of the 2D NOESY NMR spectra from both the NMR and X-ray structures shows that the active-center structure derived by X-ray crystallography is not compatible with experimental data recorded in solution. The observed torsional strain must either be a crystallization artefact or represents a conformational state that exists only to a small extent in solution. [source] Specific transcriptional responses induced by 8-methoxypsoralen and UVA in yeastFEMS YEAST RESEARCH, Issue 6 2007Michčle Dardalhon Abstract Treatment of eukaryotic cells with 8-methoxypsoralen plus UVA irradiation (8-MOP/UVA) induces pyrimidine monoadducts and interstrand crosslinks and initiates a cascade of events leading to cytotoxic, mutagenic and carcinogenic responses. Transcriptional activation plays an important part in these responses. Our previous study in Saccharomyces cerevisiae showed that the repair of these lesions involves the transient formation of DNA double-strand breaks and the enhanced expression of landmark DNA damage response genes such as RAD51, RNR2 and DUN1, as well as the Mec1/Rad53 kinase signaling cascade. We have now used DNA microarrays to examine genome-wide transcriptional changes produced after induction of 8-MOP/UVA photolesions. We found that 128 genes were strongly induced and 29 genes strongly repressed. Modifications in gene expression concern numerous biological processes. Compared to other genotoxic treatments, c. 42% of the response genes were specific to 8-MOP/UVA treatment. In addition to common DNA damage response genes and genes induced by environmental stresses, a large fraction of 8-MOP/UVA response genes correspond to membrane-related functions. [source] Increase of MCP-1 (CCL2) in myelin mutant Schwann cells is mediated by MEK-ERK signaling pathwayGLIA, Issue 8 2008Stefan Fischer Abstract Macrophages are critically involved in the pathogenesis of genetically caused demyelination, as it occurs in inherited demyelinating neuropathies. On the basis of the observation that upregulation of the Schwann cell-derived chemokine MCP-1 (CCL2) is a pathologically relevant mechanism for macrophage activation in mice heterozygously deficient for the myelin component P0 (P0+/,), we posed the question of the intracellular signaling cascade involved. By using western blot analysis of peripheral nerve lysates the MAP-kinases extracellular signal-regulated kinase 1/2 (ERK1/2) and MAP kinase/ERK kinase 1/2 (MEK1/2) showed an early and constantly increasing activation in P0 mutants. Furthermore, in nerve fibers from the P0+/, mutants, Schwann cell nuclei were much more often positive for phosphorylated ERK1/2 than in nerve fibers from wild type mice. In vitro experiments using the MEK1/2-inhibitor CI-1040 decreased ERK1/2-phosphorylation and MCP-1 expression in a Schwann cell-derived cell line. Finally, systemic application of CI-1040 lead to a decreased ERK1/2-phosphorylation and substantially reduced MCP-1-production in peripheral nerves of P0+/, mutant mice. Our study identifies MEK1/2-ERK1/2 signaling as an important intracellular pathway that connects the Schwann cell mutation with the activation of pathogenetically relevant macrophages in the peripheral nerves. These findings may have important implications for the treatment of inherited peripheral neuropathies in humans. © 2008 Wiley-Liss, Inc. [source] Transforming growth factor-activated kinase 1 induced in spinal astrocytes contributes to mechanical hypersensitivity after nerve injuryGLIA, Issue 7 2008Hirokazu Katsura Abstract Mitogen-activated protein kinase (MAPK) plays an important role in the induction and maintenance of neuropathic pain. Transforming growth factor-activated kinase 1 (TAK1), a member of the MAPK kinase kinase family, is indispensable for the activation of c-Jun N-terminal kinase (JNK) and p38 MAPK. We now show that TAK1 induced in spinal cord astrocytes is crucial for mechanical hypersensitivity after peripheral nerve injury. Nerve injury induced a striking increase in the expression of TAK1 in the ipsilateral dorsal horn, and TAK1 was increased in hyperactive astrocytes, but not in neurons or microglia. Intrathecal administration of TAK1 antisense oligodeoxynucleotide (AS-ODN) prevented and reversed nerve injury-induced mechanical, but not heat hypersensitivity. Furthermore, TAK1 AS-ODN suppressed the activation of JNK1, but not p38 MAPK, in spinal astrocytes. In contrast, there was no change in TAK1 expression in primary sensory neurons, and TAK1 AS-ODN did not attenuate the induction of transient receptor potential ion channel TRPV1 in sensory neurons. Taken together, these results demonstrate that TAK1 upregulation in spinal astrocytes has a substantial role in the development and maintenance of mechanical hypersensitivity through the JNK1 pathway. Thus, preventing the TAK1/JNK1 signaling cascade in astrocytes might provide a fruitful strategy for treating intractable neuropathic pain. © 2008 Wiley-Liss, Inc. [source] IFN-,-induced BACE1 expression is mediated by activation of JAK2 and ERK1/2 signaling pathways and direct binding of STAT1 to BACE1 promoter in astrocytesGLIA, Issue 3 2007Hyun Jin Cho Abstract ,-Site APP cleaving enzyme 1 (BACE1) is an essential enzyme for the production of , amyloid. Since we found that injection of interferon-, (IFN-,) into young mouse brains increased BACE1 expression in astrocytes, we investigated molecular mechanisms underlying this process by cloning a putative BACE1 promoter. BACE1 promoter activity was differentially regulated by IFN-, in a region specific manner and down-regulated by an inhibitor of Janus kinase 2 (JAK2). A dominant negative mutant of signal transducer and activator of transcription 1 (STAT1) expression suppressed BACE1 promoter activity, and this was rescued by transfecting wild type STAT1. Electrophoretic mobility shift assay and promoter activity assays indicated that STAT1 binds directly to the putative STAT1 binding sequence of BACE1 promoter. Because IFN-, treatment induced STAT1 phosphorylation, we examined whether the expression of a suppressor of cytokine signaling (SOCS), negative regulator of JAK2, suppresses BACE1 promoter activity. The results show that SOCS1 or SOCS3 expression suppressed BACE1 promoter by blocking phosphorylation of Tyr701 residue in STAT1. Also, because IFN-, treatment specifically potentiated extracellular signal regulated MAP kinase (ERK) 1/2 activation, pretreatment of mitogen-activated or extracellular signal-regulated protein kinase (MEK) inhibitor, PD98059, significantly attenuated IFN-,-induced BACE1 promoter activity and protein expression through blocking phosphorylation of Ser727 residue in STAT1, suggesting that ERK1/2 is associated with IFN-,-induced STAT1 signaling cascade. Taken together, our results suggest that IFN-, activates JAK2 and ERK1/2 and then phosphorylated STAT1 binds to the putative STAT1 binding sequences in BACE1 promoter region to modulate BACE1 protein expression in astrocytes. © 2006 Wiley-Liss, Inc. [source] Activation of the Raf-1/MEK/ERK cascade by bile acids occurs via the epidermal growth factor receptor in primary rat hepatocytesHEPATOLOGY, Issue 2 2002Yi-Ping Rao Bile acids have been reported to activate several different cell signaling cascades in rat hepatocytes. However, the mechanism(s) of activation of these pathways have not been determined. This study aims to determine which bile acids activate the Raf-1/MEK/ERK cascade and the mechanism of activation of this pathway. Taurodeoxycholic acid (TDCA) stimulated (+235%) the phosphorylation of p74 Raf-1 in a time (5 to 20 minutes) and concentration-dependent (10 to 100 ,mol/L) manner. Raf-1 and ERK activities were both significantly increased by most bile acids tested. Deoxycholic acid (DCA) was the best activator of ERK (3.6-fold). A dominant negative Ras (N17) construct expressed in primary hepatocytes prevented the activation of ERK by DCA. The epidermal growth factor receptor (EGFR)-specific inhibitor (AG1478) significantly inhibited (,81%) the activation of ERK by DCA. DCA rapidly (30 to 60 seconds) increased phosphorylation of the EGFR (,2-fold) and Shc (,4-fold). A dominant negative mutant of the EGFR (CD533) blocked the ability of DCA to activate ERK. In conclusion, these results show that DCA activates the Raf-1/MEK/ERK signaling cascade in primary hepatocytes primarily via an EGFR/Ras-dependent mechanism. [source] Effect of natural commensal-origin DNA on toll-like receptor 9 (TLR9) signaling cascade, chemokine IL-8 expression, and barrier integritiy of polarized intestinal epithelial cellsINFLAMMATORY BOWEL DISEASES, Issue 3 2010Darab Ghadimi Abstract Background and Aim: The intestinal epithelium is constantly exposed to high levels of genetic material like bacterial DNA. Under normal physiological conditions, the intestinal epithelial monolayer as a formidable dynamic barrier with a high-polarity structure facilitates only a controlled and selective flux on components between the lumen and the underlining mucosa and even is able to facilitate structure-based macromolecules movement. The aim of this study was to test the effect of natural commensal-origin DNA on the TLR9 signaling cascade and the barrier integrity of polarized intestinal epithelial cells (IECs). Methods: Polarized HT-29 and T84 cells were treated with TNF-, in the presence or absence of DNA from Lactobacillus rhamnosus GG (LGG) and Bifidobacterium longum. TLR9 and interleukin-8 (IL-8) mRNA expression was assessed by semiquantitative and TaqMan real-time reverse-transcription polymerase chain reaction. Expression of TLR9 protein, degradation of inhibitor of kappa B alpha (I,B,), and p38 mitogen-activated protein kinase (p38 MAP) phosphorylation were assessed by Western blotting. To further reveal the role of TLR9 signaling, the TLR9 gene was silenced by siRNA. IL-8 secretion was measured by an enzyme-linked immunosorbent assay. Nuclear factor-kappa B (NF-,B) activity was assessed by the electrophoretic mobility shift assay (EMSA) and NF-,B-dependent luciferase reporter gene assays. As an indicator of tight junction formation and monolayer integrity of epithelial cell monolayers, transepithelial electrical resistance (TER) was repetitively monitored. Transmonolayer movement of natural commensal-origin DNA across monolayers was monitored using qRT-PCR and nested PCR based on bacterial 16S rRNA genes. Results: In response to apically applied natural commensal-origin DNA, polarized HT-29 and T84 cells enhanced expression of TLR9 in a specific manner, which was subsequently associated with attenuation of TNF-,-induced NF-,B activation and NF-,B-mediated IL-8 expression. TLR9 silencing abolished this inhibitory effect. Apically applied LGG DNA attenuated TNF-,-enhanced NF-,B activity by reducing I,B, degradation and p38 phosphorylation. LGG DNA did not decrease the TER but rather diminished the TNF-,-induced TER reduction. Translocation of natural commensal-origin DNA into basolateral compartments did not occur under tested conditions. Conclusions: Our study indicates that TLR9 signaling mediates, at least in part, the anti-inflammatory effects of natural commensal-origin DNA on the gut because TLR9 silencing abolished the inhibitory effect of natural commensal-origin DNA on TNF-,-induced IL-8 secretion in polarized IECs. The nature of the TLR9 agonist, the polarity of cells, and the tight junction integrity of IECs has to be taken into account in order to predict the outcome of TLR9 signaling. (Inflamm Bowel Dis 2010) [source] Focal Adhesion Kinase pp125FAK Interacts With the Large Conductance Calcium-Activated hSlo Potassium Channel in Human Osteoblasts: Potential Role in Mechanotransduction,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2003Roger Rezzonico Abstract Molecular events of mechanotransduction in osteoblasts are poorly defined. We show that the mechanosensitive BK channels open and recruit the focal adhesion kinase FAK in osteoblasts on hypotonic shock. This could convert mechanical signals in biochemical events, leading to osteoblast activation. Introduction: Mechanical strains applied to the skeleton influence bone remodeling and architecture mainly through the osteoblast lineage. The molecular mechanisms involved in osteoblastic mechanotransduction include opening of mechanosensitive cation channels and the activation of protein tyrosine kinases, notably FAK, but their interplay remains poorly characterized. The large conductance K+ channel (BK) seems likely as a bone mechanoreceptor candidate because of its high expression in osteoblasts and its ability to open in response to membrane stretch or hypotonic shock. Propagation of the signals issued from the mechanosensitivity of BK channels inside the cell likely implies complex interactions with molecular partners involved in mechanotransduction, notably FAK. Methods: Interaction of FAK with the C terminus of the hSlo ,-subunit of BK was investigated using the yeast two-hybrid system as well as immunofluorescence microscopy and coimmunoprecipitation experiments with a rabbit anti-hslo antibody on MG63 and CAL72 human osteosarcoma cell lines and on normal human osteoblasts. Mapping of the FAK region interacting with hSlo was approached by testing the ability of hSlo to recruit mutated ot truncated FAK proteins. Results: To the best of our knowledge, we provide the first evidence of the physical association of FAK with the intracellular part of hslo. We show that FAK/hSlo interaction likely takes place through the Pro-1-rich domain situated in the C-terminal region of the kinase. FAK/hSlo association occurs constitutively at a low, but appreciable, level in human osteosarcoma cells and normal human osteoblasts that express endogenous FAK and hSlo. In addition, we found that application of an hypo-osmotic shock to these cells induced a sustained activation of BK channels associated to a marked increase in the recruitment of FAK on hSlo. Conclusions: Based on these data, we propose that BK channels might play a triggering role in the signaling cascade induced by mechanical strains in osteoblasts. [source] Silibinin attenuates cardiac hypertrophy and fibrosis through blocking EGFR-dependent signaling,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2010Wen Ai Abstract Cardiac hypertrophy is a major determinant of heart failure. The epidermal growth factor receptor (EGFR) plays an important role in cardiac hypertrophy. Since silibinin suppresses EGFR in vitro and in vivo, we hypothesized that silibinin would attenuate cardiac hypertrophy through disrupting EGFR signaling. In this study, we examined this hypothesis using neonatal cardiac myocytes and fibroblasts induced by angiotensin II (Ang II) and animal model by aortic banding (AB) mice. Our data revealed that silibinin obviously blocked cardiac hypertrophic responses induced by pressure overload. Meanwhile, silibinin markedly reduced the increased generation of EGFR. Moreover, these beneficial effects were associated with attenuation of the EGFR-dependent ERK1/2, PI3K/Akt signaling cascade. We further demonstrated silibinin decreased inflammation and fibrosis by blocking the activation of NF-,B and TGF-,1/Smad signaling pathways in vitro and in vivo. Our results indicate that silibinin has the potential to protect against cardiac hypertrophy, inflammation, and fibrosis through blocking EGFR activity and EGFR-dependent different intracellular signaling pathways. J. Cell. Biochem. 110: 1111,1122, 2010. Published 2010 Wiley-Liss, Inc. [source] FGFR1/PI3K/AKT signaling pathway is a novel target for antiangiogenic effects of the cancer drug Fumagillin (TNP-470)JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2007Gregory J. Chen Abstract Fibroblast growth factor-1 (FGF1), a prototypic member of the FGF family, is a potent angiogenic factor. Although FGF-stimulated angiogenesis has been extensively studied, the molecular mechanisms regulating FGF1-induced angiogenesis are poorly understood in vivo. Fumagillin, an antiangiogenic fungal metabolite, has the ability to inhibit FGF-stimulated angiogenesis in the chicken chorioallantoic membrane (CAM). In the current study, chicken CAMs were transfected with a signal peptide-containing version of the FGF1 gene construct (sp-FGF1). Transfected CAMs were then analyzed in the presence and absence of fumagillin treatment with respect to the mRNA expression levels and protein activity of the FGF1 receptor protein (FGFR1), phosphatidylinositol 3-kinase (PI3K), and its immediate downstream target, AKT-1 (protein kinase B). Treatment of sp-FGF1-transfected CAMs with fumagillin showed downregulation for both PI3K and AKT-1 proteins in mRNA expression and protein activity. In contrast, no major alterations in FGFR1 mRNA expression level were observed. Similar patterns of mRNA expression for the above three proteins were observed when the CAMs were treated with recombinant FGF1 protein in place of sp-FGF1 gene transfection. Investigation using biotin-labeled fumagillin showed that only the FGF1 receptor protein containing the cytoplasmic domain demonstrated binding to fumagillin. Furthermore, we demonstrated endothelial-specificity of the proposed antiangiogenic signaling cascade using an in vitro system. Based on these findings, we conclude that the binding of fumagillin to the cytoplasmic domain of the FGF1 receptor inhibited FGF1-stimulated angiogenesis both in vitro and in vivo. J. Cell. Biochem. 101: 1492,1504, 2007. © 2007 Wiley-Liss, Inc. [source] TGF-, control of cell proliferationJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2005Shuan S. Huang Abstract This article focuses on recent findings that the type V TGF-, receptor (T,R-V), which co-expresses with other TGF-, receptors (T,R-I, T,R-II, and T,R-III) in all normal cell types studied, is involved in growth inhibition by IGFBP-3 and TGF-, and that TGF-, activity is regulated by two distinct endocytic pathways (clathrin- and caveolar/lipid-raft-mediated). TGF-, is a potent growth inhibitor for most cell types, including epithelial and endothelial cells. The signaling by which TGF-, controls cell proliferation is not well understood. Many lines of evidence indicate that other signaling pathways, in addition to the prominent T,R-I/T,R-II/Smad2/3/4 signaling cascade, are required for mediating TGF-,-induced growth inhibition. Recent studies revealed that T,R-V, which is identical to LRP-1, mediates IGF-independent growth inhibition by IGFBP-3 and mediates TGF-,-induced growth inhibition in concert with T,R-I and T,R-II. In addition, IRS proteins and a Ser/Thr-specific protein phosphatase(s) are involved in the T,R-V-mediated growth inhibitory signaling cascade. The T,R-V signaling cascade appears to cross-talk with the T,R-I/T,R-II, insulin receptor (IR), IGF-I receptor (IGF-IR), integrin and c-Met signaling cascades. Attenuation or loss of the T,R-V signaling cascade may enable carcinoma cells to escape from TGF-, growth control and may contribute to the aggressiveness and invasiveness of these cells via promoting epithelial-to-mesenchymal transdifferentiation (EMT). Finally, the ratio of TGF-, binding to T,R-II and T,R-I is a signal controlling TGF-, partitioning between two distinct endocytosis pathways and resultant TGF-, responsiveness. These recent studies have provided new insights into the molecular mechanisms underlying TGF-,-induced cellular growth inhibition, cross-talk between the T,R-V and other signaling cascades, the signal that controls TGF-, responsiveness and the role of T,R-V in tumorigenesis. © 2005 Wiley-Liss, Inc. [source] Phosphorylated osteopontin promotes migration of human choriocarcinoma cells via a p70 S6 kinase-dependent pathwayJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2005Rania Al-Shami Abstract This study examined the role of osteopontin (OPN), a phosphorylated secreted glycoprotein, in the promotion of trophoblastic cell migration, an early event in the embryo implantation process. Three human choriocarcinoma cell lines, namely JAR, BeWo, and JEG-3, were treated with variants of OPN differing in the extent of phosphorylation following sequential dephosphorylation with tartrate-resistant acid phosphatase (TRAP), and their migratory response was measured. The highly phosphorylated human milk form of OPN (OPN-1) strongly triggered migration in all three cell lines, whereas the less phosphorylated variants, OPN-2a and OPN-2b, failed to stimulate migration. JAR cell migration in response to OPN-1 was accompanied by a rapid rearrangement of actin filaments to the cellular membrane. Using broad spectrum protein kinase profiling, we identified p70 S6 kinase as a major signal transduction pathway activated by OPN-1 during the migratory response in JAR cells. Activation was blocked completely by rapamycin and LY294002, thus demonstrating that OPN-1-stimulated migration occurs through mTOR and PI3K pathways, respectively. Conversely, PD98059 did not affect the activation of p70 S6 kinase by OPN-1, therefore, this response does not involve the Ras/ MAPK signaling cascade. Together, these data show that the highly phosphorylated human OPN-1 can stimulate trophoblastic cell migration and provides evidence for the involvement of the PI3K/mTOR/p70 S6 kinase pathway in the JAR cells response. Because both OPN and TRAP are expressed in the uterus during early pregnancy, it is conceivable that extracellular phosphatases such as TRAP may modify OPN charge state and thus modulate cell migration. © 2005 Wiley-Liss, Inc. [source] Basic region of residues 228,231 of protein kinase CK1, is involved in its interaction with axin: Binding to axin does not affect the kinase activity,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2005Pablo Sobrado Abstract Protein kinase CK1, also known as casein kinase 1, participates in the phosphorylation of ,-catenin, which regulates the functioning of the Wnt signaling cascade involved in embryogenesis and carcinogenesis. ,-catenin phosphorylation occurs in a multiprotein complex assembled on the scaffold protein axin. The interaction of CK1, from Danio rerio with mouse-axin has been studied using a pull-down assay that uses fragments of axin fused to glutathione S transferase, which is bound to glutathione sepharose beads. The results indicate that the three lysines present in the basic region of residues 228,231 of CK1, are necessary for the binding of CK1 to axin. Lysine 231 is particularly important in this interaction. In order to define the relevance of the axin-CK1, interaction, the effect of the presence of axin on the phosphorylating activity of CK1, was tested. It is also evident that the region of axin downstream of residues 503,562 is required for CK1, interaction. The binding of CK1, to axin fragment 292,681 does not facilitate the phosphorylation of ,-catenin despite the fact that this axin fragment can also bind ,-catenin. Binding of CK1, to axin is not required for the phosphorylation of axin itself and, likewise, axin does not affect the kinetic parameters of the CK1, towards casein or a specific peptide substrate. © 2004 Wiley-Liss, Inc. [source] JNK phosphorylates the HSF1 transcriptional activation domain: Role of JNK in the regulation of the heat shock responseJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2001Jeonghyeon Park Abstract The role of c-Jun NH2 -terminal kinase (JNK) signaling cascade in the stress-inducible phosphorylation of heat shock factor 1 (HSF1) was investigated using known agonists and antagonists of JNK. We showed that treatment of HeLa cells with MG132, a proteasome inhibitor and known JNK activator, caused the transcriptional activation domain of HSF1 to be targeted and phosphorylated by JNK2 in vivo. Dose-response and time course studies of the effects of heat shock and anisomycin treatment showed a close correlation of the activation of JNK and hyperphosphorylation of HSF1. SB203580 inhibited JNK at the 100 ,M concentration and significantly reduced the amount of hyperphosphorylated HSF1 upon heat shock or anisomycin treatment. SB203580 and dominant-negative JNK suppress hsp70 promoter-driven reporter gene expression selectively at 45°C but not at 42°C heat stress, suggesting that JNK would be preferentially associated with the protective heat shock response against severe heat stress. The possibility that JNK-mediated phosphorylation of HSF1 may selectively stabilize the HSF1 protein and confers protection to cells under conditions of severe stress is discussed. J. Cell. Biochem. 82: 326,338, 2001. © 2001 Wiley-Liss, Inc. [source] | ||||||||||||||||||||||||||||||||||||||||