Kibra functions as a tumor suppressor protein that regulates Hippo signaling in conjunction with Merlin and Expanded

PubMed Central

Yu, Jianzhong; Zheng, Yonggang; Dong, Jixin; Klusza, Stephen; Deng, Wu-Min; Pan, Duojia

2010-01-01

Summary The Hippo signaling pathway regulates organ size and tissue homeostasis from Drosophila to mammals. Central to this pathway is a kinase cascade wherein Hippo (Hpo), in complex with Salvador (Sav), phosphorylates and activates Warts (Wts), which in turn phosphorylates and inactivates the Yorkie (Yki) oncoprotein, known as the YAP coactivator in mammalian cells. The FERM domain proteins Merlin (Mer) and Expanded (Ex) are upstream components that regulate Hpo activity through unknown mechanisms. Here we identify Kibra (Kbr) as another upstream component of the Hippo signaling pathway. We show that Kbr functions together with Mer and Ex in a protein complex localized to the apical domain of epithelial cells, and that this protein complex regulates the Hippo kinase cascade via direct binding to Hpo and Sav. These results shed light on the mechanism of Ex and Mer function, and implicate Kbr as a potential tumor suppressor with relevance to neurofibromatosis. PMID:20159598

Kinetic insulation as an effective mechanism for achieving pathway specificity in intracellular signaling networks

PubMed Central

Behar, Marcelo; Dohlman, Henrik G.; Elston, Timothy C.

2007-01-01

Intracellular signaling pathways that share common components often elicit distinct physiological responses. In most cases, the biochemical mechanisms responsible for this signal specificity remain poorly understood. Protein scaffolds and cross-inhibition have been proposed as strategies to prevent unwanted cross-talk. Here, we report a mechanism for signal specificity termed “kinetic insulation.” In this approach signals are selectively transmitted through the appropriate pathway based on their temporal profile. In particular, we demonstrate how pathway architectures downstream of a common component can be designed to efficiently separate transient signals from signals that increase slowly over time. Furthermore, we demonstrate that upstream signaling proteins can generate the appropriate input to the common pathway component regardless of the temporal profile of the external stimulus. Our results suggest that multilevel signaling cascades may have evolved to modulate the temporal profile of pathway activity so that stimulus information can be efficiently encoded and transmitted while ensuring signal specificity. PMID:17913886

Cellular Organization and Cytoskeletal Regulation of the Hippo Signaling Network.

PubMed

Sun, Shuguo; Irvine, Kenneth D

2016-09-01

The Hippo signaling network integrates diverse upstream signals to control cell fate decisions and regulate organ growth. Recent studies have provided new insights into the cellular organization of Hippo signaling, its relationship to cell-cell junctions, and how the cytoskeleton modulates Hippo signaling. Cell-cell junctions serve as platforms for Hippo signaling by localizing scaffolding proteins that interact with core components of the pathway. Interactions of Hippo pathway components with cell-cell junctions and the cytoskeleton also suggest potential mechanisms for the regulation of the pathway by cell contact and cell polarity. As our understanding of the complexity of Hippo signaling increases, a future challenge will be to understand how the diverse inputs into the pathway are integrated and to define their respective contributions in vivo. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hippo signaling: growth control and beyond

PubMed Central

Halder, Georg; Johnson, Randy L.

2011-01-01

The Hippo pathway has emerged as a conserved signaling pathway that is essential for the proper regulation of organ growth in Drosophila and vertebrates. Although the mechanisms of signal transduction of the core kinases Hippo/Mst and Warts/Lats are relatively well understood, less is known about the upstream inputs of the pathway and about the downstream cellular and developmental outputs. Here, we review recently discovered mechanisms that contribute to the dynamic regulation of Hippo signaling during Drosophila and vertebrate development. We also discuss the expanding diversity of Hippo signaling functions during development, discoveries that shed light on a complex regulatory system and provide exciting new insights into the elusive mechanisms that regulate organ growth and regeneration. PMID:21138973

Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Cascade Inhibitors: How Mutations Can Result in Therapy Resistance and How to Overcome Resistance

PubMed Central

McCubrey, James A.; Steelman, Linda S.; Chappell, William H.; Abrams, Stephen L.; Franklin, Richard A.; Montalto, Giuseppe; Cervello, Melchiorre; Libra, Massimo; Candido, Saverio; Malaponte, Grazia; Mazzarino, Maria C.; Fagone, Paolo; Nicoletti, Ferdinando; Bäsecke, Jörg; Mijatovic, Sanja; Maksimovic-Ivanic, Danijela; Milella, Michele; Tafuri, Agostino; Chiarini, Francesca; Evangelisti, Camilla; Cocco, Lucio; Martelli, Alberto M.

2012-01-01

The Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades are often activated by genetic alterations in upstream signaling molecules such as receptor tyrosine kinases (RTK). Targeting these pathways is often complex and can result in pathway activation depending on the presence of upstream mutations (e.g., Raf inhibitors induce Raf activation in cells with wild type (WT) RAF in the presence of mutant, activated RAS) and rapamycin can induce Akt activation. Targeting with inhibitors directed at two constituents of the same pathway or two different signaling pathways may be a more effective approach. This review will first evaluate potential uses of Raf, MEK, PI3K, Akt and mTOR inhibitors that have been investigated in pre-clinical and clinical investigations and then discuss how cancers can become insensitive to various inhibitors and potential strategies to overcome this resistance. PMID:23085539

Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascade inhibitors: how mutations can result in therapy resistance and how to overcome resistance.

PubMed

McCubrey, James A; Steelman, Linda S; Chappell, William H; Abrams, Stephen L; Franklin, Richard A; Montalto, Giuseppe; Cervello, Melchiorre; Libra, Massimo; Candido, Saverio; Malaponte, Grazia; Mazzarino, Maria C; Fagone, Paolo; Nicoletti, Ferdinando; Bäsecke, Jörg; Mijatovic, Sanja; Maksimovic-Ivanic, Danijela; Milella, Michele; Tafuri, Agostino; Chiarini, Francesca; Evangelisti, Camilla; Cocco, Lucio; Martelli, Alberto M

2012-10-01

The Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades are often activated by genetic alterations in upstream signaling molecules such as receptor tyrosine kinases (RTK). Targeting these pathways is often complex and can result in pathway activation depending on the presence of upstream mutations (e.g., Raf inhibitors induce Raf activation in cells with wild type (WT) RAF in the presence of mutant, activated RAS) and rapamycin can induce Akt activation. Targeting with inhibitors directed at two constituents of the same pathway or two different signaling pathways may be a more effective approach. This review will first evaluate potential uses of Raf, MEK, PI3K, Akt and mTOR inhibitors that have been investigated in pre-clinical and clinical investigations and then discuss how cancers can become insensitive to various inhibitors and potential strategies to overcome this resistance.

Control of Wnt Receptor Turnover by R-spondin-ZNRF3/RNF43 Signaling Module and Its Dysregulation in Cancer.

PubMed

Hao, Huai-Xiang; Jiang, Xiaomo; Cong, Feng

2016-06-08

Aberrant activation of the Wnt/β-catenin pathway is frequently found in various cancers, often through mutations of downstream components. Inhibiting β-catenin signaling in tumors with downstream pathway mutations remains challenging, due to a lack of favorable targets. On the other hand, targeting upstream components of the Wnt pathway is rather straightforward. However, it is difficult to identify tumors addicted to autocrine or paracrine Wnt signaling. Discovery of the R-spondin-ZNRF3/RNF43 signaling module and its genetic alterations in cancers represents a breakthrough in this area. Membrane E3 ligase ZNRF3 and RNF43 are critical negative feedback regulators of the Wnt pathway, which function through promoting ubiquitination and degradation of Wnt receptors. R-spondin proteins (RSPO1-4) serve as natural antagonists of ZNRF3/RNF43. To maintain strong and sustained Wnt/β-catenin signaling, cancers need to overcome ZNRF3/RNF43-mediated feedback inhibition. Indeed, mutations of RNF43/ZNRF3 and recurrent translocations of RSPO2/RSPO3 have recently been identified in various cancers. Significantly, genetic alterations in RNF43/ZNRF3/RSPO2/RSPO3 have shown promise as predictive biomarkers in pre-clinical models for the efficacy of upstream Wnt inhibitors. In this review, we will discuss the biology of the R-spondin-ZNRF3/RNF43 signaling module, cancer-associated alterations of this signaling module, and their value as biomarkers to identify Wnt-addicted tumors.

Control of Wnt Receptor Turnover by R-spondin-ZNRF3/RNF43 Signaling Module and Its Dysregulation in Cancer

PubMed Central

Hao, Huai-Xiang; Jiang, Xiaomo; Cong, Feng

2016-01-01

Aberrant activation of the Wnt/β-catenin pathway is frequently found in various cancers, often through mutations of downstream components. Inhibiting β-catenin signaling in tumors with downstream pathway mutations remains challenging, due to a lack of favorable targets. On the other hand, targeting upstream components of the Wnt pathway is rather straightforward. However, it is difficult to identify tumors addicted to autocrine or paracrine Wnt signaling. Discovery of the R-spondin-ZNRF3/RNF43 signaling module and its genetic alterations in cancers represents a breakthrough in this area. Membrane E3 ligase ZNRF3 and RNF43 are critical negative feedback regulators of the Wnt pathway, which function through promoting ubiquitination and degradation of Wnt receptors. R-spondin proteins (RSPO1-4) serve as natural antagonists of ZNRF3/RNF43. To maintain strong and sustained Wnt/β-catenin signaling, cancers need to overcome ZNRF3/RNF43-mediated feedback inhibition. Indeed, mutations of RNF43/ZNRF3 and recurrent translocations of RSPO2/RSPO3 have recently been identified in various cancers. Significantly, genetic alterations in RNF43/ZNRF3/RSPO2/RSPO3 have shown promise as predictive biomarkers in pre-clinical models for the efficacy of upstream Wnt inhibitors. In this review, we will discuss the biology of the R-spondin-ZNRF3/RNF43 signaling module, cancer-associated alterations of this signaling module, and their value as biomarkers to identify Wnt-addicted tumors. PMID:27338477

Expanding the Substantial Interactome of NEMO Using Protein Microarrays

PubMed Central

Fenner, Beau J.; Scannell, Michael; Prehn, Jochen H. M.

2010-01-01

Signal transduction by the NF-kappaB pathway is a key regulator of a host of cellular responses to extracellular and intracellular messages. The NEMO adaptor protein lies at the top of this pathway and serves as a molecular conduit, connecting signals transmitted from upstream sensors to the downstream NF-kappaB transcription factor and subsequent gene activation. The position of NEMO within this pathway makes it an attractive target from which to search for new proteins that link NF-kappaB signaling to additional pathways and upstream effectors. In this work, we have used protein microarrays to identify novel NEMO interactors. A total of 112 protein interactors were identified, with the most statistically significant hit being the canonical NEMO interactor IKKbeta, with IKKalpha also being identified. Of the novel interactors, more than 30% were kinases, while at least 25% were involved in signal transduction. Binding of NEMO to several interactors, including CALB1, CDK2, SAG, SENP2 and SYT1, was confirmed using GST pulldown assays and coimmunoprecipitation, validating the initial screening approach. Overexpression of CALB1, CDK2 and SAG was found to stimulate transcriptional activation by NF-kappaB, while SYT1 overexpression repressed TNFalpha-dependent NF-kappaB transcriptional activation in human embryonic kidney cells. Corresponding with this finding, RNA silencing of CDK2, SAG and SENP2 reduced NF-kappaB transcriptional activation, supporting a positive role for these proteins in the NF-kappaB pathway. The identification of a host of new NEMO interactors opens up new research opportunities to improve understanding of this essential cell signaling pathway. PMID:20098747

A systematic genetic screen for genes involved in sensing inorganic phosphate availability in Saccharomyces cerevisiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Joonhyuk; Rajagopal, Abbhirami; Xu, Yi -Fan

Saccharomyces cerevisiae responds to changes in extracellular inorganic phosphate (Pi) availability by regulating the activity of the phosphate-responsive (PHO) signaling pathway, enabling cells to maintain intracellular levels of the essential nutrient P i. P i-limitation induces upregulation of inositol heptakisphosphate (IP 7) synthesized by the inositol hexakisphosphate kinase Vip1, triggering inhibition of the Pho80/Pho85 cyclin-cyclin dependent kinase (CDK) complex by the CDK inhibitor Pho81, which upregulates the PHO regulon through the CDK target and transcription factor Pho4. To identify genes that are involved in signaling upstream of the Pho80/Pho85/Pho81 complex and how they interact with each other to regulate themore » PHO pathway, we performed genome-wide screens with the synthetic genetic array method. We identified more than 300 mutants with defects in signaling upstream of the Pho80/Pho85/Pho81 complex, including AAH1, which encodes an adenine deaminase that negatively regulates the PHO pathway in a Vip1-dependent manner. Moreover, we showed that even in the absence of VIP1, the PHO pathway can be activated under prolonged periods of P i starvation, suggesting complexity in the mechanisms by which the PHO pathway is regulated.« less

A systematic genetic screen for genes involved in sensing inorganic phosphate availability in Saccharomyces cerevisiae

DOE PAGES

Choi, Joonhyuk; Rajagopal, Abbhirami; Xu, Yi -Fan; ...

2017-05-17

Saccharomyces cerevisiae responds to changes in extracellular inorganic phosphate (Pi) availability by regulating the activity of the phosphate-responsive (PHO) signaling pathway, enabling cells to maintain intracellular levels of the essential nutrient P i. P i-limitation induces upregulation of inositol heptakisphosphate (IP 7) synthesized by the inositol hexakisphosphate kinase Vip1, triggering inhibition of the Pho80/Pho85 cyclin-cyclin dependent kinase (CDK) complex by the CDK inhibitor Pho81, which upregulates the PHO regulon through the CDK target and transcription factor Pho4. To identify genes that are involved in signaling upstream of the Pho80/Pho85/Pho81 complex and how they interact with each other to regulate themore » PHO pathway, we performed genome-wide screens with the synthetic genetic array method. We identified more than 300 mutants with defects in signaling upstream of the Pho80/Pho85/Pho81 complex, including AAH1, which encodes an adenine deaminase that negatively regulates the PHO pathway in a Vip1-dependent manner. Moreover, we showed that even in the absence of VIP1, the PHO pathway can be activated under prolonged periods of P i starvation, suggesting complexity in the mechanisms by which the PHO pathway is regulated.« less

Resistance to MEK inhibitors: should we co-target upstream?

PubMed

Poulikakos, Poulikos I; Solit, David B

2011-03-29

Aberrant activation of the ERK pathway is common in human tumors. This pathway consists of a three-tiered kinase module [comprising the kinases RAF, mitogen-activated protein kinase (MAPK) kinase (MEK), and extracellular signal-regulated kinase (ERK)] that functions as a negative feedback amplifier to confer robustness and stabilization of pathway output. Because this pathway is frequently dysregulated in human cancers, intense efforts are under way to develop selective inhibitors of the ERK pathway as anticancer drugs. Although promising results have been reported in early trials for inhibitors of RAF or MEK, resistance invariably occurs. Amplification of the upstream oncogenic driver of ERK signaling has been identified as a mechanism for MEK inhibitor resistance in cells with mutant BRAF or KRAS. Increased abundance of the oncogenic driver (either KRAS or BRAF in the appropriate cellular context) in response to prolonged drug treatment results in increased flux through the ERK pathway and restoration of ERK activity above the threshold required for cell growth. For patients with BRAF mutant tumors, the results suggest that the addition of a RAF inhibitor to a MEK inhibitor may delay or overcome drug resistance. The data thus provide a mechanistic basis for ongoing trials testing concurrent treatment with RAF and MEK inhibitors.

Evolution and Design Governing Signal Precision and Amplification in a Bacterial Chemosensory Pathway

PubMed Central

Espinosa, Leon; Baronian, Grégory; Molle, Virginie; Mauriello, Emilia M. F.; Brochier-Armanet, Céline; Mignot, Tâm

2015-01-01

Understanding the principles underlying the plasticity of signal transduction networks is fundamental to decipher the functioning of living cells. In Myxococcus xanthus, a particular chemosensory system (Frz) coordinates the activity of two separate motility systems (the A- and S-motility systems), promoting multicellular development. This unusual structure asks how signal is transduced in a branched signal transduction pathway. Using combined evolution-guided and single cell approaches, we successfully uncoupled the regulations and showed that the A-motility regulation system branched-off an existing signaling system that initially only controlled S-motility. Pathway branching emerged in part following a gene duplication event and changes in the circuit structure increasing the signaling efficiency. In the evolved pathway, the Frz histidine kinase generates a steep biphasic response to increasing external stimulations, which is essential for signal partitioning to the motility systems. We further show that this behavior results from the action of two accessory response regulator proteins that act independently to filter and amplify signals from the upstream kinase. Thus, signal amplification loops may underlie the emergence of new connectivity in signal transduction pathways. PMID:26291327

Bioinformatic dissecting of TP53 regulation pathway underlying butyrate-induced histone modification in epigenetic regulation

USDA-ARS?s Scientific Manuscript database

Butyrate affects cell proliferation, differentiation and motility. Butyrate inhibits histone deacetylase (HDAC) activities and induces cell cycle arrest and apoptosis. TP53 is one of the most active upstream regulators discovered by IPA in our RNA sequencing data set. The TP53 signaling pathway pl...

Sig2GRN: a software tool linking signaling pathway with gene regulatory network for dynamic simulation.

PubMed

Zhang, Fan; Liu, Runsheng; Zheng, Jie

2016-12-23

Linking computational models of signaling pathways to predicted cellular responses such as gene expression regulation is a major challenge in computational systems biology. In this work, we present Sig2GRN, a Cytoscape plugin that is able to simulate time-course gene expression data given the user-defined external stimuli to the signaling pathways. A generalized logical model is used in modeling the upstream signaling pathways. Then a Boolean model and a thermodynamics-based model are employed to predict the downstream changes in gene expression based on the simulated dynamics of transcription factors in signaling pathways. Our empirical case studies show that the simulation of Sig2GRN can predict changes in gene expression patterns induced by DNA damage signals and drug treatments. As a software tool for modeling cellular dynamics, Sig2GRN can facilitate studies in systems biology by hypotheses generation and wet-lab experimental design. http://histone.scse.ntu.edu.sg/Sig2GRN/.

Evolution of JAK-STAT Pathway Components: Mechanisms and Role in Immune System Development

PubMed Central

Liongue, Clifford; O'Sullivan, Lynda A.; Trengove, Monique C.; Ward, Alister C.

2012-01-01

Background Lying downstream of a myriad of cytokine receptors, the Janus kinase (JAK) – Signal transducer and activator of transcription (STAT) pathway is pivotal for the development and function of the immune system, with additional important roles in other biological systems. To gain further insight into immune system evolution, we have performed a comprehensive bioinformatic analysis of the JAK-STAT pathway components, including the key negative regulators of this pathway, the SH2-domain containing tyrosine phosphatase (SHP), Protein inhibitors against Stats (PIAS), and Suppressor of cytokine signaling (SOCS) proteins across a diverse range of organisms. Results Our analysis has demonstrated significant expansion of JAK-STAT pathway components co-incident with the emergence of adaptive immunity, with whole genome duplication being the principal mechanism for generating this additional diversity. In contrast, expansion of upstream cytokine receptors appears to be a pivotal driver for the differential diversification of specific pathway components. Conclusion Diversification of JAK-STAT pathway components during early vertebrate development occurred concurrently with a major expansion of upstream cytokine receptors and two rounds of whole genome duplications. This produced an intricate cell-cell communication system that has made a significant contribution to the evolution of the immune system, particularly the emergence of adaptive immunity. PMID:22412924

Novel mechanism of JNK pathway activation by adenoviral E1A

PubMed Central

Morrison, Helen; Pospelova, Tatiana V.; Pospelov, Valery A.; Herrlich, Peter

2014-01-01

The adenoviral oncoprotein E1A influences cellular regulation by interacting with a number of cellular proteins. In collaboration with complementary oncogenes, E1A fully transforms primary cells. As part of this action, E1A inhibits transcription of c-Jun:Fos target genes while promoting that of c-Jun:ATF2-dependent genes including jun. Both c-Jun and ATF2 are hyperphosphorylated in response to E1A. In the current study, E1A was fused with the ligand binding domain of the estrogen receptor (E1A-ER) to monitor the immediate effect of E1A activation. With this approach we now show that E1A activates c-Jun N-terminal kinase (JNK), the upstream kinases MKK4 and MKK7, as well as the small GTPase Rac1. Activation of the JNK pathway requires the N-terminal domain of E1A, and, importantly, is independent of transcription. In addition, it requires the presence of ERM proteins. Downregulation of signaling components upstream of JNK inhibits E1A-dependent JNK/c-Jun activation. Taking these findings together, we show that E1A activates the JNK/c-Jun signaling pathway upstream of Rac1 in a transcription-independent manner, demonstrating a novel mechanism of E1A action. PMID:24742962

SignaLink 2 – a signaling pathway resource with multi-layered regulatory networks

PubMed Central

2013-01-01

Background Signaling networks in eukaryotes are made up of upstream and downstream subnetworks. The upstream subnetwork contains the intertwined network of signaling pathways, while the downstream regulatory part contains transcription factors and their binding sites on the DNA as well as microRNAs and their mRNA targets. Currently, most signaling and regulatory databases contain only a subsection of this network, making comprehensive analyses highly time-consuming and dependent on specific data handling expertise. The need for detailed mapping of signaling systems is also supported by the fact that several drug development failures were caused by undiscovered cross-talk or regulatory effects of drug targets. We previously created a uniformly curated signaling pathway resource, SignaLink, to facilitate the analysis of pathway cross-talks. Here, we present SignaLink 2, which significantly extends the coverage and applications of its predecessor. Description We developed a novel concept to integrate and utilize different subsections (i.e., layers) of the signaling network. The multi-layered (onion-like) database structure is made up of signaling pathways, their pathway regulators (e.g., scaffold and endocytotic proteins) and modifier enzymes (e.g., phosphatases, ubiquitin ligases), as well as transcriptional and post-transcriptional regulators of all of these components. The user-friendly website allows the interactive exploration of how each signaling protein is regulated. The customizable download page enables the analysis of any user-specified part of the signaling network. Compared to other signaling resources, distinctive features of SignaLink 2 are the following: 1) it involves experimental data not only from humans but from two invertebrate model organisms, C. elegans and D. melanogaster; 2) combines manual curation with large-scale datasets; 3) provides confidence scores for each interaction; 4) operates a customizable download page with multiple file formats (e.g., BioPAX, Cytoscape, SBML). Non-profit users can access SignaLink 2 free of charge at http://SignaLink.org. Conclusions With SignaLink 2 as a single resource, users can effectively analyze signaling pathways, scaffold proteins, modifier enzymes, transcription factors and miRNAs that are important in the regulation of signaling processes. This integrated resource allows the systems-level examination of how cross-talks and signaling flow are regulated, as well as provide data for cross-species comparisons and drug discovery analyses. PMID:23331499

SignaLink 2 - a signaling pathway resource with multi-layered regulatory networks.

PubMed

Fazekas, Dávid; Koltai, Mihály; Türei, Dénes; Módos, Dezső; Pálfy, Máté; Dúl, Zoltán; Zsákai, Lilian; Szalay-Bekő, Máté; Lenti, Katalin; Farkas, Illés J; Vellai, Tibor; Csermely, Péter; Korcsmáros, Tamás

2013-01-18

Signaling networks in eukaryotes are made up of upstream and downstream subnetworks. The upstream subnetwork contains the intertwined network of signaling pathways, while the downstream regulatory part contains transcription factors and their binding sites on the DNA as well as microRNAs and their mRNA targets. Currently, most signaling and regulatory databases contain only a subsection of this network, making comprehensive analyses highly time-consuming and dependent on specific data handling expertise. The need for detailed mapping of signaling systems is also supported by the fact that several drug development failures were caused by undiscovered cross-talk or regulatory effects of drug targets. We previously created a uniformly curated signaling pathway resource, SignaLink, to facilitate the analysis of pathway cross-talks. Here, we present SignaLink 2, which significantly extends the coverage and applications of its predecessor. We developed a novel concept to integrate and utilize different subsections (i.e., layers) of the signaling network. The multi-layered (onion-like) database structure is made up of signaling pathways, their pathway regulators (e.g., scaffold and endocytotic proteins) and modifier enzymes (e.g., phosphatases, ubiquitin ligases), as well as transcriptional and post-transcriptional regulators of all of these components. The user-friendly website allows the interactive exploration of how each signaling protein is regulated. The customizable download page enables the analysis of any user-specified part of the signaling network. Compared to other signaling resources, distinctive features of SignaLink 2 are the following: 1) it involves experimental data not only from humans but from two invertebrate model organisms, C. elegans and D. melanogaster; 2) combines manual curation with large-scale datasets; 3) provides confidence scores for each interaction; 4) operates a customizable download page with multiple file formats (e.g., BioPAX, Cytoscape, SBML). Non-profit users can access SignaLink 2 free of charge at http://SignaLink.org. With SignaLink 2 as a single resource, users can effectively analyze signaling pathways, scaffold proteins, modifier enzymes, transcription factors and miRNAs that are important in the regulation of signaling processes. This integrated resource allows the systems-level examination of how cross-talks and signaling flow are regulated, as well as provide data for cross-species comparisons and drug discovery analyses.

Integrated genomic approaches identify major pathways and upstream regulators in late onset Alzheimer’s disease

PubMed Central

Li, Xinzhong; Long, Jintao; He, Taigang; Belshaw, Robert; Scott, James

2015-01-01

Previous studies have evaluated gene expression in Alzheimer’s disease (AD) brains to identify mechanistic processes, but have been limited by the size of the datasets studied. Here we have implemented a novel meta-analysis approach to identify differentially expressed genes (DEGs) in published datasets comprising 450 late onset AD (LOAD) brains and 212 controls. We found 3124 DEGs, many of which were highly correlated with Braak stage and cerebral atrophy. Pathway Analysis revealed the most perturbed pathways to be (a) nitric oxide and reactive oxygen species in macrophages (NOROS), (b) NFkB and (c) mitochondrial dysfunction. NOROS was also up-regulated, and mitochondrial dysfunction down-regulated, in healthy ageing subjects. Upstream regulator analysis predicted the TLR4 ligands, STAT3 and NFKBIA, for activated pathways and RICTOR for mitochondrial genes. Protein-protein interaction network analysis emphasised the role of NFKB; identified a key interaction of CLU with complement; and linked TYROBP, TREM2 and DOK3 to modulation of LPS signalling through TLR4 and to phosphatidylinositol metabolism. We suggest that NEUROD6, ZCCHC17, PPEF1 and MANBAL are potentially implicated in LOAD, with predicted links to calcium signalling and protein mannosylation. Our study demonstrates a highly injurious combination of TLR4-mediated NFKB signalling, NOROS inflammatory pathway activation, and mitochondrial dysfunction in LOAD. PMID:26202100

Role of redox signaling in the autonomous proliferative response of endothelial cells to hypoxia.

PubMed

Schäfer, M; Schäfer, C; Ewald, N; Piper, H M; Noll, Th

2003-05-16

Endothelial cells exhibit an autonomous proliferative response to hypoxia, independent of paracrine effectors. In cultured endothelial cells of porcine aorta, we analyzed the signaling of this response, with a focus on the roles of redox signaling and the MEK/ERK pathway. Transient hypoxia (1 hour) stimulated proliferation by 61+/-4% (n=16; P<0.05 versus control), quantified after 24 hours normoxic postincubation. Hypoxia induced an activation of ERK2 and of NAD(P)H oxidase and a burst of reactive oxygen species (ROS), determined by DCF fluorescence. To inhibit the MEK/ERK pathway, we used PD 98059 (PD, 20 micromol/L); to downregulate NAD(P)H oxidase, we applied p22phox antisense oligonucleotides; and to inhibit mitochondrial ROS generation, we used the ubiquinone derivate mitoQ (MQ, 10 micromol/L). All three inhibitions suppressed the proliferative response: PD inhibited NAD(P)H oxidase activation; p22phox antisense transfection did not inhibit ERK2 activation, but suppressed ROS production; and MQ inhibited ERK2 activation and ROS production. The autonomous proliferative response depends on the MEK/ERK pathway and redox signaling steps upstream and downstream of ERK. Located upstream is ROS generation by mitochondria, downstream is NAD(P)H oxidase.

Jasmonate Regulates the INDUCER OF CBF EXPRESSION–C-REPEAT BINDING FACTOR/DRE BINDING FACTOR1 Cascade and Freezing Tolerance in Arabidopsis[W

PubMed Central

Hu, Yanru; Jiang, Liqun; Wang, Fang; Yu, Diqiu

2013-01-01

The INDUCER OF CBF EXPRESSION (ICE)–C-REPEAT BINDING FACTOR/DRE BINDING FACTOR1 (CBF/DREB1) transcriptional pathway plays a critical role in modulating cold stress responses in Arabidopsis thaliana. Dissecting crucial upstream regulatory signals or components of the ICE-CBF/DREB1 cascade will enhance our understanding of plant cold-tolerance mechanisms. Here, we show that jasmonate positively regulates plant responses to freezing stress in Arabidopsis. Exogenous application of jasmonate significantly enhanced plant freezing tolerance with or without cold acclimation. By contrast, blocking endogenous jasmonate biosynthesis and signaling rendered plants hypersensitive to freezing stress. Consistent with the positive role of jasmonate in freezing stress, production of endogenous jasmonate was triggered by cold treatment. In addition, cold induction of genes acting in the CBF/DREB1 signaling pathway was upregulated by jasmonate. Further investigation revealed that several JASMONATE ZIM-DOMAIN (JAZ) proteins, the repressors of jasmonate signaling, physically interact with ICE1 and ICE2 transcription factors. JAZ1 and JAZ4 repress the transcriptional function of ICE1, thereby attenuating the expression of its regulon. Consistent with this, overexpression of JAZ1 or JAZ4 represses freezing stress responses of Arabidopsis. Taken together, our study provides evidence that jasmonate functions as a critical upstream signal of the ICE-CBF/DREB1 pathway to positively regulate Arabidopsis freezing tolerance. PMID:23933884

Jasmonate regulates the inducer of cbf expression-C-repeat binding factor/DRE binding factor1 cascade and freezing tolerance in Arabidopsis.

PubMed

Hu, Yanru; Jiang, Liqun; Wang, Fang; Yu, Diqiu

2013-08-01

The inducer of cbf expression (ICE)-C-repeat binding factor/DRE binding factor1 (CBF/DREB1) transcriptional pathway plays a critical role in modulating cold stress responses in Arabidopsis thaliana. Dissecting crucial upstream regulatory signals or components of the ICE-CBF/DREB1 cascade will enhance our understanding of plant cold-tolerance mechanisms. Here, we show that jasmonate positively regulates plant responses to freezing stress in Arabidopsis. Exogenous application of jasmonate significantly enhanced plant freezing tolerance with or without cold acclimation. By contrast, blocking endogenous jasmonate biosynthesis and signaling rendered plants hypersensitive to freezing stress. Consistent with the positive role of jasmonate in freezing stress, production of endogenous jasmonate was triggered by cold treatment. In addition, cold induction of genes acting in the CBF/DREB1 signaling pathway was upregulated by jasmonate. Further investigation revealed that several jasmonate ZIM-domain (JAZ) proteins, the repressors of jasmonate signaling, physically interact with ICE1 and ICE2 transcription factors. JAZ1 and JAZ4 repress the transcriptional function of ICE1, thereby attenuating the expression of its regulon. Consistent with this, overexpression of JAZ1 or JAZ4 represses freezing stress responses of Arabidopsis. Taken together, our study provides evidence that jasmonate functions as a critical upstream signal of the ICE-CBF/DREB1 pathway to positively regulate Arabidopsis freezing tolerance.

Kibra and Merlin Activate the Hippo Pathway Spatially Distinct from and Independent of Expanded.

PubMed

Su, Ting; Ludwig, Michael Z; Xu, Jiajie; Fehon, Richard G

2017-03-13

The Hippo pathway is emerging as a key evolutionarily conserved signaling mechanism that controls organ size. Three membrane-associated proteins, Kibra, Merlin, and Expanded, regulate pathway activity, but the precise molecular mechanism by which they function is still poorly understood. Here we provide evidence that Merlin and Kibra activate Hippo signaling in parallel to Expanded at a spatially distinct cellular domain, the medial apical cortex. Merlin and Kibra together recruit the adapter protein Salvador, which in turn recruits the core kinase Hippo. In addition, we show that Crumbs has a dual effect on Hippo signaling. Crumbs promotes the ability of Expanded to activate the pathway but also sequesters Kibra to downregulate Hippo signaling. Together, our findings elucidate the mechanism of Hippo pathway activation by Merlin and Kibra, identify a subcellular domain for Hippo pathway regulation, and demonstrate differential activity of upstream regulators in different subcellular domains. Copyright © 2017 Elsevier Inc. All rights reserved.

Dysregulation of the IGF-I/PI3K/AKT/mTOR signaling pathway in autism spectrum disorders.

PubMed

Chen, Jianling; Alberts, Ian; Li, Xiaohong

2014-06-01

The IGF-I/PI3K/AKT/mTOR signaling pathway plays an important role in the regulation of cell growth, proliferation, differentiation, motility, survival, metabolism and protein synthesis. Insulin-like growth factor-I (IGF-I) is synthesized in the liver and fibroblasts, and its biological actions are mediated by the IGF-I receptor (IGF-IR). The binding of IGF-I to IGF-IR leads to the activation of phosphatidylinositol 3-kinase (PI3K). Activated PI3K stimulates the production of phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] and phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3]. The PH domain of AKT (protein kinase B, PKB) (v-AKT murine thymoma viral oncogene homolog) binds to PI(4,5)P2 and PI(3,4,5)P3, followed by phosphorylation of the Thr308 and Ser473 regulatory sites. Tuberous sclerosis complex 1 (TSC1) and TSC2 are upstream regulators of mammalian target of rapamycin (mTOR) and downstream effectors of the PI3K/AKT signaling pathway. The activation of AKT suppresses the TSC1/TSC2 heterodimer, which is an upstream regulator of mTOR. Dysregulated IGF-I/PI3K/AKT/mTOR signaling has been shown to be associated with autism spectrum disorders (ASDs). In this review, we discuss the emerging evidence for a functional relationship between the IGF-I/PI3K/AKT/mTOR pathway and ASDs, as well as a possible role of this signaling pathway in the diagnosis and treatment of ASDs. Copyright © 2014 ISDN. Published by Elsevier Ltd. All rights reserved.

Molecular mechanisms of the mammalian Hippo signaling pathway.

PubMed

Ji, Xin-yan; Zhong, Guoxuan; Zhao, Bin

2017-07-20

The Hippo pathway plays an evolutionarily conserved fundamental role in controlling organ size in multicellular organisms. Importantly, evidence from studies of patient samples and mouse models clearly indicates that deregulation of the Hippo signaling pathway plays a crucial role in the initiation and progression of many different types of human cancers. The Hippo signaling pathway is regulated by various stimuli, such as mechanical stress, G-protein coupled receptor signaling, and cellular energy status. When activated, the Hippo kinase cascade phosphorylates and inhibits the transcription co-activator YAP (Yes-associated protein), and its paralog TAZ (transcriptional coactivator with PDZ-binding motif), resulting in their cytoplasmic retention and degradation. When the Hippo signaling pathway is inactive, dephosphorylated YAP/TAZ translocate into the nucleus and activate gene transcription through binding to TEAD (TEA domain) family and other transcription factors. Such changes in gene expression promote cell proliferation and stem cell/progenitor cell self-renewal but inhibit apoptosis, thereby coordinately promote increase in organ size, tissue regeneration, and tumorigenesis. In this review, we summarize the molecular mechanisms of the mammalian Hippo signaling pathway with special emphasis on the Hippo kinase cascade and its upstream signals, the Hippo signaling pathway regulation of YAP and the mechanisms of YAP in regulation of gene transcription.

Defining the Protein–Protein Interaction Network of the Human Hippo Pathway*

PubMed Central

Wang, Wenqi; Li, Xu; Huang, Jun; Feng, Lin; Dolinta, Keithlee G.; Chen, Junjie

2014-01-01

The Hippo pathway, which is conserved from Drosophila to mammals, has been recognized as a tumor suppressor signaling pathway governing cell proliferation and apoptosis, two key events involved in organ size control and tumorigenesis. Although several upstream regulators, the conserved kinase cascade and key downstream effectors including nuclear transcriptional factors have been defined, the global organization of this signaling pathway is not been fully understood. Thus, we conducted a proteomic analysis of human Hippo pathway, which revealed the involvement of an extensive protein–protein interaction network in this pathway. The mass spectrometry data were deposited to ProteomeXchange with identifier PXD000415. Our data suggest that 550 interactions within 343 unique protein components constitute the central protein–protein interaction landscape of human Hippo pathway. Our study provides a glimpse into the global organization of Hippo pathway, reveals previously unknown interactions within this pathway, and uncovers new potential components involved in the regulation of this pathway. Understanding these interactions will help us further dissect the Hippo signaling-pathway and extend our knowledge of organ size control. PMID:24126142

FAT1 cadherin acts upstream of Hippo signalling through TAZ to regulate neuronal differentiation.

PubMed

Ahmed, Abdulrzag F; de Bock, Charles E; Lincz, Lisa F; Pundavela, Jay; Zouikr, Ihssane; Sontag, Estelle; Hondermarck, Hubert; Thorne, Rick F

2015-12-01

The Hippo pathway is emerging as a critical nexus that balances self-renewal of progenitors against differentiation; however, upstream elements in vertebrate Hippo signalling are poorly understood. High expression of Fat1 cadherin within the developing neuroepithelium and the manifestation of severe neurological phenotypes in Fat1-knockout mice suggest roles in neurogenesis. Using the SH-SY5Y model of neuronal differentiation and employing gene silencing techniques, we show that FAT1 acts to control neurite outgrowth, also driving cells towards terminal differentiation via inhibitory effects on proliferation. FAT1 actions were shown to be mediated through Hippo signalling where it activated core Hippo kinase components and antagonised functions of the Hippo effector TAZ. Suppression of FAT1 promoted the nucleocytoplasmic shuttling of TAZ leading to enhanced transcription of the Hippo target gene CTGF together with accompanying increases in nuclear levels of Smad3. Silencing of TAZ reversed the effects of FAT1 depletion thus connecting inactivation of TAZ-TGFbeta signalling with Hippo signalling mediated through FAT1. These findings establish FAT1 as a new upstream Hippo element regulating early stages of differentiation in neuronal cells.

POSH regulates Hippo signaling through ubiquitin-mediated expanded degradation.

PubMed

Ma, Xianjue; Guo, Xiaowei; Richardson, Helena E; Xu, Tian; Xue, Lei

2018-02-27

The Hippo signaling pathway is a master regulator of organ growth, tissue homeostasis, and tumorigenesis. The activity of the Hippo pathway is controlled by various upstream components, including Expanded (Ex), but the precise molecular mechanism of how Ex is regulated remains poorly understood. Here we identify Plenty of SH3s (POSH), an E3 ubiquitin ligase, as a key component of Hippo signaling in Drosophila POSH overexpression synergizes with loss of Kibra to induce overgrowth and up-regulation of Hippo pathway target genes. Furthermore, knockdown of POSH impedes dextran sulfate sodium-induced Yorkie-dependent intestinal stem cell renewal, suggesting a physiological role of POSH in modulating Hippo signaling. Mechanistically, POSH binds to the C-terminal of Ex and is essential for the Crumbs-induced ubiquitination and degradation of Ex. Our findings establish POSH as a crucial regulator that integrates the signal from the cell surface to negatively regulate Ex-mediated Hippo activation in Drosophila .

Src-dependent EGFR transactivation regulates lung inflammation via downstream signaling involving ERK1/2, PI3Kδ/Akt and NFκB induction in a murine asthma model.

PubMed

El-Hashim, Ahmed Z; Khajah, Maitham A; Renno, Waleed M; Babyson, Rhema S; Uddin, Mohib; Benter, Ibrahim F; Ezeamuzie, Charles; Akhtar, Saghir

2017-08-30

The molecular mechanisms underlying asthma pathogenesis are poorly characterized. In this study, we investigated (1) whether Src mediates epidermal growth factor receptor (EGFR) transactivation; (2) if ERK1/2, PI3Kδ/Akt and NF-κB are signaling effectors downstream of Src/EGFR activation; and (3) if upstream inhibition of Src/EGFR is more effective in downregulating the allergic inflammation than selective inhibition of downstream signaling pathways. Allergic inflammation resulted in increased phosphorylation of EGFR, Akt, ERK1/2 and IκB in the lung tissues from ovalbumin (OVA)-challenged BALB/c mice. Treatment with inhibitors of Src (SU6656) or EGFR (AG1478) reduced EGFR phosphorylation and downstream signaling which resulted in the inhibition of the OVA-induced inflammatory cell influx in bronchoalveolar lavage fluid (BALF), perivascular and peribronchial inflammation, fibrosis, goblet cell hyper/metaplasia and airway hyper-responsiveness. Treatment with pathway-selective inhibitors for ERK1/2 (PD89059) and PI3Kδ/Akt (IC-87114) respectively, or an inhibitor of NF-κB (BAY11-7085) also reduced the OVA-induced asthmatic phenotype but to a lesser extent compared to Src/EGFR inhibition. Thus, Src via EGFR transactivation and subsequent downstream activation of multiple pathways regulates the allergic airway inflammatory response. Furthermore, a broader upstream inhibition of Src/EGFR offers an attractive therapeutic alternative in the treatment of asthma relative to selectively targeting the individual downstream signaling effectors.

Novel targets for prostate cancer chemoprevention

PubMed Central

Sarkar, Fazlul H; Li, Yiwei; Wang, Zhiwei; Kong, Dejuan

2010-01-01

Among many endocrine-related cancers, prostate cancer (PCa) is the most frequent male malignancy, and it is the second most common cause of cancer-related death in men in the United States. Therefore, this review focuses on summarizing the knowledge of molecular signaling pathways in PCa because, in order to better design new preventive strategies for the fight against PCa, documentation of the knowledge on the pathogenesis of PCa at the molecular level is very important. Cancer cells are known to have alterations in multiple cellular signaling pathways; indeed, the development and the progression of PCa are known to be caused by the deregulation of several selective signaling pathways such as the androgen receptor, Akt, nuclear factor-κB, Wnt, Hedgehog, and Notch. Therefore, strategies targeting these important pathways and their upstream and downstream signaling could be promising for the prevention of PCa progression. In this review, we summarize the current knowledge regarding the alterations in cell signaling pathways during the development and progression of PCa, and document compelling evidence showing that these are the targets of several natural agents against PCa progression and its metastases. PMID:20576802

Producing the Ethylene Signal: Regulation and Diversification of Ethylene Biosynthetic Enzymes1

PubMed Central

Booker, Matthew A.; DeLong, Alison

2015-01-01

Strictly controlled production of ethylene gas lies upstream of the signaling activities of this crucial regulator throughout the plant life cycle. Although the biosynthetic pathway is enzymatically simple, the regulatory circuits that modulate signal production are fine tuned to allow integration of responses to environmental and intrinsic cues. Recently identified posttranslational mechanisms that control ethylene production converge on one family of biosynthetic enzymes and overlay several independent reversible phosphorylation events and distinct mediators of ubiquitin-dependent protein degradation. Although the core pathway is conserved throughout seed plants, these posttranslational regulatory mechanisms may represent evolutionarily recent innovations. The evolutionary origins of the pathway and its regulators are not yet clear; outside the seed plants, numerous biochemical and phylogenetic questions remain to be addressed. PMID:26134162

mAKAP – A Master Scaffold for Cardiac Remodeling

PubMed Central

Passariello, Catherine L.; Li, Jinliang; Dodge-Kafka, Kimberly; Kapiloff, Michael S.

2014-01-01

Cardiac remodeling is regulated by an extensive intracellular signal transduction network. Each of the many signaling pathways in this network contributes uniquely to the control of cellular adaptation. In the last few years, it has become apparent that multimolecular signaling complexes or ‘signalosomes’ are important for fidelity in intracellular signaling and for mediating crosstalk between the different signaling pathways. These complexes integrate upstream signals and control downstream effectors. In the cardiac myocyte, the protein mAKAPβ serves as a scaffold for a large signalosome that is responsive to cAMP, calcium, hypoxia, and mitogen-activated protein kinase signaling. The main function of mAKAPβ signalosomes is to modulate stress-related gene expression regulated by the transcription factors NFATc, MEF2 and HIF-1α and type II histone deacetylases that control pathological cardiac hypertrophy. PMID:25551320

Ras Signaling Regulates Stem Cells and Amelogenesis in the Mouse Incisor.

PubMed

Zheng, X; Goodwin, A F; Tian, H; Jheon, A H; Klein, O D

2017-11-01

The role of Ras signaling during tooth development is poorly understood. Ras proteins-which are activated by many upstream pathways, including receptor tyrosine kinase cascades-signal through multiple effectors, such as the mitogen-activated protein kinase (MAPK) and PI3K pathways. Here, we utilized the mouse incisor as a model to study how the MAPK and PI3K pathways regulate dental epithelial stem cells and amelogenesis. The rodent incisor-which grows continuously throughout the life of the animal due to the presence of epithelial and mesenchymal stem cells-provides a model for the study of ectodermal organ renewal and regeneration. Utilizing models of Ras dysregulation as well as inhibitors of the MAPK and PI3K pathways, we found that MAPK and PI3K regulate dental epithelial stem cell activity, transit-amplifying cell proliferation, and enamel formation in the mouse incisor.

The role of phosphoinositide 3-kinase and phosphatidic acid in the regulation of mammalian target of rapamycin following eccentric contractions

PubMed Central

O’Neil, T K; Duffy, L R; Frey, J W; Hornberger, T A

2009-01-01

Resistance exercise induces a hypertrophic response in skeletal muscle and recent studies have begun to shed light on the molecular mechanisms involved in this process. For example, several studies indicate that signalling by the mammalian target of rapamycin (mTOR) is necessary for a hypertrophic response. Furthermore, resistance exercise has been proposed to activate mTOR signalling through an upstream pathway involving the phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB); however, this hypothesis has not been thoroughly tested. To test this hypothesis, we first evaluated the temporal pattern of signalling through PI3K–PKB and mTOR following a bout of resistance exercise with eccentric contractions (EC). Our results indicated that the activation of signalling through PI3K–PKB is a transient event (<15 min), while the activation of mTOR is sustained for a long duration (>12 h). Furthermore, inhibition of PI3K–PKB activity did not prevent the activation of mTOR signalling by ECs, indicating that PI3K–PKB is not part of the upstream regulatory pathway. These observations led us to investigate an alternative pathway for the activation of mTOR signalling involving the synthesis of phosphatidic acid (PA) by phospholipase D (PLD). Our results demonstrate that ECs induce a sustained elevation in [PA] and inhibiting the synthesis of PA by PLD prevented the activation of mTOR. Furthermore, we determined that similar to ECs, PA activates mTOR signalling through a PI3K–PKB-independent mechanism. Combined, the results of this study indicate that the activation of mTOR following eccentric contractions occurs through a PI3K–PKB-independent mechanism that requires PLD and PA. PMID:19470781

mTOR signaling for biological control and cancer.

PubMed

Alayev, Anya; Holz, Marina K

2013-08-01

Mammalian target of rapamycin (mTOR) is a major intersection that connects signals from the extracellular milieu to corresponding changes in intracellular processes. When abnormally regulated, the mTOR signaling pathway is implicated in a wide spectrum of cancers, neurological diseases, and proliferative disorders. Therefore, pharmacological agents that restore the regulatory balance of the mTOR pathway could be beneficial for a great number of diseases. This review summarizes current understanding of mTOR signaling and some unanswered questions in the field. We describe the composition of the mTOR complexes, upstream signals that activate mTOR, and physiological processes that mTOR regulates. We also discuss the role of mTOR and its downstream effectors in cancer, obesity and diabetes, and autism. Copyright © 2013 Wiley Periodicals, Inc.

Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways

PubMed Central

Lee, Jun Ho; Patel, Kalpesh; Tae, Hyun Jin; Lustig, Ana; Kim, Jie Wan; Mattson, Mark P.; Taub, Dennis D.

2014-01-01

Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levelsand impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo. PMID:25447526

BDNF-TrkB signaling through Erk1/2MAPK phosphorylation mediates the enhancement of fear memory induced by glucocorticoids

PubMed Central

Revest, J-M; Le Roux, A; Roullot-Lacarrière, V; Kaouane, N; Vallée, M; Kasanetz, F; Rougé-Pont, F; Tronche, F; Desmedt, A; Piazza, P V

2014-01-01

Activation of glucocorticoid receptors (GR) by glucocorticoid hormones (GC) enhances contextual fear memories through the activation of the Erk1/2MAPK signaling pathway. However, the molecular mechanism mediating this effect of GC remains unknown. Here we used complementary molecular and behavioral approaches in mice and rats and in genetically modified mice in which the GR was conditionally deleted (GRNesCre). We identified the tPA-BDNF-TrkB signaling pathway as the upstream molecular effectors of GR-mediated phosphorylation of Erk1/2MAPK responsible for the enhancement of contextual fear memory. These findings complete our knowledge of the molecular cascade through which GC enhance contextual fear memory and highlight the role of tPA-BDNF-TrkB-Erk1/2MAPK signaling pathways as one of the core effectors of stress-related effects of GC. PMID:24126929

Scaling of Optogenetically Evoked Signaling in a Higher-Order Corticocortical Pathway in the Anesthetized Mouse.

PubMed

Li, Xiaojian; Yamawaki, Naoki; Barrett, John M; Körding, Konrad P; Shepherd, Gordon M G

2018-01-01

Quantitative analysis of corticocortical signaling is needed to understand and model information processing in cerebral networks. However, higher-order pathways, hodologically remote from sensory input, are not amenable to spatiotemporally precise activation by sensory stimuli. Here, we combined parametric channelrhodopsin-2 (ChR2) photostimulation with multi-unit electrophysiology to study corticocortical driving in a parietofrontal pathway from retrosplenial cortex (RSC) to posterior secondary motor cortex (M2) in mice in vivo . Ketamine anesthesia was used both to eliminate complex activity associated with the awake state and to enable stable recordings of responses over a wide range of stimulus parameters. Photostimulation of ChR2-expressing neurons in RSC, the upstream area, produced local activity that decayed quickly. This activity in turn drove downstream activity in M2 that arrived rapidly (5-10 ms latencies), and scaled in amplitude across a wide range of stimulus parameters as an approximately constant fraction (~0.1) of the upstream activity. A model-based analysis could explain the corticocortically driven activity with exponentially decaying kernels (~20 ms time constant) and small delay. Reverse (antidromic) driving was similarly robust. The results show that corticocortical signaling in this pathway drives downstream activity rapidly and scalably, in a mostly linear manner. These properties, identified in anesthetized mice and represented in a simple model, suggest a robust basis for supporting complex non-linear dynamic activity in corticocortical circuits in the awake state.

The Core Molecular Machinery Used for Engulfment of Apoptotic Cells Regulates the JNK Pathway Mediating Axon Regeneration in Caenorhabditis elegans.

PubMed

Pastuhov, Strahil Iv; Fujiki, Kota; Tsuge, Anna; Asai, Kazuma; Ishikawa, Sho; Hirose, Kazuya; Matsumoto, Kunihiro; Hisamoto, Naoki

2016-09-14

The mechanisms that govern the ability of specific neurons to regenerate their axons after injury are not well understood. In Caenorhabditis elegans, the initiation of axon regeneration is positively regulated by the JNK-MAPK pathway. In this study, we identify two components functioning upstream of the JNK pathway: the Ste20-related protein kinase MAX-2 and the Rac-type GTPase CED-10. CED-10, when bound by GTP, interacts with MAX-2 and functions as its upstream regulator in axon regeneration. CED-10, in turn, is activated by axon injury via signals initiated from the integrin α-subunit INA-1 and the nonreceptor tyrosine kinase SRC-1 and transmitted via the signaling module CED-2/CrkII-CED-5/Dock180-CED-12/ELMO. This module is also known to regulate the engulfment of apoptotic cells during development. Our findings thus reveal that the molecular machinery used for engulfment of apoptotic cells also promotes axon regeneration through activation of the JNK pathway. The molecular mechanisms of axon regeneration after injury remain poorly understood. In Caenorhabditis elegans, the initiation of axon regeneration is positively regulated by the JNK-MAPK pathway. In this study, we show that integrin, Rac-GTPase, and several other molecules, all of which are known to regulate engulfment of apoptotic cells during development, also regulate axon regeneration. This signaling module activates the JNK-MAPK cascade via MAX-2, a PAK-like protein kinase that binds Rac. Our findings thus reveal that the molecular machinery used for engulfment of apoptotic cells also promotes axon regeneration through activation of the JNK pathway. Copyright © 2016 the authors 0270-6474/16/369710-12$15.00/0.

Activation of the Cph1-Dependent MAP Kinase Signaling Pathway Induces White-Opaque Switching in Candida albicans

PubMed Central

Ramírez-Zavala, Bernardo; Weyler, Michael; Gildor, Tsvia; Schmauch, Christian; Kornitzer, Daniel; Arkowitz, Robert; Morschhäuser, Joachim

2013-01-01

Depending on the environmental conditions, the pathogenic yeast Candida albicans can undergo different developmental programs, which are controlled by dedicated transcription factors and upstream signaling pathways. C. albicans strains that are homozygous at the mating type locus can switch from the normal yeast form (white) to an elongated cell type (opaque), which is the mating-competent form of this fungus. Both white and opaque cells use the Ste11-Hst7-Cek1/Cek2 MAP kinase signaling pathway to react to the presence of mating pheromone. However, while opaque cells employ the transcription factor Cph1 to induce the mating response, white cells recruit a different downstream transcription factor, Tec1, to promote the formation of a biofilm that facilitates mating of opaque cells in the population. The switch from the white to the opaque cell form is itself induced by environmental signals that result in the upregulation of the transcription factor Wor1, the master regulator of white-opaque switching. To get insight into the upstream signaling pathways controlling the switch, we expressed all C. albicans protein kinases from a tetracycline-inducible promoter in a switching-competent strain. Screening of this library of strains showed that a hyperactive form of Ste11 lacking its N-terminal domain (Ste11ΔN467) efficiently stimulated white cells to switch to the opaque phase, a behavior that did not occur in response to pheromone. Ste11ΔN467-induced switching specifically required the downstream MAP kinase Cek1 and its target transcription factor Cph1, but not Cek2 and Tec1, and forced expression of Cph1 also promoted white-opaque switching in a Wor1-dependent manner. Therefore, depending on the activation mechanism, components of the pheromone-responsive MAP kinase pathway can be reconnected to stimulate an alternative developmental program, switching of white cells to the mating-competent opaque phase. PMID:24130492

Cryptotanshinone suppresses the proliferation and induces the apoptosis of pancreatic cancer cells via the STAT3 signaling pathway.

PubMed

Ge, Yuqing; Yang, Bo; Chen, Zhe; Cheng, Rubin

2015-11-01

Pancreatic cancer remains a challenging disease worldwide. Cryptotanshinone (CPT) is one of the active constituents of Salvia miltiorrhiza Bunge and exhibits significant antitumor activities in several human cancer cells. However, the efficacy and molecular mechanism of CPT in pancreatic cancer remains to be elucidated. In the present study, the effect of CPT on the proliferation, apoptosis and cell cycle of human pancreatic cancer cell BxPC‑3 cells was evaluated. The results demonstrated that CPT inhibited proliferation of the BxPC‑3 cells in a concentration‑dependent manner, and significantly induced cell apoptosis and cell cycle arrest. The protein levels of cleaved caspase‑3, caspase‑9 and poly ADP ribose polymerase were upregulated, while the levels of c‑myc, survivin and cyclin D1 were downregulated following treatment with CPT. In addition, CPT decreased the activities of signal transducer and activator of transcription 3 (STAT3) and several upstream regulatory signaling pathways after 24 h. However, CPT only inhibited the phosphorylation of STAT3 Tyr705 within 30 min, without marked effects on the phosphorylation of the other proteins. These results suggested that the inhibition of STAT3 activity by CPT was directly and independent of the upstream regulators in human pancreatic cancer. The present study demonstrated that CPT exerts anticancer effects by inducing apoptosis and cell cycle arrest via inhibition of the STAT3 signaling pathway in human BxPC-3 cells.

Intraflagellar transport protein 122 antagonizes Sonic Hedgehog signaling and controls ciliary localization of pathway components.

PubMed

Qin, Jian; Lin, Yulian; Norman, Ryan X; Ko, Hyuk W; Eggenschwiler, Jonathan T

2011-01-25

Primary cilia are required for proper Sonic Hedgehog (Shh) signaling in mammals. However, their role in the signal transduction process remains unclear. We have identified sister of open brain (sopb), a null allele of mouse Intraflagellar transport protein 122 (Ift122). IFT122 negatively regulates the Shh pathway in the cilium at a step downstream of the Shh ligand and the transmembrane protein Smoothened, but upstream of the Gli2 transcription factor. Ift122(sopb) mutants generate primary cilia, but they show features of defective retrograde intraflagellar transport. IFT122 controls the ciliary localization of Shh pathway regulators in different ways. Disruption of IFT122 leads to accumulation of Gli2 and Gli3 at cilia tips while blocking the ciliary localization of the antagonist TULP3. Suppressor of Fused and Smoothened localize to the cilium through an IFT122-independent mechanism. We propose that the balance between positive and negative regulators of the Shh pathway at the cilium tip controls the output of the pathway and that Shh signaling regulates this balance through intraflagellar transport.

PTPRS Regulates Colorectal Cancer RAS Pathway Activity by Inactivating Erk and Preventing Its Nuclear Translocation.

PubMed

Davis, Thomas B; Yang, Mingli; Schell, Michael J; Wang, Heiman; Ma, Le; Pledger, W Jack; Yeatman, Timothy J

2018-06-18

Colorectal cancer (CRC) growth and progression is frequently driven by RAS pathway activation through upstream growth factor receptor activation or through mutational activation of KRAS or BRAF. Here we describe an additional mechanism by which the RAS pathway may be modulated in CRC. PTPRS, a receptor-type protein tyrosine phosphatase, appears to regulate RAS pathway activation through ERK. PTPRS modulates ERK phosphorylation and subsequent translocation to the nucleus. Native mutations in PTPRS, present in ~10% of CRC, may reduce its phosphatase activity while increasing ERK activation and downstream transcriptional signaling.

Bilateral crosstalk of rho- and extracellular-signal-regulated-kinase (ERK) pathways is confined to an unidirectional mode in spinal muscular atrophy (SMA).

PubMed

Hensel, Niko; Stockbrügger, Inga; Rademacher, Sebastian; Broughton, Natasha; Brinkmann, Hella; Grothe, Claudia; Claus, Peter

2014-03-01

Rho-kinase (ROCK) as well as extracellular signal regulated kinase (ERK) control actin cytoskeletal organization thereby regulating dynamic changes of cellular morphology. In neurons, motility processes such as axonal guidance and neurite outgrowth demand a fine regulation of upstream pathways. Here we demonstrate a bilateral ROCK-ERK information flow in neurons. This process is shifted towards an unidirectional crosstalk in a model of the neurodegenerative disease Spinal Muscular Atrophy (SMA), ultimately leading to neurite outgrowth dysregulations. As both pathways are of therapeutic relevance for SMA, our results argue for a combinatorial ROCK/ERK-targeting as a future treatment strategy. Copyright © 2013 Elsevier Inc. All rights reserved.

A Role for MORE AXILLARY GROWTH1 (MAX1) in Evolutionary Diversity in Strigolactone Signaling Upstream of MAX21[C][W][OA

PubMed Central

Challis, Richard J.; Hepworth, Jo; Mouchel, Céline; Waites, Richard; Leyser, Ottoline

2013-01-01

Strigolactones (SLs) are carotenoid-derived phytohormones with diverse roles. They are secreted from roots as attractants for arbuscular mycorrhizal fungi and have a wide range of endogenous functions, such as regulation of root and shoot system architecture. To date, six genes associated with SL synthesis and signaling have been molecularly identified using the shoot-branching mutants more axillary growth (max) of Arabidopsis (Arabidopsis thaliana) and dwarf (d) of rice (Oryza sativa). Here, we present a phylogenetic analysis of the MAX/D genes to clarify the relationships of each gene with its wider family and to allow the correlation of events in the evolution of the genes with the evolution of SL function. Our analysis suggests that the notion of a distinct SL pathway is inappropriate. Instead, there may be a diversity of SL-like compounds, the response to which requires a D14/D14-like protein. This ancestral system could have been refined toward distinct ligand-specific pathways channeled through MAX2, the most downstream known component of SL signaling. MAX2 is tightly conserved among land plants and is more diverged from its nearest sister clade than any other SL-related gene, suggesting a pivotal role in the evolution of SL signaling. By contrast, the evidence suggests much greater flexibility upstream of MAX2. The MAX1 gene is a particularly strong candidate for contributing to diversification of inputs upstream of MAX2. Our functional analysis of the MAX1 family demonstrates the early origin of its catalytic function and both redundancy and functional diversification associated with its duplication in angiosperm lineages. PMID:23424248

Modulation of C. elegans Touch Sensitivity Is Integrated at Multiple Levels

PubMed Central

Chen, Xiaoyin

2014-01-01

Sensory systems can adapt to different environmental signals. Here we identify four conditions that modulate anterior touch sensitivity in Caenorhabditis elegans after several hours and demonstrate that such sensory modulation is integrated at multiple levels to produce a single output. Prolonged vibration involving integrin signaling directly sensitizes the touch receptor neurons (TRNs). In contrast, hypoxia, the dauer state, and high salt reduce touch sensitivity by preventing the release of long-range neuroregulators, including two insulin-like proteins. Integration of these latter inputs occurs at upstream neurohormonal cells and at the insulin signaling cascade within the TRNs. These signals and those from integrin signaling converge to modulate touch sensitivity by regulating AKT kinases and DAF-16/FOXO. Thus, activation of either the integrin or insulin pathways can compensate for defects in the other pathway. This modulatory system integrates conflicting signals from different modalities, and adapts touch sensitivity to both mechanical and non-mechanical conditions. PMID:24806678

A LATS biosensor screen identifies VEGFR as a regulator of the Hippo pathway in angiogenesis.

PubMed

Azad, T; Janse van Rensburg, H J; Lightbody, E D; Neveu, B; Champagne, A; Ghaffari, A; Kay, V R; Hao, Y; Shen, H; Yeung, B; Croy, B A; Guan, K L; Pouliot, F; Zhang, J; Nicol, C J B; Yang, X

2018-03-13

The Hippo pathway is a central regulator of tissue development and homeostasis, and has been reported to have a role during vascular development. Here we develop a bioluminescence-based biosensor that monitors the activity of the Hippo core component LATS kinase. Using this biosensor and a library of small molecule kinase inhibitors, we perform a screen for kinases modulating LATS activity and identify VEGFR as an upstream regulator of the Hippo pathway. We find that VEGFR activation by VEGF triggers PI3K/MAPK signaling, which subsequently inhibits LATS and activates the Hippo effectors YAP and TAZ. We further show that the Hippo pathway is a critical mediator of VEGF-induced angiogenesis and tumor vasculogenic mimicry. Thus, our work offers a biosensor tool for the study of the Hippo pathway and suggests a role for Hippo signaling in regulating blood vessel formation in physiological and pathological settings.

Differential Cellular Responses to Hedgehog Signalling in Vertebrates—What is the Role of Competence?

PubMed Central

Kiecker, Clemens; Graham, Anthony; Logan, Malcolm

2016-01-01

A surprisingly small number of signalling pathways generate a plethora of cellular responses ranging from the acquisition of multiple cell fates to proliferation, differentiation, morphogenesis and cell death. These diverse responses may be due to the dose-dependent activities of signalling factors, or to intrinsic differences in the response of cells to a given signal—a phenomenon called differential cellular competence. In this review, we focus on temporal and spatial differences in competence for Hedgehog (HH) signalling, a signalling pathway that is reiteratively employed in embryos and adult organisms. We discuss the upstream signals and mechanisms that may establish differential competence for HHs in a range of different tissues. We argue that the changing competence for HH signalling provides a four-dimensional framework for the interpretation of the signal that is essential for the emergence of functional anatomy. A number of diseases—including several types of cancer—are caused by malfunctions of the HH pathway. A better understanding of what provides differential competence for this signal may reveal HH-related disease mechanisms and equip us with more specific tools to manipulate HH signalling in the clinic. PMID:29615599

Experimentally-Derived Fibroblast Gene Signatures Identify Molecular Pathways Associated with Distinct Subsets of Systemic Sclerosis Patients in Three Independent Cohorts

PubMed Central

Johnson, Michael E.; Mahoney, J. Matthew; Taroni, Jaclyn; Sargent, Jennifer L.; Marmarelis, Eleni; Wu, Ming-Ru; Varga, John; Hinchcliff, Monique E.; Whitfield, Michael L.

2015-01-01

Genome-wide expression profiling in systemic sclerosis (SSc) has identified four ‘intrinsic’ subsets of disease (fibroproliferative, inflammatory, limited, and normal-like), each of which shows deregulation of distinct signaling pathways; however, the full set of pathways contributing to this differential gene expression has not been fully elucidated. Here we examine experimentally derived gene expression signatures in dermal fibroblasts for thirteen different signaling pathways implicated in SSc pathogenesis. These data show distinct and overlapping sets of genes induced by each pathway, allowing for a better understanding of the molecular relationship between profibrotic and immune signaling networks. Pathway-specific gene signatures were analyzed across a compendium of microarray datasets consisting of skin biopsies from three independent cohorts representing 80 SSc patients, 4 morphea, and 26 controls. IFNα signaling showed a strong association with early disease, while TGFβ signaling spanned the fibroproliferative and inflammatory subsets, was associated with worse MRSS, and was higher in lesional than non-lesional skin. The fibroproliferative subset was most strongly associated with PDGF signaling, while the inflammatory subset demonstrated strong activation of innate immune pathways including TLR signaling upstream of NF-κB. The limited and normal-like subsets did not show associations with fibrotic and inflammatory mediators such as TGFβ and TNFα. The normal-like subset showed high expression of genes associated with lipid signaling, which was absent in the inflammatory and limited subsets. Together, these data suggest a model by which IFNα is involved in early disease pathology, and disease severity is associated with active TGFβ signaling. PMID:25607805

Fragment-based drug discovery of potent and selective MKK3/6 inhibitors.

PubMed

Adams, Mark; Kobayashi, Toshitake; Lawson, J David; Saitoh, Morihisa; Shimokawa, Kenichiro; Bigi, Simone V; Hixon, Mark S; Smith, Christopher R; Tatamiya, Takayuki; Goto, Masayuki; Russo, Joseph; Grimshaw, Charles E; Swann, Steven

2016-02-01

The MAPK signaling cascade, comprised of several linear and intersecting pathways, propagates signaling into the nucleus resulting in cytokine and chemokine release. The Map Kinase Kinase isoforms 3 and 6 (MKK3 and MKK6) are responsible for the phosphorylation and activation of p38, and are hypothesized to play a key role in regulating this pathway without the redundancy seen in downstream effectors. Using FBDD, we have discovered efficient and selective inhibitors of MKK3 and MKK6 that can serve as tool molecules to help further understand the role of these kinases in MAPK signaling, and the potential impact of inhibiting kinases upstream of p38. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Role of Notch Signaling Pathway in Breast Cancer Pathogenesis

DTIC Science & Technology

2005-07-01

breast cancer cells, I tested whether ErbB2 overexpression will cooperate with Notch in HMLE cells. While overexpression of activated Notch1 failed to...tyrosine kinase upstream of Ras normally found overexpressed in many breast cancers , also failed to transform HMLE cells. These observations suggested...cooperation between Notch1IC and ErbB2 signaling in transforming HMLE cells. Breast cancers typically do not harbor oncogenic Ras mutations; nevertheless

Cancer cachexia: mediators, signaling, and metabolic pathways.

PubMed

Fearon, Kenneth C H; Glass, David J; Guttridge, Denis C

2012-08-08

Cancer cachexia is characterized by a significant reduction in body weight resulting predominantly from loss of adipose tissue and skeletal muscle. Cachexia causes reduced cancer treatment tolerance and reduced quality and length of life, and remains an unmet medical need. Therapeutic progress has been impeded, in part, by the marked heterogeneity of mediators, signaling, and metabolic pathways both within and between model systems and the clinical syndrome. Recent progress in understanding conserved, molecular mechanisms of skeletal muscle atrophy/hypertrophy has provided a downstream platform for circumventing the variations and redundancy in upstream mediators and may ultimately translate into new targeted therapies. Copyright © 2012 Elsevier Inc. All rights reserved.

Signaling Architectures that Transmit Unidirectional Information Despite Retroactivity.

PubMed

Shah, Rushina; Del Vecchio, Domitilla

2017-08-08

A signaling pathway transmits information from an upstream system to downstream systems, ideally in a unidirectional fashion. A key obstacle to unidirectional transmission is retroactivity, the additional reaction flux that affects a system once its species interact with those of downstream systems. This raises the fundamental question of whether signaling pathways have developed specialized architectures that overcome retroactivity and transmit unidirectional signals. Here, we propose a general procedure based on mathematical analysis that provides an answer to this question. Using this procedure, we analyze the ability of a variety of signaling architectures to transmit one-way (from upstream to downstream) signals, as key biological parameters are tuned. We find that single stage phosphorylation and phosphotransfer systems that transmit signals from a kinase show a stringent design tradeoff that hampers their ability to overcome retroactivity. Interestingly, cascades of these architectures, which are highly represented in nature, can overcome this tradeoff and thus enable unidirectional transmission. By contrast, phosphotransfer systems, and single and double phosphorylation cycles that transmit signals from a substrate, are unable to mitigate retroactivity effects, even when cascaded, and hence are not well suited for unidirectional information transmission. These results are largely independent of the specific reaction-rate constant values, and depend on the topology of the architectures. Our results therefore identify signaling architectures that, allowing unidirectional transmission of signals, embody modular processes that conserve their input/output behavior across multiple contexts. These findings can be used to decompose natural signal transduction networks into modules, and at the same time, they establish a library of devices that can be used in synthetic biology to facilitate modular circuit design. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Regulation of the Hippo-YAP Pathway by Glucose Sensor O-GlcNAcylation.

PubMed

Peng, Changmin; Zhu, Yue; Zhang, Wanjun; Liao, Qinchao; Chen, Yali; Zhao, Xinyuan; Guo, Qiang; Shen, Pan; Zhen, Bei; Qian, Xiaohong; Yang, Dong; Zhang, Jin-San; Xiao, Dongguang; Qin, Weijie; Pei, Huadong

2017-11-02

The Hippo pathway is crucial in organ size control and tissue homeostasis, with deregulation leading to cancer. An extracellular nutrition signal, such as glucose, regulates the Hippo pathway activation. However, the mechanisms are still not clear. Here, we found that the Hippo pathway is directly regulated by the hexosamine biosynthesis pathway (HBP) in response to metabolic nutrients. Mechanistically, the core component of Hippo pathway (YAP) is O-GlcNAcylated by O-GlcNAc transferase (OGT) at serine 109. YAP O-GlcNAcylation disrupts its interaction with upstream kinase LATS1, prevents its phosphorylation, and activates its transcriptional activity. And this activation is not dependent on AMPK. We also identified OGT as a YAP-regulated gene that forms a feedback loop. Finally, we confirmed that glucose-induced YAP O-GlcNAcylation and activation promoted tumorigenesis. Together, our data establish a molecular mechanism and functional significance of the HBP in directly linking extracellular glucose signal to the Hippo-YAP pathway and tumorigenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Regulatory network rewiring for secondary metabolism in Arabidopsis thaliana under various conditions

PubMed Central

2014-01-01

Background Plant secondary metabolites are critical to various biological processes. However, the regulations of these metabolites are complex because of regulatory rewiring or crosstalk. To unveil how regulatory behaviors on secondary metabolism reshape biological processes, we constructed and analyzed a dynamic regulatory network of secondary metabolic pathways in Arabidopsis. Results The dynamic regulatory network was constructed through integrating co-expressed gene pairs and regulatory interactions. Regulatory interactions were either predicted by conserved transcription factor binding sites (TFBSs) or proved by experiments. We found that integrating two data (co-expression and predicted regulatory interactions) enhanced the number of highly confident regulatory interactions by over 10% compared with using single data. The dynamic changes of regulatory network systematically manifested regulatory rewiring to explain the mechanism of regulation, such as in terpenoids metabolism, the regulatory crosstalk of RAV1 (AT1G13260) and ATHB1 (AT3G01470) on HMG1 (hydroxymethylglutaryl-CoA reductase, AT1G76490); and regulation of RAV1 on epoxysqualene biosynthesis and sterol biosynthesis. Besides, we investigated regulatory rewiring with expression, network topology and upstream signaling pathways. Regulatory rewiring was revealed by the variability of genes’ expression: pathway genes and transcription factors (TFs) were significantly differentially expressed under different conditions (such as terpenoids biosynthetic genes in tissue experiments and E2F/DP family members in genotype experiments). Both network topology and signaling pathways supported regulatory rewiring. For example, we discovered correlation among the numbers of pathway genes, TFs and network topology: one-gene pathways (such as δ-carotene biosynthesis) were regulated by a fewer TFs, and were not critical to metabolic network because of their low degrees in topology. Upstream signaling pathways of 50 TFs were identified to comprehend the underlying mechanism of TFs’ regulatory rewiring. Conclusion Overall, this dynamic regulatory network largely improves the understanding of perplexed regulatory rewiring in secondary metabolism in Arabidopsis. PMID:24993737

Phosphorylation of basic helix-loop-helix transcription factor Twist in development and disease.

PubMed

Xue, Gongda; Hemmings, Brian A

2012-02-01

The transcription factor Twist plays vital roles during embryonic development through regulating/controlling cell migration. However, postnatally, in normal physiological settings, Twist is either not expressed or inactivated. Increasing evidence shows a strong correlation between Twist reactivation and both cancer progression and malignancy, where the transcriptional activities of Twist support cancer cells to disseminate from primary tumours and subsequently establish a secondary tumour growth in distant organs. However, it is largely unclear how this signalling programme is reactivated or what signalling pathways regulate its activity. The present review discusses recent advances in Twist regulation and activity, with a focus on phosphorylation-dependent Twist activity, potential upstream kinases and the contribution of these factors in transducing biological signals from upstream signalling complexes. The recent advances in these areas have shed new light on how phosphorylation-dependent regulation of the Twist proteins promotes or suppresses Twist activity, leading to differential regulation of Twist transcriptional targets and thereby influencing cell fate.

The mTOR signalling pathway in cancer and the potential mTOR inhibitory activities of natural phytochemicals.

PubMed

Tan, Heng Kean; Moad, Ahmed Ismail Hassan; Tan, Mei Lan

2014-01-01

The mammalian target of rapamycin (mTOR) kinase plays an important role in regulating cell growth and cell cycle progression in response to cellular signals. It is a key regulator of cell proliferation and many upstream activators and downstream effectors of mTOR are known to be deregulated in various types of cancers. Since the mTOR signalling pathway is commonly activated in human cancers, many researchers are actively developing inhibitors that target key components in the pathway and some of these drugs are already on the market. Numerous preclinical investigations have also suggested that some herbs and natural phytochemicals, such as curcumin, resveratrol, timosaponin III, gallic acid, diosgenin, pomegranate, epigallocatechin gallate (EGCC), genistein and 3,3'-diindolylmethane inhibit the mTOR pathway either directly or indirectly. Some of these natural compounds are also in the clinical trial stage. In this review, the potential anti-cancer and chemopreventive activities and the current status of clinical trials of these phytochemicals are discussed.

Zyxin-Siah2–Lats2 axis mediates cooperation between Hippo and TGF-β signalling pathways

PubMed Central

Ma, Biao; Cheng, Hongcheng; Gao, Ruize; Mu, Chenglong; Chen, Ling; Wu, Shian; Chen, Quan; Zhu, Yushan

2016-01-01

The evolutionarily conserved Hippo pathway is a regulator that controls organ size, cell growth and tissue homeostasis. Upstream signals of the Hippo pathway have been widely studied, but how microenvironmental factors coordinately regulate this pathway remains unclear. In this study, we identify LIM domain protein Zyxin, as a scaffold protein, that in response to hypoxia and TGF-β stimuli, forms a ternary complex with Lats2 and Siah2 and stabilizes their interaction. This interaction facilitates Lats2 ubiquitination and degradation, Yap dephosphorylation and subsequently activation. We show that Zyxin is required for TGF-β and hypoxia-induced Lats2 downregulation and deactivation of Hippo signalling in MDA-MB-231 cells. Depletion of Zyxin impairs the capability of cell migration, proliferation and tumourigenesis in a xenograft model. Zyxin is upregulated in human breast cancer and positively correlates with histological stages and metastasis. Our study demonstrates that Zyxin-Lats2–Siah2 axis may serve as a potential therapeutic target in cancer treatment. PMID:27030211

Acrylamide up-regulates cyclooxygenase-2 expression through the MEK/ERK signaling pathway in mouse epidermal cells.

PubMed

Lim, Tae-Gyu; Lee, Bo Kyung; Kwon, Jung Yeon; Jung, Sung Keun; Lee, Ki Won

2011-06-01

Acrylamide is formed during cooking processes and is present in many foods. Accumulating evidence suggests that AA is carcinogenic, but the underlying mechanism remains unclear. Here, we investigated the carcinogenesis mechanisms of AA. AA increased the COX-2 expression. Two major transcription factors, AP-1 and NF-κB, were activated by AA treatment. AA induced the ERK phosphorylation, and this was abolished by the treatment of U0126, a pharmacological inhibitor of MEK, an upstream kinase of ERK. AA-induced expression and promoter activity of COX-2 were suppressed by U0126. U0126 treatment attenuated AA-induced transactivation of AP-1 and NF-κB, suggesting that the MEK/ERK signaling pathway regulates COX-2 expression. In addition, myricetin, a natural inhibitor of the MEK/ERK signal pathway, reduced AA-induced activation of the COX-2 promoter as well as activation of AP-1 and NF-κB. Collectively, these results suggest that the ability of AA to up-regulate COX-2 expression through the MEK/ERK signaling pathway underlies AA carcinogenicity. Copyright © 2011 Elsevier Ltd. All rights reserved.

Biliverdin Reductase: More than a Namesake – The Reductase, Its Peptide Fragments, and Biliverdin Regulate Activity of the Three Classes of Protein Kinase C

PubMed Central

Gibbs, Peter E. M.; Tudor, Cicerone; Maines, Mahin. D.

2012-01-01

The expanse of human biliverdin reductase (hBVR) functions in the cells is arguably unmatched by any single protein. hBVR is a Ser/Thr/Tyr-kinase, a scaffold protein, a transcription factor, and an intracellular transporter of gene regulators. hBVR is an upstream activator of the insulin/IGF-1 signaling pathway and of protein kinase C (PKC) kinases in the two major arms of the pathway. In addition, it is the sole means for generating the antioxidant bilirubin-IXα. hBVR is essential for activation of ERK1/2 kinases by upstream MAPKK-MEK and by PKCδ, as well as the nuclear import and export of ERK1/2. Small fragments of hBVR are potent activators and inhibitors of the ERK kinases and PKCs: as such, they suggest the potential application of BVR-based technology in therapeutic settings. Presently, we have reviewed the function of hBVR in cell signaling with an emphasis on regulation of PKCδ activity. PMID:22419908

Biliverdin reductase: more than a namesake - the reductase, its Peptide fragments, and biliverdin regulate activity of the three classes of protein kinase C.

PubMed

Gibbs, Peter E M; Tudor, Cicerone; Maines, Mahin D

2012-01-01

The expanse of human biliverdin reductase (hBVR) functions in the cells is arguably unmatched by any single protein. hBVR is a Ser/Thr/Tyr-kinase, a scaffold protein, a transcription factor, and an intracellular transporter of gene regulators. hBVR is an upstream activator of the insulin/IGF-1 signaling pathway and of protein kinase C (PKC) kinases in the two major arms of the pathway. In addition, it is the sole means for generating the antioxidant bilirubin-IXα. hBVR is essential for activation of ERK1/2 kinases by upstream MAPKK-MEK and by PKCδ, as well as the nuclear import and export of ERK1/2. Small fragments of hBVR are potent activators and inhibitors of the ERK kinases and PKCs: as such, they suggest the potential application of BVR-based technology in therapeutic settings. Presently, we have reviewed the function of hBVR in cell signaling with an emphasis on regulation of PKCδ activity.

Signaling intermediates (MAPK and PI3K) as therapeutic targets in NSCLC.

PubMed

Ciuffreda, Ludovica; Incani, Ursula Cesta; Steelman, Linda S; Abrams, Stephen L; Falcone, Italia; Curatolo, Anais Del; Chappell, William H; Franklin, Richard A; Vari, Sabrina; Cognetti, Francesco; McCubrey, James A; Milella, Michele

2014-01-01

The RAS/RAF/MEK/ ERK and the PI3K/AKT/mTOR pathways govern fundamental physiological processes, such as cell proliferation, differentiation, metabolism, cytoskeleton reorganization and cell death and survival. Constitutive activation of these signal transduction pathways is a required hallmark of cancer and dysregulation, on either genetic or epigenetic grounds, of these pathways has been implicated in the initiation, progression and metastastic spread of lung cances. Targeting components of the MAPK and PI3K cascades is thus an attractive strategy in the development of novel therapeutic approaches to treat lung cancer, although the use of single pathway inhibitors has met with limited clinical success so far. Indeed, the presence of intra- and inter-pathway compensatory loops that re-activate the very same cascade, either upstream or downstream the point of pharmacological blockade, or activate the alternate pathway following the blockade of one signaling cascade has been demonstrated, potentially driving preclinical (and possibly clinical) resistance. Therefore, the blockade of both pathways with combinations of signaling inhibitors might result in a more efficient anti-tumor effect, and thus potentially overcome and/or delay clinical resistance, as compared with single agent. The current review aims at summarizing the current status of preclinical and clinical research with regard to pathway crosstalks between the MAPK and PI3K cascades in NSCLC and the rationale for combined therapeutic pathway targeting.

Time-dependent LXR/RXR pathway modulation characterizes capillary remodeling in inflammatory corneal neovascularization.

PubMed

Mukwaya, Anthony; Lennikov, Anton; Xeroudaki, Maria; Mirabelli, Pierfrancesco; Lachota, Mieszko; Jensen, Lasse; Peebo, Beatrice; Lagali, Neil

2018-05-01

Inflammation in the normally immune-privileged cornea can initiate a pathologic angiogenic response causing vision-threatening corneal neovascularization. Inflammatory pathways, however, are numerous, complex and are activated in a time-dependent manner. Effective resolution of inflammation and associated angiogenesis in the cornea requires knowledge of these pathways and their time dependence, which has, to date, remained largely unexplored. Here, using a model of endogenous resolution of inflammation-induced corneal angiogenesis, we investigate the time dependence of inflammatory genes in effecting capillary regression and the return of corneal transparency. Endogenous capillary regression was characterized by a progressive thinning and remodeling of angiogenic capillaries and inflammatory cell retreat in vivo in the rat cornea. By whole-genome longitudinal microarray analysis, early suppression of VEGF ligand-receptor signaling and inflammatory pathways preceded an unexpected later-phase preferential activation of LXR/RXR, PPARα/RXRα and STAT3 canonical pathways, with a concurrent attenuation of LPS/IL-1 inhibition of RXR function and Wnt/β-catenin signaling pathways. Potent downstream inflammatory cytokines such as Cxcl5, IL-1β, IL-6 and Ccl2 were concomitantly downregulated during the remodeling phase. Upstream regulators of the inflammatory pathways included Socs3, Sparc and ApoE. A complex and coordinated time-dependent interplay between pro- and anti-inflammatory signaling pathways highlights a potential anti-inflammatory role of LXR/RXR, PPARα/RXRα and STAT3 signaling pathways in resolving inflammatory corneal angiogenesis.

Tetramethylpyrazine attenuates TNF-α-induced iNOS expression in human endothelial cells: Involvement of Syk-mediated activation of PI3K-IKK-IκB signaling pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Zhen; Li, Zhiliang; Chen, Song

2013-08-15

Endothelial cells produce nitric oxide (NO) by activation of constitutive nitric oxide synthase (NOS) and transcription of inducible NO synthase (iNOS). We explored the effect of tetramethylpyrazine (TMP), a compound derived from chuanxiong, on tumor necrosis factor (TNF)-α-induced iNOS in human umbilical vein endothelial cells (HUVECs) and explored the signal pathways involved by using RT-PCR and Western blot. TMP suppressed TNF-α-induced expression of iNOS by inhibiting IκB kinase (IKK) phosphorylation, IκB degradation and nuclear factor κB (NF-κB) nuclear translocation, which were required for NO gene transcription. Exposure to wortmannin abrogated IKK/IκB/NF-κB-mediated iNOS expression, suggesting activation of such a signal pathwaymore » might be phosphoinositide-3-kinase (PI3K) dependent. Spleen tyrosine kinase (Syk) inhibitor piceatannol significantly inhibited NO production. Furthermore, piceatannol obviously suppressed TNF-α-induced IκB phosphorylation and the downstream NF-κB activation, suggesting that Syk is an upstream key regulator in the activation of PI3K/IKK/IκB-mediated signaling. TMP significantly inhibited TNF-α-induced phosphorylation of Syk and PI3K. Our data indicate that TMP might repress iNOS expression, at least in part, through its inhibitory effect of Syk-mediated PI3K phosphorylation in TNF-α-stimulated HUVECs. -- Highlights: •TMP suppressed TNF-α-induced expression of iNOS by inhibiting IKK/IκB/NF-κB pathway. •PI3K inhibitor wortmannin abrogated IKK/IκB/NF-κB-mediated iNOS expression. •Syk inhibitor piceatannol repressed PI3K/IKK/IκB mediated NO production. •Syk is an upstream regulator in the activation of PI3K/IKK/IκB-mediated signaling. •TMP might repress iNOS expression through Syk-mediated PI3K pathway.« less

PI3K: A Crucial Piece in the RAS Signaling Puzzle.

PubMed

Krygowska, Agata Adelajda; Castellano, Esther

2018-06-01

RAS proteins are key signaling switches essential for control of proliferation, differentiation, and survival of eukaryotic cells. RAS proteins are mutated in 30% of human cancers. In addition, mutations in upstream or downstream signaling components also contribute to oncogenic activation of the pathway. RAS proteins exert their functions through activation of several signaling pathways and dissecting the contributions of these effectors in normal cells and in cancer is an ongoing challenge. In this review, we summarize our current knowledge about how RAS regulates type I phosphatidylinositol 3-kinase (PI3K), one of the main RAS effectors. RAS signaling through PI3K is necessary for normal lymphatic vasculature development and for RAS-induced transformation in vitro and in vivo, especially in lung cancer, where it is essential for tumor initiation and necessary for tumor maintenance. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

C-Jun N-terminal Kinase and Apoptotic Signaling in Prostate Cancer

DTIC Science & Technology

2002-01-01

determine cell fate. Curcumin (diferuloylmethane), a dietary pigment from Curcuma longa , gives the golden-yellow color and unique flavor to curry...suggesting that p53 is not required for JNK-mediated apoptosis. 4-HPR-induced apoptosis in LNCaP cells was suppressed by curcumin , which inhibits JNK...Previously, we found that curcumin may affect the JNK pathway by interfering with the signaling molecule(s) at the same level or proximally upstream of the

Left-right asymmetry specification in amphioxus: review and prospects.

PubMed

Soukup, Vladimir

2017-01-01

Extant bilaterally symmetrical animals usually show asymmetry in the arrangement of their inner organs. However, the exaggerated left-right (LR) asymmetry in amphioxus represents a true peculiarity among them. The amphioxus larva shows completely disparate fates of left and right body sides, so that organs associated with pharynx are either positioned exclusively on the left or on the right side. Moreover, segmented paraxial structures such as muscle blocks and their neuronal innervation show offset arrangement between the sides making it difficult to propose any explanation or adaptivity to larval and adult life. First LR asymmetries can be traced back to an early embryonic period when morphological asymmetries are preceded by molecular asymmetries driven by the action of the Nodal signaling pathway. This review sums up recent advances in understanding LR asymmetry specification in amphioxus and proposes upstream events that may regulate asymmetric Nodal signaling. These events include the presence of the vertebrate-like LR organizer and a cilia-driven fluid flow that may be involved in the breaking of bilateral symmetry. The upstream pathways comprising the ion flux, Delta/Notch, Wnt/β-catenin and Wnt/PCP are hypothesized to regulate both formation of the LR organizer and expression of the downstream Nodal signaling pathway genes. These suggestions are in line with what we know from vertebrate and ambulacrarian LR axis specification and are directly testable by experimental manipulations. Thanks to the phylogenetic position of amphioxus, the proposed mechanisms may be helpful in understanding the evolution of LR axis specification across deuterostomes.

Reverse Engineering a Signaling Network Using Alternative Inputs

PubMed Central

Tanaka, Hiromasa; Yi, Tau-Mu

2009-01-01

One of the goals of systems biology is to reverse engineer in a comprehensive fashion the arrow diagrams of signal transduction systems. An important tool for ordering pathway components is genetic epistasis analysis, and here we present a strategy termed Alternative Inputs (AIs) to perform systematic epistasis analysis. An alternative input is defined as any genetic manipulation that can activate the signaling pathway instead of the natural input. We introduced the concept of an “AIs-Deletions matrix” that summarizes the outputs of all combinations of alternative inputs and deletions. We developed the theory and algorithms to construct a pairwise relationship graph from the AIs-Deletions matrix capturing both functional ordering (upstream, downstream) and logical relationships (AND, OR), and then interpreting these relationships into a standard arrow diagram. As a proof-of-principle, we applied this methodology to a subset of genes involved in yeast mating signaling. This experimental pilot study highlights the robustness of the approach and important technical challenges. In summary, this research formalizes and extends classical epistasis analysis from linear pathways to more complex networks, facilitating computational analysis and reconstruction of signaling arrow diagrams. PMID:19898612

PAR1 participates in the ability of multidrug resistance and tumorigenesis by controlling Hippo-YAP pathway.

PubMed

Fujimoto, Daisuke; Ueda, Yuki; Hirono, Yasuo; Goi, Takanori; Yamaguchi, Akio

2015-10-27

The Hippo pathway significantly correlates with organ size control and tumorigenesis. The activity of YAP/TAZ, a transducer of the Hippo pathway, is required to sustain self-renewal and tumor-initiation capacities in cancer stem cells (CSCs). But, upstream signals that control the mammalian Hippo pathway have not been well understood. Here, we reveal a connection between the Protease-activated receptor 1 (PAR1) signaling pathway and the Hippo-YAP pathway in gastric cancer stem-like cells. The selective PAR1 agonist TFLLR-NH2 induces an increase in the fraction of side population cells which is enriched in CSCs, and promotes tumorigenesis, multi cancer drug resistance, cell morphological change, and cell invasion which are characteristics of CSCs. In addition, PAR1 activation inhibits the Hippo-YAP pathway kinase Lats via Rho GTPase. Lats kinase inhibition in turn results in increased nuclear localization of dephosphorylated YAP. Furthermore, PAR1 activation confers CSCs related traits via the Hippo-YAP pathway, and the Hippo-YAP pathway correlates with epithelial mesenchymal transition which is induced by PAR1 activation. Our research suggests that the PAR1 signaling deeply participates in the ability of multi drug resistance and tumorigenesis through interactions with the Hippo-YAP pathway signaling in gastric cancer stem-like cells. We presume that inhibited YAP is a new therapeutic target in the treatment human gastric cancer invasion and metastasis by dysregulated PAR1 or its agonists.

The external amino acid signaling pathway promotes activation of Stp1 and Uga35/Dal81 transcription factors for induction of the AGP1 gene in Saccharomyces cerevisiae.

PubMed Central

Abdel-Sater, Fadi; Iraqui, Ismaïl; Urrestarazu, Antonio; André, Bruno

2004-01-01

Yeast cells respond to the presence of amino acids in their environment by inducing transcription of several amino acid permease genes including AGP1, BAP2, and BAP3. The signaling pathway responsible for this induction involves Ssy1, a permease-like sensor of external amino acids, and culminates with proteolytic cleavage and translocation to the nucleus of the zinc-finger proteins Stp1 and Stp2, the lack of which abolishes induction of BAP2 and BAP3. Here we show that Stp1-but not Stp2-plays an important role in AGP1 induction, although significant induction of AGP1 by amino acids persists in stp1 and stp1 stp2 mutants. This residual induction depends on the Uga35/Dal81 transcription factor, indicating that the external amino acid signaling pathway activates not only Stp1 and Stp2, but also another Uga35/Dal81-dependent transcriptional circuit. Analysis of the AGP1 gene's upstream region revealed that Stp1 and Uga35/Dal81 act synergistically through a 21-bp cis-acting sequence similar to the UAS(AA) element previously found in the BAP2 and BAP3 upstream regions. Although cells growing under poor nitrogen-supply conditions display much higher induction of AGP1 expression than cells growing under good nitrogen-supply conditions, the UAS(AA) itself is totally insensitive to nitrogen availability. Nitrogen-source control of AGP1 induction is mediated by the GATA factor Gln3, likely acting through adjacent 5'-GATA-3' sequences, to amplify the positive effect of UAS(AA). Our data indicate that Stp1 may act in combination with distinct sets of transcription factors, according to the gene context, to promote induction of transcription in response to external amino acids. The data also suggest that Uga35/Dal81 is yet another transcription factor under the control of the external amino acid sensing pathway. Finally, the data show that the TOR pathway mediating global nitrogen control of transcription does not interfere with the external amino acid signaling pathway. PMID:15126393

Coordination of the recruitment of the FANCD2 and PALB2 Fanconi anemia proteins by an ubiquitin signaling network.

PubMed

Bick, Gregory; Zhang, Fan; Meetei, A Ruhikanta; Andreassen, Paul R

2017-06-01

Fanconi anemia (FA) is a chromosome instability syndrome and the 20 identified FA proteins are organized into two main arms which are thought to function at distinct steps in the repair of DNA interstrand crosslinks (ICLs). These two arms include the upstream FA pathway, which culminates in the monoubiquitination of FANCD2 and FANCI, and downstream breast cancer (BRCA)-associated proteins that interact in protein complexes. How, and whether, these two groups of FA proteins are integrated is unclear. Here, we show that FANCD2 and PALB2, as indicators of the upstream and downstream arms, respectively, colocalize independently of each other in response to DNA damage induced by mitomycin C (MMC). We also show that ubiquitin chains are induced by MMC and colocalize with both FANCD2 and PALB2. Our finding that the RNF8 E3 ligase has a role in recruiting FANCD2 and PALB2 also provides support for the hypothesis that the two branches of the FA-BRCA pathway are coordinated by ubiquitin signaling. Interestingly, we find that the RNF8 partner, MDC1, as well as the ubiquitin-binding protein, RAP80, specifically recruit PALB2, while a different ubiquitin-binding protein, FAAP20, functions only in the recruitment of FANCD2. Thus, FANCD2 and PALB2 are not recruited in a single linear pathway, rather we define how their localization is coordinated and integrated by a network of ubiquitin-related proteins. We propose that such regulation may enable upstream and downstream FA proteins to act at distinct steps in the repair of ICLs.

The Hippo pathway in heart development, regeneration, and diseases

PubMed Central

Zhou, Qi; Li, Li; Zhao, Bin; Guan, Kun-Liang

2015-01-01

The heart is the first organ formed during mammalian development. A properly sized and functional heart is vital throughout the entire lifespan. Loss of cardiomyocytes due to injury or diseases leads to heart failure, which is a major cause of human morbidity and mortality. Unfortunately, regenerative potential of the adult heart is very limited. The Hippo pathway is a recently identified signaling cascade that plays an evolutionarily conserved role in organ size control by inhibiting cell proliferation, promoting apoptosis, regulating fates of stem/ progenitor cells, and in some circumstances, limiting cell size. Interestingly, research indicates a key role of this pathway in regulation of cardiomyocyte proliferation and heart size. Inactivation of the Hippo pathway or activation of its downstream effector, the Yes-associated protein (YAP) transcription co-activator, improves cardiac regeneration. Several known upstream signals of the Hippo pathway such as mechanical stress, G-protein-coupled receptor (GPCR) signaling, and oxidative stress, are known to play critical roles in cardiac physiology. In addition, YAP has been shown to regulate cardiomyocyte fate through multiple transcriptional mechanisms. In this review, we summarize and discuss current findings regarding the roles and mechanisms of the Hippo pathway in heart development, injury, and regeneration. PMID:25858067

The hippo pathway in heart development, regeneration, and diseases.

PubMed

Zhou, Qi; Li, Li; Zhao, Bin; Guan, Kun-Liang

2015-04-10

The heart is the first organ formed during mammalian development. A properly sized and functional heart is vital throughout the entire lifespan. Loss of cardiomyocytes because of injury or diseases leads to heart failure, which is a major cause of human morbidity and mortality. Unfortunately, regenerative potential of the adult heart is limited. The Hippo pathway is a recently identified signaling cascade that plays an evolutionarily conserved role in organ size control by inhibiting cell proliferation, promoting apoptosis, regulating fates of stem/progenitor cells, and in some circumstances, limiting cell size. Interestingly, research indicates a key role of this pathway in regulation of cardiomyocyte proliferation and heart size. Inactivation of the Hippo pathway or activation of its downstream effector, the Yes-associated protein transcription coactivator, improves cardiac regeneration. Several known upstream signals of the Hippo pathway such as mechanical stress, G-protein-coupled receptor signaling, and oxidative stress are known to play critical roles in cardiac physiology. In addition, Yes-associated protein has been shown to regulate cardiomyocyte fate through multiple transcriptional mechanisms. In this review, we summarize and discuss current findings on the roles and mechanisms of the Hippo pathway in heart development, injury, and regeneration. © 2015 American Heart Association, Inc.

Microarray pathway analysis indicated that mitogen-activated protein kinase/extracellular signal-regulated kinase and insulin growth factor 1 signaling pathways were inhibited by small interfering RNA against AT-rich interactive domain 1A in endometrial cancer

PubMed Central

Yang, Ye; Bao, Wei; Sang, Zhengyu; Yang, Yongbing; Lu, Meng; Xi, Xiaowei

2018-01-01

Mutations in the gene encoding AT-rich interactive domain 1A (ARID1A) are frequently observed in endometrial cancer (EC) but the molecular mechanisms linking the genetic changes remain to be fully understood. The present study aimed to elucidate the influence of ARID1A mutations on signaling pathways. Missense, synonymous and nonsense heterozygous ARID1A mutations in the EC HEC-1-A cell line were verified by Sanger sequencing. Mutated ARID1A small interfering RNA was transfected into HEC-1-A cells. Biochemical microarray analysis revealed 13 upregulated pathways, 17 downregulated pathways, 14 significantly affected disease states and functions, 662 upstream and 512 downstream genes in mutated ARID1A-depleted HEC-1-A cells, among which the mitogen-activated protein kinase/extracellular signal-regulated kinase and insulin-like growth factor-1 (IGF1) signaling pathways were the 2 most downregulated pathways. Furthermore, the forkhead box protein O1 pathway was upregulated, while the IGF1 receptor, insulin receptor substrate 1 and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit b pathways were downregulated. Carcinoma tumorigenesis, tumor cell mitosis and tumor cell death were significantly upregulated disease states and functions, while cell proliferation and tumor growth were significantly downregulated. The results of the present study suggested that ARID1A may be a potential prognostic and therapeutic molecular drug target for the prevention of EC progression. PMID:29399196

Signaling through protein kinases and transcriptional regulators in Candida albicans.

PubMed

Dhillon, Navneet K; Sharma, Sadhna; Khuller, G K

2003-01-01

The human fungal pathogen Candida albicans switches from a budding yeast form to a polarized hyphal form in response to various external signals. This morphogenetic switching has been implicated in the development of pathogenicity. Several signaling pathways that regulate morphogenesis have been identified, including various transcription factors that either activate or repress hypha-specific genes. Two well-characterized pathways include the MAP kinase cascade and cAMP-dependent protein kinase pathway that regulate the transcription factors Cph1p and Efg1p, respectively. cAMP also appears to interplay with other second messengers: Ca2+, inositol tri-phosphates in regulating yeast-hyphal transition. Other, less-characterized pathways include two component histidine kinases, cyclin-dependent kinase pathway, and condition specific pathways such as pH and embedded growth conditions. Nrg1 and Rfg1 function as transcriptional repressors of hyphal genes via recruitment of Tup1 co-repressor complex. Different upstream signals converge into a common downstream output during hyphal switch. The levels of expression of several genes have been shown to be associated with hyphal morphogenesis rather than with a specific hypha-inducing condition. Hyphal development is also linked to the expression of a range of other virulence factors. This review explains the relative contribution of multiple pathways that could be used by Candida albican cells to sense subtle differences in the growth conditions of its native host environment.

Autocrine motility factor (neuroleukin, phosphohexose isomerase) induces cell movement through 12-lipoxygenase-dependent tyrosine phosphorylation and serine dephosphorylation events.

PubMed

Timár, J; Tóth, S; Tóvári, J; Paku, S; Raz, A

1999-01-01

Autocrine motility factor (AMF) is one of the motility cytokines regulating tumor cell migration, therefore identification of the signaling pathway coupled with it has critical importance. Previous studies revealed several elements of this pathway predominated by lipoxygenase-PKC activations but the role for tyrosine kinases remained questionable. Motility cytokines frequently have mitogenic effect as well, producing activation of overlapping signaling pathways therefore we have used B16a melanoma cells as models where AMF has exclusive motility effect. Our studies revealed that in B16a cells AMF initiated rapid (1-5 min) activation of the protein tyrosine kinase (PTK) cascade inducing phosphorylation of 179, 125, 95 and 40/37 kD proteins which was mediated by upstream cyclo- and lipoxygenases. The phosphorylated proteins were localized to the cortical actin-stress fiber attachment zones in situ by confocal microscopy. On the other hand, AMF receptor activation induced significant decrease in overall serine-phosphorylation level of cellular proteins accompanied by serine phosphorylation of 200, 90, 78 and 65 kd proteins. The decrease in serine phosphorylation was independent of PTKs, PKC as well as cyclo- and lipoxygenases. However, AMF induced robust translocation of PKCalpha to the stress fibers and cortical actin suggesting a critical role for this kinase in the generation of the motility signal. Based on the significant decrease in serine phosphorylation after AMF stimulus in B16a cells we postulated the involvement of putative serine/threonine phosphatase(s) upstream lipoxygenase and activation of the protein tyrosine kinase cascade downstream cyclo- and lipoxygenase(s) in the previously identified autocrine motility signal.

Single-Cell Analysis Reveals that Insulation Maintains Signaling Specificity between Two Yeast MAPK Pathways with Common Components

PubMed Central

Patterson, Jesse C.; Klimenko, Evguenia S.; Thorner, Jeremy

2014-01-01

Eukaryotic cells use multiple mitogen-activated protein kinase (MAPK) cascades to evoke appropriate responses to external stimuli. In Saccharomyces cerevisiae, the MAPK Fus3 is activated by pheromone-binding G protein-coupled receptors to promote mating, whereas the MAPK Hog1 is activated by hyperosmotic stress to elicit the high osmolarity glycerol (HOG) response. Although these MAPK pathways share several upstream components, exposure to either pheromone or osmolyte alone triggers only the appropriate response. We used fluorescent localization- and transcription-specific reporters to assess activation of these pathways in individual cells on the minute and hour timescale, respectively. Dual activation of these two MAPK pathways occurred over a broad range of stimulant concentrations and temporal regimes in wild-type cells subjected to co-stimulation. Thus, signaling specificity is achieved through an “insulation” mechanism, not a “cross-inhibition” mechanism. Furthermore, we showed that there was a critical period during which Hog1 activity had to occur for proper insulation of the HOG pathway. PMID:20959523

The Caenorhabditis elegans EGL-15 Signaling Pathway Implicates a DOS-Like Multisubstrate Adaptor Protein in Fibroblast Growth Factor Signal Transduction

PubMed Central

Schutzman, Jennifer L.; Borland, Christina Z.; Newman, John C.; Robinson, Matthew K.; Kokel, Michelle; Stern, Michael J.

2001-01-01

EGL-15 is a fibroblast growth factor receptor in the nematode Caenorhabditis elegans. Components that mediate EGL-15 signaling have been identified via mutations that confer a Clear (Clr) phenotype, indicative of hyperactivity of this pathway, or a suppressor-of-Clr (Soc) phenotype, indicative of reduced pathway activity. We have isolated a gain-of-function allele of let-60 ras that confers a Clr phenotype and implicated both let-60 ras and components of a mitogen-activated protein kinase cascade in EGL-15 signaling by their Soc phenotype. Epistasis analysis indicates that the gene soc-1 functions in EGL-15 signaling by acting either upstream of or independently of LET-60 RAS. soc-1 encodes a multisubstrate adaptor protein with an amino-terminal pleckstrin homology domain that is structurally similar to the DOS protein in Drosophila and mammalian GAB1. DOS is known to act with the cytoplasmic tyrosine phosphatase Corkscrew (CSW) in signaling pathways in Drosophila. Similarly, the C. elegans CSW ortholog PTP-2 was found to be involved in EGL-15 signaling. Structure-function analysis of SOC-1 and phenotypic analysis of single and double mutants are consistent with a model in which SOC-1 and PTP-2 act together in a pathway downstream of EGL-15 and the Src homology domain 2 (SH2)/SH3-adaptor protein SEM-5/GRB2 contributes to SOC-1-independent activities of EGL-15. PMID:11689700

Calcium sequestration by fungal melanin inhibits calcium-calmodulin signalling to prevent LC3-associated phagocytosis.

PubMed

Kyrmizi, Irene; Ferreira, Helena; Carvalho, Agostinho; Figueroa, Julio Alberto Landero; Zarmpas, Pavlos; Cunha, Cristina; Akoumianaki, Tonia; Stylianou, Kostas; Deepe, George S; Samonis, George; Lacerda, João F; Campos, António; Kontoyiannis, Dimitrios P; Mihalopoulos, Nikolaos; Kwon-Chung, Kyung J; El-Benna, Jamel; Valsecchi, Isabel; Beauvais, Anne; Brakhage, Axel A; Neves, Nuno M; Latge, Jean-Paul; Chamilos, Georgios

2018-05-30

LC3-associated phagocytosis (LAP) is a non-canonical autophagy pathway regulated by Rubicon, with an emerging role in immune homeostasis and antifungal host defence. Aspergillus cell wall melanin protects conidia (spores) from killing by phagocytes and promotes pathogenicity through blocking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent activation of LAP. However, the signalling regulating LAP upstream of Rubicon and the mechanism of melanin-induced inhibition of this pathway remain incompletely understood. Herein, we identify a Ca 2+ signalling pathway that depends on intracellular Ca 2+ sources from endoplasmic reticulum, endoplasmic reticulum-phagosome communication, Ca 2+ release from phagosome lumen and calmodulin (CaM) recruitment, as a master regulator of Rubicon, the phagocyte NADPH oxidase NOX2 and other molecular components of LAP. Furthermore, we provide genetic evidence for the physiological importance of Ca 2+ -CaM signalling in aspergillosis. Finally, we demonstrate that Ca 2+ sequestration by Aspergillus melanin inside the phagosome abrogates activation of Ca 2+ -CaM signalling to inhibit LAP. These findings reveal the important role of Ca 2+ -CaM signalling in antifungal immunity and identify an immunological function of Ca 2+ binding by melanin pigments with broad physiological implications beyond fungal disease pathogenesis.

Anti-inflammatory effects of novel polygonum multiflorum compound via inhibiting NF-κB/MAPK and upregulating the Nrf2 pathways in LPS-stimulated microglia.

PubMed

Park, Sun Young; Jin, Mei Ling; Kang, Nam Jun; Park, Geuntae; Choi, Young-Whan

2017-06-09

The incorporation of Polygonum multiflorum into the diet can result in anti-aging effects owing to its wide range of biological and pharmaceutical properties. We investigated the anti-neuroinflammatory properties of CRPE56IGIH isolated from P. multiflorum by focusing on its role in the induction of phase II antioxidant enzymes and the modulation of upstream signaling pathways. In microglia, CRPE56IGIH significantly inhibited lipopolysaccharide (LPS)-stimulated nitric oxide and prostaglandin E 2 production with nonspecific cytotoxicity. CRPE56IGIH also markedly inhibited LPS-inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 protein and mRNA expression in the same manner as it inhibited nitric oxide and prostaglandin E 2 production. In the control cells, NF-κB transactivation and nuclear translocation occurred at a baseline level, which was significantly increased in response to LPS. However, pretreatment with CRPE56IGIH concentration-dependently inhibited the LPS-induced NF-κB transactivation and nuclear translocation. The phosphorylation of Janus kinase-signal transducers and activators of transcription and mitogen-activated protein kinases was markedly upregulated by LPS, but considerably and dose-dependently inhibited by pretreatment with CRPE56IGIH. Furthermore, CRPE56IGIH induced the expression of phase II antioxidant enzymes, including heme oxygenase-1 (HO-1) and NADPH dehydrogenase quinone-1 (NQO-1). The activation of upstream signaling pathways, such as the Nrf2 pathway, was significantly increased following CRPE56IGIH treatment. Furthermore, the anti-neuroinflammatory effect of CRPE56IGIH was reversed by transfection of Nrf2, HO-1, and NQO-1 siRNA. Our results indicated that CRPE56IGIH isolated from P. multiflorum could be used as a natural anti-neuroinflammatory agent that induces phase II antioxidant enzymes via Nrf2 signaling. Copyright © 2017 Elsevier B.V. All rights reserved.

Wnt/β-catenin signaling integrates patterning and metabolism of the insect growth zone.

PubMed

Oberhofer, Georg; Grossmann, Daniela; Siemanowski, Janna L; Beissbarth, Tim; Bucher, Gregor

2014-12-01

Wnt/β-catenin and hedgehog (Hh) signaling are essential for transmitting signals across cell membranes in animal embryos. Early patterning of the principal insect model, Drosophila melanogaster, occurs in the syncytial blastoderm, where diffusion of transcription factors obviates the need for signaling pathways. However, in the cellularized growth zone of typical short germ insect embryos, signaling pathways are predicted to play a more fundamental role. Indeed, the Wnt/β-catenin pathway is required for posterior elongation in most arthropods, although which target genes are activated in this context remains elusive. Here, we use the short germ beetle Tribolium castaneum to investigate two Wnt and Hh signaling centers located in the head anlagen and in the growth zone of early embryos. We find that Wnt/β-catenin signaling acts upstream of Hh in the growth zone, whereas the opposite interaction occurs in the head. We determine the target gene sets of the Wnt/β-catenin and Hh pathways and find that the growth zone signaling center activates a much greater number of genes and that the Wnt and Hh target gene sets are essentially non-overlapping. The Wnt pathway activates key genes of all three germ layers, including pair-rule genes, and Tc-caudal and Tc-twist. Furthermore, the Wnt pathway is required for hindgut development and we identify Tc-senseless as a novel hindgut patterning gene required in the early growth zone. At the same time, Wnt acts on growth zone metabolism and cell division, thereby integrating growth with patterning. Posterior Hh signaling activates several genes potentially involved in a proteinase cascade of unknown function. © 2014. Published by The Company of Biologists Ltd.

Wnt/β-catenin signaling integrates patterning and metabolism of the insect growth zone

PubMed Central

Oberhofer, Georg; Grossmann, Daniela; Siemanowski, Janna L.; Beissbarth, Tim; Bucher, Gregor

2014-01-01

Wnt/β-catenin and hedgehog (Hh) signaling are essential for transmitting signals across cell membranes in animal embryos. Early patterning of the principal insect model, Drosophila melanogaster, occurs in the syncytial blastoderm, where diffusion of transcription factors obviates the need for signaling pathways. However, in the cellularized growth zone of typical short germ insect embryos, signaling pathways are predicted to play a more fundamental role. Indeed, the Wnt/β-catenin pathway is required for posterior elongation in most arthropods, although which target genes are activated in this context remains elusive. Here, we use the short germ beetle Tribolium castaneum to investigate two Wnt and Hh signaling centers located in the head anlagen and in the growth zone of early embryos. We find that Wnt/β-catenin signaling acts upstream of Hh in the growth zone, whereas the opposite interaction occurs in the head. We determine the target gene sets of the Wnt/β-catenin and Hh pathways and find that the growth zone signaling center activates a much greater number of genes and that the Wnt and Hh target gene sets are essentially non-overlapping. The Wnt pathway activates key genes of all three germ layers, including pair-rule genes, and Tc-caudal and Tc-twist. Furthermore, the Wnt pathway is required for hindgut development and we identify Tc-senseless as a novel hindgut patterning gene required in the early growth zone. At the same time, Wnt acts on growth zone metabolism and cell division, thereby integrating growth with patterning. Posterior Hh signaling activates several genes potentially involved in a proteinase cascade of unknown function. PMID:25395458

Analysis of corkscrew signaling in the Drosophila epidermal growth factor receptor pathway during myogenesis.

PubMed

Johnson Hamlet, M R; Perkins, L A

2001-11-01

The Drosophila nonreceptor protein tyrosine phosphatase, Corkscrew (Csw), functions positively in multiple receptor tyrosine kinase (RTK) pathways, including signaling by the epidermal growth factor receptor (EGFR). Detailed phenotypic analyses of csw mutations have revealed that Csw activity is required in many of the same developmental processes that require EGFR function. However, it is still unclear where in the signaling hierarchy Csw functions relative to other proteins whose activities are also required downstream of the receptor. To address this issue, genetic interaction experiments were performed to place csw gene activity relative to the EGFR, spitz (spi), rhomboid (rho), daughter of sevenless (DOS), kinase-suppressor of ras (ksr), ras1, D-raf, pointed (pnt), and moleskin. We followed the EGFR-dependent formation of VA2 muscle precursor cells as a sensitive assay for these genetic interaction studies. First, we established that Csw has a positive function during mesoderm development. Second, we found that tissue-specific expression of a gain-of-function csw construct rescues loss-of-function mutations in other positive signaling genes upstream of rolled (rl)/MAPK in the EGFR pathway. Third, we were able to infer levels of EGFR signaling in various mutant backgrounds during myogenesis. This work extends previous studies of Csw during Torso and Sevenless RTK signaling to include an in-depth analysis of the role of Csw in the EGFR signaling pathway.

Analysis of corkscrew signaling in the Drosophila epidermal growth factor receptor pathway during myogenesis.

PubMed Central

Johnson Hamlet, M R; Perkins, L A

2001-01-01

The Drosophila nonreceptor protein tyrosine phosphatase, Corkscrew (Csw), functions positively in multiple receptor tyrosine kinase (RTK) pathways, including signaling by the epidermal growth factor receptor (EGFR). Detailed phenotypic analyses of csw mutations have revealed that Csw activity is required in many of the same developmental processes that require EGFR function. However, it is still unclear where in the signaling hierarchy Csw functions relative to other proteins whose activities are also required downstream of the receptor. To address this issue, genetic interaction experiments were performed to place csw gene activity relative to the EGFR, spitz (spi), rhomboid (rho), daughter of sevenless (DOS), kinase-suppressor of ras (ksr), ras1, D-raf, pointed (pnt), and moleskin. We followed the EGFR-dependent formation of VA2 muscle precursor cells as a sensitive assay for these genetic interaction studies. First, we established that Csw has a positive function during mesoderm development. Second, we found that tissue-specific expression of a gain-of-function csw construct rescues loss-of-function mutations in other positive signaling genes upstream of rolled (rl)/MAPK in the EGFR pathway. Third, we were able to infer levels of EGFR signaling in various mutant backgrounds during myogenesis. This work extends previous studies of Csw during Torso and Sevenless RTK signaling to include an in-depth analysis of the role of Csw in the EGFR signaling pathway. PMID:11729154

Insulin resistance, glycemic control and adiposity: key determinants of healthy lifespan.

PubMed

DiStefano, Peter S; Curtis, Rory; Geddes, Bradley J

2007-04-01

Identification of genes and pathways that alter lifespan has allowed for new insights into factors that control the aging process as well as disease. While strong molecular links exist between aging and metabolism, we hypothesize that targeting the mechanisms involved in aging will also give rise to therapeutics that treat other devastating age-related diseases, such as neurodegeneration, cancer, inflammation and cardiovascular disease. Insulin sensitivity, glycemic control and adiposity are not only hallmarks of the major metabolic diseases, type 2 diabetes and obesity, but they also represent significant risk factors for the development of Alzheimer's Disease and cognitive impairment. Insulin/IGF-1 signaling is an important pathway regulating aging and disease in a variety of species, including mammals. Here we describe an important role for the gut-derived peptide ghrelin in upstream signaling through the insulin/IGF-1 pathway and exemplify modulation of ghrelin signaling as an approach to mechanistic treatment of multiple age-related diseases by virtue of its ability to regulate key metabolic functions.

Monoamine oxidase A and repressor R1 are involved in apoptotic signaling pathway.

PubMed

Ou, Xiao-Ming; Chen, Kevin; Shih, Jean C

2006-07-18

Monoamine oxidase A (MAO A) degrades serotonin, norepinephrine, and dopamine and produces reactive oxygen that may cause neuronal cell death. We have previously reported that a novel transcription factor R1 (RAM2/CDCA7L/JPO2) inhibits the MAO A promoter and enzymatic activities. This study reports the roles of MAO A and R1 in apoptosis and proliferation. We have found that in serum starvation-induced apoptosis, p38 kinase, MAO A, and caspase-3 were increased, whereas Bcl-2 and R1 were reduced. Using a p38 kinase inhibitor, R1 overexpression, and MAO A inhibitor, we have shown that MAO A and R1 are downstream of p38 kinase and Bcl-2, but upstream of caspase-3. Inhibition of MAO A prevents cell apoptosis. This notion was further supported by the finding that serum starvation-induced apoptosis is reduced in cortical brain cells from MAO A-deficient mice compared with WT. In addition, we found that MAO A and R1 are involved in the c-Myc-induced proliferative signaling pathway in the presence of serum. Immunoprecipitation and immunohistochemistry experiments indicate that the oncogene c-Myc colocalizes with R1 and induces R1 gene expression. Using R1 overexpression, R1 small interfering RNA, and a MAO A inhibitor, we found that R1 and MAO A act upstream of cyclin D1 and E2F1. In summary, this study demonstrates the functions of MAO A and its repressor R1 in apoptotic signaling pathways.

Response to Hyperosmotic Stress

PubMed Central

Saito, Haruo; Posas, Francesc

2012-01-01

An appropriate response and adaptation to hyperosmolarity, i.e., an external osmolarity that is higher than the physiological range, can be a matter of life or death for all cells. It is especially important for free-living organisms such as the yeast Saccharomyces cerevisiae. When exposed to hyperosmotic stress, the yeast initiates a complex adaptive program that includes temporary arrest of cell-cycle progression, adjustment of transcription and translation patterns, and the synthesis and retention of the compatible osmolyte glycerol. These adaptive responses are mostly governed by the high osmolarity glycerol (HOG) pathway, which is composed of membrane-associated osmosensors, an intracellular signaling pathway whose core is the Hog1 MAP kinase (MAPK) cascade, and cytoplasmic and nuclear effector functions. The entire pathway is conserved in diverse fungal species, while the Hog1 MAPK cascade is conserved even in higher eukaryotes including humans. This conservation is illustrated by the fact that the mammalian stress-responsive p38 MAPK can rescue the osmosensitivity of hog1Δ mutations in response to hyperosmotic challenge. As the HOG pathway is one of the best-understood eukaryotic signal transduction pathways, it is useful not only as a model for analysis of osmostress responses, but also as a model for mathematical analysis of signal transduction pathways. In this review, we have summarized the current understanding of both the upstream signaling mechanism and the downstream adaptive responses to hyperosmotic stress in yeast. PMID:23028184

Drosophila melanogaster auxilin regulates the internalization of Delta to control activity of the Notch signaling pathway

PubMed Central

Hagedorn, Elliott J.; Bayraktar, Jennifer L.; Kandachar, Vasundhara R.; Bai, Ting; Englert, Dane M.; Chang, Henry C.

2006-01-01

We have isolated mutations in the Drosophila melanogaster homologue of auxilin, a J-domain–containing protein known to cooperate with Hsc70 in the disassembly of clathrin coats from clathrin-coated vesicles in vitro. Consistent with this biochemical role, animals with reduced auxilin function exhibit genetic interactions with Hsc70 and clathrin. Interestingly, the auxilin mutations interact specifically with Notch and disrupt several Notch-mediated processes. Genetic evidence places auxilin function in the signal-sending cells, upstream of Notch receptor activation, suggesting that the relevant cargo for this auxilin-mediated endocytosis is the Notch ligand Delta. Indeed, the localization of Delta protein is disrupted in auxilin mutant tissues. Thus, our data suggest that auxilin is an integral component of the Notch signaling pathway, participating in the ubiquitin-dependent endocytosis of Delta. Furthermore, the fact that auxilin is required for Notch signaling suggests that ligand endocytosis in the signal-sending cells needs to proceed past coat disassembly to activate Notch. PMID:16682530

Divergent cellular pathways of hippocampal memory consolidation and reconsolidation

PubMed Central

Lee, Jonathan L. C.; Hynds, Robert E.

2013-01-01

The reconsolidation of memories after their retrieval involves cellular mechanisms that recapitulate much of the initial consolidation process. However, we have previously demonstrated that there are independent cellular mechanisms of consolidation and reconsolidation in the dorsal hippocampus for contextual fear memories. Expression of BDNF was required for consolidation, while Zif268 expression was necessary for reconsolidation. Given the dichotomy between the obvious mechanistic similarity and notable dissociations between consolidation and reconsolidation, we sought to determine whether the separation at the level of gene expression reflected either parallel and independent upstream signalling pathways, or common upstream mechanisms that diverge by the level of transcriptional activation. Here we show that while consolidation and reconsolidation are commonly dependent upon NMDA receptor activation in the dorsal hippocampus there is a double dissociation between the effects of the MEK inhibitor U0126 and the IKK inhibitor sulfasalazine. Moreover, rescue experiments and western blot analyses show that there are functional NMDA receptor–ERK1–BDNF and NMDA receptor–IKKα–Zif268 pathways for consolidation and reconsolidation, respectively. Therefore, there are divergent pathways of hippocampal memory consolidation and reconsolidation, involving commonality at the cell surface, but separable downstream kinase cascades and transcriptional regulation. PMID:23197404

Coordination of receptor signaling in multiple hematopoietic cell lineages by the adaptor protein SLP-76.

PubMed

Jordan, Martha S; Koretzky, Gary A

2010-04-01

The adaptor protein SLP-76 is expressed in multiple hematopoietic lineages including T cells, platelets, and neutrophils. SLP-76 mediated signaling is dependent on its multiple protein interaction domains, as it creates a scaffold on which key signaling complexes are built. SLP-76 is critical for supporting signaling downstream of both immunoreceptors and integrins. The signaling molecules used both upstream and downstream of SLP-76 are similar among these receptors and across cell types; however, important differences exist. Appreciating how SLP-76 coordinates signal transduction across different cell and receptor types provides insights into the complex interplay of pathways critical for activation of cells of the immune system that are essential for host defense.

TAK1 regulates NF-{Kappa}B and AP-1 activation in airway epithelial cells following RSV infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dey, Nilay; Liu Tianshuang; Garofalo, Roberto P.

2011-09-30

Respiratory syncytial virus (RSV) is the most common cause of epidemic respiratory diseases in infants and young children. RSV infection of airway epithelial cells induces the expression of immune/inflammatory genes through the activation of a subset of transcription factors, including Nuclear Factor-{kappa}B (NF-{kappa}B) and AP-1. In this study, we have investigated the signaling pathway leading to activation of these two transcription factors in response to RSV infection. Our results show that IKK{beta} plays a key role in viral-induced NF-{kappa}B activation, while JNK regulates AP-1-dependent gene transcription, as demonstrated by using kinase inactive proteins and chemical inhibitors of the two kinases.more » Inhibition of TAK1 activation, by overexpression of kinase inactive TAK1 or using cells lacking TAK1 expression, significantly reduced RSV-induced NF-{kappa}B and AP-1 nuclear translocation and DNA-binding activity, as well as NF-{kappa}B-dependent gene expression, identifying TAK1 as an important upstream signaling molecule regulating RSV-induced NF-{kappa}B and AP-1 activation. - Highlights: > IKK{beta} is a major kinase involved in RSV-induced NF-{kappa}B activation. > JNK regulates AP-1-dependent gene transcription in RSV infection. > TAK1 is a critical upstream signaling molecule for both pathways in infected cells.« less

eXpression2Kinases (X2K) Web: linking expression signatures to upstream cell signaling networks.

PubMed

Clarke, Daniel J B; Kuleshov, Maxim V; Schilder, Brian M; Torre, Denis; Duffy, Mary E; Keenan, Alexandra B; Lachmann, Alexander; Feldmann, Axel S; Gundersen, Gregory W; Silverstein, Moshe C; Wang, Zichen; Ma'ayan, Avi

2018-05-25

While gene expression data at the mRNA level can be globally and accurately measured, profiling the activity of cell signaling pathways is currently much more difficult. eXpression2Kinases (X2K) computationally predicts involvement of upstream cell signaling pathways, given a signature of differentially expressed genes. X2K first computes enrichment for transcription factors likely to regulate the expression of the differentially expressed genes. The next step of X2K connects these enriched transcription factors through known protein-protein interactions (PPIs) to construct a subnetwork. The final step performs kinase enrichment analysis on the members of the subnetwork. X2K Web is a new implementation of the original eXpression2Kinases algorithm with important enhancements. X2K Web includes many new transcription factor and kinase libraries, and PPI networks. For demonstration, thousands of gene expression signatures induced by kinase inhibitors, applied to six breast cancer cell lines, are provided for fetching directly into X2K Web. The results are displayed as interactive downloadable vector graphic network images and bar graphs. Benchmarking various settings via random permutations enabled the identification of an optimal set of parameters to be used as the default settings in X2K Web. X2K Web is freely available from http://X2K.cloud.

A Notch-dependent transcriptional hierarchy promotes mesenchymal transdifferentiation in the cardiac cushion.

PubMed

Chang, Alex C Y; Garside, Victoria C; Fournier, Michele; Smrz, Justin; Vrljicak, Pavle; Umlandt, Patricia; Fuller, Megan; Robertson, Gordon; Zhao, Yongjun; Tam, Angela; Jones, Steven J M; Marra, Marco A; Hoodless, Pamela A; Karsan, Aly

2014-07-01

Valvuloseptal defects are the most common congenital heart defects. Notch signaling-induced endothelial-to-mesenchymal transition (EMT) in the atrioventricular canal (AVC) cushions at murine embryonic day (E)9.5 is a required step during early valve development. Insights to the transcriptional network that is activated in endocardial cells (EC) during EMT and how these pathways direct valve maturation are lacking. We show that at E11.5, AVC-EC retain the ability to undergo Notch-dependent EMT when explanted on collagen. EC-Notch inhibition at E10.5 blocks expression of known mesenchymal genes in E11.5 AVC-EC. To understand the genetic network and AVC development downstream of Notch signaling beyond E9.5, we constructed Tag-Seq libraries corresponding to different cell types of the E11.5 AVC and atrium in wild-type mice and in EC-Notch inhibited mice. We identified 1,400 potential Notch targets in the AVC-EC, of which 124 are transcription factors (TF). From the 124 TFs, we constructed a transcriptional hierarchy and identify 10 upstream TFs within the network. We validated 4 of the upstream TFs as Notch targets that are enriched in AVC-EC. Functionally, we show these 4 TFs regulate EMT in AVC explant assays. These novel signaling pathways downstream of Notch are potentially relevant to valve development. © 2014 Wiley Periodicals, Inc.

Src is a major signaling component for CTGF induction by TGF-β1 in osteoblasts

PubMed Central

X, Zhang; JA, Arnott; S, Rehman; WG, DeLong; A, Sanjay; FF, Safadi; SN, Popoff

2010-01-01

Connective tissue growth factor (CTGF/CCN2) is induced by transforming growth factor beta 1(TGF-β1) where it acts as a downstream mediator of TGF-β1 induced matrix production in osteoblasts. We have shown the requirement of Src, Erk and Smad signaling for CTGF induction by TGF-β1 in osteoblasts, however the potential interaction among these signaling pathways remains undetermined. In this study we demonstrate that TGF-β1 activates Src kinase in ROS17/2.8 cells and that treatment with the Src family kinase inhibitor PP2 prevents Src activation and CTGF induction by TGF-β1. Additionally, inhibiting Src activation prevented Erk activation, Smad 2 & 3 activation and nuclear translocation by TGF-β1, demonstrating that Src is an essential upstream signaling partner of both Erk and Smads in osteoblasts. MAPKs such as Erk can modulate the Smad pathway through directly mediating the phosphorylation of Smads or indirectly through activation/inactivation of required nuclear co-activators that mediate Smad DNA binding. When we treated cells with the Erk inhibitor, PD98059 it inhibited TGF-β1-induced CTGF protein expression but had no effect on Src activation, Smad activation or Smad nuclear translocation. However PD98059 impaired transcriptional complex formation on the Smad binding element (SBE) on the CTGF promoter, demonstrating that Erk activation was required for SBE transactivation. This data demonstrates that Src is an essential upstream signaling transducer of Erk and Smad signaling with respect to TGF-β1 in osteoblasts and that Smads and Erk function independently but are both essential for forming a transcriptionally active complex on the CTGF promoter in osteoblasts. PMID:20432467

MAPK signaling promotes axonal degeneration by speeding the turnover of the axonal maintenance factor NMNAT2

PubMed Central

Walker, Lauren J; Summers, Daniel W; Sasaki, Yo; Brace, EJ; Milbrandt, Jeffrey; DiAntonio, Aaron

2017-01-01

Injury-induced (Wallerian) axonal degeneration is regulated via the opposing actions of pro-degenerative factors such as SARM1 and a MAPK signal and pro-survival factors, the most important of which is the NAD+ biosynthetic enzyme NMNAT2 that inhibits activation of the SARM1 pathway. Here we investigate the mechanism by which MAPK signaling facilitates axonal degeneration. We show that MAPK signaling promotes the turnover of the axonal survival factor NMNAT2 in cultured mammalian neurons as well as the Drosophila ortholog dNMNAT in motoneurons. The increased levels of NMNAT2 are required for the axonal protection caused by loss of MAPK signaling. Regulation of NMNAT2 by MAPK signaling does not require SARM1, and so cannot be downstream of SARM1. Hence, pro-degenerative MAPK signaling functions upstream of SARM1 by limiting the levels of the essential axonal survival factor NMNAT2 to promote injury-dependent SARM1 activation. These findings are consistent with a linear molecular pathway for the axonal degeneration program. DOI: http://dx.doi.org/10.7554/eLife.22540.001 PMID:28095293

High humidity suppresses ssi4-mediated cell death and disease resistance upstream of MAP kinase activation, H2O2 production and defense gene expression.

PubMed

Zhou, Fasong; Menke, Frank L H; Yoshioka, Keiko; Moder, Wolfgang; Shirano, Yumiko; Klessig, Daniel F

2004-09-01

The Arabidopsis ssi4 mutant, which exhibits spontaneous lesion formation, constitutive expression of pathogenesis-related (PR) genes and enhanced resistance to virulent bacterial and oomycete pathogens, contains a gain-of-function mutation in a TIR-NBS-LRR type R gene. Epistatic analyses revealed that both PR gene expression and disease resistance are activated via a salicylic acid (SA)- and EDS1-dependent, but NPR1- and NDR1-independent signaling pathway. In this study, we demonstrate that in moderate relative humidity (RH; 60%), the ssi4 mutant accumulates H(2)O(2) and SA prior to lesion formation and displays constitutive activation of the MAP kinases AtMPK6 and AtMPK3. It also constitutively expresses a variety of defense-associated genes, including those encoding the WRKY transcription factors AtWRKY29 and AtWRKY6, the MAP kinases AtMPK6 and AtMPK3, the powdery mildew R proteins RPW8.1 and RPW8.2, EDS1 and PR proteins. All of these ssi4-induced responses, as well as the chlorotic, stunted morphology and enhanced disease resistance phenotype, are suppressed by high RH (95%) growth conditions. Thus, a humidity sensitive factor (HSF) appears to function at an early point in the ssi4 signaling pathway. All ssi4 phenotypes, except for MAP kinase activation, also were suppressed by the eds1-1 mutation. Thus, ssi4-induced MAP kinase activation occurs downstream of the HSF but either upstream of EDS1 or on a separate branch of the ssi4 signaling pathway. SA is a critical signaling component in ssi4-mediated defense responses. However, exogenously supplied SA failed to restore lesion formation in high RH-grown ssi4 plants, although it induced defense gene expression. Thus, additional signals also are involved.

Effects of imatinib and nilotinib on the whole transcriptome of cultured murine osteoblasts.

PubMed

Kirschner, Gyöngyi; Balla, Bernadett; Horváth, Péter; Kövesdi, Andrea; Lakatos, Gergely; Takács, István; Nagy, Zsolt; Tóbiás, Bálint; Árvai, Kristóf; Kósa, János Pál; Lakatos, Péter

2016-09-01

Numerous clinical observations have confirmed that breakpoint cluster region-abelson fusion oncoprotein tyrosine kinase inhibitors used in leukemia treatment alter bone physiology in a complex manner. The aim of the present study was to analyze the whole transcriptome of cultured murine osteoblasts and determine the changes following treatment with imatinib and nilotinib using Sequencing by Oligonucleotide Ligation and Detection next generation RNA sequencing. This study also aimed to identify candidate signaling pathways and network regulators by multivariate Ingenuity Pathway Analysis. Based on the right-tailed Fisher's exact test, significantly altered pathways including upstream regulators were defined for each drug. The correlation between these pathways and bone metabolism was also examined. The preliminary results suggest the two drugs have different mechanisms of action on osteoblasts, and imatinib was shown to have a greater effect on gene expression. Data also indicated the potential role of a number of genes and signaling cascades that may contribute to identifying novel targets for the treatment of metabolic bone diseases.

Huntingtin-interacting protein 1-mediated neuronal cell death occurs through intrinsic apoptotic pathways and mitochondrial alterations.

PubMed

Choi, Shin Ae; Kim, Steven J; Chung, Kwang Chul

2006-10-02

Huntingtin interacting protein-1 (Hip1) is known to be associated with the N-terminal domain of huntingtin. Although Hip1 can induce apoptosis, the exact upstream signal transduction pathways have not been clarified yet. In the present study, we examined whether activation of intrinsic and/or extrinsic apoptotic pathways occurs during Hip1-mediated neuronal cell death. Overexpression of Hip1 induced cell death through caspase-3 activation in immortalized hippocampal neuroprogenitor cells. Interestingly, proteolytic processing of Hip1 into partial fragments was observed in response to Hip1 transfection and apoptosis-inducing drugs. Moreover, Hip1 was found to directly bind to and activate caspase-9. This promoted cytosolic release of cytochrome c and apoptosis-inducing factor via mitochondrial membrane perturbation. Furthermore, Hip1 could directly bind to Apaf-1, suggesting that the neurotoxic signals of Hip1 transmit through the intrinsic mitochondrial apoptotic pathways and the formation of apoptosome complex.

Lysophosphatidylcholine up-regulates human endothelial nitric oxide synthase gene transactivity by c-Jun N-terminal kinase signalling pathway

PubMed Central

Xing, Feiyue; Liu, Jing; Mo, Yongyan; Liu, Zhifeng; Qin, Qinghe; Wang, Jingzhen; Fan, Zhenhua; Long, Yutian; Liu, Na; Zhao, Kesen; Jiang, Yong

2009-01-01

Human endothelial nitric oxide synthase (eNOS) plays a pivotal role in maintaining blood pressure homeostasis and vascular integrity. It has recently been reported that mitogen-activated protein kinases (MAPKs) are intimately implicated in expression of eNOS. However detailed mechanism mediated by them remains to be clarified. In this study, eNOS gene transactivity in human umbilical vein endothelial cells was up-regulated by stimulation of lysophosphatidylcholine (LPC). The stimulation of LPC highly activated both extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK), with differences in the dynamic processes of activation between them. Unexpectedly, p38 MAPK could not be activated by the stimulation of LPC. The activation of JNK signalling pathway by overexpression of JNK or its upstream kinase active mutant up-regulated the transactivity of eNOS significantly, but the activation of p38 signalling pathway down-regulated it largely. The inhibition of either ERK1/2 or JNK signalling pathway by kinase-selective inhibitors could markedly block the induction of the transactivity by LPC. It was observed by electrophoretic mobility shift assay that LPC stimulated both SP1 and AP1 DNA binding activity to go up. Additionally using decoy oligonucleotides proved that SP1 was necessary for maintaining the basal or stimulated transactivity, whereas AP1 contributed mainly to the increase of the stimulated transactivity. These findings indicate that the up-regulation of the eNOS gene transactivity by LPC involves the enhancement of SP1 transcription factor by the activation of JNK and ERK1/2 signalling pathways and AP1 transcription factor by the activation of JNK signalling pathway. PMID:18624763

Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling.

PubMed Central

Denhardt, D T

1996-01-01

The features of three distinct protein phosphorylation cascades in mammalian cells are becoming clear. These signalling pathways link receptor-mediated events at the cell surface or intracellular perturbations such as DNA damage to changes in cytoskeletal structure, vesicle transport and altered transcription factor activity. The best known pathway, the Ras-->Raf-->MEK-->ERK cascade [where ERK is extracellular-signal-regulated kinase and MEK is mitogen-activated protein (MAP) kinase/ERK kinase], is typically stimulated strongly by mitogens and growth factors. The other two pathways, stimulated primarily by assorted cytokines, hormones and various forms of stress, predominantly utilize p21 proteins of the Rho family (Rho, Rac and CDC42), although Ras can also participate. Diagnostic of each pathway is the MAP kinase component, which is phosphorylated by a unique dual-specificity kinase on both tyrosine and threonine in one of three motifs (Thr-Glu-Tyr, Thr-Phe-Tyr or Thr-Gly-Tyr), depending upon the pathway. In addition to activating one or more protein phosphorylation cascades, the initiating stimulus may also mobilize a variety of other signalling molecules (e.g. protein kinase C isoforms, phospholipid kinases, G-protein alpha and beta gamma subunits, phospholipases, intracellular Ca2+). These various signals impact to a greater or lesser extent on multiple downstream effectors. Important concepts are that signal transmission often entails the targeted relocation of specific proteins in the cell, and the reversible formation of protein complexes by means of regulated protein phosphorylation. The signalling circuits may be completed by the phosphorylation of upstream effectors by downstream kinases, resulting in a modulation of the signal. Signalling is terminated and the components returned to the ground state largely by dephosphorylation. There is an indeterminant amount of cross-talk among the pathways, and many of the proteins in the pathways belong to families of closely related proteins. The potential for more than one signal to be conveyed down a pathway simultaneously (multiplex signalling) is discussed. The net effect of a given stimulus on the cell is the result of a complex intracellular integration of the intensity and duration of activation of the individual pathways. The specific outcome depends on the particular signalling molecules expressed by the target cells and on the dynamic balance among the pathways. PMID:8836113

Epigenetic Regulation of Myeloid Cells

PubMed Central

IVASHKIV, LIONEL B.; PARK, SUNG HO

2017-01-01

Epigenetic regulation in myeloid cells is crucial for cell differentiation and activation in response to developmental and environmental cues. Epigenetic control involves posttranslational modification of DNA or chromatin, and is also coupled to upstream signaling pathways and transcription factors. In this review, we summarize key epigenetic events and how dynamics in the epigenetic landscape of myeloid cells shape the development, immune activation, and innate immune memory. PMID:27337441

Roles of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways in controlling growth and sensitivity to therapy-implications for cancer and aging

PubMed Central

Steelman, Linda S.; Chappell, William H.; Abrams, Stephen L.; Kempf, C. Ruth; Long, Jacquelyn; Laidler, Piotr; Mijatovic, Sanja; Maksimovic-Ivanic, Danijela; Stivala, Franca; Mazzarino, Maria C.; Donia, Marco; Fagone, Paolo; Malaponte, Graziella; Nicoletti, Ferdinando; Libra, Massimo; Milella, Michele; Tafuri, Agostino; Bonati, Antonio; Bäsecke, Jörg; Cocco, Lucio; Evangelisti, Camilla; Martelli, Alberto M.; Montalto, Giuseppe; Cervello, Melchiorre; McCubrey, James A.

2011-01-01

Dysregulated signaling through the Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways is often the result of genetic alterations in critical components in these pathways or upstream activators. Unrestricted cellular proliferation and decreased sensitivity to apoptotic-inducing agents are typically associated with activation of these pro-survival pathways. This review discusses the functions these pathways have in normal and neoplastic tissue growth and how they contribute to resistance to apoptotic stimuli. Crosstalk and commonly identified mutations that occur within these pathways that contribute to abnormal activation and cancer growth will also be addressed. Finally the recently described roles of these pathways in cancer stem cells, cellular senescence and aging will be evaluated. Controlling the expression of these pathways could ameliorate human health. PMID:21422497

The serine protease inhibitor TLCK attenuates intrinsic death pathways in neurons upstream of mitochondrial demise.

PubMed

Reuther, C; Ganjam, G K; Dolga, A M; Culmsee, C

2014-11-01

It is well-established that activation of proteases, such as caspases, calpains and cathepsins are essential components in signaling pathways of programmed cell death (PCD). Although these proteases have also been linked to mechanisms of neuronal cell death, they are dispensable in paradigms of intrinsic death pathways, e.g. induced by oxidative stress. However, emerging evidence implicated a particular role for serine proteases in mechanisms of PCD in neurons. Here, we investigated the role of trypsin-like serine proteases in a model of glutamate toxicity in HT-22 cells. In these cells glutamate induces oxytosis, a form of caspase-independent cell death that involves activation of the pro-apoptotic protein BH3 interacting-domain death agonist (Bid), leading to mitochondrial demise and ensuing cell death. In this model system, the trypsin-like serine protease inhibitor Nα-tosyl-l-lysine chloromethyl ketone hydrochloride (TLCK) inhibited mitochondrial damage and cell death. Mitochondrial morphology alterations, the impairment of the mitochondrial membrane potential and ATP depletion were prevented and, moreover, lipid peroxidation induced by glutamate was completely abolished. Strikingly, truncated Bid-induced cell death was not affected by TLCK, suggesting a detrimental activity of serine proteases upstream of Bid activation and mitochondrial demise. In summary, this study demonstrates the protective effect of serine protease inhibition by TLCK against oxytosis-induced mitochondrial damage and cell death. These findings indicate that TLCK-sensitive serine proteases play a crucial role in cell death mechanisms upstream of mitochondrial demise and thus, may serve as therapeutic targets in diseases, where oxidative stress and intrinsic pathways of PCD mediate neuronal cell death.

Targeting HIF-2α as therapy for advanced cancers.

PubMed

Murugesan, Thanabal; Rajajeyabalachandran, Gurukumari; Kumar, Swetha; Nagaraju, Shruthi; Kumar, Sooriya

2018-05-14

Hypoxia-inducible factors (HIF-1α, -2α -3α, and -β) are key factors that control hypoxia-induced carcinogenic pathways. HIF-1α is predominantly involved in the early stages of cancer, whereas HIF-2α is actively involved in the later stages; in addition, chronic (prolonged) rather than acute (short) hypoxia is a feature of metastasis and chemoresistance that occur during the later stages of cancer. Oncometabolites, onco-miRNAs, glucose deprivation, pseudohypoxia, cytokine/chemokine secretion, and some unique upstream proteins are involved in the signaling switch from HIF-1α to HIF-2α; thus, understanding this signaling switch is critical for the treatment of advanced cancer. In this review, we highlight data relating to HIF-2α rather than HIF-1α signaling in cancer pathways and discuss prospective drugs that target this important factor. Copyright © 2018 Elsevier Ltd. All rights reserved.

Neuroendocrine regulation of somatic growth in fishes.

PubMed

Dai, XiangYan; Zhang, Wei; Zhuo, ZiJian; He, JiangYan; Yin, Zhan

2015-02-01

Growth is a polygenic trait that is under the influence of multiple physiological pathways regulating energy metabolism and muscle growth. Among the possible growth-regulating pathways in vertebrates, components of the somatotropic axis are thought to have the greatest influence. There is growing body of literature focusing on the somatotropic axis and its role regulating growth in fish. This includes research into growth hormone, upstream hypothalamic hormones, insulin-like growth factors, and downstream signaling molecules. Many of these signals have both somatic effects stimulating the growth of tissues and metabolic effects that play a role in nutrient metabolism. Signals of other endocrine axes exhibit profound effects on the function of the somatotropic axis in vivo. In this review we highlight recent advances in our understanding of the teleost fish endocrine somatotropic axis, including emerging research using genetic modified models. These studies have revealed new aspects and challenges associated with regulation of the important steps of somatic growth.

The visual orientation memory of Drosophila requires Foraging (PKG) upstream of Ignorant (RSK2) in ring neurons of the central complex

PubMed Central

Kuntz, Sara; Poeck, Burkhard; Sokolowski, Marla B.; Strauss, Roland

2012-01-01

Orientation and navigation in a complex environment requires path planning and recall to exert goal-driven behavior. Walking Drosophila flies possess a visual orientation memory for attractive targets which is localized in the central complex of the adult brain. Here we show that this type of working memory requires the cGMP-dependent protein kinase encoded by the foraging gene in just one type of ellipsoid-body ring neurons. Moreover, genetic and epistatic interaction studies provide evidence that Foraging functions upstream of the Ignorant Ribosomal-S6 Kinase 2, thus revealing a novel neuronal signaling pathway necessary for this type of memory in Drosophila. PMID:22815538

Identification of novel biomarker and therapeutic target candidates for acute intracerebral hemorrhage by quantitative plasma proteomics.

PubMed

Li, Guo-Chun; Zhang, Lina; Yu, Ming; Jia, Haiyu; Tian, Ting; Wang, Junqin; Wang, Fuqiang; Zhou, Ling

2017-01-01

The systematic mechanisms of acute intracerebral hemorrhage are still unknown and unverified, although many recent researches have indicated the secondary insults. This study was aimed to disclose the pathological mechanism and identify novel biomarker and therapeutic target candidates by plasma proteome. Patients with AICH (n = 8) who demographically matched healthy controls (n = 4) were prospectively enrolled, and their plasma samples were obtained. The TMT-LC-MS/MS-based proteomics approach was used to quantify the differential proteome across plasma samples, and the results were analyzed by Ingenuity Pathway Analysis to explore canonical pathways and the relationship involved in the uploaded data. Compared with healthy controls, there were 31 differentially expressed proteins in the ICH group ( P  < 0.05), of which 21 proteins increased while 10 proteins decreased in abundance. These proteins are involved in 21 canonical pathways. One network with high confidence level was selected by the function network analysis, in which 23 proteins, P38MAPK and NFκB signaling pathways participated. Upstream regulator analysis found two regulators, IL6 and TNF, with an activation z -score. Seven biomarker candidates: APCS, FGB, LBP, MGMT, IGFBP2, LYZ, and APOA4 were found. Six candidate proteins were selected to assess the validity of the results by subsequent Western blotting analysis. Our analysis provided several intriguing pathways involved in ICH, like LXR/RXR activation, acute phase response signaling, and production of NO and ROS in macrophages pathways. The three upstream regulators: IL-6, TNF, LPS, and seven biomarker candidates: APCS, APOA4, FGB, IGFBP2, LBP, LYZ, and MGMT were uncovered. LPS, APOA4, IGFBP2, LBP, LYZ, and MGMT are novel potential biomarkers in ICH development. The identified proteins and pathways provide new perspectives to the potential pathological mechanism and therapeutic targets underlying ICH.

LIF-activated Jak signaling determines Esrrb expression during late-stage reprogramming

PubMed Central

Huang, Delun; Wang, Ling; Duan, Jingyue; Huang, Chang; Tian, Xiuchun (Cindy); Zhang, Ming

2018-01-01

ABSTRACT The regulatory process of naïve-state induced pluripotent stem cell (iPSC) generation is not well understood. Leukemia inhibitory factor (LIF)-activated Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) is the master regulator for naïve-state pluripotency achievement and maintenance. The estrogen-related receptor beta (Esrrb) serves as a naïve-state marker gene regulating self-renewal of embryonic stem cells (ESCs). However, the interconnection between Esrrb and LIF signaling for pluripotency establishment in reprogramming is unclear. We screened the marker genes critical for complete reprogramming during mouse iPSC generation, and identified genes including Esrrb that are responsive to LIF/Jak pathway signaling. Overexpression of Esrrb resumes the reprogramming halted by inhibition of Jak activity in partially reprogrammed cells (pre-iPSCs), and leads to the generation of pluripotent iPSCs. We further show that neither overexpression of Nanog nor stimulation of Wnt signaling, two upstream regulators of Esrrb in ESCs, stimulates the expression of Esrrb in reprogramming when LIF or Jak activity is blocked. Our study demonstrates that Esrrb is a specific reprogramming factor regulated downstream of the LIF/Jak signaling pathway. These results shed new light on the regulatory role of LIF pathway on complete pluripotency establishment during iPSC generation. PMID:29212799

Reciprocal Signaling between the Ectoderm and a Mesendodermal Left-Right Organizer Directs Left-Right Determination in the Sea Urchin Embryo

PubMed Central

Bessodes, Nathalie; Haillot, Emmanuel; Duboc, Véronique; Röttinger, Eric; Lahaye, François; Lepage, Thierry

2012-01-01

During echinoderm development, expression of nodal on the right side plays a crucial role in positioning of the rudiment on the left side, but the mechanisms that restrict nodal expression to the right side are not known. Here we show that establishment of left-right asymmetry in the sea urchin embryo relies on reciprocal signaling between the ectoderm and a left-right organizer located in the endomesoderm. FGF/ERK and BMP2/4 signaling are required to initiate nodal expression in this organizer, while Delta/Notch signaling is required to suppress formation of this organizer on the left side of the archenteron. Furthermore, we report that the H+/K+-ATPase is critically required in the Notch signaling pathway upstream of the S3 cleavage of Notch. Our results identify several novel players and key early steps responsible for initiation, restriction, and propagation of left-right asymmetry during embryogenesis of a non-chordate deuterostome and uncover a functional link between the H+/K+-ATPase and the Notch signaling pathway. PMID:23271979

Deficiency in methionine, tryptophan, isoleucine, or choline induces apoptosis in cultured cells.

PubMed

Yen, Chi-Liang E; Mar, Mei-Heng; Craciunescu, Corneliu N; Edwards, Lloyd J; Zeisel, Steven H

2002-07-01

Cells in culture die by apoptosis when deprived of the essential nutrient choline. We now report that cells (both proliferating PC12 cells and postmitotic neurons isolated from fetal rat brains) undergo apoptosis when deprived of other individual essential nutrients (methionine, tryptophan or isoleucine). In PC12 cells, deficiencies of each nutrient independently led to ceramide accumulation and to caspase activation, both recognized signals of several apoptotic pathways. A similar profile of caspases was activated in PC12 cells deprived of choline, methionine, tryptophan or isoleucine. More than one caspase was involved and these caspases appeared to transmit parallel signals for apoptosis induction because only broad-spectrum caspase inhibitors, but not inhibitors for specific individual caspases inhibited apoptosis in choline- or methionine-deprived cells. The induction of these caspase-dependent apoptosis pathways likely did not involve the same upstream signals. Choline deficiency perturbed choline metabolism but did not affect protein synthesis, whereas amino acid deficiencies inhibited protein synthesis but did not perturb choline metabolism. In addition, a subclone of PC12 cells that was resistant to choline deficiency-induced apoptosis was not resistant to tryptophan deficiency-induced apoptosis. These observations suggest that deficiency of each studied nutrient activates different pathways for signaling apoptosis that ultimately converge on a common execution pathway.

Inhibition of the Ras-Net (Elk-3) pathway by a novel pyrazole that affects microtubules.

PubMed

Wasylyk, Christine; Zheng, Hong; Castell, Christelle; Debussche, Laurent; Multon, Marie-Christine; Wasylyk, Bohdan

2008-03-01

Net (Elk-3/SAP-2/Erp) is a transcription factor that is phosphorylated and activated by the Ras-extracellular signal-regulated kinase (Erk) signaling pathway and is involved in wound healing, angiogenesis, and tumor growth. In a cell-based screen for small molecule inhibitors of Ras activation of Net transcriptional activity, we identified a novel pyrazole, XRP44X. XRP44X inhibits fibroblast growth factor 2 (FGF-2)-induced Net phosphorylation by the Ras-Erk signaling upstream from Ras. It also binds to the colchicine-binding site of tubulin, depolymerizes microtubules, stimulates cell membrane blebbing, and affects the morphology of the actin skeleton. Interestingly, Combretastin-A4, which produces similar effects on the cytoskeleton, also inhibits FGF-2 Ras-Net signaling. This differs from other classes of agents that target microtubules, which have either little effect (vincristine) or no effect (docetaxel and nocodazole) on the Ras-Net pathway. XRP44X inhibits various cellular properties, including cell growth, cell cycle progression, and aortal sprouting, similar to other molecules that bind to the tubulin colchicine site. XRP44X has the potentially interesting property of connecting two important pathways involved in cell transformation and may thereby represent an interesting class of molecules that could be developed for cancer treatment.

LncRNA mediated regulation of aging pathways in Drosophila melanogaster during dietary restriction.

PubMed

Yang, Deying; Lian, Ting; Tu, Jianbo; Gaur, Uma; Mao, Xueping; Fan, Xiaolan; Li, Diyan; Li, Ying; Yang, Mingyao

2016-09-27

Dietary restriction (DR) extends lifespan in many species which is a well-known phenomenon. Long non-coding RNAs (lncRNAs) play an important role in regulation of cell senescence and important age-related signaling pathways. Here, we profiled the lncRNA and mRNA transcriptome of fruit flies at 7 day and 42 day during DR and fully-fed conditions, respectively. In general, 102 differentially expressed lncRNAs and 1406 differentially expressed coding genes were identified. Most informatively we found a large number of differentially expressed lncRNAs and their targets enriched in GO and KEGG analysis. We discovered some new aging related signaling pathways during DR, such as hippo signaling pathway-fly, phototransduction-fly and protein processing in endoplasmic reticulum etc. Novel lncRNAs XLOC_092363 and XLOC_166557 are found to be located in 10 kb upstream sequences of hairy and ems promoters, respectively. Furthermore, tissue specificity of some novel lncRNAs had been analyzed at 7 day of DR in fly head, gut and fat body. Also the silencing of lncRNA XLOC_076307 resulted in altered expression level of its targets including Gadd45 (involved in FoxO signaling pathway). Together, the results implicated many lncRNAs closely associated with dietary restriction, which could provide a resource for lncRNA in aging and age-related disease field.

Rho-associated protein kinase regulates subcellular localisation of Angiomotin and Hippo-signalling during preimplantation mouse embryo development.

PubMed

Mihajlović, Aleksandar I; Bruce, Alexander W

2016-09-01

The differential activity of the Hippo-signalling pathway between the outer- and inner-cell populations of the developing preimplantation mouse embryo directs appropriate formation of trophectoderm and inner cell mass (ICM) lineages. Such distinct signalling activity is under control of intracellular polarization, whereby Hippo-signalling is either supressed in polarized outer cells or activated in apolar inner cells. The central role of apical-basolateral polarization to such differential Hippo-signalling regulation prompted us to reinvestigate the role of potential upstream molecular regulators affecting apical-basolateral polarity. This study reports that the chemical inhibition of Rho-associated kinase (Rock) is associated with failure to form morphologically distinct blastocysts, indicative of compromised trophectoderm differentiation, and defects in the localization of both apical and basolateral polarity factors associated with malformation of tight junctions. Moreover, Rock-inhibition mediates mislocalization of the Hippo-signalling activator Angiomotin (Amot), to the basolateral regions of outer cells and is concomitant with aberrant activation of the pathway. The Rock-inhibition phenotype is mediated by Amot, as RNAi-based Amot knockdown totally rescues the normal suppression of Hippo-signalling in outer cells. In conclusion, Rock, via regulating appropriate apical-basolateral polarization in outer cells, regulates the appropriate activity of the Hippo-signalling pathway, by ensuring correct subcellular localization of Amot protein in outer cells. Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

A Protein Scaffold Coordinates SRC-Mediated JNK Activation in Response to Metabolic Stress.

PubMed

Kant, Shashi; Standen, Claire L; Morel, Caroline; Jung, Dae Young; Kim, Jason K; Swat, Wojciech; Flavell, Richard A; Davis, Roger J

2017-09-19

Obesity is a major risk factor for the development of metabolic syndrome and type 2 diabetes. How obesity contributes to metabolic syndrome is unclear. Free fatty acid (FFA) activation of a non-receptor tyrosine kinase (SRC)-dependent cJun NH 2 -terminal kinase (JNK) signaling pathway is implicated in this process. However, the mechanism that mediates SRC-dependent JNK activation is unclear. Here, we identify a role for the scaffold protein JIP1 in SRC-dependent JNK activation. SRC phosphorylation of JIP1 creates phosphotyrosine interaction motifs that bind the SH2 domains of SRC and the guanine nucleotide exchange factor VAV. These interactions are required for SRC-induced activation of VAV and the subsequent engagement of a JIP1-tethered JNK signaling module. The JIP1 scaffold protein, therefore, plays a dual role in FFA signaling by coordinating upstream SRC functions together with downstream effector signaling by the JNK pathway. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Function-specific intracellular signaling pathways downstream of heparin-binding EGF-like growth factor utilized by human trophoblasts.

PubMed

Jessmon, Philip; Kilburn, Brian A; Romero, Roberto; Leach, Richard E; Armant, D Randall

2010-05-01

Heparin-binding EGF-like growth factor (HBEGF) is expressed by trophoblast cells throughout gestation. First-trimester cytotrophoblast cells are protected from hypoxia-induced apoptosis because of the accumulation of HBEGF through a posttranscriptional autocrine mechanism. Exogenous application of HBEGF is cytoprotective in a hypoxia/reoxygenation (H/R) injury model and initiates trophoblast extravillous differentiation to an invasive phenotype. The downstream signaling pathways induced by HBEGF that mediate these various cellular activities were identified using two human first-trimester cytotrophoblast cell lines, HTR-8/SVneo and SW.71, with similar results. Recombinant HBEGF (1 nM) induced transient phosphorylation of MAPK3/1 (ERK), MAPK14 (p38), and AKT within 15 min and JNK after 1-2 h. To determine which downstream pathways regulate the various functions of HBEGF, cells were treated with specific inhibitors of the ERK upstream regulator MEK (U0126), the AKT upstream regulator phosphoinositide-3 (PI3)-kinase (LY294002), MAPK14 (SB203580), and JNK (SP600125), as well as with inactive structural analogues. Only SB203580 specifically prevented HBEGF-mediated rescue during H/R, while each inhibitor attenuated HBEGF-stimulated cell migration. Accumulation of HBEGF at reduced oxygen was blocked only by a combination of U0126, SB203580, and SP600125. We conclude that HBEGF advances trophoblast extravillous differentiation through coordinate activation of PI3 kinase, ERK, MAPK14, and JNK, while only MAPK14 is required for its antiapoptotic activity. Additionally, hypoxia induces an autocrine increase in HBEGF protein levels through MAPK14, JNK or ERK. These experiments reveal a complexity of the intracellular signaling circuitry that regulates trophoblast functions critical for implantation and placentation.

Function-Specific Intracellular Signaling Pathways Downstream of Heparin-Binding EGF-Like Growth Factor Utilized by Human Trophoblasts1

PubMed Central

Jessmon, Philip; Kilburn, Brian A.; Romero, Roberto; Leach, Richard E.; Armant, D. Randall

2010-01-01

Heparin-binding EGF-like growth factor (HBEGF) is expressed by trophoblast cells throughout gestation. First-trimester cytotrophoblast cells are protected from hypoxia-induced apoptosis because of the accumulation of HBEGF through a posttranscriptional autocrine mechanism. Exogenous application of HBEGF is cytoprotective in a hypoxia/reoxygenation (H/R) injury model and initiates trophoblast extravillous differentiation to an invasive phenotype. The downstream signaling pathways induced by HBEGF that mediate these various cellular activities were identified using two human first-trimester cytotrophoblast cell lines, HTR-8/SVneo and SW.71, with similar results. Recombinant HBEGF (1 nM) induced transient phosphorylation of MAPK3/1 (ERK), MAPK14 (p38), and AKT within 15 min and JNK after 1–2 h. To determine which downstream pathways regulate the various functions of HBEGF, cells were treated with specific inhibitors of the ERK upstream regulator MEK (U0126), the AKT upstream regulator phosphoinositide-3 (PI3)-kinase (LY294002), MAPK14 (SB203580), and JNK (SP600125), as well as with inactive structural analogues. Only SB203580 specifically prevented HBEGF-mediated rescue during H/R, while each inhibitor attenuated HBEGF-stimulated cell migration. Accumulation of HBEGF at reduced oxygen was blocked only by a combination of U0126, SB203580, and SP600125. We conclude that HBEGF advances trophoblast extravillous differentiation through coordinate activation of PI3 kinase, ERK, MAPK14, and JNK, while only MAPK14 is required for its antiapoptotic activity. Additionally, hypoxia induces an autocrine increase in HBEGF protein levels through MAPK14, JNK or ERK. These experiments reveal a complexity of the intracellular signaling circuitry that regulates trophoblast functions critical for implantation and placentation. PMID:20130271

Characterization of miRNA-regulated networks, hubs of signaling, and biomarkers in obstruction-induced bladder dysfunction

PubMed Central

Kiss, Bernhard; Moltzahn, Felix; Keller, Irene; Rehrauer, Hubert; Fournier, Catharine Aquino; Burkhard, Fiona C.

2017-01-01

Bladder outlet obstruction (BOO) induces significant organ remodeling, leading to lower urinary tract symptoms accompanied by urodynamic changes in bladder function. Here, we report mRNA and miRNA transcriptome sequencing of bladder samples from human patients with different urodynamically defined states of BOO. Patients’ miRNA and mRNA expression profiles correlated with urodynamic findings. Validation of RNA sequencing results in an independent patient cohort identified combinations of 3 mRNAs (NRXN3, BMP7, UPK1A) and 3 miRNAs (miR-103a-3p, miR-10a-5p, miR-199a-3p) sufficient to discriminate between bladder functional states. All BOO patients shared cytokine and immune response pathways, TGF-β and NO signaling pathways, and hypertrophic PI3K/AKT signaling pathways. AP-1 and NFkB were dominant transcription factors, and TNF-α was the top upstream regulator. Integrated miRNA-mRNA expression analysis identified pathways and molecules targeted by differentially expressed miRNAs. Molecular changes in BOO suggest an increasing involvement of miRNAs in the control of bladder function from the overactive to underactive/acontractile states. PMID:28138557

Scaffold Protein Ahk1, Which Associates with Hkr1, Sho1, Ste11, and Pbs2, Inhibits Cross Talk Signaling from the Hkr1 Osmosensor to the Kss1 Mitogen-Activated Protein Kinase

PubMed Central

Nishimura, Akiko; Yamamoto, Katsuyoshi; Oyama, Masaaki; Kozuka-Hata, Hiroko

2016-01-01

In the budding yeast Saccharomyces cerevisiae, osmostress activates the Hog1 mitogen-activated protein kinase (MAPK), which regulates diverse osmoadaptive responses. Hkr1 is a large, highly glycosylated, single-path transmembrane protein that is a putative osmosensor in one of the Hog1 upstream pathways termed the HKR1 subbranch. The extracellular region of Hkr1 contains both a positive and a negative regulatory domain. However, the function of the cytoplasmic domain of Hkr1 (Hkr1-cyto) is unknown. Here, using a mass spectrometric method, we identified a protein, termed Ahk1 (Associated with Hkr1), that binds to Hkr1-cyto. Deletion of the AHK1 gene (in the absence of other Hog1 upstream branches) only partially inhibited osmostress-induced Hog1 activation. In contrast, Hog1 could not be activated by constitutively active mutants of the Hog1 pathway signaling molecules Opy2 or Ste50 in ahk1Δ cells, whereas robust Hog1 activation occurred in AHK1+ cells. In addition to Hkr1-cyto binding, Ahk1 also bound to other signaling molecules in the HKR1 subbranch, including Sho1, Ste11, and Pbs2. Although osmotic stimulation of Hkr1 does not activate the Kss1 MAPK, deletion of AHK1 allowed Hkr1 to activate Kss1 by cross talk. Thus, Ahk1 is a scaffold protein in the HKR1 subbranch and prevents incorrect signal flow from Hkr1 to Kss1. PMID:26787842

A novel peptide, colivelin, prevents alcohol-induced apoptosis in fetal brain of C57BL/6 mice: signaling pathway investigations

PubMed Central

Sari, Youssef; Chiba, Tomohiro; Yamada, Marina; Rebec, George V.; Aiso, Sadakazu

2009-01-01

Fetal alcohol exposure is known to induce cell death through apoptosis. We found that colivelin (CLN), a novel peptide with the sequence SALLRSIPAPAGASRLLLLTGEIDLP, prevents this apoptosis. Our initial experiment revealed that CLN enhanced the viability of primary cortical neurons exposed to alcohol. We then used a mouse model of fetal alcohol exposure to identify the intracellular mechanisms underlying these neuroprotective effects. On embryonic day 7 (E7), weight-matched pregnant females were assigned to the following groups: (1) ethanol liquid diet (ALC) 25% (4.49%, v/v) ethanol derived calories; (2) pair-fed control; (3) normal chow; (4) ALC combined with administration (i.p.) of CLN (20 μg/20 g body weight); and (5) pair-fed combined with administration (i.p.) of CLN (20 μg/20 g body weight). On E13, fetal brains were collected and assayed for TUNEL staining, caspase-3 colorimetric assay, ELISA, and MSD electrochemiluminescence. CLN blocked the alcohol-induced decline in brain weight and prevented alcohol-induced: apoptosis, activation of caspase-3 and increases of cytosolic cytochrome c, and decreases of mitochondrial cytochrome c. Analysis of proteins in the upstream signaling pathway revealed that CLN down-regulated the phosphorylation of the c-Jun N-terminal kinase. Moreover, CLN prevented alcohol-induced reduction in phosphorylation of BAD protein. Thus, CLN appears to act directly on upstream signaling proteins to prevent alcohol-induced apoptosis. Further assessment of these proteins and their signaling mechanisms is likely to enhance development of neuroprotective therapies. PMID:19782727

Perturbed rhythmic activation of signaling pathways in mice deficient for Sterol Carrier Protein 2-dependent diurnal lipid transport and metabolism

PubMed Central

Jouffe, Céline; Gobet, Cédric; Martin, Eva; Métairon, Sylviane; Morin-Rivron, Delphine; Masoodi, Mojgan; Gachon, Frédéric

2016-01-01

Through evolution, most of the living species have acquired a time keeping system to anticipate daily changes caused by the rotation of the Earth. In all of the systems this pacemaker is based on a molecular transcriptional/translational negative feedback loop able to generate rhythmic gene expression with a period close to 24 hours. Recent evidences suggest that post-transcriptional regulations activated mostly by systemic cues play a fundamental role in the process, fine tuning the time keeping system and linking it to animal physiology. Among these signals, we consider the role of lipid transport and metabolism regulated by SCP2. Mice harboring a deletion of the Scp2 locus present a modulated diurnal accumulation of lipids in the liver and a perturbed activation of several signaling pathways including PPARα, SREBP, LRH-1, TORC1 and its upstream regulators. This defect in signaling pathways activation feedbacks upon the clock by lengthening the circadian period of animals through post-translational regulation of core clock regulators, showing that rhythmic lipid transport is a major player in the establishment of rhythmic mRNA and protein expression landscape. PMID:27097688

Perturbed rhythmic activation of signaling pathways in mice deficient for Sterol Carrier Protein 2-dependent diurnal lipid transport and metabolism.

PubMed

Jouffe, Céline; Gobet, Cédric; Martin, Eva; Métairon, Sylviane; Morin-Rivron, Delphine; Masoodi, Mojgan; Gachon, Frédéric

2016-04-21

Through evolution, most of the living species have acquired a time keeping system to anticipate daily changes caused by the rotation of the Earth. In all of the systems this pacemaker is based on a molecular transcriptional/translational negative feedback loop able to generate rhythmic gene expression with a period close to 24 hours. Recent evidences suggest that post-transcriptional regulations activated mostly by systemic cues play a fundamental role in the process, fine tuning the time keeping system and linking it to animal physiology. Among these signals, we consider the role of lipid transport and metabolism regulated by SCP2. Mice harboring a deletion of the Scp2 locus present a modulated diurnal accumulation of lipids in the liver and a perturbed activation of several signaling pathways including PPARα, SREBP, LRH-1, TORC1 and its upstream regulators. This defect in signaling pathways activation feedbacks upon the clock by lengthening the circadian period of animals through post-translational regulation of core clock regulators, showing that rhythmic lipid transport is a major player in the establishment of rhythmic mRNA and protein expression landscape.

R-spondin 1 is required for specification of hematopoietic stem cells through Wnt16 and Vegfa signaling pathways

PubMed Central

2017-01-01

Hematopoietic stem cells (HSCs) are the therapeutic component of bone marrow transplants, but finding immune-compatible donors limits treatment availability and efficacy. Recapitulation of endogenous specification during development is a promising approach to directing HSC specification in vitro, but current protocols are not capable of generating authentic HSCs with high efficiency. Across phyla, HSCs arise from hemogenic endothelium in the ventral floor of the dorsal aorta concurrent with arteriovenous specification and intersegmental vessel (ISV) sprouting, processes regulated by Notch and Wnt. We hypothesized that coordination of HSC specification with vessel patterning might involve modulatory regulatory factors such as R-spondin 1 (Rspo1), an extracellular protein that enhances β-catenin-dependent Wnt signaling and has previously been shown to regulate ISV patterning. We find that Rspo1 is required for HSC specification through control of parallel signaling pathways controlling HSC specification: Wnt16/DeltaC/DeltaD and Vegfa/Tgfβ1. Our results define Rspo1 as a key upstream regulator of two crucial pathways necessary for HSC specification. PMID:28087636

Regulation of the grapevine polygalacturonase-inhibiting protein encoding gene: expression pattern, induction profile and promoter analysis.

PubMed

Joubert, D Albert; de Lorenzo, Giulia; Vivier, Melané A

2013-03-01

Regulation of defense in plants is a complex process mediated by various signaling pathways. Promoter analysis of defense-related genes is useful to understand these signaling pathways involved in regulation. To this end, the regulation of the polygalacturonase-inhibiting protein encoding gene from Vitis vinifera L. (Vvpgip1) was analyzed with regard to expression pattern and induction profile as well as the promoter in terms of putative regulatory elements present, core promoter size and the start of transcription. Expression of Vvpgip1 is tissue-specific and developmentally regulated. Vvpgip1 expression was induced in response to auxin, salicylic acid and sugar treatment, wounding and pathogen infection. The start of transcription was mapped to 17 bp upstream of the ATG and the core promoter was mapped to the 137 bp upstream of the ATG. Fructose- and Botrytis responsiveness were identified in the region between positions -3.1 and -1.5 kb. The analyses showed induction in water when the leaves were submersed and this response and the response to wounding mapped to the region between positions -1.1 and -0.1 kb. In silico analyses revealed putative cis-acting elements in these areas that correspond well to the induction stimuli tested.

G protein-coupled receptors Flop1 and Flop2 inhibit Wnt/β-catenin signaling and are essential for head formation in Xenopus.

PubMed

Miyagi, Asuka; Negishi, Takefumi; Yamamoto, Takamasa S; Ueno, Naoto

2015-11-01

Patterning of the vertebrate anterior-posterior axis is regulated by the coordinated action of growth factors whose effects can be further modulated by upstream and downstream mediators and the cross-talk of different intracellular pathways. In particular, the inhibition of the Wnt/β-catenin signaling pathway by various factors is critically required for anterior specification. Here, we report that Flop1 and Flop2 (Flop1/2), G protein-coupled receptors related to Gpr4, contribute to the regulation of head formation by inhibiting Wnt/β-catenin signaling in Xenopus embryos. Using whole-mount in situ hybridization, we showed that flop1 and flop2 mRNAs were expressed in the neural ectoderm during early gastrulation. Both the overexpression and knockdown of Flop1/2 resulted in altered embryonic head phenotypes, while the overexpression of either Flop1/2 or the small GTPase RhoA in the absence of bone morphogenetic protein (BMP) signaling resulted in ectopic head induction. Examination of the Flops' function in Xenopus embryo animal cap cells showed that they inhibited Wnt/β-catenin signaling by promoting β-catenin degradation through both RhoA-dependent and -independent pathways in a cell-autonomous manner. These results suggest that Flop1 and Flop2 are essential regulators of Xenopus head formation that act as novel inhibitory components of the Wnt/β-catenin signaling pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

Small GTPase CDC-42 promotes apoptotic cell corpse clearance in response to PAT-2 and CED-1 in C. elegans

PubMed Central

Neukomm, L J; Zeng, S; Frei, A P; Huegli, P A; Hengartner, M O

2014-01-01

The rapid clearance of dying cells is important for the well-being of multicellular organisms. In C. elegans, cell corpse removal is mainly mediated by three parallel engulfment signaling cascades. These pathways include two small GTPases, MIG-2/RhoG and CED-10/Rac1. Here we present the identification and characterization of CDC-42 as a third GTPase involved in the regulation of cell corpse clearance. Genetic analyses performed by both loss of cdc-42 function and cdc-42 overexpression place cdc-42 in parallel to the ced-2/5/12 signaling module, in parallel to or upstream of the ced-10 module, and downstream of the ced-1/6/7 module. CDC-42 accumulates in engulfing cells at membranes surrounding apoptotic corpses. The formation of such halos depends on the integrins PAT-2/PAT-3, UNC-112 and the GEF protein UIG-1, but not on the canonical ced-1/6/7 or ced-2/5/12 signaling modules. Together, our results suggest that the small GTPase CDC-42 regulates apoptotic cell engulfment possibly upstream of the canonical Rac GTPase CED-10, by polarizing the engulfing cell toward the apoptotic corpse in response to integrin signaling and ced-1/6/7 signaling in C. elegans. PMID:24632947

Small GTPase CDC-42 promotes apoptotic cell corpse clearance in response to PAT-2 and CED-1 in C. elegans.

PubMed

Neukomm, L J; Zeng, S; Frei, A P; Huegli, P A; Hengartner, M O

2014-06-01

The rapid clearance of dying cells is important for the well-being of multicellular organisms. In C. elegans, cell corpse removal is mainly mediated by three parallel engulfment signaling cascades. These pathways include two small GTPases, MIG-2/RhoG and CED-10/Rac1. Here we present the identification and characterization of CDC-42 as a third GTPase involved in the regulation of cell corpse clearance. Genetic analyses performed by both loss of cdc-42 function and cdc-42 overexpression place cdc-42 in parallel to the ced-2/5/12 signaling module, in parallel to or upstream of the ced-10 module, and downstream of the ced-1/6/7 module. CDC-42 accumulates in engulfing cells at membranes surrounding apoptotic corpses. The formation of such halos depends on the integrins PAT-2/PAT-3, UNC-112 and the GEF protein UIG-1, but not on the canonical ced-1/6/7 or ced-2/5/12 signaling modules. Together, our results suggest that the small GTPase CDC-42 regulates apoptotic cell engulfment possibly upstream of the canonical Rac GTPase CED-10, by polarizing the engulfing cell toward the apoptotic corpse in response to integrin signaling and ced-1/6/7 signaling in C. elegans.

Human Monocytes Engage an Alternative Inflammasome Pathway.

PubMed

Gaidt, Moritz M; Ebert, Thomas S; Chauhan, Dhruv; Schmidt, Tobias; Schmid-Burgk, Jonathan L; Rapino, Francesca; Robertson, Avril A B; Cooper, Matthew A; Graf, Thomas; Hornung, Veit

2016-04-19

Interleukin-1β (IL-1β) is a cytokine whose bioactivity is controlled by activation of the inflammasome. However, in response to lipopolysaccharide, human monocytes secrete IL-1β independently of classical inflammasome stimuli. Here, we report that this constituted a species-specific response that is not observed in the murine system. Indeed, in human monocytes, lipopolysaccharide triggered an "alternative inflammasome" that relied on NLRP3-ASC-caspase-1 signaling, yet was devoid of any classical inflammasome characteristics including pyroptosome formation, pyroptosis induction, and K(+) efflux dependency. Genetic dissection of the underlying signaling pathway in a monocyte transdifferentiation system revealed that alternative inflammasome activation was propagated by TLR4-TRIF-RIPK1-FADD-CASP8 signaling upstream of NLRP3. Importantly, involvement of this signaling cascade was limited to alternative inflammasome activation and did not extend to classical NLRP3 activation. Because alternative inflammasome activation embraces both sensitivity and promiscuity of TLR4, we propose a pivotal role for this signaling cascade in TLR4-driven, IL-1β-mediated immune responses and immunopathology in humans. Copyright © 2016 Elsevier Inc. All rights reserved.

dbl-1/TGF-β and daf-12/NHR Signaling Mediate Cell-Nonautonomous Effects of daf-16/FOXO on Starvation-Induced Developmental Arrest.

PubMed

Kaplan, Rebecca E W; Chen, Yutao; Moore, Brad T; Jordan, James M; Maxwell, Colin S; Schindler, Adam J; Baugh, L Ryan

2015-12-01

Nutrient availability has profound influence on development. In the nematode C. elegans, nutrient availability governs post-embryonic development. L1-stage larvae remain in a state of developmental arrest after hatching until they feed. This "L1 arrest" (or "L1 diapause") is associated with increased stress resistance, supporting starvation survival. Loss of the transcription factor daf-16/FOXO, an effector of insulin/IGF signaling, results in arrest-defective and starvation-sensitive phenotypes. We show that daf-16/FOXO regulates L1 arrest cell-nonautonomously, suggesting that insulin/IGF signaling regulates at least one additional signaling pathway. We used mRNA-seq to identify candidate signaling molecules affected by daf-16/FOXO during L1 arrest. dbl-1/TGF-β, a ligand for the Sma/Mab pathway, daf-12/NHR and daf-36/oxygenase, an upstream component of the daf-12 steroid hormone signaling pathway, were up-regulated during L1 arrest in a daf-16/FOXO mutant. Using genetic epistasis analysis, we show that dbl-1/TGF-β and daf-12/NHR steroid hormone signaling pathways are required for the daf-16/FOXO arrest-defective phenotype, suggesting that daf-16/FOXO represses dbl-1/TGF-β, daf-12/NHR and daf-36/oxygenase. The dbl-1/TGF-β and daf-12/NHR pathways have not previously been shown to affect L1 development, but we found that disruption of these pathways delayed L1 development in fed larvae, consistent with these pathways promoting development in starved daf-16/FOXO mutants. Though the dbl-1/TGF-β and daf-12/NHR pathways are epistatic to daf-16/FOXO for the arrest-defective phenotype, disruption of these pathways does not suppress starvation sensitivity of daf-16/FOXO mutants. This observation uncouples starvation survival from developmental arrest, indicating that DAF-16/FOXO targets distinct effectors for each phenotype and revealing that inappropriate development during starvation does not cause the early demise of daf-16/FOXO mutants. Overall, this study shows that daf-16/FOXO promotes developmental arrest cell-nonautonomously by repressing pathways that promote larval development.

dbl-1/TGF-β and daf-12/NHR Signaling Mediate Cell-Nonautonomous Effects of daf-16/FOXO on Starvation-Induced Developmental Arrest

PubMed Central

Moore, Brad T.; Jordan, James M.; Maxwell, Colin S.; Schindler, Adam J.; Baugh, L. Ryan

2015-01-01

Nutrient availability has profound influence on development. In the nematode C. elegans, nutrient availability governs post-embryonic development. L1-stage larvae remain in a state of developmental arrest after hatching until they feed. This “L1 arrest” (or "L1 diapause") is associated with increased stress resistance, supporting starvation survival. Loss of the transcription factor daf-16/FOXO, an effector of insulin/IGF signaling, results in arrest-defective and starvation-sensitive phenotypes. We show that daf-16/FOXO regulates L1 arrest cell-nonautonomously, suggesting that insulin/IGF signaling regulates at least one additional signaling pathway. We used mRNA-seq to identify candidate signaling molecules affected by daf-16/FOXO during L1 arrest. dbl-1/TGF-β, a ligand for the Sma/Mab pathway, daf-12/NHR and daf-36/oxygenase, an upstream component of the daf-12 steroid hormone signaling pathway, were up-regulated during L1 arrest in a daf-16/FOXO mutant. Using genetic epistasis analysis, we show that dbl-1/TGF-β and daf-12/NHR steroid hormone signaling pathways are required for the daf-16/FOXO arrest-defective phenotype, suggesting that daf-16/FOXO represses dbl-1/TGF-β, daf-12/NHR and daf-36/oxygenase. The dbl-1/TGF-β and daf-12/NHR pathways have not previously been shown to affect L1 development, but we found that disruption of these pathways delayed L1 development in fed larvae, consistent with these pathways promoting development in starved daf-16/FOXO mutants. Though the dbl-1/TGF-β and daf-12/NHR pathways are epistatic to daf-16/FOXO for the arrest-defective phenotype, disruption of these pathways does not suppress starvation sensitivity of daf-16/FOXO mutants. This observation uncouples starvation survival from developmental arrest, indicating that DAF-16/FOXO targets distinct effectors for each phenotype and revealing that inappropriate development during starvation does not cause the early demise of daf-16/FOXO mutants. Overall, this study shows that daf-16/FOXO promotes developmental arrest cell-nonautonomously by repressing pathways that promote larval development. PMID:26656736

Tbx6-mediated Notch signaling controls somite-specific Mesp2 expression.

PubMed

Yasuhiko, Yukuto; Haraguchi, Seiki; Kitajima, Satoshi; Takahashi, Yu; Kanno, Jun; Saga, Yumiko

2006-03-07

Mesp2 is a transcription factor that plays fundamental roles in somitogenesis, and its expression is strictly restricted to the anterior presomitic mesoderm just before segment border formation. The transcriptional on-off cycle is linked to the segmentation clock. In our current study, we show that a T-box transcription factor, Tbx6, is essential for Mesp2 expression. Tbx6 directly binds to the Mesp2 gene upstream region and mediates Notch signaling, and subsequent Mesp2 transcription, in the anterior presomitic mesoderm. Our data therefore reveal that a mechanism, via Tbx6-dependent Notch signaling, acts on the transcriptional regulation of Mesp2. This finding uncovers an additional component of the interacting network of various signaling pathways that are involved in somitogenesis.

Antimicrobial inflammasomes: unified signalling against diverse bacterial pathogens.

PubMed

Eldridge, Matthew J G; Shenoy, Avinash R

2015-02-01

Inflammasomes - molecular platforms for caspase-1 activation - have emerged as common hubs for a number of pathways that detect and respond to bacterial pathogens. Caspase-1 activation results in the secretion of bioactive IL-1β and IL-18 and pyroptosis, and thus launches a systemic immune and inflammatory response. In this review we discuss signal transduction leading to 'canonical' and 'non-canonical' activation of caspase-1 through the involvement of upstream caspases. Recent studies have identified a growing number of regulatory networks involving guanylate binding proteins, protein kinases, ubiquitylation and necroptosis related pathways that modulate inflammasome responses and immunity to bacterial infection. By being able to respond to extracellular, vacuolar and cytosolic bacteria, their cytosolic toxins or ligands for cell surface receptors, inflammasomes have emerged as important sentinels of infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

Prediction of target genes for miR-140-5p in pulmonary arterial hypertension using bioinformatics methods.

PubMed

Li, Fangwei; Shi, Wenhua; Wan, Yixin; Wang, Qingting; Feng, Wei; Yan, Xin; Wang, Jian; Chai, Limin; Zhang, Qianqian; Li, Manxiang

2017-12-01

The expression of microRNA (miR)-140-5p is known to be reduced in both pulmonary arterial hypertension (PAH) patients and monocrotaline-induced PAH models in rat. Identification of target genes for miR-140-5p with bioinformatics analysis may reveal new pathways and connections in PAH. This study aimed to explore downstream target genes and relevant signaling pathways regulated by miR-140-5p to provide theoretical evidences for further researches on role of miR-140-5p in PAH. Multiple downstream target genes and upstream transcription factors (TFs) of miR-140-5p were predicted in the analysis. Gene ontology (GO) enrichment analysis indicated that downstream target genes of miR-140-5p were enriched in many biological processes, such as biological regulation, signal transduction, response to chemical stimulus, stem cell proliferation, cell surface receptor signaling pathways. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis found that downstream target genes were mainly located in Notch, TGF-beta, PI3K/Akt, and Hippo signaling pathway. According to TF-miRNA-mRNA network, the important downstream target genes of miR-140-5p were PPI, TGF-betaR1, smad4, JAG1, ADAM10, FGF9, PDGFRA, VEGFA, LAMC1, TLR4, and CREB. After thoroughly reviewing published literature, we found that 23 target genes and seven signaling pathways were truly inhibited by miR-140-5p in various tissues or cells; most of these verified targets were in accordance with our present prediction. Other predicted targets still need further verification in vivo and in vitro .

A Fat-Facets-Dscam1-JNK Pathway Enhances Axonal Growth in Development and after Injury

PubMed Central

Koch, Marta; Nicolas, Maya; Zschaetzsch, Marlen; de Geest, Natalie; Claeys, Annelies; Yan, Jiekun; Morgan, Matthew J.; Erfurth, Maria-Luise; Holt, Matthew; Schmucker, Dietmar; Hassan, Bassem A.

2018-01-01

Injury to the adult central nervous systems (CNS) can result in severe long-term disability because damaged CNS connections fail to regenerate after trauma. Identification of regulators that enhance the intrinsic growth capacity of severed axons is a first step to restore function. Here, we conducted a gain-of-function genetic screen in Drosophila to identify strong inducers of axonal growth after injury. We focus on a novel axis the Down Syndrome Cell Adhesion Molecule (Dscam1), the de-ubiquitinating enzyme Fat Facets (Faf)/Usp9x and the Jun N-Terminal Kinase (JNK) pathway transcription factor Kayak (Kay)/Fos. Genetic and biochemical analyses link these genes in a common signaling pathway whereby Faf stabilizes Dscam1 protein levels, by acting on the 3′-UTR of its mRNA, and Dscam1 acts upstream of the growth-promoting JNK signal. The mammalian homolog of Faf, Usp9x/FAM, shares both the regenerative and Dscam1 stabilizing activities, suggesting a conserved mechanism. PMID:29472843

Expression profiling associates blood and brain glucocorticoid receptor signaling with trauma-related individual differences in both sexes.

PubMed

Daskalakis, Nikolaos P; Cohen, Hagit; Cai, Guiqing; Buxbaum, Joseph D; Yehuda, Rachel

2014-09-16

Delineating the molecular basis of individual differences in the stress response is critical to understanding the pathophysiology and treatment of posttraumatic stress disorder (PTSD). In this study, 7 d after predator-scent-stress (PSS) exposure, male and female rats were classified into vulnerable (i.e., "PTSD-like") and resilient (i.e., minimally affected) phenotypes on the basis of their performance on a variety of behavioral measures. Genome-wide expression profiling in blood and two limbic brain regions (amygdala and hippocampus), followed by quantitative PCR validation, was performed in these two groups of animals, as well as in an unexposed control group. Differentially expressed genes were identified in blood and brain associated with PSS-exposure and with distinct behavioral profiles postexposure. There was a small but significant between-tissue overlap (4-21%) for the genes associated with exposure-related individual differences, indicating convergent gene expression in both sexes. To uncover convergent signaling pathways across tissue and sex, upstream activated/deactivated transcription factors were first predicted for each tissue and then the respective pathways were identified. Glucocorticoid receptor (GR) signaling was the only convergent pathway associated with individual differences when using the most stringent statistical threshold. Corticosterone treatment 1 h after PSS-exposure prevented anxiety and hyperarousal 7 d later in both sexes, confirming the GR involvement in the PSS behavioral response. In conclusion, genes and pathways associated with extreme differences in the traumatic stress behavioral response can be distinguished from those associated with trauma exposure. Blood-based biomarkers can predict aspects of brain signaling. GR signaling is a convergent signaling pathway, associated with trauma-related individual differences in both sexes.

Genes and (Common) Pathways Underlying Drug Addiction

PubMed Central

Li, Chuan-Yun; Mao, Xizeng; Wei, Liping

2008-01-01

Drug addiction is a serious worldwide problem with strong genetic and environmental influences. Different technologies have revealed a variety of genes and pathways underlying addiction; however, each individual technology can be biased and incomplete. We integrated 2,343 items of evidence from peer-reviewed publications between 1976 and 2006 linking genes and chromosome regions to addiction by single-gene strategies, microrray, proteomics, or genetic studies. We identified 1,500 human addiction-related genes and developed KARG (http://karg.cbi.pku.edu.cn), the first molecular database for addiction-related genes with extensive annotations and a friendly Web interface. We then performed a meta-analysis of 396 genes that were supported by two or more independent items of evidence to identify 18 molecular pathways that were statistically significantly enriched, covering both upstream signaling events and downstream effects. Five molecular pathways significantly enriched for all four different types of addictive drugs were identified as common pathways which may underlie shared rewarding and addictive actions, including two new ones, GnRH signaling pathway and gap junction. We connected the common pathways into a hypothetical common molecular network for addiction. We observed that fast and slow positive feedback loops were interlinked through CAMKII, which may provide clues to explain some of the irreversible features of addiction. PMID:18179280

bantam miRNA is important for Drosophila blood cell homeostasis and a regulator of proliferation in the hematopoietic progenitor niche

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lam, Victoria; Tokusumi, Tsuyoshi; Tokusumi, Yumiko

Highlights: • bantam miRNA is endogenously expressed in the hematopoietic progenitor niche. • bantam is necessary and sufficient to induce cellular proliferation in the PSC. • bantam is upstream of the Insulin Receptor signaling pathway. • A model for positive regulation of hematopoietic niche growth is proposed. - Abstract: The Drosophila hematopoietic system is utilized in this study to gain novel insights into the process of growth control of the hematopoietic progenitor niche in blood development. The niche microenvironment is an essential component controlling the balance between progenitor populations and differentiated, mature blood cells and has been shown to leadmore » to hematopoietic malignancies in humans when misregulated. MicroRNAs are one class of regulators associated with blood malignancies; however, there remains a relative paucity of information about the role of miRNAs in the niche. Here we demonstrate that bantam miRNA is endogenously active in the Drosophila hematopoietic progenitor niche, the posterior signaling center (PSC), and functions in the primary hematopoietic organ, the lymph gland, as a positive regulator of growth. Loss of bantam leads to a significant reduction in the PSC and overall lymph gland size, as well as a loss of the progenitor population and correlative premature differentiation of mature hemocytes. Interestingly, in addition to being essential for proper lymph gland development, we have determined bantam to be a novel upstream component of the insulin signaling cascade in the PSC and have unveiled dMyc as one factor central to bantam activity. These important findings identify bantam as a new hematopoietic regulator, place it in an evolutionarily conserved signaling pathway, present one way in which it is regulated, and provide a mechanism through which it facilitates cellular proliferation in the hematopoietic niche.« less

VLDL-activated cell signaling pathways that stimulate adrenal cell aldosterone production

PubMed Central

Tsai, Ying-Ying; Rainey, William E.; Johnson, Maribeth H.; Bollag, Wendy B.

2016-01-01

Aldosterone plays an important role in regulating ion and fluid homeostasis and thus blood pressure, and hyperaldosteronism results in hypertension. Hypertension is also observed with obesity, which is associated with additional health risks, including cardiovascular disease. Obese individuals have high serum levels of very low-density lipoprotein (VLDL), which has been shown to stimulate aldosterone production; however, the mechanisms underlying VLDL-induced aldosterone production are still unclear. Here we demonstrate in human adrenocortical carcinoma (HAC15) cells that submaximal concentrations of angiotensin II and VLDL stimulate aldosterone production in an additive fashion, suggesting the possibility of common mechanisms of action. We show using inhibitors that VLDL-induced aldosterone production is mediated by the PLC/IP3/PKC signaling pathway. Our results suggest that PKC is upstream of the extracellular signal-regulated kinase (ERK) activation previously observed with VLDL. An understanding of the mechanisms mediating VLDL-induced aldosterone production may provide insights into therapies to treat obesity-associated hypertension. PMID:27222295

VLDL-activated cell signaling pathways that stimulate adrenal cell aldosterone production.

PubMed

Tsai, Ying-Ying; Rainey, William E; Johnson, Maribeth H; Bollag, Wendy B

2016-09-15

Aldosterone plays an important role in regulating ion and fluid homeostasis and thus blood pressure, and hyperaldosteronism results in hypertension. Hypertension is also observed with obesity, which is associated with additional health risks, including cardiovascular disease. Obese individuals have high serum levels of very low-density lipoprotein (VLDL), which has been shown to stimulate aldosterone production; however, the mechanisms underlying VLDL-induced aldosterone production are still unclear. Here we demonstrate in human adrenocortical carcinoma (HAC15) cells that submaximal concentrations of angiotensin II and VLDL stimulate aldosterone production in an additive fashion, suggesting the possibility of common mechanisms of action. We show using inhibitors that VLDL-induced aldosterone production is mediated by the PLC/IP3/PKC signaling pathway. Our results suggest that PKC is upstream of the extracellular signal-regulated kinase (ERK) activation previously observed with VLDL. An understanding of the mechanisms mediating VLDL-induced aldosterone production may provide insights into therapies to treat obesity-associated hypertension. Published by Elsevier Ireland Ltd.

Redox implications of AMPK-mediated signal transduction beyond energetic clues.

PubMed

Cardaci, Simone; Filomeni, Giuseppe; Ciriolo, Maria Rosa

2012-05-01

Since the discovery of AMP-dependent protein kinase (AMPK), its fundamental role in regulating metabolic pathways and the molecular mechanism underlying the regulation of its activity by adenine nucleotides has been widely studied. AMPK is not only an energy-responsive enzyme, but it also senses redox signals. This review aims at recapitulating the recent lines of evidence that demonstrate the responsiveness of this kinase to metabolic and nitroxidative imbalance, thus providing new insights into the intimate networks of redox-based signals upstream of AMPK. In particular, we discuss its well-recognized activation downstream of mitochondrial dysfunction, debate the recent findings that AMPK is directly targeted by pro-oxidant species, and question alternative redox pathways that allow AMPK to be included into the large class of redox-sensing proteins. The possible therapeutic implications of the role of AMPK in redox-associated pathologies, such as cancer and neurodegeneration, are also discussed in light of recent advances that suggest a role for AMPK in the tuning of redox-dependent processes, such as apoptosis and autophagy.

Dynamic regulation of VEGF-inducible genes by an ERK/ERG/p300 transcriptional network.

PubMed

Fish, Jason E; Cantu Gutierrez, Manuel; Dang, Lan T; Khyzha, Nadiya; Chen, Zhiqi; Veitch, Shawn; Cheng, Henry S; Khor, Melvin; Antounians, Lina; Njock, Makon-Sébastien; Boudreau, Emilie; Herman, Alexander M; Rhyner, Alexander M; Ruiz, Oscar E; Eisenhoffer, George T; Medina-Rivera, Alejandra; Wilson, Michael D; Wythe, Joshua D

2017-07-01

The transcriptional pathways activated downstream of vascular endothelial growth factor (VEGF) signaling during angiogenesis remain incompletely characterized. By assessing the signals responsible for induction of the Notch ligand delta-like 4 (DLL4) in endothelial cells, we find that activation of the MAPK/ERK pathway mirrors the rapid and dynamic induction of DLL4 transcription and that this pathway is required for DLL4 expression. Furthermore, VEGF/ERK signaling induces phosphorylation and activation of the ETS transcription factor ERG, a prerequisite for DLL4 induction. Transcription of DLL4 coincides with dynamic ERG-dependent recruitment of the transcriptional co-activator p300. Genome-wide gene expression profiling identified a network of VEGF-responsive and ERG-dependent genes, and ERG chromatin immunoprecipitation (ChIP)-seq revealed the presence of conserved ERG-bound putative enhancer elements near these target genes. Functional experiments performed in vitro and in vivo confirm that this network of genes requires ERK, ERG and p300 activity. Finally, genome-editing and transgenic approaches demonstrate that a highly conserved ERG-bound enhancer located upstream of HLX (which encodes a transcription factor implicated in sprouting angiogenesis) is required for its VEGF-mediated induction. Collectively, these findings elucidate a novel transcriptional pathway contributing to VEGF-dependent angiogenesis. © 2017. Published by The Company of Biologists Ltd.

GLABROUS INFLORESCENCE STEMS (GIS) is required for trichome branching through gibberellic acid signaling in Arabidopsis.

PubMed

An, Lijun; Zhou, Zhongjing; Su, Sha; Yan, An; Gan, Yinbo

2012-02-01

Cell differentiation generally corresponds to the cell cycle, typically forming a non-dividing cell with a unique differentiated morphology, and Arabidopsis trichome is an excellent model system to study all aspects of cell differentiation. Although gibberellic acid is reported to be involved in trichome branching in Arabidopsis, the mechanism for such signaling is unclear. Here, we demonstrated that GLABROUS INFLORESCENCE STEMS (GIS) is required for the control of trichome branching through gibberellic acid signaling. The phenotypes of a loss-of-function gis mutant and an overexpressor showed that GIS acted as a repressor to control trichome branching. Our results also show that GIS is not required for cell endoreduplication, and our molecular and genetic study results have shown that GIS functions downstream of the key regulator of trichome branching, STICHEL (STI), to control trichome branching through the endoreduplication-independent pathway. Furthermore, our results also suggest that GIS controls trichome branching in Arabidopsis through two different pathways and acts either upstream or downstream of the negative regulator of gibbellic acid signaling SPINDLY (SPY).

MAP Kinase-Mediated Negative Regulation of Symbiotic Nodule Formation in Medicago truncatula.

PubMed

Ryu, Hojin; Laffont, Carole; Frugier, Florian; Hwang, Ildoo

2017-01-01

Mitogen-activated protein kinase (MAPK) signaling cascades play critical roles in various cellular events in plants, including stress responses, innate immunity, hormone signaling, and cell specificity. MAPK-mediated stress signaling is also known to negatively regulate nitrogen-fixing symbiotic interactions, but the molecular mechanism of the MAPK signaling cascades underlying the symbiotic nodule development remains largely unknown. We show that the MtMKK5-MtMPK3/6 signaling module negatively regulates the early symbiotic nodule formation, probably upstream of ERN1 (ERF Required for Nodulation 1) and NSP1 (Nod factor Signaling Pathway 1) in Medicago truncatula . The overexpression of MtMKK5 stimulated stress and defense signaling pathways but also reduced nodule formation in M. truncatula roots. Conversely, a MAPK specific inhibitor, U0126, enhanced nodule formation and the expression of an early nodulation marker gene, MtNIN . We found that MtMKK5 directly activates MtMPK3/6 by phosphorylating the TEY motif within the activation loop and that the MtMPK3/6 proteins physically interact with the early nodulation-related transcription factors ERN1 and NSP1. These data suggest that the stress signaling-mediated MtMKK5/MtMPK3/6 module suppresses symbiotic nodule development via the action of early nodulation transcription factors.

MAP Kinase-Mediated Negative Regulation of Symbiotic Nodule Formation in Medicago truncatula

PubMed Central

Ryu, Hojin; Laffont, Carole; Frugier, Florian; Hwang, Ildoo

2017-01-01

Mitogen-activated protein kinase (MAPK) signaling cascades play critical roles in various cellular events in plants, including stress responses, innate immunity, hormone signaling, and cell specificity. MAPK-mediated stress signaling is also known to negatively regulate nitrogen-fixing symbiotic interactions, but the molecular mechanism of the MAPK signaling cascades underlying the symbiotic nodule development remains largely unknown. We show that the MtMKK5-MtMPK3/6 signaling module negatively regulates the early symbiotic nodule formation, probably upstream of ERN1 (ERF Required for Nodulation 1) and NSP1 (Nod factor Signaling Pathway 1) in Medicago truncatula. The overexpression of MtMKK5 stimulated stress and defense signaling pathways but also reduced nodule formation in M. truncatula roots. Conversely, a MAPK specific inhibitor, U0126, enhanced nodule formation and the expression of an early nodulation marker gene, MtNIN. We found that MtMKK5 directly activates MtMPK3/6 by phosphorylating the TEY motif within the activation loop and that the MtMPK3/6 proteins physically interact with the early nodulation-related transcription factors ERN1 and NSP1. These data suggest that the stress signaling-mediated MtMKK5/MtMPK3/6 module suppresses symbiotic nodule development via the action of early nodulation transcription factors. PMID:28152300

Tbx6-mediated Notch signaling controls somite-specific Mesp2 expression

PubMed Central

Yasuhiko, Yukuto; Haraguchi, Seiki; Kitajima, Satoshi; Takahashi, Yu; Kanno, Jun; Saga, Yumiko

2006-01-01

Mesp2 is a transcription factor that plays fundamental roles in somitogenesis, and its expression is strictly restricted to the anterior presomitic mesoderm just before segment border formation. The transcriptional on–off cycle is linked to the segmentation clock. In our current study, we show that a T-box transcription factor, Tbx6, is essential for Mesp2 expression. Tbx6 directly binds to the Mesp2 gene upstream region and mediates Notch signaling, and subsequent Mesp2 transcription, in the anterior presomitic mesoderm. Our data therefore reveal that a mechanism, via Tbx6-dependent Notch signaling, acts on the transcriptional regulation of Mesp2. This finding uncovers an additional component of the interacting network of various signaling pathways that are involved in somitogenesis. PMID:16505380

Optical signal suppression by a cascaded SOA/RSOA for wavelength reusing reflective PON upstream transmission.

PubMed

Jung, Sang Min; Mun, Kyoung Hak; Kang, Soo Min; Han, Sang Kook

2017-09-18

An optical signal suppression technique based on a cascaded SOA and RSOA is proposed for the reflective passive optical networks (PONs) with wavelength division multiplexing (WDM). By suppressing the downstream signal of the optical carrier, the proposed reflective PON effectively reuses the downstream optical carrier for upstream signal transmission. As an experimental demonstration, we show that the proposed optical signal suppression technique is effective in terms of the signal bandwidth and bit-error-rate (BER) performance of the remodulated upstream transmission.

NF-κB pathways are involved in M1 polarization of RAW 264.7 macrophage by polyporus polysaccharide in the tumor microenvironment

PubMed Central

Liu, Chun-Ping; Zhang, Xian; Tan, Qing-Long; Xu, Wen-Xing; Zhou, Chang-Yuan; Luo, Min; Li, Xiong; Zeng, Xing

2017-01-01

Bladder cancer is one of the most malignant tumors closely associated with macrophages. Polyporus polysaccharide (PPS) has shown excellent efficacy in treating bladder cancer with minimal side effects. However, the molecular mechanisms underlying the effects of PPS in inhibiting bladder cancer remain unclear. In this study, we used macrophages cultured alone or with T24 human bladder cancer cell culture supernatant as study models. We found that PPS enhanced the activities of IFN-γ-stimulated RAW 264.7 macrophages, as shown by the release of inducible nitric oxide synthase (INOS), secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-6, phagocytosis activity, as well as expression of M1 phenotype indicators, such as CD40, CD284 and CD86. PPS acted upstream in activation cascade of nuclear factor (NF)-κB signaling pathways by interfering with IκB phosphorylation. In addition, PPS regulated NF-κB (P65) signaling by interfering with Toll-like receptor (TLR)-4, INOS and cyclooxygenase (COX)-2. Our results indicate that PPS activates macrophages through TLR4/NF-κB signaling pathways. PMID:29155869

LPS-induced chemokine expression in both MyD88-dependent and -independent manners is regulated by Cot/Tpl2-ERK axis in macrophages.

PubMed

Bandow, Kenjiro; Kusuyama, Joji; Shamoto, Mitsuo; Kakimoto, Kyoko; Ohnishi, Tomokazu; Matsuguchi, Tetsuya

2012-05-21

LPS signaling is mediated through MyD88-dependent and -independent pathways, activating NF-?B, MAP kinases and IRF3. Cot/Tpl2 is an essential upstream kinase in LPS-mediated activation of ERKs. Here we explore the roles of MyD88 and Cot/Tpl2 in LPS-induced chemokine expression by studying myd88(-/-) and cot/tpl2(-/-) macrophages. Among the nine LPS-responsive chemokines examined, mRNA induction of ccl5, cxcl10, and cxcl13 is mediated through the MyD88-independent pathway. Notably, Cot/Tpl2-ERK signaling axis exerts negative effects on the expression of these three chemokines. In contrast, LPS-induced gene expression of ccl2, ccl7, cxcl2, cxcl3, ccl8, and cxcl9 is mediated in the MyD88-dependent manner. The Cot/Tpl2-ERK axis promotes the expression of the first four and inhibits the expression of the latter two. Thus, LPS induces expression of multiple chemokines through various signaling pathways in macrophages. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Signaling in Human and Murine Lymphocytes in Microgravity: Parallels and Contrasts

NASA Technical Reports Server (NTRS)

Neal, Pellis; Alamelu, Sundaresan; Kulkarni, A. D.; Yamauchi, K.

2006-01-01

Immune function in space undergoes dramatic changes, some of which are detrimental to lymphocyte function. These changes may lead to significant immune suppression. Studies with human lymphocytes both in space flight and with ground-based models (NASA in vitro ground-based microgravity analog) indicate that T cell activation is inhibited in microgravity. Other lymphocyte functions, such as locomotion, are also inhibited. There is about an 80 percent homology in the immune response of mice to that of humans. A murine model was investigated because of its ability to parallel some microgravity using hind limb suspension. In in vivo antiorthostatically (AOS)-suspended mice, T cell activation is greatly suppressed, with the majority of activation related cytokines being inhibited. PHA activation in lymphocytes derived from AOS mice (in vivo ground-based microgravity analog) is also suppressed. Calcium ionophore studies in human lymphocytes exposed to modeled microgravity indicate that the calcium pathways are probably unaffected in microgravity. IP3 (inositol triphosphate) receptor expression in both human and mouse lymphocytes cultured in modeled microgravity indicate no suppression of calcium signaling. In the human system, microgravity seems to inhibit signaling cascades either at the level of, or up-stream of, Protein Kinase C (PKC). In particular, a membrane event, such as phospholipase C gamma 1 activity in human lymphocytes is affected, with its direct upstream effector, LAT, being deficiently expressed. In the mouse pathway, LAT is undiminished while another critical intermediate, SLP-76, is diminished significantly. This study identifies critical stages in the human and mouse immune systems and in lymphocytes as a function of microgravity.

Downstream components of RhoA required for signal pathway of superoxide formation during phagocytosis of serum opsonized zymosans in macrophages.

PubMed

Kim, Jun Sub; Kim, Jae Gyu; Jeon, Chan Young; Won, Ha Young; Moon, Mi Young; Seo, Ji Yeon; Kim, Jong Il; Kim, Jaebong; Lee, Jae Yong; Choi, Soo Young; Park, Jinseu; Yoon Park, Jung Han; Ha, Kwon Soo; Kim, Pyeung Hyeun; Park, Jae Bong

2005-12-31

Rac1 and Rac2 are essential for the control of oxidative burst catalyzed by NADPH oxidase. It was also documented that Rho is associated with the superoxide burst reaction during phagocytosis of serum- (SOZ) and IgG-opsonized zymosan particles (IOZ). In this study, we attempted to reveal the signal pathway components in the superoxide formation regulated by Rho GTPase. Tat-C3 blocked superoxide production, suggesting that RhoA is essentially involved in superoxide formation during phagocytosis of SOZ. Conversely SOZ activated both RhoA and Rac1/2. Inhibition of RhoA-activated kinase (ROCK), an important downstream effector of RhoA, by Y27632 and myosin light chain kinase (MLCK) by ML-7 abrogated superoxide production by SOZ. Extracellular signaling-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were activated during phagocytosis of SOZ, and Tat-C3 and SB203580 reduced ERK1/2 and p38 MAPK activation, suggesting that RhoA and p38 MAPK may be upstream regulators of ERK1/2. Inhibition of ERK1/2, p38 MAPK, phosphatidyl inositol 3-kinase did not block translocation of RhoA to membranes, suggesting that RhoA is upstream to these kinases. Inhibition of RhoA by Tat-C3 blocked phosphorylation of p47(PHOX). Taken together, RhoA, ROCK, p38MAPK, ERK1/2, and p47(PHOX) may be subsequently activated, leading to activation of NADPH oxidase to produce superoxide.

G protein-coupled receptors: bridging the gap from the extracellular signals to the Hippo pathway.

PubMed

Zhou, Xin; Wang, Zhen; Huang, Wei; Lei, Qun-Ying

2015-01-01

The Hippo pathway is crucial in organ size control, whereas its dysregulation contributes to organ degeneration or tumorigenesis. The kinase cascade of MST1/2 and LATS1/2 and the coupling transcription co-activators YAP/TAZ represent the core components of the Hippo pathway. Extensive studies have identified a number of upstream regulators of the Hippo pathway, including contact inhibition, mechanic stress, extracellular matrix stiffness, cytoskeletal rearrangement, and some molecules of cell polarity and cell junction. However, how the diffuse extracellular signals regulate the Hippo pathway puzzles the researchers for a long time. Unexpectedly, recent elegant studies demonstrated that stimulation of some G protein-coupled receptors (GPCRs), such as lysophosphatidic acid receptor, sphingosine-1-phosphate receptor, and the protease activated receptor PAR1, causes potent YAP/TAZ dephosphorylation and activation by promoting actin cytoskeleton assemble. In this review, we briefly describe the components of the Hippo pathway and focus on the recent progress with respect to the regulation of the Hippo pathway by GPCRs and G proteins in cancer cells. In addition, we also discuss the potential therapeutic roles targeting the Hippo pathway in human cancers. © The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Models of crk adaptor proteins in cancer.

PubMed

Bell, Emily S; Park, Morag

2012-05-01

The Crk family of adaptor proteins (CrkI, CrkII, and CrkL), originally discovered as the oncogene fusion product, v-Crk, of the CT10 chicken retrovirus, lacks catalytic activity but engages with multiple signaling pathways through their SH2 and SH3 domains. Crk proteins link upstream tyrosine kinase and integrin-dependent signals to downstream effectors, acting as adaptors in diverse signaling pathways and cellular processes. Crk proteins are now recognized to play a role in the malignancy of many human cancers, stimulating renewed interest in their mechanism of action in cancer progression. The contribution of Crk signaling to malignancy has been predominantly studied in fibroblasts and in hematopoietic models and more recently in epithelial models. A mechanistic understanding of Crk proteins in cancer progression in vivo is still poorly understood in part due to the highly pleiotropic nature of Crk signaling. Recent advances in the structural organization of Crk domains, new roles in kinase regulation, and increased knowledge of the mechanisms and frequency of Crk overexpression in human cancers have provided an incentive for further study in in vivo models. An understanding of the mechanisms through which Crk proteins act as oncogenic drivers could have important implications in therapeutic targeting.

The Hippo Pathway Targets Rae1 to Regulate Mitosis and Organ Size and to Feed Back to Regulate Upstream Components Merlin, Hippo, and Warts.

PubMed

Jahanshahi, Maryam; Hsiao, Kuangfu; Jenny, Andreas; Pfleger, Cathie M

2016-08-01

Hippo signaling acts as a master regulatory pathway controlling growth, proliferation, and apoptosis and also ensures that variations in proliferation do not alter organ size. How the pathway coordinates restricting proliferation with organ size control remains a major unanswered question. Here we identify Rae1 as a highly-conserved target of the Hippo Pathway integrating proliferation and organ size. Genetic and biochemical studies in Drosophila cells and tissues and in mammalian cells indicate that Hippo signaling promotes Rae1 degradation downstream of Warts/Lats. In proliferating cells, Rae1 loss restricts cyclin B levels and organ size while Rae1 over-expression increases cyclin B levels and organ size, similar to Hippo Pathway over-activation or loss-of-function, respectively. Importantly, Rae1 regulation by the Hippo Pathway is crucial for its regulation of cyclin B and organ size; reducing Rae1 blocks cyclin B accumulation and suppresses overgrowth caused by Hippo Pathway loss. Surprisingly, in addition to suppressing overgrowth, reducing Rae1 also compromises survival of epithelial tissue overgrowing due to loss of Hippo signaling leading to a tissue "synthetic lethality" phenotype. Excitingly, Rae1 plays a highly conserved role to reduce the levels and activity of the Yki/YAP oncogene. Rae1 increases activation of the core kinases Hippo and Warts and plays a post-transcriptional role to increase the protein levels of the Merlin, Hippo, and Warts components of the pathway; therefore, in addition to Rae1 coordinating organ size regulation with proliferative control, we propose that Rae1 also acts in a feedback circuit to regulate pathway homeostasis.

The Hippo Pathway Targets Rae1 to Regulate Mitosis and Organ Size and to Feed Back to Regulate Upstream Components Merlin, Hippo, and Warts

PubMed Central

Jenny, Andreas; Pfleger, Cathie M.

2016-01-01

Hippo signaling acts as a master regulatory pathway controlling growth, proliferation, and apoptosis and also ensures that variations in proliferation do not alter organ size. How the pathway coordinates restricting proliferation with organ size control remains a major unanswered question. Here we identify Rae1 as a highly-conserved target of the Hippo Pathway integrating proliferation and organ size. Genetic and biochemical studies in Drosophila cells and tissues and in mammalian cells indicate that Hippo signaling promotes Rae1 degradation downstream of Warts/Lats. In proliferating cells, Rae1 loss restricts cyclin B levels and organ size while Rae1 over-expression increases cyclin B levels and organ size, similar to Hippo Pathway over-activation or loss-of-function, respectively. Importantly, Rae1 regulation by the Hippo Pathway is crucial for its regulation of cyclin B and organ size; reducing Rae1 blocks cyclin B accumulation and suppresses overgrowth caused by Hippo Pathway loss. Surprisingly, in addition to suppressing overgrowth, reducing Rae1 also compromises survival of epithelial tissue overgrowing due to loss of Hippo signaling leading to a tissue “synthetic lethality” phenotype. Excitingly, Rae1 plays a highly conserved role to reduce the levels and activity of the Yki/YAP oncogene. Rae1 increases activation of the core kinases Hippo and Warts and plays a post-transcriptional role to increase the protein levels of the Merlin, Hippo, and Warts components of the pathway; therefore, in addition to Rae1 coordinating organ size regulation with proliferative control, we propose that Rae1 also acts in a feedback circuit to regulate pathway homeostasis. PMID:27494403

Sensor kinase KinB and its pathway-associated key factors sense the signal of nutrition starvation in sporulation of Bacillus subtilis.

PubMed

Liu, Weipeng; He, Zeying; Gao, Feng; Yan, Jinyuan; Huang, Xiaowei

2018-01-03

Bacillus subtilis responds to environmental stress cues and develops endospores for survival. In the process of endospore formation, sporulation initiation is a vital stage and this stage is governed by autophosphorylation of the sensor histidine kinases. The second major sensor kinase KinB perceives the intracellular changes of GTP and ATP during sporulation. However, determination of the environmental signals as well as its related signaling pathway of KinB requires further elucidation. Our current study found that, contrary to the sporulation failure induced by ΔkinA in the nutrient-rich 2× SG medium, the sensor kinase KinB sensed the environmental cues in the nutrient-poor MM medium. Two other membrane proteins, KapB and KbaA, also responded similarly to the same external signal as KinB. Both KapB and KbaA acted upstream of KinB, but they exerted their regulation upon KinB independently. Furthermore, we demonstrated that both the SH3 domain and the α-helix structure in KapB are required for sensing or transducing the signal of sporulation initiation. Collectively, our work here supplied the direct evidences that KinB and its pathway sense the external signal of nutrient starvation in MM medium, and further analyzes the interrelationship among KinB, KbaA, and KapB. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Characterization of Hippo Pathway Components by Gene Inactivation.

PubMed

Plouffe, Steven W; Meng, Zhipeng; Lin, Kimberly C; Lin, Brian; Hong, Audrey W; Chun, Justin V; Guan, Kun-Liang

2016-12-01

The Hippo pathway is important for regulating tissue homeostasis, and its dysregulation has been implicated in human cancer. However, it is not well understood how the Hippo pathway becomes dysregulated because few mutations in core Hippo pathway components have been identified. Therefore, much work in the Hippo field has focused on identifying upstream regulators, and a complex Hippo interactome has been identified. Nevertheless, it is not always clear which components are the most physiologically relevant in regulating YAP/TAZ. To provide an overview of important Hippo pathway components, we created knockout cell lines for many of these components and compared their relative contributions to YAP/TAZ regulation in response to a wide range of physiological signals. By this approach, we provide an overview of the functional importance of many Hippo pathway components and demonstrate NF2 and RHOA as important regulators of YAP/TAZ and TAOK1/3 as direct kinases for LATS1/2. Copyright © 2016 Elsevier Inc. All rights reserved.

MiRNA-335 suppresses neuroblastoma cell invasiveness by direct targeting of multiple genes from the non-canonical TGF-β signalling pathway

PubMed Central

Lynch, Jennifer; Fay, Joanna; Meehan, Maria; Bryan, Kenneth; Watters, Karen M.; Murphy, Derek M.; Stallings, Raymond L.

2012-01-01

Transforming growth factor-β (TGF-β) signaling regulates many diverse cellular activities through both canonical (SMAD-dependent) and non-canonical branches, which includes the mitogen-activated protein kinase (MAPK), Rho-like guanosine triphosphatase and phosphatidylinositol-3-kinase/AKT pathways. Here, we demonstrate that miR-335 directly targets and downregulates genes in the TGF-β non-canonical pathways, including the Rho-associated coiled-coil containing protein (ROCK1) and MAPK1, resulting in reduced phosphorylation of downstream pathway members. Specifically, inhibition of ROCK1 and MAPK1 reduces phosphorylation levels of the motor protein myosin light chain (MLC) leading to a significant inhibition of the invasive and migratory potential of neuroblastoma cells. Additionally, miR-335 targets the leucine-rich alpha-2-glycoprotein 1 (LRG1) messenger RNA, which similarly results in a significant reduction in the phosphorylation status of MLC and a decrease in neuroblastoma cell migration and invasion. Thus, we link LRG1 to the migratory machinery of the cell, altering its activity presumably by exerting its effect within the non-canonical TGF-β pathway. Moreover, we demonstrate that the MYCN transcription factor, whose coding sequence is highly amplified in a particularly clinically aggressive neuroblastoma tumor subtype, directly binds to a region immediately upstream of the miR-335 transcriptional start site, resulting in transcriptional repression. We conclude that MYCN contributes to neuroblastoma cell migration and invasion, by directly downregulating miR-335, resulting in the upregulation of the TGF-β signaling pathway members ROCK1, MAPK1 and putative member LRG1, which positively promote this process. Our results provide novel insight into the direct regulation of TGF-β non-canonical signaling by miR-335, which in turn is downregulated by MYCN. PMID:22382496

Characterization of New Otic Enhancers of the Pou3f4 Gene Reveal Distinct Signaling Pathway Regulation and Spatio-Temporal Patterns

PubMed Central

Robert-Moreno, Àlex; Naranjo, Silvia; de la Calle-Mustienes, Elisa; Gómez-Skarmeta, José Luis; Alsina, Berta

2010-01-01

POU3F4 is a member of the POU-homedomain transcription factor family with a prominent role in inner ear development. Mutations in the human POU3F4 coding unit leads to X-linked deafness type 3 (DFN3), characterized by conductive hearing loss and progressive sensorineural deafness. Microdeletions found 1 Mb 5′ upstream of the coding region also displayed the same phenotype, suggesting that cis-regulatory elements might be present in that region. Indeed, we and others have recently identified several enhancers at the 1 Mb 5′ upstream interval of the pou3f4 locus. Here we characterize the spatio-temporal patterns of these regulatory elements in zebrafish transgenic lines. We show that the most distal enhancer (HCNR 81675) is activated earlier and drives GFP reporter expression initially to a broad ear domain to progressively restrict to the sensory patches. The proximal enhancer (HCNR 82478) is switched later during development and promotes expression, among in other tissues, in sensory patches from its onset. The third enhancer (HCNR 81728) is also active at later stages in the otic mesenchyme and in the otic epithelium. We also characterize the signaling pathways regulating these enhancers. While HCNR 81675 is regulated by very early signals of retinoic acid, HCNR 82478 is regulated by Fgf activity at a later stage and the HCNR 81728 enhancer is under the control of Hh signaling. Finally, we show that Sox2 and Pax2 transcription factors are bound to HCNR 81675 genomic region during otic development and specific mutations to these transcription factor binding sites abrogates HCNR 81675 enhancer activity. Altogether, our results suggest that pou3f4 expression in inner ear might be under the control of distinct regulatory elements that fine-tune the spatio-temporal activity of this gene and provides novel data on the signaling mechanisms controlling pou3f4 function. PMID:21209840

NMDA receptor activation upstream of methyl farnesoate signaling for short day-induced male offspring production in the water flea, Daphnia pulex.

PubMed

Toyota, Kenji; Miyakawa, Hitoshi; Yamaguchi, Katsushi; Shigenobu, Shuji; Ogino, Yukiko; Tatarazako, Norihisa; Miyagawa, Shinichi; Iguchi, Taisen

2015-03-14

The cladoceran crustacean Daphnia pulex produces female offspring by parthenogenesis under favorable conditions, but in response to various unfavorable external stimuli, it produces male offspring (environmental sex determination: ESD). We recently established an innovative system for ESD studies using D. pulex WTN6 strain, in which the sex of the offspring can be controlled simply by changes in the photoperiod: the long-day and short-day conditions can induce female and male offspring, respectively. Taking advantage of this system, we demonstrated that de novo methyl farnesoate (MF) synthesis is necessary for male offspring production. These results indicate the key role of innate MF signaling as a conductor between external environmental stimuli and the endogenous male developmental pathway. Despite these findings, the molecular mechanisms underlying up- and downstream signaling of MF have not yet been well elucidated in D. pulex. To elucidate up- and downstream events of MF signaling during sex determination processes, we compared the transcriptomes of daphnids reared under the long-day (female) condition with short-day (male) and MF-treated (male) conditions. We found that genes involved in ionotropic glutamate receptors, known to mediate the vast majority of excitatory neurotransmitting processes in various organisms, were significantly activated in daphnids by the short-day condition but not by MF treatment. Administration of specific agonists and antagonists, especially for the N-methyl-D-aspartic acid (NMDA) receptor, strongly increased or decreased, respectively, the proportion of male-producing mothers. Moreover, we also identified genes responsible for male production (e.g., protein kinase C pathway-related genes). Such genes were generally shared between the short-day reared and MF-treated daphnids. We identified several candidate genes regulating ESD which strongly suggests that these genes may be essential factors for male offspring production as an upstream regulator of MF signaling in D. pulex. This study provides new insight into the fundamental mechanisms underlying how living organisms alter their phenotypes in response to various external environments.

Vinpocetine alleviate cerebral ischemia/reperfusion injury by down-regulating TLR4/MyD88/NF-κB signaling

PubMed Central

Wu, Li-Rong; Liu, Liang; Xiong, Xiao-Yi; Zhang, Qin; Wang, Fa-Xiang; Gong, Chang-Xiong; Zhong, Qi; Yang, Yuan-Rui; Meng, Zhao-You; Yang, Qing-Wu

2017-01-01

Inflammatory responses play crucial roles in cerebral ischemia/reperfusion injury. Toll-like receptor 4 (TLR4) is an important mediator of the neuroinflammatory response to cerebral ischemia/reperfusion injury. Vinpocetine is a derivative of the alkaloid vincamine and exerts an anti-inflammatory effect by inhibiting NF-κB activation. However, the effects of vinpocetine on pathways upstream of NF-κB signaling, such as TLR4, have not been fully elucidated. Here, we used mouse middle cerebral artery occlusion (MCAO) and cell-based oxygen-glucose deprivation (OGD) models to evaluate the therapeutic effects and mechanisms of vinpocetine treatment. The vinpocetine treatment significantly reduced mice cerebral infarct volumes and neurological scores. Moreover, the numbers of TUNEL+ and Fluoro-Jade B+ cells were significantly decreased in the ischemic brain tissues after vinpocetine treatment. In the OGD model, the vinpocetine treatment also increased the viability of cultured cortical neurons. Interestingly, vinpocetine exerted a neuroprotective effect on the mouse MCAO model and cell-based OGD model by inhibiting TLR4-mediated inflammatory responses and decreasing proinflammatory cytokine release through the MyD88-dependent signaling pathway, independent of TRIF signaling pathway. In conclusion, vinpocetine exerts anti-inflammatory effects to ameliorate cerebral ischemia/reperfusion injury in vitro and in vivo. Vinpocetine may inhibit inflammatory responses through the TLR4/MyD88/NF-κB signaling pathway, independent of TRIF-mediated inflammatory responses. Thus, vinpocetine may be an attractive therapeutic candidate for the treatment of ischemic cerebral injury or other inflammatory diseases. PMID:29113305

Vinpocetine alleviate cerebral ischemia/reperfusion injury by down-regulating TLR4/MyD88/NF-κB signaling.

PubMed

Wu, Li-Rong; Liu, Liang; Xiong, Xiao-Yi; Zhang, Qin; Wang, Fa-Xiang; Gong, Chang-Xiong; Zhong, Qi; Yang, Yuan-Rui; Meng, Zhao-You; Yang, Qing-Wu

2017-10-06

Inflammatory responses play crucial roles in cerebral ischemia/reperfusion injury. Toll-like receptor 4 (TLR4) is an important mediator of the neuroinflammatory response to cerebral ischemia/reperfusion injury. Vinpocetine is a derivative of the alkaloid vincamine and exerts an anti-inflammatory effect by inhibiting NF-κB activation. However, the effects of vinpocetine on pathways upstream of NF-κB signaling, such as TLR4, have not been fully elucidated. Here, we used mouse middle cerebral artery occlusion (MCAO) and cell-based oxygen-glucose deprivation (OGD) models to evaluate the therapeutic effects and mechanisms of vinpocetine treatment. The vinpocetine treatment significantly reduced mice cerebral infarct volumes and neurological scores. Moreover, the numbers of TUNEL+ and Fluoro-Jade B+ cells were significantly decreased in the ischemic brain tissues after vinpocetine treatment. In the OGD model, the vinpocetine treatment also increased the viability of cultured cortical neurons. Interestingly, vinpocetine exerted a neuroprotective effect on the mouse MCAO model and cell-based OGD model by inhibiting TLR4-mediated inflammatory responses and decreasing proinflammatory cytokine release through the MyD88-dependent signaling pathway, independent of TRIF signaling pathway. In conclusion, vinpocetine exerts anti-inflammatory effects to ameliorate cerebral ischemia/reperfusion injury in vitro and in vivo. Vinpocetine may inhibit inflammatory responses through the TLR4/MyD88/NF-κB signaling pathway, independent of TRIF-mediated inflammatory responses. Thus, vinpocetine may be an attractive therapeutic candidate for the treatment of ischemic cerebral injury or other inflammatory diseases.

Epigenetic restriction of Hippo signaling by MORC2 underlies stemness of hepatocellular carcinoma cells.

PubMed

Wang, Tao; Qin, Zhong-Yi; Wen, Liang-Zhi; Guo, Yan; Liu, Qin; Lei, Zeng-Jie; Pan, Wei; Liu, Kai-Jun; Wang, Xing-Wei; Lai, Shu-Jie; Sun, Wen-Jing; Wei, Yan-Ling; Liu, Lei; Guo, Ling; Chen, Yu-Qin; Wang, Jun; Xiao, Hua-Liang; Bian, Xiu-Wu; Chen, Dong-Feng; Wang, Bin

2018-03-19

The evolutionarily conserved Hippo signaling pathway is a key regulator of stem cell self-renewal, differentiation, and organ size. While alterations in Hippo signaling are causally linked to uncontrolled cell growth and a broad range of malignancies, genetic mutations in the Hippo pathway are uncommon and it is unclear how the tumor suppressor function of the Hippo pathway is disrupted in human cancers. Here, we report a novel epigenetic mechanism of Hippo inactivation in the context of hepatocellular carcinoma (HCC). We identify a member of the microrchidia (MORC) protein family, MORC2, as an inhibitor of the Hippo pathway by controlling upstream Hippo regulators, neurofibromatosis 2 (NF2) and kidney and brain protein (KIBRA). Mechanistically, MORC2 forms a complex with DNA methyltransferase 3A (DNMT3A) at the promoters of NF2 and KIBRA, leading to their DNA hyper-methylation and transcriptional repression. As a result, NF2 and KIBRA are crucial targets of MORC2 to regulate confluence-induced activation of Hippo signaling and contact inhibition of cell growth under both physiological and pathological conditions. The MORC2-NF2/KIBRA axis is critical for maintaining self-renewal, sorafenib resistance, and oncogenicity of HCC cells in vitro and in nude mice. Furthermore, MORC2 expression is elevated in HCC tissues, associated with stem-like properties of cancer cells, and disease progression in patients. Collectively, MORC2 promotes cancer stemness and tumorigenesis by facilitating DNA methylation-dependent silencing of Hippo signaling and could be a potential molecular target for cancer therapeutics.

Identification of DEAD-Box RNA Helicase DDX41 as a Trafficking Protein That Involves in Multiple Innate Immune Signaling Pathways in a Zebrafish Model.

PubMed

Ma, Jun-Xia; Li, Jiang-Yuan; Fan, Dong-Dong; Feng, Wei; Lin, Ai-Fu; Xiang, Li-Xin; Shao, Jian-Zhong

2018-01-01

DDX41 is an important sensor for host recognition of DNA viruses and initiation of nuclear factor-κB (NF-κB) and IFN signaling pathways in mammals. However, its occurrence and functions in other vertebrates remain poorly defined. Here, a DDX41 ortholog [ Danio rerio DDX41 ( Dr DDX41)] with various conserved structural features to its mammalian counterparts was identified from a zebrafish model. This Dr DDX41 was found to be a trafficking protein distributed in the nucleus of resting cells but transported into the cytoplasm under DNA stimulation. Two nuclear localization signal motifs were localized beside the coiled-coil domain, whereas one nuclear export signal motif existed in the DEADc domain. Dr DDX41 acts as an initiator for the activation of NF-κB and IFN signaling pathways in a Danio rerio STING ( Dr STING)-dependent manner through its DEADc domain, which is a typical performance of mammalian DDX41. These observations suggested the conservation of DDX41 proteins throughout the vertebrate evolution, making zebrafish an alternative model in understanding DDX41-mediated immunology. With this model system, we found that Dr DDX41 contributes to Dr STING- Danio rerio STAT6 ( Dr STAT6)-mediated chemokine ( Danio rerio CCL20) production through its DEADc domain. To the best of our knowledge, this work is the first report showing that DDX41 is an upstream initiator in this newly identified signaling pathway. The Dr DDX41-mediated signaling pathways play important roles in innate antibacterial immunity because knockdown of either Dr DDX41 or Dr STING/ Dr STAT6 significantly reduced the survival of zebrafish under Aeromonas hydrophilia or Edwardsiella tarda infection. Our findings would enrich the current knowledge of DDX41-mediated immunology and the evolutionary history of the DDX41 family.

Steroidal androgens and nonsteroidal, tissue-selective androgen receptor modulator, S-22, regulate androgen receptor function through distinct genomic and nongenomic signaling pathways.

PubMed

Narayanan, Ramesh; Coss, Christopher C; Yepuru, Muralimohan; Kearbey, Jeffrey D; Miller, Duane D; Dalton, James T

2008-11-01

Androgen receptor (AR) ligands are important for the development and function of several tissues and organs. However, the poor oral bioavailability, pharmacokinetic properties, and receptor cross-reactivity of testosterone, coupled with side effects, place limits on its clinical use. Selective AR modulators (SARMs) elicit anabolic effects in muscle and bone, sparing reproductive organs like the prostate. However, molecular mechanisms underlying the tissue selectivity remain ambiguous. We performed a variety of in vitro studies to compare and define the molecular mechanisms of an aryl propionamide SARM, S-22, as compared with dihydrotestosterone (DHT). Studies indicated that S-22 increased levator ani muscle weight but decreased the size of prostate in rats. Analysis of the upstream intracellular signaling events indicated that S-22 and DHT mediated their actions through distinct pathways. Modulation of these pathways altered the recruitment of AR and its cofactors to the PSA enhancer in a ligand-dependent fashion. In addition, S-22 induced Xenopus laevis oocyte maturation and rapid phosphorylation of several kinases, through pathways distinct from steroids. These studies reveal novel differences in the molecular mechanisms by which S-22, a nonsteroidal SARM, and DHT mediate their pharmacological effects.

Dampened Hedgehog signaling but normal Wnt signaling in zebrafish without cilia

PubMed Central

Huang, Peng; Schier, Alexander F.

2009-01-01

Summary Cilia have been implicated in Hedgehog (Hh) and Wnt signaling in mouse but not in Drosophila. To determine whether the role of cilia is conserved in zebrafish, we generated maternal-zygotic (MZ) oval (ovl; ift88) mutants that lack all cilia. MZovl mutants display normal canonical and non-canonical Wnt signaling but show defects in Hh signaling. As in mouse, zebrafish cilia are required to mediate the activities of Hh, Ptc, Smo and PKA. However, in contrast to mouse Ift88 mutants, which show a dramatic reduction in Hh signaling, zebrafish MZovl mutants display dampened, but expanded, Hh pathway activity. This activity is largely due to gli1, the expression of which is fully dependent on Hh signaling in mouse but not in zebrafish. These results reveal a conserved requirement for cilia in transducing the activity of upstream regulators of Hh signaling but distinct phenotypic effects due to differential regulation and differing roles of transcriptional mediators. PMID:19700616

Phosphorylated 4E binding protein 1: a hallmark of cell signaling that correlates with survival in ovarian cancer.

PubMed

Castellvi, Josep; Garcia, Angel; Rojo, Federico; Ruiz-Marcellan, Carmen; Gil, Antonio; Baselga, Jose; Ramon y Cajal, Santiago

2006-10-15

Growth factor receptors and cell signaling factors play a crucial role in human carcinomas and have been studied in ovarian tumors with varying results. Cell signaling involves multiple pathways and a myriad of factors that can be mutated or amplified. Cell signaling is driven through the mammalian target of rapamycin (mTOR) and extracellular regulated kinase (ERK) pathways and by some downstream molecules, such as 4E binding protein 1 (4EBP1), eukaryotic initiation factor 4E, and p70 ribosomal protein S6 kinase (p70S6K). The objectives of this study were to analyze the real role that these pathways play in ovarian cancer, to correlate them with clinicopathologic characteristics, and to identify the factors that transmit individual proliferation signals and are associated with pathologic grade and prognosis, regardless specific oncogenic alterations upstream. One hundred twenty-nine ovarian epithelial tumors were studied, including 20 serous cystadenomas, 7 mucinous cystadenomas, 11 serous borderline tumors, 16 mucinous borderline tumors, 29 serous carcinomas, 16 endometrioid carcinomas, 15 clear cell carcinomas, and 15 mucinous carcinomas. Tissue microarrays were constructed, and immunohistochemistry for the receptors epidermal growth factor receptor (EGFR) and c-erb-B2 was performed and with phosphorylated antibodies for protein kinase B (AKT), 4EBP1, p70S6K, S6, and ERK. Among 129 ovarian neoplasms, 17.8% were positive for c-erb-B2, 9.3% were positive for EGFR, 47.3% were positive for phosphorylated AKT (p-AKT), 58.9% were positive for p-ERK, 41.1% were positive for p-4EBP1, 26.4% were positive for p70S6K, and 15.5% were positive for p-S6. Although EGFR, p-AKT, and p-ERK expression did not differ between benign, borderline, or malignant tumors, c-erb-B2, p-4EBP1, p-p70S6K, and p-S6 were expressed significantly more often in malignant tumors. Only p-4EBP1 expression demonstrated prognostic significance (P = .005), and only surgical stage and p-4EBP1 expression had statistical significance in the multivariate analysis. In patients with ovarian carcinoma, significant expression of p-4EBP1 was associated with high-grade tumors and a poor prognosis, regardless other oncogenic alterations upstream. This finding supports the study of this factor as a hallmark or pivotal factor in cell signaling in ovarian carcinoma that may crucial in the transmission of the proliferation cell signal and may reflect the real oncogenic role of this pathway in ovarian tumors. 2006 American Cancer Society

Revisiting the X:A signal that specifies Caenorhabditis elegans sexual fate.

PubMed

Gladden, John M; Farboud, Behnom; Meyer, Barbara J

2007-11-01

In Caenorhabditis elegans, sex is determined by the opposing actions of X-signal elements (XSEs) and autosomal signal elements (ASEs), which communicate the ratio of X chromosomes to sets of autosomes (X:A signal). This study delves more deeply into the mechanism by which XSEs transmit X chromosome dose. We determined the relative contributions of individual XSEs to the X:A signal and showed the order of XSE strength to be sex-1 > sex-2 > fox-1 > ceh-39 >/= region 1 XSE. sex-1 exerts a more potent influence on sex determination and dosage compensation than any other XSE by functioning in two separate capacities in the pathway: sex-1 acts upstream as an XSE to repress xol-1 and downstream as an activator of hermaphrodite development and dosage compensation. Furthermore, the process of dosage compensation affects expression of the very XSEs that control it; XSEs become fully dosage compensated once sex is determined. The X:A signal is then equivalent between XO and XX animals, causing sexual differentiation to be controlled by genes downstream of xol-1 in the sex-determination pathway. Prior to the onset of dosage compensation, the difference in XSE expression between XX and XO embryos appears to be greater than twofold, making X chromosome counting a robust process.

Integrated Stress Response Mediates Epithelial Injury in Mechanical Ventilation.

PubMed

Dolinay, Tamas; Himes, Blanca E; Shumyatcher, Maya; Lawrence, Gladys Gray; Margulies, Susan S

2017-08-01

Ventilator-induced lung injury (VILI) is a severe complication of mechanical ventilation that can lead to acute respiratory distress syndrome. VILI is characterized by damage to the epithelial barrier with subsequent pulmonary edema and profound hypoxia. Available lung-protective ventilator strategies offer only a modest benefit in preventing VILI because they cannot impede alveolar overdistension and concomitant epithelial barrier dysfunction in the inflamed lung regions. There are currently no effective biochemical therapies to mitigate injury to the alveolar epithelium. We hypothesize that alveolar stretch activates the integrated stress response (ISR) pathway and that the chemical inhibition of this pathway mitigates alveolar barrier disruption during stretch and mechanical ventilation. Using our established rat primary type I-like alveolar epithelial cell monolayer stretch model and in vivo rat mechanical ventilation that mimics the alveolar overdistension seen in acute respiratory distress syndrome, we studied epithelial responses to mechanical stress. Our studies revealed that the ISR signaling pathway is a key modulator of epithelial permeability. We show that prolonged epithelial stretch and injurious mechanical ventilation activate the ISR, leading to increased alveolar permeability, cell death, and proinflammatory signaling. Chemical inhibition of protein kinase RNA-like endoplasmic reticulum kinase, an upstream regulator of the pathway, resulted in decreased injury signaling and improved barrier function after prolonged cyclic stretch and injurious mechanical ventilation. Our results provide new evidence that therapeutic targeting of the ISR can mitigate VILI.

Insulin-like growth factor-mediated muscle differentiation: collaboration between phosphatidylinositol 3-kinase-Akt-signaling pathways and myogenin.

PubMed

Tureckova, J; Wilson, E M; Cappalonga, J L; Rotwein, P

2001-10-19

The differentiation and maturation of skeletal muscle require interactions between signaling pathways activated by hormones and growth factors and an intrinsic regulatory network controlled by myogenic transcription factors. Insulin-like growth factors (IGFs) play key roles in muscle development in the embryo and in regeneration in the adult. To study mechanisms of IGF action in muscle, we developed a myogenic cell line that overexpresses IGF-binding protein-5. C2BP5 cells remain quiescent in low serum differentiation medium until the addition of IGF-I. Here we use this cell line to identify signaling pathways controlling IGF-mediated differentiation. Induction of myogenin by IGF-I and myotube formation were prevented by the phosphatidylinositol (PI) 3-kinase inhibitor, LY294002, even when included 2 days after growth factor addition, whereas expression of active PI 3-kinase could promote differentiation in the absence of IGF-I. Differentiation also was induced by myogenin but was blocked by LY294002. The differentiation-promoting effects of IGF-I were mimicked by a modified membrane-targeted inducible Akt-1 (iAkt), and iAkt was able to stimulate differentiation of C2 myoblasts and primary mouse myoblasts incubated with otherwise inhibitory concentrations of LY294002. These results show that an IGF-regulated PI 3-kinase-Akt pathway controls muscle differentiation by mechanisms acting both upstream and downstream of myogenin.

Transcription factor AP-2γ induces early Cdx2 expression and represses HIPPO signaling to specify the trophectoderm lineage

PubMed Central

Cao, Zubing; Carey, Timothy S.; Ganguly, Avishek; Wilson, Catherine A.; Paul, Soumen; Knott, Jason G.

2015-01-01

Cell fate decisions are fundamental to the development of multicellular organisms. In mammals the first cell fate decision involves segregation of the pluripotent inner cell mass and the trophectoderm, a process regulated by cell polarity proteins, HIPPO signaling and lineage-specific transcription factors such as CDX2. However, the regulatory mechanisms that operate upstream to specify the trophectoderm lineage have not been established. Here we report that transcription factor AP-2γ (TFAP2C) functions as a novel upstream regulator of Cdx2 expression and position-dependent HIPPO signaling in mice. Loss- and gain-of-function studies and promoter analysis revealed that TFAP2C binding to an intronic enhancer is required for activation of Cdx2 expression during early development. During the 8-cell to morula transition TFAP2C potentiates cell polarity to suppress HIPPO signaling in the outside blastomeres. TFAP2C depletion triggered downregulation of PARD6B, loss of apical cell polarity, disorganization of F-actin, and activation of HIPPO signaling in the outside blastomeres. Rescue experiments using Pard6b mRNA restored cell polarity but only partially corrected position-dependent HIPPO signaling, suggesting that TFAP2C negatively regulates HIPPO signaling via multiple pathways. Several genes involved in regulation of the actin cytoskeleton (including Rock1, Rock2) were downregulated in TFAP2C-depleted embryos. Inhibition of ROCK1 and ROCK2 activity during the 8-cell to morula transition phenocopied TFAP2C knockdown, triggering a loss of position-dependent HIPPO signaling and decrease in Cdx2 expression. Altogether, these results demonstrate that TFAP2C facilitates trophectoderm lineage specification by functioning as a key regulator of Cdx2 transcription, cell polarity and position-dependent HIPPO signaling. PMID:25858457

Tetramethylpyrazine induces SH-SY5Y cell differentiation toward the neuronal phenotype through activation of the PI3K/Akt/Sp1/TopoIIβ pathway.

PubMed

Yan, Yong-Xin; Zhao, Jun-Xia; Han, Shuo; Zhou, Na-Jing; Jia, Zhi-Qiang; Yao, Sheng-Jie; Cao, Cui-Li; Wang, Yan-Ling; Xu, Yan-Nan; Zhao, Juan; Yan, Yun-Li; Cui, Hui-Xian

2015-12-01

Tetramethylpyrazine (TMP) is an active compound extracted from the traditional Chinese medicinal herb Chuanxiong. Previously, we have shown that TMP induces human SH-SY5Y neuroblastoma cell differentiation toward the neuronal phenotype by targeting topoisomeraseIIβ (TopoIIβ), a protein implicated in neural development. In the present study, we aimed to elucidate whether the transcriptional factors specificity protein 1 (Sp1) and nuclear factor Y (NF-Y), in addition to the upstream signaling pathways ERK1/2 and PI3K/Akt, are involved in modulating TopoIIβ expression in the neuronal differentiation process. We demonstrated that SH-SY5Y cells treated with TMP (80μM) terminally differentiated into neurons, characterized by increased neuronal markers, tubulin βIII and microtubule associated protein 2 (MAP2), and increased neurite outgrowth, with no negative effect on cell survival. TMP also increased the expression of TopoIIβ, which was accompanied by increased expression of Sp1 in the differentiated neuron-like cells, whereas NF-Y protein levels remained unchanged following the differentiation progression. We also found that the phosphorylation level of Akt, but not ERK1/2, was significantly increased as a result of TMP stimulation. Furthermore, as established by chromatin immunoprecipitation (ChIP) assay, activation of the PI3K/Akt pathway increased Sp1 binding to the promoter of the TopoIIβ gene. Blockage of PI3K/Akt was shown to lead to subsequent inhibition of TopoIIβ expression and neuronal differentiation. Collectively, the results indicate that the PI3K/Akt/Sp1/TopoIIβ signaling pathway is necessary for TMP-induced neuronal differentiation. Our findings offer mechanistic insights into understanding the upstream regulation of TopoIIβ in neuronal differentiation, and suggest potential applications of TMP both in neuroscience research and clinical practice to treat relevant diseases of the nervous system. Copyright © 2015 Elsevier GmbH. All rights reserved.

Feedback Activation of Basic Fibroblast Growth Factor Signaling via the Wnt/β-Catenin Pathway in Skin Fibroblasts

PubMed Central

Wang, Xu; Zhu, Yuting; Sun, Congcong; Wang, Tao; Shen, Yingjie; Cai, Wanhui; Sun, Jia; Chi, Lisha; Wang, Haijun; Song, Na; Niu, Chao; Shen, Jiayi; Cong, Weitao; Zhu, Zhongxin; Xuan, Yuanhu; Li, Xiaokun; Jin, Litai

2017-01-01

Skin wound healing is a complex process requiring the coordinated behavior of many cell types, especially in the proliferation and migration of fibroblasts. Basic fibroblast growth factor (bFGF) is a member of the FGF family that promotes fibroblast migration, but the underlying molecular mechanism remains elusive. The present RNA sequencing study showed that the expression levels of several canonical Wnt pathway genes, including Wnt2b, Wnt3, Wnt11, T-cell factor 7 (TCF7), and Frizzled 8 (FZD8) were modified by bFGF stimulation in fibroblasts. Enzyme-linked immunosorbent assay (ELISA) analysis also showed that Wnt pathway was activated under bFGF treatment. Furthermore, treatment of fibroblasts with lithium chloride or IWR-1, an inducer and inhibitor of the Wnt signaling pathway, respectively, promoted and inhibited cell migration. Also, levels of cytosolic glycogen synthase kinase 3 beta phosphorylated at serine9 (pGSK3β Ser9) and nuclear β-catenin were increased upon exposure to bFGF. Molecular and biochemical assays indicated that phosphoinositide 3-kinase (PI3K) signaling activated the GSK3β/β-catenin/Wnt signaling pathway via activation of c-Jun N-terminal kinase (JNK), suggesting that PI3K and JNK act at the upstream of β-catenin. In contrast, knock-down of β-catenin delayed fibroblast cell migration even under bFGF stimulation. RNA sequencing analysis of β-catenin knock-down fibroblasts demonstrated that β-catenin positively regulated the transcription of bFGF and FGF21. Moreover, FGF21 treatment activated AKT and JNK, and accelerated fibroblast migration to a similar extent as bFGF does. In addition, ELISA analysis demonstrated that both of bFGF and FGF21 were auto secretion factor and be regulated by Wnt pathway stimulators. Taken together, our analyses define a feedback regulatory loop between bFGF (FGF21) and Wnt signaling acting through β-catenin in skin fibroblasts. PMID:28217097

Crosstalk of the EphA2 Receptor with a Serine/Threonine Phosphatase Suppresses the Akt-mTORC1 Pathway in Cancer Cells

PubMed Central

Yang, Nai-Ying; Fernandez, Carlos; Richter, Melanie; Xiao, Zhan; Valencia, Fatima; Tice, David A.; Pasquale, Elena B.

2010-01-01

Receptor tyrosine kinases of the Eph family play multiple roles in the physiological regulation of tissue homeostasis and in the pathogenesis of various diseases, including cancer. The EphA2 receptor is highly expressed in most cancer cell types, where it has disparate activities that are not well understood. It has been reported that interplay of EphA2 with oncogenic signaling pathways promotes cancer cell malignancy independently of ephrin ligand binding and receptor kinase activity. In contrast, stimulation of EphA2 signaling with ephrin-A ligands can suppress malignancy by inhibiting the Ras-MAP kinase pathway, integrin-mediated adhesion, and epithelial to mesenchymal transition. Here we show that ephrin-A1 ligand-dependent activation of EphA2 decreases the growth of PC3 prostate cancer cells and profoundly inhibits the Akt-mTORC1 pathway, which is hyperactivated due to loss of the PTEN tumor suppressor. Our results do not implicate changes in the activity of Akt upstream regulators (such as Ras family GTPases, PI3 kinase, integrins, or the Ship2 lipid phosphatase) in the observed loss of Akt T308 and S473 phosphorylation downstream of EphA2. Indeed, EphA2 can inhibit Akt phosphorylation induced by oncogenic mutations of not only PTEN but also PI3 kinase. Furthermore, it can decrease the hyperphosphorylation induced by constitutive membrane-targeting of Akt. Our data suggest a novel signaling mechanism whereby EphA2 inactivates the Akt-mTORC1 oncogenic pathway through Akt dephosphorylation mediated by a serine/threonine phosphatase. Ephrin-A1-induced Akt dephosphorylation was observed not only in PC3 prostate cancer cells but also in other cancer cell types. Thus, activation of EphA2 signaling represents a possible new avenue for anti-cancer therapies that exploit the remarkable ability of this receptor to counteract multiple oncogenic signaling pathways. PMID:20837138

Transcriptional regulation of hepatic lipogenesis.

PubMed

Wang, Yuhui; Viscarra, Jose; Kim, Sun-Joong; Sul, Hei Sook

2015-11-01

Fatty acid and fat synthesis in the liver is a highly regulated metabolic pathway that is important for very low-density lipoprotein (VLDL) production and thus energy distribution to other tissues. Having common features at their promoter regions, lipogenic genes are coordinately regulated at the transcriptional level. Transcription factors, such as upstream stimulatory factors (USFs), sterol regulatory element-binding protein 1C (SREBP1C), liver X receptors (LXRs) and carbohydrate-responsive element-binding protein (ChREBP) have crucial roles in this process. Recently, insights have been gained into the signalling pathways that regulate these transcription factors. After feeding, high blood glucose and insulin levels activate lipogenic genes through several pathways, including the DNA-dependent protein kinase (DNA-PK), atypical protein kinase C (aPKC) and AKT-mTOR pathways. These pathways control the post-translational modifications of transcription factors and co-regulators, such as phosphorylation, acetylation or ubiquitylation, that affect their function, stability and/or localization. Dysregulation of lipogenesis can contribute to hepatosteatosis, which is associated with obesity and insulin resistance.

Lipid-induced insulin resistance mediated by the proinflammatory receptor TLR4 requires saturated fatty acid-induced ceramide biosynthesis in mice.

PubMed

Holland, William L; Bikman, Benjamin T; Wang, Li-Ping; Yuguang, Guan; Sargent, Katherine M; Bulchand, Sarada; Knotts, Trina A; Shui, Guanghou; Clegg, Deborah J; Wenk, Markus R; Pagliassotti, Michael J; Scherer, Philipp E; Summers, Scott A

2011-05-01

Obesity is associated with an enhanced inflammatory response that exacerbates insulin resistance and contributes to diabetes, atherosclerosis, and cardiovascular disease. One mechanism accounting for the increased inflammation associated with obesity is activation of the innate immune signaling pathway triggered by TLR4 recognition of saturated fatty acids, an event that is essential for lipid-induced insulin resistance. Using in vitro and in vivo systems to model lipid induction of TLR4-dependent inflammatory events in rodents, we show here that TLR4 is an upstream signaling component required for saturated fatty acid-induced ceramide biosynthesis. This increase in ceramide production was associated with the upregulation of genes driving ceramide biosynthesis, an event dependent of the activity of the proinflammatory kinase IKKβ. Importantly, increased ceramide production was not required for TLR4-dependent induction of inflammatory cytokines, but it was essential for TLR4-dependent insulin resistance. These findings suggest that sphingolipids such as ceramide might be key components of the signaling networks that link lipid-induced inflammatory pathways to the antagonism of insulin action that contributes to diabetes.

Possible involvement of G-proteins and cAMP in the induction of progesterone hydroxylating enzyme system in the vascular wilt fungus Fusarium oxysporum.

PubMed

Poli, Anna; Di Pietro, Antonio; Zigon, Dusan; Lenasi, Helena

2009-02-01

Fungi present the ability to hydroxylate steroids. In some filamentous fungi, progesterone induces an enzyme system which converts the compound into a less toxic hydroxylated product. We investigated the progesterone response in the vascular wilt pathogen Fusarium oxysporum, using mass spectrometry and high performance liquid chromatography (HPLC). Progesterone was mainly transformed into 15alpha-hydroxyprogesterone, which was found predominantly in the extracellular medium. The role of two conserved fungal signaling cascades in the induction of the progesterone-transforming enzyme system was studied, using knockout mutants lacking the mitogen-activated protein kinase Fmk1 or the heterotrimeric G-protein beta subunit Fgb1 functioning upstream of the cyclic adenosine monophosphate (cAMP) pathway. No steroid hydroxylation was induced in the Deltafgb1 strain, suggesting a role for the G-protein beta subunit in progesterone signaling. Exogenous cAMP restored the induction of progesterone-transforming activity in the Deltafgb1 strain, suggesting that steroid signaling in F. oxysporum is mediated by the cAMP-PKA pathway.

Drosophila Spidey/Kar Regulates Oenocyte Growth via PI3-Kinase Signaling

PubMed Central

Cinnamon, Einat; Sawala, Annick; Tittiger, Claus; Paroush, Ze'ev

2016-01-01

Cell growth and proliferation depend upon many different aspects of lipid metabolism. One key signaling pathway that is utilized in many different anabolic contexts involves Phosphatidylinositide 3-kinase (PI3K) and its membrane lipid products, the Phosphatidylinositol (3,4,5)-trisphosphates. It remains unclear, however, which other branches of lipid metabolism interact with the PI3K signaling pathway. Here, we focus on specialized fat metabolizing cells in Drosophila called larval oenocytes. In the presence of dietary nutrients, oenocytes undergo PI3K-dependent cell growth and contain very few lipid droplets. In contrast, during starvation, oenocytes decrease PI3K signaling, shut down cell growth and accumulate abundant lipid droplets. We now show that PI3K in larval oenocytes, but not in fat body cells, functions to suppress lipid droplet accumulation. Several enzymes of fatty acid, triglyceride and hydrocarbon metabolism are required in oenocytes primarily for lipid droplet induction rather than for cell growth. In contrast, a very long chain fatty-acyl-CoA reductase (FarO) and a putative lipid dehydrogenase/reductase (Spidey, also known as Kar) not only promote lipid droplet induction but also inhibit oenocyte growth. In the case of Spidey/Kar, we show that the growth suppression mechanism involves inhibition of the PI3K signaling pathway upstream of Akt activity. Together, the findings in this study show how Spidey/Kar and FarO regulate the balance between the cell growth and lipid storage of larval oenocytes. PMID:27500738

Mechanisms for type-II vitellogenesis-inhibiting hormone suppression of vitellogenin transcription in shrimp hepatopancreas: Crosstalk of GC/cGMP pathway with different MAPK-dependent cascades.

PubMed

Chen, Ting; Ren, Chunhua; Jiang, Xiao; Zhang, Lvping; Li, Hongmei; Huang, Wen; Hu, Chaoqun

2018-01-01

Vitellogenesis is the process of yolk formation via accumulating vitellin (Vn) with nutrients in the oocytes. Expression of vitellogenin (Vg), the precursor of Vn, is one of the indicators for the start of vitellogenesis. In Pacific white shrimp (Litopenaeus vannamei), the type-II vitellogenesis-inhibiting hormone (VIH-2) effectively suppresses hepatopancreatic Vg mRNA expression. In this study, we demonstrate the increasing transcript levels of hepatopancreatic Vg during L. vannamei ovarian development, suggesting that the hepatopancreas-derived Vg/Vn may also contribute to vitellogenesis in this species. Using a combination of in vivo injections and in vitro primary cell cultures, we provide evidences that the inhibition of VIH-2 on hepatopancreatic Vg gene expression is mediated through a functional coupling of the GC/cGMP pathway with different MAPK-dependent cascades in female shrimp. In VIH-2 signaling, the NO-independent GC/cGMP/PKG cascades were upstream of the MAPKs. Activations of the MAPK signal by VIH-2 include the phosphorylation of JNK and the mRNA/protein expression of P38MAPK. Additionally, the cAMP/PKA pathway is another positive intracellular signal for hepatopancreatic Vg mRNA expression but is independent of its VIH-2 regulation. Our findings establish a model for the signal transduction mechanism of Vg regulation by VIH and shed light on the biological functions and signaling of the CHH family in crustaceans.

Mechanisms for type-II vitellogenesis-inhibiting hormone suppression of vitellogenin transcription in shrimp hepatopancreas: Crosstalk of GC/cGMP pathway with different MAPK-dependent cascades

PubMed Central

Ren, Chunhua; Jiang, Xiao; Zhang, Lvping; Li, Hongmei; Huang, Wen; Hu, Chaoqun

2018-01-01

Vitellogenesis is the process of yolk formation via accumulating vitellin (Vn) with nutrients in the oocytes. Expression of vitellogenin (Vg), the precursor of Vn, is one of the indicators for the start of vitellogenesis. In Pacific white shrimp (Litopenaeus vannamei), the type-II vitellogenesis-inhibiting hormone (VIH-2) effectively suppresses hepatopancreatic Vg mRNA expression. In this study, we demonstrate the increasing transcript levels of hepatopancreatic Vg during L. vannamei ovarian development, suggesting that the hepatopancreas-derived Vg/Vn may also contribute to vitellogenesis in this species. Using a combination of in vivo injections and in vitro primary cell cultures, we provide evidences that the inhibition of VIH-2 on hepatopancreatic Vg gene expression is mediated through a functional coupling of the GC/cGMP pathway with different MAPK-dependent cascades in female shrimp. In VIH-2 signaling, the NO-independent GC/cGMP/PKG cascades were upstream of the MAPKs. Activations of the MAPK signal by VIH-2 include the phosphorylation of JNK and the mRNA/protein expression of P38MAPK. Additionally, the cAMP/PKA pathway is another positive intracellular signal for hepatopancreatic Vg mRNA expression but is independent of its VIH-2 regulation. Our findings establish a model for the signal transduction mechanism of Vg regulation by VIH and shed light on the biological functions and signaling of the CHH family in crustaceans. PMID:29590153

Sulfur mustard analog induces oxidative stress and activates signaling cascades in the skin of SKH-1 hairless mice.

PubMed

Pal, Arttatrana; Tewari-Singh, Neera; Gu, Mallikarjuna; Agarwal, Chapla; Huang, Jie; Day, Brian J; White, Carl W; Agarwal, Rajesh

2009-12-01

A monofunctional analog of the chemical warfare agent sulfur mustard (HD), 2-chloroethyl ethyl sulfide (CEES), induces tissue damage similar to HD. Herein we studied the molecular mechanisms associated with CEES-induced skin inflammation and toxicity in SKH-1 hairless mice. Topical CEES exposure caused an increase in oxidative stress as observed by enhanced 4-hydroxynonenal and 5,5-dimethyl-2-(8-octanoic acid)-1-pyrroline N-oxide protein adduct formation and an increase in protein oxidation. The CEES-induced increase in the formation of 8-oxo-2-deoxyguanosine indicated DNA oxidation. CEES exposure instigated an increase in the phosphorylation of mitogen-activated protein kinases (MAPKs; ERK1/2, JNK, and p38). After CEES exposure, a significant increase in the phosphorylation of Akt at Ser473 and Thr308 was observed as well as upregulation of its upstream effector, PDK1, in mouse skin tissue. Subsequently, CEES exposure caused activation of AP-1 family proteins and the NF-kappaB pathway, including phosphorylation and degradation of IkappaBalpha in addition to phosphorylation of the NF-kappaB essential modulator. Collectively, our results indicate that CEES induces oxidative stress and the activation of the transcription factors AP-1 and NF-kappaB via upstream signaling pathways including MAPKs and Akt in SKH-1 hairless mouse skin. These novel molecular targets could be supportive in the development of prophylactic and therapeutic interventions against HD-related skin injury.

Prospero-related homeobox 1 (Prox1) at the crossroads of diverse pathways during adult neural fate specification

PubMed Central

Stergiopoulos, Athanasios; Elkouris, Maximilianos; Politis, Panagiotis K.

2015-01-01

Over the last decades, adult neurogenesis in the central nervous system (CNS) has emerged as a fundamental process underlying physiology and disease. Recent evidence indicates that the homeobox transcription factor Prox1 is a critical intrinsic regulator of neurogenesis in the embryonic CNS and adult dentate gyrus (DG) of the hippocampus, acting in multiple ways and instructed by extrinsic cues and intrinsic factors. In the embryonic CNS, Prox1 is mechanistically involved in the regulation of proliferation vs. differentiation decisions of neural stem cells (NSCs), promoting cell cycle exit and neuronal differentiation, while inhibiting astrogliogenesis. During the complex differentiation events in adult hippocampal neurogenesis, Prox1 is required for maintenance of intermediate progenitors (IPs), differentiation and maturation of glutamatergic interneurons, as well as specification of DG cell identity over CA3 pyramidal fate. The mechanism by which Prox1 exerts multiple functions involves distinct signaling pathways currently not fully highlighted. In this mini-review, we thoroughly discuss the Prox1-dependent phenotypes and molecular pathways in adult neurogenesis in relation to different upstream signaling cues and cell fate determinants. In addition, we discuss the possibility that Prox1 may act as a cross-talk point between diverse signaling cascades to achieve specific outcomes during adult neurogenesis. PMID:25674048

Src-family-tyrosine kinase Lyn is critical for TLR2-mediated NF-κB activation through the PI 3-kinase signaling pathway.

PubMed

Toubiana, Julie; Rossi, Anne-Lise; Belaidouni, Nadia; Grimaldi, David; Pene, Frederic; Chafey, Philippe; Comba, Béatrice; Camoin, Luc; Bismuth, Georges; Claessens, Yann-Erick; Mira, Jean-Paul; Chiche, Jean-Daniel

2015-10-01

TLR2 has a prominent role in host defense against a wide variety of pathogens. Stimulation of TLR2 triggers MyD88-dependent signaling to induce NF-κB translocation, and activates a Rac1-PI 3-kinase dependent pathway that leads to transactivation of NF-κB through phosphorylation of the P65 NF-κB subunit. This transactivation pathway involves tyrosine phosphorylations. The role of the tyrosine kinases in TLR signaling is controversial, with discrepancies between studies using only chemical inhibitors and knockout mice. Here, we show the involvement of the tyrosine-kinase Lyn in TLR2-dependent activation of NF-κB in human cellular models, by using complementary inhibition strategies. Stimulation of TLR2 induces the formation of an activation cluster involving TLR2, CD14, PI 3-kinase and Lyn, and leads to the activation of AKT. Lyn-dependent phosphorylation of the p110 catalytic subunit of PI 3-kinase is essential to the control of PI 3-kinase biological activity upstream of AKT and thereby to the transactivation of NF-κB. Thus, Lyn kinase activity is crucial in TLR2-mediated activation of the innate immune response in human mononuclear cells. © The Author(s) 2015.

Ferulic acid regulates the AKT/GSK-3β/CRMP-2 signaling pathway in a middle cerebral artery occlusion animal model

PubMed Central

Gim, Sang-A; Sung, Jin-Hee; Shah, Fawad-Ali; Kim, Myeong-Ok

2013-01-01

Ferulic acid, a component of the plants Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort, exerts a neuroprotective effect by regulating various signaling pathways. This study showed that ferulic acid treatment prevents the injury-induced increase of collapsin response mediator protein 2 (CRMP-2) in focal cerebral ischemia. Glycogen synthase kinase-3β (GSK-3β) regulates CRMP-2 function through phosphorylation of CRMP-2. Moreover, the pro-apoptotic activity of GSK-3β is inactivated by phosphorylation by Akt. This study investigated whether ferulic acid modulates the expression of CRMP-2 and its upstream targets, Akt and GSK-3β, in focal cerebral ischemia. Male rats were treated immediately with ferulic acid (100 mg/kg, i.v.) or vehicle after middle cerebral artery occlusion (MCAO), and then cerebral cortices were collected 24 hr after MCAO. MCAO resulted in decreased levels of phospho-Akt and phospho-GSK-3β, while ferulic acid treatment prevented the decrease in the levels of these proteins. Moreover, phospho-CRMP-2 and CRMP-2 levels increased during MCAO, whereas ferulic acid attenuated these injury-induced increases. These results demonstrate that ferulic acid regulates the Akt/GSK-3β/CRMP-2 signaling pathway in focal cerebral ischemic injury, thereby protecting against brain injury. PMID:23825478

Resveratrol prevents cognitive deficits induced by chronic unpredictable mild stress: Sirt1/miR-134 signalling pathway regulates CREB/BDNF expression in hippocampus in vivo and in vitro.

PubMed

Shen, Jun; Xu, Linling; Qu, Chujie; Sun, Huimin; Zhang, Junjian

2018-04-30

Chronic unpredictable mild stress (CUMS) leads to neuropsychiatric disorders, such as depression, anxiety and cognitive impairment. Resveratrol is a natural polyphenol existed in polygonum cuspidatum and has been demonstrated to be a potent activator of Sirtuin 1 (Sirt1). Previous studies reported that resveratrol treatment ameliorated CUMS-induced depressive-like behavior and cognitive deficits through upregulating cAMP response element-binding protein (CREB) and brain derived neurotrophic factor (BDNF) expression. However, the upstream signalling pathway mediating CREB/BDNF expression and then exerting a protective role on cognitive function remains unclear. The present study aims to investigate the possible mechanism of resveratrol on CUMS-induced cognitive deficits. Male Sprague Dawley rats were adminstrated resveratrol (40 and 80 mg/kg) every day for 4 consecutive weeks before exposure to CUMS procedure. Morris Water Maze test was used to appraise spatial learing and memory of rats. Sirt1/miR-134 signalling pathway and CREB/BDNF expression in hippocampus of rats were measured. We also explored Sirt1/miR-134 signalling pathway and CREB/BDNF expression in primary cultured hippocampus neurons with resveratrol (25, 50 and 100 μmol/L) treatment. We found that resveratrol treatment prevented spatial learing and memory impairment induced by CUMS. Meanwhile the potential mechanism of resveratrol was associated with increased levels of Sirt1, CREB phosphorylation (p-CREB), CREB, BDNF and decreased levels of miR-134 in vivo and in vitro. In conclusion, our study showed that the neuroprotective effect of resveratrol on CUMS-induced cognitive impairment may rely on activating Sirt1/miR-134 pathway and then upregulating its downstream CREB/BDNF expression in hippocampus. Copyright © 2018 Elsevier B.V. All rights reserved.

Retinoic Acid Signaling Mediates Hair Cell Regeneration by Repressing p27kip and sox2 in Supporting Cells.

PubMed

Rubbini, Davide; Robert-Moreno, Àlex; Hoijman, Esteban; Alsina, Berta

2015-11-25

During development, otic sensory progenitors give rise to hair cells and supporting cells. In mammalian adults, differentiated and quiescent sensory cells are unable to generate new hair cells when these are lost due to various insults, leading to irreversible hearing loss. Retinoic acid (RA) has strong regenerative capacity in several organs, but its role in hair cell regeneration is unknown. Here, we use genetic and pharmacological inhibition to show that the RA pathway is required for hair cell regeneration in zebrafish. When regeneration is induced by laser ablation in the inner ear or by neomycin treatment in the lateral line, we observe rapid activation of several components of the RA pathway, with dynamics that position RA signaling upstream of other signaling pathways. We demonstrate that blockade of the RA pathway impairs cell proliferation of supporting cells in the inner ear and lateral line. Moreover, in neuromast, RA pathway regulates the transcription of p27(kip) and sox2 in supporting cells but not fgf3. Finally, genetic cell-lineage tracing using Kaede photoconversion demonstrates that de novo hair cells derive from FGF-active supporting cells. Our findings reveal that RA has a pivotal role in zebrafish hair cell regeneration by inducing supporting cell proliferation, and shed light on the underlying transcriptional mechanisms involved. This signaling pathway might be a promising approach for hearing recovery. Hair cells are the specialized mechanosensory cells of the inner ear that capture auditory and balance sensory input. Hair cells die after acoustic trauma, ototoxic drugs or aging diseases, leading to progressive hearing loss. Mammals, in contrast to zebrafish, lack the ability to regenerate hair cells. Here, we find that retinoic acid (RA) pathway is required for hair cell regeneration in vivo in the zebrafish inner ear and lateral line. RA pathway is activated very early upon hair cell loss, promotes cell proliferation of progenitor cells, and regulates two key genes, p27(kip) and sox2. Our results position RA as an essential signal for hair cell regeneration with relevance in future regenerative strategies in mammals. Copyright © 2015 the authors 0270-6474/15/3515752-15$15.00/0.

Self-potential monitoring of a thermal pulse advecting through a preferential flow path

NASA Astrophysics Data System (ADS)

Ikard, S. J.; Revil, A.

2014-11-01

There is a need to develop new non-intrusive geophysical methods to detect preferential flow paths in heterogeneous porous media. A laboratory experiment is performed to non-invasively localize a preferential flow pathway in a sandbox using a heat pulse monitored by time-lapse self-potential measurements. Our goal is to investigate the amplitude of the intrinsic thermoelectric self-potential anomalies and the ability of this method to track preferential flow paths. A negative self-potential anomaly (-10 to -15 mV with respect to the background signals) is observed at the surface of the tank after hot water is injected in the upstream reservoir during steady state flow between the upstream and downstream reservoirs of the sandbox. Repeating the same experiment with the same volume of water injected upstream, but at the same temperature as the background pore water, produces a negligible self-potential anomaly. The negative self-potential anomaly is possibly associated with an intrinsic thermoelectric effect, with the temperature dependence of the streaming potential coupling coefficient, or with an apparent thermoelectric effect associated with the temperature dependence of the electrodes themselves. We model the experiment in 3D using a finite element code. Our results show that time-lapse self-potential signals can be used to track the position of traveling heat flow pulses in saturated porous materials, and therefore to find preferential flow pathways, especially in a very permeable environment and in real time. The numerical model and the data allows quantifying the intrinsic thermoelectric coupling coefficient, which is on the order of -0.3 to -1.8 mV per degree Celsius. The temperature dependence of the streaming potential during the experiment is negligible with respect to the intrinsic thermoelectric coupling. However, the temperature dependence of the potential of the electrodes needs to be accounted for and is far from being negligible if the electrodes experience temperature changes.

The Orphan G Protein-coupled Receptor Gpr175 (Tpra40) Enhances Hedgehog Signaling by Modulating cAMP Levels.

PubMed

Singh, Jaskirat; Wen, Xiaohui; Scales, Suzie J

2015-12-04

The Hedgehog (Hh) signaling pathway plays an essential role in vertebrate embryonic tissue patterning of many developing organs. Signaling occurs predominantly in primary cilia and is initiated by the entry of the G protein-coupled receptor (GPCR)-like protein Smoothened into cilia and culminates in gene transcription via the Gli family of transcription factors upon their nuclear entry. Here we identify an orphan GPCR, Gpr175 (also known as Tpra1 or Tpra40: transmembrane protein, adipocyte associated 1 or of 40 kDa), which also localizes to primary cilia upon Hh stimulation and positively regulates Hh signaling. Interaction experiments place Gpr175 at the level of PKA and upstream of the Gαi component of heterotrimeric G proteins, which itself localizes to cilia and can modulate Hh signaling. Gpr175 or Gαi1 depletion leads to increases in cellular cAMP levels and in Gli3 processing into its repressor form. Thus we propose that Gpr175 coupled to Gαi1 normally functions to inhibit the production of cAMP by adenylyl cyclase upon Hh stimulation, thus maximizing signaling by turning off PKA activity and hence Gli3 repressor formation. Taken together our data suggest that Gpr175 is a novel positive regulator of the Hh signaling pathway. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A Discrete Dynamical System Approach to Pathway Activation Profiles of Signaling Cascades.

PubMed

Catozzi, S; Sepulchre, J-A

2017-08-01

In living organisms, cascades of covalent modification cycles are one of the major intracellular signaling mechanisms, allowing to transduce physical or chemical stimuli of the external world into variations of activated biochemical species within the cell. In this paper, we develop a novel method to study the stimulus-response of signaling cascades and overall the concept of pathway activation profile which is, for a given stimulus, the sequence of activated proteins at each tier of the cascade. Our approach is based on a correspondence that we establish between the stationary states of a cascade and pieces of orbits of a 2D discrete dynamical system. The study of its possible phase portraits in function of the biochemical parameters, and in particular of the contraction/expansion properties around the fixed points of this discrete map, as well as their bifurcations, yields a classification of the cascade tiers into three main types, whose biological impact within a signaling network is examined. In particular, our approach enables to discuss quantitatively the notion of cascade amplification/attenuation from this new perspective. The method allows also to study the interplay between forward and "retroactive" signaling, i.e., the upstream influence of an inhibiting drug bound to the last tier of the cascade.

A WDM-PON with DPSK modulated downstream and OOK modulated upstream signals based on symmetric 10 Gbit/s wavelength reused bidirectional reflective SOA

NASA Astrophysics Data System (ADS)

El-Nahal, Fady I.

2017-01-01

We investigate a wavelength-division-multiplexing passive optical network (WDM-PON) with centralized lightwave and direct detection. The system is demonstrated for symmetric 10 Gbit/s differential phase-shift keying (DPSK) downstream signals and on-off keying (OOK) upstream signals, respectively. A wavelength reused scheme is employed to carry the upstream data by using a reflective semiconductor optical amplifier (RSOA) as an intensity modulator at the optical network unit (ONU). The constant-intensity property of the DPSK modulation format can keep high extinction ratio ( ER) of downstream signal and reduce the crosstalk to the upstream signal. The bit error rate ( BER) performance of our scheme shows that the proposed 10 Gbit/s symmetric WDM-PON can achieve error free transmission over 25-km-long fiber transmission with low power penalty.

Cryptochromes and Hormone Signal Transduction under Near-Zero Magnetic Fields: New Clues to Magnetic Field Effects in a Rice Planthopper.

PubMed

Wan, Gui-Jun; Wang, Wen-Jing; Xu, Jing-Jing; Yang, Quan-Feng; Dai, Ming-Jiang; Zhang, Feng-Jiao; Sword, Gregory A; Pan, Wei-Dong; Chen, Fa-Jun

2015-01-01

Although there are considerable reports of magnetic field effects (MFE) on organisms, very little is known so far about the MFE-related signal transduction pathways. Here we establish a manipulative near-zero magnetic field (NZMF) to investigate the potential signal transduction pathways involved in MFE. We show that exposure of migratory white-backed planthopper, Sogatella furcifera, to the NZMF results in delayed egg and nymphal development, increased frequency of brachypterous females, and reduced longevity of macropterous female adults. To understand the changes in gene expression underlying these phenotypes, we examined the temporal patterns of gene expression of (i) CRY1 and CRY2 as putative magnetosensors, (ii) JHAMT, FAMeT and JHEH in the juvenile hormone pathway, (iii) CYP307A1 in the ecdysone pathway, and (iv) reproduction-related Vitellogenin (Vg). The significantly altered gene expression of CRY1 and CRY2 under the NZMF suggest their developmental stage-specific patterns and potential upstream location in magnetic response. Gene expression patterns of JHAMT, JHEH and CYP307A1 were consistent with the NZMF-triggered delay in nymphal development, higher proportion of brachypterous female adults, and the shortened longevity of macropterous female adults, which show feasible links between hormone signal transduction and phenotypic MFE. By conducting manipulative NZMF experiments, our study suggests an important role of the geomagnetic field (GMF) in modulating development and physiology of insects, provides new insights into the complexity of MFE-magnetosensitivity interactions, and represents an initial but crucial step forward in understanding the molecular basis of cryptochromes and hormone signal transduction involved in MFE.

The gram-negative sensing receptor PGRP-LC contributes to grooming induction in Drosophila

PubMed Central

Neyen, Claudine; Lemaitre, Bruno; Marion-Poll, Frédéric

2017-01-01

Behavioral resistance protects insects from microbial infection. However, signals inducing insect hygiene behavior are still relatively unexplored. Our previous study demonstrated that olfactory signals from microbes enhance insect hygiene behavior, and gustatory signals even induce the behavior. In this paper, we postulated a cross-talk between behavioral resistance and innate immunity. To examine this hypothesis, we employed a previously validated behavioral test to examine the function of taste signals in inducing a grooming reflex in decapitated flies. Microbes, which activate different pattern recognition systems upstream of immune pathways, were applied to see if there was any correlation between microbial perception and grooming reflex. To narrow down candidate elicitors, the grooming induction tests were conducted with highly purified bacterial components. Lastly, the role of DAP-type peptidoglycan in grooming induction was confirmed. Our results demonstrate that cleaning behavior can be triggered through recognition of DAP-type PGN by its receptor PGRP-LC. PMID:29121087

The BMP pathway acts to directly regulate Tbx20 in the developing heart

PubMed Central

Mandel, Elizabeth M.; Kaltenbrun, Erin; Callis, Thomas E.; Zeng, Xin-Xin I.; Marques, Sara R.; Yelon, Deborah; Wang, Da-Zhi; Conlon, Frank L.

2010-01-01

TBX20 has been shown to be essential for vertebrate heart development. Mutations within the TBX20 coding region are associated with human congenital heart disease, and the loss of Tbx20 in a wide variety of model systems leads to cardiac defects and eventually heart failure. Despite the crucial role of TBX20 in a range of cardiac cellular processes, the signal transduction pathways that act upstream of Tbx20 remain unknown. Here, we have identified and characterized a conserved 334 bp Tbx20 cardiac regulatory element that is directly activated by the BMP/SMAD1 signaling pathway. We demonstrate that this element is both necessary and sufficient to drive cardiac-specific expression of Tbx20 in Xenopus, and that blocking SMAD1 signaling in vivo specifically abolishes transcription of Tbx20, but not that of other cardiac factors, such as Tbx5 and MHC, in the developing heart. We further demonstrate that activation of Tbx20 by SMAD1 is mediated by a set of novel, non-canonical, high-affinity SMAD-binding sites located within this regulatory element and that phospho-SMAD1 directly binds a non-canonical SMAD1 site in vivo. Finally, we show that these non-canonical sites are necessary and sufficient for Tbx20 expression in Xenopus, and that reporter constructs containing these sites are expressed in a cardiac-specific manner in zebrafish and mouse. Collectively, our findings define Tbx20 as a direct transcriptional target of the BMP/SMAD1 signaling pathway during cardiac maturation. PMID:20460370

KIT Suppresses BRAFV600E-Mutant Melanoma by Attenuating Oncogenic RAS/MAPK Signaling.

PubMed

Neiswender, James V; Kortum, Robert L; Bourque, Caitlin; Kasheta, Melissa; Zon, Leonard I; Morrison, Deborah K; Ceol, Craig J

2017-11-01

The receptor tyrosine kinase KIT promotes survival and migration of melanocytes during development, and excessive KIT activity hyperactivates the RAS/MAPK pathway and can drive formation of melanomas, most notably of rare melanomas that occur on volar and mucosal surfaces of the skin. The much larger fraction of melanomas that occur on sun-exposed skin is driven primarily by BRAF- or NRAS-activating mutations, but these melanomas exhibit a surprising loss of KIT expression, which raises the question of whether loss of KIT in these tumors facilitates tumorigenesis. To address this question, we introduced a kit(lf) mutation into a strain of Tg(mitfa:BRAF V600E ); p53(lf) melanoma-prone zebrafish. Melanoma onset was accelerated in kit(lf); Tg(mitfa:BRAF V600E ); p53(lf) fish. Tumors from kit(lf) animals were more invasive and had higher RAS/MAPK pathway activation. KIT knockdown also increased RAS/MAPK pathway activation in a BRAF V600E -mutant human melanoma cell line. We found that pathway stimulation upstream of BRAF V600E could paradoxically reduce signaling downstream of BRAF V600E , and wild-type BRAF was necessary for this effect, suggesting that its activation can dampen oncogenic BRAF V600E signaling. In vivo , expression of wild-type BRAF delayed melanoma onset, but only in a kit -dependent manner. Together, these results suggest that KIT can activate signaling through wild-type RAF proteins, thus interfering with oncogenic BRAF V600E -driven melanoma formation. Cancer Res; 77(21); 5820-30. ©2017 AACR . ©2017 American Association for Cancer Research.

Auxin, the organizer of the hormonal/environmental signals for root hair growth

PubMed Central

Lee, Richard D.-W.; Cho, Hyung-Taeg

2013-01-01

The root hair development is controlled by diverse factors such as fate-determining developmental cues, auxin-related environmental factors, and hormones. In particular, the soil environmental factors are important as they maximize their absorption by modulating root hair development. These environmental factors affect the root hair developmental process by making use of diverse hormones. These hormonal factors interact with each other to modulate root hair development in which auxin appears to form the most intensive networks with the pathways from environmental factors and hormones. Moreover, auxin action for root hair development is genetically located immediately upstream of the root hair-morphogenetic genes. These observations suggest that auxin plays as an organizing node for environmental/hormonal pathways to modulate root hair growth. PMID:24273547

Regulation of Gap Junction Dynamics by UNC-44/ankyrin and UNC-33/CRMP through VAB-8 in C. elegans Neurons

PubMed Central

Yan, Dong

2016-01-01

Gap junctions are present in both vertebrates and invertebrates from nematodes to mammals. Although the importance of gap junctions has been documented in many biological processes, the molecular mechanisms underlying gap junction dynamics remain unclear. Here, using the C. elegans PLM neurons as a model, we show that UNC-44/ankyrin acts upstream of UNC-33/CRMP in regulation of a potential kinesin VAB-8 to control gap junction dynamics, and loss-of-function in the UNC-44/UNC-33/VAB-8 pathway suppresses the turnover of gap junction channels. Therefore, we first show a signal pathway including ankyrin, CRMP, and kinesin in regulating gap junctions. PMID:27015090

Increased Expression of Interleukin-6 Family Members and Receptors in Urinary Bladder with Cyclophosphamide-Induced Bladder Inflammation in Female Rats

PubMed Central

Girard, Beatrice M.; Cheppudira, Bopaiah P.; Malley, Susan E.; Schutz, Kristin C.; May, Victor; Vizzard, Margaret A.

2011-01-01

Recent studies suggest that janus-activated kinases–signal transducer and activator of transcription signaling pathways contribute to increased voiding frequency and referred pain of cyclophosphamide (CYP)-induced cystitis in rats. Potential upstream chemical mediator(s) that may be activated by CYP-induced cystitis to stimulate JAK/STAT signaling are not known in detail. In these studies, members of the interleukin (IL)-6 family of cytokines including, leukemia inhibitory factor (LIF), IL-6, and ciliary neurotrophic factor (CNTF) and associated receptors, IL-6 receptor (R) α, LIFR, and gp130 were examined in the urinary bladder in control and CYP-treated rats. Cytokine and receptor transcript and protein expression and distribution were determined in urinary bladder after CYP-induced cystitis using quantitative, real-time polymerase chain reaction (Q-PCR), western blotting, and immunohistochemistry. Acute (4 h; 150 mg/kg; i.p.), intermediate (48 h; 150 mg/kg; i.p.), or chronic (75 mg/kg; i.p., once every 3 days for 10 days) cystitis was induced in adult, female Wistar rats with CYP treatment. Q-PCR analyses revealed significant (p ≤ 0.01) CYP duration- and tissue- (e.g., urothelium, detrusor) dependent increases in LIF, IL-6, IL-6Rα, LIFR, and gp130 mRNA expression. Western blotting demonstrated significant (p ≤ 0.01) increases in IL-6, LIF, and gp130 protein expression in whole urinary bladder with CYP treatment. CYP-induced cystitis significantly (p ≤ 0.01) increased LIF-immunoreactivity (IR) in urothelium, detrusor, and suburothelial plexus whereas increased gp130-IR was only observed in urothelium and detrusor. These studies suggest that IL-6 and LIF may be potential upstream chemical mediators that activate JAK/STAT signaling in urinary bladder pathways. PMID:21373362

Proteome and phosphoproteome analysis of commensally induced dendritic cell maturation states.

PubMed

Korkmaz, Ali Giray; Popov, Todor; Peisl, Loulou; Codrea, Marius Cosmin; Nahnsen, Sven; Steimle, Alexander; Velic, Ana; Macek, Boris; von Bergen, Martin; Bernhardt, Joerg; Frick, Julia-Stefanie

2018-05-30

Dendritic cells (DCs) can shape the immune system towards an inflammatory or tolerant state depending on the bacterial antigens and the environment they encounter. In this study we provide a proteomic catalogue of differentially expressed proteins between distinct DC maturation states, brought about by bacteria that differ in their endotoxicity. To achieve this, we have performed proteomics and phosphoproteomics on murine DC cultures. Symbiont and pathobiont bacteria were used to direct dendritic cells into a semi-mature and fully-mature state, respectively. The comparison of semi-mature and fully-mature DCs revealed differential expression in 103 proteins and differential phosphorylation in 118 phosphosites, including major regulatory factors of central immune processes. Our analyses predict that these differences are mediated by upstream elements such as SOCS1, IRF3, ABCA1, TLR4, and PTGER4. Our analyses indicate that the symbiont bacterial strain affects DC proteome in a distinct way, by downregulating inflammatory proteins and activating anti-inflammatory upstream regulators. Biological significance In this study we have investigated the responses of immune cells to distinct bacterial stimuli. We have used the symbiont bacterial strain B. vulgatus and the pathobiont E. coli strain to stimulate cultured primary dendritic cells and performed a shotgun proteome analysis to investigate the protein expression and phosphorylation level differences on a genome level. We have observed expression and phosphorylation level differences in key immune regulators, transcription factors and signal transducers. Moreover, our subsequent bioinformatics analysis indicated regulation at several signaling pathways such as PPAR signaling, LXR/RXR activation and glucocorticoid signaling pathways, which are not studied in detail in an inflammation and DC maturation context. Our phosphoproteome analysis showed differential phosphorylation in 118 phosphosites including those belonging to epigenetic regulators, transcription factors and major cell cycle regulators. We anticipate that our study will facilitate further investigation of immune cell proteomes under different inflammatory and non-inflammatory conditions. Copyright © 2017. Published by Elsevier B.V.

P-glycoprotein (ABCB1) inhibited network of mitochondrion transport along microtubule and BMP signal-induced cell shape in chimpanzee left cerebrum by systems-theoretical analysis.

PubMed

Lin, Hong; Wang, Lin; Jiang, Minghu; Huang, Juxiang; Qi, Lianxiu

2012-10-01

We constructed the significant low-expression P-glycoprotein (ABCB1) inhibited transport and signal network in chimpanzee compared with high-expression (fold change ≥2) the human left cerebrum in GEO data set, by using integration of gene regulatory activated and inhibited network inference method with gene ontology (GO) analysis. Our result showed that ABCB1 transport and signal upstream network RAB2A inhibited ABCB1, and downstream ABCB1-inhibited SMAD1_2, NCK2, SLC25A46, GDF10, RASGRP1, EGFR, LRPPRC, RASSF2, RASA4, CA2, CBLB, UBR5, SLC25A16, ITGB3BP, DDIT4, PDPN, RAB2A in chimpanzee left cerebrum. We obtained that the different biological processes of ABCB1 inhibited transport and signal network repressed carbon dioxide transport, ER to Golgi vesicle-mediated transport, folic acid transport, mitochondrion transport along microtubule, water transport, BMP signaling pathway, Ras protein signal transduction, transforming growth factor beta receptor signaling pathway in chimpanzee compared with the inhibited network of the human left cerebrum, as a result of inducing inhibition of mitochondrion transport along microtubule and BMP signal-induced cell shape in chimpanzee left cerebrum. Our hypothesis was verified by the same and different biological processes of ABCB1 inhibited transport and signal network of chimpanzee compared with the corresponding activated network of chimpanzee and the human left cerebrum, respectively. Copyright © 2012 John Wiley & Sons, Ltd.

Toll-Like Receptor 4 Is an Essential Upstream Regulator of On-Time Parturition and Perinatal Viability in Mice.

PubMed

Wahid, Hanan H; Dorian, Camilla L; Chin, Peck Yin; Hutchinson, Mark R; Rice, Kenner C; Olson, David M; Moldenhauer, Lachlan M; Robertson, Sarah A

2015-10-01

An inflammatory response is instrumental in the physiological process of parturition but the upstream signals initiating inflammation are undefined. Because endogenous ligands for Toll-like receptor 4 (TLR4) are released in late gestation, we hypothesized that on-time labor requires TLR4 signaling, to trigger a cytokine and leukocyte response and accelerate the parturition cascade. In pregnant TLR4-deficient (Tlr4-/-) mice, average gestation length was extended by 13 hours and increased perinatal mortality was seen compared with wild-type controls. Quantification of cytokine and uterine activation gene expression showed that late gestation induction of Il1b, Il6, Il12b, and Tnf expression seen in control placenta and fetal membranes was disrupted in Tlr4-/- mice, and accompanied by a transient delay in expression of uterine activation genes, including prostaglandin F receptor, oxytocin receptor, and connexin-43. Leukocyte populations were altered before birth in TLR4-deficient females, with fewer neutrophils and macrophages in the placenta, and fewer dendritic cells and more regulatory T cells in the myometrium. Administration of TLR4 ligand lipopolysaccharide to pregnant wild-type mice induced cytokine expression and fetal loss, whereas Tlr4-/- pregnancies were protected. The small molecule TLR4 antagonist (+)-naloxone increased mean duration of gestation by 16 hours in wild-type mice. Collectively, these data demonstrate that TLR4 is a key upstream regulator of the inflammatory response acting to drive uterine activation and control the timing of labor. Because causal pathways for term and preterm labor converge with TLR4, interventions to manipulate TLR4 signaling may have therapeutic utility for women at risk of preterm labor, or in postterm pregnancy.

Toll-Like Receptor 4 Is an Essential Upstream Regulator of On-Time Parturition and Perinatal Viability in Mice

PubMed Central

Wahid, Hanan H.; Dorian, Camilla L.; Chin, Peck Yin; Hutchinson, Mark R.; Rice, Kenner C.; Olson, David M.; Moldenhauer, Lachlan M.

2015-01-01

An inflammatory response is instrumental in the physiological process of parturition but the upstream signals initiating inflammation are undefined. Because endogenous ligands for Toll-like receptor 4 (TLR4) are released in late gestation, we hypothesized that on-time labor requires TLR4 signaling, to trigger a cytokine and leukocyte response and accelerate the parturition cascade. In pregnant TLR4-deficient (Tlr4−/−) mice, average gestation length was extended by 13 hours and increased perinatal mortality was seen compared with wild-type controls. Quantification of cytokine and uterine activation gene expression showed that late gestation induction of Il1b, Il6, Il12b, and Tnf expression seen in control placenta and fetal membranes was disrupted in Tlr4−/− mice, and accompanied by a transient delay in expression of uterine activation genes, including prostaglandin F receptor, oxytocin receptor, and connexin-43. Leukocyte populations were altered before birth in TLR4-deficient females, with fewer neutrophils and macrophages in the placenta, and fewer dendritic cells and more regulatory T cells in the myometrium. Administration of TLR4 ligand lipopolysaccharide to pregnant wild-type mice induced cytokine expression and fetal loss, whereas Tlr4−/− pregnancies were protected. The small molecule TLR4 antagonist (+)-naloxone increased mean duration of gestation by 16 hours in wild-type mice. Collectively, these data demonstrate that TLR4 is a key upstream regulator of the inflammatory response acting to drive uterine activation and control the timing of labor. Because causal pathways for term and preterm labor converge with TLR4, interventions to manipulate TLR4 signaling may have therapeutic utility for women at risk of preterm labor, or in postterm pregnancy. PMID:26151355

Upstream regulators and downstream effectors of NF-κB in Alzheimer's disease.

PubMed

Shi, Zhe-Min; Han, Ya-Wei; Han, Xiao-Hui; Zhang, Kun; Chang, Ya-Nan; Hu, Zhi-Mei; Qi, Hai-Xia; Ting, Chen; Zhen, Zhang; Hong, Wei

2016-07-15

Since Alzheimer's disease (AD) is becoming the prevalent dementia in the whole world, more underlying mechanisms are emerging. Long time has the transcription factor NF-κB been identified to participate in AD pathogenesis, various studies have focused on the causes and effects of AD that are linked to NF-κB. In this review we discuss diverse environmental stimuli including oxidative stress, neuroinflammation and metabolism, involved signaling pathways such as PI3K/AKT, MAPK and AGE/RAGE/GSK-3 and newly found ncRNAs that mediate neuron toxicity or neuron protection through NF-κB activation and the following response associated with the same factors in AD. These may provide future orientation of investigation at transcription level and support efficient treatment to AD by a better understanding of the upstream regulators and downstream effectors of NF-κB. Copyright © 2016 Elsevier B.V. All rights reserved.

Alternative Mating Type Configurations (a/α versus a/a or α/α) of Candida albicans Result in Alternative Biofilms Regulated by Different Pathways

PubMed Central

Srikantha, Thyagarajan; Huang, Guanghua; Garnaas, Adam M.; Soll, David R.

2011-01-01

Similar multicellular structures can evolve within the same organism that may have different evolutionary histories, be controlled by different regulatory pathways, and play similar but nonidentical roles. In the human fungal pathogen Candida albicans, a quite extraordinary example of this has occurred. Depending upon the configuration of the mating type locus (a/α versus a/a or α/α), C. albicans forms alternative biofilms that appear similar morphologically, but exhibit dramatically different characteristics and are regulated by distinctly different signal transduction pathways. Biofilms formed by a/α cells are impermeable to molecules in the size range of 300 Da to 140 kDa, are poorly penetrated by human polymorphonuclear leukocytes (PMNs), and are resistant to antifungals. In contrast, a/a or α/α biofilms are permeable to molecules in this size range, are readily penetrated by PMNs, and are susceptible to antifungals. By mutational analyses, a/α biofilms are demonstrated to be regulated by the Ras1/cAMP pathway that includes Ras1→Cdc35→cAMP(Pde2—|)→Tpk2(Tpk1)→Efg1→Tec1→Bcr1, and a/a biofilms by the MAP kinase pathway that includes Mfα→Ste2→ (Ste4, Ste18, Cag1)→Ste11→Hst7→Cek2(Cek1)→Tec1. These observations suggest the hypothesis that while the upstream portion of the newly evolved pathway regulating a/a and α/α cell biofilms was derived intact from the upstream portion of the conserved pheromone-regulated pathway for mating, the downstream portion was derived through modification of the downstream portion of the conserved pathway for a/α biofilm formation. C. albicans therefore forms two alternative biofilms depending upon mating configuration. PMID:21829325

[Expression of angiopoietin-like proteins for animal breeding: a review].

PubMed

Fu, Weiwei; Ma, Yun; Chen, Ningbo; Li, He; Bai, Yueyu

2015-11-01

Angiopoietin-like proteins are a family of proteins that are closely related to lipid, glucose and energy metabolism, as well as angiogenesis. To date, eight Angptls have been discovered, namely Angptl1 to Angptl8 that play key roles in metabolic regulation and marker assisted selection. In this review, we summarized current progress on the structure, signaling pathways, upstream regulatory genes and metabolic network of Angptl1-8. Finally, in combination with our work, the status and problems of animal breeding as well as the future prospects for Angptls were discussed.

The emerging role of mTORC1 signaling in placental nutrient-sensing.

PubMed

Jansson, T; Aye, I L M H; Goberdhan, D C I

2012-11-01

Nutrient-sensing signaling pathways regulate cell metabolism and growth in response to altered nutrient levels and growth factor signaling. Because trophoblast cell metabolism and associated signaling influence fetal nutrient availability, trophoblast nutrient sensors may have a unique role in regulating fetal growth. We review data in support of a role for mammalian target of rapamycin complex 1 (mTORC1) in placental nutrient-sensing. Placental insulin/IGF-I signaling and fetal levels of oxygen, glucose and amino acids (AAs) are altered in pregnancy complications such as intrauterine growth restriction, and all these factors are well-established upstream regulators of mTORC1. Furthermore, mTORC1 is a positive regulator of placental AA transporters, suggesting that trophoblast mTORC1 modulates AA transfer across the placenta. In addition, placental mTORC1 signaling is also known to be modulated in pregnancy complications associated with altered fetal growth and in animal models in which maternal nutrient availability has been altered experimentally. Recently, significant progress has been made in identifying the molecular mechanisms by which mTORC1 senses AAs, a process requiring shuttling of mTOR to late endosomal and lysosomal compartments (LELs). We recently identified members of the proton-assisted amino acid transporter (PAT/SLC36) family as critical components of the AA-sensing system or 'nutrisome' that regulates mTORC1 on LEL membranes, placing AA transporters and their subcellular regulation both upstream and downstream of mTORC1-driven processes. We propose a model in which placental mTORC1 signaling constitutes a critical link between maternal nutrient availability and fetal growth, thereby influencing the long-term health of the fetus. Copyright © 2012 Elsevier Ltd. All rights reserved.

Estrogen modulates mesenchyme-epidermis interactions in the adult nipple

PubMed Central

Wu, Hsing-Jung; Oh, Ji Won; Spandau, Dan F.; Tholpady, Sunil; Diaz, Jesus; Schroeder, Laura J.; Offutt, Carlos D.; Glick, Adam B.; Plikus, Maksim V.; Koyama, Sachiko

2017-01-01

Maintenance of specialized epidermis requires signals from the underlying mesenchyme; however, the specific pathways involved remain to be identified. By recombining cells from the ventral skin of the K14-PTHrP transgenic mice [which overexpress parathyroid hormone-related protein (PTHrP) in their developing epidermis and mammary glands] with those from wild type, we show that transgenic stroma is sufficient to reprogram wild-type keratinocytes into nipple-like epidermis. To identify candidate nipple-specific signaling factors, we compared gene expression signatures of sorted Pdgfrα-positive ventral K14-PTHrP and wild-type fibroblasts, identifying differentially expressed transcripts that are involved in WNT, HGF, TGFβ, IGF, BMP, FGF and estrogen signaling. Considering that some of the growth factor pathways are targets for estrogen regulation, we examined the upstream role of this hormone in maintaining the nipple. Ablation of estrogen signaling through ovariectomy produced nipples with abnormally thin epidermis, and we identified TGFβ as a negatively regulated target of estrogen signaling. Estrogen treatment represses Tgfβ1 at the transcript and protein levels in K14-PTHrP fibroblasts in vitro, while ovariectomy increases Tgfb1 levels in K14-PTHrP ventral skin. Moreover, ectopic delivery of Tgfβ1 protein into nipple connective tissue reduced epidermal proliferation. Taken together, these results show that specialized nipple epidermis is maintained by estrogen-induced repression of TGFβ signaling in the local fibroblasts. PMID:28289136

Endogenous voltage gradients as mediators of cell-cell communication: strategies for investigating bioelectrical signals during pattern formation

PubMed Central

Adams, Dany S.; Levin, Michael

2013-01-01

Alongside the well-known chemical modes of cell-cell communication, we find an important and powerful system of bioelectrical signaling: changes in the resting voltage potential (Vmem) of the plasma membrane driven by ion channels, pumps and gap junctions. Slow Vmem changes in all cells serve as a highly conserved, information-bearing pathway that regulates cell proliferation, migration and differentiation. In embryonic and regenerative pattern formation and in the disorganization of neoplasia, bioelectrical cues serve as mediators of large-scale anatomical polarity, organ identity and positional information. Recent developments have resulted in tools that enable a high-resolution analysis of these biophysical signals and their linkage with upstream and downstream canonical genetic pathways. Here, we provide an overview for the study of bioelectric signaling, focusing on state-of-the-art approaches that use molecular physiology and developmental genetics to probe the roles of bioelectric events functionally. We highlight the logic, strategies and well-developed technologies that any group of researchers can employ to identify and dissect ionic signaling components in their own work and thus to help crack the bioelectric code. The dissection of bioelectric events as instructive signals enabling the orchestration of cell behaviors into large-scale coherent patterning programs will enrich on-going work in diverse areas of biology, as biophysical factors become incorporated into our systems-level understanding of cell interactions. PMID:22350846

Incoherent feedforward control governs adaptation of activated ras in a eukaryotic chemotaxis pathway.

PubMed

Takeda, Kosuke; Shao, Danying; Adler, Micha; Charest, Pascale G; Loomis, William F; Levine, Herbert; Groisman, Alex; Rappel, Wouter-Jan; Firtel, Richard A

2012-01-03

Adaptation in signaling systems, during which the output returns to a fixed baseline after a change in the input, often involves negative feedback loops and plays a crucial role in eukaryotic chemotaxis. We determined the dynamical response to a uniform change in chemoattractant concentration of a eukaryotic chemotaxis pathway immediately downstream from G protein-coupled receptors. The response of an activated Ras showed near-perfect adaptation, leading us to attempt to fit the results using mathematical models for the two possible simple network topologies that can provide perfect adaptation. Only the incoherent feedforward network accurately described the experimental results. This analysis revealed that adaptation in this Ras pathway is achieved through the proportional activation of upstream components and not through negative feedback loops. Furthermore, these results are consistent with a local excitation, global inhibition mechanism for gradient sensing, possibly with a Ras guanosine triphosphatase-activating protein acting as a global inhibitor.

Assessing co-regulation of directly linked genes in biological networks using microarray time series analysis.

PubMed

Del Sorbo, Maria Rosaria; Balzano, Walter; Donato, Michele; Draghici, Sorin

2013-11-01

Differential expression of genes detected with the analysis of high throughput genomic experiments is a commonly used intermediate step for the identification of signaling pathways involved in the response to different biological conditions. The impact analysis was the first approach for the analysis of signaling pathways involved in a certain biological process that was able to take into account not only the magnitude of the expression change of the genes but also the topology of signaling pathways including the type of each interactions between the genes. In the impact analysis, signaling pathways are represented as weighted directed graphs with genes as nodes and the interactions between genes as edges. Edges weights are represented by a β factor, the regulatory efficiency, which is assumed to be equal to 1 in inductive interactions between genes and equal to -1 in repressive interactions. This study presents a similarity analysis between gene expression time series aimed to find correspondences with the regulatory efficiency, i.e. the β factor as found in a widely used pathway database. Here, we focused on correlations among genes directly connected in signaling pathways, assuming that the expression variations of upstream genes impact immediately downstream genes in a short time interval and without significant influences by the interactions with other genes. Time series were processed using three different similarity metrics. The first metric is based on the bit string matching; the second one is a specific application of the Dynamic Time Warping to detect similarities even in presence of stretching and delays; the third one is a quantitative comparative analysis resulting by an evaluation of frequency domain representation of time series: the similarity metric is the correlation between dominant spectral components. These three approaches are tested on real data and pathways, and a comparison is performed using Information Retrieval benchmark tools, indicating the frequency approach as the best similarity metric among the three, for its ability to detect the correlation based on the correspondence of the most significant frequency components. Copyright © 2013. Published by Elsevier Ireland Ltd.

Genome Analysis Reveals Interplay between 5′UTR Introns and Nuclear mRNA Export for Secretory and Mitochondrial Genes

PubMed Central

Cenik, Can; Chua, Hon Nian; Zhang, Hui; Tarnawsky, Stefan P.; Akef, Abdalla; Derti, Adnan; Tasan, Murat; Moore, Melissa J.; Palazzo, Alexander F.; Roth, Frederick P.

2011-01-01

In higher eukaryotes, messenger RNAs (mRNAs) are exported from the nucleus to the cytoplasm via factors deposited near the 5′ end of the transcript during splicing. The signal sequence coding region (SSCR) can support an alternative mRNA export (ALREX) pathway that does not require splicing. However, most SSCR–containing genes also have introns, so the interplay between these export mechanisms remains unclear. Here we support a model in which the furthest upstream element in a given transcript, be it an intron or an ALREX–promoting SSCR, dictates the mRNA export pathway used. We also experimentally demonstrate that nuclear-encoded mitochondrial genes can use the ALREX pathway. Thus, ALREX can also be supported by nucleotide signals within mitochondrial-targeting sequence coding regions (MSCRs). Finally, we identified and experimentally verified novel motifs associated with the ALREX pathway that are shared by both SSCRs and MSCRs. Our results show strong correlation between 5′ untranslated region (5′UTR) intron presence/absence and sequence features at the beginning of the coding region. They also suggest that genes encoding secretory and mitochondrial proteins share a common regulatory mechanism at the level of mRNA export. PMID:21533221

Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2.

PubMed

Konopatskaya, Olga; Matthews, Sharon A; Harper, Matthew T; Gilio, Karen; Cosemans, Judith M E M; Williams, Christopher M; Navarro, Maria N; Carter, Deborah A; Heemskerk, Johan W M; Leitges, Michael; Cantrell, Doreen; Poole, Alastair W

2011-07-14

Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.

Rab proteins: The key regulators of intracellular vesicle transport

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhuin, Tanmay; Roy, Jagat Kumar, E-mail: jkroy@bhu.ac.in

2014-10-15

Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied bymore » cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future.« less

Effects of Germline Mutations in the Ras/MAPK Signaling Pathway on Adaptive Behavior: Cardiofaciocutaneous Syndrome and Noonan Syndrome

PubMed Central

Pierpont, Elizabeth I.; Pierpont, Mary Ella; Mendelsohn, Nancy J.; Roberts, Amy E.; Tworog-Dube, Erica; Rauen, Katherine A.; Seidenberg, Mark S.

2011-01-01

Cardiofaciocutaneous syndrome (CFC) and Noonan syndrome (NS) are two phenotypically overlapping genetic disorders whose underlying molecular etiologies affect a common signaling pathway. Mutations in the BRAF, MEK1 and MEK2 genes cause most cases of CFC and mutations in PTPN11, SOS1, KRAS and RAF1 typically cause NS. Although both syndromes are associated with developmental delays of varying severity, the extent to which the behavioral profiles differ may shed light on the different roles these respective genes play in development of skills necessary for everyday functioning. In this study, profiles of adaptive behavior of individuals with CFC and NS who had confirmed pathogenic mutations in Ras/MAPK pathway genes were investigated. Patterns of strengths and weaknesses, age-related differences, and risk factors for difficulties in adaptive skills were assessed. Although genes acting more downstream in the Ras/MAPK pathway were associated with more difficulties in adaptive functioning than genes more upstream in the pathway, several inconsistencies highlight the wide spectrum of possible developmental courses in CFC and NS. Along with clinical and genetic factors, variables such as chronological age, gestational age at birth and parental education levels accounted for significant variance in adaptive skills. Results indicate that there is wide heterogeneity in adaptive ability in CFC and NS, but that these abilities are correlated to some extent with the specific disease-causing genes. PMID:20186801

Ethylene Responses in Rice Roots and Coleoptiles Are Differentially Regulated by a Carotenoid Isomerase-Mediated Abscisic Acid Pathway[OPEN

PubMed Central

Yin, Cui-Cui; Ma, Biao; Collinge, Derek Phillip; Pogson, Barry James; He, Si-Jie; Xiong, Qing; Duan, Kai-Xuan; Chen, Hui; Yang, Chao; Lu, Xiang; Wang, Yi-Qin; Zhang, Wan-Ke; Chu, Cheng-Cai; Sun, Xiao-Hong; Fang, Shuang; Chu, Jin-Fang; Lu, Tie-Gang; Chen, Shou-Yi; Zhang, Jin-Song

2015-01-01

Ethylene and abscisic acid (ABA) act synergistically or antagonistically to regulate plant growth and development. ABA is derived from the carotenoid biosynthesis pathway. Here, we analyzed the interplay among ethylene, carotenoid biogenesis, and ABA in rice (Oryza sativa) using the rice ethylene response mutant mhz5, which displays a reduced ethylene response in roots but an enhanced ethylene response in coleoptiles. We found that MHZ5 encodes a carotenoid isomerase and that the mutation in mhz5 blocks carotenoid biosynthesis, reduces ABA accumulation, and promotes ethylene production in etiolated seedlings. ABA can largely rescue the ethylene response of the mhz5 mutant. Ethylene induces MHZ5 expression, the production of neoxanthin, an ABA biosynthesis precursor, and ABA accumulation in roots. MHZ5 overexpression results in enhanced ethylene sensitivity in roots and reduced ethylene sensitivity in coleoptiles. Mutation or overexpression of MHZ5 also alters the expression of ethylene-responsive genes. Genetic studies revealed that the MHZ5-mediated ABA pathway acts downstream of ethylene signaling to inhibit root growth. The MHZ5-mediated ABA pathway likely acts upstream but negatively regulates ethylene signaling to control coleoptile growth. Our study reveals novel interactions among ethylene, carotenogenesis, and ABA and provides insight into improvements in agronomic traits and adaptive growth through the manipulation of these pathways in rice. PMID:25841037

Multidimensional Single-Cell Analysis of BCR Signaling Reveals Proximal Activation Defect As a Hallmark of Chronic Lymphocytic Leukemia B Cells

PubMed Central

Palomba, M. Lia; Piersanti, Kelly; Ziegler, Carly G. K.; Decker, Hugo; Cotari, Jesse W.; Bantilan, Kurt; Rijo, Ivelise; Gardner, Jeff R.; Heaney, Mark; Bemis, Debra; Balderas, Robert; Malek, Sami N.; Seymour, Erlene; Zelenetz, Andrew D.

2014-01-01

Purpose Chronic Lymphocytic Leukemia (CLL) is defined by a perturbed B-cell receptor-mediated signaling machinery. We aimed to model differential signaling behavior between B cells from CLL and healthy individuals to pinpoint modes of dysregulation. Experimental Design We developed an experimental methodology combining immunophenotyping, multiplexed phosphospecific flow cytometry, and multifactorial statistical modeling. Utilizing patterns of signaling network covariance, we modeled BCR signaling in 67 CLL patients using Partial Least Squares Regression (PLSR). Results from multidimensional modeling were validated using an independent test cohort of 38 patients. Results We identified a dynamic and variable imbalance between proximal (pSYK, pBTK) and distal (pPLCγ2, pBLNK, ppERK) phosphoresponses. PLSR identified the relationship between upstream tyrosine kinase SYK and its target, PLCγ2, as maximally predictive and sufficient to distinguish CLL from healthy samples, pointing to this juncture in the signaling pathway as a hallmark of CLL B cells. Specific BCR pathway signaling signatures that correlate with the disease and its degree of aggressiveness were identified. Heterogeneity in the PLSR response variable within the B cell population is both a characteristic mark of healthy samples and predictive of disease aggressiveness. Conclusion Single-cell multidimensional analysis of BCR signaling permitted focused analysis of the variability and heterogeneity of signaling behavior from patient-to-patient, and from cell-to-cell. Disruption of the pSYK/pPLCγ2 relationship is uncovered as a robust hallmark of CLL B cell signaling behavior. Together, these observations implicate novel elements of the BCR signal transduction as potential therapeutic targets. PMID:24489640

Silibinin inhibits translation initiation: implications for anticancer therapy.

PubMed

Lin, Chen-Ju; Sukarieh, Rami; Pelletier, Jerry

2009-06-01

Silibinin is a nontoxic flavonoid reported to have anticancer properties. In this study, we show that silibinin exhibits antiproliferative activity on MCF-7 breast cancer cells. Exposure to silibinin leads to a concentration-dependent decrease in global protein synthesis associated with reduced levels of eukaryotic initiation factor 4F complex. Moreover, polysome profile analysis of silibinin-treated cells shows a decrease in polysome content and translation of cyclin D1 mRNA. Silibinin exerts its effects on translation initiation by inhibiting the mammalian target of rapamycin signaling pathway by acting upstream of TSC2. Our results show that silibinin blocks mammalian target of rapamycin signaling with a concomitant reduction in translation initiation, thus providing a possible molecular mechanism of how silibinin can inhibit growth of transformed cells.

Hepatitis B virus X protein suppresses virus-triggered IRF3 activation and IFN-beta induction by disrupting the VISA-associated complex.

PubMed

Wang, Xianmiao; Li, Ying; Mao, Aiping; Li, Chao; Li, Yongkui; Tien, Po

2010-09-01

Viral RNAs produced during viral infection are recognized by the cytoplasmic RNA helicases retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). A central adapter protein downstream of RIG-I and MDA5 is the mitochondrial membrane protein virus-induced signaling adaptor (VISA), which mediates the induction of type I interferons (IFNs) through the activation of transcription factors such as nuclear factor-kappaB (NF-kappaB) and IFN-regulatory factor-3 (IRF3). Here we found that hepatitis B virus (HBV)-encoded X protein (HBx) acts as an inhibitor of virus-triggered IRF3 activation and IFN-beta induction. Reporter and plaque assays indicate that HBx inhibits signaling by components upstream but not downstream of VISA. Immunoprecipitation experiments indicate that HBx interacts with VISA and disrupts the association of VISA with its upstream and downstream components. These findings suggest that HBx acts as a suppressor of virus-triggered induction of type I IFNs, which explains the observation that HBV causes transient and chronic infection in hepatocytes but fails to activate the pattern recognition receptor-mediated IFN induction pathways.

Regulatory Mechanisms of Fear Extinction and Depression-Like Behavior

PubMed Central

Tronson, Natalie C; Schrick, Christina; Fischer, Andre; Sananbenesi, Farahnaz; Pagès, Gilles; Pouysségur, Jacques; Radulovic, Jelena

2008-01-01

Human anxiety is frequently accompanied by depression, and when they co-occur both conditions exhibit greater severity and resistance to treatment. Little is known, however, about the molecular processes linking these emotional and mood disorders. Based on previously reported phosphorylation patterns of extracellular signal-regulated kinase (ERK) in the brain, we hypothesized that ERK’s upstream activators intertwine fear and mood regulation through their hippocampal actions. We tested this hypothesis by studying the upstream regulation of ERK signaling in behavioral models of fear and depression. Wild-type and ERK1-deficient mice were used to study the dorsohippocampal actions of the putative ERK activators: mitogen-activated and extracellular signal-regulated kinase (MEK), protein kinase C (PKC), and cAMP-dependent protein kinase (PKA). Mice lacking ERK1 exhibited enhanced fear extinction and reduced depression caused by overactivation of ERK2. Both behaviors were reversed by inhibition of MEK, however the extinction phenotype depended on hippocampal, whereas the depression phenotype predominantly involved extrahippocampal MEK. Unexpectedly, inhibition of PKC accelerated extinction and decreased depression by ERK-independent mechanisms, whereas inhibition of PKA did not produce detectable molecular or behavioral effects in the employed paradigm. These results indicate that, contrary to fear conditioning but similar to mood stabilization, extinction of fear required upregulation of MEK/ERK and downregulation of ERK-independent PKC signaling. The dissociation of these pathways may thus represent a common mechanism for fear and mood regulation, and a potential therapeutic option for comorbid anxiety and depression. PMID:17712345

HCV upregulates Bim through the ROS/JNK signalling pathway, leading to Bax-mediated apoptosis.

PubMed

Deng, Lin; Chen, Ming; Tanaka, Motofumi; Ku, Yonson; Itoh, Tomoo; Shoji, Ikuo; Hotta, Hak

2015-09-01

We previously reported that hepatitis C virus (HCV) infection induces Bax-triggered, mitochondrion-mediated apoptosis by using the HCV J6/JFH1 strain and Huh-7.5 cells. However, it was still unclear how HCV-induced Bax activation. In this study, we showed that the HCV-induced activation and mitochondrial accumulation of Bax were significantly attenuated by treatment with a general antioxidant, N-acetyl cysteine (NAC), or a specific c-Jun N-terminal kinase (JNK) inhibitor, SP600125, with the result suggesting that the reactive oxygen species (ROS)/JNK signalling pathway is upstream of Bax activation in HCV-induced apoptosis. We also demonstrated that HCV infection transcriptionally activated the gene for the pro-apoptotic protein Bim and the protein expression of three major splice variants of Bim (BimEL, BimL and BimS). The HCV-induced increase in the Bim mRNA and protein levels was significantly counteracted by treatment with NAC or SP600125, suggesting that the ROS/JNK signalling pathway is involved in Bim upregulation. Moreover, HCV infection led to a marked accumulation of Bim on the mitochondria to facilitate its interaction with Bax. On the other hand, downregulation of Bim by siRNA (small interfering RNA) significantly prevented HCV-mediated activation of Bax and caspase 3. Taken together, these observations suggest that HCV-induced ROS/JNK signalling transcriptionally activates Bim expression, which leads to Bax activation and apoptosis induction.

IR/IGF1R signaling as potential target for treatment of high-grade osteosarcoma

PubMed Central

2013-01-01

Background High-grade osteosarcoma is an aggressive tumor most often developing in the long bones of adolescents, with a second peak in the 5th decade of life. Better knowledge on cellular signaling in this tumor may identify new possibilities for targeted treatment. Methods We performed gene set analysis on previously published genome-wide gene expression data of osteosarcoma cell lines (n=19) and pretreatment biopsies (n=84). We characterized overexpression of the insulin-like growth factor receptor (IGF1R) signaling pathways in human osteosarcoma as compared with osteoblasts and with the hypothesized progenitor cells of osteosarcoma – mesenchymal stem cells. This pathway plays a key role in the growth and development of bone. Since most profound differences in mRNA expression were found at and upstream of the receptor of this pathway, we set out to inhibit IR/IGF1R using OSI-906, a dual inhibitor for IR/IGF1R, on four osteosarcoma cell lines. Inhibitory effects of this drug were measured by Western blotting and cell proliferation assays. Results OSI-906 had a strong inhibitory effect on proliferation of 3 of 4 osteosarcoma cell lines, with IC50s below 100 nM at 72 hrs of treatment. Phosphorylation of IRS-1, a direct downstream target of IGF1R signaling, was inhibited in the responsive osteosarcoma cell lines. Conclusions This study provides an in vitro rationale for using IR/IGF1R inhibitors in preclinical studies of osteosarcoma. PMID:23688189

Time course of gene expression during mouse skeletal muscle hypertrophy

PubMed Central

Lee, Jonah D.; England, Jonathan H.; Esser, Karyn A.; McCarthy, John J.

2013-01-01

The purpose of this study was to perform a comprehensive transcriptome analysis during skeletal muscle hypertrophy to identify signaling pathways that are operative throughout the hypertrophic response. Global gene expression patterns were determined from microarray results on days 1, 3, 5, 7, 10, and 14 during plantaris muscle hypertrophy induced by synergist ablation in adult mice. Principal component analysis and the number of differentially expressed genes (cutoffs ≥2-fold increase or ≥50% decrease compared with control muscle) revealed three gene expression patterns during overload-induced hypertrophy: early (1 day), intermediate (3, 5, and 7 days), and late (10 and 14 days) patterns. Based on the robust changes in total RNA content and in the number of differentially expressed genes, we focused our attention on the intermediate gene expression pattern. Ingenuity Pathway Analysis revealed a downregulation of genes encoding components of the branched-chain amino acid degradation pathway during hypertrophy. Among these genes, five were predicted by Ingenuity Pathway Analysis or previously shown to be regulated by the transcription factor Kruppel-like factor-15, which was also downregulated during hypertrophy. Moreover, the integrin-linked kinase signaling pathway was activated during hypertrophy, and the downregulation of muscle-specific micro-RNA-1 correlated with the upregulation of five predicted targets associated with the integrin-linked kinase pathway. In conclusion, we identified two novel pathways that may be involved in muscle hypertrophy, as well as two upstream regulators (Kruppel-like factor-15 and micro-RNA-1) that provide targets for future studies investigating the importance of these pathways in muscle hypertrophy. PMID:23869057

Time course of gene expression during mouse skeletal muscle hypertrophy.

PubMed

Chaillou, Thomas; Lee, Jonah D; England, Jonathan H; Esser, Karyn A; McCarthy, John J

2013-10-01

The purpose of this study was to perform a comprehensive transcriptome analysis during skeletal muscle hypertrophy to identify signaling pathways that are operative throughout the hypertrophic response. Global gene expression patterns were determined from microarray results on days 1, 3, 5, 7, 10, and 14 during plantaris muscle hypertrophy induced by synergist ablation in adult mice. Principal component analysis and the number of differentially expressed genes (cutoffs ≥2-fold increase or ≥50% decrease compared with control muscle) revealed three gene expression patterns during overload-induced hypertrophy: early (1 day), intermediate (3, 5, and 7 days), and late (10 and 14 days) patterns. Based on the robust changes in total RNA content and in the number of differentially expressed genes, we focused our attention on the intermediate gene expression pattern. Ingenuity Pathway Analysis revealed a downregulation of genes encoding components of the branched-chain amino acid degradation pathway during hypertrophy. Among these genes, five were predicted by Ingenuity Pathway Analysis or previously shown to be regulated by the transcription factor Kruppel-like factor-15, which was also downregulated during hypertrophy. Moreover, the integrin-linked kinase signaling pathway was activated during hypertrophy, and the downregulation of muscle-specific micro-RNA-1 correlated with the upregulation of five predicted targets associated with the integrin-linked kinase pathway. In conclusion, we identified two novel pathways that may be involved in muscle hypertrophy, as well as two upstream regulators (Kruppel-like factor-15 and micro-RNA-1) that provide targets for future studies investigating the importance of these pathways in muscle hypertrophy.

Effect Of Simulated Microgravity On Activated T Cell Gene Transcription

NASA Technical Reports Server (NTRS)

Morrow, Maureen A.

2003-01-01

Studies of T lymphocytes under the shear stress environment of clinorotation have demonstrated an inhibition of activation in response to TCR mediated signaling. These results mimic those observed during space flight. This work investigates the molecular signaling events of T lymphocyte activation with clinorotation. Purified human T lymphocytes and the T cell clone Jurkat exhibit an uncoupling of signaling as mediated through the TCR. Activation of the transcription factor AP-1 is inhibited while activation of NFAT occurs. NFAT dephosphorylation and activation is dependent on sustained Ca(++) influx. Alternatively, AP-1, which consists of two transcription factors, jun and fos, is activated by PKC and Ras mediated pathways. TCR signaling is known to be dependent on cytoskeletal rearrangements, in particular, raft aggregation is critical. Raft aggregation, as mediated through GM, crosslinking, overcomes the inhibition of T lymphocyte activation with clinorotation, indicating that the block is occurring upstream of raft aggregation. Clinorotation is shown to have an effect similar to a weak TCR signal.

JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships

PubMed Central

Zeke, András; Misheva, Mariya

2016-01-01

SUMMARY The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. PMID:27466283

Bruton’s Tyrosine Kinase, a Component of B Cell Signaling Pathways, Has Multiple Roles in the Pathogenesis of Lupus

PubMed Central

Satterthwaite, Anne B.

2018-01-01

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the loss of adaptive immune tolerance to nucleic acid-containing antigens. The resulting autoantibodies form immune complexes that promote inflammation and tissue damage. Defining the signals that drive pathogenic autoantibody production is an important step in the development of more targeted therapeutic approaches for lupus, which is currently treated primarily with non-specific immunosuppression. Here, we review the contribution of Bruton’s tyrosine kinase (Btk), a component of B and myeloid cell signaling pathways, to disease in murine lupus models. Both gain- and loss-of-function genetic studies have revealed that Btk plays multiple roles in the production of autoantibodies. These include promoting the activation, plasma cell differentiation, and class switching of autoreactive B cells. Small molecule inhibitors of Btk are effective at reducing autoantibody levels, B cell activation, and kidney damage in several lupus models. These studies suggest that Btk may promote end-organ damage both by facilitating the production of autoantibodies and by mediating the inflammatory response of myeloid cells to these immune complexes. While Btk has not been associated with SLE in GWAS studies, SLE B cells display signaling defects in components both upstream and downstream of Btk consistent with enhanced activation of Btk signaling pathways. Taken together, these observations indicate that limiting Btk activity is critical for maintaining B cell tolerance and preventing the development of autoimmune disease. Btk inhibitors, generally well-tolerated and approved to treat B cell malignancy, may thus be a useful therapeutic approach for SLE. PMID:29403475

Lipoxygenase-mediated pro-radical effect of melatonin via stimulation of arachidonic acid metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Radogna, F.; Sestili, P.; Martinelli, C.

We have shown that melatonin immediately and transiently stimulates intracellular free radical production on a set of leukocytes, possibly as a consequence of calmodulin binding. We show here that melatonin-induced ROS are produced by lipoxygenase (LOX), since they are prevented by a set of LOX inhibitors, and are accompanied by increase of the 5-LOX product 5-HETE. LOX activation is accompanied by strong liberation of AA; inhibition of Ca{sup 2+}-independent, but not Ca{sup 2+}-dependent, phospholipase A2 (PLA2), prevents both melatonin-induced arachidonic acid and ROS production, whereas LOX inhibition only prevents ROS, indicating that PLA2 is upstream with respect to LOX, asmore » occurs in many signaling pathways. Chlorpromazine, an inhibitor of melatonin-calmodulin interaction, inhibits both ROS and arachidonic acid production, thus possibly placing calmodulin at the origin of a melatonin-induced pro-radical pathway. Interestingly, it is known that Ca{sup 2+}-independent PLA2 binds to calmodulin: our results are compatible with PLA2 being liberated by melatonin from a steady-state calmodulin sequestration, thus initiating an arachidonate signal transduction. These results delineate a novel molecular pathway through which melatonin may participate to the inflammatory response.« less

Endogenous RNAi Pathways Are Required in Neurons for Dauer Formation in Caenorhabditis elegans.

PubMed

Bharadwaj, Pallavi S; Hall, Sarah E

2017-04-01

Animals can adapt to unfavorable environments through changes in physiology or behavior. In the nematode, Caenorhabditis elegans , environmental conditions perceived early in development determine whether the animal enters either the reproductive cycle, or enters into an alternative diapause stage named dauer. Here, we show that endogenous RNAi pathways play a role in dauer formation in crowding (high pheromone), starvation, and high temperature conditions. Disruption of the Mutator proteins or the nuclear Argonaute CSR-1 result in differential dauer-deficient phenotypes that are dependent upon the experienced environmental stress. We provide evidence that the RNAi pathways function in chemosensory neurons for dauer formation, upstream of the TGF-β and insulin signaling pathways. In addition, we show that Mutator MUT-16 expression in a subset of individual pheromone-sensing neurons is sufficient for dauer formation in high pheromone conditions, but not in starvation or high temperature conditions. Furthermore, we also show that MUT-16 and CSR-1 are required for expression of a subset of G proteins with functions in the detection of pheromone components. Together, our data suggest a model where Mutator -amplified siRNAs that associate with the CSR-1 pathway promote expression of genes required for the detection and signaling of environmental conditions to regulate development and behavior in C. elegans This study highlights a mechanism whereby RNAi pathways mediate the link between environmental stress and adaptive phenotypic plasticity in animals. Copyright © 2017 by the Genetics Society of America.

Lipoxin A4 suppresses osteoclastogenesis in RAW264.7 cells and prevents ovariectomy-induced bone loss

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Changyu; Guan, Hanfeng; Cai, Cong

Lipoxin A4 (LXA4; 5S, 6R, 15Strihydroxy- 7,9,13-trans-11-eicosatetraenoic acid) is a metabolic product of arachidonic acid under the action of lipoxidase. This lipid molecule plays important roles in several biological functions, especially inflammatory processes. In vivo, LXA4 regulates the inflammatory response through several signaling pathways. Its mechanism suggests that it might have an effect on osteoclastogenesis and bone loss. Using both in vitro and in vivo studies, it was here observed that LXA4 could significantly inhibit the formation and function of osteoclasts and these effects could be blocked by Boc-2, the specific inhibitor of FPR2/ALX (the receptor of LXA4). Meanwhile, LXA4more » reduce the amount of ovariectomy-induced bone loss. These protective effects was found to be associated with inhibition of nuclear factor-κB (NF-κB), activator protein-1 (AP-1), PI3K-AKT, and p-38, ERK, and JNK in MAPKs. The expression of the receptor activator of the NF-κB ligand RANKL:osteoprotegerin ratio and serum levels of TNF-α, IL-1β, and IL-6 were decreased by LXA4. Moreover, LXA4 prevented the production of reactive oxygen species (ROS), the expression of osteoclast-specific genes, including tartrate-resistant acid phosphatase (TRAP), cathepsin K (CK), matrix metalloproteinase (MMP)-9, RANK, and osteoclastic related transcription factors of c-Fos, NFATc1 could also be significantly inhibited by LXA4 in a dose-dependent manner. Studies have demonstrated that LXA4 can inhibit the formation and function of osteoclasts through modulation of several pathways both upstream and downstream of RANKL signaling and FPR2/ALX was involved in the procedures. This shows that LXA4 may be used as a new strategy for the treatment of osteoclast-related diseases. - Highlights: • Lipoxin A4 can significantly inhibit the formation and function of osteoclasts. • Several pathways both upstream and downstream of RANKL signaling can be inhibit by Lipoxin A4. • Lipoxin A4 can alleviate ovariectomy-induced bone loss effectively.« less

Suppression of transforming growth factor-beta-induced apoptosis through a phosphatidylinositol 3-kinase/Akt-dependent pathway.

PubMed

Chen, R H; Su, Y H; Chuang, R L; Chang, T Y

1998-10-15

Insulin and insulin receptor substrate 1 (IRS-1) are capable of protecting liver cells from apoptosis induced by transforming growth factor-beta1 (TGF-beta). The Ras/mitogen-activated protein kinase (MAP kinase) and the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathways are both activated upon insulin stimulation and can protect against apoptosis under certain circumstances. We investigated which of these pathways is responsible for the protective effect of insulin on TGF-beta-induced apoptosis. An activated Ras, although elicited a strong mitogenic effect, could not protect Hep3B cells from TGF-beta-induced apoptosis. Furthermore, PD98059, a selective inhibitor of MEK, did not suppress the antiapoptotic effect of insulin. In contrast, the PI 3-kinase inhibitor, LY294002, efficiently blocked the effect of insulin. Protection against TGF-beta-induced apoptosis conferred by PI 3-kinase was further verified by stable transfection of an activated PI 3-kinase. Downstream targets of PI 3-kinase involved in this protection was further investigated. An activated Akt mimicked the antiapoptotic effect of insulin, whereas a dominant-negative Akt inhibited such effect. However, rapamycin, the p70S6 kinase inhibitor, had no effect on the protectivity of insulin against TGF-beta-induced apoptosis, suggesting that the antiapoptotic target of PI 3-kinase/Akt pathway is independent or lies upstream of the p70S6 kinase. The mechanism by which PI 3-kinase/Akt pathway interferes with the apoptotic signaling of TGF-beta was explored. Activation of PI 3-kinase did not lead to a suppression of Smad hetero-oligomerization or nuclear translocation but blocked TGF-beta-induced caspase-3-like activity. In summary, the PI 3-kinase/Akt pathway, but not the Ras/MAP kinase pathway, protects against TGF-beta-induced apoptosis by inhibiting a step downstream of Smad but upstream of caspase-3.

Competence in Streptococcus pneumoniae is regulated by the rate of ribosomal decoding errors.

PubMed

Stevens, Kathleen E; Chang, Diana; Zwack, Erin E; Sebert, Michael E

2011-01-01

Competence for genetic transformation in Streptococcus pneumoniae develops in response to accumulation of a secreted peptide pheromone and was one of the initial examples of bacterial quorum sensing. Activation of this signaling system induces not only expression of the proteins required for transformation but also the production of cellular chaperones and proteases. We have shown here that activity of this pathway is sensitively responsive to changes in the accuracy of protein synthesis that are triggered by either mutations in ribosomal proteins or exposure to antibiotics. Increasing the error rate during ribosomal decoding promoted competence, while reducing the error rate below the baseline level repressed the development of both spontaneous and antibiotic-induced competence. This pattern of regulation was promoted by the bacterial HtrA serine protease. Analysis of strains with the htrA (S234A) catalytic site mutation showed that the proteolytic activity of HtrA selectively repressed competence when translational fidelity was high but not when accuracy was low. These findings redefine the pneumococcal competence pathway as a response to errors during protein synthesis. This response has the capacity to address the immediate challenge of misfolded proteins through production of chaperones and proteases and may also be able to address, through genetic exchange, upstream coding errors that cause intrinsic protein folding defects. The competence pathway may thereby represent a strategy for dealing with lesions that impair proper protein coding and for maintaining the coding integrity of the genome. The signaling pathway that governs competence in the human respiratory tract pathogen Streptococcus pneumoniae regulates both genetic transformation and the production of cellular chaperones and proteases. The current study shows that this pathway is sensitively controlled in response to changes in the accuracy of protein synthesis. Increasing the error rate during ribosomal decoding induced competence, while decreasing the error rate repressed competence. This pattern of regulation was promoted by the HtrA protease, which selectively repressed competence when translational fidelity was high but not when accuracy was low. Our findings demonstrate that this organism is able to monitor the accuracy of information used for protein biosynthesis and suggest that errors trigger a response addressing both the immediate challenge of misfolded proteins and, through genetic exchange, upstream coding errors that may underlie protein folding defects. This pathway may represent an evolutionary strategy for maintaining the coding integrity of the genome.

Yes-associated protein (YAP) in pancreatic cancer: at the epicenter of a targetable signaling network associated with patient survival.

PubMed

Rozengurt, Enrique; Sinnett-Smith, James; Eibl, Guido

2018-01-01

Pancreatic ductal adenocarcinoma (PDAC) is generally a fatal disease with no efficacious treatment modalities. Elucidation of signaling mechanisms that will lead to the identification of novel targets for therapy and chemoprevention is urgently needed. Here, we review the role of Yes-associated protein (YAP) and WW-domain-containing Transcriptional co-Activator with a PDZ-binding motif (TAZ) in the development of PDAC. These oncogenic proteins are at the center of a signaling network that involves multiple upstream signals and downstream YAP-regulated genes. We also discuss the clinical significance of the YAP signaling network in PDAC using a recently published interactive open-access database (www.proteinatlas.org/pathology) that allows genome-wide exploration of the impact of individual proteins on survival outcomes. Multiple YAP/TEAD-regulated genes, including AJUBA , ANLN , AREG , ARHGAP29 , AURKA , BUB1 , CCND1 , CDK6, CXCL5 , EDN2 , DKK1 , FOSL1,FOXM1 , HBEGF , IGFBP2 , JAG1 , NOTCH2 , RHAMM , RRM2 , SERP1 , and ZWILCH , are associated with unfavorable survival of PDAC patients. Similarly, components of AP-1 that synergize with YAP ( FOSL1 ), growth factors (TGFα, EPEG, and HBEGF), a specific integrin ( ITGA2 ), heptahelical receptors ( P2Y 2 R , GPR87 ) and an inhibitor of the Hippo pathway ( MUC1 ), all of which stimulate YAP activity, are associated with unfavorable survival of PDAC patients. By contrast, YAP inhibitory pathways (STRAD/LKB-1/AMPK, PKA/LATS, and TSC/mTORC1) indicate a favorable prognosis. These associations emphasize that the YAP signaling network correlates with poor survival of pancreatic cancer patients. We conclude that the YAP pathway is a major determinant of clinical aggressiveness in PDAC patients and a target for therapeutic and preventive strategies in this disease.

Hedgehog signaling contributes to basic fibroblast growth factor-regulated fibroblast migration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Zhong Xin; Sun, Cong Cong; Wenzhou People's Hospital, Wenzhou, Zhejiang

Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Westernmore » blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-β-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of β-catenin, suggesting that β-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy. - Highlights: • bFGF regulates Hedgehog (Hh) signaling in fibroblasts. • The Smo and Gli two master regulators of Hh signaling positively regulate fibroblast migration. • Smo facilitates β-catenin nuclear translocation via activation PI3K/JNK/GSK3β. • β-catenin positively regulates fibroblast cell migration and the expression of Hh signaling genes including Smo and Gli.« less

DNA-repair scaffolds dampen checkpoint signalling by counteracting the adaptor Rad9.

PubMed

Ohouo, Patrice Y; Bastos de Oliveira, Francisco M; Liu, Yi; Ma, Chu Jian; Smolka, Marcus B

2013-01-03

In response to genotoxic stress, a transient arrest in cell-cycle progression enforced by the DNA-damage checkpoint (DDC) signalling pathway positively contributes to genome maintenance. Because hyperactivated DDC signalling can lead to a persistent and detrimental cell-cycle arrest, cells must tightly regulate the activity of the kinases involved in this pathway. Despite their importance, the mechanisms for monitoring and modulating DDC signalling are not fully understood. Here we show that the DNA-repair scaffolding proteins Slx4 and Rtt107 prevent the aberrant hyperactivation of DDC signalling by lesions that are generated during DNA replication in Saccharomyces cerevisiae. On replication stress, cells lacking Slx4 or Rtt107 show hyperactivation of the downstream DDC kinase Rad53, whereas activation of the upstream DDC kinase Mec1 remains normal. An Slx4-Rtt107 complex counteracts the checkpoint adaptor Rad9 by physically interacting with Dpb11 and phosphorylated histone H2A, two positive regulators of Rad9-dependent Rad53 activation. A decrease in DDC signalling results from hypomorphic mutations in RAD53 and H2A and rescues the hypersensitivity to replication stress of cells lacking Slx4 or Rtt107. We propose that the Slx4-Rtt107 complex modulates Rad53 activation by a competition-based mechanism that balances the engagement of Rad9 at replication-induced lesions. Our findings show that DDC signalling is monitored and modulated through the direct action of DNA-repair factors.

Hyperthyroidism, but not hypertension, impairs PITX2 expression leading to Wnt-microRNA-ion channel remodeling

PubMed Central

Lozano-Velasco, Estefanía; Wangensteen, Rosemary; Quesada, Andrés; Garcia-Padilla, Carlos; Osorio, Julia A.; Ruiz-Torres, María Dolores; Aranega, Amelia

2017-01-01

PITX2 is a homeobox transcription factor involved in embryonic left/right signaling and more recently has been associated to cardiac arrhythmias. Genome wide association studies have pinpointed PITX2 as a major player underlying atrial fibrillation (AF). We have previously described that PITX2 expression is impaired in AF patients. Furthermore, distinct studies demonstrate that Pitx2 insufficiency leads to complex gene regulatory network remodeling, i.e. Wnt>microRNAs, leading to ion channel impairment and thus to arrhythmogenic events in mice. Whereas large body of evidences has been provided in recent years on PITX2 downstream signaling pathways, scarce information is available on upstream pathways influencing PITX2 in the context of AF. Multiple risk factors are associated to the onset of AF, such as e.g. hypertension (HTN), hyperthyroidism (HTD) and redox homeostasis impairment. In this study we have analyzed whether HTN, HTD and/or redox homeostasis impact on PITX2 and its downstream signaling pathways. Using rat models for spontaneous HTN (SHR) and experimentally-induced HTD we have observed that both cardiovascular risk factors lead to severe Pitx2 downregulation. Interesting HTD, but not SHR, leads to up-regulation of Wnt signaling as well as deregulation of multiple microRNAs and ion channels as previously described in Pitx2 insufficiency models. In addition, redox signaling is impaired in HTD but not SHR, in line with similar findings in atrial-specific Pitx2 deficient mice. In vitro cell culture analyses using gain- and loss-of-function strategies demonstrate that Pitx2, Zfhx3 and Wnt signaling influence redox homeostasis in cardiomyocytes. Thus, redox homeostasis seems to play a pivotal role in this setting, providing a regulatory feedback loop. Overall these data demonstrate that HTD, but not HTN, can impair Pitx2>>Wnt pathway providing thus a molecular link to AF. PMID:29194452

Rab7: roles in membrane trafficking and disease.

PubMed

Zhang, Ming; Chen, Li; Wang, Shicong; Wang, Tuanlao

2009-06-01

The endocytosis pathway controls multiple cellular and physiological events. The lysosome is the destination of newly synthesized lysosomal hydrolytic enzymes. Internalized molecules or particles are delivered to the lysosome for degradation through sequential transport along the endocytic pathway. The endocytic pathway is also emerging as a signalling platform, in addition to the well-known role of the plasma membrane for signalling. Rab7 is a late endosome-/lysosome-associated small GTPase, perhaps the only lysosomal Rab protein identified to date. Rab7 plays critical roles in the endocytic processes. Through interaction with its partners (including upstream regulators and downstream effectors), Rab7 participates in multiple regulation mechanisms in endosomal sorting, biogenesis of lysosome [or LRO (lysosome-related organelle)] and phagocytosis. These processes are closely related to substrates degradation, antigen presentation, cell signalling, cell survival and microbial pathogen infection. Consistently, mutations or dysfunctions of Rab7 result in traffic disorders, which cause various diseases, such as neuropathy, cancer and lipid metabolism disease. Rab7 also plays important roles in microbial pathogen infection and survival, as well as in participating in the life cycle of viruses. Here, we give a brief review on the central role of Rab7 in endosomal traffic and summarize the studies focusing on the participation of Rab7 in disease pathogenesis. The underlying mechanism governed by Rab7 and its partners will also be discussed.

A water-soluble polysaccharide from Grifola frondosa induced macrophages activation via TLR4-MyD88-IKKβ-NF-κB p65 pathways

PubMed Central

Hou, Lihua; Meng, Meng; Chen, Yuanyuan; Wang, Chunling

2017-01-01

Here, the immunomodulatory effects of water-soluble polysaccharide from Grifola frondosa on RAW264.7 macrophages and its molecular mechanisms were investigated. G. frondosa polysaccharide could obviously enhance immunostimulatory activity such as the release of nitric oxide and cytokine production. Western blotting results showed that G. frondosa polysaccharide elevated the TLR4, which might act as an upstream regulator of MyD88 induced G. frondosa polysaccharide. MyD88 promoted IKKβ in endochylema and translocate NF-κB p65 subunit into the nucleus which increased the NO production and cytokine/chemokines level. The results suggested that G. frondosa polysaccharide activated macrophages through TLR4-MyD88-IKKβ-NF-κBp65 signaling pathways. PMID:29156820

Extracellular Signal–Regulated Kinase in the Ventromedial Hypothalamus Mediates Leptin-Induced Glucose Uptake in Red-Type Skeletal Muscle

PubMed Central

Toda, Chitoku; Shiuchi, Tetsuya; Kageyama, Haruaki; Okamoto, Shiki; Coutinho, Eulalia A.; Sato, Tatsuya; Okamatsu-Ogura, Yuko; Yokota, Shigefumi; Takagi, Kazuyo; Tang, Lijun; Saito, Kumiko; Shioda, Seiji; Minokoshi, Yasuhiko

2013-01-01

Leptin is a key regulator of glucose metabolism in mammals, but the mechanisms of its action have remained elusive. We now show that signaling by extracellular signal–regulated kinase (ERK) and its upstream kinase MEK in the ventromedial hypothalamus (VMH) mediates the leptin-induced increase in glucose utilization as well as its insulin sensitivity in the whole body and in red-type skeletal muscle of mice through activation of the melanocortin receptor (MCR) in the VMH. In contrast, activation of signal transducer and activator of transcription 3 (STAT3), but not the MEK-ERK pathway, in the VMH by leptin enhances the insulin-induced suppression of endogenous glucose production in an MCR-independent manner, with this effect of leptin occurring only in the presence of an increased plasma concentration of insulin. Given that leptin requires 6 h to increase muscle glucose uptake, the transient activation of the MEK-ERK pathway in the VMH by leptin may play a role in the induction of synaptic plasticity in the VMH, resulting in the enhancement of MCR signaling in the nucleus and leading to an increase in insulin sensitivity in red-type muscle. PMID:23530005

Global Phosphoproteomics of Activated B Cells Using Complementary Metal Ion Functionalized Soluble Nanopolymers

PubMed Central

2015-01-01

Engagement of the B cell receptor for antigen (BCR) leads to immune responses through a cascade of intracellular signaling events. Most studies to date have focused on the BCR and protein tyrosine phosphorylation. Because spleen tyrosine kinase, Syk, is an upstream kinase in multiple BCR-regulated signaling pathways, it also affects many downstream events that are modulated through the phosphorylation of proteins on serine and threonine residues. Here, we report a novel phosphopeptide enrichment strategy and its application to a comprehensive quantitative phosphoproteomics analysis of Syk-dependent downstream signaling events in B cells, focusing on serine and threonine phosphorylation. Using a combination of the Syk inhibitor piceatannol, SILAC quantification, peptide fractionation, and complementary PolyMAC-Ti and PolyMAC-Zr enrichment techniques, we analyzed changes in BCR-stimulated protein phosphorylation that were dependent on the activity of Syk. We identified and quantified over 13 000 unique phosphopeptides, with a large percentage dependent on Syk activity in BCR-stimulated B cells. Our results not only confirmed many known functions of Syk, but more importantly, suggested many novel roles, including in the ubiquitin proteasome pathway, that warrant further exploration. PMID:24905233

Lipid-induced insulin resistance mediated by the proinflammatory receptor TLR4 requires saturated fatty acid–induced ceramide biosynthesis in mice

PubMed Central

Holland, William L.; Bikman, Benjamin T.; Wang, Li-Ping; Yuguang, Guan; Sargent, Katherine M.; Bulchand, Sarada; Knotts, Trina A.; Shui, Guanghou; Clegg, Deborah J.; Wenk, Markus R.; Pagliassotti, Michael J.; Scherer, Philipp E.; Summers, Scott A.

2011-01-01

Obesity is associated with an enhanced inflammatory response that exacerbates insulin resistance and contributes to diabetes, atherosclerosis, and cardiovascular disease. One mechanism accounting for the increased inflammation associated with obesity is activation of the innate immune signaling pathway triggered by TLR4 recognition of saturated fatty acids, an event that is essential for lipid-induced insulin resistance. Using in vitro and in vivo systems to model lipid induction of TLR4-dependent inflammatory events in rodents, we show here that TLR4 is an upstream signaling component required for saturated fatty acid–induced ceramide biosynthesis. This increase in ceramide production was associated with the upregulation of genes driving ceramide biosynthesis, an event dependent of the activity of the proinflammatory kinase IKKβ. Importantly, increased ceramide production was not required for TLR4-dependent induction of inflammatory cytokines, but it was essential for TLR4-dependent insulin resistance. These findings suggest that sphingolipids such as ceramide might be key components of the signaling networks that link lipid-induced inflammatory pathways to the antagonism of insulin action that contributes to diabetes. PMID:21490391

Platelet-derived growth factor receptor mediates activation of ras through different signaling pathways in different cell types.

PubMed Central

Satoh, T; Fantl, W J; Escobedo, J A; Williams, L T; Kaziro, Y

1993-01-01

A series of pieces of evidence have shown that Ras protein acts as a transducer of the platelet-derived growth factor (PDGF) receptor-mediated signaling pathway: (i) formation of Ras.GTP is detected immediately on PDGF stimulation, and (ii) a dominant inhibitory mutant Ras, as well as a neutralizing anti-Ras antibody, can interfere with PDGF-induced responses. On the other hand, several signal transducing molecules including phosphatidylinositol 3-kinase (PI3-K), GTPase-activating protein (GAP), and phospholipase C gamma (PLC gamma) bind directly to the PDGF receptor and become tyrosine phosphorylated. Recently, it was shown that specific phosphorylated tyrosines of the PDGF receptor are responsible for interaction between the receptor and each signaling molecule. However, the roles of these signaling molecules have not been elucidated, and it remains unclear which molecules are implicated in the Ras pathway. In this study, we measured Ras activation in cell lines expressing mutant PDGF receptors that are deficient in coupling with specific molecules. In fibroblast CHO cells, a mutant receptor (Y708F/Y719F [PI3-K-binding sites]) was unable to stimulate Ras, whereas another mutant (Y739F [the GAP-binding site]) could do so, suggesting an indispensable role of PI3-K or a protein that binds to the same sites as PI3-K for PDGF-stimulated Ras activation. By contrast, both of the above mutants were capable of stimulating Ras protein in a pro-B-cell line, BaF3. Furthermore, a mutant receptor (Y977F/Y989F [PLC gamma-binding sites]) could fully activate Ras, and the direct activation of protein kinase C and calcium mobilization had almost no effect on the GDP/GTP state of Ras in this cell line. These results suggest that, in the pro-B-cell transfectants, each of the above pathways (PI3-K, GAP, and PLC gamma) can be eliminated without a loss of Ras activation. It remains unclear whether another unknown essential pathway which regulates Ras protein exists within BaF3 cells. Therefore, it is likely that several different PDGF receptor-mediated signaling pathways function upstream of Ras, and the extent of the contribution of each pathway for the regulation of Ras may differ among different cell types. Images PMID:8388543

Soybean Homologs of MPK4 Negatively Regulate Defense Responses and Positively Regulate Growth and Development1[W][OA

PubMed Central

Liu, Jian-Zhong; Horstman, Heidi D.; Braun, Edward; Graham, Michelle A.; Zhang, Chunquan; Navarre, Duroy; Qiu, Wen-Li; Lee, Yeunsook; Nettleton, Dan; Hill, John H.; Whitham, Steven A.

2011-01-01

Mitogen-activated protein kinase (MAPK) cascades play important roles in disease resistance in model plant species such as Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum). However, the importance of MAPK signaling pathways in the disease resistance of crops is still largely uninvestigated. To better understand the role of MAPK signaling pathways in disease resistance in soybean (Glycine max), 13, nine, and 10 genes encoding distinct MAPKs, MAPKKs, and MAPKKKs, respectively, were silenced using virus-induced gene silencing mediated by Bean pod mottle virus. Among the plants silenced for various MAPKs, MAPKKs, and MAPKKKs, those in which GmMAPK4 homologs (GmMPK4s) were silenced displayed strong phenotypes including stunted stature and spontaneous cell death on the leaves and stems, the characteristic hallmarks of activated defense responses. Microarray analysis showed that genes involved in defense responses, such as those in salicylic acid (SA) signaling pathways, were significantly up-regulated in GmMPK4-silenced plants, whereas genes involved in growth and development, such as those in auxin signaling pathways and in cell cycle and proliferation, were significantly down-regulated. As expected, SA and hydrogen peroxide accumulation was significantly increased in GmMPK4-silenced plants. Accordingly, GmMPK4-silenced plants were more resistant to downy mildew and Soybean mosaic virus compared with vector control plants. Using bimolecular fluorescence complementation analysis and in vitro kinase assays, we determined that GmMKK1 and GmMKK2 might function upstream of GmMPK4. Taken together, our results indicate that GmMPK4s negatively regulate SA accumulation and defense response but positively regulate plant growth and development, and their functions are conserved across plant species. PMID:21878550

TGF-β induction of FGF-2 expression in stromal cells requires integrated smad3 and MAPK pathways.

PubMed

Strand, Douglas W; Liang, Yao-Yun; Yang, Feng; Barron, David A; Ressler, Steven J; Schauer, Isaiah G; Feng, Xin-Hua; Rowley, David R

2014-01-01

Transforming Growth Factor-β (TGF-β) regulates the reactive stroma microenvironment associated with most carcinomas and mediates expression of many stromal derived factors important for tumor progression, including FGF-2 and CTGF. TGF-β is over-expressed in most carcinomas, and FGF-2 action is important in tumor-induced angiogenesis. The signaling mechanisms of how TGF-β regulates FGF-2 expression in the reactive stroma microenvironment are not understood. Accordingly, we have assessed key signaling pathways that mediate TGF-β1-induced FGF-2 expression in prostate stromal fibroblasts and mouse embryo fibroblasts (MEFs) null for Smad2 and Smad3. TGF-β1 induced phosphorylation of Smad2, Smad3, p38 and ERK1/2 proteins in both control MEFs and prostate fibroblasts. Of these, Smad3, but not Smad2 was found to be required for TGF-β1 induction of FGF-2 expression in stromal cells. ChIP analysis revealed a Smad3/Smad4 complex was associated with the -1.9 to -2.3 kb upstream proximal promoter of the FGF-2 gene, further suggesting a Smad3-specific regulation. In addition, chemical inhibition of p38 or ERK1/2 MAPK activity also blocked TGF-β1-induced FGF-2 expression in a Smad3-independent manner. Conversely, inhibition of JNK signaling enhanced FGF-2 expression. Together, these data indicate that expression of FGF-2 in fibroblasts in the tumor stromal cell microenvironment is coordinately dependent on both intact Smad3 and MAP kinase signaling pathways. These pathways and key downstream mediators of TGF-β action in the tumor reactive stroma microenvironment, may evolve as putative targets for therapeutic intervention.

Reactivation of cocaine reward memory engages the Akt/GSK3/mTOR signaling pathway and can be disrupted by GSK3 inhibition.

PubMed

Shi, Xiangdang; Miller, Jonathan S; Harper, Lauren J; Poole, Rachel L; Gould, Thomas J; Unterwald, Ellen M

2014-08-01

Memories return to a labile state following their retrieval and must undergo a process of reconsolidation to be maintained. Thus, disruption of cocaine reward memories by interference with reconsolidation may be therapeutically beneficial in the treatment of cocaine addiction. The objectives were to elucidate the signaling pathway involved in reconsolidation of cocaine reward memory and to test whether targeting this pathway could disrupt cocaine-associated contextual memory. Using a mouse model of conditioned place preference, regulation of the activity of glycogen synthase kinase-3 (GSK3), mammalian target of Rapamycin complex 1 (mTORC1), P70S6K, β-catenin, and the upstream signaling molecule Akt, was studied in cortico-limbic-striatal circuitry after re-exposure to an environment previously paired with cocaine. Levels of phosporylated Akt-Thr308, GSK3α-Ser21, GSK3β-Ser9, mTORC1, and P70S6K were reduced in the nucleus accumbens and hippocampus 10 min after the reactivation of cocaine cue memories. Levels of pAkt and pGSK3 were also reduced in the prefrontal cortex. Since reduced phosphorylation of GSK3 indicates heightened enzyme activity, the effect of a selective GSK3 inhibitor, SB216763, on reconsolidation was tested. Administration of SB216763 immediately after exposure to an environment previously paired with cocaine abrogated a previously established place preference, suggesting that GSK3 inhibition interfered with reconsolidation of cocaine-associated reward memories. These findings suggest that the Akt/GSK3/mTORC1 signaling pathway in the nucleus accumbens, hippocampus, and/or prefrontal cortex is critically involved in the reconsolidation of cocaine contextual reward memory. Inhibition of GSK3 activity during memory retrieval can erase an established cocaine place preference.

Fungal Communication Requires the MAK-2 Pathway Elements STE-20 and RAS-2, the NRC-1 Adapter STE-50 and the MAP Kinase Scaffold HAM-5

PubMed Central

Dettmann, Anne; Heilig, Yvonne; Valerius, Oliver; Ludwig, Sarah; Seiler, Stephan

2014-01-01

Intercellular communication is critical for the survival of unicellular organisms as well as for the development and function of multicellular tissues. Cell-to-cell signaling is also required to develop the interconnected mycelial network characteristic of filamentous fungi and is a prerequisite for symbiotic and pathogenic host colonization achieved by molds. Somatic cell–cell communication and subsequent cell fusion is governed by the MAK-2 mitogen activated protein kinase (MAPK) cascade in the filamentous ascomycete model Neurospora crassa, yet the composition and mode of regulation of the MAK-2 pathway are currently unclear. In order to identify additional components involved in MAK-2 signaling we performed affinity purification experiments coupled to mass spectrometry with strains expressing functional GFP-fusion proteins of the MAPK cascade. This approach identified STE-50 as a regulatory subunit of the Ste11p homolog NRC-1 and HAM-5 as cell-communication-specific scaffold protein of the MAPK cascade. Moreover, we defined a network of proteins consisting of two Ste20-related kinases, the small GTPase RAS-2 and the adenylate cyclase capping protein CAP-1 that function upstream of the MAK-2 pathway and whose signals converge on the NRC-1/STE-50 MAP3K complex and the HAM-5 scaffold. Finally, our data suggest an involvement of the striatin interacting phosphatase and kinase (STRIPAK) complex, the casein kinase 2 heterodimer, the phospholipid flippase modulators YPK-1 and NRC-2 and motor protein-dependent vesicle trafficking in the regulation of MAK-2 pathway activity and function. Taken together, these data will have significant implications for our mechanistic understanding of MAPK signaling and for homotypic cell–cell communication in fungi and higher eukaryotes. PMID:25411845

Fungal communication requires the MAK-2 pathway elements STE-20 and RAS-2, the NRC-1 adapter STE-50 and the MAP kinase scaffold HAM-5.

PubMed

Dettmann, Anne; Heilig, Yvonne; Valerius, Oliver; Ludwig, Sarah; Seiler, Stephan

2014-11-01

Intercellular communication is critical for the survival of unicellular organisms as well as for the development and function of multicellular tissues. Cell-to-cell signaling is also required to develop the interconnected mycelial network characteristic of filamentous fungi and is a prerequisite for symbiotic and pathogenic host colonization achieved by molds. Somatic cell-cell communication and subsequent cell fusion is governed by the MAK-2 mitogen activated protein kinase (MAPK) cascade in the filamentous ascomycete model Neurospora crassa, yet the composition and mode of regulation of the MAK-2 pathway are currently unclear. In order to identify additional components involved in MAK-2 signaling we performed affinity purification experiments coupled to mass spectrometry with strains expressing functional GFP-fusion proteins of the MAPK cascade. This approach identified STE-50 as a regulatory subunit of the Ste11p homolog NRC-1 and HAM-5 as cell-communication-specific scaffold protein of the MAPK cascade. Moreover, we defined a network of proteins consisting of two Ste20-related kinases, the small GTPase RAS-2 and the adenylate cyclase capping protein CAP-1 that function upstream of the MAK-2 pathway and whose signals converge on the NRC-1/STE-50 MAP3K complex and the HAM-5 scaffold. Finally, our data suggest an involvement of the striatin interacting phosphatase and kinase (STRIPAK) complex, the casein kinase 2 heterodimer, the phospholipid flippase modulators YPK-1 and NRC-2 and motor protein-dependent vesicle trafficking in the regulation of MAK-2 pathway activity and function. Taken together, these data will have significant implications for our mechanistic understanding of MAPK signaling and for homotypic cell-cell communication in fungi and higher eukaryotes.

The Response of wnt/ ß-Catenin Signaling Pathway in Osteocytes Under Simulated Microgravity

NASA Astrophysics Data System (ADS)

Yang, Xiao; Sun, Lian-Wen; Liang, Meng; Wang, Xiao-Nan; Fan, Yu-Bo

2015-11-01

Osteocytes were considered as potential sensors of mechanical loading and orchestrate the bone remodeling adapted to mechanical loading. On the other hand, osteocytes are also considered as the unloading sensors in vivo. Previous studies showed that the mechanosensation and mechanotransduction of osteocytes may play an essential role in mediating bone response to microgravity, and one of the most important molecular signaling pathway involved in the mechanotransduction is the Wnt/ ß-catenin signaling pathway. In order to investigate the effect of simulated microgravity on the Wnt/ ß-catenin signaling pathway in osteocytes, MLO-Y4 cells (an osteocyte-like cell line) were cultured under controlled rotation to simulate microgravity for 5 days. The cytoskeleton and ß-catenin nuclear translocation of MLO-Y4 cells were detected by laser scanning confocal microscope and the fluorescence intensity was quantified; the mRNA expressions of upstream and downstream key components in Wnt canonical signaling were detected with RT-PCR. Two regulators of the Wnt/ ß-catenin pathway, NMP4/CIZ and Smads, were also investigated by RT-PCR; finally the expression of Wnt target genes and Sost protein level were detected with the absence or presence of the Sclerostin antibody (Scl-AbI) under simulated microgravity. The results showed that under simulated microgravity, (1) F-actin filaments were disassembled and some short dendritic processes appeared at the cell periphery; (2) the gene expression of Wnt3a, Wnt5a, DKK1, CyclinD1, LEF-1 and CX43 in the simulated microgravity group were significantly lower whereas Wnt1 and Sost in the simulated microgravity group were significantly higher than the control group; (3) the gene and protein level of ß-catenin were reduced, and no ß-catenin nuclear translocation observed; (4) the gene expression of Smad1, Smad4 and Smad7 were significantly lower whereas NMP4/CIZ and Smad3 in the simulated microgravity were significantly higher than the control group; (5) Scl-AbI partially inhibited the down-regulation of simulated microgravity to Wnt target gene expression and Sclerostin protein expression. The results suggested that firstly the cytoskeleton was disturbed in MLO-Y4 by simulated microgravity; secondly the activity of Wnt/ ß-catenin signaling pathway was depressed, with the nuclear translocation of ß-catenin suppressed by simulated microgravity; thirdly the Wnt/ ß-catenin signaling pathway positive regulators (Smads) were decreased, while the negative regulator (NMP4/CIZ) was increased under simulated microgravity; finally Scl-AbI could partially restore the adverse effect of simulated microgravity to Wnt signaling. This study may help us to understand the mechanotransduction alteration of Wnt/ ß-catenin signaling pathway in osteocytes under simulated microgravity, and further may partly clarify the mechanism of microgravity-induced osteoporosis.

Pathway enrichment analysis approach based on topological structure and updated annotation of pathway.

PubMed

Yang, Qian; Wang, Shuyuan; Dai, Enyu; Zhou, Shunheng; Liu, Dianming; Liu, Haizhou; Meng, Qianqian; Jiang, Bin; Jiang, Wei

2017-08-16

Pathway enrichment analysis has been widely used to identify cancer risk pathways, and contributes to elucidating the mechanism of tumorigenesis. However, most of the existing approaches use the outdated pathway information and neglect the complex gene interactions in pathway. Here, we first reviewed the existing widely used pathway enrichment analysis approaches briefly, and then, we proposed a novel topology-based pathway enrichment analysis (TPEA) method, which integrated topological properties and global upstream/downstream positions of genes in pathways. We compared TPEA with four widely used pathway enrichment analysis tools, including database for annotation, visualization and integrated discovery (DAVID), gene set enrichment analysis (GSEA), centrality-based pathway enrichment (CePa) and signaling pathway impact analysis (SPIA), through analyzing six gene expression profiles of three tumor types (colorectal cancer, thyroid cancer and endometrial cancer). As a result, we identified several well-known cancer risk pathways that could not be obtained by the existing tools, and the results of TPEA were more stable than that of the other tools in analyzing different data sets of the same cancer. Ultimately, we developed an R package to implement TPEA, which could online update KEGG pathway information and is available at the Comprehensive R Archive Network (CRAN): https://cran.r-project.org/web/packages/TPEA/. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Mechanism of arginine sensing by CASTOR1 upstream of mTORC1

PubMed Central

Saxton, Robert A.; Chantranupong, Lynne; Knockenhauer, Kevin E.; Schwartz, Thomas U.; Sabatini, David M.

2016-01-01

Summary The mechanistic Target of Rapamycin Complex 1 (mTORC1) is a major regulator of eukaryotic growth that coordinates anabolic and catabolic cellular processes with inputs such as growth factors and nutrients, including amino acids1–3. In mammals, arginine is particularly important and promotes diverse physiological effects including immune cell activation, insulin secretion, and muscle growth, largely through activation of mTORC14–7. Arginine activates mTORC1 upstream of the Rag GTPases8, through either the lysosomal amino acid transporter SLC38A9 or the GATOR2-interacting CASTOR1 (Cellular Arginine Sensor for mTORC1)9–12. However, the mechanism by which the mTORC1 pathway detects and transmits the arginine signal has been elusive. Here, we present the 1.8 Å crystal structure of arginine-bound CASTOR1. Homodimeric CASTOR1 binds arginine at the interface of two ACT domains, enabling allosteric control of the adjacent GATOR2-binding site to trigger dissociation from GATOR2 and the downstream activation of mTORC1. Our data reveal that CASTOR1 shares substantial structural homology with the lysine-binding regulatory domain of prokaryotic aspartate kinases, suggesting that the mTORC1 pathway exploited an ancient amino-acid-dependent allosteric mechanism to acquire arginine sensitivity. Together, these results establish a structural basis for arginine sensing by the mTORC1 pathway and provide insights into the evolution of a mammalian nutrient sensor. PMID:27487210

Transcription factor AP-2γ induces early Cdx2 expression and represses HIPPO signaling to specify the trophectoderm lineage.

PubMed

Cao, Zubing; Carey, Timothy S; Ganguly, Avishek; Wilson, Catherine A; Paul, Soumen; Knott, Jason G

2015-05-01

Cell fate decisions are fundamental to the development of multicellular organisms. In mammals the first cell fate decision involves segregation of the pluripotent inner cell mass and the trophectoderm, a process regulated by cell polarity proteins, HIPPO signaling and lineage-specific transcription factors such as CDX2. However, the regulatory mechanisms that operate upstream to specify the trophectoderm lineage have not been established. Here we report that transcription factor AP-2γ (TFAP2C) functions as a novel upstream regulator of Cdx2 expression and position-dependent HIPPO signaling in mice. Loss- and gain-of-function studies and promoter analysis revealed that TFAP2C binding to an intronic enhancer is required for activation of Cdx2 expression during early development. During the 8-cell to morula transition TFAP2C potentiates cell polarity to suppress HIPPO signaling in the outside blastomeres. TFAP2C depletion triggered downregulation of PARD6B, loss of apical cell polarity, disorganization of F-actin, and activation of HIPPO signaling in the outside blastomeres. Rescue experiments using Pard6b mRNA restored cell polarity but only partially corrected position-dependent HIPPO signaling, suggesting that TFAP2C negatively regulates HIPPO signaling via multiple pathways. Several genes involved in regulation of the actin cytoskeleton (including Rock1, Rock2) were downregulated in TFAP2C-depleted embryos. Inhibition of ROCK1 and ROCK2 activity during the 8-cell to morula transition phenocopied TFAP2C knockdown, triggering a loss of position-dependent HIPPO signaling and decrease in Cdx2 expression. Altogether, these results demonstrate that TFAP2C facilitates trophectoderm lineage specification by functioning as a key regulator of Cdx2 transcription, cell polarity and position-dependent HIPPO signaling. © 2015. Published by The Company of Biologists Ltd.

Methamphetamine exposure triggers apoptosis and autophagy in neuronal cells by activating the C/EBPβ-related signaling pathway.

PubMed

Xu, Xiang; Huang, Enping; Luo, Baoying; Cai, Dunpeng; Zhao, Xu; Luo, Qin; Jin, Yili; Chen, Ling; Wang, Qi; Liu, Chao; Lin, Zhoumeng; Xie, Wei-Bing; Wang, Huijun

2018-06-25

Methamphetamine (Meth) is a widely abused psychoactive drug that primarily damages the nervous system, notably causing dopaminergic neuronal apoptosis. CCAAT-enhancer binding protein (C/EBPβ) is a transcription factor and an important regulator of cell apoptosis and autophagy. Insulin-like growth factor binding protein (IGFBP5) is a proapoptotic factor that mediates Meth-induced neuronal apoptosis, and Trib3 (tribbles pseudokinase 3) is an endoplasmic reticulum (ER) stress-inducible gene involved in autophagic cell death through the mammalian target of rapamycin (mTOR) signaling pathway. To test the hypothesis that C/EBPβ is involved in Meth-induced IGFBP5-mediated neuronal apoptosis and Trib3-mediated neuronal autophagy, we measured the protein expression of C/EBPβ after Meth exposure and evaluated the effects of silencing C/EBPβ, IGFBP5, or Trib3 on Meth-induced apoptosis and autophagy in neuronal cells and in the rat striatum after intrastriatal Meth injection. We found that, at relatively high doses, Meth exposure increased C/EBPβ protein expression, which was accompanied by increased neuronal apoptosis and autophagy; triggered the IGFBP5-mediated, p53-up-regulated modulator of apoptosis (PUMA)-related mitochondrial apoptotic signaling pathway; and stimulated the Trib3-mediated ER stress signaling pathway through the Akt-mTOR signaling axis. We also found that autophagy is an early response to Meth-induced stress upstream of apoptosis and plays a detrimental role in Meth-induced neuronal cell death. These results suggest that Meth exposure induces C/EBPβ expression, which plays an essential role in the neuronal apoptosis and autophagy induced by relatively high doses of Meth; however, relatively low concentrations of Meth did not change the expression of C/EBPβ in vitro. Further studies are needed to elucidate the role of C/EBPβ in low-dose Meth-induced neurotoxicity.-Xu, X., Huang, E., Luo, B., Cai, D., Zhao, X., Luo, Q., Jin, Y., Chen, L., Wang, Q., Liu, C., Lin, Z., Xie, W.-B., Wang, H. Methamphetamine exposure triggers apoptosis and autophagy in neuronal cells by activating the C/EBPβ-related signaling pathway.

Hydration status regulates sodium flux and inflammatory pathways through epithelial sodium channel (ENaC) in the skin.

PubMed

Xu, Wei; Hong, Seok Jong; Zeitchek, Michael; Cooper, Garry; Jia, Shengxian; Xie, Ping; Qureshi, Hannan A; Zhong, Aimei; Porterfield, Marshall D; Galiano, Robert D; Surmeier, D James; Mustoe, Thomas A

2015-03-01

Although it is known that the inflammatory response that results from disruption of epithelial barrier function after injury results in excessive scarring, the upstream signals remain unknown. It has also been observed that epithelial disruption results in reduced hydration status and that the use of occlusive dressings that prevent water loss from wounds decreases scar formation. We hypothesized that hydration status changes sodium homeostasis and induces sodium flux in keratinocytes, which result in activation of pathways responsible for keratinocyte-fibroblast signaling and ultimately lead to activation of fibroblasts. Here, we demonstrate that perturbations in epithelial barrier function lead to increased sodium flux in keratinocytes. We identified that sodium flux in keratinocytes is mediated by epithelial sodium channels (ENaCs) and causes increased secretion of proinflammatory cytokines, which activate fibroblast via the cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2) pathway. Similar changes in signal transduction and sodium flux occur by increased sodium concentration, which simulates reduced hydration, in the media in epithelial cultures or human ex vivo skin cultures. Blockade of ENaC, prostaglandin synthesis, or PGE2 receptors all reduce markers of fibroblast activation and collagen synthesis. In addition, employing a validated in vivo excessive scar model in the rabbit ear, we demonstrate that utilization of either an ENaC blocker or a COX-2 inhibitor results in a marked reduction in scarring. Other experiments demonstrate that the activation of COX-2 in response to increased sodium flux is mediated through the PIK3/Akt pathway. Our results indicate that ENaC responds to small changes in sodium concentration with inflammatory mediators and suggest that the ENaC pathway is a potential target for a strategy to prevent fibrosis.

Transcriptional control of Sost in bone [Transcriptional control of Sclerostin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sebastian, Aimy; Loots, Gabriela G.

Sclerostin is an osteocyte derived negative regulator of bone formation. A highly specific expression pattern and the exclusive bone phenotype have made Sclerostin an attractive target for therapeutic intervention in treating metabolic bone diseases such as osteoporosis and in facilitating fracture repair. Understanding the molecular mechanisms that regulate Sclerostin transcription is of great interest as it may unveil new avenues for therapeutic approaches. Such studies may also elucidate how various signaling pathways intersect to modulate bone metabolism. Furthermore we review the current understanding of the upstream molecular mechanisms that regulate Sost/SOST transcription, in bone.

Transcriptional control of Sost in bone [Transcriptional control of Sclerostin

DOE PAGES

Sebastian, Aimy; Loots, Gabriela G.

2016-10-19

Sclerostin is an osteocyte derived negative regulator of bone formation. A highly specific expression pattern and the exclusive bone phenotype have made Sclerostin an attractive target for therapeutic intervention in treating metabolic bone diseases such as osteoporosis and in facilitating fracture repair. Understanding the molecular mechanisms that regulate Sclerostin transcription is of great interest as it may unveil new avenues for therapeutic approaches. Such studies may also elucidate how various signaling pathways intersect to modulate bone metabolism. Furthermore we review the current understanding of the upstream molecular mechanisms that regulate Sost/SOST transcription, in bone.

Biophysically Inspired Rational Design of Structured Chimeric Substrates for DNAzyme Cascade Engineering

PubMed Central

Lakin, Matthew R.; Brown, Carl W.; Horwitz, Eli K.; Fanning, M. Leigh; West, Hannah E.; Stefanovic, Darko; Graves, Steven W.

2014-01-01

The development of large-scale molecular computational networks is a promising approach to implementing logical decision making at the nanoscale, analogous to cellular signaling and regulatory cascades. DNA strands with catalytic activity (DNAzymes) are one means of systematically constructing molecular computation networks with inherent signal amplification. Linking multiple DNAzymes into a computational circuit requires the design of substrate molecules that allow a signal to be passed from one DNAzyme to another through programmed biochemical interactions. In this paper, we chronicle an iterative design process guided by biophysical and kinetic constraints on the desired reaction pathways and use the resulting substrate design to implement heterogeneous DNAzyme signaling cascades. A key aspect of our design process is the use of secondary structure in the substrate molecule to sequester a downstream effector sequence prior to cleavage by an upstream DNAzyme. Our goal was to develop a concrete substrate molecule design to achieve efficient signal propagation with maximal activation and minimal leakage. We have previously employed the resulting design to develop high-performance DNAzyme-based signaling systems with applications in pathogen detection and autonomous theranostics. PMID:25347066

Intestinal Insulin Signaling Encodes Two Different Molecular Mechanisms for the Shortened Longevity Induced by Graphene Oxide in Caenorhabditis elegans

NASA Astrophysics Data System (ADS)

Zhao, Yunli; Yang, Ruilong; Rui, Qi; Wang, Dayong

2016-04-01

Graphene oxide (GO) has been shown to cause multiple toxicities in various organisms. However, the underlying molecular mechanisms for GO-induced shortened longevity are still unclear. We employed Caenorhabditis elegans to investigate the possible involvement of insulin signaling pathway in the control of GO toxicity and its underlying molecular mechanisms. Mutation of daf-2, age-1, akt-1, or akt-2 gene induced a resistant property of nematodes to GO toxicity, while mutation of daf-16 gene led to a susceptible property of nematodes to GO toxicity, suggesting that GO may dysregulate the functions of DAF-2/IGF-1 receptor, AGE-1, AKT-1 and AKT-2-mediated kinase cascade, and DAF-16/FOXO transcription factor. Genetic interaction analysis suggested the involvement of signaling cascade of DAF-2-AGE-1-AKT-1/2-DAF-16 in the control of GO toxicity on longevity. Moreover, intestinal RNA interference (RNAi) analysis demonstrated that GO reduced longevity by affecting the functions of signaling cascade of DAF-2-AGE-1-AKT-1/2-DAF-16 in the intestine. DAF-16 could also regulate GO toxicity on longevity by functioning upstream of SOD-3, which encodes an antioxidation system that prevents the accumulation of oxidative stress. Therefore, intestinal insulin signaling may encode two different molecular mechanisms responsible for the GO toxicity in inducing the shortened longevity. Our results highlight the key role of insulin signaling pathway in the control of GO toxicity in organisms.

Emerging Insights into Wnt/β-catenin Signaling in Head and Neck Cancer.

PubMed

Alamoud, K A; Kukuruzinska, M A

2018-06-01

Head and neck cancer presents primarily as head and neck squamous cell carcinoma (HNSCC), a debilitating malignancy fraught with high morbidity, poor survival rates, and limited treatment options. Mounting evidence indicates that the Wnt/β-catenin signaling pathway plays important roles in the pathobiology of HNSCC. Wnt/β-catenin signaling affects multiple cellular processes that endow cancer cells with the ability to maintain and expand immature stem-like phenotypes, proliferate, extend survival, and acquire aggressive characteristics by adopting mesenchymal traits. A central component of canonical Wnt signaling is β-catenin, which balances its role as a structural component of E-cadherin junctions with its function as a transcriptional coactivator of numerous target genes. Recent genomic characterization of head and neck cancer revealed that while β-catenin is not frequently mutated in HNSCC, its activity is unchecked by more common mutations in genes encoding upstream regulators of β-catenin, NOTCH1, FAT1, and AJUBA. Wnt/β-catenin signaling affects a wide range epigenetic and transcriptional activities, mediated by the interaction of β-catenin with different transcription factors and transcriptional coactivators and corepressors. Furthermore, Wnt/β-catenin functions in a network with many signaling and metabolic pathways that modulate its activity. In addition to its effects on tumor epithelia, β-catenin activity regulates the tumor microenvironment by regulating extracellular matrix remodeling, fibrotic processes, and immune response. These multifunctional oncogenic effects of β-catenin make it an attractive bona fide target for HNSCC therapy.

Estradiol-induced object recognition memory consolidation is dependent on activation of mTOR signaling in the dorsal hippocampus

PubMed Central

Fortress, Ashley M.; Fan, Lu; Orr, Patrick T.; Zhao, Zaorui; Frick, Karyn M.

2013-01-01

The mammalian target of rapamycin (mTOR) signaling pathway is an important regulator of protein synthesis and is essential for various forms of hippocampal memory. Here, we asked whether the enhancement of object recognition memory consolidation produced by dorsal hippocampal infusion of 17β-estradiol (E2) is dependent on mTOR signaling in the dorsal hippocampus, and whether E2-induced mTOR signaling is dependent on dorsal hippocampal phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) activation. We first demonstrated that the enhancement of object recognition induced by E2 was blocked by dorsal hippocampal inhibition of ERK, PI3K, or mTOR activation. We then showed that an increase in dorsal hippocampal ERK phosphorylation 5 min after intracerebroventricular (ICV) E2 infusion was also blocked by dorsal hippocampal infusion of the three cell signaling inhibitors. Next, we found that ICV infusion of E2 increased phosphorylation of the downstream mTOR targets S6K (Thr-421) and 4E-BP1 in the dorsal hippocampus 5 min after infusion, and that this phosphorylation was blocked by dorsal hippocampal infusion of inhibitors of ERK, PI3K, and mTOR. Collectively, these data demonstrate for the first time that activation of the dorsal hippocampal mTOR signaling pathway is necessary for E2 to enhance object recognition memory consolidation and that E2-induced mTOR activation is dependent on upstream activation of ERK and PI3K signaling. PMID:23422279

Continuous tooth generation in mouse is induced by activated epithelial Wnt/β-catenin signaling

PubMed Central

Järvinen, Elina; Salazar-Ciudad, Isaac; Birchmeier, Walter; Taketo, Makoto M.; Jernvall, Jukka; Thesleff, Irma

2006-01-01

The single replacement from milk teeth to permanent teeth makes mammalian teeth different from teeth of most nonmammalian vertebrates and other epithelial organs such as hair and feathers, whose continuous replacement has been linked to Wnt signaling. Here we show that mouse tooth buds expressing stabilized β-catenin in epithelium give rise to dozens of teeth. The molar crowns, however, are typically simplified unicusped cones. We demonstrate that the supernumerary teeth develop by a renewal process where new signaling centers, the enamel knots, bud off from the existing dental epithelium. The basic aspects of the unlocked tooth renewal can be reproduced with a computer model on tooth development by increasing the intrinsic level of activator production, supporting the role of β-catenin pathway as an upstream activator of enamel knot formation. These results may implicate Wnt signaling in tooth renewal, a capacity that was all but lost when mammals evolved progressively more complicated tooth shapes. PMID:17121988

JNK1 Mediates Lipopolysaccharide-Induced CD14 and SR-AI Expression and Macrophage Foam Cell Formation.

PubMed

An, Dong; Hao, Feng; Hu, Chen; Kong, Wei; Xu, Xuemin; Cui, Mei-Zhen

2017-01-01

Foam cell formation is the key process in the development of atherosclerosis. The uptake of oxidized low-density lipoprotein (oxLDL) converts macrophages into foam cells. We recently reported that lipopolysaccharide (LPS)-induced foam cell formation is regulated by CD14 and scavenger receptor AI (SR-AI). In this study, we employed pharmaceutical and gene knockdown approaches to determine the upstream molecular mediators, which control LPS-induced foam cell formation. Our results demonstrated that the specific c-Jun N-terminal kinase (JNK) pathway inhibitor, SP600125, but neither the specific inhibitor of extracellular signaling-regulated kinase (ERK) kinase MEK1/2, U0126, nor the specific inhibitor of p38 MAPK, SB203580, significantly blocks LPS-induced oxLDL uptake, suggesting that the JNK pathway is the upstream mediator of LPS-induced oxLDL uptake/foam cell formation. To address whether JNK pathway mediates LPS-induced oxLDL uptake is due to JNK pathway-regulated CD14 and SR-AI expression, we assessed whether the pharmaceutical inhibitor of JNK influences LPS-induced expression of CD14 and SR-AI. Our results indicate that JNK pathway mediates LPS-induced CD14 and SR-AI expression. To conclusively address the isoform role of JNK family, we depleted JNK isoforms using the JNK isoform-specific siRNA. Our data showed that the depletion of JNK1, but not JNK2 blocked LPS-induced CD14/SR-AI expression and foam cell formation. Taken together, our results reveal for the first time that JNK1 is the key mediator of LPS-induced CD14 and SR-AI expression in macrophages, leading to LPS-induced oxLDL uptake/foam cell formation. We conclude that the novel JNK1/CD14/SR-AI pathway controls macrophage oxLDL uptake/foam cell formation.

Improving upstream transmission performance using a receiver with decision threshold level adjustment in a loopback WDM-PON

NASA Astrophysics Data System (ADS)

Cho, Seung-Hyun; Lee, Sang-Soo; Shin, Dong-Wook

2010-06-01

We have experimentally demonstrated that the use of an optical receiver with decision threshold level adjustment (DTLA) improved the performance of an upstream transmission in reflective semiconductor optical amplifier (RSOA)-based loopback wavelength division multiplexing-passive optical network (WDM-PON). Even though the extinction ratio (ER) of the downstream signal was as much as 9 dB and the injection power into the RSOA at the optical network unit was about -24 dBm, we successfully obtained error-free transmission results for the upstream signal through careful control of the decision threshold value in the optical receiver located at optical line terminal (OLT). Using an optical receiver with DTLA for upstream signal detection overcame significant obstacles related to the injection power into the RSOA and the ER of the downstream signal, which were previously considered limitations of the wavelength remodulation scheme. This technique is expected to provide flexibility for the optical link design in the practical deployment of a WDM-PON.

Pathway-specific effect of caffeine on protection against UV irradiation-induced apoptosis in corneal epithelial cells.

PubMed

Wang, Ling; Lu, Luo

2007-02-01

To define the role of molecular interaction between the UV-induced JNK (c-Jun N-terminal kinase) cascade and corneal epithelial cell apoptosis and protection against apoptosis by caffeine. Rabbit and human corneal epithelial cells were cultured in DMEM/F12 medium containing 10% FBS and 5 microg/mL insulin at 37 degrees C in 5% CO(2). DNA fragmentation and ethidium bromide/acridine orange (EB/AO) nuclear staining were performed to detect cell death. Western blot, immunoprecipitation, and kinase assays were used to measure UV-induced mitogen-activated protein (MAP) kinase activity. UV irradiation-induced apoptosis through apoptosis signal-regulating kinase 1 (ASK1) and MAKK4 (SEK1) upstream from JNK was caffeine sensitive. Caffeine (1,3,7-trimethylxanthine), an agent that is one of the most popular additions to food consumed in the world and a potential enhancer of chemotherapy, effectively protected corneal epithelial cells against apoptosis by its specific effect on the JNK cascade. Theophylline (1,3-dimethylxanthine) exhibited an effect similar to that of caffeine on prevention of UV irradiation-induced apoptosis. However, alterations of either intracellular cAMP or Ca(2+) levels did not alter the effect of caffeine on the JNK signaling pathway. In addition, the blockade of PI3K-like kinases by wortmannin had no impact on the protective effect of caffeine against UV irradiation-induced apoptosis, suggesting that the protective effect of caffeine acts through a specific mechanism involving UV irradiation-induced activation of ASK1 and SEK1. In contrast, caffeine had no effects on melphalan-, hyperosmotic stress-, or IL-1beta-induced activation of the JNK signaling pathway in these cells. UV irradiation stress-induced activation of the ASK1-SEK1-JNK signaling pathway leading to apoptosis is a caffeine-sensitive process, and caffeine, as a multifunctional agent in cells, can specifically interact with the pathway to protect against apoptosis.

Porphyromonas gingivalis lipopolysaccharide activates canonical Wnt/β-catenin and p38 MAPK signalling in stem cells from the apical papilla.

PubMed

Wang, Jia; Dai, Jiewen; Liu, Bin; Gu, Shensheng; Cheng, Lan; Liang, Jingping

2013-12-01

As dental precursor cells, stem cells from the apical papilla (SCAP) are capable of forming roots and undergoing apexogenesis, which are impaired upon exposure to bacterial infection. Porphyromonas gingivalis is a common Gram-negative bacterium that is involved in pulpal and periapical infection. The purpose of this study was to investigate the effects of P. gingivalis lipopolysaccharide (LPS) on the Wnt/β-catenin and p38 mitogen-activated protein kinase (MAPK) signalling pathways in SCAP. As indicated by the IL-1β and TNF-α mRNA levels, P. gingivalis LPS induced the expression of pro-inflammatory cytokines in a dose-dependent manner. In addition, activation of the p38 MAPK and Wnt/β-catenin pathways was confirmed by the augmentation of phospho-p38 and β-catenin protein expression and increased expression of c-myc and cyclin D1 mRNA. Despite no significant increase in β-catenin mRNA expression, increased phosphorylation of glycogen synthase kinase (GSK)-3β suggested that GSK-3β was responsible for the accumulation of β-catenin in the cytoplasm and translocation to the nucleus. Previous studies have shown that GSK-3β plays a critical role in crosstalk between the Wnt/β-catenin and p38 MAPK pathways. In the present study, we showed that the level of p38 phosphorylation decreased upon pretreatment with a p38 MAPK inhibitor for 1 h before stimulating SCAP with 10 μg/ml P. gingivalis LPS. However, the levels of GSK-3β and β-catenin phosphorylation in the cytoplasm and nucleus were not significantly altered. Our results suggest that the p38 MAPK and canonical Wnt/β-catenin signalling pathways are activated by P. gingivalis LPS in SCAP, but we have no evidence that p38 MAPK is upstream of GSK-3β in the Wnt/β-catenin signalling pathway.

Novel Mechanisms of Target Cell Death and Survival and of Therapeutic Action of IVIg in Pemphigus

PubMed Central

Arredondo, Juan; Chernyavsky, Alexander I.; Karaouni, Ali; Grando, Sergei A.

2005-01-01

Pemphigus vulgaris (PV) is a potentially lethal mucocutaneous blistering disease characterized by cell-cell detachment within the stratified epithelium (acantholysis) caused by IgG autoantibodies. Intravenous immunoglobulin (IVIg) therapy effectively treats PV, but the mechanism is not fully understood. To further understand acantholysis and the efficacy of IVIg, we measured effects of IgG fractions from PV patients on keratinocyte death processes. Using IgGs from representative PV patients who improved with IVIg, we identified apoptotic and oncotic signaling pathways in in vitro and in vivo PV models. We identified two groups of PV patients, each producing autoantibodies activating predominantly either apoptotic or oncotic cell death pathway. Experimental treatments with caspase 3 or calpain inhibitors demonstrated that PV IgGs induced acantholysis through both pathways. Upstream, the apoptotic signaling involved activation of caspases 8 and 3 and up-regulation of Fas ligand mRNA, whereas calpain-mediated cell death depended on elevated intracellular free Ca2+. IVIg reduced PV IgG-mediated acantholysis and cell death and up-regulated the caspase inhibitor FLIP and the calpain inhibitor calpastatin. These results indicate that in different PV patients, IgG-induced acantholysis proceeds predominantly via distinct, yet complementary, pathways of programmed cell death differentially mediated by apoptosis and oncosis effectors, with IVIg protecting target cells by up-regulating endogenous caspase and calpain inhibitors. PMID:16314468

Modelling the structure of a ceRNA-theoretical, bipartite microRNA-mRNA interaction network regulating intestinal epithelial cellular pathways using R programming.

PubMed

Robinson, J M; Henderson, W A

2018-01-12

We report a method using functional-molecular databases and network modelling to identify hypothetical mRNA-miRNA interaction networks regulating intestinal epithelial barrier function. The model forms a data-analysis component of our cell culture experiments, which produce RNA expression data from Nanostring Technologies nCounter ® system. The epithelial tight-junction (TJ) and actin cytoskeleton interact as molecular components of the intestinal epithelial barrier. Upstream regulation of TJ-cytoskeleton interaction is effected by the Rac/Rock/Rho signaling pathway and other associated pathways which may be activated or suppressed by extracellular signaling from growth factors, hormones, and immune receptors. Pathway activations affect epithelial homeostasis, contributing to degradation of the epithelial barrier associated with osmotic dysregulation, inflammation, and tumor development. The complexity underlying miRNA-mRNA interaction networks represents a roadblock for prediction and validation of competing-endogenous RNA network function. We developed a network model to identify hypothetical co-regulatory motifs in a miRNA-mRNA interaction network related to epithelial function. A mRNA-miRNA interaction list was generated using KEGG and miRWalk2.0 databases. R-code was developed to quantify and visualize inherent network structures. We identified a sub-network with a high number of shared, targeting miRNAs, of genes associated with cellular proliferation and cancer, including c-MYC and Cyclin D.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Penrose, Harrison; Heller, Sandra; Cable, Chloe

The proliferation of colon cancer cells is mediated in part by epidermal growth factor receptor (EGFR) signaling and requires sustained levels of cellular energy to meet its high metabolic needs. Intracellular lipid droplets (LDs) are a source of energy used for various cellular functions and they are elevated in density in human cancer, yet their regulation and function are not well understood. Here, in human colon cancer cells, EGF stimulates increases in LD density, which depends on EGFR expression and activation as well as the individual cellular capacity for lipid synthesis. Increases in LDs are blockaded by inhibition of PI3K/mTORmore » and PGE2 synthesis, supporting their dependency on select upstream pathways. In colon cancer cells, silencing of the FOXO3 transcription factor leads to down regulation of SIRT6, a negative regulator of lipid synthesis, and consequent increases in the LD coat protein PLIN2, revealing that increases in LDs depend on loss of FOXO3/SIRT6. Moreover, EGF stimulates loss of FOXO3/SIRT6, which is blockaded by the inhibition of upstream pathways as well as lipid synthesis, revealing existence of a negative regulatory loop between LDs and FOXO3/SIRT6. Elevated LDs are utilized by EGF treatment and their depletion through the inhibition of lipid synthesis or silencing of PLIN2 significantly attenuates proliferation. This novel mechanism of proliferative EGFR signaling leading to elevated LD density in colon cancer cells could potentially be therapeutically targeted for the treatment of tumor progression. - Highlights: • In colon cancer cells, EGFR activation leads to increases in LD density. • EGFR signaling includes PI3K/mTOR and PGE2 leading to lipid synthesis. • Increases in LDs are controlled by a negative regulatory loop with FOXO3/SIRT6. • EGFR mediated colon cancer cell proliferation depends on increased LD density.« less

JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships.

PubMed

Zeke, András; Misheva, Mariya; Reményi, Attila; Bogoyevitch, Marie A

2016-09-01

The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Silymarin and its active component silibinin act as novel therapeutic alternatives for salivary gland cancer by targeting the ERK1/2-Bim signaling cascade.

PubMed

Choi, Eun-Sun; Oh, Sejun; Jang, Boonsil; Yu, Hyun-Ju; Shin, Ji-Ae; Cho, Nam-Pyo; Yang, In-Hyoung; Won, Dong-Hoon; Kwon, Hye-Jeong; Hong, Seong Doo; Cho, Sung-Dae

2017-06-01

Approximately 20% of all salivary gland cancer patients who are treated with current treatment modalities will ultimately develop metastases. Its most common form, mucoepidermoid carcinoma (MEC) is a highly aggressive tumor with an overall 5-year survival rate of ~30%. Until now, several chemotherapeutic drugs have been tested for the treatment of salivary gland tumors, but the results have been disappointing and the drugs often cause unwanted side effects. Therefore, several recent studies have focused on the potential of alternative and/or complementary therapeutic options, including the use of silymarin. The effects of silymarin and its active component silibinin on salivary gland cancer-derived MC3 and HN22 cells and their underlying molecular mechanisms were examined using trypan blue exclusion, 4'-6-diamidino-2-phenylindole (DAPI) staining, Live/Dead, Annexin V/PI staining, mitochondrial membrane potential (ΔΨm) measurement, quantitative RT-PCR, soft agar colony formation and Western blotting analyses. We found that silymarin and silibinin dramatically increased the expression of the pro-apoptotic protein Bim in a concentration- and time-dependent manner and, concomitantly, induced apoptosis in MC3 and HN22 cells. We also found that ERK1/2 signaling inhibition successfully sensitized these cells to the apoptotic effects of silymarin and silibinin, which indicates that the ERK1/2 signaling pathway may act as an upstream regulator that modulates the silymarin/silibinin-induced Bim signaling pathway. Taken together, we conclude that ERK1/2 signaling pathway inhibition by silymarin and silibinin increases the expression of the pro-apoptotic Bcl-2 family member Bim which, subsequently, induces mitochondria-mediated apoptosis in salivary gland cancer-derived cells.

MAP4-regulated dynein-dependent trafficking of BTN3A1 controls the TBK1–IRF3 signaling axis

PubMed Central

Seo, Minji; Lee, Seong-Ok; Kim, Ji-Hoon; Hong, Yujin; Kim, Seongchan; Kim, Yeumin; Min, Dal-Hee; Kong, Young-Yun; Shin, Jinwook; Ahn, Kwangseog

2016-01-01

The innate immune system detects viral nucleic acids and induces type I interferon (IFN) responses. The RNA- and DNA-sensing pathways converge on the protein kinase TANK-binding kinase 1 (TBK1) and the transcription factor IFN-regulatory factor 3 (IRF3). Activation of the IFN signaling pathway is known to trigger the redistribution of key signaling molecules to punctate perinuclear structures, but the mediators of this spatiotemporal regulation have yet to be defined. Here we identify butyrophilin 3A1 (BTN3A1) as a positive regulator of nucleic acid-mediated type I IFN signaling. Depletion of BTN3A1 inhibits the cytoplasmic nucleic acid- or virus-triggered activation of IFN-β production. In the resting state, BTN3A1 is constitutively associated with TBK1. Stimulation with nucleic acids induces the redistribution of the BTN3A1–TBK1 complex to the perinuclear region, where BTN3A1 mediates the interaction between TBK1 and IRF3, leading to the phosphorylation of IRF3. Furthermore, we show that microtubule-associated protein 4 (MAP4) controls the dynein-dependent transport of BTN3A1 in response to nucleic acid stimulation, thereby identifying MAP4 as an upstream regulator of BTN3A1. Thus, the depletion of either MAP4 or BTN3A1 impairs cytosolic DNA- or RNA-mediated type I IFN responses. Our findings demonstrate a critical role for MAP4 and BTN3A1 in the spatiotemporal regulation of TBK1, a central player in the intracellular nucleic acid-sensing pathways involved in antiviral signaling. PMID:27911820

Thinking Upstream: A 25-Year Retrospective and Conceptual Model Aimed at Reducing Health Inequities.

PubMed

Butterfield, Patricia G

Thinking upstream was first introduced into the nursing vernacular in 1990 with the goal of advancing broad and context-rich perspectives of health. Initially invoked as conceptual framing language, upstream precepts were subsequently adopted and adapted by a generation of thoughtful nursing scholars. Their work reduced health inequities by redirecting actions further up etiologic pathways and by emphasizing economic, political, and environmental health determinants. US health care reform has fostered a much broader adoption of upstream language in policy documents. This article includes a semantic exploration of thinking upstream and a new model, the Butterfield Upstream Model for Population Health (BUMP Health).

NECA and bradykinin at reperfusion reduce infarction in rabbit hearts by signaling through PI3K, ERK, and NO.

PubMed

Yang, Xi-Ming; Krieg, Thomas; Cui, Lin; Downey, James M; Cohen, Michael V

2004-03-01

The adenosine A1/A2 adenosine agonist 5'-(N-ethylcarboxamido) adenosine (NECA) and bradykinin both limit infarction when administered at reperfusion in rabbits. This study compares the signal transduction pathways responsible for their anti-infarct effect. Receptor agonists were administered to isolated rabbit hearts starting 25 min after the onset of a 30-min period of ischemia and continued into the 2-h reperfusion period. Infarct size was measured. Both NECA and bradykinin decreased infarction from 31.5 +/- 2.4% of the risk zone in untreated hearts to 11.8 +/- 2.0% and 15.4 +/- 2.4%, respectively (P<0.05). Protection from both agents was blocked by PD98059, wortmannin, and Nomega-nitro-L-arginine methyl ester (L-NAME), thus demonstrating dependence on activation of extracellular regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K) and stimulation of nitric oxide synthase (NOS). Both wortmannin and PD98059 prevented phosphorylation of ERK 1/2 in NECA-treated hearts, whereas only wortmannin and not PD98059 blocked Akt phosphorylation. These data suggest Akt is upstream of ERK 1/2. In addition, 8-(3-chlorostyryl) caffeine blocked NECA's protection indicating that A2 adenosine receptors trigger NECA's anti-infarct effect. Of note, both bradykinin and acetylcholine (ACh) administered before ischemia to trigger preconditioning's cardioprotection use PI3K and NOS in their signaling pathway. Curiously, however, ACh, unlike bradykinin, was not protective when administered at reperfusion. Hence, both NECA and bradykinin administered at reperfusion protect through a common signaling pathway that includes PI3K, NO, and ERK.

A novel interaction between calcium-modulating cyclophilin ligand and Basigin regulates calcium signaling and matrix metalloproteinase activities in human melanoma cells.

PubMed

Long, Tingting; Su, Juan; Tang, Wen; Luo, Zhongling; Liu, Shuang; Liu, Zhaoqian; Zhou, Honghao; Qi, Min; Zeng, Weiqi; Zhang, Jianglin; Chen, Xiang

2013-10-01

Intracellular free calcium is a ubiquitous second messenger regulating a multitude of normal and pathogenic cellular responses, including the development of melanoma. Upstream signaling pathways regulating the intracellular free calcium concentration ([Ca2+]i) may therefore have a significant impact on melanoma growth and metastasis. In this study, we demonstrate that the endoplasmic reticulum (ER)-associated protein calcium-modulating cyclophilin ligand (CAML) is bound to Basigin, a widely expressed integral plasma membrane glycoprotein and extracellular matrix metalloproteinase inducer (EMMPRIN, or CD147) implicated in melanoma proliferation, invasiveness, and metastasis. This interaction between CAML and Basigin was first identified using yeast two-hybrid screening and further confirmed by co-immunoprecipitation. In human A375 melanoma cells, CAML and Basigin were co-localized to the ER. Knockdown of Basigin in melanoma cells by siRNA significantly decreased resting [Ca2+]i and the [Ca2+]i increase induced by the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin (TG), indicating that the interaction between CAML and Basigin regulates ER-dependent [Ca2+]i signaling. Meanwhile upregulating the [Ca2+]i either by TG or phorbol myristate acetate (PMA) could stimulate the production of MMP-9 in A375 cells with the expression of Basigin. Our study has revealed a previously uncharacterized [Ca2+]i signaling pathway that may control melanoma invasion, and metastasis. Disruption of this pathway may be a novel therapeutic strategy for melanoma treatment. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Development of a bioassay to screen for chemicals mimicking the anti-aging effects of calorie restriction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chiba, Takuya, E-mail: takuya@nagasaki-u.ac.jp; Tsuchiya, Tomoshi; Komatsu, Toshimitsu

2010-10-15

Research highlights: {yields} We identified four sequence motifs lying upstream of putative pro-longevity genes. {yields} One of these motifs binds to HNF-4{alpha}. {yields} HNF-4{alpha}/PGC-1{alpha} could up-regulate the transcription of a reporter gene linked to this motif. {yields} The reporter system described here could be used to screen candidate anti-aging molecules. -- Abstract: Suppression of the growth hormone/insulin-like growth factor-I pathway in Ames dwarf (DF) mice, and caloric restriction (CR) in normal mice extends lifespan and delays the onset of age-related disorders. In combination, these interventions have an additive effect on lifespan in Ames DF mice. Therefore, common signaling pathways regulatedmore » by DF and CR could have additive effects on longevity. In this study, we tried to identity the signaling mechanism and develop a system to assess pro-longevity status in cells and mice. We previously identified genes up-regulated in the liver of DF and CR mice by DNA microarray analysis. Motif analysis of the upstream sequences of those genes revealed four major consensus sequence motifs, which have been named dwarfism and calorie restriction-responsive elements (DFCR-REs). One of the synthesized sequences bound to hepatocyte nuclear factor-4{alpha} (HNF-4{alpha}), an important transcription factor involved in liver metabolism. Furthermore, using this sequence information, we developed a highly sensitive bioassay to identify chemicals mimicking the anti-aging effects of CR. When the reporter construct, containing an element upstream of a secreted alkaline phosphatase (SEAP) gene, was co-transfected with HNF-4{alpha} and its regulator peroxisome proliferator-activated receptor (PPAR) {gamma} coactivator-1{alpha} (PGC-1{alpha}), SEAP activity was increased compared with untransfected controls. Moreover, transient transgenic mice established using this construct showed increased SEAP activity in CR mice compared with ad libitum-fed mice. These data suggest that because of its rapidity, ease of use, and specificity, our bioassay will be more useful than the systems currently employed to screen for CR mimetics, which mimic the beneficial effects of CR. Our system will be particularly useful for high-throughput screening of natural and synthetic candidate molecules.« less

Bone Morphogenetic Protein 15 (BMP15) Acts as a BMP and Wnt Inhibitor during Early Embryogenesis*

PubMed Central

Di Pasquale, Elisa; Brivanlou, Ali H.

2009-01-01

Bone morphogenetic protein 15 (BMP15) belongs to an unusual subgroup of the transforming growth factor β (TGFβ) superfamily of signaling ligands as it lacks a key cysteine residue in the mature region required for proper intermolecular dimerization. Naturally occurring BMP15 mutation leads to early ovarian failure in humans, and BMP15 has been shown to activate the Smad1/5/8 pathway in that context. Despite its important role in germ cell specification, the embryological function of BMP15 remains unknown. Surprisingly, we find that during early Xenopus embryogenesis BMP15 acts solely as an inhibitor of the Smad1/5/8 pathway and the Wnt pathway. BMP15 gain-of-function leads to embryos with secondary ectopic heads and to direct neural induction in intact explants. BMP15 inhibits BMP4-mediated epidermal induction in dissociated explants. BMP15 strongly inhibits BRE response induced by BMP4 and blocks phosphorylation and activation of Smad1/5/8 MH2-domain. Mechanistically, BMP15 protein specifically interacts with BMP4 protein, suggesting inhibition upstream of receptor binding. Loss-of-function experiments using morpholinos or a naturally occurring human BMP15 dominant-negative mutant (BMP15-Y235C) leads to embryos lacking head. BMP15-Y235C also eliminates the inhibitory activity of BMP15 on BRE (BMP-responsive element). Finally, we show that BMP15 inhibits the canonical branch of the Wnt pathway, upstream of β-catenin. We, thus, demonstrate that BMP15 is necessary and sufficient for the specification of dorso-anterior structures and highlight novel mechanisms of BMP15 function that strongly suggest a reinterpretation of its function in ovaries specially for ovarian failure. PMID:19553676

cPLA2α Gene Activation by IL-1β is Dependent on an Upstream Kinase pathway, Enzymatic Activation and Downstream 15-lipoxygenase Activity: A Positive Feedback Loop

PubMed Central

Walters, Jewell N.; Bickford, Justin S.; Beachy, Dawn E.; Newsom, Kimberly J.; Herlihy, John-David H.; Peck, Molly V.; Qiu, Xiaolei; Nick, Harry S.

2011-01-01

Cytosolic phospholipase A2α (cPLA2α) is the most widely studied member of the Group IV PLA2 family. The enzyme is Ca2+-dependent with specificity for phospholipid substrates containing arachidonic acid. As the pinnacle of the arachidonic acid pathway, cPLA2α has a primary role in the biosynthesis of a diverse family of eicosanoid metabolites, with potent physiological, inflammatory and pathological consequences. cPLA2α activity is regulated by pro-inflammatory stimuli through pathways involving increased intracellular Ca2+ levels, phosphorylation coupled to increased enzymatic activity and de novo gene transcription. This study addresses the signal transduction pathways for protein phosphorylation and gene induction following IL-1β stimulation in human fetal lung fibroblasts. Our results utilizing both inhibitors and kinase-deficient cells demonstrate that cPLA2α is phosphorylated within 10 min of IL-1β treatment, an event requiring p38 MAPK as well as the upstream kinase, MKK3/MKK6. Inhibition of p38 MAPK also blocks the phosphorylation of a downstream, nuclear kinase, MSK-1. Our results further demonstrate that the activities of both cPLA2α and a downstream lipoxygenase (15-LOX2) are required for IL-1β-dependent induction of cPLA2α mRNA expression. Overall, these data support an MKK3/MKK6→p38 MAPK→MSK-1→cPLA2α→15-LOX2-dependent, positive feedback loop where a protein’s enzymatic activity is required to regulate its own gene induction by a pro-inflammatory stimulus. PMID:21771656

Emodin induces neurite outgrowth through PI3K/Akt/GSK-3β-mediated signaling pathways in Neuro2a cells.

PubMed

Park, Shin-Ji; Jin, Mei Ling; An, Hyun-Kyu; Kim, Kyoung-Sook; Ko, Min Jung; Kim, Cheol Min; Choi, Young Whan; Lee, Young-Choon

2015-02-19

In this study, a neurite outgrowth-inducing substance was isolated from the ethylacetate extract of the Polygonum multiflorum roots and identified as emodin by gas-liquid chromatography-mass spectrometry and (1)H NMR and (13)C NMR. Emodin displayed remarkable neurite outgrowth-inducing activity in Neuro2a cells, as demonstrated by morphological changes and immunocytochemistry for class III β-tubulin. Emodin exhibited a stronger neutrophic activity than retinoic acid (RA) known as inducer of neurite outgrowth in Neuro2a cells. Emodin treatment resulted in marked increases in phosphorylation of Akt a direct downstream signaling molecule of phosphatidylinositol 3-kinase (PI3K), but upstream of glycogen synthase kinase-3β (GSK-3β) and cAMP response element-binding protein (CREB). These augmentations and neurite-bearing cells induced by emodin were remarkably reduced by the addition of PI3K inhibitor LY294002. These results demonstrate that emodin induces neuronal differentiation of Neuro2a cells via PI3K/Akt/GSK-3β pathway. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

p38β, A novel regulatory target of Pokemon in hepatic cells.

PubMed

Chen, Zhe; Liu, Feng; Zhang, Nannan; Cao, Deliang; Liu, Min; Tan, Ying; Jiang, Yuyang

2013-06-27

Pokemon is an important proto-oncogene involved in various biological processes and cancer development, such as cell differentiation, tumorigenesis and metastasis. Pokemon is recognized as a transcription factor localized upstream of several oncogenes, regulating their expression. p38MAPKs act as key regulatory factors in cellular signaling pathways associated with inflammatory responses, cell proliferation, differentiation and survival. p38β, a member of p38MAPK family, is closely correlated with tumorigenesis, but the mechanism of activation remains unclear. In this study, we found overexpression of Pokemon promoted the growth, migration and invasion of HepG2 cells. However, a p38 inhibitor SB202190 efficiently attenuated the promoting effect of Pokemon in the HepG2 cells. Targeted expression or silencing of Pokemon changed cellular p38β protein level and phosphorylation of downstream ATF2 in the p38 signaling pathway. Both dual luciferase report assay and ChIP assay suggested that p38β is a novel regulatory target of the transcription factor Pokemon and positively regulated by Pokemon in hepatic cells.

p38β, A Novel Regulatory Target of Pokemon in Hepatic Cells

PubMed Central

Chen, Zhe; Liu, Feng; Zhang, Nannan; Cao, Deliang; Liu, Min; Tan, Ying; Jiang, Yuyang

2013-01-01

Pokemon is an important proto-oncogene involved in various biological processes and cancer development, such as cell differentiation, tumorigenesis and metastasis. Pokemon is recognized as a transcription factor localized upstream of several oncogenes, regulating their expression. p38MAPKs act as key regulatory factors in cellular signaling pathways associated with inflammatory responses, cell proliferation, differentiation and survival. p38β, a member of p38MAPK family, is closely correlated with tumorigenesis, but the mechanism of activation remains unclear. In this study, we found overexpression of Pokemon promoted the growth, migration and invasion of HepG2 cells. However, a p38 inhibitor SB202190 efficiently attenuated the promoting effect of Pokemon in the HepG2 cells. Targeted expression or silencing of Pokemon changed cellular p38β protein level and phosphorylation of downstream ATF2 in the p38 signaling pathway. Both dual luciferase report assay and ChIP assay suggested that p38β is a novel regulatory target of the transcription factor Pokemon and positively regulated by Pokemon in hepatic cells. PMID:23807508

The flavonoid fisetin as an anticancer agent targeting the growth signaling pathways.

PubMed

Rengarajan, Thamaraiselvan; Yaacob, Nik Soriani

2016-10-15

Epidemiological studies show that consumption of diets rich in fruits and vegetables is associated with lower risks of cancer. This evidence has kindled interest into research on bioactive food components and has till date resulted in the identification of many compounds with cancer preventive and therapeutic potential. Among such compounds is fisetin (3,7,3,4-tetrahydroxyflavone), a flavonol that is commonly found in many fruits and vegetables such as apples, persimmons, grapes, kiwis, strawberries, onions and cucumbers. Fisetin has been shown to inhibit or retard the growth of various cancer cells in culture and implanted tumors in vivo. Fisetin targets many components of intracellular signaling pathways including regulators of cell survival and apoptosis, tumor angiogenic and metastatic switches by modulating a distinct set of upstream kinases, transcription factors and their regulators. Current evidence supports the idea that fisetin is a promising agent for cancer treatment. This review summarizes reported anticancer effects of fisetin, and re-emphasizes its potential therapeutic role in the treatment of cancer. Copyright © 2016 Elsevier B.V. All rights reserved.

PGRP-SD, an Extracellular Pattern-Recognition Receptor, Enhances Peptidoglycan-Mediated Activation of the Drosophila Imd Pathway.

PubMed

Iatsenko, Igor; Kondo, Shu; Mengin-Lecreulx, Dominique; Lemaitre, Bruno

2016-11-15

Activation of the innate immune response in Metazoans is initiated through the recognition of microbes by host pattern-recognition receptors. In Drosophila, diaminopimelic acid (DAP)-containing peptidoglycan from Gram-negative bacteria is detected by the transmembrane receptor PGRP-LC and by the intracellular receptor PGRP-LE. Here, we show that PGRP-SD acted upstream of PGRP-LC as an extracellular receptor to enhance peptidoglycan-mediated activation of Imd signaling. Consistent with this, PGRP-SD mutants exhibited impaired activation of the Imd pathway and increased susceptibility to DAP-type bacteria. PGRP-SD enhanced the localization of peptidoglycans to the cell surface and hence promoted signaling. Moreover, PGRP-SD antagonized the action of PGRP-LB, an extracellular negative regulator, to fine-tune the intensity of the immune response. These data reveal that Drosophila PGRP-SD functions as an extracellular receptor similar to mammalian CD14 and demonstrate that, comparable to lipopolysaccharide sensing in mammals, Drosophila relies on both intra- and extracellular receptors for the detection of bacteria. Copyright © 2016 Elsevier Inc. All rights reserved.

Sex-specific processing of social cues in the medial amygdala

PubMed Central

Bergan, Joseph F; Ben-Shaul, Yoram; Dulac, Catherine

2014-01-01

Animal–animal recognition within, and across species, is essential for predator avoidance and social interactions. Despite its essential role in orchestrating responses to animal cues, basic principles of information processing by the vomeronasal system are still unknown. The medial amygdala (MeA) occupies a central position in the vomeronasal pathway, upstream of hypothalamic centers dedicated to defensive and social responses. We have characterized sensory responses in the mouse MeA and uncovered emergent properties that shed new light onto the transformation of vomeronasal information into sex- and species-specific responses. In particular, we show that the MeA displays a degree of stimulus selectivity and a striking sexually dimorphic sensory representation that are not observed in the upstream relay of the accessory olfactory bulb (AOB). Furthermore, our results demonstrate that the development of sexually dimorphic circuits in the MeA requires steroid signaling near the time of puberty to organize the functional representation of sensory stimuli. DOI: http://dx.doi.org/10.7554/eLife.02743.001 PMID:24894465

Analyzing gene perturbation screens with nested effects models in R and bioconductor.

PubMed

Fröhlich, Holger; Beissbarth, Tim; Tresch, Achim; Kostka, Dennis; Jacob, Juby; Spang, Rainer; Markowetz, F

2008-11-01

Nested effects models (NEMs) are a class of probabilistic models introduced to analyze the effects of gene perturbation screens visible in high-dimensional phenotypes like microarrays or cell morphology. NEMs reverse engineer upstream/downstream relations of cellular signaling cascades. NEMs take as input a set of candidate pathway genes and phenotypic profiles of perturbing these genes. NEMs return a pathway structure explaining the observed perturbation effects. Here, we describe the package nem, an open-source software to efficiently infer NEMs from data. Our software implements several search algorithms for model fitting and is applicable to a wide range of different data types and representations. The methods we present summarize the current state-of-the-art in NEMs. Our software is written in the R language and freely avail-able via the Bioconductor project at http://www.bioconductor.org.

MAP3K1 May be a Promising Susceptibility Gene for Type 2 Diabetes Mellitus in an Iranian Population

PubMed Central

Torkamandi, Shahram; Bastami, Milad; Ghaedi, Hamid; Moghadam, Fateme; Mirfakhraie, Reza; Omrani, Mir Davood

2016-01-01

Considering that MAPK (mitogen- activated protein kinase) signaling pathway has an important role in the progression of inflammatory cytokine secretion in type 2 diabetes mellitus (T2DM), we have recently investigated the reported genetic polymorphism from genome wide association study in MAP3K1 (mitogen-activated protein kinase kinase kinase 1) in diabetes as an important member of MAPK signaling. This study aimed to investigate the possible association of rs10461617 at the upstream of MAP3K1 gene in an Iranian case-control study with the risk of T2DM. The study population was comprised of 342 unrelated Iranian individuals including 177 patients with T2DM and 165 unrelated healthy control subjects. Genotyping was performed using PCR-RFLP and confirmed with sequencing. In a logistic regression analysis, the rs10461617A allele was associated with a significantly higher risk of T2DM assuming the log- additive model (OR: 1.44, 95% CI: 1.01-2.05, P = 0.039). In conclusion, we provided the first evidence for the association of rs10461617 at the upstream of MAP3K1 with the risk of T2DM in an Iranian population. PMID:27942499

RELM-β promotes human pulmonary artery smooth muscle cell proliferation via FAK-stimulated surviving

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Chunlong, E-mail: lclmd@sina.com; Li, Xiaohui; Luo, Qiong

Resistin-like molecule-β (RELM-β), focal adhesion kinase (FAK), and survivin may be involved in the proliferation of cultured human pulmonary artery smooth muscle cells (HPAMSCs), which is involved in pulmonary hypertension. HPAMSCs were treated with human recombinant RELM-β (rhRELM-β). siRNAs against FAK and survivin were transfected into cultured HPASMCs. Expression of FAK and survivin were examined by RT-PCR and western blot. Immunofluorescence was used to localize FAK. Flow cytometry was used to examine cell cycle distribution and cell death. Compared to the control group, all rhRELM-β-treated groups demonstrated significant increases in the expression of FAK and survivin (P<0.05). rhRELM-β significantly increasedmore » the proportion of HPASMCs in the S phase and decreased the proportion in G0/G1. FAK siRNA down-regulated survivin expression while survivin siRNA did not affect FAK expression. FAK siRNA effectively inhibited FAK and survivin expression in RELM-β-treated HPASMCs and partially suppressed cell proliferation. RELM-β promoted HPASMC proliferation and upregulated FAK and survivin expression. In conclusion, results suggested that FAK is upstream of survivin in the signaling pathway mediating cell proliferation. FAK seems to be important in RELM-β-induced HPASMC proliferation, partially by upregulating survivin expression. - Highlights: • rhRELM-β increased the expression of FAK and survivin. • rhRELM-β increased the proportion of HPASMCs in the S phase. • FAK is upstream of survivin in the signaling pathway mediating cell proliferation. • FAK is important in RELM-β-induced HPASMC proliferation, partly via survivin.« less

Physical Exercise Modulates L-DOPA-Regulated Molecular Pathways in the MPTP Mouse Model of Parkinson's Disease.

PubMed

Klemann, Cornelius J H M; Xicoy, Helena; Poelmans, Geert; Bloem, Bas R; Martens, Gerard J M; Visser, Jasper E

2018-07-01

Parkinson's disease (PD) is characterized by the degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc), resulting in motor and non-motor dysfunction. Physical exercise improves these symptoms in PD patients. To explore the molecular mechanisms underlying the beneficial effects of physical exercise, we exposed 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrimidine (MPTP)-treated mice to a four-week physical exercise regimen, and subsequently explored their motor performance and the transcriptome of multiple PD-linked brain areas. MPTP reduced the number of DA neurons in the SNpc, whereas physical exercise improved beam walking, rotarod performance, and motor behavior in the open field. Further, enrichment analyses of the RNA-sequencing data revealed that in the MPTP-treated mice physical exercise predominantly modulated signaling cascades that are regulated by the top upstream regulators L-DOPA, RICTOR, CREB1, or bicuculline/dalfampridine, associated with movement disorders, mitochondrial dysfunction, and epilepsy-related processes. To elucidate the molecular pathways underlying these cascades, we integrated the proteins encoded by the exercise-induced differentially expressed mRNAs for each of the upstream regulators into a molecular landscape, for multiple key brain areas. Most notable was the opposite effect of physical exercise compared to previously reported effects of L-DOPA on the expression of mRNAs in the SN and the ventromedial striatum that are involved in-among other processes-circadian rhythm and signaling involving DA, neuropeptides, and endocannabinoids. Altogether, our findings suggest that physical exercise can improve motor function in PD and may, at the same time, counteract L-DOPA-mediated molecular mechanisms. Further, we hypothesize that physical exercise has the potential to improve non-motor symptoms of PD, some of which may be the result of (chronic) L-DOPA use.

Transcriptional Profiling Identifies Functional Interactions of TGFβ and PPARβ/δ Signaling

PubMed Central

Kaddatz, Kerstin; Adhikary, Till; Finkernagel, Florian; Meissner, Wolfgang; Müller-Brüsselbach, Sabine; Müller, Rolf

2010-01-01

Peroxisome proliferator-activated receptors (PPARs) not only play a key role in regulating metabolic pathways but also modulate inflammatory processes, pointing to a functional interaction between PPAR and cytokine signaling pathways. In this study, we show by genome-wide transcriptional profiling that PPARβ/δ and transforming growth factor-β (TGFβ) pathways functionally interact in human myofibroblasts and that a subset of these genes is cooperatively activated by TGFβ and PPARβ/δ. Using the angiopoietin-like 4 (ANGPTL4) gene as a model, we demonstrate that two enhancer regions cooperate to mediate the observed synergistic response. A TGFβ-responsive enhancer located ∼8 kb upstream of the transcriptional start site is regulated by a mechanism involving SMAD3, ETS1, RUNX, and AP-1 transcription factors that interact with multiple contiguous binding sites. A second enhancer (PPAR-E) consisting of three juxtaposed PPAR response elements is located in the third intron ∼3.5 kb downstream of the transcriptional start site. The PPAR-E is strongly activated by all three PPAR subtypes, with a novel type of PPAR response element motif playing a central role. Although the PPAR-E is not regulated by TGFβ, it interacts with SMAD3, ETS1, RUNX2, and AP-1 in vivo, providing a possible mechanistic explanation for the observed synergism. PMID:20595396

DEG9, a serine protease, modulates cytokinin and light signaling by regulating the level of ARABIDOPSIS RESPONSE REGULATOR 4.

PubMed

Chi, Wei; Li, Jing; He, Baoye; Chai, Xin; Xu, Xiumei; Sun, Xuwu; Jiang, Jingjing; Feng, Peiqiang; Zuo, Jianru; Lin, Rongcheng; Rochaix, Jean-David; Zhang, Lixin

2016-06-21

Cytokinin is an essential phytohormone that controls various biological processes in plants. A number of response regulators are known to be important for cytokinin signal transduction. ARABIDOPSIS RESPONSE REGULATOR 4 (ARR4) mediates the cross-talk between light and cytokinin signaling through modulation of the activity of phytochrome B. However, the mechanism that regulates the activity and stability of ARR4 is unknown. Here we identify an ATP-independent serine protease, degradation of periplasmic proteins 9 (DEG9), which localizes to the nucleus and regulates the stability of ARR4. Biochemical evidence shows that DEG9 interacts with ARR4, thereby targeting ARR4 for degradation, which suggests that DEG9 regulates the stability of ARR4. Moreover, genetic evidence shows that DEG9 acts upstream of ARR4 and regulates the activity of ARR4 in cytokinin and light-signaling pathways. This study thus identifies a role for a ubiquitin-independent selective protein proteolysis in the regulation of the stability of plant signaling components.

Transforming Growth Factor-β Is an Upstream Regulator of Mammalian Target of Rapamycin Complex 2–Dependent Bladder Cancer Cell Migration and Invasion

PubMed Central

Gupta, Sounak; Hau, Andrew M.; Al-Ahmadie, Hikmat A.; Harwalkar, Jyoti; Shoskes, Aaron C.; Elson, Paul; Beach, Jordan R.; Hussey, George S.; Schiemann, William P.; Egelhoff, Thomas T.; Howe, Philip H.; Hansel, Donna E.

2017-01-01

Our prior work identified the mammalian target of rapamycin complex 2 (mTORC2) as a key regulator of bladder cancer cell migration and invasion, although upstream growth factor mediators of this pathway in bladder cancer have not been well delineated. We tested whether transforming growth factor (TGF)-β, which can function as a promotility factor in bladder cancer cells, could regulate mTORC2-dependent bladder cancer cell motility and invasion. In human bladder cancers, the highest levels of phosphorylated SMAD2, a TGF-β signaling intermediate, were present in high-grade invasive bladder cancers and associated with more frequent recurrence and decreased disease-specific survival. Increased expression of TGF-β isoforms, receptors, and signaling components was detected in invasive high-grade bladder cancer cells that expressed Vimentin and lacked E-cadherin. Application of TGF-β induced phosphorylation of the Ser473 residue of AKT, a selective target of mTORC2, in a SMAD2- and SMAD4-independent manner and increased bladder cancer cell migration in a modified scratch wound assay and invasion through Matrigel. Inhibition of TGF-β receptor I using SB431542 ablated TGF-β–induced migration and invasion. A similar effect was seen when Rictor, a key mTORC2 component, was selectively silenced. Our results suggest that TGF-β can induce bladder cancer cell invasion via mTORC2 signaling, which may be applicable in most bladder cancers. PMID:26988652

JNK signaling pathway regulates sorbitol-induced Tau proteolysis and apoptosis in SH-SY5Y cells by targeting caspase-3.

PubMed

Olivera Santa-Catalina, Marta; Caballero Bermejo, Montaña; Argent, Ricardo; Alonso, Juan C; Centeno, Francisco; Lorenzo, María J

2017-12-15

Growing evidence suggests that Diabetes Mellitus increases the risk of developing Alzheimer's disease. It is well known that hyperglycemia, a key feature of Diabetes Mellitus, may induce plasma osmolarity disturbances. Both hyperglycemia and hyperosmolarity promote the altered post-translational regulation of microtubule-associated protein Tau. Interestingly, abnormal hyperphosphorylation and cleavage of Tau have been proven to lead to the genesis of filamentous structures referred to as neurofibrillary tangles, the main pathological hallmark of Alzheimer's disease. We have previously described that hyperosmotic stress induced by sorbitol promotes Tau proteolysis and apoptosis in SH-SY5Y cells via caspase-3 activation. In order to gain insights into the regulatory mechanisms of such processes, in this work we explored the intracellular signaling pathways that regulate these events. We found that sorbitol treatment significantly enhanced the activation of conventional families of MAPK in SH-SY5Y cells. Tau proteolysis was completely prevented by JNK inhibition but not affected by either ERK1/2 or p38 MAPK blockade. Moreover, inhibition of JNK, but not ERK1/2 or p38 MAPK, efficiently prevented sorbitol-induced apoptosis and caspase-3 activation. In summary, we provide evidence that JNK signaling pathway is an upstream regulator of hyperosmotic stress-induced Tau cleavage and apoptosis in SH-SY5Y through the control of caspase-3 activation. Copyright © 2017 Elsevier Inc. All rights reserved.

Lycopene acts through inhibition of IκB kinase to suppress NF-κB signaling in human prostate and breast cancer cells.

PubMed

Assar, Emelia A; Vidalle, Magdalena Castellano; Chopra, Mridula; Hafizi, Sassan

2016-07-01

We studied the effect of the potent dietary antioxidant lycopene on multiple points along the nuclear factor kappa B (NF-κB) signaling pathway in prostate and breast cancer cells. Lycopene significantly inhibited prostate and breast cancer cell growth at physiologically relevant concentrations of ≥1.25 μM. Similar concentrations also caused a 30-40 % reduction in inhibitor of kappa B (IκB) phosphorylation in the cells, as determined by western blotting. Furthermore, the same degree of inhibition by lycopene was observed for NF-κB transcriptional activity, as determined by reporter gene assay. Concomitant with this, immunofluorescence staining of lycopene-treated cells showed a significant suppression (≥25 %) of TNF-induced NF-κB p65 subunit nuclear translocation. Further probing of lycopene's effects on upstream elements of the NF-κB pathway showed a 25 % inhibition of both activity of recombinant IκB kinase β (IKKβ) kinase in a cell-free in vitro assay, as well as activity of IKKβ immunoprecipitated from MDA-MB-231 cells treated with lycopene. In conclusion, the anticancer properties of lycopene may occur through inhibition of the NF-κB signaling pathway, beginning at the early stage of cytoplasmic IKK kinase activity, which then leads to reduced NF-κB-responsive gene regulation. Furthermore, these effects in cancer cells were observed at concentrations of lycopene that are relevant and achievable in vivo.

Palomid 529, a Novel Small-Molecule Drug, Is a TORC1/TORC2 Inhibitor That Reduces Tumor Growth, Tumor Angiogenesis, and Vascular Permeability

PubMed Central

Xue, Qi; Hopkins, Benjamin; Perruzzi, Carole; Udayakumar, Durga; Sherris, David; Benjamin, Laura E.

2009-01-01

It has become clear that the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is central for promoting both tumor and tumor stroma and is therefore a major target for anticancer drug development. First- and second-generation rapalogs (prototypical mTOR inhibitors) have shown promise but, due to the complex nature of mTOR signaling, can result in counterproductive feedback signaling to potentiate upstream Akt signaling. We present a novel PI3K/Akt/mTOR inhibitor, Palomid 529 (P529), which inhibits the TORC1 and TORC2 complexes and shows both inhibition of Akt signaling and mTOR signaling similarly in tumor and vasculature. We show that P529 inhibits tumor growth, angiogenesis, and vascular permeability. It retains the beneficial aspects of tumor vascular normalization that rapamycin boasts. However, P529 has the additional benefit of blocking pAktS473 signaling consistent with blocking TORC2 in all cells and thus bypassing feedback loops that lead to increased Akt signaling in some tumor cells. [Cancer Res 2008;68(22):9551–7] PMID:19010932

Mitogen-activated protein kinase kinase 5 (MKK5)-mediated signalling cascade regulates expression of iron superoxide dismutase gene in Arabidopsis under salinity stress

PubMed Central

Xing, Yu; Chen, Wei-hua; Jia, Wensuo; Zhang, Jianhua

2015-01-01

Superoxide dismutases (SODs) are involved in plant adaptive responses to biotic and abiotic stresses but the upstream signalling process that modulates their expression is not clear. Expression of two iron SODs, FSD2 and FSD3, was significantly increased in Arabidopsis in response to NaCl treatment but blocked in transgenic MKK5-RNAi plant, mkk5. Using an assay system for transient expression in protoplasts, it was found that mitogen-activated protein kinase kinase 5 (MKK5) was also activated in response to salt stress. Overexpression of MKK5 in wild-type plants enhanced their tolerance to salt treatments, while mkk5 mutant exhibited hypersensitivity to salt stress in germination on salt-containing media. Moreover, another kinase, MPK6, was also involved in the MKK5-mediated iron superoxide dismutase (FSD) signalling pathway in salt stress. The kinase activity of MPK6 was totally turned off in mkk5, whereas the activity of MPK3 was only partially blocked. MKK5 interacted with the MEKK1 protein that was also involved in the salt-induced FSD signalling pathway. These data suggest that salt-induced FSD2 and FSD3 expressions are influenced by MEKK1 via MKK5–MPK6-coupled signalling. This MAP kinase cascade (MEKK1, MKK5, and MPK6) mediates the salt-induced expression of iron superoxide dismutases. PMID:26136265

Intrinsic attenuation of post-irradiation calcium and ER stress imparts significant radioprotection to lepidopteran insect cells.

PubMed

Guleria, Ayushi; Thukral, Neha; Chandna, Sudhir

2018-04-15

Sf9 lepidopteran insect cells are 100-200 times more radioresistant than mammalian cells. This distinctive feature thus makes them suitable for studies exploring radioprotective molecular mechanisms. It has been established from previous studies of our group that downstream mitochondrial apoptotic signaling pathways in Sf9 cells are quite similar to mammalian cells, implicating the upstream signaling pathways in their extensive radioresistance. In the present study, intracellular and mitochondrial calcium levels remained unaltered in Sf9 cells in response to radiation, in sharp contrast to human (HEK293T) cells. The isolated mitochondria from Sf9 cells exhibited nearly 1.5 times greater calcium retention capacity than mammalian cells, highlighting their inherent stress resilience. Importantly, UPR/ER stress marker proteins (p-eIF2α, GRP4 and SERCA) remained unaltered by radiation and suggested highly attenuated ER and calcium stress. Lack of SERCA induction further corroborates the lack of radiation-induced calcium mobilization in these cells. The expression of CaMKII, an important effector molecule of calcium signaling, did not alter in response to radiation. Inhibiting CaMKII by KN-93 or suppressing CaM by siRNA failed to alter Sf9 cells response to radiation and suggests CaM-CaMKII independent radiation signaling. Therefore, this study suggests that attenuated calcium signaling/ER stress is an important determinant of lepidopteran cell radioresistance. Copyright © 2018 Elsevier Inc. All rights reserved.

Regulation of cardiac excitation and contraction by p21 activated kinase-1.

PubMed

Ke, Yunbo; Lei, Ming; Solaro, R John

2008-01-01

Cardiac excitation and contraction are regulated by a variety of signaling molecules. Central to the regulatory scheme are protein kinases and phosphatases that carry out reversible phosphorylation of different effectors. The process of beta-adrenergic stimulation mediated by cAMP dependent protein kinase (PKA) forms a well-known pathway considered as the most significant control mechanism in excitation and contraction as well as many other regulatory mechanisms in cardiac function. However, although dephosphorylation pathways are critical to these regulatory processes, signaling to phosphatases is relatively poorly understood. Emerging evidence indicates that regulation of phosphatases, which dampen the effect of beta-adrenergic stimulation, is also important. We review here functional studies of p21 activated kinase-1 (Pak1) and its potential role as an upstream signal for protein phosphatase PP2A in the heart. Pak1 is a serine/threonine protein kinase directly activated by the small GTPases Cdc42 and Rac1. Pak1 is highly expressed in different regions of the heart and modulates the activities of ion channels, sarcomeric proteins, and other phosphoproteins through up-regulation of PP2A activity. Coordination of Pak1 and PP2A activities is not only potentially involved in regulation of normal cardiac function, but is likely to be important in patho-physiological conditions.

A Catharanthus roseus BPF-1 homologue interacts with an elicitor-responsive region of the secondary metabolite biosynthetic gene Str and is induced by elicitor via a JA-independent signal transduction pathway.

PubMed

van der Fits, L; Zhang, H; Menke, F L; Deneka, M; Memelink, J

2000-11-01

Plants respond to pathogen attack by induction of various defence responses, including the biosynthesis of protective secondary metabolites. In Catharanthus roseus, the elicitor-induced expression of the terpenoid indole alkaloid biosynthetic gene Strictosidine synthase (Str) is mediated via the plant stress hormonejasmonate. In the promoters of several defence-related genes, cis-acting elements have been identified that are important for transcriptional regulation upon stress signals. Here we show that an upstream region in the Str promoter confers responsiveness to partially purified yeast elicitor and jasmonate. Yeast one-hybrid screening with this element as a bait identified a MYB-like protein, which shows high homology to parsley box P-binding factor-1 (PcBPF-1). In vitro analyses showed that the Str promoter fragment contained a novel binding site for BPF-1-like proteins with higher binding affinity than the previously described box P. CrBPF-1 mRNA accumulated rapidly in elicitor-treated C. roseus suspension cells, whereas no induction was observed with jasmonate. Inhibitor studies indicated that CrBPF-1 plays a role in an elicitor-responsive but jasmonate-independent signal transduction pathway, acting downstream of protein phosphorylation and calcium influx.

COT phosphorylates prolyl-isomerase Pin1 to promote tumorigenesis in breast cancer.

PubMed

Kim, Garam; Khanal, Prem; Kim, Jin Young; Yun, Hyo-Jeong; Lim, Sung-Chul; Shim, Jung-Hyun; Choi, Hong Seok

2015-06-01

Pin1, a conserved eukaryotic Peptidyl-prolyl cis/trans isomerase, has profound effects on numerous key-signaling molecules, and its deregulation contributes to disease, particularly cancer. Although Pin1-mediated prolyl isomerization is an essential and novel regulatory mechanism for protein phosphorylation, little is known about the upstream signaling pathway(s) that regulates Pin1 activity. Here, we identify MAP3K-related serine-threonine kinase (the gene encoding COT/Tpl2) as a kinase responsible for phosphorylation of Pin1 Ser16. COT interacts with and phosphorylates Pin1 on Ser16. Consequently, Pin1 Ser16 phosphorylation by COT increases cyclin D1 abundance and enhances tumorigenecity of MCF7 cells. In contrast, depletion of COT in MCF7 cells leads to downregulation of Pin1 Ser16 phosphorylation, which subsequently decrease cyclin D1 levels, inhibiting tumorigenecity of MCF7 cells. In a xenograft model, treatment of TKI, a COT inhibitor, and Juglone, a Pin1 inhibitor, abrogates tumor growth. In human breast cancer patients, immunohistochemical staining shows that Pin1 pSer16 levels are positively correlated with COT levels, providing strong evidence for an essential role of the COT/Pin1 axis in conveying oncogenic signals to promote aggressiveness in human breast cancer. © 2013 Wiley Periodicals, Inc.

5-Hydroxytryptamine1A receptor/Gibetagamma stimulates mitogen-activated protein kinase via NAD(P)H oxidase and reactive oxygen species upstream of src in chinese hamster ovary fibroblasts.

PubMed Central

Mukhin, Y V; Garnovskaya, M N; Collinsworth, G; Grewal, J S; Pendergrass, D; Nagai, T; Pinckney, S; Greene, E L; Raymond, J R

2000-01-01

The hypothesis of this work is that the 'serotonin' or 5-hydroxytryptamine (5-HT)(1A) receptor, which activates the extracellular signal-regulated kinase (ERK) through a G(i)betagamma-mediated pathway, does so through the intermediate actions of reactive oxygen species (ROS). Five criteria were shown to support a key role for ROS in the activation of ERK by the 5-HT(1A) receptor. (1) Antioxidants inhibit activation of ERK by 5-HT. (2) Application of cysteine-reactive oxidant molecules activates ERK. (3) The 5-HT(1A) receptor alters cellular redox properties, and generates both superoxide and hydrogen peroxide. (4) A specific ROS-producing enzyme [NAD(P)H oxidase] is involved in the activation of ERK. (5) There is specificity both in the effects of various chemical oxidizers, and in the putative location of the ROS in the ERK activation pathway. We propose that NAD(P)H oxidase is located in the ERK activation pathway stimulated by the transfected 5-HT(1A) receptor in Chinese hamster ovary (CHO) cells downstream of G(i)betagamma subunits and upstream of or at the level of the non-receptor tyrosine kinase, Src. Moreover, these experiments provide confirmation that the transfected human 5-HT(1A) receptor induces the production of ROS (superoxide and hydrogen peroxide) in CHO cells, and support the possibility that an NAD(P)H oxidase-like enzyme might be involved in the 5-HT-mediated generation of both superoxide and hydrogen peroxide. PMID:10727402

The Ste20 Family Kinases MAP4K4, MINK1, and TNIK Converge to Regulate Stress-Induced JNK Signaling in Neurons.

PubMed

Larhammar, Martin; Huntwork-Rodriguez, Sarah; Rudhard, York; Sengupta-Ghosh, Arundhati; Lewcock, Joseph W

2017-11-15

The c-Jun- N -terminal kinase (JNK) signaling pathway regulates nervous system development, axon regeneration, and neuronal degeneration after acute injury or in chronic neurodegenerative disease. Dual leucine zipper kinase (DLK) is required for stress-induced JNK signaling in neurons, yet the factors that initiate DLK/JNK pathway activity remain poorly defined. In the present study, we identify the Ste20 kinases MAP4K4, misshapen-like kinase 1 (MINK1 or MAP4K6) and TNIK Traf2- and Nck-interacting kinase (TNIK or MAP4K7), as upstream regulators of DLK/JNK signaling in neurons. Using a trophic factor withdrawal-based model of neurodegeneration in both male and female embryonic mouse dorsal root ganglion neurons, we show that MAP4K4, MINK1, and TNIK act redundantly to regulate DLK activation and downstream JNK-dependent phosphorylation of c-Jun in response to stress. Targeting MAP4K4, MINK1, and TNIK, but not any of these kinases individually, is sufficient to protect neurons potently from degeneration. Pharmacological inhibition of MAP4Ks blocks stabilization and phosphorylation of DLK within axons and subsequent retrograde translocation of the JNK signaling complex to the nucleus. These results position MAP4Ks as important regulators of the DLK/JNK signaling pathway. SIGNIFICANCE STATEMENT Neuronal degeneration occurs in disparate circumstances: during development to refine neuronal connections, after injury to clear damaged neurons, or pathologically during disease. The dual leucine zipper kinase (DLK)/c-Jun- N -terminal kinase (JNK) pathway represents a conserved regulator of neuronal injury signaling that drives both neurodegeneration and axon regeneration, yet little is known about the factors that initiate DLK activity. Here, we uncover a novel role for a subfamily of MAP4 kinases consisting of MAP4K4, Traf2- and Nck-interacting kinase (TNIK or MAP4K7), and misshapen-like kinase 1 (MINK1 or MAP4K6) in regulating DLK/JNK signaling in neurons. Inhibition of these MAP4Ks blocks stress-induced retrograde JNK signaling and protects from neurodegeneration, suggesting that these kinases may represent attractive therapeutic targets. Copyright © 2017 the authors 0270-6474/17/3711074-11$15.00/0.

The proteins encoded by the Drosophila Planar Polarity Effector genes inturned, fuzzy and fritz interact physically and can re-pattern the accumulation of “upstream” Planar Cell Polarity proteins

PubMed Central

Wang, Ying; Yan, Jie; Lee, Haeryun; Lu, Qiuheng; Adler, Paul N.

2014-01-01

The frizzled/starry night pathway regulates planar cell polarity in a wide variety of tissues in many types of animals. It was discovered and has been most intensively studied in the Drosophila wing where it controls the formation of the array of distally pointing hairs that cover the wing. The pathway does this by restricting the activation of the cytoskeleton to the distal edge of wing cells. This results in hairs initiating at the distal edge and growing in the distal direction. All of the proteins encoded by genes in the pathway accumulate asymmetrically in wing cells. The pathway is a hierarchy with the Planar Cell Polarity (PCP) genes (aka the core genes) functioning as a group upstream of the Planar Polarity Effector (PPE) genes which in turn function as a group upstream of multiple wing hairs. Upstream proteins, such as Frizzled accumulate on either the distal and/or proximal edges of wing cells. Downstream PPE proteins accumulate on the proximal edge under the instruction of the upstream proteins. A variety of types of data support this hierarchy, however, we have found that when over expressed the PPE proteins can alter both the subcellular location and level of accumulation of the upstream proteins. Thus, the epistatic relationship is context dependent. We further show that the PPE proteins interact physically and can modulate the accumulation of each other in wing cells. We also find that over expression of Frtz results in a marked delay in hair initiation suggesting that it has a separate role/activity in regulating the cytoskeleton that is not shared by other members of the group. PMID:25072625

HNF4α is a therapeutic target that links AMPK to WNT signalling in early-stage gastric cancer

PubMed Central

Chang, Hae Ryung; Nam, Seungyoon; Kook, Myeong-Cherl; Kim, Kyung-Tae; Liu, Xiuping; Yao, Hui; Jung, Hae Rim; Lemos, Robert; Seo, Hye Hyun; Park, Hee Seo; Gim, Youme; Hong, Dongwan; Huh, Iksoo; Kim, Young-Woo; Tan, Dongfeng; Liu, Chang-Gong; Powis, Garth; Park, Taesung; Liang, Han; Kim, Yon Hui

2016-01-01

Background Worldwide, gastric cancer (GC) is the fourth most common malignancy and the most common cancer in East Asia. Development of targeted therapies for this disease has focused on a few known oncogenes but has had limited effects. Objective To determine oncogenic mechanisms and novel therapeutic targets specific for GC by identifying commonly dysregulated genes from the tumours of both Asian-Pacific and Caucasian patients. Methods We generated transcriptomic profiles of 22 Caucasian GC tumours and their matched non-cancerous samples and performed an integrative analysis across different GC gene expression datasets. We examined the inhibition of commonly overexpressed oncogenes and their constituent signalling pathways by RNAi and/or pharmacological inhibition. Results Hepatocyte nuclear factor-4α (HNF4α) upregulation was a key signalling event in gastric tumours from both Caucasian and Asian patients, and HNF4α antagonism was antineoplastic. Perturbation experiments in GC tumour cell lines and xenograft models further demonstrated that HNF4α is downregulated by AMPKα signalling and the AMPK agonist metformin; blockade of HNF4α activity resulted in cyclin downregulation, cell cycle arrest and tumour growth inhibition. HNF4α also regulated WNT signalling through its target gene WNT5A, a potential prognostic marker of diffuse type gastric tumours. Conclusions Our results indicate that HNF4α is a targetable oncoprotein in GC, is regulated by AMPK signalling through AMPKα and resides upstream of WNT signalling. HNF4α may regulate ‘metabolic switch’ characteristic of a general malignant phenotype and its target WNT5A has potential prognostic values. The AMPKα-HNF4α-WNT5A signalling cascade represents a potentially targetable pathway for drug development. PMID:25410163

A New Strategy to Control and Eradicate "Undruggable" Oncogenic K-RAS-Driven Pancreatic Cancer: Molecular Insights and Core Principles Learned from Developmental and Evolutionary Biology.

PubMed

Van Sciver, Robert E; Lee, Michael P; Lee, Caroline Dasom; Lafever, Alex C; Svyatova, Elizaveta; Kanda, Kevin; Colliver, Amber L; Siewertsz van Reesema, Lauren L; Tang-Tan, Angela M; Zheleva, Vasilena; Bwayi, Monicah N; Bian, Minglei; Schmidt, Rebecca L; Matrisian, Lynn M; Petersen, Gloria M; Tang, Amy H

2018-05-14

Oncogenic K-RAS mutations are found in virtually all pancreatic cancers, making K-RAS one of the most targeted oncoproteins for drug development in cancer therapies. Despite intense research efforts over the past three decades, oncogenic K-RAS has remained largely "undruggable". Rather than targeting an upstream component of the RAS signaling pathway (i.e., EGFR/HER2) and/or the midstream effector kinases (i.e., RAF/MEK/ERK/PI3K/mTOR), we propose an alternative strategy to control oncogenic K-RAS signal by targeting its most downstream signaling module, Seven-In-Absentia Homolog (SIAH). SIAH E3 ligase controls the signal output of oncogenic K-RAS hyperactivation that drives unchecked cell proliferation, uncontrolled tumor growth, and rapid cancer cell dissemination in human pancreatic cancer. Therefore, SIAH is an ideal therapeutic target as it is an extraordinarily conserved downstream signaling gatekeeper indispensable for proper RAS signaling. Guided by molecular insights and core principles obtained from developmental and evolutionary biology, we propose an anti-SIAH-centered anti-K-RAS strategy as a logical and alternative anticancer strategy to dampen uncontrolled K-RAS hyperactivation and halt tumor growth and metastasis in pancreatic cancer. The clinical utility of developing SIAH as both a tumor-specific and therapy-responsive biomarker, as well as a viable anti-K-RAS drug target, is logically simple and conceptually innovative. SIAH clearly constitutes a major tumor vulnerability and K-RAS signaling bottleneck in pancreatic ductal adenocarcinoma (PDAC). Given the high degree of evolutionary conservation in the K-RAS/SIAH signaling pathway, an anti-SIAH-based anti-PDAC therapy will synergize with covalent K-RAS inhibitors and direct K-RAS targeted initiatives to control and eradicate pancreatic cancer in the future.

Expression patterns of members of the ethylene signaling-related gene families in response to dehydration stresses in cassava.

PubMed

Ren, Meng Yun; Feng, Ren Jun; Shi, Hou Rui; Lu, Li Fang; Yun, Tian Yan; Peng, Ming; Guan, Xiao; Zhang, Heng; Wang, Jing Yi; Zhang, Xi Yan; Li, Cheng Liang; Chen, Yan Jun; He, Peng; Zhang, Yin Dong; Xie, Jiang Hui

2017-01-01

Drought is the one of the most important environment stresses that restricts crop yield worldwide. Cassava (Manihot esculenta Crantz) is an important food and energy crop that has many desirable traits such as drought, heat and low nutrients tolerance. However, the mechanisms underlying drought tolerance in cassava are unclear. Ethylene signaling pathway, from the upstream receptors to the downstream transcription factors, plays important roles in environmental stress responses during plant growth and development. In this study, we used bioinformatics approaches to identify and characterize candidate Manihot esculenta ethylene receptor genes and transcription factor genes. Using computational methods, we localized these genes on cassava chromosomes, constructed phylogenetic trees and identified stress-responsive cis-elements within their 5' upstream regions. Additionally, we measured the trehalose and proline contents in cassava fresh leaves after drought, osmotic, and salt stress treatments, and then it was found that the regulation patterns of contents of proline and trehalose in response to various dehydration stresses were differential, or even the opposite, which shows that plant may take different coping strategies to deal with different stresses, when stresses come. Furthermore, expression profiles of these genes in different organs and tissues under non-stress and abiotic stress were investigated through quantitative real-time PCR (qRT-PCR) analyses in cassava. Expression profiles exhibited clear differences among different tissues under non-stress and various dehydration stress conditions. We found that the leaf and tuberous root tissues had the greatest and least responses, respectively, to drought stress through the ethylene signaling pathway in cassava. Moreover, tuber and root tissues had the greatest and least reponses to osmotic and salt stresses through ethylene signaling in cassava, respectively. These results show that these plant tissues had differential expression levels of genes involved in ethylene signaling in response to the stresses tested. Moreover, after several gene duplication events, the spatiotemporally differential expression pattern of homologous genes in response to abiotic and biotic stresses may imply their functional diversity as a mechanism for adapting to the environment. Our data provide a framework for further research on the molecular mechanisms of cassava resistance to drought stress and provide a foundation for breeding drought-resistant new cultivars.

Emerging role of Hippo signalling in pancreatic biology: YAP re-expression and plausible link to islet cell apoptosis and replication.

PubMed

Sharma, Anjana; Yerra, Veera Ganesh; Kumar, Ashutosh

2017-02-01

Diabetes mellitus is an ailment that develops when the functional capacity of the pancreas does not meet the metabolic requirements of the whole body, either due to insulin insufficiency or resistance to insulin action. Current therapies that control glycaemia are limited by their unwanted effects or their inability to prevent the development of long-term complications. Regeneration and replacement of beta cell therapies are shaping the goals of future management of diabetes. The Hippo pathway, first discovered in Drosophila melanogaster, plays a vital role in controlling the organ size. Nuclear recruitment of YAP/TAZ (Yes-associated protein/transcriptional co-activator with PDZ-binding motif), a mammalian analogue of Yorkie protein found in Drosophila, activates cell proliferation and inhibits apoptosis. YAP was found to regulate early pancreatic development followed by downregulation during Ngn3-specific endocrine lineage maturation corresponding to their mitotic quiescence. Recent evidences have shown that optimum modulation of upstream kinases in the Hippo signalling pathway may lead to apoptosis inhibition and renewal of progenitor as well as stem cells in case of tissue or cell injury. This article reviews the evidences linking the role of various components of the Hippo pathway to pancreatic regeneration. In particular, the focus is on the beneficial role of induced YAP expression and its nuclear distribution on apoptosis and replication of adult pancreatic β islets. This approach may be of immense significance towards our fight against diabetes; thus, more insightful research is warranted in the area of Hippo signalling pathway and its involvement in pancreatic regeneration. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Caged mudsnail Potamopyrgus antipodarum (Gray) as an integrated field biomonitoring tool: exposure assessment and reprotoxic effects of water column contamination.

PubMed

Gust, M; Gagné, F; Berlioz-Barbier, A; Besse, J P; Buronfosse, T; Tournier, M; Tutundjian, R; Garric, J; Cren-Olivé, C

2014-05-01

This study highlights the usefulness of gastropods for water quality monitoring. Gastropods were caged upstream and downstream of an effluent discharge. Exposure was assessed by measurement of organic contaminants in water. Contamination of the Potamopyrgus antipodarum mudsnail was also measured using innovative techniques at the end of the 42 days of exposure. Biological effects were measured at the individual level (growth, reproduction) and subindividual level (energy reserves, vitellin-like proteins, steroid levels, expression of genes involved in estrogen signaling pathways), thus providing a better understanding of reprotoxic effects. The effluent was mainly contaminated by pharmaceutical compounds, as was the mudsnail. The highest concentrations were measured for oxazepam and were higher than 2 mg/kg downstream of the effluent discharge. Alkylphenols, bisphenol A, and vertebrate-like sex-steroid hormones were also bioaccumulated by the mudsnail downstream of the effluent. The combined use of water and snail contamination provided a complete exposure assessment. Exposure was further linked to biological effects. The mudsnail was shown to be a better adapted species for in situ exposures than Valvata piscinalis. Reproduction was sharply decreased after 6 weeks of exposure in the mudsnail. Feeding issues were excluded, confirming the toxic origin. These effects were related to estrogen signaling pathways using genomic analysis. Genes coding for proteins involved in nongenomic signaling pathways were inhibited, and those of genomic pathway repressors were induced. These results suggest that the chemical contamination due to the effluent discharge altered steroid control of reproduction and blocked the transition between oocyte and unshelled embryo, resulting in a drastic decrease of embryo production, while survival was not affected. Copyright © 2014 Elsevier Ltd. All rights reserved.

Anti-inflammatory effects of fimasartan via Akt, ERK, and NFκB pathways on astrocytes stimulated by hemolysate.

PubMed

Yang, Xiu-Li; Kim, Chi Kyung; Kim, Tae Jung; Sun, Jing; Rim, Doeun; Kim, Young-Ju; Ko, Sang-Bae; Jang, Hyunduk; Yoon, Byung-Woo

2016-02-01

The aim of this study was to investigate whether fimasartan, a novel angiotensin II receptor blocker, modulates hemolysate-induced inflammation in astrocytes. We stimulated astrocytes with hemolysate to induce hemorrhagic inflammation in vitro. Astrocytes were pretreated with fimasartan and then incubated with hemolysate at different durations. Anti-inflammatory cell signaling molecules including Akt, extracellular signal regulated kinase (ERK), NFκB and cyclooxygenase-2 (COX-2) were assessed by western blotting. Pro-inflammatory mediators were evaluated by real-time RT-PCR and ELISA. The stimulation by hemolysate generated a robust activation of inflammatory signaling pathways in astrocytes. Hemolysate increased the phosphorylation of Akt at 1 h, and ERK1/2 at 20 min compared with the control group and promoted the degradation of IκBα. Pretreated fimasartan significantly decreased hemolysate-induced phosphorylation of Akt and ERK1/2. In addition, fimasartan also suppressed NFκB-related inflammatory pathways induced by hemolysate, including reduction of the gene expression of NFκB, and decreased nuclear translocation of NFκB and degradation of IκB. This reduction of inflammatory upstream pathways decreased the expression of inflammatory end-products: COX-2 and interleukin-1 (IL-1β). Furthermore, the expression of COX-2 was attenuated by both Akt inhibitor (LY294002) and ERK inhibitor (U0126), and IκBα degradation was suppressed by LY294002. These results demonstrate that pretreatment with fimasartan to astrocytes suppresses the inflammatory responses induced by hemolysate. Akt, ERK and NFκB were associated with hemolysate-induced COX-2 and IL-1β expression. Based on these mechanisms, fimasartan could be a candidate anti-inflammatory regulator for the treatment of intracerebral hemorrhage.

The MAPKK FgMkk1 of Fusarium graminearum regulates vegetative differentiation, multiple stress response, and virulence via the cell wall integrity and high-osmolarity glycerol signaling pathways.

PubMed

Yun, Yingzi; Liu, Zunyong; Zhang, Jingze; Shim, Won-Bo; Chen, Yun; Ma, Zhonghua

2014-07-01

Mitogen-activated protein (MAP) kinases play crucial roles in regulating fungal development, growth and pathogenicity, and in responses to the environment. In this study, we characterized a MAP kinase kinase FgMkk1 in Fusarium graminearum, the causal agent of wheat head blight. Phenotypic analyses of the FgMKK1 mutant (ΔFgMKK1) showed that FgMkk1 is involved in the regulation of hyphal growth, pigmentation, conidiation, deoxynivalenol biosynthesis and virulence of F. graminearum. ΔFgMKK1 also showed increased sensitivity to cell wall-damaging agents, and to osmotic and oxidative stresses, but exhibited decreased sensitivity to the fungicides iprodione and fludioxonil. In addition, the mutant revealed increased sensitivity to a biocontrol agent, Trichoderma atroviride. Western blot assays revealed that FgMkk1 positively regulates phosphorylation of the MAP kinases Mgv1 and FgOs-2, the key component in the cell wall integrity (CWI) and high-osmolarity glycerol (HOG) signalling pathway respectively. Yeast two-hybrid assay indicated that Mgv1 interacts with a transcription factor FgRlm1. The FgRLM1 mutant (ΔFgRLM1) showed increased sensitivity to cell wall-damaging agents and exhibited decreased virulence. Taken together, our data indicated that FgMkk1 is an upstream component of Mgv1, and regulates vegetative differentiation, multiple stress response and virulence via the CWI and HOG signalling pathways. FgRlm1 may be a downstream component of Mgv1 in the CWI pathway in F. graminearum. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Integrative Computational Network Analysis Reveals Site-Specific Mediators of Inflammation in Alzheimer's Disease

PubMed Central

Ravichandran, Srikanth; Michelucci, Alessandro; del Sol, Antonio

2018-01-01

Alzheimer's disease (AD) is a major neurodegenerative disease and is one of the most common cause of dementia in older adults. Among several factors, neuroinflammation is known to play a critical role in the pathogenesis of chronic neurodegenerative diseases. In particular, studies of brains affected by AD show a clear involvement of several inflammatory pathways. Furthermore, depending on the brain regions affected by the disease, the nature and the effect of inflammation can vary. Here, in order to shed more light on distinct and common features of inflammation in different brain regions affected by AD, we employed a computational approach to analyze gene expression data of six site-specific neuronal populations from AD patients. Our network based computational approach is driven by the concept that a sustained inflammatory environment could result in neurotoxicity leading to the disease. Thus, our method aims to infer intracellular signaling pathways/networks that are likely to be constantly activated or inhibited due to persistent inflammatory conditions. The computational analysis identified several inflammatory mediators, such as tumor necrosis factor alpha (TNF-a)-associated pathway, as key upstream receptors/ligands that are likely to transmit sustained inflammatory signals. Further, the analysis revealed that several inflammatory mediators were mainly region specific with few commonalities across different brain regions. Taken together, our results show that our integrative approach aids identification of inflammation-related signaling pathways that could be responsible for the onset or the progression of AD and can be applied to study other neurodegenerative diseases. Furthermore, such computational approaches can enable the translation of clinical omics data toward the development of novel therapeutic strategies for neurodegenerative diseases. PMID:29551980

Integrative Computational Network Analysis Reveals Site-Specific Mediators of Inflammation in Alzheimer's Disease.

PubMed

Ravichandran, Srikanth; Michelucci, Alessandro; Del Sol, Antonio

2018-01-01

Alzheimer's disease (AD) is a major neurodegenerative disease and is one of the most common cause of dementia in older adults. Among several factors, neuroinflammation is known to play a critical role in the pathogenesis of chronic neurodegenerative diseases. In particular, studies of brains affected by AD show a clear involvement of several inflammatory pathways. Furthermore, depending on the brain regions affected by the disease, the nature and the effect of inflammation can vary. Here, in order to shed more light on distinct and common features of inflammation in different brain regions affected by AD, we employed a computational approach to analyze gene expression data of six site-specific neuronal populations from AD patients. Our network based computational approach is driven by the concept that a sustained inflammatory environment could result in neurotoxicity leading to the disease. Thus, our method aims to infer intracellular signaling pathways/networks that are likely to be constantly activated or inhibited due to persistent inflammatory conditions. The computational analysis identified several inflammatory mediators, such as tumor necrosis factor alpha (TNF-a)-associated pathway, as key upstream receptors/ligands that are likely to transmit sustained inflammatory signals. Further, the analysis revealed that several inflammatory mediators were mainly region specific with few commonalities across different brain regions. Taken together, our results show that our integrative approach aids identification of inflammation-related signaling pathways that could be responsible for the onset or the progression of AD and can be applied to study other neurodegenerative diseases. Furthermore, such computational approaches can enable the translation of clinical omics data toward the development of novel therapeutic strategies for neurodegenerative diseases.

A novel compound DT-010 protects against doxorubicin-induced cardiotoxicity in zebrafish and H9c2 cells by inhibiting reactive oxygen species-mediated apoptotic and autophagic pathways.

PubMed

Tang, Fan; Zhou, Xinhua; Wang, Liang; Shan, Luchen; Li, Chuwen; Zhou, Hefeng; Lee, Simon Ming-Yuen; Hoi, Maggie Pui-Man

2018-02-05

Doxorubicin (Dox) is an effective anti-cancer agent but limited by its cardiotoxicity, thus the search for pharmacological agents for enhancing anti-cancer activities and protecting against cardiotoxicity has been a subject of great interest. We have previously reported the synergistic anti-cancer effects of a novel compound DT-010. In the present study, we further investigated the cardioprotective effects of DT-010 in zebrafish embryos in vivo and the molecular underlying mechanisms in H9c2 cardiomyocytes in vitro. We showed that DT-010 prevented the Dox-induced morphological distortions in the zebrafish heart and the associated cardiac impairments, and especially improved ventricular functions. By using H9c2 cells model, we showed that DT-010 directly inhibited the generation of reactive oxygen species by Dox and protected cell death and cellular damage. We further observed that DT-010 protected against Dox-induced myocardiopathy via inhibiting downstream molecular pathways in response to oxidative stress, including reactive oxygen species-mediated MAPK signaling pathways ERK and JNK, and apoptotic pathways involving the activation of caspase 3, caspase 7, and PARP signaling. Recent studies also suggest the importance of alterations in cardiac autophagy in Dox cardiotoxicity. We further showed that DT-010 could inhibit the induction of autophagosomes formation by Dox via regulating the upstream Akt/AMPK/mTOR signaling. Since Dox-induced cardiotoxicity is multifactorial, our results suggest that multi-functional agent such as DT-010 might be an effective therapeutic agent for combating cardiotoxicity associated with chemotherapeutic agents such as Dox. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of dFOXO binding sites upstream of the Insulin Receptor P2 promoter across the Drosophila phylogeny

PubMed Central

Orengo, Dorcas J.; Aguadé, Montserrat

2017-01-01

The insulin/TOR signal transduction pathway plays a critical role in determining such important traits as body and organ size, metabolic homeostasis and life span. Although this pathway is highly conserved across the animal kingdom, the affected traits can exhibit important differences even between closely related species. Evolutionary studies of regulatory regions require the reliable identification of transcription factor binding sites. Here we have focused on the Insulin Receptor (InR) expression from its P2 promoter in the Drosophila genus, which in D. melanogaster is up-regulated by hypophosphorylated Drosophila FOXO (dFOXO). We have finely characterized this transcription factor binding sites in vitro along the 1.3 kb region upstream of the InR P2 promoter in five Drosophila species. Moreover, we have tested the effect of mutations in the characterized dFOXO sites of D. melanogaster in transgenic flies. The number of experimentally established binding sites varies across the 1.3 kb region of any particular species, and their distribution also differs among species. In D. melanogaster, InR expression from P2 is differentially affected by dFOXO binding sites at the proximal and distal halves of the species 1.3 kb fragment. The observed uneven distribution of binding sites across this fragment might underlie their differential contribution to regulate InR transcription. PMID:29200426

Dynein-mediated trafficking negatively regulates LET-23 EGFR signaling

PubMed Central

Skorobogata, Olga; Meng, Jassy; Gauthier, Kimberley; Rocheleau, Christian E.

2016-01-01

Epidermal growth factor receptor (EGFR) signaling is essential for animal development, and increased signaling underlies many human cancers. Identifying the genes and cellular processes that regulate EGFR signaling in vivo will help to elucidate how this pathway can become inappropriately activated. Caenorhabditis elegans vulva development provides an in vivo model to genetically dissect EGFR signaling. Here we identified a mutation in dhc-1, the heavy chain of the cytoplasmic dynein minus end–directed microtubule motor, in a genetic screen for regulators of EGFR signaling. Despite the many cellular functions of dynein, DHC-1 is a strong negative regulator of EGFR signaling during vulva induction. DHC-1 is required in the signal-receiving cell and genetically functions upstream or in parallel to LET-23 EGFR. LET-23 EGFR accumulates in cytoplasmic foci in dhc-1 mutants, consistent with mammalian cell studies in which dynein is shown to regulate late endosome trafficking of EGFR with the Rab7 GTPase. However, we found different distributions of LET-23 EGFR foci in rab-7 versus dhc-1 mutants, suggesting that dynein functions at an earlier step of LET-23 EGFR trafficking to the lysosome than RAB-7. Our results demonstrate an in vivo role for dynein in limiting LET-23 EGFR signaling via endosomal trafficking. PMID:27654944

Brazilian Red Propolis Attenuates Inflammatory Signaling Cascade in LPS-Activated Macrophages

PubMed Central

Bueno-Silva, Bruno; Kawamoto, Dione; Ando-Suguimoto, Ellen S.; Alencar, Severino M.; Rosalen, Pedro L.; Mayer, Marcia P. A.

2015-01-01

Although previous studies suggested an anti-inflammatory property of Brazilian red propolis (BRP), the mechanisms involved in the anti-inflammatory effects of BRP and its activity on macrophages were still not elucidated. This study aimed to evaluate whether BRP attenuates the inflammatory effect of LPS on macrophages and to investigate its underlying mechanisms. BRP was added to RAW 264.7 murine macrophages after activation with LPS. NO production, cell viability, cytokines profile were evaluated. Activation of inflammatory signaling pathways and macrophage polarization were determined by RT-qPCR and Western blot. BRP at 50 μg/ml inhibited NO production by 78% without affecting cell viability. Cd80 and Cd86 were upregulated whereas mrc1 was down regulated by BRP indicating macrophage polarization at M1. BRP attenuated the production of pro-inflammatory mediators IL-12, GM-CSF, IFN-Ɣ, IL-1β in cell supernatants although levels of TNF- α and IL-6 were slightly increased after BRP treatment. Levels of IL-4, IL-10 and TGF-β were also reduced by BRP. BRP significantly reduced the up-regulation promoted by LPS of transcription of genes in inflammatory signaling (Pdk1, Pak1, Nfkb1, Mtcp1, Gsk3b, Fos and Elk1) and of Il1β and Il1f9 (fold-change rate > 5), which were further confirmed by the inhibition of NF-κB and MAPK signaling pathways. Furthermore, the upstream adaptor MyD88 adaptor-like (Mal), also known as TIRAP, involved in TLR2 and TLR4 signaling, was down- regulated in BRP treated LPS-activated macrophages. Given that BRP inhibited multiple signaling pathways in macrophages involved in the inflammatory process activated by LPS, our data indicated that BRP is a noteworthy food-source for the discovery of new bioactive compounds and a potential candidate to attenuate exhacerbated inflammatory diseases. PMID:26660901

Hole-in-One Mutant Phenotypes Link EGFR/ERK Signaling to Epithelial Tissue Repair in Drosophila

PubMed Central

Campos, Isabel; Santos, Ana Catarina; Jacinto, Antonio

2011-01-01

Background Epithelia act as physical barriers protecting living organisms and their organs from the surrounding environment. Simple epithelial tissues have the capacity to efficiently repair wounds through a resealing mechanism. The known molecular mechanisms underlying this process appear to be conserved in both vertebrates and invertebrates, namely the involvement of the transcription factors Grainy head (Grh) and Fos. In Drosophila, Grh and Fos lead to the activation of wound response genes required for epithelial repair. ERK is upstream of this pathway and known to be one of the first kinases to be activated upon wounding. However, it is still unclear how ERK activation contributes to a proper wound response and which molecular mechanisms regulate its activation. Methodology/Principal Findings In a previous screen, we isolated mutants with defects in wound healing. Here, we describe the role of one of these genes, hole-in-one (holn1), in the wound healing process. Holn1 is a GYF domain containing protein that we found to be required for the activation of several Grh and Fos regulated wound response genes at the wound site. We also provide evidence suggesting that Holn1 may be involved in the Ras/ERK signaling pathway, by acting downstream of ERK. Finally, we show that wound healing requires the function of EGFR and ERK signaling. Conclusions/Significance Based on these data, we conclude that holn1 is a novel gene required for a proper wound healing response. We further propose and discuss a model whereby Holn1 acts downstream of EGFR and ERK signaling in the Grh/Fos mediated wound closure pathway. PMID:22140578

BAD: undertaker by night, candyman by day.

PubMed

Danial, N N

2008-12-01

The BH3-only pro-apoptotic proteins are upstream sensors of cellular damage that selectively respond to specific, proximal death and survival signals. Genetic models and biochemical studies indicate that these molecules are latent killers until activated through transcriptional or post-translational mechanisms in a tissue-restricted and signal-specific manner. The large number of BH3-only proteins, their unique subcellular localization, protein-interaction network and diverse modes of activation suggest specialization of their damage-sensing function, ensuring that the core apoptotic machinery is poised to receive input from a wide range of cellular stress signals. The apoptotic response initiated by the activation of BH3-only proteins ultimately culminates in allosteric activation of pro-apoptotic BAX and BAK, the gateway proteins to the mitochondrial pathway of apoptosis. From activation of BH3-only proteins to oligomerization of BAX and BAK and mitochondrial outer membrane permeabilization, an intricate network of interactions between the pro- and anti-apoptotic members of the BCL-2 family orchestrates the decision to undergo apoptosis. Beyond regulation of apoptosis, multiple BCL-2 proteins have recently emerged as active components of select homeostatic pathways carrying other cellular functions. This review focuses on BAD, which was the first BH3-only protein linked to proximal survival signals through phosphorylation by survival kinases. In addition to findings that delineated the physiological role of BAD in apoptosis and its dynamic regulation by phosphorylation, studies pointing to new roles for this protein in other physiological pathways, such as glucose metabolism, are highlighted. By executing its 'day' and 'night' jobs in metabolism and apoptosis, respectively, BAD helps coordinate mitochondrial fuel metabolism and the apoptotic machinery.

STAT3 inhibition as a therapeutic strategy for leukemia.

PubMed

Kanna, Rubashruti; Choudhary, Gaurav; Ramachandra, Nandini; Steidl, Ulrich; Verma, Amit; Shastri, Aditi

2017-11-22

Leukemia is characterized by selective overgrowth of malignant hematopoietic stem cells (HSC's) that interfere with HSC differentiation. Cytoreductive chemotherapy can kill rapidly dividing cancerous cells but cannot eradicate the malignant HSC pool leading to relapses. Leukemic stem cells have several dysregulated pathways and the Janus kinases (JAKs) and signal transducer and activator of transcription (STAT) pathway are prominent among them. STAT3 is an important transcription factor that regulates cell growth, proliferation, and inhibits apoptosis. High STAT3 expression in leukemia has been associated with an increased risk for relapse and decreased overall survival. Multiple strategies for interfering with STAT3 activity in leukemic cells include inhibition of STAT3 phosphorylation, interfering with STAT3 interactions, preventing nuclear transfer, inhibiting transcription and causing interference in STAT: DNA binding. A better understanding of key interactions and upstream mediators of STAT3 activity will help facilitate the development of effective cancer therapies and may result in durable remissions.

Lipid signalling couples translational surveillance to systemic detoxification in Caenorhabditis elegans

PubMed Central

Govindan, J. Amaranath; Jayamani, Elamparithi; Zhang, Xinrui; Breen, Peter; Larkins-Ford, Jonah; Mylonakis, Eleftherios

2015-01-01

Translation in eukaryotes is surveilled to detect toxins and virulence factors and coupled to the induction of defense pathways. C. elegans germline-specific mutations in translation components are detected by this system to induce detoxification and immune responses in distinct somatic cells. An RNAi screen revealed gene inactivations that act at multiple steps in lipid biosynthetic and kinase pathways that act upstream of MAP kinase to mediate the systemic communication of translation-defects to induce detoxification genes. Mammalian bile acids can rescue the defect in detoxification gene induction caused by C. elegans lipid biosynthetic gene inactivations. Extracts prepared from C. elegans with translation deficits but not from wild type can also rescue detoxification gene induction in lipid biosynthetic defective strains. These eukaryotic antibacterial countermeasures are not ignored by bacteria: particular bacterial species suppress normal C. elegans detoxification responses to mutations in translation factors. PMID:26322678

Hyphal formation of Candida albicans is controlled by electron transfer system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, Toshihiko; Ogasawara, Ayako; Mikami, Takeshi

2006-09-15

Most Candida albicans cells cultured in RPMI1640 medium at 37 deg. C grow in hyphal form in aerobic conditions, but they grow in yeast form in anaerobic conditions. The hyphal growth of C. albicans was inhibited in glucose-deficient conditions. Malonic acid, an inhibitor of succinate dehydrogenase, enhanced the yeast proliferation of C. albicans, indicating that the hyphal-formation signal was derived from the glycolysis system and the signal was transmitted to the electron transfer system via the citric acid cycle. Thenoyl trifluoro acetone (TTFA), an inhibitor of the signal transmission between complex II and Co Q, significantly inhibited the hyphal growthmore » of C. albicans. Antimycin, KCN, and oligomycin, inhibitors of complex III, IV, and V, respectively, did not inhibit the hyphal growth of C. albicans. The production of mRNAs for the hyphal formation signal was completely inhibited in anaerobic conditions. These results indicate that the electron transfer system functions upstream of the RAS1 signal pathway and activates the expression of the hyphal formation signal. Since the electron transfer system is inactivated in anaerobic conditions, C. albicans grew in yeast form in this condition.« less

Recent Progress on Liver Kinase B1 (LKB1): Expression, Regulation, Downstream Signaling and Cancer Suppressive Function

PubMed Central

Gan, Ren-You; Li, Hua-Bin

2014-01-01

Liver kinase B1 (LKB1), known as a serine/threonine kinase, has been identified as a critical cancer suppressor in many cancer cells. It is a master upstream kinase of 13 AMP-activated protein kinase (AMPK)-related protein kinases, and possesses versatile biological functions. LKB1 gene is mutated in many cancers, and its protein can form different protein complexes with different cellular localizations in various cell types. The expression of LKB1 can be regulated through epigenetic modification, transcriptional regulation and post-translational modification. LKB1 dowcnstream pathways mainly include AMPK, microtubule affinity regulating kinase (MARK), salt-inducible kinase (SIK), sucrose non-fermenting protein-related kinase (SNRK) and brain selective kinase (BRSK) signalings, etc. This review, therefore, mainly discusses recent studies about the expression, regulation, downstream signaling and cancer suppressive function of LKB1, which can be helpful for better understanding of this molecular and its significance in cancers. PMID:25244018

Hexavalent Chromium Cr(VI) Up-Regulates COX-2 Expression through an NFκB/c-Jun/AP-1–Dependent Pathway

PubMed Central

Zuo, Zhenghong; Cai, Tongjian; Li, Jingxia; Zhang, Dongyun; Yu, Yonghui

2012-01-01

Background: Hexavalent chromium [Cr(VI)] is recognized as a human carcinogen via inhalation. However, the molecular mechanisms by which Cr(VI) causes cancers are not well understood. Objectives: We evaluated cyclooxygenase-2 (COX-2) expression and the signaling pathway leading to this induction due to Cr(VI) exposure in cultured cells. Methods: We used the luciferase reporter assay and Western blotting to determine COX-2 induction by Cr(VI). We used dominant negative mutant, genetic knockout, gene knockdown, and chromatin immunoprecipitation approaches to elucidate the signaling pathway leading to COX-2 induction. Results: We found that Cr(VI) exposure induced COX-2 expression in both normal human bronchial epithelial cells and mouse embryonic fibroblasts in a concentration- and time-dependent manner. Deletion of IKKβ [inhibitor of transcription factor NFκB (IκB) kinase β; an upstream kinase responsible for nuclear factor κB (NFκB) activation] or overexpression of TAM67 (a dominant-negative mutant of c-Jun) dramatically inhibited the COX-2 induction due to Cr(VI), suggesting that both NFκB and c-Jun/AP-1 pathways were required for Cr(VI)-induced COX-2 expression. Our results show that p65 and c-Jun are two major components involved in NFκB and AP-1 activation, respectively. Moreover, our studies suggest crosstalk between NFκB and c-Jun/AP-1 pathways in cellular response to Cr(VI) exposure for COX-2 induction. Conclusion: We demonstrate for the first time that Cr(VI) is able to induce COX-2 expression via an NFκB/c-Jun/AP-1–dependent pathway. Our results provide novel insight into the molecular mechanisms linking Cr(VI) exposure to lung inflammation and carcinogenesis. PMID:22472290

Reactive oxygen species upregulate expression of muscle atrophy-associated ubiquitin ligase Cbl-b in rat L6 skeletal muscle cells.

PubMed

Uchida, Takayuki; Sakashita, Yoshihiro; Kitahata, Kanako; Yamashita, Yui; Tomida, Chisato; Kimori, Yuki; Komatsu, Akio; Hirasaka, Katsuya; Ohno, Ayako; Nakao, Reiko; Higashitani, Atsushi; Higashibata, Akira; Ishioka, Noriaki; Shimazu, Toru; Kobayashi, Takeshi; Okumura, Yuushi; Choi, Inho; Oarada, Motoko; Mills, Edward M; Teshima-Kondo, Shigetada; Takeda, Shin'ichi; Tanaka, Eiji; Tanaka, Keiji; Sokabe, Masahiro; Nikawa, Takeshi

2018-06-01

Unloading-mediated muscle atrophy is associated with increased reactive oxygen species (ROS) production. We previously demonstrated that elevated ubiquitin ligase casitas B-lineage lymphoma-b (Cbl-b) resulted in the loss of muscle volume (Nakao R, Hirasaka K, Goto J, Ishidoh K, Yamada C, Ohno A, Okumura Y, Nonaka I, Yasutomo K, Baldwin KM, Kominami E, Higashibata A, Nagano K, Tanaka K, Yasui N, Mills EM, Takeda S, Nikawa T. Mol Cell Biol 29: 4798-4811, 2009). However, the pathological role of ROS production associated with unloading-mediated muscle atrophy still remains unknown. Here, we showed that the ROS-mediated signal transduction caused by microgravity or its simulation contributes to Cbl-b expression. In L6 myotubes, the assessment of redox status revealed that oxidized glutathione was increased under microgravity conditions, and simulated microgravity caused a burst of ROS, implicating ROS as a critical upstream mediator linking to downstream atrophic signaling. ROS generation activated the ERK1/2 early-growth response protein (Egr)1/2-Cbl-b signaling pathway, an established contributing pathway to muscle volume loss. Interestingly, antioxidant treatments such as N-acetylcysteine and TEMPOL, but not catalase, blocked the clinorotation-mediated activation of ERK1/2. The increased ROS induced transcriptional activity of Egr1 and/or Egr2 to stimulate Cbl-b expression through the ERK1/2 pathway in L6 myoblasts, since treatment with Egr1/2 siRNA and an ERK1/2 inhibitor significantly suppressed clinorotation-induced Cbl-b and Egr expression, respectively. Promoter and gel mobility shift assays revealed that Cbl-b was upregulated via an Egr consensus oxidative responsive element at -110 to -60 bp of the Cbl-b promoter. Together, this indicates that under microgravity conditions, elevated ROS may be a crucial mechanotransducer in skeletal muscle cells, regulating muscle mass through Cbl-b expression activated by the ERK-Egr signaling pathway.

Ras1 interacts with multiple new signaling and cytoskeletal loci in Drosophila eggshell patterning and morphogenesis.

PubMed Central

Schnorr, J D; Holdcraft, R; Chevalier, B; Berg, C A

2001-01-01

Little is known about the genes that interact with Ras signaling pathways to regulate morphogenesis. The synthesis of dorsal eggshell structures in Drosophila melanogaster requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. We took advantage of this process to identify genes that link patterning and morphogenesis; we screened lethal mutations on the second chromosome for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. Our genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61beta, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway; these are Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a "neurospecific" receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype. PMID:11606538

Ras1 interacts with multiple new signaling and cytoskeletal loci in Drosophila eggshell patterning and morphogenesis.

PubMed

Schnorr, J D; Holdcraft, R; Chevalier, B; Berg, C A

2001-10-01

Little is known about the genes that interact with Ras signaling pathways to regulate morphogenesis. The synthesis of dorsal eggshell structures in Drosophila melanogaster requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. We took advantage of this process to identify genes that link patterning and morphogenesis; we screened lethal mutations on the second chromosome for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. Our genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61beta, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway; these are Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a "neurospecific" receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype.

Activation of YUCCA5 by the Transcription Factor TCP4 Integrates Developmental and Environmental Signals to Promote Hypocotyl Elongation in Arabidopsis.

PubMed

Challa, Krishna Reddy; Aggarwal, Pooja; Nath, Utpal

2016-09-05

Cell expansion is an essential process in plant morphogenesis and is regulated by the coordinated action of environmental stimuli and endogenous factors, such as the phytohormones auxin and brassinosteroid. Although the biosynthetic pathways that generate these hormones and their downstream signaling mechanisms have been extensively studied, the upstream transcriptional network that modulates their levels and connects their action to cell morphogenesis is less clear. Here we show that the miR319-regulated TCP (TEOSINTE BRANCHED 1, CYCLODEA, PROLIFERATING CELL FACTORS) transcription factors, notably TCP4, directly activate YUCCA5 transcription and integrate the auxin response to a brassinosteroid-dependent molecular circuit that promotes cell elongation in Arabidopsis hypocotyls. Further, TCP4 modulates the common transcriptional network downstream to auxin-BR signaling, which is also triggered by environmental cues, such as light, to promote cell expansion. Our study links TCP function with the hormone response during cell morphogenesis and shows that developmental and environmental signals converge on a common transcriptional network to promote cell elongation. {copyright, serif} 2016 American Society of Plant Biologists. All rights reserved.

p16(INK4A) inhibits the pro-metastatic potentials of osteosarcoma cells through targeting the ERK pathway and TGF-β1.

PubMed

Silva, Gabriela; Aboussekhra, Abdelilah

2016-05-01

Extracellular signal-regulated kinase (ERK) is a downstream component of the evolutionarily conserved mitogen-activated protein kinase-signaling pathway, which controls the expression of a plethora of genes implicated in various physiological processes. This pathway is often hyper-activated by mutations or abnormal extracellular signaling in different types of human cancer, including the most common primary malignant bone tumor osteosarcomas. p16(INK4A) is an important tumor suppressor gene frequently lost in osteosarcomas, and is associated with the progression of these malignancies. We have shown, here, that the ERK1/2 protein kinase is also activated by p16(INK4A) down-regulation in osteosarcoma cells and normal human as well as mouse cells. This inhibitory effect is associated with the suppression of the upstream kinase MEK1/2, and is mediated via the repression of miR-21-5p and the consequent up-regulation of the MEK/ERK antagonist SPRY2 in osteosarcoma cells. Furthermore, we have shown that p16(INK4) inhibits the migration/invasion abilities of these cells through miR-21-5p-dependent inhibition of ERK1/2. In addition, we present clear evidence that p16(INK4) represses the paracrine pro-migratory effect of osteosarcoma cells on stromal fibroblasts through the inhibition of the TGF-β1 expression/secretion. This effect is also ERK1/2-dependent, indicating that in addition to their cell-autonomous actions, p16(INK4) and ERK1/2 have also non-cell-autonomous cancer-related functions. Together, these results indicate that the tumor suppressor p16(INK4) protein represses the carcinogenic process of osteosarcoma cells not only as a cell cycle regulator, but also as a negative regulator of pro-carcinogenic/-metastatic pathways. This indicates that targeting the ERK pathway is of utmost therapeutic value. © 2015 Wiley Periodicals, Inc.

SGK2 promotes hepatocellular carcinoma progression and mediates GSK-3β/β-catenin signaling in HCC cells.

PubMed

Liu, Junying; Zhang, Guangdong; Lv, Yanping; Zhang, Xiaoyang; Ying, Cui; Yang, Suocheng; Kong, Xin; Yu, Yanzhang

2017-06-01

The phosphoinositide 3-kinase pathway is one of the most commonly altered pathways in human cancers. The serum/glucocorticoid-regulated kinase (SGK) family of serine/threonine kinases consists of three isoforms, SGK1, SGK2, and SGK3. This family of kinases is highly homologous to the AKT kinase family, sharing similar upstream activators and downstream targets. Few studies have investigated the role of SGK2 in hepatocellular carcinoma. Here, we report that SGK2 expression levels were upregulated in hepatocellular carcinoma tissues and human hepatoma cell lines compared to the adjacent normal liver tissues and a normal hepatocyte line, respectively. We found that downregulated SGK2 inhibits cell migration and invasive potential of hepatocellular carcinoma cell lines (SMMC-7721 and Huh-7).We also found that downregulated SGK2 suppressed the expression level of unphosphorylated (activated) glycogen synthase kinase 3 beta. In addition, SGK2 downregulation decreased the dephosphorylation (activation) of β-catenin by preventing its proteasomal degradation in the hepatocellular carcinoma cell lines. These findings suggest that SGK2 promotes hepatocellular carcinoma progression and mediates glycogen synthase kinase 3 beta/β-catenin signaling in hepatocellular carcinoma cells.

Rutin-Mediated Priming of Plant Resistance to Three Bacterial Pathogens Initiating the Early SA Signal Pathway

PubMed Central

Li, Yang; Wang, Yingzi; Li, Ming; Wang, Yong; Ding, Xinhua; Chu, Zhaohui

2016-01-01

Flavonoids are ubiquitous in the plant kingdom and have many diverse functions, including UV protection, auxin transport inhibition, allelopathy, flower coloring and insect resistance. Here we show that rutin, a proud member of the flavonoid family, could be functional as an activator to improve plant disease resistances. Three plant species pretreated with 2 mM rutin were found to enhance resistance to Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, and Pseudomonas syringae pv. tomato strain DC3000 in rice, tobacco and Arabidopsis thaliana respectively. While they were normally propagated on the cultural medium supplemented with 2 mM rutin for those pathogenic bacteria. The enhanced resistance was associated with primed expression of several pathogenesis-related genes. We also demonstrated that the rutin-mediated priming resistance was attenuated in npr1, eds1, eds5, pad4-1, ndr1 mutants, and NahG transgenic Arabidopsis plant, while not in either snc1-11, ein2-5 or jar1 mutants. We concluded that the rutin-priming defense signal was modulated by the salicylic acid (SA)-dependent pathway from an early stage upstream of NDR1 and EDS1. PMID:26751786

Transcriptome analysis demonstrates that long noncoding RNA is involved in the hypoxic response in Larimichthys crocea.

PubMed

Liu, Wei; Liu, Xiaoxu; Wu, Changwen; Jiang, Lihua

2018-06-15

The large yellow croaker (Larimichthys crocea) has low hypoxia tolerance compared with other fish species, and the mRNA levels of hypoxia-inducible factor (HIF)-1α in its brain do not change markedly under hypoxic conditions. In this study, we investigated noncoding transcription in the hypoxic response mechanism of L. crocea. We generated a catalog of long noncoding RNAs (lncRNAs) from the brain of L. crocea individuals under hypoxic stress, investigated lncRNA expression patterns, and analyzed the HIF signaling pathway by RNA sequencing. Prolyl hydroxylase domain 2 (PHD2) expression significantly increased after 6 and 12 h of hypoxia, and a lncRNA (Linc_06633.1) was found in the upstream, antisense region of PHD2. Linc_06633.1 may be an important regulator that promotes PDH2 expression under hypoxia in L. crocea, and we constructed a regulatory profile of L. crocea under hypoxic conditions. To the best of our knowledge, it is the first study that has been conducted on hypoxia signaling pathway regulation by lncRNAs in L. crocea and elucidates the role played by lncRNAs in the regulation of the hypoxia stress response in teleost fish.

Regulation of Stomatal Immunity by Interdependent Functions of a Pathogen-Responsive MPK3/MPK6 Cascade and Abscisic Acid

PubMed Central

Zhang, Lawrence; Sun, Tiefeng

2017-01-01

Activation of mitogen-activated protein kinases (MAPKs) is one of the earliest responses after plants sense an invading pathogen. Here, we show that MPK3 and MPK6, two Arabidopsis thaliana pathogen-responsive MAPKs, and their upstream MAPK kinases, MKK4 and MKK5, are essential to both stomatal and apoplastic immunity. Loss of function of MPK3 and MPK6, or their upstream MKK4 and MKK5, abolishes pathogen/microbe-associated molecular pattern- and pathogen-induced stomatal closure. Gain-of-function activation of MPK3/MPK6 induces stomatal closure independently of abscisic acid (ABA) biosynthesis and signaling. In contrast, exogenously applied organic acids such as malate or citrate are able to reverse the stomatal closure induced by MPK3/MPK6 activation. Gene expression analysis and in situ enzyme activity staining revealed that malate metabolism increases in guard cells after activation of MPK3/MPK6 or inoculation of pathogen. In addition, pathogen-induced malate metabolism requires functional MKK4/MKK5 and MPK3/MPK6. We propose that the pathogen-responsive MPK3/MPK6 cascade and ABA are two essential signaling pathways that control, respectively, the organic acid metabolism and ion channels, two main branches of osmotic regulation in guard cells that function interdependently to control stomatal opening/closure. PMID:28254778

Transforming Growth Factor-β Is an Upstream Regulator of Mammalian Target of Rapamycin Complex 2-Dependent Bladder Cancer Cell Migration and Invasion.

PubMed

Gupta, Sounak; Hau, Andrew M; Al-Ahmadie, Hikmat A; Harwalkar, Jyoti; Shoskes, Aaron C; Elson, Paul; Beach, Jordan R; Hussey, George S; Schiemann, William P; Egelhoff, Thomas T; Howe, Philip H; Hansel, Donna E

2016-05-01

Our prior work identified the mammalian target of rapamycin complex 2 (mTORC2) as a key regulator of bladder cancer cell migration and invasion, although upstream growth factor mediators of this pathway in bladder cancer have not been well delineated. We tested whether transforming growth factor (TGF)-β, which can function as a promotility factor in bladder cancer cells, could regulate mTORC2-dependent bladder cancer cell motility and invasion. In human bladder cancers, the highest levels of phosphorylated SMAD2, a TGF-β signaling intermediate, were present in high-grade invasive bladder cancers and associated with more frequent recurrence and decreased disease-specific survival. Increased expression of TGF-β isoforms, receptors, and signaling components was detected in invasive high-grade bladder cancer cells that expressed Vimentin and lacked E-cadherin. Application of TGF-β induced phosphorylation of the Ser473 residue of AKT, a selective target of mTORC2, in a SMAD2- and SMAD4-independent manner and increased bladder cancer cell migration in a modified scratch wound assay and invasion through Matrigel. Inhibition of TGF-β receptor I using SB431542 ablated TGF-β-induced migration and invasion. A similar effect was seen when Rictor, a key mTORC2 component, was selectively silenced. Our results suggest that TGF-β can induce bladder cancer cell invasion via mTORC2 signaling, which may be applicable in most bladder cancers. Copyright © 2016. Published by Elsevier Inc.

The Hedgehog processing pathway is required for NSCLC growth and survival

PubMed Central

Rodriguez-Blanco, Jezabel; Schilling, Neal S.; Tokhunts, Robert; Giambelli, Camilla; Long, Jun; Liang Fei, Dennis; Singh, Samer; Black, Kendall E.; Wang, Zhiqiang; Galimberti, Fabrizio; Bejarano, Pablo A.; Elliot, Sharon; Glassberg, Marilyn K.; Nguyen, Dao M.; Lockwood, William W.; Lam, Wan L.; Dmitrovsky, Ethan; Capobianco, Anthony J.; Robbins, David J.

2013-01-01

Considerable interest has been generated from the results of recent clinical trials using SMOOTHENED (SMO) antagonists to inhibit the growth of HEDGEHOG (HH) signaling dependent tumors. This interest is tempered by the discovery of SMO mutations mediating resistance, underscoring the rationale for developing therapeutic strategies that interrupt HH signaling at levels distinct from those inhibiting SMO function. Here, we demonstrate that HH dependent non-small cell lung carcinoma (NSCLC) growth is sensitive to blockade of the HH pathway upstream of SMO, at the level of HH ligand processing. Individually, the use of different lentivirally delivered shRNA constructs targeting two functionally distinct HH-processing proteins, SKINNY HEDGEHOG (SKN) or DISPATCHED-1 (DISP-1), in NSCLC cell lines produced similar decreases in cell proliferation and increased cell death. Further, providing either an exogenous source of processed HH or a SMO agonist reverses these effects. The attenuation of HH processing, by knocking down either of these gene products, also abrogated tumor growth in mouse xenografts. Finally, we extended these findings to primary clinical specimens, showing that SKN is frequently over-expressed in NSCLC and that higher DISP-1 expression is associated with an unfavorable clinical outcome. Our results show a critical role for HH processing in HH-dependent tumors, identifies two potential druggable targets in the HH pathway, and suggest that similar therapeutic strategies could be explored to treat patients harboring HH ligand dependent cancers. PMID:22733134

Use of restrained molecular dynamics to predict the conformations of phosphorylated receiver domains in two-component signaling systems.

PubMed

Foster, Clay A; West, Ann H

2017-01-01

Two-component signaling (TCS) is the primary means by which bacteria, as well as certain plants and fungi, respond to external stimuli. Signal transduction involves stimulus-dependent autophosphorylation of a sensor histidine kinase and phosphoryl transfer to the receiver domain of a downstream response regulator. Phosphorylation acts as an allosteric switch, inducing structural and functional changes in the pathway's components. Due to their transient nature, phosphorylated receiver domains are challenging to characterize structurally. In this work, we provide a methodology for simulating receiver domain phosphorylation to predict conformations that are nearly identical to experimental structures. Using restrained molecular dynamics, phosphorylated conformations of receiver domains can be reliably sampled on nanosecond timescales. These simulations also provide data on conformational dynamics that can be used to identify regions of functional significance related to phosphorylation. We first validated this approach on several well-characterized receiver domains and then used it to compare the upstream and downstream components of the fungal Sln1 phosphorelay. Our results demonstrate that this technique provides structural insight, obtained in the absence of crystallographic or NMR information, regarding phosphorylation-induced conformational changes in receiver domains that regulate the output of their associated signaling pathway. To our knowledge, this is the first time such a protocol has been described that can be broadly applied to TCS proteins for predictive purposes. Proteins 2016; 85:155-176. © 2016 Wiley Periodicals, Inc. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Vitamin K epoxide reductase regulation of androgen receptor activity

PubMed Central

Tew, Ben Yi; Hong, Teresa B.; Otto-Duessel, Maya; Elix, Catherine; Castro, Egbert; He, Miaoling; Wu, Xiwei; Pal, Sumanta K.; Kalkum, Markus; Jones, Jeremy O.

2017-01-01

Long-term use of warfarin has been shown to be associated with a reduced risk of prostate cancer. Warfarin belongs to the vitamin K antagonist class of anticoagulants, which inhibit vitamin K epoxide reductase (VKOR). The vitamin K cycle is primarily known for its role in γ-carboxylation, a rare post-translational modification important in blood coagulation. Here we show that warfarin inhibits the transcriptional activity of the androgen receptor (AR), an important driver of prostate cancer development and progression. Warfarin treatment or knockdown of its target VKOR inhibits the activity of AR both in cell lines and in mouse prostate tissue. We demonstrate that AR can be γ-carboxylated, and mapped the γ-carboxylation to glutamate residue 2 (E2) using mass spectrometry. However, mutation of E2 and other glutamates on AR failed to suppress the effects of warfarin on AR suggesting that inhibition of AR is γ-carboxylation independent. To identify pathways upstream of AR signaling that are affected by warfarin, we performed RNA-seq on prostates of warfarin-treated mice. We found that warfarin inhibited peroxisome proliferator-activated receptor gamma (PPARγ) signaling, which in turn, inhibited AR signaling. Although warfarin is unfit for use as a chemopreventative due to its anticoagulatory effects, our data suggest that its ability to reduce prostate cancer risk is independent of its anticoagulation properties. Furthermore, our data show that warfarin inhibits PPARγ and AR signaling, which suggests that inhibition of these pathways could be used to reduce the risk of developing prostate cancer. PMID:28099154

Signaling Role of Fructose Mediated by FINS1/FBP in Arabidopsis thaliana

PubMed Central

Cho, Young-Hee; Yoo, Sang-Dong

2011-01-01

Sugars are evolutionarily conserved signaling molecules that regulate the growth and development of both unicellular and multicellular organisms. As sugar-producing photosynthetic organisms, plants utilize glucose as one of their major signaling molecules. However, the details of other sugar signaling molecules and their regulatory factors have remained elusive, due to the complexity of the metabolite and hormone interactions that control physiological and developmental programs in plants. We combined information from a gain-of-function cell-based screen and a loss-of-function reverse-genetic analysis to demonstrate that fructose acts as a signaling molecule in Arabidopsis thaliana. Fructose signaling induced seedling developmental arrest and interacted with plant stress hormone signaling in a manner similar to that of glucose. For fructose signaling responses, the plant glucose sensor HEXOKINASE1 (HXK1) was dispensable, while FRUCTOSE INSENSITIVE1 (FINS1), a putative FRUCTOSE-1,6-BISPHOSPHATASE, played a crucial role. Interestingly, FINS1 function in fructose signaling appeared to be independent of its catalytic activity in sugar metabolism. Genetic analysis further indicated that FINS1–dependent fructose signaling may act downstream of the abscisic acid pathway, in spite of the fact that HXK1–dependent glucose signaling works upstream of hormone synthesis. Our findings revealed that multiple layers of controls by fructose, glucose, and abscisic acid finely tune the plant autotrophic transition and modulate early seedling establishment after seed germination. PMID:21253566

Matrix-specific protein kinase A signaling regulates p21 activated kinase activation by flow in endothelial cells

PubMed Central

Funk, Steven Daniel; Yurdagul, Arif; Green, Jonette M.; Jhaveri, Krishna A.; Schwartz, Martin Alexander; Orr, A. Wayne

2010-01-01

Rationale Atherosclerosis is initiated by blood flow patterns that activate inflammatory pathways in endothelial cells. Activation of inflammatory signaling by fluid shear stress is highly dependent on the composition of the subendothelial extracellular matrix. The basement membrane proteins laminin and collagen found in normal vessels suppress flow-induced p21 activated kinase (PAK) and NF-κB activation. By contrast, the provisional matrix proteins fibronectin and fibrinogen found in wounded or inflamed vessels support flow-induced PAK and NF-κB activation. PAK mediates both flow-induced permeability and matrix-specific activation of NF-κB. Objective To elucidate the mechanisms regulating matrix-specific PAK activation. Methods and Results We now show that matrix composition does not affect the upstream pathway by which flow activates PAK (integrin activation, Rac). Instead basement membrane proteins enhance flow-induced protein kinase A (PKA) activation, which suppresses PAK. Inhibiting PKA restored flow-induced PAK and NF-κB activation in cells on basement membrane proteins, whereas stimulating PKA inhibited flow-induced activation of inflammatory signaling in cells on fibronectin. PKA suppressed inflammatory signaling through PAK inhibition. Activating PKA by injection of the PGI2 analog iloprost reduced PAK activation and inflammatory gene expression at sites of disturbed flow in vivo, whereas inhibiting PKA by PKI injection enhanced PAK activation and inflammatory gene expression. Inhibiting PAK prevented the enhancement of inflammatory gene expression by PKI. Conclusions Basement membrane proteins inhibit inflammatory signaling in endothelial cells via PKA-dependent inhibition of PAK. PMID:20224042

Role of Ca2+ signaling in initiation of stretch-induced apoptosis in neonatal heart cells.

PubMed

Liao, Xu Dong; Tang, Ai Hui; Chen, Quan; Jin, Hai Jing; Wu, Cai Hong; Chen, Lan-Ying; Wang, Shi Qiang

2003-10-17

Abnormal mechanical load, as seen in hypertension, is found to induce heart cell apoptosis, yet the signaling link between cell stretch and apoptotic pathways is not known. Using an in vitro stretch model mimicking diastolic pressure stress, here we show that Ca(2+) signaling participates essentially in the early stage of stretch-induced apoptosis. In neonatal rat cardiomyocytes, the moderate 20% stretch resulted in tonic elevation of intracellular free Ca(2+) ([Ca(2+)](i)). Buffering [Ca(2+)](i) by EGTA-AM, suppressing ryanodine-sensitive Ca(2+) release, and blocking L-type Ca(2+) channels all prevented the stretch-induced apoptosis as assessed by phosphatidylserine exposure and nuclear fragmentation. Notably, Ca(2+) suppression also prevented known stretch-activated apoptotic events, including caspase-3/-9 activation, mitochondrial membrane potential corruption, and reactive oxygen species production, suggesting that Ca(2+) signaling is the upstream of these events. Since [Ca(2+)](i) did not change without activating mechanosensitive Ca(2+) entry, we conclude that stretch-induced Ca(2+) entry, via the Ca(2+)-induced Ca(2+) release mechanism, plays an important role in initiating apoptotic signaling during mechanical stress.

SH2/SH3 adaptor proteins can link tyrosine kinases to a Ste20-related protein kinase, HPK1.

PubMed

Anafi, M; Kiefer, F; Gish, G D; Mbamalu, G; Iscove, N N; Pawson, T

1997-10-31

Ste20-related protein kinases have been implicated as regulating a range of cellular responses, including stress-activated protein kinase pathways and the control of cytoskeletal architecture. An important issue involves the identities of the upstream signals and regulators that might control the biological functions of mammalian Ste20-related protein kinases. HPK1 is a protein-serine/threonine kinase that possesses a Ste20-like kinase domain, and in transfected cells activates a protein kinase pathway leading to the stress-activated protein kinase SAPK/JNK. Here we have investigated candidate upstream regulators that might interact with HPK1. HPK1 possesses an N-terminal catalytic domain and an extended C-terminal tail with four proline-rich motifs. The SH3 domains of Grb2 bound in vitro to specific proline-rich motifs in the HPK1 tail and functioned synergistically to direct the stable binding of Grb2 to HPK1 in transfected Cos1 cells. Epidermal growth factor (EGF) stimulation did not affect the binding of Grb2 to HPK1 but induced recruitment of the Grb2.HPK1 complex to the autophosphorylated EGF receptor and to the Shc docking protein. Several activated receptor and cytoplasmic tyrosine kinases, including the EGF receptor, stimulated the tyrosine phosphorylation of the HPK1 serine/threonine kinase. These results suggest that HPK1, a mammalian Ste20-related protein-serine/threonine kinase, can potentially associate with protein-tyrosine kinases through interactions mediated by SH2/SH3 adaptors such as Grb2. Such interaction may provide a possible mechanism for cross-talk between distinct biochemical pathways following the activation of tyrosine kinases.

Trithorax Group Protein Oryza sativa Trithorax1 Controls Flowering Time in Rice via Interaction with Early heading date31[W][OPEN

PubMed Central

Choi, Sang Chul; Lee, Shinyoung; Kim, Sung-Ryul; Lee, Yang-Seok; Liu, Chunyan; Cao, Xiaofeng; An, Gynheung

2014-01-01

Trithorax group proteins are chromatin-remodeling factors that activate target gene expression by antagonistically functioning against the Polycomb group. In Arabidopsis (Arabidopsis thaliana), Arabidopsis Trithorax protein1 (ATX1) regulates flowering time and floral organ identity. Here, we observed that suppression of Oryza sativa Trithorax1 (OsTrx1), an ortholog of ATX1, delayed flowering time in rice (Oryza sativa). Because the delay occurred only under long-day conditions, we evaluated the flowering signal pathways that specifically function under long-day conditions. Among them, the OsMADS50 and Heading date1 pathways were not affected by the mutation. However, the Grain number, plant height, and heading date7 (Ghd7) pathway was altered in ostrx1. Transcript levels of OsGI, phytochrome genes, and Early heading date3 (Ehd3), which function upstream of Ghd7, were unchanged in the mutant. Because Trx group proteins form a complex with other proteins to modify the chromatin structure of target genes, we investigated whether OsTrx1 interacts with a previously identified protein that functions upstream of Ghd7. We demonstrated that the plant homeodomain motif of OsTrx1 binds to native histone H3 from the calf thymus and that OsTrx1 binds to Ehd3 through the region between the plant homeodomain and SET domains. Finally, we showed that the SET domain at the C-terminal end of OsTrx1 has histone H3 methyltransferase activity when incubated with oligonucleosomes. Our results suggest that OsTrx1 plays an important role in regulating flowering time in rice by modulating chromatin structure. PMID:24420930

Relationship between nitric oxide- and calcium-dependent signal transduction pathways in growth hormone release from dispersed goldfish pituitary cells.

PubMed

Chang, John P; Sawisky, Grant R; Davis, Philip J; Pemberton, Joshua G; Rieger, Aja M; Barreda, Daniel R

2014-09-15

Nitric oxide (NO) and Ca(2+) are two of the many intracellular signal transduction pathways mediating the control of growth hormone (GH) secretion from somatotropes by neuroendocrine factors. We have previously shown that the NO donor sodium nitroprusside (SNP) elicits Ca(2+) signals in identified goldfish somatotropes. In this study, we examined the relationships between NO- and Ca(2+)-dependent signal transduction mechanisms in GH secretion from primary cultures of dispersed goldfish pituitary cells. Morphologically identified goldfish somatotropes stained positively for an NO-sensitive dye indicating they may be a source of NO production. In 2h static incubation experiments, GH release responses to the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) were attenuated by CoCl2, nifedipine, verapamil, TMB-8, BHQ, and KN62. In column perifusion experiments, the ability of SNP to induce GH release was impaired in the presence of TMB-8, BHQ, caffeine, and thapsigargin, but not ryanodine. Caffeine-elicited GH secretion was not affected by the NO scavenger PTIO. These results suggest that NO-stimulated GH release is dependent on extracellular Ca(2+) availability and voltage-sensitive Ca(2+) channels, as well as intracellular Ca(2+) store(s) that possess BHQ- and/or thapsigargin-inhibited sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases, as well as TMB-8- and/or caffeine-sensitive, but not ryanodine-sensitive, Ca(2+)-release channels. Calmodulin kinase-II also likely participates in NO-elicited GH secretion but caffeine-induced GH release is not upstream of NO production. These findings provide insights into how NO actions many integrate with Ca(2+)-dependent signalling mechanisms in goldfish somatotropes and how such interactions may participate in the GH-releasing actions of regulators that utilize both NO- and Ca(2+)-dependent transduction pathways. Copyright © 2014 Elsevier Inc. All rights reserved.

Rho2 Palmitoylation Is Required for Plasma Membrane Localization and Proper Signaling to the Fission Yeast Cell Integrity Mitogen-Activated Protein Kinase Pathway

PubMed Central

Sánchez-Mir, Laura; Franco, Alejandro; Martín-García, Rebeca; Madrid, Marisa; Vicente-Soler, Jero; Soto, Teresa; Gacto, Mariano; Pérez, Pilar

2014-01-01

The fission yeast small GTPase Rho2 regulates morphogenesis and is an upstream activator of the cell integrity pathway, whose key element, mitogen-activated protein kinase (MAPK) Pmk1, becomes activated by multiple environmental stimuli and controls several cellular functions. Here we demonstrate that farnesylated Rho2 becomes palmitoylated in vivo at cysteine-196 within its carboxyl end and that this modification allows its specific targeting to the plasma membrane. Unlike that of other palmitoylated and prenylated GTPases, the Rho2 control of morphogenesis and Pmk1 activity is strictly dependent upon plasma membrane localization and is not found in other cellular membranes. Indeed, artificial plasma membrane targeting bypassed the Rho2 need for palmitoylation in order to signal. Detailed functional analysis of Rho2 chimeras fused to the carboxyl end from the essential GTPase Rho1 showed that GTPase palmitoylation is partially dependent on the prenylation context and confirmed that Rho2 signaling is independent of Rho GTP dissociation inhibitor (GDI) function. We further demonstrate that Rho2 is an in vivo substrate for DHHC family acyltransferase Erf2 palmitoyltransferase. Remarkably, Rho3, another Erf2 target, negatively regulates Pmk1 activity in a Rho2-independent fashion, thus revealing the existence of cross talk whereby both GTPases antagonistically modulate the activity of this MAPK cascade. PMID:24820419

The prostaglandin receptor EP2 activates multiple signaling pathways and β-arrestin1 complex formation during mouse skin papilloma development

PubMed Central

Chun, Kyung-Soo; Lao, Huei-Chen; Trempus, Carol S.; Okada, Manabu; Langenbach, Robert

2009-01-01

Prostaglandin E2 (PGE2) is elevated in many tumor types, but PGE2's contributions to tumor growth are largely unknown. To investigate PGE2's roles, the contributions of one of its receptors, EP2, were studied using the mouse skin initiation/promotion model. Initial studies indicated that protein kinase A (PKA), epidermal growth factor receptor (EGFR) and several effectors—cyclic adenosine 3′,5′-monophosphate response element-binding protein (CREB), H-Ras, Src, protein kinase B (AKT) and extracellular signal-regulated kinase (ERK)1/2—were activated in 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted papillomas and that PKA and EGFR inhibition (H89 and AG1478, respectively) decreased papilloma formation. EP2's contributions to the activation of these pathways and papilloma development were determined by inhibiting endogenous TPA-induced PGE2 production with indomethacin (Indo) and concomitantly treating with the EP2 agonist, CAY10399 (CAY). CAY treatment restored papilloma formation in TPA/Indo-treated mice and increased cyclic adenosine 3′,5′-monophosphate and PKA activation as measured by p-CREB formation. CAY treatment also increased EGFR and Src activation and their inhibition by AG1478 and PP2 indicated that Src was upstream of EGFR. CAY also increased H-Ras, ERK1/2 and AKT activation, and AG1478 decreased their activation indicating EGFR being upstream. Supporting EP2's contribution, EP2−/− mice exhibited 65% fewer papillomas and reduced Src, EGFR, H-Ras, AKT and ERK1/2 activation. G protein-coupled receptor (GPCR) activation of EGFR has been reported to involve Src's activation via a GPCR–β-arrestin–Src complex. Indeed, immunoprecipitation of β-arrestin1 or p-Src indicated the presence of an EP2–β-arrestin1–p-Src complex in papillomas. The data indicated that EP2 contributed to tumor formation via activation of PKA and EGFR and that EP2 formed a complex with β-arrestin1 and Src that contributed to signaling and/or EP2 desensitization. PMID:19587094

Cost-effective TCM-based WDM-PON for highly asymmetric traffic conditions.

PubMed

Lee, Danbi; Kwon, Won-Bae; Chae, Chang-Joon; Park, Chang-Soo

2015-11-16

A time compression multiplexing (TCM)-based wavelength division multiplexing passive optical network (WDM-PON) using a reflective semiconductor optical amplifier (RSOA) is proposed, and its feasibility is experimentally demonstrated. In the proposed system, the RSOA pre-amplifies a 10 Gb/s downstream signal and modulates the RSOA output, wavelength-locked to the downstream signal, with a 1.25 Gb/s upstream signal simultaneously. The sensitivity of the downstream signal is improved by about 3 dB through the RSOA. The downstream and upstream signals have power penalties of about 0.1 dB and 1.1 dB, respectively, at bit error rates (BERs) of 10(-9) after 20 km transmission.

Void/particulate detector

DOEpatents

Claytor, Thomas N.; Karplus, Henry B.

1985-01-01

Voids and particulates are detected in a flowing stream of fluid contained in a pipe by a detector which includes three transducers spaced about the pipe. A first transducer at a first location on the pipe transmits an ultrasonic signal into the stream. A second transducer detects the through-transmission of the signal at a second location and a third transducer at a third location upstream from the first location detects the back-scattering of the signal from any voids or particulates. To differentiate between voids and particulates a fourth transducer is positioned at a fourth location which is also upstream from the first location. The back-scattered signals are normalized with the through-transmission signal to minimize temperature fluctuations.

Expression patterns of members of the ethylene signaling–related gene families in response to dehydration stresses in cassava

PubMed Central

Shi, Hou Rui; Lu, Li Fang; Yun, Tian Yan; Peng, Ming; Guan, Xiao; Zhang, Heng; Wang, Jing Yi; Zhang, Xi Yan; Li, Cheng Liang; Chen, Yan Jun; He, Peng; Zhang, Yin Dong; Xie, Jiang Hui

2017-01-01

Drought is the one of the most important environment stresses that restricts crop yield worldwide. Cassava (Manihot esculenta Crantz) is an important food and energy crop that has many desirable traits such as drought, heat and low nutrients tolerance. However, the mechanisms underlying drought tolerance in cassava are unclear. Ethylene signaling pathway, from the upstream receptors to the downstream transcription factors, plays important roles in environmental stress responses during plant growth and development. In this study, we used bioinformatics approaches to identify and characterize candidate Manihot esculenta ethylene receptor genes and transcription factor genes. Using computational methods, we localized these genes on cassava chromosomes, constructed phylogenetic trees and identified stress-responsive cis-elements within their 5’ upstream regions. Additionally, we measured the trehalose and proline contents in cassava fresh leaves after drought, osmotic, and salt stress treatments, and then it was found that the regulation patterns of contents of proline and trehalose in response to various dehydration stresses were differential, or even the opposite, which shows that plant may take different coping strategies to deal with different stresses, when stresses come. Furthermore, expression profiles of these genes in different organs and tissues under non-stress and abiotic stress were investigated through quantitative real-time PCR (qRT-PCR) analyses in cassava. Expression profiles exhibited clear differences among different tissues under non-stress and various dehydration stress conditions. We found that the leaf and tuberous root tissues had the greatest and least responses, respectively, to drought stress through the ethylene signaling pathway in cassava. Moreover, tuber and root tissues had the greatest and least reponses to osmotic and salt stresses through ethylene signaling in cassava, respectively. These results show that these plant tissues had differential expression levels of genes involved in ethylene signaling in response to the stresses tested. Moreover, after several gene duplication events, the spatiotemporally differential expression pattern of homologous genes in response to abiotic and biotic stresses may imply their functional diversity as a mechanism for adapting to the environment. Our data provide a framework for further research on the molecular mechanisms of cassava resistance to drought stress and provide a foundation for breeding drought-resistant new cultivars. PMID:28542282

Contrasting dynamic responses in vivo of the Bcl-xL and Bim erythropoietic survival pathways

PubMed Central

Koulnis, Miroslav; Porpiglia, Ermelinda; Porpiglia, P. Alberto; Liu, Ying; Hallstrom, Kelly; Hidalgo, Daniel

2012-01-01

Survival signaling by the erythropoietin (Epo) receptor (EpoR) is essential for erythropoiesis and for its acceleration in hypoxic stress. Several apparently redundant EpoR survival pathways were identified in vitro, raising the possibility of their functional specialization in vivo. Here we used mouse models of acute and chronic stress, including a hypoxic environment and β-thalassemia, to identify two markedly different response dynamics for two erythroblast survival pathways in vivo. Induction of the antiapoptotic protein Bcl-xL is rapid but transient, while suppression of the proapoptotic protein Bim is slower but persistent. Similar to sensory adaptation, however, the Bcl-xL pathway “resets,” allowing it to respond afresh to acute stress superimposed on a chronic stress stimulus. Using “knock-in” mouse models expressing mutant EpoRs, we found that adaptation in the Bcl-xL response occurs because of adaptation of its upstream regulator Stat5, both requiring the EpoR distal cytoplasmic domain. We conclude that survival pathways show previously unsuspected functional specialization for the acute and chronic phases of the stress response. Bcl-xL induction provides a “stop-gap” in acute stress, until slower but permanent pathways are activated. Furthermore, pathologic elevation of Bcl-xL may be the result of impaired adaptation, with implications for myeloproliferative disease mechanisms. PMID:22086418

Integrated Transcriptomic and Epigenomic Analysis of Primary Human Lung Epithelial Cell Differentiation

PubMed Central

Marconett, Crystal N.; Zhou, Beiyun; Rieger, Megan E.; Selamat, Suhaida A.; Dubourd, Mickael; Fang, Xiaohui; Lynch, Sean K.; Stueve, Theresa Ryan; Siegmund, Kimberly D.; Berman, Benjamin P.

2013-01-01

Elucidation of the epigenetic basis for cell-type specific gene regulation is key to gaining a full understanding of how the distinct phenotypes of differentiated cells are achieved and maintained. Here we examined how epigenetic changes are integrated with transcriptional activation to determine cell phenotype during differentiation. We performed epigenomic profiling in conjunction with transcriptomic profiling using in vitro differentiation of human primary alveolar epithelial cells (AEC). This model recapitulates an in vivo process in which AEC transition from one differentiated cell type to another during regeneration following lung injury. Interrogation of histone marks over time revealed enrichment of specific transcription factor binding motifs within regions of changing chromatin structure. Cross-referencing of these motifs with pathways showing transcriptional changes revealed known regulatory pathways of distal alveolar differentiation, such as the WNT and transforming growth factor beta (TGFB) pathways, and putative novel regulators of adult AEC differentiation including hepatocyte nuclear factor 4 alpha (HNF4A), and the retinoid X receptor (RXR) signaling pathways. Inhibition of the RXR pathway confirmed its functional relevance for alveolar differentiation. Our incorporation of epigenetic data allowed specific identification of transcription factors that are potential direct upstream regulators of the differentiation process, demonstrating the power of this approach. Integration of epigenomic data with transcriptomic profiling has broad application for the identification of regulatory pathways in other models of differentiation. PMID:23818859

Compilation of mRNA Polyadenylation Signals in Arabidopsis Revealed a New Signal Element and Potential Secondary Structures1[w

PubMed Central

Loke, Johnny C.; Stahlberg, Eric A.; Strenski, David G.; Haas, Brian J.; Wood, Paul Chris; Li, Qingshun Quinn

2005-01-01

Using a novel program, SignalSleuth, and a database containing authenticated polyadenylation [poly(A)] sites, we analyzed the composition of mRNA poly(A) signals in Arabidopsis (Arabidopsis thaliana), and reevaluated previously described cis-elements within the 3′-untranslated (UTR) regions, including near upstream elements and far upstream elements. As predicted, there are absences of high-consensus signal patterns. The AAUAAA signal topped the near upstream elements patterns and was found within the predicted location to only approximately 10% of 3′-UTRs. More importantly, we identified a new set, named cleavage elements, of poly(A) signals flanking both sides of the cleavage site. These cis-elements were not previously revealed by conventional mutagenesis and are contemplated as a cluster of signals for cleavage site recognition. Moreover, a single-nucleotide profile scan on the 3′-UTR regions unveiled a distinct arrangement of alternate stretches of U and A nucleotides, which led to a prediction of the formation of secondary structures. Using an RNA secondary structure prediction program, mFold, we identified three main types of secondary structures on the sequences analyzed. Surprisingly, these observed secondary structures were all interrupted in previously constructed mutations in these regions. These results will enable us to revise the current model of plant poly(A) signals and to develop tools to predict 3′-ends for gene annotation. PMID:15965016

Particle Velocity Measuring System

NASA Technical Reports Server (NTRS)

Arndt, G. Dickey (Inventor); Carl, James R. (Inventor)

1998-01-01

Method and apparatus are provided for determining the velocity of individual food particles within a liquid/solid food mixture that is cooked by an aseptic cooking method whereby the food mixture is heated as it flows through a flowline. At least one upstream and at least one downstream microwave transducer are provided to determine the minimum possible travel time of the fastest food particle through the flowline. In one embodiment, the upstream detector is not required. In another embodiment, a plurality of small dipole antenna markers are secured to a plurality of food particles to provide a plurality of signals as the markers pass the upstream and downstream transducers. The dipole antenna markers may also include a non-linear element to reradiate a harmonic frequency of a transmitter frequency. Upstream and downstream transducers include dipole antennas that are matched to the impedance of the food slurry and a signal transmission cable by various impedance matching means including unbalanced feed to the antennas.

Involvement of cyclin D1/CDK4 and pRb mediated by PI3K/AKT pathway activation in Pb{sup 2+}-induced neuronal death in cultured hippocampal neurons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li Chenchen; Xing Tairan; Tang Mingliang

2008-06-15

Lead (Pb) is widely recognized as a neurotoxicant. One of the suggested mechanisms of lead neurotoxicity is apoptotic cell death. And the mechanism by which Pb{sup 2+} causes neuronal death is not well understood. The present study sought to examine the obligate nature of cyclin D1/cyclin-dependent kinase 4 (CDK4), phosphorylation of its substrate retinoblastoma protein (pRb) and its select upstream signal phosphoinositide 3-kinase (PI3K)/AKT pathway in the death of primary cultured rat hippocampal neurons evoked by Pb{sup 2+}. Our data showed that lead treatment of primary hippocampal cultures results in dose-dependent cell death. Inhibition of CDK4 prevented Pb{sup 2+}-induced neuronalmore » death significantly but was incomplete. In addition, we demonstrated that the levels of cyclin D1 and pRb/p107 were increased during Pb{sup 2+} treatment. These elevated expression persisted up to 48 h, returning to control levels after 72 h. We also presented pharmacological and morphological evidences that cyclin D1/CDK4 and pRb/p107 were required for such kind of neuronal death. Addition of the PI3K inhibitor LY294002 (30 {mu}M) or wortmannin (100 nM) significantly rescued the cultured hippocampal neurons from death caused by Pb{sup 2+}. And that Pb{sup 2+}-elicited phospho-AKT (Ser473) participated in the induction of cyclin D1 and partial pRb/p107 expression. These results provide evidences that cell cycle elements play a required role in the death of neurons evoked by Pb{sup 2+} and suggest that certain signaling elements upstream of cyclin D1/CDK4 are modified and/or required for this form of neuronal death.« less

Upstream kinases of plant SnRKs are involved in salt stress tolerance.

PubMed

Barajas-Lopez, Juan de Dios; Moreno, Jose Ramon; Gamez-Arjona, Francisco M; Pardo, Jose M; Punkkinen, Matleena; Zhu, Jian-Kang; Quintero, Francisco J; Fujii, Hiroaki

2018-01-01

Sucrose non-fermenting 1-related protein kinases (SnRKs) are important for plant growth and stress responses. This family has three clades: SnRK1, SnRK2 and SnRK3. Although plant SnRKs are thought to be activated by upstream kinases, the overall mechanism remains obscure. Geminivirus Rep-Interacting Kinase (GRIK)1 and GRIK2 phosphorylate SnRK1s, which are involved in sugar/energy sensing, and the grik1-1 grik2-1 double mutant shows growth retardation under regular growth conditions. In this study, we established another Arabidopsis mutant line harbouring a different allele of gene GRIK1 (grik1-2 grik2-1) that grows similarly to the wild-type, enabling us to evaluate the function of GRIKs under stress conditions. In the grik1-2 grik2-1 double mutant, phosphorylation of SnRK1.1 was reduced, but not eliminated, suggesting that the grik1-2 mutation is a weak allele. In addition to high sensitivity to glucose, the grik1-2 grik2-1 mutant was sensitive to high salt, indicating that GRIKs are also involved in salinity signalling pathways. Salt Overly Sensitive (SOS)2, a member of the SnRK3 subfamily, is a critical mediator of the response to salinity. GRIK1 phosphorylated SOS2 in vitro, resulting in elevated kinase activity of SOS2. The salt tolerance of sos2 was restored to normal levels by wild-type SOS2, but not by a mutated form of SOS2 lacking the T168 residue phosphorylated by GRIK1. Activation of SOS2 by GRIK1 was also demonstrated in a reconstituted system in yeast. Our results indicate that GRIKs phosphorylate and activate SnRK1 and other members of the SnRK3 family, and that they play important roles in multiple signalling pathways in vivo. © 2017 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

Diabetes abolishes the cardioprotection induced by sevoflurane postconditioning in the rat heart in vivo: roles of glycogen synthase kinase-3β and its upstream pathways.

PubMed

Tai, Wenjun; Shi, Enyi; Yan, Lihui; Jiang, Xiaojing; Ma, Hong; Ai, Chunyu

2012-11-01

We measured the cardioprotection afforded by sevoflurane postconditioning in streptozotocin-induced diabetic rats (DRs) and determined the roles of glycogen synthase kinase (GSK), phosphatidylinositol-3-kinase/Akt, and extracellular signal-regulated kinase (ERK1/2) in such a procedure. DRs and nondiabetic rats (NDRs) were subjected to a 30-min coronary artery occlusion followed by a 120-min reperfusion. Postconditioning was achieved by inhalation of 1 minimum alveolar concentration sevoflurane at the first 5 min of reperfusion. The infarct size was determined by triphenyltetrazolium chloride staining. Expressions of GSK-3β, Akt, and ERK1/2 were measured using Western blotting. In NDRs, the infarct size was significantly decreased from 53.4% ± 7.6% to 34.9% ± 5.6% by sevoflurane postconditioning (P < 0.01). Such an anti-infarct effect was abolished completely in the DRs, as evidenced by a similar infarct size observed between the sevoflurane-treated and untreated DRs (49.3% ± 8.6% and 49.6% ± 9.3%, respectively, P > 0.05). Direct inhibition of GSK-3β by injection of SB216763 just before the start of reperfusion induced equivalent infarct-sparing effects in both NDRs (37.8% ± 3.9% and 53.4% ± 7.6% in SB216763-treated and untreated NDRs, respectively; P < 0.01) and DRs (38.8% ± 3.2% and 49.3% ± 8.6% in SB216763-treated and untreated DRs, respectively; P < 0.05). Sevoflurane postconditioning remarkably enhanced the phosphorylation of GSK-3β Ser(9), Akt Ser(473), and ERK1/2 in NDRs, which were blocked in DRs. The cardioprotection induced by sevoflurane postconditioning is abolished by diabetes. This might be due to the impairment of phosphorylation of GSK-3β and its upstream signaling pathways of phosphatidylinositol-3-kinase/Akt and ERK1/2 in the presence of diabetes. Copyright © 2012 Elsevier Inc. All rights reserved.

Bisphosphonate Inhibitors Reveal a Large Elasticity of Plastidic Isoprenoid Synthesis Pathway in Isoprene-Emitting Hybrid Aspen1

PubMed Central

2015-01-01

Recently, a feedback inhibition of the chloroplastic 1-deoxy-d-xylulose 5-phosphate (DXP)/2-C-methyl-d-erythritol 4-phosphate (MEP) pathway of isoprenoid synthesis by end products dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP) was postulated, but the extent to which DMADP and IDP can build up is not known. We used bisphosphonate inhibitors, alendronate and zoledronate, that inhibit the consumption of DMADP and IDP by prenyltransferases to gain insight into the extent of end product accumulation and possible feedback inhibition in isoprene-emitting hybrid aspen (Populus tremula × Populus tremuloides). A kinetic method based on dark release of isoprene emission at the expense of substrate pools accumulated in light was used to estimate the in vivo pool sizes of DMADP and upstream metabolites. Feeding with fosmidomycin, an inhibitor of DXP reductoisomerase, alone or in combination with bisphosphonates was used to inhibit carbon input into DXP/MEP pathway or both input and output. We observed a major increase in pathway intermediates, 3- to 4-fold, upstream of DMADP in bisphosphonate-inhibited leaves, but the DMADP pool was enhanced much less, 1.3- to 1.5-fold. In combined fosmidomycin/bisphosphonate treatment, pathway intermediates accumulated, reflecting cytosolic flux of intermediates that can be important under strong metabolic pull in physiological conditions. The data suggested that metabolites accumulated upstream of DMADP consist of phosphorylated intermediates and IDP. Slow conversion of the huge pools of intermediates to DMADP was limited by reductive energy supply. These data indicate that the DXP/MEP pathway is extremely elastic, and the presence of a significant pool of phosphorylated intermediates provides an important valve for fine tuning the pathway flux. PMID:25926480

Protein Kinase C-δ Mediates Neuronal Apoptosis in the Retinas of Diabetic Rats via the Akt Signaling Pathway

PubMed Central

Kim, Young-Hee; Kim, Yoon-Sook; Park, Chang-Hwan; Chung, In-Yong; Yoo, Ji-Myong; Kim, Jae-Geun; Lee, Byung-Ju; Kang, Sang-Soo; Cho, Gyeong-Jae; Choi, Wan-Sung

2008-01-01

OBJECTIVE—Protein kinase C (PKC)-δ, an upstream regulator of the Akt survival pathway, contributes to cellular dysfunction in the pathogenesis of diabetes. Herein, we examined the role of PKC-δ in neuronal apoptosis through Akt in the retinas of diabetic rats. RESEARCH DESIGN AND METHODS—We used retinas from 24- and 35-week-old male Otsuka Long-Evans Tokushima fatty (OLETF) diabetic and Long-Evans Tokushima Otsuka (LETO) nondiabetic rats. To assess whether PKC-δ affects Akt signaling and cell death in OLETF rat retinas, we examined 1) PKC-δ activity and apoptosis; 2) protein levels of phosphatidylinositol 3-kinase (PI 3-kinase) p85, heat shock protein 90 (HSP90), and protein phosphatase 2A (PP2A); 3) Akt phosphorylation; and 4) Akt binding to HSP90 or PP2A in LETO and OLETF retinas in the presence or absence of rottlerin, a highly specific PKC-δ inhibitor, or small interfering RNAs (siRNAs) for PKC-δ and HSP90. RESULTS—In OLETF retinas from 35-week-old rats, ganglion cell death, PKC-δ and PP2A activity, and Akt-PP2A binding were significantly increased and Akt phosphorylation and Akt-HSP90 binding were decreased compared with retinas from 24-week-old OLETF and LETO rats. Rottlerin and PKC-δ siRNA abrogated these effects in OLETF retinas from 35-week-old rats. HSP90 siRNA significantly increased ganglion cell death and Akt-PP2A complexes and markedly decreased HSP90-Akt binding and Akt phosphorylation in LETO retinas from 35-week-old rats compared with those from nontreated LETO rats. CONCLUSIONS—PKC-δ activation contributes to neuro-retinal apoptosis in diabetic rats by inhibiting Akt-mediated signaling pathways. PMID:18443201

Glutathione peroxidase mimic ebselen improves glucose-stimulated insulin secretion in murine islets.

PubMed

Wang, Xinhui; Yun, Jun-Won; Lei, Xin Gen

2014-01-10

Glutathione peroxidase (GPX) mimic ebselen and superoxide dismutase (SOD) mimic copper diisopropylsalicylate (CuDIPs) were used to rescue impaired glucose-stimulated insulin secretion (GSIS) in islets of GPX1 and(or) SOD1-knockout mice. Ebselen improved GSIS in islets of all four tested genotypes. The rescue in the GPX1 knockout resulted from a coordinated transcriptional regulation of four key GSIS regulators and was mediated by the peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α)-mediated signaling pathways. In contrast, CuDIPs improved GSIS only in the SOD1 knockout and suppressed gene expression of the PGC-1α pathway. Islets from the GPX1 and(or) SOD1 knockout mice provided metabolically controlled intracellular hydrogen peroxide (H2O2) and superoxide conditions for the present study to avoid confounding effects. Bioinformatics analyses of gene promoters and expression profiles guided the search for upstream signaling pathways to link the ebselen-initiated H2O2 scavenging to downstream key events of GSIS. The RNA interference was applied to prove PGC-1α as the main mediator for that link. Our study revealed a novel metabolic use and clinical potential of ebselen in rescuing GSIS in the GPX1-deficient islets and mice, along with distinct differences between the GPX and SOD mimics in this regard. These findings highlight the necessities and opportunities of discretional applications of various antioxidant enzyme mimics in treating insulin secretion disorders. REBOUND TRACK: This work was rejected during standard peer review and rescued by Rebound Peer Review (Antioxid Redox Signal 16: 293-296, 2012) with the following serving as open reviewers: Regina Brigelius-Flohe, Vadim Gladyshev, Dexing Hou, and Holger Steinbrenner.

Phosphorylated 4EBP1 is associated with tumor progression and poor prognosis in Xp11.2 translocation renal cell carcinoma

PubMed Central

Qu, Yuanyuan; Zhao, Rui; Wang, Hongkai; Chang, Kun; Yang, Xiaoqun; Zhou, Xiaoyan; Dai, Bo; Zhu, Yao; Shi, Guohai; Zhang, Hailiang; Ye, Dingwei

2016-01-01

Two main signaling pathways, PI3K-AKT-mTOR and RAS-MAPK, are involved in transmitting the proliferative signals which play critical roles in human cancers. However, the functions of these pathways in Xp11.2 RCC remain undefined. This study aimed to explore the expression of mTOR and MAPK cascades in Xp11.2 RCC and to assess the prognostic significance of proteins evaluated. Immunohistochemistry was performed to evaluate the expression of 4EBP1, p-4EBP1, p-mTOR, p-S6K and p-MAPK in 36 adult Xp11.2 RCC patients who were confirmed by FISH assay. Cox regression models were used to evaluate the prognostic value of all covariates. Among 36 assessed patients, 14 (38.9%), 26 (72.2%), 16 (44.4%), 19 (52.8%), and 9 (25.0%) patients showed high expression of 4EBP1, p-4EBP1, p-mTOR, p-S6K, and p-MAPK, respectively. We noted that p-4EBP1 expression was significantly correlated with lymph node metastases (P = 0.027). Multivariate analysis showed that high p-4EBP1 expression was an independent adverse prognostic factor for both PFS (HR = 33.750, P = 0.017) and OS (HR = 56.111, P = 0.026). Our findings suggest that p-4EBP1 may serve as a funnel factor that converge the upstream proliferative oncogenic signals. Effective inhibition of the pathways responsible for 4E-BP1 phosphorylation might be a useful strategy to improve the outcome of Xp11.2 RCC patients. PMID:27026382

Phosphorylated 4EBP1 is associated with tumor progression and poor prognosis in Xp11.2 translocation renal cell carcinoma.

PubMed

Qu, Yuanyuan; Zhao, Rui; Wang, Hongkai; Chang, Kun; Yang, Xiaoqun; Zhou, Xiaoyan; Dai, Bo; Zhu, Yao; Shi, Guohai; Zhang, Hailiang; Ye, Dingwei

2016-03-30

Two main signaling pathways, PI3K-AKT-mTOR and RAS-MAPK, are involved in transmitting the proliferative signals which play critical roles in human cancers. However, the functions of these pathways in Xp11.2 RCC remain undefined. This study aimed to explore the expression of mTOR and MAPK cascades in Xp11.2 RCC and to assess the prognostic significance of proteins evaluated. Immunohistochemistry was performed to evaluate the expression of 4EBP1, p-4EBP1, p-mTOR, p-S6K and p-MAPK in 36 adult Xp11.2 RCC patients who were confirmed by FISH assay. Cox regression models were used to evaluate the prognostic value of all covariates. Among 36 assessed patients, 14 (38.9%), 26 (72.2%), 16 (44.4%), 19 (52.8%), and 9 (25.0%) patients showed high expression of 4EBP1, p-4EBP1, p-mTOR, p-S6K, and p-MAPK, respectively. We noted that p-4EBP1 expression was significantly correlated with lymph node metastases (P = 0.027). Multivariate analysis showed that high p-4EBP1 expression was an independent adverse prognostic factor for both PFS (HR = 33.750, P = 0.017) and OS (HR = 56.111, P = 0.026). Our findings suggest that p-4EBP1 may serve as a funnel factor that converge the upstream proliferative oncogenic signals. Effective inhibition of the pathways responsible for 4E-BP1 phosphorylation might be a useful strategy to improve the outcome of Xp11.2 RCC patients.

Calcium oxalate crystals induces tight junction disruption in distal renal tubular epithelial cells by activating ROS/Akt/p38 MAPK signaling pathway.

PubMed

Yu, Lei; Gan, Xiuguo; Liu, Xukun; An, Ruihua

2017-11-01

Tight junction plays important roles in regulating paracellular transports and maintaining cell polarity. Calcium oxalate monohydrate (COM) crystals, the major crystalline composition of kidney stones, have been demonstrated to be able to cause tight junction disruption to accelerate renal cell injury. However, the cellular signaling involved in COM crystal-induced tight junction disruption remains largely to be investigated. In the present study, we proved that COM crystals induced tight junction disruption by activating ROS/Akt/p38 MAPK pathway. Treating Madin-Darby canine kidney (MDCK) cells with COM crystals induced a substantial increasing of ROS generation and activation of Akt that triggered subsequential activation of ASK1 and p38 mitogen-activated protein kinase (MAPK). Western blot revealed a significantly decreased expression of ZO-1 and occludin, two important structural proteins of tight junction. Besides, redistribution and dissociation of ZO-1 were observed by COM crystals treatment. Inhibition of ROS by N-acetyl-l-cysteine (NAC) attenuated the activation of Akt, ASK1, p38 MAPK, and down-regulation of ZO-1 and occludin. The redistribution and dissociation of ZO-1 were also alleviated by NAC treatment. These results indicated that ROS were involved in the regulation of tight junction disruption induced by COM crystals. In addition, the down-regulation of ZO-1 and occludin, the phosphorylation of ASK1 and p38 MAPK were also attenuated by MK-2206, an inhibitor of Akt kinase, implying Akt was involved in the disruption of tight junction upstream of p38 MAPK. Thus, these results suggested that ROS-Akt-p38 MAPK signaling pathway was activated in COM crystal-induced disruption of tight junction in MDCK cells.

Adam10 Mediates the Choice between Principal Cells and Intercalated Cells in the Kidney

PubMed Central

Guo, Qiusha; Wang, Yinqiu; Tripathi, Piyush; Manda, Kalyan R.; Mukherjee, Malini; Chaklader, Malay; Austin, Paul F.

2015-01-01

A disintegrin and metalloproteinase domain 10 (Adam10), a member of the ADAM family of cell membrane–anchored proteins, has been linked to the regulation of the Notch, EGF, E-cadherin, and other signaling pathways. However, it is unclear what role Adam10 has in the kidney in vivo. In this study, we showed that Adam10 deficiency in ureteric bud (UB) derivatives leads to a decrease in urinary concentrating ability, polyuria, and hydronephrosis in mice. Furthermore, Adam10 deficiency led to a reduction in the percentage of aquaporin 2 (Aqp2)+ principal cells (PCs) in the collecting ducts that was accompanied by a proportional increase in the percentage of intercalated cells (ICs). This increase was more prominent in type A ICs than in type B ICs. Foxi1, a transcription factor important for the differentiation of ICs, was upregulated in the Adam10 mutants. The observed reduction of Notch activity in Adam10 mutant collecting duct epithelium and the similar reduction of PC/IC ratios in the collecting ducts in mice deficient for mindbomb E3 ubiquitin protein ligase 1, a key regulator of the Notch and Wnt/receptor-like tyrosine kinase signaling pathways, suggest that Adam10 regulates cell fate determination through the activation of Notch signaling, probably through the regulation of Foxi1 expression. However, phenotypic differences between the Adam10 mutants, the Mib1 mutants, and the Foxi1 mutants suggest that the functions of Adam10 in determining the fate of collecting duct cells are more complex than those of a simple upstream factor in a linear pathway involving Notch and Foxi1. PMID:24904084

Recruitment of the proneural gene scute to the Drosophila sex-determination pathway.

PubMed Central

Wrischnik, Lisa A; Timmer, John R; Megna, Lisa A; Cline, Thomas W

2003-01-01

In flies, scute (sc) works with its paralogs in the achaete-scute-complex (ASC) to direct neuronal development. However, in the family Drosophilidae, sc also acquired a role in the primary event of sex determination, X chromosome counting, by becoming an X chromosome signal element (XSE)-an evolutionary step shown here to have occurred after sc diverged from its closest paralog, achaete (ac). Two temperature-sensitive alleles, sc(sisB2) and sc(sisB3), which disrupt only sex determination, were recovered in a powerful F1 genetic selection and used to investigate how sc was recruited to the sex-determination pathway. sc(sisB2) revealed 3' nontranscribed regulatory sequences likely to be involved. The sc(sisB2) lesion abolished XSE activity when combined with mutations engineered in a sequence upstream of all XSEs. In contrast, changes in Sc protein sequence seem not to have been important for recruitment. The observation that the other new allele, sc(sisB3), eliminates the C-terminal half of Sc without affecting neurogenesis and that sc(sisB1), the most XSE-specific allele previously available, is a nonsense mutant, would seem to suggest the opposite, but we show that housefly Sc can substitute for fruit fly Sc in sex determination, despite lacking Drosophilidae-specific conserved residues in its C-terminal half. Lack of synergistic lethality among mutations in sc, twist, and dorsal argue against a proposed role for sc in mesoderm formation that had seemed potentially relevant to sex-pathway recruitment. The screen that yielded new sc alleles also generated autosomal duplications that argue against the textbook view that fruit fly sex signal evolution recruited a set of autosomal signal elements comparable to the XSEs. PMID:14704182

Leflunomide or A77 1726 protect from acetaminophen-induced cell injury through inhibition of JNK-mediated mitochondrial permeability transition in immortalized human hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Latchoumycandane, Calivarathan; Seah, Quee Ming; Tan, Rachel C.H.

2006-11-15

Leflunomide, a disease-modifying anti-rheumatic drug, protects against T-cell-mediated liver injury by poorly understood mechanisms. The active metabolite of leflunomide, A77 1726 (teriflunomide) has been shown to inhibit stress-activated protein kinases (JNK pathway), which are key regulators of mitochondria-mediated cell death. Therefore, we hypothesized that leflunomide may protect from drugs that induce the mitochondrial permeability transition (mPT) by blocking the JNK signaling pathway. To this end, we exposed cultured immortalized human hepatocytes (HC-04) to the standard protoxicant drug acetaminophen (APAP), which induces CsA-sensitive mPT-mediated cell death. We determined the effects of leflunomide on the extent of APAP-induced hepatocyte injury and themore » upstream JNK-mediated mitochondrial signaling pathways. We found that leflunomide or A77 1726 concentration-dependently protected hepatocytes from APAP (1 mM)-induced mitochondrial permeabilization and lethal cell injury. This was not due to proximal inhibition of CYP-catalyzed APAP bioactivation to its thiol-reactive metabolite. Instead, we demonstrate that leflunomide (20 {mu}M) inhibited the APAP-induced early (3 h) activation (phosphorylation) of JNK1/2, thus inhibiting phosphorylation of the anti-apoptotic protein Bcl-2 and preventing P-Bcl-2-mediated induction of the mPT. This greatly attenuated mitochondrial cytochrome c release, which we used as a marker for mitochondrial permeabilization. The specific JNK2 inhibitor SP600125 similarly protected from APAP-induced cell death. In conclusion, these findings are consistent with our hypothesis that leflunomide protects from protoxicant-induced hepatocyte injury by inhibiting JNK signaling and preventing mPT induction.« less

A New Strategy to Control and Eradicate “Undruggable” Oncogenic K-RAS-Driven Pancreatic Cancer: Molecular Insights and Core Principles Learned from Developmental and Evolutionary Biology

PubMed Central

Lee, Michael P.; Lee, Caroline Dasom; Lafever, Alex C.; Svyatova, Elizaveta; Kanda, Kevin; Collier, Amber L.; Siewertsz van Reesema, Lauren L.; Tang-Tan, Angela M.; Zheleva, Vasilena; Bwayi, Monicah N.; Bian, Minglei; Schmidt, Rebecca L.; Petersen, Gloria M.

2018-01-01

Oncogenic K-RAS mutations are found in virtually all pancreatic cancers, making K-RAS one of the most targeted oncoproteins for drug development in cancer therapies. Despite intense research efforts over the past three decades, oncogenic K-RAS has remained largely “undruggable”. Rather than targeting an upstream component of the RAS signaling pathway (i.e., EGFR/HER2) and/or the midstream effector kinases (i.e., RAF/MEK/ERK/PI3K/mTOR), we propose an alternative strategy to control oncogenic K-RAS signal by targeting its most downstream signaling module, Seven-In-Absentia Homolog (SIAH). SIAH E3 ligase controls the signal output of oncogenic K-RAS hyperactivation that drives unchecked cell proliferation, uncontrolled tumor growth, and rapid cancer cell dissemination in human pancreatic cancer. Therefore, SIAH is an ideal therapeutic target as it is an extraordinarily conserved downstream signaling gatekeeper indispensable for proper RAS signaling. Guided by molecular insights and core principles obtained from developmental and evolutionary biology, we propose an anti-SIAH-centered anti-K-RAS strategy as a logical and alternative anticancer strategy to dampen uncontrolled K-RAS hyperactivation and halt tumor growth and metastasis in pancreatic cancer. The clinical utility of developing SIAH as both a tumor-specific and therapy-responsive biomarker, as well as a viable anti-K-RAS drug target, is logically simple and conceptually innovative. SIAH clearly constitutes a major tumor vulnerability and K-RAS signaling bottleneck in pancreatic ductal adenocarcinoma (PDAC). Given the high degree of evolutionary conservation in the K-RAS/SIAH signaling pathway, an anti-SIAH-based anti-PDAC therapy will synergize with covalent K-RAS inhibitors and direct K-RAS targeted initiatives to control and eradicate pancreatic cancer in the future. PMID:29757973

Phospholipase Cϵ Scaffolds to Muscle-specific A Kinase Anchoring Protein (mAKAPβ) and Integrates Multiple Hypertrophic Stimuli in Cardiac Myocytes*

PubMed Central

Zhang, Lianghui; Malik, Sundeep; Kelley, Grant G.; Kapiloff, Michael S.; Smrcka, Alan V.

2011-01-01

To define a role for phospholipase Cϵ (PLCϵ) signaling in cardiac myocyte hypertrophic growth, PLCϵ protein was depleted from neonatal rat ventricular myocytes (NRVMs) using siRNA. NRVMs with PLCϵ depletion were stimulated with endothelin (ET-1), norepinephrine, insulin-like growth factor-1 (IGF-1), or isoproterenol and assessed for development of hypertrophy. PLCϵ depletion dramatically reduced hypertrophic growth and gene expression induced by all agonists tested. PLCϵ catalytic activity was required for hypertrophy development, yet PLCϵ depletion did not reduce global agonist-stimulated inositol phosphate production, suggesting a requirement for localized PLC activity. PLCϵ was found to be scaffolded to a muscle-specific A kinase anchoring protein (mAKAPβ) in heart and NRVMs, and mAKAPβ localizes to the nuclear envelope in NRVMs. PLCϵ-mAKAP interaction domains were defined and overexpressed to disrupt endogenous mAKAPβ-PLCϵ complexes in NRVMs, resulting in significantly reduced ET-1-dependent NRVM hypertrophy. We propose that PLCϵ integrates multiple upstream signaling pathways to generate local signals at the nucleus that regulate hypertrophy. PMID:21550986

Scalloped and Yorkie are required for cell cycle re-entry of quiescent cells after tissue damage.

PubMed

Meserve, Joy H; Duronio, Robert J

2015-08-15

Regeneration of damaged tissues typically requires a population of active stem cells. How damaged tissue is regenerated in quiescent tissues lacking a stem cell population is less well understood. We used a genetic screen in the developing Drosophila melanogaster eye to investigate the mechanisms that trigger quiescent cells to re-enter the cell cycle and proliferate in response to tissue damage. We discovered that Hippo signaling regulates compensatory proliferation after extensive cell death in the developing eye. Scalloped and Yorkie, transcriptional effectors of the Hippo pathway, drive Cyclin E expression to induce cell cycle re-entry in cells that normally remain quiescent in the absence of damage. Ajuba, an upstream regulator of Hippo signaling that functions as a sensor of epithelial integrity, is also required for cell cycle re-entry. Thus, in addition to its well-established role in modulating proliferation during periods of tissue growth, Hippo signaling maintains homeostasis by regulating quiescent cell populations affected by tissue damage. © 2015. Published by The Company of Biologists Ltd.

Myelination and mTOR

PubMed Central

Figlia, Gianluca; Gerber, Daniel

2017-01-01

Abstract Myelinating cells surround axons to accelerate the propagation of action potentials, to support axonal health, and to refine neural circuits. Myelination is metabolically demanding and, consistent with this notion, mTORC1—a signaling hub coordinating cell metabolism—has been implicated as a key signal for myelination. Here, we will discuss metabolic aspects of myelination, illustrate the main metabolic processes regulated by mTORC1, and review advances on the role of mTORC1 in myelination of the central nervous system and the peripheral nervous system. Recent progress has revealed a complex role of mTORC1 in myelinating cells that includes, besides positive regulation of myelin growth, additional critical functions in the stages preceding active myelination. Based on the available evidence, we will also highlight potential nonoverlapping roles between mTORC1 and its known main upstream pathways PI3K‐Akt, Mek‐Erk1/2, and AMPK in myelinating cells. Finally, we will discuss signals that are already known or hypothesized to be responsible for the regulation of mTORC1 activity in myelinating cells. PMID:29210103

The function of the Mediator complex in plant immunity.

PubMed

An, Chuanfu; Mou, Zhonglin

2013-03-01

Upon pathogen infection, plants undergo dramatic transcriptome reprogramming to shift from normal growth and development to immune response. During this rapid process, the multiprotein Mediator complex has been recognized as an important player to fine-tune gene-specific and pathway-specific transcriptional reprogramming by acting as an adaptor/coregulator between sequence-specific transcription factor and RNA polymerase II (RNAPII). Here, we review current understanding of the role of five functionally characterized Mediator subunits (MED8, MED15, MED16, MED21 and MED25) in plant immunity. All these Mediator subunits positively regulate resistance against leaf-infecting biotrophic bacteria or necrotrophic fungi. While MED21 appears to regulate defense against fungal pathogens via relaying signals from upstream regulators and chromatin modification to RNAPII, the other four Mediator subunits locate at different positions of the defense network to convey phytohormone signal(s). Fully understanding the role of Mediator in plant immunity needs to characterize more Mediator subunits in both Arabidopsis and other plant species. Identification of interacting proteins of Mediator subunits will further help to reveal their specific regulatory mechanisms in plant immunity.

Mitochondrial Energy and Redox Signaling in Plants

PubMed Central

Schwarzländer, Markus

2013-01-01

Abstract Significance: For a plant to grow and develop, energy and appropriate building blocks are a fundamental requirement. Mitochondrial respiration is a vital source for both. The delicate redox processes that make up respiration are affected by the plant's changing environment. Therefore, mitochondrial regulation is critically important to maintain cellular homeostasis. This involves sensing signals from changes in mitochondrial physiology, transducing this information, and mounting tailored responses, by either adjusting mitochondrial and cellular functions directly or reprogramming gene expression. Recent Advances: Retrograde (RTG) signaling, by which mitochondrial signals control nuclear gene expression, has been a field of very active research in recent years. Nevertheless, no mitochondrial RTG-signaling pathway is yet understood in plants. This review summarizes recent advances toward elucidating redox processes and other bioenergetic factors as a part of RTG signaling of plant mitochondria. Critical Issues: Novel insights into mitochondrial physiology and redox-regulation provide a framework of upstream signaling. On the other end, downstream responses to modified mitochondrial function have become available, including transcriptomic data and mitochondrial phenotypes, revealing processes in the plant that are under mitochondrial control. Future Directions: Drawing parallels to chloroplast signaling and mitochondrial signaling in animal systems allows to bridge gaps in the current understanding and to deduce promising directions for future research. It is proposed that targeted usage of new technical approaches, such as quantitative in vivo imaging, will provide novel leverage to the dissection of plant mitochondrial signaling. Antioxid. Redox Signal. 18, 2122–2144. PMID:23234467

Potential upstream regulators of cannabinoid receptor 1 signaling in prostate cancer: a Bayesian network analysis of data from a tissue microarray.

PubMed

Häggström, Jenny; Cipriano, Mariateresa; Forshell, Linus Plym; Persson, Emma; Hammarsten, Peter; Stella, Nephi; Fowler, Christopher J

2014-08-01

The endocannabinoid system regulates cancer cell proliferation, and in prostate cancer a high cannabinoid CB1 receptor expression is associated with a poor prognosis. Down-stream mediators of CB1 receptor signaling in prostate cancer are known, but information on potential upstream regulators is lacking. Data from a well-characterized tumor tissue microarray were used for a Bayesian network analysis using the max-min hill-climbing method. In non-malignant tissue samples, a directionality of pEGFR (the phosphorylated form of the epidermal growth factor receptor) → CB1 receptors were found regardless as to whether the endocannabinoid metabolizing enzyme fatty acid amide hydrolase (FAAH) was included as a parameter. A similar result was found in the tumor tissue, but only when FAAH was included in the analysis. A second regulatory pathway, from the growth factor receptor ErbB2 → FAAH was also identified in the tumor samples. Transfection of AT1 prostate cancer cells with CB1 receptors induced a sensitivity to the growth-inhibiting effects of the CB receptor agonist CP55,940. The sensitivity was not dependent upon the level of receptor expression. Thus a high CB1 receptor expression alone does not drive the cells towards a survival phenotype in the presence of a CB receptor agonist. The data identify two potential regulators of the endocannabinoid system in prostate cancer and allow the construction of a model of a dysregulated endocannabinoid signaling network in this tumor. Further studies should be designed to test the veracity of the predictions of the network analysis in prostate cancer and other solid tumors. © 2014 The Authors. The Prostate published by Wiley Periodicals, Inc.

Modeled Microgravity-Induced Protein Kinase C Isoform Expression in Human Lymphocytes

NASA Technical Reports Server (NTRS)

Sundaresan, A.; Risin, D.; Pellis, N. R.

2003-01-01

In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited both in microgravity and modeled microgravity (MMG) as reflected in diminished DNA synthess in peripheral blood lymphocytes and their locomotion through gelled type 1 collagen. Direct activation of Protein Kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 19 and MMG-culture. Human lymphocytes were cultured and harvested at 24, 48, 72 and 96 hours and serial samples assessed for locomotion using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta and -epsilon was assessed by RT-PCR, flow cytometry and immunoblotting. Results indicated that PKC isoforms delta and epsilon were down-regulated by more than 50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 19 controls. Events upstream of PKC such as phosphorylation of Phospholipase C(gamma) (PLC-gamma) in MMG, revealed accumulation of inactive enzyme. Depressed Ca++ -independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than, but after ligand-receptor interaction. Keywords: Signal transduction, locomotion, immunity

The daidzein metabolite, 6,7,4'-Trihydroxyisoflavone, is a novel inhibitor of PKCα in suppressing solar UV-induced matrix metalloproteinase 1.

PubMed

Lim, Tae-Gyu; Kim, Jong-Eun; Lee, Sung-Young; Park, Jun Seong; Yeom, Myung Hun; Chen, Hanyong; Bode, Ann M; Dong, Zigang; Lee, Ki Won

2014-11-19

Soy isoflavone is an attractive source of functional cosmetic materials with anti-wrinkle, whitening and skin hydration effects. After consumption, the majority of soy isoflavones are converted to their metabolites in the human gastrointestinal tract. To understand the physiological impact of soy isoflavone on the human body, it is necessary to evaluate and address the biological function of its metabolites. In this study, we investigated the effect of 6,7,4'-trihydroxyisoflavone (6,7,4'-THIF), a major metabolite of daidzein, against solar UV (sUV)-induced matrix metalloproteinases (MMPs) in normal human dermal fibroblasts. MMPs play a critical role in the degradation of collagen in skin, thereby accelerating the aging process of skin. The mitogen-activated protein/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MKK)3/6/p38 and MKK4/c-Jun N-terminal kinases (JNK) signaling pathways are known to modulate MMP-1 function, and their activation by sUV was significantly reduced by 6,7,4'-THIF pretreatment. Our results also indicated that the enzyme activity of protein kinase C (PKC)α, an upstream regulator of MKKs signaling, is suppressed by 6,7,4'-THIF using the in vitro kinase assay. Furthermore, the direct interaction between 6,7,4'-THIF and endogenous PKCα was confirmed using the pull-down assay. Not only sUV-induced MMP-1 expression, but also sUV-induced signaling pathway activation were decreased in PKCα knockdown cells. Overall, we elucidated the inhibitory effect of 6,7,4'-THIF on sUV-induced MMPs and suggest PKCα as its direct molecular target.

A novel pre-clinical strategy for identifying cardiotoxic kinase inhibitors and mechanisms of cardiotoxicity

PubMed Central

Cheng, Hui; Kari, Gabor; Dicker, Adam P; Rodeck, Ulrich; Koch, Walter J; Force, Thomas

2011-01-01

Rationale 1) Despite intense interest in strategies to predict which kinase inhibitor (KI) cancer therapeutics may be associated with cardiotoxicity, current approaches are inadequate. 2) Sorafenib is a KI of concern since it inhibits growth factor receptors and Raf-1/B-Raf, kinases that are upstream of ERKs and signal cardiomyocyte survival in the setting of stress. Objectives 1) Explore the potential use of zebrafish as a pre-clinical model to predict cardiotoxicity. 2) Determine whether sorafenib has associated cardiotoxicity and, if so, define the mechanisms. Methods and Results We find that the zebrafish model is readily able to discriminate a KI with little or no cardiotoxicity (gefitinib) from one with demonstrated cardiotoxicity (sunitinib). Sorafenib, like sunitinib, leads to cardiomyocyte apoptosis, a reduction in total myocyte number per heart, contractile dysfunction and ventricular dilatation in zebrafish. In cultured rat cardiomyocytes, sorafenib induces cell death. This can be rescued by adenovirus-mediated gene transfer of constitutively active MEK1 which restores ERK activity even in the presence of sorafenib. While growth factor-induced activation of ERKs requires Raf, α-adrenergic agonist-induced activation of ERKs does not. Consequently, activation of α-adrenergic signaling markedly decreases sorafenib-induced cell death. Consistent with these in vitro data, inhibition of α-adrenergic signaling with the receptor antagonist prazosin worsens sorafenib-induced cardiomyopathy in zebrafish. Conclusions 1) Zebrafish may be a valuable pre-clinical tool to predict cardiotoxicity. 2) The α-adrenergic signaling pathway is an important modulator of sorafenib cardiotoxicity in vitro and in vivo and appears to act via a here-to-fore unrecognized signaling pathway downstream of α-adrenergic activation that bypasses Raf to activate ERKs. PMID:21998323

Sestrin-2, a repressor of PDGFRβ signalling, promotes cigarette-smoke-induced pulmonary emphysema in mice and is upregulated in individuals with COPD

PubMed Central

Heidler, Juliana; Fysikopoulos, Athanasios; Wempe, Frank; Seimetz, Michael; Bangsow, Thorsten; Tomasovic, Ana; Veit, Florian; Scheibe, Susan; Pichl, Alexandra; Weisel, Friederike; Lloyd, K. C. Kent; Jaksch, Peter; Klepetko, Walter; Weissmann, Norbert; von Melchner, Harald

2013-01-01

SUMMARY Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. COPD is caused by chronic exposure to cigarette smoke and/or other environmental pollutants that are believed to induce reactive oxygen species (ROS) that gradually disrupt signalling pathways responsible for maintaining lung integrity. Here we identify the antioxidant protein sestrin-2 (SESN2) as a repressor of PDGFRβ signalling, and PDGFRβ signalling as an upstream regulator of alveolar maintenance programmes. In mice, the mutational inactivation of Sesn2 prevents the development of cigarette-smoke-induced pulmonary emphysema by upregulating PDGFRβ expression via a selective accumulation of intracellular superoxide anions (O2−). We also show that SESN2 is overexpressed and PDGFRβ downregulated in the emphysematous lungs of individuals with COPD and to a lesser extent in human lungs of habitual smokers without COPD, implicating a negative SESN2-PDGFRβ interrelationship in the pathogenesis of COPD. Taken together, our results imply that SESN2 could serve as both a biomarker and as a drug target in the clinical management of COPD. PMID:24046361

A Role for Barley CRYPTOCHROME1 in Light Regulation of Grain Dormancy and Germination[W][OPEN

PubMed Central

Barrero, Jose M.; Downie, A. Bruce; Xu, Qian; Gubler, Frank

2014-01-01

It is well known that abscisic acid (ABA) plays a central role in the regulation of seed dormancy and that transcriptional regulation of genes encoding ABA biosynthetic and degradation enzymes is responsible for determining ABA content. However, little is known about the upstream signaling pathways impinging on transcription to ultimately regulate ABA content or how environmental signals (e.g., light and cold) might direct such expression in grains. Our previous studies indicated that light is a key environmental signal inhibiting germination in dormant grains of barley (Hordeum vulgare), wheat (Triticum aestivum), and Brachypodium distachyon and that this effect attenuates as after-ripening progresses further. We found that the blue component of the light spectrum inhibits completion of germination in barley by inducing the expression of the ABA biosynthetic gene 9-cis-epoxycarotenoid dioxygenase and dampening expression of ABA 8’-hydroxylase, thus increasing ABA content in the grain. We have now created barley transgenic lines downregulating the genes encoding the blue light receptors CRYTOCHROME (CRY1) and CRY2. Our results demonstrate that CRY1 is the key receptor perceiving and transducing the blue light signal in dormant grains. PMID:24642944

Abscisic Acid–Responsive Guard Cell Metabolomes of Arabidopsis Wild-Type and gpa1 G-Protein Mutants[C][W

PubMed Central

Jin, Xiaofen; Wang, Rui-Sheng; Zhu, Mengmeng; Jeon, Byeong Wook; Albert, Reka; Chen, Sixue; Assmann, Sarah M.

2013-01-01

Individual metabolites have been implicated in abscisic acid (ABA) signaling in guard cells, but a metabolite profile of this specialized cell type is lacking. We used liquid chromatography–multiple reaction monitoring mass spectrometry for targeted analysis of 85 signaling-related metabolites in Arabidopsis thaliana guard cell protoplasts over a time course of ABA treatment. The analysis utilized ∼350 million guard cell protoplasts from ∼30,000 plants of the Arabidopsis Columbia accession (Col) wild type and the heterotrimeric G-protein α subunit mutant, gpa1, which has ABA-hyposensitive stomata. These metabolomes revealed coordinated regulation of signaling metabolites in unrelated biochemical pathways. Metabolites clustered into different temporal modules in Col versus gpa1, with fewer metabolites showing ABA-altered profiles in gpa1. Ca2+-mobilizing agents sphingosine-1-phosphate and cyclic adenosine diphosphate ribose exhibited weaker ABA-stimulated increases in gpa1. Hormone metabolites were responsive to ABA, with generally greater responsiveness in Col than in gpa1. Most hormones also showed different ABA responses in guard cell versus mesophyll cell metabolomes. These findings suggest that ABA functions upstream to regulate other hormones, and are also consistent with G proteins modulating multiple hormonal signaling pathways. In particular, indole-3-acetic acid levels declined after ABA treatment in Col but not gpa1 guard cells. Consistent with this observation, the auxin antagonist α-(phenyl ethyl-2-one)-indole-3-acetic acid enhanced ABA-regulated stomatal movement and restored partial ABA sensitivity to gpa1. PMID:24368793

Leptin activates hypothalamic acetyl-CoA carboxylase to inhibit food intake

PubMed Central

Gao, Su; Kinzig, Kimberly P.; Aja, Susan; Scott, Karen A.; Keung, Wendy; Kelly, Sandra; Strynadka, Ken; Chohnan, Shigeru; Smith, Wanli W.; Tamashiro, Kellie L. K.; Ladenheim, Ellen E.; Ronnett, Gabriele V.; Tu, Yajun; Birnbaum, Morris J.; Lopaschuk, Gary D.; Moran, Timothy H.

2007-01-01

Hypothalamic fatty acid metabolism has recently been implicated in the controls of food intake and energy homeostasis. We report that intracerebroventricular (ICV) injection of leptin, concomitant with inhibiting AMP-activated kinase (AMPK), activates acetyl-CoA carboxylase (ACC), the key regulatory enzyme in fatty acid biosynthesis, in the arcuate nucleus (Arc) and paraventricular nucleus (PVN) in the hypothalamus. Arc overexpression of constitutively active AMPK prevents the Arc ACC activation in response to ICV leptin, supporting the hypothesis that AMPK lies upstream of ACC in leptin's Arc intracellular signaling pathway. Inhibiting hypothalamic ACC with 5-tetradecyloxy-2-furoic acid, a specific ACC inhibitor, blocks leptin-mediated decreases in food intake, body weight, and mRNA level of the orexigenic neuropeptide NPY. These results show that hypothalamic ACC activation makes an important contribution to leptin's anorectic effects. Furthermore, we find that ICV leptin up-regulates the level of malonyl-CoA (the intermediate of fatty acid biosynthesis) specifically in the Arc and increases the level of palmitoyl-CoA (a major product of fatty acid biosynthesis) specifically in the PVN. The rises of both levels are blocked by 5-tetradecyloxy-2-furoic acid along with the blockade of leptin-mediated hypophagia. These data suggest malonyl-CoA as a downstream mediator of ACC in leptin's signaling pathway in the Arc and imply that palmitoyl-CoA, instead of malonyl-CoA, could be an effector in relaying ACC signaling in the PVN. Together, these findings highlight site-specific impacts of hypothalamic ACC activation in leptin's anorectic signaling cascade. PMID:17956983

The UPR reduces glucose metabolism via IRE1 signaling.

PubMed

van der Harg, Judith M; van Heest, Jessica C; Bangel, Fabian N; Patiwael, Sanne; van Weering, Jan R T; Scheper, Wiep

2017-04-01

Neurons are highly dependent on glucose. A disturbance in glucose homeostasis therefore poses a severe risk that is counteracted by activation of stress responses to limit damage and restore the energy balance. A major stress response that is activated under conditions of glucose deprivation is the unfolded protein response (UPR) that is aimed to restore proteostasis in the endoplasmic reticulum. The key signaling of the UPR involves the transient activation of a transcriptional program and an overall reduction of protein synthesis. Since the UPR is strategically positioned to sense and integrate metabolic stress signals, it is likely that - apart from its adaptive response to restore proteostasis - it also directly affects metabolic pathways. Here we investigate the direct role of the UPR in glucose homeostasis. O-GlcNAc is a post-translational modification that is highly responsive to glucose fluctuations. We find that UPR activation results in decreased O-GlcNAc modification, in line with reduced glucose metabolism. Our data indicate that UPR activation has no direct impact on the upstream processes in glucose metabolism; glucose transporter expression, glucose uptake and hexokinase activity. In contrast, prolonged UPR activation decreases glycolysis and mitochondrial metabolism. Decreased mitochondrial respiration is not accompanied by apoptosis or a structural change in mitochondria indicating that the reduction in metabolic rate upon UPR activation is a physiological non-apoptotic response. Metabolic decrease is prevented if the IRE1 pathway of the UPR is inhibited. This indicates that activation of IRE1 signaling induces a reduction in glucose metabolism, as part of an adaptive response. Copyright © 2017 Elsevier B.V. All rights reserved.

The regulation of vascular endothelial growth factor-induced microvascular permeability requires Rac and reactive oxygen species.

PubMed

Monaghan-Benson, Elizabeth; Burridge, Keith

2009-09-18

Vascular permeability is a complex process involving the coordinated regulation of multiple signaling pathways in the endothelial cell. It has long been documented that vascular endothelial growth factor (VEGF) greatly enhances microvascular permeability; however, the molecular mechanisms controlling VEGF-induced permeability remain unknown. Treatment of microvascular endothelial cells with VEGF led to an increase in reactive oxygen species (ROS) production. ROS are required for VEGF-induced permeability as treatment with the free radical scavenger, N-acetylcysteine, inhibited this effect. Additionally, treatment with VEGF caused ROS-dependent tyrosine phosphorylation of both vascular-endothelial (VE)-cadherin and beta-catenin. Rac1 was required for the VEGF-induced increase in permeability and adherens junction protein phosphorylation. Knockdown of Rac1 inhibited VEGF-induced ROS production consistent with Rac lying upstream of ROS in this pathway. Collectively, these data suggest that VEGF leads to a Rac-mediated generation of ROS, which, in turn, elevates the tyrosine phosphorylation of VE-cadherin and beta-catenin, ultimately regulating adherens junction integrity.

Crosstalk between AhR and wnt/β-catenin signal pathways in the cardiac developmental toxicity of PM2.5 in zebrafish embryos.

PubMed

Zhang, Hang; Yao, Yugang; Chen, Yang; Yue, Cong; Chen, Jiahong; Tong, Jian; Jiang, Yan; Chen, Tao

2016-04-29

Recent studies have shown an association between congenital heart defects and air fine particle matter (PM2.5), but the molecular mechanisms remain elusive. It is well known that a number of organic compounds in PM2.5 can act as AhR agonists, and activation of AhR can antagonize Wnt/β-catenin signaling. Therefore, we hypothesized that PM2.5 could activate AhR and then repress the expression of wnt/β-catenin targeted genes essential for cardiogenesis, resulting in heart defects. To test this hypothesis, we investigated the effects of extractable organic matter (EOM) from PM2.5 on AhR and Wnt/β-catenin signal pathways in zebrafish embryos. We confirmed that EOM could cause malformations in the heart and decreased heart rate in zebrafish embryos at 72hpf, and found that the EOM-induced heart defects were rescued in embryos co-exposed with EOM plus AhR antagonist CH223191 or β-catenin agonist CHIR99021. We further found that EOM had increased the expression levels of AhR targeted genes (Cyp1a1, Cyp1b1 and Ahrra) and reduced the mRNA levels of β-catenin targeted genes (axin2, nkx2.5 and sox9b). The mRNA expression level of Rspo2, a β-catenin upstream gene, was also decreased in embryos exposed to EOM. Supplementation with CH223191 or CHIR99021 attenuated most of the EOM-induced expression changes of genes involved in both AhR and wnt/β-catenin signal pathways. However, the mRNA expression level of AhR inhibitor Ahrrb, which did not change by EOM treatment alone, was increased in embryos co-exposed to EOM plus CH223191 or CHIR99021. We conclude that the activation of AhR by EOM from PM2.5 might repress wnt/β-catenin signaling, leading to heart defects in zebrafish embryos. Furthermore, our results indicate that the cardiac developmental toxicity of PM2.5 might be prevented by targeting AhR or wnt/β-catenin signaling. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Quorum Sensing Signaling Molecules Produced by Reference and Emerging Soft-Rot Bacteria (Dickeya and Pectobacterium spp.)

PubMed Central

Crépin, Alexandre; Barbey, Corinne; Beury-Cirou, Amélie; Hélias, Valérie; Taupin, Laure; Reverchon, Sylvie; Nasser, William; Faure, Denis; Dufour, Alain; Orange, Nicole; Feuilloley, Marc; Heurlier, Karin; Burini, Jean-François; Latour, Xavier

2012-01-01

Background Several small diffusible molecules are involved in bacterial quorum sensing and virulence. The production of autoinducers-1 and -2, quinolone, indole and γ-amino butyrate signaling molecules was investigated in a set of soft-rot bacteria belonging to six Dickeya or Pectobacterium species including recent or emerging potato isolates. Methodology/Principal Findings Using bacterial biosensors, immunoassay, and chromatographic analysis, we showed that soft-rot bacteria have the common ability to produce transiently during their exponential phase of growth the N-3-oxo-hexanoyl- or the N-3-oxo-octanoyl-l-homoserine lactones and a molecule of the autoinducer-2 family. Dickeya spp. produced in addition the indole-3-acetic acid in tryptophan-rich conditions. All these signaling molecules have been identified for the first time in the novel Dickeya solani species. In contrast, quinolone and γ-amino butyrate signals were not identified and the corresponding synthases are not present in the available genomes of soft-rot bacteria. To determine if the variations of signal production according to growth phase could result from expression modifications of the corresponding synthase gene, the respective mRNA levels were estimated by reverse transcriptase-PCR. While the N-acyl-homoserine lactone production is systematically correlated to the synthase expression, that of the autoinducer-2 follows the expression of an enzyme upstream in the activated methyl cycle and providing its precursor, rather than the expression of its own synthase. Conclusions/Significance Despite sharing the S-adenosylmethionine precursor, no strong link was detected between the production kinetics or metabolic pathways of autoinducers-1 and -2. In contrast, the signaling pathway of autoinducer-2 seems to be switched off by the indole-3-acetic acid pathway under tryptophan control. It therefore appears that the two genera of soft-rot bacteria have similarities but also differences in the mechanisms of communication via the diffusible molecules. Our results designate autoinducer-1 lactones as the main targets for a global biocontrol of soft-rot bacteria communications, including those of emerging isolates. PMID:22539957

Intragraft Molecular Pathways Associated with Tolerance Induction in Renal Transplantation.

PubMed

Gallon, Lorenzo; Mathew, James M; Bontha, Sai Vineela; Dumur, Catherine I; Dalal, Pranav; Nadimpalli, Lakshmi; Maluf, Daniel G; Shetty, Aneesha A; Ildstad, Suzanne T; Leventhal, Joseph R; Mas, Valeria R

2018-02-01

The modern immunosuppression regimen has greatly improved short-term allograft outcomes but not long-term allograft survival. Complications associated with immunosuppression, specifically nephrotoxicity and infection risk, significantly affect graft and patient survival. Inducing and understanding pathways underlying clinical tolerance after transplantation are, therefore, necessary. We previously showed full donor chimerism and immunosuppression withdrawal in highly mismatched allograft recipients using a bioengineered stem cell product (FCRx). Here, we evaluated the gene expression and microRNA expression profiles in renal biopsy samples from tolerance-induced FCRx recipients, paired donor organs before implant, and subjects under standard immunosuppression (SIS) without rejection and with acute rejection. Unlike allograft samples showing acute rejection, samples from FCRx recipients did not show upregulation of T cell- and B cell-mediated rejection pathways. Gene expression pathways differed slightly between FCRx samples and the paired preimplantation donor organ samples, but most of the functional gene networks overlapped. Notably, compared with SIS samples, FCRx samples showed upregulation of genes involved in pathways, like B cell receptor signaling. Additionally, prediction analysis showed inhibition of proinflammatory regulators and activation of anti-inflammatory pathways in FCRx samples. Furthermore, integrative analyses (microRNA and gene expression profiling from the same biopsy sample) identified the induction of regulators with demonstrated roles in the downregulation of inflammatory pathways and maintenance of tissue homeostasis in tolerance-induced FCRx samples compared with SIS samples. This pilot study highlights the utility of molecular intragraft evaluation of pathways related to FCRx-induced tolerance and the use of integrative analyses for identifying upstream regulators of the affected downstream molecular pathways. Copyright © 2018 by the American Society of Nephrology.

Reactive Oxygen Species-Producing Myeloid Cells Act as a Bone Marrow Niche for Sterile Inflammation-Induced Reactive Granulopoiesis.

PubMed

Zhu, Haiyan; Kwak, Hyun-Jeong; Liu, Peng; Bajrami, Besnik; Xu, Yuanfu; Park, Shin-Young; Nombela-Arrieta, Cesar; Mondal, Subhanjan; Kambara, Hiroto; Yu, Hongbo; Chai, Li; Silberstein, Leslie E; Cheng, Tao; Luo, Hongbo R

2017-04-01

Both microbial infection and sterile inflammation augment bone marrow (BM) neutrophil production, but whether the induced accelerated granulopoiesis is mediated by a common pathway and the nature of such a pathway are poorly defined. We recently established that BM myeloid cell-derived reactive oxygen species (ROS) externally regulate myeloid progenitor proliferation and differentiation in bacteria-elicited emergency granulopoiesis. In this article, we show that BM ROS levels are also elevated during sterile inflammation. Similar to in microbial infection, ROS were mainly generated by the phagocytic NADPH oxidase in Gr1 + myeloid cells. The myeloid cells and their ROS were uniformly distributed in the BM when visualized by multiphoton intravital microscopy, and ROS production was both required and sufficient for sterile inflammation-elicited reactive granulopoiesis. Elevated granulopoiesis was mediated by ROS-induced phosphatase and tensin homolog oxidation and deactivation, leading to upregulated PtdIns(3,4,5)P3 signaling and increased progenitor cell proliferation. Collectively, these results demonstrate that, although infection-induced emergency granulopoiesis and sterile inflammation-elicited reactive granulopoiesis are triggered by different stimuli and are mediated by distinct upstream signals, the pathways converge to NADPH oxidase-dependent ROS production by BM myeloid cells. Thus, BM Gr1 + myeloid cells represent a key hematopoietic niche that supports accelerated granulopoiesis in infective and sterile inflammation. This niche may be an excellent target in various immune-mediated pathologies or immune reconstitution after BM transplantation. Copyright © 2017 by The American Association of Immunologists, Inc.

Reactive oxygen species-producing myeloid cells act as a bone marrow niche for sterile inflammation-induced reactive granulopoiesis

PubMed Central

Zhu, Haiyan; Kwak, Hyun-Jeong; Liu, Peng; Bajrami, Besnik; Xu, Yuanfu; Park, Shin-Young; Nombela-Arrieta, Cesar; Mondal, Subhanjan; Kambara, Hiroto; Yu, Hongbo; Chai, Li; Silberstein, Leslie E.; Cheng, Tao; Luo, Hongbo R.

2017-01-01

Summary Both microbial infection and sterile inflammation augment bone marrow (BM) neutrophil production, but whether the induced accelerated granulopoiesis is mediated by a common pathway and the nature of such a pathway are poorly defined. We recently established that BM myeloid cell-derived reactive oxygen species (ROS) externally regulate myeloid progenitor proliferation and differentiation in bacteria-elicited emergency granulopoiesis. Here we show that BM ROS levels are also elevated during sterile inflammation. Similar to in microbial infection, ROS were mainly generated by the phagocytic NADPH oxidase in Gr1+ myeloid cells. The myeloid cells and their ROS were uniformly distributed in the BM when visualized by multi-photon intravital microscopy, and ROS production was both required and sufficient for sterile inflammation-elicited reactive granulopoiesis. Elevated granulopoiesis was mediated by ROS-induced PTEN oxidation and deactivation leading to upregulated PtdIns(3,4,5)P3 signaling and increased progenitor cell proliferation. Collectively, these results demonstrate that although infection-induced emergency granulopoiesis and sterile inflammation-elicited reactive granulopoiesis are triggered by different stimuli and are mediated by distinct upstream signals, the pathways converge to NADPH oxidase-dependent ROS production by BM myeloid cells. Thus, BM Gr1+ myeloid cells represent a key hematopoietic niche that supports accelerated granulopoiesis in both infective and sterile inflammation. This niche may be an excellent target in various immune-mediated pathologies or immune reconstitution after BM transplantation. PMID:28235862

The Metastasis Suppressor, N-MYC Downstream-regulated Gene-1 (NDRG1), Down-regulates the ErbB Family of Receptors to Inhibit Downstream Oncogenic Signaling Pathways*

PubMed Central

Kovacevic, Zaklina; Menezes, Sharleen V.; Sahni, Sumit; Kalinowski, Danuta S.; Bae, Dong-Hun; Lane, Darius J. R.; Richardson, Des R.

2016-01-01

N-MYC downstream-regulated gene-1 (NDRG1) is a potent growth and metastasis suppressor that acts through its inhibitory effects on a wide variety of cellular signaling pathways, including the TGF-β pathway, protein kinase B (AKT)/PI3K pathway, RAS, etc. To investigate the hypothesis that its multiple effects could be regulated by a common upstream effector, the role of NDRG1 on the epidermal growth factor receptor (EGFR) and other members of the ErbB family, namely human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 3 (HER3), was examined. We demonstrate that NDRG1 markedly decreased the expression and activation of EGFR, HER2, and HER3 in response to the epidermal growth factor (EGF) ligand, while also inhibiting formation of the EGFR/HER2 and HER2/HER3 heterodimers. In addition, NDRG1 also decreased activation of the downstream MAPKK in response to EGF. Moreover, novel anti-tumor agents of the di-2-pyridylketone class of thiosemicarbazones, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, which markedly up-regulate NDRG1, were found to inhibit EGFR, HER2, and HER3 expression and phosphorylation in cancer cells. However, the mechanism involved appeared dependent on NDRG1 for di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone, but was independent of this metastasis suppressor for di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone. This observation demonstrates that small structural changes in thiosemicarbazones result in marked alterations in molecular targeting. Collectively, these results reveal a mechanism for the extensive downstream effects on cellular signaling attributed to NDRG1. Furthermore, this study identifies a novel approach for the treatment of tumors resistant to traditional EGFR inhibitors. PMID:26534963

Isobutyrylshikonin inhibits lipopolysaccharide-induced nitric oxide and prostaglandin E2 production in BV2 microglial cells by suppressing the PI3K/Akt-mediated nuclear transcription factor-κB pathway.

PubMed

Jayasooriya, Rajapaksha Gedara Prasad Tharanga; Lee, Kyoung-Tae; Kang, Chang-Hee; Dilshara, Matharage Gayani; Lee, Hak-Ju; Choi, Yung Hyun; Choi, Il-Whan; Kim, Gi-Young

2014-12-01

Microglia are important macrophages to defend against pathogens in the central nervous system (CNS); however, persistent or acute inflammation of microglia lead to CNS disorders via neuronal cell death. Therefore, we theorized that a good strategy for the treatment of CNS disorders would be to target inflammatory mediators from microglia in disease. Consequently, we investigated whether isobutyrylshikonin (IBS) attenuates the production of proinflammatory mediators, such as nitric oxide (NO) and prostaglandin E2, in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Treatment with IBS inhibited the secretion of NO and prostaglandin E2 (as well as the expression of their key regulatory genes), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2). Isobutyrylshikonin also suppressed LPS-induced DNA-binding activity of nuclear transcription factor-κB (NF-κB), by inhibiting the nuclear translocation of p50 and p65 in addition to blocking the phosphorylation and degradation of IκBα. Pretreatment with pyrrolidine dithiocarbamate, a specific NF-κB inhibitor, showed the down-regulation of LPS-induced iNOS and COX-2 messenger RNA by suppressing NF-κB activity. This indirectly suggests that IBS-mediated NF-κB inhibition is the main signaling pathway involved in the inhibition of iNOS and COX-2 expression. In addition, IBS attenuated LPS-induced phosphorylation of PI3K and Akt, which are upstream molecules of NF-κB, in LPS-stimulated BV2 microglial cells. The functional aspects of the PI3K/Akt signaling pathway were analyzed with LY294002, which is a specific PI3K/Akt inhibitor that attenuated LPS-induced iNOS and COX-2 expression by suppressing NF-κB activity. These data suggest that an IBS-mediated anti-inflammatory effect may be involved in suppressing the PI3K/Akt-mediated NF-κB signaling pathway. Copyright © 2014 Elsevier Inc. All rights reserved.

Linking γ-aminobutyric acid A receptor to epidermal growth factor receptor pathways activation in human prostate cancer.

PubMed

Wu, Weijuan; Yang, Qing; Fung, Kar-Ming; Humphreys, Mitchell R; Brame, Lacy S; Cao, Amy; Fang, Yu-Ting; Shih, Pin-Tsen; Kropp, Bradley P; Lin, Hsueh-Kung

2014-03-05

Neuroendocrine (NE) differentiation has been attributed to the progression of castration-resistant prostate cancer (CRPC). Growth factor pathways including the epidermal growth factor receptor (EGFR) signaling have been implicated in the development of NE features and progression to a castration-resistant phenotype. However, upstream molecules that regulate the growth factor pathway remain largely unknown. Using androgen-insensitive bone metastasis PC-3 cells and androgen-sensitive lymph node metastasis LNCaP cells derived from human prostate cancer (PCa) patients, we demonstrated that γ-aminobutyric acid A receptor (GABA(A)R) ligand (GABA) and agonist (isoguvacine) stimulate cell proliferation, enhance EGF family members expression, and activate EGFR and a downstream signaling molecule, Src, in both PC-3 and LNCaP cells. Inclusion of a GABA(A)R antagonist, picrotoxin, or an EGFR tyrosine kinase inhibitor, Gefitinib (ZD1839 or Iressa), blocked isoguvacine and GABA-stimulated cell growth, trans-phospohorylation of EGFR, and tyrosyl phosphorylation of Src in both PCa cell lines. Spatial distributions of GABAAR α₁ and phosphorylated Src (Tyr416) were studied in human prostate tissues by immunohistochemistry. In contrast to extremely low or absence of GABA(A)R α₁-positive immunoreactivity in normal prostate epithelium, elevated GABA(A)R α₁ immunoreactivity was detected in prostate carcinomatous glands. Similarly, immunoreactivity of phospho-Src (Tyr416) was specifically localized and limited to the nucleoli of all invasive prostate carcinoma cells, but negative in normal tissues. Strong GABAAR α₁ immunoreactivity was spatially adjacent to the neoplastic glands where strong phospho-Src (Tyr416)-positive immunoreactivity was demonstrated, but not in adjacent to normal glands. These results suggest that the GABA signaling is linked to the EGFR pathway and may work through autocrine or paracine mechanism to promote CRPC progression. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Wavelength reused bidirectional transmission of adaptively modulated optical OFDM signals in WDM-PONs incorporating SOA and RSOA intensity modulators.

PubMed

Wei, J L; Hugues-Salas, E; Giddings, R P; Jin, X Q; Zheng, X; Mansoor, S; Tang, J M

2010-05-10

Detailed numerical investigations are undertaken of wavelength reused bidirectional transmission of adaptively modulated optical OFDM (AMOOFDM) signals over a single SMF in a colorless WDM-PON incorporating a semiconductor optical amplifier (SOA) intensity modulator and a reflective SOA (RSOA) intensity modulator in the optical line termination and optical network unit, respectively. A comprehensive theoretical model describing the performance of such network scenarios is, for the first time, developed, taking into account dynamic optical characteristics of SOA and RSOA intensity modulators as well as the effects of Rayleigh backscattering (RB) and residual downstream signal-induced crosstalk. The developed model is rigorously verified experimentally in RSOA-based real-time end-to-end OOFDM systems at 7.5 Gb/s. It is shown that the RB noise and crosstalk effects are dominant factors limiting the maximum achievable downstream and upstream transmission performance. Under optimum SOA and RSOA operating conditions as well as practical downstream and upstream optical launch powers, 10 Gb/s downstream and 6 Gb/s upstream over 40 km SMF transmissions of conventional double sideband AMOOFDM signals are feasible without utilizing in-line optical amplification and chromatic dispersion compensation. In particular, the aforementioned transmission performance can be improved to 23 Gb/s downstream and 8 Gb/s upstream over 40 km SMFs when single sideband subcarrier modulation is adopted in the downstream systems. (c) 2010 Optical Society of America.

Eicosapentaenoic Acid (EPA) Induced Macrophages Activation through GPR120-Mediated Raf-ERK1/2-IKKβ-NF-κB p65 Signaling Pathways

PubMed Central

Han, Lirong; Song, Shumin; Niu, Yabing; Meng, Meng; Wang, Chunling

2017-01-01

Objectives: To investigate the immunomodulatory effect and molecular mechanisms of Eicosapentaenoic acid (EPA, a typical kind of n-3PUFAs) on RAW264.7 cells. Methods: A variety of research methods, including the RAW264.7 cells culture, cell proliferation assays, morphologic observations, measurements of NO production, cytokine assays, nuclear protein extractions, western blot analyses and NF-κB p65 immunofluorescence assays were used in this study. Results: The results showed that EPA could increase the proliferation index and enhance the release of nitric oxide (NO) and cytokines in RAW264.7 cells. Western blotting results revealed that the protein level of GPR120 increased significantly in RAW264.7 cells after EPA treatment. Meanwhile, EPA elevated the phosphorylation status of Raf, which may act as an upstream regulator of EPA-induced phosphorylated ERK1/2. In addition, the phosphorylated ERK1/2 may then promote IKKβ in endochylema and translocate the NF-κB p65 subunit into the nucleus, thus regulating the production of inducible nitric oxide synthase (iNOS) and cytokines. Conclusions: EPA (0.6–3.0 μmol) activates RAW264.7 cells through GPR120-mediated Raf-ERK1/2-IKKβ-NF-κB p65 signaling pathways. PMID:28841192

Eicosapentaenoic Acid (EPA) Induced Macrophages Activation through GPR120-Mediated Raf-ERK1/2-IKKβ-NF-κB p65 Signaling Pathways.

PubMed

Han, Lirong; Song, Shumin; Niu, Yabing; Meng, Meng; Wang, Chunling

2017-08-25

Objectives: To investigate the immunomodulatory effect and molecular mechanisms of Eicosapentaenoic acid (EPA, a typical kind of n-3PUFAs) on RAW264.7 cells. Methods: A variety of research methods, including the RAW264.7 cells culture, cell proliferation assays, morphologic observations, measurements of NO production, cytokine assays, nuclear protein extractions, western blot analyses and NF-κB p65 immunofluorescence assays were used in this study. Results: The results showed that EPA could increase the proliferation index and enhance the release of nitric oxide (NO) and cytokines in RAW264.7 cells. Western blotting results revealed that the protein level of GPR120 increased significantly in RAW264.7 cells after EPA treatment. Meanwhile, EPA elevated the phosphorylation status of Raf, which may act as an upstream regulator of EPA-induced phosphorylated ERK1/2. In addition, the phosphorylated ERK1/2 may then promote IKKβ in endochylema and translocate the NF-κB p65 subunit into the nucleus, thus regulating the production of inducible nitric oxide synthase (iNOS) and cytokines. Conclusions: EPA (0.6-3.0 μmol) activates RAW264.7 cells through GPR120-mediated Raf-ERK1/2-IKKβ-NF-κB p65 signaling pathways.

Arabidopsis AIP1-2 restricted by WER-mediated patterning modulates planar polarity

PubMed Central

Kiefer, Christian S.; Claes, Andrea R.; Nzayisenga, Jean-Claude; Pietra, Stefano; Stanislas, Thomas; Hüser, Anke; Ikeda, Yoshihisa; Grebe, Markus

2015-01-01

The coordination of cell polarity within the plane of the tissue layer (planar polarity) is crucial for the development of diverse multicellular organisms. Small Rac/Rho-family GTPases and the actin cytoskeleton contribute to planar polarity formation at sites of polarity establishment in animals and plants. Yet, upstream pathways coordinating planar polarity differ strikingly between kingdoms. In the root of Arabidopsis thaliana, a concentration gradient of the phytohormone auxin coordinates polar recruitment of Rho-of-plant (ROP) to sites of polar epidermal hair initiation. However, little is known about cytoskeletal components and interactions that contribute to this planar polarity or about their relation to the patterning machinery. Here, we show that ACTIN7 (ACT7) represents a main actin isoform required for planar polarity of root hair positioning, interacting with the negative modulator ACTIN-INTERACTING PROTEIN1-2 (AIP1-2). ACT7, AIP1-2 and their genetic interaction are required for coordinated planar polarity of ROP downstream of ethylene signalling. Strikingly, AIP1-2 displays hair cell file-enriched expression, restricted by WEREWOLF (WER)-dependent patterning and modified by ethylene and auxin action. Hence, our findings reveal AIP1-2, expressed under control of the WER-dependent patterning machinery and the ethylene signalling pathway, as a modulator of actin-mediated planar polarity. PMID:25428588

Cholecystokinin induces caspase activation and mitochondrial dysfunction in pancreatic acinar cells. Roles in cell injury processes of pancreatitis.

PubMed

Gukovskaya, Anna S; Gukovsky, Ilya; Jung, Yoon; Mouria, Michelle; Pandol, Stephen J

2002-06-21

Apoptosis and necrosis are critical parameters of pancreatitis, the mechanisms of which remain unknown. Many characteristics of pancreatitis can be studied in vitro in pancreatic acini treated with high doses of cholecystokinin (CCK). We show here that CCK stimulates apoptosis and death signaling pathways in rat pancreatic acinar cells, including caspase activation, cytochrome c release, and mitochondrial depolarization. The mitochondrial dysfunction is mediated by upstream caspases (possibly caspase-8) and, in turn, leads to activation of caspase-3. CCK causes mitochondrial alterations through both permeability transition pore-dependent (cytochrome c release) and permeability transition pore-independent (mitochondrial depolarization) mechanisms. Caspase activation and mitochondrial alterations also occur in untreated pancreatic acinar cells; however, the underlying mechanisms are different. In particular, caspases protect untreated acinar cells from mitochondrial damage. We found that caspases not only mediate apoptosis but also regulate other parameters of CCK-induced acinar cell injury that are characteristic of pancreatitis; in particular, caspases negatively regulate necrosis and trypsin activation in acinar cells. The results suggest that the observed signaling pathways regulate parenchymal cell injury and death in CCK-induced pancreatitis. Protection against necrosis and trypsin activation by caspases can explain why the severity of pancreatitis in experimental models correlates inversely with the extent of apoptosis.

Lysosomal membrane permeabilization in cell death: new evidence and implications for health and disease.

PubMed

Serrano-Puebla, Ana; Boya, Patricia

2016-05-01

Recent studies have demonstrated that, in addition to their central role in cellular catabolic reactions, lysosomes are implicated in many cellular processes, including metabolism, membrane repair, and cell death. Lysosomal membrane permeabilization (LMP) has emerged as a pathway by which cell demise is regulated under physiological conditions and contributes to cell death in many pathological situations. Here, we review the latest evidence on LMP-mediated cell death, the upstream and downstream signals involved, and the role of LMP in the normal physiology of organisms. We also discuss the contributions of lysosomal damage and LMP to the pathogenic features of several disease states, such as lysosomal storage disorders and other neurodegenerative conditions. © 2015 New York Academy of Sciences.

ER-stress and apoptosis: molecular mechanisms and potential relevance in infection.

PubMed

Häcker, Georg

2014-10-01

During ER-stress, one of the responses a cell can choose is apoptosis. Apoptosis generally is a cell's preferred response when other control mechanisms are overwhelmed. We now have a reasonably clear molecular picture what is happening once the apoptotic apparatus has been started. Unclear however are the majority of the upstream pathways that connect other signalling to apoptosis. During ER-stress, confirmed apoptosis-regulating targets are pro- and anti-apoptotic proteins of the Bcl-2-family, whose concerted action induces apoptosis. I will here discuss how mitochondrial apoptosis is triggered, how this is linked to the ER-stress response and in what way this may be relevant during microbial infections. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Activation of HIV-1 pre-mRNA 3' processing in vitro requires both an upstream element and TAR.

PubMed Central

Gilmartin, G M; Fleming, E S; Oetjen, J

1992-01-01

The architecture of the human immunodeficiency virus type 1 (HIV-1) genome presents an intriguing dilemma for the 3' processing of viral transcripts--to disregard a canonical 'core' poly(A) site processing signal present at the 5' end of the transcript and yet to utilize efficiently an identical signal that resides at the 3' end of the message. The choice of processing sites in HIV-1 appears to be influenced by two factors: (i) proximity to the cap site, and (ii) sequences upstream of the core poly(A) site. We now demonstrate that an in vivo-defined upstream element that resides within the U3 region, 76 nucleotides upstream of the AAUAAA hexamer, acts specifically to enhance 3' processing at the HIV-1 core poly(A) site in vitro. We furthermore show that efficient in vitro 3' processing requires the RNA stem-loop structure of TAR, which serves to juxtapose spatially the upstream element and the core poly(A) site. An analysis of the stability of 3' processing complexes formed at the HIV-1 poly(A) site in vitro suggests that the upstream element may function by increasing processing complex stability at the core poly(A) site. Images PMID:1425577

The role of complement system in septic shock.

PubMed

Charchaflieh, Jean; Wei, Jiandong; Labaze, Georges; Hou, Yunfang Joan; Babarsh, Benjamin; Stutz, Helen; Lee, Haekyung; Worah, Samrat; Zhang, Ming

2012-01-01

Septic shock is a critical clinical condition with a high mortality rate. A better understanding of the underlying mechanisms is important to develop effective therapies. Basic and clinical studies suggest that activation of complements in the common cascade, for example, complement component 3 (C3) and C5, is involved in the development of septic shock. The involvement of three upstream complement pathways in septic shock is more complicated. Both the classical and alternative pathways appear to be activated in septic shock, but the alternative pathway may be activated earlier than the classical pathway. Activation of these two pathways is essential to clear endotoxin. Recent investigations have shed light on the role of lectin complement pathway in septic shock. Published reports suggest a protective role of mannose-binding lectin (MBL) against sepsis. Our preliminary study of MBL-associated serine protease-2 (MASP-2) in septic shock patients indicated that acute decrease of MASP-2 in the early phase of septic shock might correlate with in-hospital mortality. It is unknown whether excessive activation of these three upstream complement pathways may contribute to the detrimental effects in septic shock. This paper also discusses additional complement-related pathogenic mechanisms and intervention strategies for septic shock.

Mini-dystrophin Expression Down-regulates Overactivation of G Protein–mediated IP3 Signaling Pathway in Dystrophin-deficient Muscle Cells

PubMed Central

Balghi, Haouaria; Sebille, Stéphane; Constantin, Bruno; Patri, Sylvie; Thoreau, Vincent; Mondin, Ludivine; Mok, Elise; Kitzis, Alain; Raymond, Guy; Cognard, Christian

2006-01-01

We present here evidence for the enhancement of an inositol 1,4,5-trisphosphate (IP3) mediated calcium signaling pathway in myotubes from dystrophin-deficient cell lines (SolC1(−)) as compared to a cell line from the same origin but transfected with mini-dystrophin (SolD(+)). With confocal microscopy, we demonstrated that calcium rise, induced by the perifusion of a solution containing a high potassium concentration, was higher in SolC1(−) than in SolD(+) myotubes. The analysis of amplitude and kinetics of the calcium increase in SolC1(−) and in SolD(+) myotubes during the exposure with SR Ca2+ channel inhibitors (ryanodine and 2-APB) suggested the presence of two mechanisms of SR calcium release: (1) a fast SR calcium release that depended on ryanodine receptors and (2) a slow SR calcium release mediated by IP3 receptors. Detection analyses of mRNAs (reverse transcriptase [RT]-PCR) and proteins (Western blot and immunolocalization) demonstrated the presence of the three known isoforms of IP3 receptors in both SolC1(−) and SolD(+) myotubes. Furthermore, analysis of the kinetics of the rise in calcium revealed that the slow IP3-dependent release may be increased in the SolC1(−) as compared to the SolD(+), suggesting an inhibitory effect of mini-dystrophin in this signaling pathway. Upon incubation with pertussis toxin (PTX), an inhibitory effect similar to that of the IP3R inhibitor (2-APB) was observed on K+-evoked calcium release. This result suggests the involvement of a Gi protein upstream of the IP3 pathway in these stimulation conditions. A hypothetical model is depicted in which both Gi protein and IP3 production could be involved in K+-evoked calcium release as well as a possible interaction with mini-dystrophin. Our findings demonstrate the existence of a potential relationship between mini-dystrophin and SR calcium release as well as a regulatory role of mini-dystrophin on intracellular signaling. PMID:16446505

The Crosstalk of Pathways Involved in Immune Response Maybe the Shared Molecular Basis of Rheumatoid Arthritis and Type 2 Diabetes

PubMed Central

Ge, Na; Jiang, Miao; Li, Li; Bian, Yanqin; Xu, Gang; Bian, Zhaoxiang; Zhang, Ge; Lu, Aiping

2015-01-01

Rheumatoid arthritis (RA) and Type 2 diabetes (T2D) are both systemic diseases linked with altered immune response, moderate mortality when present together. The treatment for both RA and T2D are not satisfied, partly because of the linkage between them has not yet been appreciated. A comprehensive study for the potential associations between the two disorders is needed. In this study, we used RNA sequencing to explore the differently expressed genes (DEGs) in peripheral blood mononuclear cells (PBMC) of 10 RA and 10 T2D patients comparing with 10 healthy volunteers (control). We used bioinformatics analysis and the Ingenuity Pathways Analysis (IPA) to predict the commonalities on signaling pathways and molecular networks between those two diseases. 212 DEGs in RA and 114 DEGs in T2D patients were identified compared with healthy controls, respectively. 32 DEGs were shared between the two comparisons. The top 10 shared pathways interacted in cross-talking networks, regulated by 5 shared predicted upstream regulators, leading to the activated immune response were explored, which was considered as partly of the association mechanism of this two disorders. These discoveries would be considered as new understanding on the associations between RA and T2D, and provide novel treatment or prevention strategy. PMID:26252209

Curcumin sensitizes human lung cancer cells to apoptosis and metastasis synergistically combined with carboplatin.

PubMed

Kang, Ji Ho; Kang, Hye Seon; Kim, In Kyoung; Lee, Hwa Young; Ha, Jick Hwan; Yeo, Chang Dong; Kang, Hyun Hui; Moon, Hwa Sik; Lee, Sang Haak

2015-11-01

Although carboplatin is one of the standard chemotherapeutic agents for non-small cell lung cancer (NSCLC), it has limited therapeutic efficacy due to activation of a survival signaling pathway and the induction of multidrug resistance. Curcumin, a natural compound isolated from the plant Curcuma longa, is known to sensitize tumors to different chemotherapeutic agents. The aim of this study is to evaluate whether curcumin can chemosensitize lung cancer cells to carboplatin and to analyze the signaling pathway underlying this synergism. We investigated the synergistic effect of both agents on cell proliferation, apoptosis, invasion, migration, and expression of related signaling proteins using the human NSCLC cell line, A549. A549 cell was treated with different concentrations of curcumin and carboplatin alone and in combination. Combined treatment with curcumin and carboplatin inhibited tumor cell growth, migration, and invasion compared with either drug alone. Matrix metalloproteinase (MMP)-2 and MMP-9 were more efficiently downregulated by co-treatment than by each treatment alone. mRNA and protein expression of caspase-3 and caspase-9 and proapoptotic genes was increased in cells treated with a combination of curcumin and carboplatin, whereas expression of the antiapoptotic Bcl-2 gene was suppressed. Co-treatment of both agents substantially suppressed NF-κB activation and increased expression of p53. Phosphorylation of Akt, a protein upstream of NF-κB, was reduced, resulting in inhibition of the degradation of inhibitor of κB(IκBα), whereas the activity of extracellular signal-regulated kinase (ERK1/2) was enhanced. Our study demonstrated that the synergistic antitumor activity of curcumin combined with carboplatin is mediated by multiple mechanisms involving suppression of NF-κB via inhibition of the Akt/IKKα pathway and enhanced ERK1/2 activity. Based on this mechanism, curcumin has potential as a chemosensitizer for carboplatin in the treatment of patients with NSCLC. © 2015 by the Society for Experimental Biology and Medicine.

The Crosstalk Between Nrf2 and AMPK Signal Pathways Is Important for the Anti-Inflammatory Effect of Berberine in LPS-Stimulated Macrophages and Endotoxin-Shocked Mice

PubMed Central

Mo, Chunfen; Wang, Ling; Zhang, Jie; Numazawa, Satoshi; Tang, Hong; Tang, Xiaoqiang; Han, XiaoJuan; Li, Junhong; Yang, Ming; Wang, Zhe; Wei, Dandan

2014-01-01

Abstract Aims: The response of AMP-activated protein kinase (AMPK) to oxidative stress has been recently reported but the downstream signals of this response are largely unknown. Meanwhile, the upstream events for the activation of nuclear factor erythroid-2-related factor-2 (Nrf2), a critical transcriptional activator for antioxidative responses, remain unclear. In the present study, we investigated the relationship between AMPK and Nrf2 signal pathways in lipopolysaccharide (LPS)-triggered inflammatory system, in which berberine (BBR), a known AMPK activator, was used for inflammation suppression. Results and Innovation: In inflammatory macrophages, BBR attenuated LPS-induced expression of inflammatory genes (inducible nitric oxide synthase [iNOS], cyclooxygenase-2 [COX2], interleukin [IL]-6), and the generation of nitric oxide and reactive oxygen species, but increased the transcription of Nrf2-targeted antioxidative genes (NADPH quinone oxidoreductase-1 [NQO-1], heme oxygenase-1 [HO-1]), as well as the nuclear localization and phosphorylation of Nrf2 protein. Importantly, we found BBR-induced activation of Nrf2 is AMPK-dependent, as either pharmacologically or genetically inactivating AMPK blocked the activation of Nrf2. Consistent with in vitro experiments, BBR down-regulated the expression of proinflammatory genes but upregulated those of Nrf2-targeted genes in lungs of LPS-injected mice, and these effects were attenuated in Nrf2-deficient mice. Moreover, the effect of BBR on survival time extension and plasma redox regulation in endotoxin-shocked mice was largely weakened when Nrf2-depleted. Conclusions: Our results demonstrate convergence between AMPK and Nrf2 pathways and this intersection is essential for anti-inflammatory effect of BBR in LPS-stimulated macrophages and endotoxin-shocked mice. Uncovering this intersection is significant for understanding the relationship between energy homeostasis and antioxidative responses and may be beneficial for developing new therapeutic strategies against inflammatory diseases. Antioxid. Redox Signal. 20, 574–588. PMID:23875776

Curcumin sensitizes human lung cancer cells to apoptosis and metastasis synergistically combined with carboplatin

PubMed Central

Kang, Ji Ho; Kang, Hye Seon; Kim, In Kyoung; Lee, Hwa Young; Ha, Jick Hwan; Yeo, Chang Dong; Kang, Hyun Hui; Moon, Hwa Sik

2015-01-01

Although carboplatin is one of the standard chemotherapeutic agents for non-small cell lung cancer (NSCLC), it has limited therapeutic efficacy due to activation of a survival signaling pathway and the induction of multidrug resistance. Curcumin, a natural compound isolated from the plant Curcuma longa, is known to sensitize tumors to different chemotherapeutic agents. The aim of this study is to evaluate whether curcumin can chemosensitize lung cancer cells to carboplatin and to analyze the signaling pathway underlying this synergism. We investigated the synergistic effect of both agents on cell proliferation, apoptosis, invasion, migration, and expression of related signaling proteins using the human NSCLC cell line, A549. A549 cell was treated with different concentrations of curcumin and carboplatin alone and in combination. Combined treatment with curcumin and carboplatin inhibited tumor cell growth, migration, and invasion compared with either drug alone. Matrix metalloproteinase (MMP)-2 and MMP-9 were more efficiently downregulated by co-treatment than by each treatment alone. mRNA and protein expression of caspase-3 and caspase-9 and proapoptotic genes was increased in cells treated with a combination of curcumin and carboplatin, whereas expression of the antiapoptotic Bcl-2 gene was suppressed. Co-treatment of both agents substantially suppressed NF-κB activation and increased expression of p53. Phosphorylation of Akt, a protein upstream of NF-κB, was reduced, resulting in inhibition of the degradation of inhibitor of κB(IκBα), whereas the activity of extracellular signal-regulated kinase (ERK1/2) was enhanced. Our study demonstrated that the synergistic antitumor activity of curcumin combined with carboplatin is mediated by multiple mechanisms involving suppression of NF-κB via inhibition of the Akt/IKKα pathway and enhanced ERK1/2 activity. Based on this mechanism, curcumin has potential as a chemosensitizer for carboplatin in the treatment of patients with NSCLC. PMID:25716014

Symmetric 40-Gb/s TWDM-PON with 51-dB loss budget by using a single SOA as preamplifier, booster and format converter in ONU.

PubMed

Li, Zhengxuan; Yi, Lilin; Hu, Weisheng

2014-10-06

In this paper, we propose to use a semiconductor optical amplifier (SOA) in the optical network unit (ONU) to improve the loss budget in time and wavelength division multiplexed-passive optical network (TWDM-PON) systems. The SOA boosts the upstream signal to increase the output power of the electro-absorption modulated laser (EML) and simultaneously pre-amplifies the downstream signal for sensitivity improvement. The penalty caused by cross gain modulation (XGM) effect is negligible due to the low extinction ratio (ER) of upstream signal and the large wavelength difference between upstream and downstream links. In order to achieve a higher output power, the SOA is driven into its saturation region, where the self-phase modulation (SPM) effect converts the intensity into phase information and realizes on-off-keying (OOK) to phase-shifted-keying (PSK) format conversion. In this way, the pattern effect is eliminated, which releases the requirement of gain-clamping on SOA. To further improve the loss budget of upstream link, an Erbium doped fiber amplifier (EDFA) is used in the optical line terminal (OLT) to pre-amplify the received signal. For the downstream direction, directly modulated laser (DML) is used as the laser source. Taking advantage of its carrier-less characteristic, directly modulated signal shows high tolerance to fiber nonlinearity, which could support a downstream launch power as high as + 16 dBm per channel. In addition, the signal is pre-amplified by the SOA in ONU before being detected, so the sensitivity limitation for downstream link is also removed. As a result, a truly passive symmetric 40-Gb/s TWDM-PON was demonstrated, achieving a link loss budget of 51 dB.

Sulfur dioxide inhibits vascular smooth muscle cell proliferation via suppressing the Erk/MAP kinase pathway mediated by cAMP/PKA signaling

PubMed Central

Liu, D; Huang, Y; Bu, D; Liu, A D; Holmberg, L; Jia, Y; Tang, C; Du, J; Jin, H

2014-01-01

The present study was designed to investigate the role of endogenous sulfur dioxide (SO2) in vascular smooth muscle cell (VSMC) proliferation, and explore the possible role of cross-talk between cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and extracellular signal-regulated kinase (Erk)/mitogen-activated protein kinase (MAPK) pathways in this action. By cell counting, growth curve depict, flow cytometry and bromodeoxyuridine (BrdU) labeling assays, we found that SO2 inhibited VSMC proliferation by preventing cell cycle progression from G1 to S phase and by reducing DNA synthesis. SO2 synthase aspartate aminotransferase (AAT1 and AAT2) overexpression significantly inhibited serum-induced proliferating cell nuclear antigen (PCNA) protein expression in VSMCs, demonstrated by western blot analysis. Moreover, overexpression of AAT1 or AAT2 markedly reduced incorporation of BrdU in serum-treated VSMCs. By contrast, either AAT1 or AAT2 knockdown significantly exacerbated serum-stimulated VSMC proliferation. Thus, both exogenous- and endogenous-derived SO2 suppressed serum-induced VSMC proliferation. However, annexin V-propidium iodide (PI) staining and cell cycle analysis demonstrated that SO2 did not influence VSMC apoptosis in the serum-induced proliferation model. In a platelet-derived growth factor (PDGF)-BB-stimulated VSMC proliferation model, SO2 dephosphorylated the active sites of Erk1/2, MAPK kinase 1/2 and RAF proto-oncogene serine/threonine-protein kinase (c-Raf) induced by PDGF-BB. However, the inactivation of the three kinases of the Erk/MAPK pathway was not due to the separate interferences on them by SO2 simultaneously, but a consequence of the influence on the upstream activity of the c-Raf molecule. Hence, we examined the cAMP/PKA pathway, which could inhibit Erk/MAPK transduction in VSMCs. The results showed that SO2 could stimulate the cAMP/PKA pathway to block c-Raf activation, whereas the Ser259 site on c-Raf had an important role in SO2-induced suppression of Erk/MAPK pathway. The present study firstly demonstrated that SO2 exerted a negative regulation of VSMC proliferation via suppressing the Erk/MAPK pathway mediated by cAMP/PKA signaling. PMID:24853429

TGF-β Coordinately Activates TAK1/MEK/AKT/NFkB and Smad Pathways to Promote Osteoclast Survival

PubMed Central

Gingery, Anne; Bradley, Elizabeth W.; Pederson, Larry; Ruan, Ming; Horwood, Nikki J.; Oursler, Merry Jo

2008-01-01

To better understand the roles of TGF-β in bone metabolism, we investigated osteoclast survival in response TGF-β and found that TGF-β inhibited apoptosis. We examined the receptors involved in promotion of osteoclast survival and found that the canonical TGF-β receptor complex is involved in the survival response. The upstream MEK kinase TAK1 was rapidly activated following TGF-β treatment. Since osteoclast survival involves MEK, AKT, and NFκB activation, we examined TGF-β effects on activation of these pathways and observed rapid phosphorylation of MEK, AKT, IKK, IκB, and NFκB. The timing of activation coincided with SMAD activation and dominant negative SMAD expression did not inhibit NFκB activation, indicating that kinase pathway activation is independent of SMAD signaling. Inhibition of TAK1, MEK, AKT, NIK, IKK, or NFκB repressed TGF-β-mediated osteoclast survival. Adenoviral-mediated TAK1 or MEK inhibition eliminated TGF-β-mediated kinase pathway activation and constitutively active AKT expression overcame apoptosis induction following MEK inhibition. TAK1/MEK activation induces pro-survival BclXL expression and TAK1/MEK and SMAD pathway activation induces pro-survival Mcl-1 expression. These data show that TGF-β-induced NFκB activation is through TAK1/MEK-mediated AKT activation, which is essential for TGF-β to support of osteoclast survival. PMID:18586026

Multiple-Localization and Hub Proteins

PubMed Central

Ota, Motonori; Gonja, Hideki; Koike, Ryotaro; Fukuchi, Satoshi

2016-01-01

Protein-protein interactions are fundamental for all biological phenomena, and protein-protein interaction networks provide a global view of the interactions. The hub proteins, with many interaction partners, play vital roles in the networks. We investigated the subcellular localizations of proteins in the human network, and found that the ones localized in multiple subcellular compartments, especially the nucleus/cytoplasm proteins (NCP), the cytoplasm/cell membrane proteins (CMP), and the nucleus/cytoplasm/cell membrane proteins (NCMP), tend to be hubs. Examinations of keywords suggested that among NCP, those related to post-translational modifications and transcription functions are the major contributors to the large number of interactions. These types of proteins are characterized by a multi-domain architecture and intrinsic disorder. A survey of the typical hub proteins with prominent numbers of interaction partners in the type revealed that most are either transcription factors or co-regulators involved in signaling pathways. They translocate from the cytoplasm to the nucleus, triggered by the phosphorylation and/or ubiquitination of intrinsically disordered regions. Among CMP and NCMP, the contributors to the numerous interactions are related to either kinase or ubiquitin ligase activity. Many of them reside on the cytoplasmic side of the cell membrane, and act as the upstream regulators of signaling pathways. Overall, these hub proteins function to transfer external signals to the nucleus, through the cell membrane and the cytoplasm. Our analysis suggests that multiple-localization is a crucial concept to characterize groups of hub proteins and their biological functions in cellular information processing. PMID:27285823

Dabrafenib; Preclinical Characterization, Increased Efficacy when Combined with Trametinib, while BRAF/MEK Tool Combination Reduced Skin Lesions

PubMed Central

King, Alastair J.; Arnone, Marc R.; Bleam, Maureen R.; Moss, Katherine G.; Yang, Jingsong; Fedorowicz, Kelly E.; Smitheman, Kimberly N.; Erhardt, Joseph A.; Hughes-Earle, Angela; Kane-Carson, Laurie S.; Sinnamon, Robert H.; Qi, Hongwei; Rheault, Tara R.; Uehling, David E.; Laquerre, Sylvie G.

2013-01-01

Mitogen-Activated Protein Kinase (MAPK) pathway activation has been implicated in many types of human cancer. BRAF mutations that constitutively activate MAPK signalling and bypass the need for upstream stimuli occur with high prevalence in melanoma, colorectal carcinoma, ovarian cancer, papillary thyroid carcinoma, and cholangiocarcinoma. In this report we characterize the novel, potent, and selective BRAF inhibitor, dabrafenib (GSK2118436). Cellular inhibition of BRAFV600E kinase activity by dabrafenib resulted in decreased MEK and ERK phosphorylation and inhibition of cell proliferation through an initial G1 cell cycle arrest, followed by cell death. In a BRAFV600E-containing xenograft model of human melanoma, orally administered dabrafenib inhibited ERK activation, downregulated Ki67, and upregulated p27, leading to tumor growth inhibition. However, as reported for other BRAF inhibitors, dabrafenib also induced MAPK pathway activation in wild-type BRAF cells through CRAF (RAF1) signalling, potentially explaining the squamous cell carcinomas and keratoacanthomas arising in patients treated with BRAF inhibitors. In addressing this issue, we showed that concomitant administration of BRAF and MEK inhibitors abrogated paradoxical BRAF inhibitor-induced MAPK signalling in cells, reduced the occurrence of skin lesions in rats, and enhanced the inhibition of human tumor xenograft growth in mouse models. Taken together, our findings offer preclinical proof of concept for dabrafenib as a specific and highly efficacious BRAF inhibitor and provide evidence for its potential clinical benefits when used in combination with a MEK inhibitor. PMID:23844038

Requirement and Redundancy of the Src Family Kinases Fyn and Lyn in Perforin-Dependent Killing of Cryptococcus neoformans by NK Cells

PubMed Central

Oykhman, Paul; Timm-McCann, Martina; Xiang, Richard F.; Islam, Anowara; Li, Shu Shun; Stack, Danuta; Huston, Shaunna M.; Ma, Ling Ling

2013-01-01

Natural killer (NK) cells directly recognize and kill fungi, such as the pathogenic fungus Cryptococcus neoformans, via cytolytic mechanisms. However, the precise signaling pathways governing this NK cell microbicidal activity and the implications for fungal recognition are still unknown. Previously, it was reported that NK cell anticryptococcal activity is mediated through a conserved phosphatidylinositol 3-kinase–extracellular signal-regulated kinase 1/2 (PI3K-ERK1/2) pathway. Using YT (a human NK-like cell line) and primary human NK cells, we sought to identify the upstream, receptor-proximal signaling elements that led to fungal cytolysis. We demonstrate that Src family kinases were activated in response to C. neoformans. Furthermore, pharmacologic inhibition with an Src kinase inhibitor blocked C. neoformans-induced downstream activation of PI3K and ERK1/2 and abrogated cryptococcal killing. At the same time, the inhibitor disrupted the polarization of perforin-containing granules toward the NK cell-cryptococcal synapse but had no effect on conjugate formation between the organism and the NK cell. Finally, small interfering RNA (siRNA) double (but not single) knockdown of two Src family kinases, Fyn and Lyn, blocked cryptococcal killing. Together these data demonstrate a mechanism whereby the Src family kinases, Fyn and Lyn, redundantly mediate anticryptococcal activity through the activation of PI3K and ERK1/2, which in turn facilitates killing by inducing the polarization of perforin-containing granules to the NK cell-cryptococcal synapse. PMID:23918783

Heterotrimeric G proteins-mediated resistance to necrotrophic pathogens includes mechanisms independent of salicylic acid-, jasmonic acid/ethylene- and abscisic acid-mediated defense signaling.

PubMed

Trusov, Yuri; Sewelam, Nasser; Rookes, James Edward; Kunkel, Matt; Nowak, Ekaterina; Schenk, Peer Martin; Botella, José Ramón

2009-04-01

Heterotrimeric G proteins are involved in the defense response against necrotrophic fungi in Arabidopsis. In order to elucidate the resistance mechanisms involving heterotrimeric G proteins, we analyzed the effects of the Gβ (subunit deficiency in the mutant agb1-2 on pathogenesis-related gene expression, as well as the genetic interaction between agb1-2 and a number of mutants of established defense pathways. Gβ-mediated signaling suppresses the induction of salicylic acid (SA)-, jasmonic acid (JA)-, ethylene (ET)- and abscisic acid (ABA)-dependent genes during the initial phase of the infection with Fusarium oxysporum (up to 48 h after inoculation). However, at a later phase it enhances JA/ET-dependent genes such as PDF1.2 and PR4. Quantification of the Fusarium wilt symptoms revealed that Gβ- and SA-deficient mutants were more susceptible than wild-type plants, whereas JA- and ET-insensitive and ABA-deficient mutants demonstrated various levels of resistance. Analysis of the double mutants showed that the Gβ-mediated resistance to F. oxysporum and Alternaria brassicicola was mostly independent of all of the previously mentioned pathways. However, the progressive decay of agb1-2 mutants was compensated by coi1-21 and jin1-9 mutations, suggesting that at this stage of F. oxysporum infection Gβ acts upstream of COI1 and ATMYC2 in JA signaling. © 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd.

Stromal liver kinase B1 [STK11] signaling loss induces oviductal adenomas and endometrial cancer by activating mammalian Target of Rapamycin Complex 1.

PubMed

Tanwar, Pradeep S; Kaneko-Tarui, Tomoko; Zhang, Lihua; Tanaka, Yoshihiro; Crum, Christopher P; Teixeira, Jose M

2012-01-01

Germline mutations of the Liver Kinase b1 (LKB1/STK11) tumor suppressor gene have been linked to Peutz-Jeghers Syndrome (PJS), an autosomal-dominant, cancer-prone disorder in which patients develop neoplasms in several organs, including the oviduct, ovary, and cervix. We have conditionally deleted Lkb1 in Müllerian duct mesenchyme-derived cells of the female reproductive tract and observed expansion of the stromal compartment and hyperplasia and/or neoplasia of adjacent epithelial cells throughout the reproductive tract with paratubal cysts and adenomyomas in oviducts and, eventually, endometrial cancer. Examination of the proliferation marker phospho-histone H3 and mammalian Target Of Rapamycin Complex 1 (mTORC1) pathway members revealed increased proliferation and mTORC1 activation in stromal cells of both the oviduct and uterus. Treatment with rapamycin, an inhibitor of mTORC1 activity, decreased tumor burden in adult Lkb1 mutant mice. Deletion of the genes for Tuberous Sclerosis 1 (Tsc1) or Tsc2, regulators of mTORC1 that are downstream of LKB1 signaling, in the oviductal and uterine stroma phenocopies some of the defects observed in Lkb1 mutant mice, confirming that dysregulated mTORC1 activation in the Lkb1-deleted stroma contributes to the phenotype. Loss of PTEN, an upstream regulator of mTORC1 signaling, along with Lkb1 deletion significantly increased tumor burden in uteri and induced tumorigenesis in the cervix and vagina. These studies show that LKB1/TSC1/TSC2/mTORC1 signaling in mesenchymal cells is important for the maintenance of epithelial integrity and suppression of carcinogenesis in adjacent epithelial cells. Because similar changes in the stromal population are also observed in human oviductal/ovarian adenoma and endometrial adenocarcinoma patients, we predict that dysregulated mTORC1 activity by upstream mechanisms similar to those described in these model systems contributes to the pathogenesis of these human diseases.

Stromal Liver Kinase B1 [STK11] Signaling Loss Induces Oviductal Adenomas and Endometrial Cancer by Activating Mammalian Target of Rapamycin Complex 1

PubMed Central

Zhang, LiHua; Tanaka, Yoshihiro; Crum, Christopher P.; Teixeira, Jose M.

2012-01-01

Germline mutations of the Liver Kinase b1 (LKB1/STK11) tumor suppressor gene have been linked to Peutz-Jeghers Syndrome (PJS), an autosomal-dominant, cancer-prone disorder in which patients develop neoplasms in several organs, including the oviduct, ovary, and cervix. We have conditionally deleted Lkb1 in Müllerian duct mesenchyme-derived cells of the female reproductive tract and observed expansion of the stromal compartment and hyperplasia and/or neoplasia of adjacent epithelial cells throughout the reproductive tract with paratubal cysts and adenomyomas in oviducts and, eventually, endometrial cancer. Examination of the proliferation marker phospho-histone H3 and mammalian Target Of Rapamycin Complex 1 (mTORC1) pathway members revealed increased proliferation and mTORC1 activation in stromal cells of both the oviduct and uterus. Treatment with rapamycin, an inhibitor of mTORC1 activity, decreased tumor burden in adult Lkb1 mutant mice. Deletion of the genes for Tuberous Sclerosis 1 (Tsc1) or Tsc2, regulators of mTORC1 that are downstream of LKB1 signaling, in the oviductal and uterine stroma phenocopies some of the defects observed in Lkb1 mutant mice, confirming that dysregulated mTORC1 activation in the Lkb1-deleted stroma contributes to the phenotype. Loss of PTEN, an upstream regulator of mTORC1 signaling, along with Lkb1 deletion significantly increased tumor burden in uteri and induced tumorigenesis in the cervix and vagina. These studies show that LKB1/TSC1/TSC2/mTORC1 signaling in mesenchymal cells is important for the maintenance of epithelial integrity and suppression of carcinogenesis in adjacent epithelial cells. Because similar changes in the stromal population are also observed in human oviductal/ovarian adenoma and endometrial adenocarcinoma patients, we predict that dysregulated mTORC1 activity by upstream mechanisms similar to those described in these model systems contributes to the pathogenesis of these human diseases. PMID:22916036

Transcriptional activation of Mina by Sp1/3 factors.

PubMed

Lian, Shangli; Potula, Hari Hara S K; Pillai, Meenu R; Van Stry, Melanie; Koyanagi, Madoka; Chung, Linda; Watanabe, Makiko; Bix, Mark

2013-01-01

Mina is an epigenetic gene regulatory protein known to function in multiple physiological and pathological contexts, including pulmonary inflammation, cell proliferation, cancer and immunity. We showed previously that the level of Mina gene expression is subject to natural genetic variation linked to 21 SNPs occurring in the Mina 5' region. In order to explore the mechanisms regulating Mina gene expression, we set out to molecularly characterize the Mina promoter in the region encompassing these SNPs. We used three kinds of assays--reporter, gel shift and chromatin immunoprecipitation--to analyze a 2 kb genomic fragment spanning the upstream and intron 1 regions flanking exon 1. Here we discovered a pair of Mina promoters (P1 and P2) and a P1-specific enhancer element (E1). Pharmacologic inhibition and siRNA knockdown experiments suggested that Sp1/3 transcription factors trigger Mina expression through additive activity targeted to a cluster of four Sp1/3 binding sites forming the P1 promoter. These results set the stage for comprehensive analysis of Mina gene regulation from the context of tissue specificity, the impact of inherited genetic variation and the nature of upstream signaling pathways.

Transcriptional Activation of Mina by Sp1/3 Factors

PubMed Central

Lian, Shangli; Potula, Hari Hara S. K.; Pillai, Meenu R.; Van Stry, Melanie; Koyanagi, Madoka; Chung, Linda; Watanabe, Makiko; Bix, Mark

2013-01-01

Mina is an epigenetic gene regulatory protein known to function in multiple physiological and pathological contexts, including pulmonary inflammation, cell proliferation, cancer and immunity. We showed previously that the level of Mina gene expression is subject to natural genetic variation linked to 21 SNPs occurring in the Mina 5′ region [1]. In order to explore the mechanisms regulating Mina gene expression, we set out to molecularly characterize the Mina promoter in the region encompassing these SNPs. We used three kinds of assays – reporter, gel shift and chromatin immunoprecipitation – to analyze a 2 kb genomic fragment spanning the upstream and intron 1 regions flanking exon 1. Here we discovered a pair of Mina promoters (P1 and P2) and a P1-specific enhancer element (E1). Pharmacologic inhibition and siRNA knockdown experiments suggested that Sp1/3 transcription factors trigger Mina expression through additive activity targeted to a cluster of four Sp1/3 binding sites forming the P1 promoter. These results set the stage for comprehensive analysis of Mina gene regulation from the context of tissue specificity, the impact of inherited genetic variation and the nature of upstream signaling pathways. PMID:24324617

Glycogen Synthase Kinase 3 Inactivation Drives T-bet-Mediated Downregulation of Co-receptor PD-1 to Enhance CD8+ Cytolytic T Cell Responses

PubMed Central

Taylor, Alison; Harker, James A.; Chanthong, Kittiphat; Stevenson, Philip G.; Zuniga, Elina I.; Rudd, Christopher E.

2016-01-01

Summary Despite the importance of the co-receptor PD-1 in T cell immunity, the upstream signaling pathway that regulates PD-1 expression has not been defined. Glycogen synthase kinase 3 (GSK-3, isoforms α and β) is a serine-threonine kinase implicated in cellular processes. Here, we identified GSK-3 as a key upstream kinase that regulated PD-1 expression in CD8+ T cells. GSK-3 siRNA downregulation, or inhibition by small molecules, blocked PD-1 expression, resulting in increased CD8+ cytotoxic T lymphocyte (CTL) function. Mechanistically, GSK-3 inactivation increased Tbx21 transcription, promoting enhanced T-bet expression and subsequent suppression of Pdcd1 (encodes PD-1) transcription in CD8+ CTLs. Injection of GSK-3 inhibitors in mice increased in vivo CD8+ OT-I CTL function and the clearance of murine gamma-herpesvirus 68 and lymphocytic choriomeningitis clone 13 and reversed T cell exhaustion. Our findings identify GSK-3 as a regulator of PD-1 expression and demonstrate the applicability of GSK-3 inhibitors in the modulation of PD-1 in immunotherapy. PMID:26885856

Mechanistic Target of Rapamycin-Independent Antidepressant Effects of (R)-Ketamine in a Social Defeat Stress Model.

PubMed

Yang, Chun; Ren, Qian; Qu, Youge; Zhang, Ji-Chun; Ma, Min; Dong, Chao; Hashimoto, Kenji

2018-01-01

The role of the mechanistic target of rapamycin (mTOR) signaling in the antidepressant effects of ketamine is controversial. In addition to mTOR, extracellular signal-regulated kinase (ERK) is a key signaling molecule in prominent pathways that regulate protein synthesis. (R)-Ketamine has a greater potency and longer-lasting antidepressant effects than (S)-ketamine. Here we investigated whether mTOR signaling and ERK signaling play a role in the antidepressant effects of two enantiomers. The effects of mTOR inhibitors (rapamycin and AZD8055) and an ERK inhibitor (SL327) on the antidepressant effects of ketamine enantiomers in the chronic social defeat stress (CSDS) model (n = 7 or 8) and on those of ketamine enantiomers in these signaling pathways in mouse brain regions were examined. The intracerebroventricular infusion of rapamycin or AZD8055 blocked the antidepressant effects of (S)-ketamine, but not (R)-ketamine, in the CSDS model. Furthermore, (S)-ketamine, but not (R)-ketamine, significantly attenuated the decreased phosphorylation of mTOR and its downstream effector, ribosomal protein S6 kinase, in the prefrontal cortex of susceptible mice after CSDS. Pretreatment with SL327 blocked the antidepressant effects of (R)-ketamine but not (S)-ketamine. Moreover, (R)-ketamine, but not (S)-ketamine, significantly attenuated the decreased phosphorylation of ERK and its upstream effector, mitogen-activated protein kinase/ERK kinase, in the prefrontal cortex and hippocampal dentate gyrus of susceptible mice after CSDS. This study suggests that mTOR plays a role in the antidepressant effects of (S)-ketamine, but not (R)-ketamine, and that ERK plays a role in (R)-ketamine's antidepressant effects. Thus, it is unlikely that the activation of mTOR signaling is necessary for antidepressant actions of (R)-ketamine. Copyright © 2017 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Optical power equalization for upstream traffic with injection-locked Fabry-Perot lasers in TDM-PON

NASA Astrophysics Data System (ADS)

Huang, Ting-Tsan; Sheu, Lih-Gen; Chi, Sien

2010-10-01

An optical power equalization of upstream traffic in time-division-multiplexed passive optical network (TDM-PON) based on injection-locked Fabry-Perot lasers has been experimentally investigated. The upstream transmitters with stable spectrum are achieved by using an external injection light source in the optical line terminal (OLT). The different upstream powers can be equalized by injection locking a Fabry-Perot laser diode (FP-LD) biased below threshold current in OLT. The dynamic upstream power range from - 8.5 to - 19.5 db m is reduced to a 1.6 dB maximal power variation, when the uplink signal is directly modulated at 1.25 Gb/s.

IL-4 and IL-13 employ discrete signaling pathways for target gene expression in alternatively activated monocytes/macrophages.

PubMed

Bhattacharjee, Ashish; Shukla, Meenakshi; Yakubenko, Valentin P; Mulya, Anny; Kundu, Suman; Cathcart, Martha K

2013-01-01

Monocytes/macrophages are innate immune cells that play a crucial role in the resolution of inflammation. In the presence of the Th2 cytokines interleukin-4 (IL-4) and interleukin-13 (IL-13), they display an anti-inflammatory profile and this activation pathway is known as alternative activation. In this study we compare and differentiate pathways mediated by IL-4 and IL-13 activation of human monocytes/macrophages. Here we report differential regulation of IL-4 and IL-13 signaling in monocytes/macrophages starting from IL-4/IL-13 cytokine receptors to Jak/Stat-mediated signaling pathways that ultimately control expression of several inflammatory genes. Our data demonstrate that although the receptor-associated tyrosine kinases Jak2 and Tyk2 are activated after the recruitment of IL-13 to its receptor (containing IL-4Rα and IL-13Rα1), IL-4 stimulates Jak1 activation. We further show that Jak2 is upstream of Stat3 activation and Tyk2 controls Stat1 and Stat6 activation in response to IL-13 stimulation. In contrast, Jak1 regulates Stat3 and Stat6 activation in IL-4-induced monocytes. Our results further reveal that although IL-13 utilizes both IL-4Rα/Jak2/Stat3 and IL-13Rα1/Tyk2/Stat1/Stat6 signaling pathways, IL-4 can use only the IL-4Rα/Jak1/Stat3/Stat6 cascade to regulate the expression of some critical inflammatory genes, including 15-lipoxygenase, monoamine oxidase A (MAO-A), and the scavenger receptor CD36. Moreover, we demonstrate here that IL-13 and IL-4 can uniquely affect the expression of particular genes such as dual-specificity phosphatase 1 and tissue inhibitor of metalloprotease-3 and do so through different Jaks. As evidence of differential regulation of gene function by IL-4 and IL-13, we further report that MAO-A-mediated reactive oxygen species generation is influenced by different Jaks. Collectively, these results have major implications for understanding the mechanism and function of alternatively activated monocytes/macrophages by IL-4 and IL-13 and add novel insights into the pathogenesis and potential treatment of various inflammatory diseases. Copyright © 2012 Elsevier Inc. All rights reserved.

NF-κB deregulation in splenic marginal zone lymphoma.

PubMed

Spina, Valeria; Rossi, Davide

2016-08-01

Splenic marginal zone lymphoma is a rare mature B-cell malignancy involving the spleen, bone marrow and blood. Over the past years, the rapid expansion of sequencing technologies allowing the genome-wide assessment of genomic, epigenetic and transcriptional changes has revolutionized our understanding of the biological basis of splenic marginal zone lymphoma by providing a comprehensive and unbiased view of the genes/pathways that are deregulated in this disease. NF-κB is a family of transcription factors that plays critical roles in development, survival, and activation of B lymphocytes. Consistent with the physiological involvement of NF-κB signalling in proliferation and commitment of mature B-cells to the marginal zone of the spleen, many oncogenic mutations involved in constitutive activation of the NF-κB pathway were recently identified in splenic marginal zone lymphoma. This review describes the progress in understanding the mechanism of NF-κB activation in splenic marginal zone lymphoma, including molecular, epigenetic and post-transcriptional modifications of NF-κB genes and of upstream pathways, and discusses how information gained from these efforts has provided new insights on potential targets of diagnostic, prognostic and therapeutic relevance for splenic marginal zone lymphoma. Copyright © 2016. Published by Elsevier Ltd.

Rme1 is necessary for Mi-1-mediated resistance and acts early in the resistance pathway.

PubMed

Martinez de Ilarduya, Oscar; Nombela, Gloria; Hwang, Chin-Feng; Williamson, Valerie M; Muñiz, Mariano; Kaloshian, Isgouhi

2004-01-01

The tomato gene Mi-1 confers resistance to root-knot nematodes (Meloidogyne spp.), potato aphid, and whitefly. Using genetic screens, we have isolated a mutant, rme1 (resistance to Meloidogyne spp.), compromised in resistance to M. javanica and potato aphid. Here, we show that the rme1 mutant is also compromised in resistance to M. incognita, M. arenaria, and whitefly. In addition, using an Agrobacterium-mediated transient assay in leaves to express constitutive gain-of-function mutant Pto(L205D), we demonstrated that the rme1 mutation is not compromised in Pto-mediated hypersensitive response. Moreover, the mutation in rme1 does not result in increased virulence of pathogenic Pseudomonas syringae or Mi-1-virulent M. incognita. Using a chimeric Mi-1 construct, Mi-DS4, which confers constitutive cell death phenotype and A. rhizogenes root transformation, we showed that the Mi-1-mediated cell death pathway is intact in this mutant. Our results indicate that Rme1 is required for Mi-1-mediated resistance and acts either at the same step in the signal transduction pathway as Mi-1 or upstream of Mi-1.

Differential effects of black raspberry and strawberry extracts on BaPDE-induced activation of transcription factors and their target genes.

PubMed

Li, Jingxia; Zhang, Dongyun; Stoner, Gary D; Huang, Chuanshu

2008-04-01

The chemopreventive properties of edible berries have been demonstrated both in vitro and in vivo, however, the specific molecular mechanisms underlying their anti-cancer effects are largely unknown. Our previous studies have shown that a methanol extract fraction of freeze-dried black raspberries inhibits benzoapyrene (BaP)-induced transformation of Syrian hamster embryo cells. This fraction also blocks activation of activator protein-1 (AP-1) and nuclear factor kappaB (NF-kappaB) induced by benzoapyrene diol-epoxide (BaPDE) in mouse epidermal JB6 Cl 41 cells. To determine if different berry types exhibit specific mechanisms for their anti-cancer effects, we compared the effects of extract fractions from both black raspberries and strawberries on BaPDE-induced activation of various signaling pathways in Cl 41 cells. Black raspberry fractions inhibited the activation of AP-1, NF-kappaB, and nuclear factor of activated T cells (NFAT) by BaPDE as well as their upstream PI-3K/Akt-p70(S6K) and mitogen-activated protein kinase pathways. In contrast, strawberry fractions inhibited NFAT activation, but did not inhibit the activation of AP-1, NF-kappaB or the PI-3K/Akt-p70(S6K) and mitogen-activated protein kinase pathways. Consistent with the effects on NFAT activation, tumor necrosis factor-alpha (TNF-alpha) induction by BaPDE was blocked by extract fractions of both black raspberries and strawberries, whereas vascular endothelial growth factor (VEGF) expression, which depends on AP-1 activation, was suppressed by black raspberry fractions but not strawberry fractions. These results suggest that black raspberry and strawberry components may target different signaling pathways in exerting their anti-carcinogenic effects. (c) 2007 Wiley-Liss, Inc.

Differential Effects of Black Raspberry and Strawberry Extracts on BaPDE-Induced Activation of Transcription Factors and Their Target Genes

PubMed Central

Li, Jingxia; Zhang, Dongyun; Stoner, Gary D.; Huang, Chuanshu

2013-01-01

The chemopreventive properties of edible berries have been demonstrated both in vitro and in vivo, however, the specific molecular mechanisms underlying their anti-cancer effects are largely unknown. Our previous studies have shown that a methanol extract fraction of freeze-dried black raspberries inhibits benzoapyrene (BaP)-induced transformation of Syrian hamster embryo cells. This fraction also blocks activation of activator protein-1 (AP-1) and nuclear factor κB (NF-κB) induced by benzoapyrene diol-epoxide (BaPDE) in mouse epidermal JB6 Cl 41 cells. To determine if different berry types exhibit specific mechanisms for their anti-cancer effects, we compared the effects of extract fractions from both black raspberries and strawberries on BaPDE-induced activation of various signaling pathways in Cl 41 cells. Black raspberry fractions inhibited the activation of AP-1, NF-κB, and nuclear factor of activated T cells (NFAT) by BaPDE as well as their upstream PI-3K/Akt-p70S6K and mitogen-activated protein kinase pathways. In contrast, strawberry fractions inhibited NFAT activation, but did not inhibit the activation of AP-1, NF-κB or the PI-3K/Akt-p70S6K and mitogen-activated protein kinase pathways. Consistent with the effects on NFAT activation, tumor necrosis factor-α (TNF-α) induction by BaPDE was blocked by extract fractions of both black raspberries and strawberries, whereas vascular endothelial growth factor (VEGF) expression, which depends on AP-1 activation, was suppressed by black raspberry fractions but not strawberry fractions. These results suggest that black raspberry and strawberry components may target different signaling pathways in exerting their anti-carcinogenic effects. PMID:18085529

New insights into the roles of NADPH oxidases in sexual development and ascospore germination in Sordaria macrospora.

PubMed

Dirschnabel, Daniela Elisabeth; Nowrousian, Minou; Cano-Domínguez, Nallely; Aguirre, Jesus; Teichert, Ines; Kück, Ulrich

2014-03-01

NADPH oxidase (NOX)-derived reactive oxygen species (ROS) act as signaling determinants that induce different cellular processes. To characterize NOX function during fungal development, we utilized the genetically tractable ascomycete Sordaria macrospora. Genome sequencing of a sterile mutant led us to identify the NADPH oxidase encoding nox1 as a gene required for fruiting body formation, regular hyphal growth, and hyphal fusion. These phenotypes are shared by nor1, lacking the NOX regulator NOR1. Further phenotypic analyses revealed a high correlation between increased ROS production and hyphal fusion deficiencies in nox1 and other sterile mutants. A genome-wide transcriptional profiling analysis of mycelia and isolated protoperithecia from wild type and nox1 revealed that nox1 inactivation affects the expression of genes related to cytoskeleton remodeling, hyphal fusion, metabolism, and mitochondrial respiration. Genetic analysis of nox2, lacking the NADPH oxidase 2 gene, nor1, and transcription factor deletion mutant ste12, revealed a strict melanin-dependent ascospore germination defect, indicating a common genetic pathway for these three genes. We report that gsa3, encoding a G-protein α-subunit, and sac1, encoding cAMP-generating adenylate cyclase, act in a separate pathway during the germination process. The finding that cAMP inhibits ascospore germination in a melanin-dependent manner supports a model in which cAMP inhibits NOX2 activity, thus suggesting a link between both pathways. Our results expand the current knowledge on the role of NOX enzymes in fungal development and provide a frame to define upstream and downstream components of the NOX signaling pathways in fungi.

New Insights Into the Roles of NADPH Oxidases in Sexual Development and Ascospore Germination in Sordaria macrospora

PubMed Central

Dirschnabel, Daniela Elisabeth; Nowrousian, Minou; Cano-Domínguez, Nallely; Aguirre, Jesus; Teichert, Ines; Kück, Ulrich

2014-01-01

NADPH oxidase (NOX)-derived reactive oxygen species (ROS) act as signaling determinants that induce different cellular processes. To characterize NOX function during fungal development, we utilized the genetically tractable ascomycete Sordaria macrospora. Genome sequencing of a sterile mutant led us to identify the NADPH oxidase encoding nox1 as a gene required for fruiting body formation, regular hyphal growth, and hyphal fusion. These phenotypes are shared by ∆nor1, lacking the NOX regulator NOR1. Further phenotypic analyses revealed a high correlation between increased ROS production and hyphal fusion deficiencies in ∆nox1 and other sterile mutants. A genome-wide transcriptional profiling analysis of mycelia and isolated protoperithecia from wild type and ∆nox1 revealed that nox1 inactivation affects the expression of genes related to cytoskeleton remodeling, hyphal fusion, metabolism, and mitochondrial respiration. Genetic analysis of ∆nox2, lacking the NADPH oxidase 2 gene, ∆nor1, and transcription factor deletion mutant ∆ste12, revealed a strict melanin-dependent ascospore germination defect, indicating a common genetic pathway for these three genes. We report that gsa3, encoding a G-protein α-subunit, and sac1, encoding cAMP-generating adenylate cyclase, act in a separate pathway during the germination process. The finding that cAMP inhibits ascospore germination in a melanin-dependent manner supports a model in which cAMP inhibits NOX2 activity, thus suggesting a link between both pathways. Our results expand the current knowledge on the role of NOX enzymes in fungal development and provide a frame to define upstream and downstream components of the NOX signaling pathways in fungi. PMID:24407906

Pancreatic glucagon-like peptide-1 receptor couples to multiple G proteins and activates mitogen-activated protein kinase pathways in Chinese hamster ovary cells.

PubMed

Montrose-Rafizadeh, C; Avdonin, P; Garant, M J; Rodgers, B D; Kole, S; Yang, H; Levine, M A; Schwindinger, W; Bernier, M

1999-03-01

Chinese hamster ovary (CHO) cells stably expressing the human insulin receptor and the rat glucagon-like peptide-1 (GLP-1) receptor (CHO/GLPR) were used to study the functional coupling of the GLP-1 receptor with G proteins and to examine the regulation of the mitogen-activated protein (MAP) kinase signaling pathway by GLP-1. We showed that ligand activation of GLP-1 receptor led to increased incorporation of GTP-azidoanilide into Gs alpha, Gq/11 alpha, and Gi1,2 alpha, but not Gi3 alpha. GLP-1 increased p38 MAP kinase activity 2.5- and 2.0-fold over the basal level in both CHO/GLPR cells and rat insulinoma cells (RIN 1046-38), respectively. Moreover, GLP-1 induced phosphorylation of the immediate upstream kinases of p38, MKK3/MKK6, in CHO/GLPR and RIN 1046-38 cells. Ligand-stimulated GLP-1 receptor produced 1.45- and 2.7-fold increases in tyrosine phosphorylation of 42-kDa extracellular signal-regulated kinase (ERK) in CHO/GLPR and RIN 1046-38 cells, respectively. In CHO/GLPR cells, these effects of GLP-1 on the ERK and p38 MAP kinase pathways were inhibited by pretreatment with cholera toxin (CTX), but not with pertussis toxin. The combination of insulin and GLP-1 resulted in an additive response (1.6-fold over insulin alone) that was attenuated by CTX. In contrast, the ability of insulin alone to activate these pathways was insensitive to either toxin. Our study indicates a direct coupling between the GLP-1 receptor and several G proteins, and that CTX-sensitive proteins are required for GLP-1-mediated activation of MAP kinases.

A macrophage NBR1-MEKK3 complex triggers JNK-mediated adipose-tissue inflammation in obesity

PubMed Central

Hernandez, Eloy D.; Lee, Sang Jun; Kim, Ji Young; Duran, Angeles; Linares, Juan F.; Yajima, Tomoko; Müller, Timo D.; Tschöp, Matthias H.; Smith, Steven R.; Diaz-Meco, Maria T.; Moscat, Jorge

2014-01-01

SUMMARY The c-Jun NH(2)-terminal kinase (JNK) is a critical determinant of obesity-associated inflammation and glucose intolerance. The upstream mechanisms controlling this pathway are still unknown. Here we report that the levels of the PB1 domain-containing adapter NBR1 correlated with the expression of pro-inflammatory molecules in adipose tissue from human patients with metabolic syndrome, suggesting that NBR1 plays a key role in adipose-tissue inflammation. We also show that NBR1 inactivation in the myeloid compartment impairs the function, M1 polarization and chemotactic activity of macrophages, prevents inflammation of adipose tissue, and improves glucose tolerance in obese mice. Furthermore, we demonstrate that an interaction between the PB1 domains of NBR1 and the mitogen-activated kinase kinase 3 (MEKK3) enables the formation of a signaling complex required for the activation of JNK. Together these discoveries identify an NBR1-MEKK3 complex as a key regulator of JNK signaling and adipose-tissue inflammation in obesity. PMID:25043814

Mad1 kinetochore recruitment by Mps1-mediated phosphorylation of Bub1 signals the spindle checkpoint.

PubMed

London, Nitobe; Biggins, Sue

2014-01-15

The spindle checkpoint is a conserved signaling pathway that ensures genomic integrity by preventing cell division when chromosomes are not correctly attached to the spindle. Checkpoint activation depends on the hierarchical recruitment of checkpoint proteins to generate a catalytic platform at the kinetochore. Although Mad1 kinetochore localization is the key regulatory downstream event in this cascade, its receptor and mechanism of recruitment have not been conclusively identified. Here, we demonstrate that Mad1 kinetochore association in budding yeast is mediated by phosphorylation of a region within the Bub1 checkpoint protein by the conserved protein kinase Mps1. Tethering this region of Bub1 to kinetochores bypasses the checkpoint requirement for Mps1-mediated kinetochore recruitment of upstream checkpoint proteins. The Mad1 interaction with Bub1 and kinetochores can be reconstituted in the presence of Mps1 and Mad2. Together, this work reveals a critical mechanism that determines kinetochore activation of the spindle checkpoint.

A role for the tyrosine kinase ACK1 in neurotrophin signaling and neuronal extension and branching

PubMed Central

La Torre, A; del Mar Masdeu, M; Cotrufo, T; Moubarak, R S; del Río, J A; Comella, J X; Soriano, E; Ureña, J M

2013-01-01

Neurotrophins are involved in many crucial cellular functions, including neurite outgrowth, synapse formation, and plasticity. Although these events have long been known, the molecular determinants underlying neuritogenesis have not been fully characterized. Ack1 (activated Cdc42-associated tyrosine kinase) is a non-receptor tyrosine kinase that is highly expressed in the brain. Here, we demonstrate that Ack1 is a molecular constituent of neurotrophin signaling cascades in neurons and PC12 cells. We report that Ack1 interacts with Trk receptors and becomes tyrosine phosphorylated and its kinase activity is increased in response to neurotrophins. Moreover, our data indicate that Ack1 acts upstream of the Akt and MAPK pathways. We show that Ack1 overexpression induces neuritic outgrowth and promotes branching in neurotrophin-treated neuronal cells, whereas the expression of Ack1 dominant negatives or short-hairpin RNAs counteract neurotrophin-stimulated differentiation. Our results identify Ack1 as a novel regulator of neurotrophin-mediated events in primary neurons and in PC12 cells. PMID:23598414

Colorless ONU implementation for WDM-PON using direct-detection optical OFDM

NASA Astrophysics Data System (ADS)

Feng, Min; Luo, Qing-long; Bai, Cheng-lin

2013-03-01

A novel architecture for the colorless optical network unit (ONU) is proposed and experimentally demonstrated with direct-detection optical orthogonal frequency division multiplexing (DDO-OFDM). In this architecture, polarization-division multiplexing is used to reduce the cost at ONU. In optical line terminal (OLT), quadrature amplitude modulation (QAM) intensity-modulated OFDM signal with x-polarization at 10 Gbit/s is transmitted as downstream. At each ONU, the optical OFDM signal is demodulated with direct detection, and γ-polarization signal is modulated for upstream on-off keying (OOK) data at 5 Gbit/s. Simulation results show that the power penalty is negligible for both optical OFDM downstream and the on-off keying upstream signals after over 50 km single-mode fiber (SMF) transmission.

Mapping the functional versatility and fragility of Ras GTPase signaling circuits through in vitro network reconstitution.

PubMed

Coyle, Scott M; Lim, Wendell A

2016-01-14

The Ras-superfamily GTPases are central controllers of cell proliferation and morphology. Ras signaling is mediated by a system of interacting molecules: upstream enzymes (GEF/GAP) regulate Ras's ability to recruit multiple competing downstream effectors. We developed a multiplexed, multi-turnover assay for measuring the dynamic signaling behavior of in vitro reconstituted H-Ras signaling systems. By including both upstream regulators and downstream effectors, we can systematically map how different network configurations shape the dynamic system response. The concentration and identity of both upstream and downstream signaling components strongly impacted the timing, duration, shape, and amplitude of effector outputs. The distorted output of oncogenic alleles of Ras was highly dependent on the balance of positive (GAP) and negative (GEF) regulators in the system. We found that different effectors interpreted the same inputs with distinct output dynamics, enabling a Ras system to encode multiple unique temporal outputs in response to a single input. We also found that different Ras-to-GEF positive feedback mechanisms could reshape output dynamics in distinct ways, such as signal amplification or overshoot minimization. Mapping of the space of output behaviors accessible to Ras provides a design manual for programming Ras circuits, and reveals how these systems are readily adapted to produce an array of dynamic signaling behaviors. Nonetheless, this versatility comes with a trade-off of fragility, as there exist numerous paths to altered signaling behaviors that could cause disease.

Binary agonist surface patterns prime platelets for downstream adhesion in flowing whole blood.

PubMed

Eichinger, Colin D; Hlady, Vladimir

2017-04-28

As platelets encounter damaged vessels or biomaterials, they interact with a complex milieu of surface-bound agonists, from exposed subendothelium to adsorbed plasma proteins. It has been shown that an upstream, surface-immobilized agonist is capable of priming platelets for enhanced adhesion downstream. In this study, binary agonists were integrated into the upstream position of flow cells and the platelet priming response was measured by downstream adhesion in flowing whole blood. A nonadditive response was observed in which platelets transiently exposed to two agonists exhibited greater activation and downstream adhesion than that from the sum of either agonist alone. Antibody blocking of one of the two upstream agonists eliminated nonadditive activation and downstream adhesion. Crosstalk between platelet activation pathways likely led to a synergistic effect which created an enhanced activation response in the platelet population. The existence of synergy between platelet priming pathways is a concept that has broad implications for the field of biomaterials hemocompatibility and platelet activity testing.

Experimental demonstration of wavelength domain rogue-free ONU based on wavelength-pairing for TDM/WDM optical access networks.

PubMed

Lee, Jie Hyun; Park, Heuk; Kang, Sae-Kyoung; Lee, Joon Ki; Chung, Hwan Seok

2015-11-30

In this study, we propose and experimentally demonstrate a wavelength domain rogue-free ONU based on wavelength-pairing of downstream and upstream signals for time/wavelength division-multiplexed optical access networks. The wavelength-pairing tunable filter is aligned to the upstream wavelength channel by aligning it to one of the downstream wavelength channels. Wavelength-pairing is implemented with a compact and cyclic Si-AWG integrated with a Ge-PD. The pairing filter covered four 100 GHz-spaced wavelength channels. The feasibility of the wavelength domain rogue-free operation is investigated by emulating malfunction of the misaligned laser. The wavelength-pairing tunable filter based on the Si-AWG blocks the upstream signal in the non-assigned wavelength channel before data collision with other ONUs.

[Chrysin inhibits lipopolysaccharide-induced inflammatory responses of macrophages via JAK-STATs signaling pathway].

PubMed

Qi, Shi-Mei; Li, Qiang; Jiang, Qi; Qi, Zhi-Lin; Zhang, Yao

2018-03-20

To investigate the mechanism of chrysin in regulating lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells. RAW264.7 cells were treated with different concentrations (0, 5, 10, 20, 40, 60, 80, 100, 150, and 200 µg/mL) of chrysin for 24 h, and the cell viability was measured using CCK-8. RAW264.7 cells were pre-treated with 10, 30, or 60 µg/mL chrysin for 2 h before stimulation with LPS for different times. The levels of TNF-α, IL-6 and MCP-1 were detected by ELISA, and Western blotting was used to detect the phosphorylation of JAK- 1, JAK-2, STAT-1 and STAT-3. The level of reactive oxygen species in RAW264.7 cells was detected by CM-H2DCFDA fluorescence probe. The effect of ROS on LPS-induced JAK-STATs signal and the inflammatory response of RAW264.7 cells was detected by ROS scavenger NAC. The transcription factors STAT-1 and STAT-3 nuclear translocation were observed by laser confocal microscopy. Chrysin below 60 µg/mL did not significantly affect the viability of RAW264.7 cells. At 10, 30, and 60 µg/mL, chrysin dose-dependently inhibited the expression of iNOS induced by LPS. Chrysin treatment also inhibited LPS-induced phosphorylation of JAK-STATs, nuclear translocation of STAT1 and STAT3, release of TNF-α, IL-6 and MCP-1, and the production of ROS in RAW264.7 cells; ROS acted as an upstream signal to mediate the activation of JAK-STATs signaling pathway. Chrysin blocks the activity of JAK-STATs mediated by ROS to inhibit LPS-induced inflammatory response in RAW264.7 cells.

ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling*

PubMed Central

Kanamarlapudi, Venkateswarlu; Thompson, Aiysha; Kelly, Eamonn; López Bernal, Andrés

2012-01-01

The luteinizing hormone chorionic gonadotropin receptor (LHCGR) is a Gs-coupled GPCR that is essential for the maturation and function of the ovary and testis. LHCGR is internalized following its activation, which regulates the biological responsiveness of the receptor. Previous studies indicated that ADP-ribosylation factor (ARF)6 and its GTP-exchange factor (GEF) cytohesin 2 regulate LHCGR internalization in follicular membranes. However, the mechanisms by which ARF6 and cytohesin 2 regulate LHCGR internalization remain incompletely understood. Here we investigated the role of the ARF6 signaling pathway in the internalization of heterologously expressed human LHCGR (HLHCGR) in intact cells using a combination of pharmacological inhibitors, siRNA and the expression of mutant proteins. We found that human CG (HCG)-induced HLHCGR internalization, cAMP accumulation and ARF6 activation were inhibited by Gallein (βγ inhibitor), Wortmannin (PI 3-kinase inhibitor), SecinH3 (cytohesin ARF GEF inhibitor), QS11 (an ARF GAP inhibitor), an ARF6 inhibitory peptide and ARF6 siRNA. However, Dynasore (dynamin inhibitor), the dominant negative mutants of NM23-H1 (dynamin activator) and clathrin, and PBP10 (PtdIns 4,5-P2-binding peptide) inhibited agonist-induced HLHCGR and cAMP accumulation but not ARF6 activation. These results indicate that heterotrimeric G-protein, phosphatidylinositol (PI) 3-kinase (PI3K), cytohesin ARF GEF and ARF GAP function upstream of ARF6 whereas dynamin and clathrin act downstream of ARF6 in the regulation of HCG-induced HLHCGR internalization and signaling. In conclusion, we have identified the components and molecular details of the ARF6 signaling pathway required for agonist-induced HLHCGR internalization. PMID:22523074

Reciprocal repression between Fgf8 and miR-133 regulates cardiac induction through Bmp2 signaling.

PubMed

Lopez-Sanchez, Carmen; Franco, Diego; Bonet, Fernando; Garcia-Lopez, Virginio; Aranega, Amelia; Garcia-Martinez, Virginio

2015-12-01

This data article contains complementary figures and results related to the research article entitled "Negative Fgf8-Bmp2 feed-back is controlled by miR-130 during early cardiac specification" [15], which reveals what specific role miR-130 plays during the cardiac induction process. This study evidenced miR-130 a putative microRNA that targets Erk1/2 (Mapk1) 3'UTR- as a necessary linkage in the control of Fgf8 signaling, mediated by Bmp2. Thus, miR-130 regulates a negative Fgf8-Bmp2 feed-back loop responsible to achieve early cardiac specification. A significant aspect supporting our conclusions is given by the expression pattern of miR-130 during early cardiac specification, as well as by those results obtained after the designed experimental procedures. The data presented here reveal that miR-133 is also expressed within the precardiac areas during early cardiogenesis, pattern which is comparable to that of FGFR1, receptor involved in the Fgf8/ERK signaling pathway. Interestingly, our miR-133 overexpression experiments resulted in a decrease of Fgf8 expression, whereas we observed an increase of Bmp2 and subsequently of cardiac specific markers Nkx-2.5 and Gata4. Additionally, our loss-of-function experiments -through Fgf8 siRNA electroporation- showed an increase of miR-133 expression. Finally, after our Bmp2 experiments, we observed that miR-133 is upstream-regulated by Bmp2. All those results suggest that miR-133 also constitutes a crucial linkage in the crosstalk between Fgf8 and Bmp2 signaling by regulating the Fgf8/ERK pathway during cardiac induction.

Gremlin 2 promotes differentiation of embryonic stem cells to atrial fate by activation of the JNK signaling pathway

PubMed Central

Tanwar, Vineeta; Bylund, Jeffery B.; Hu, Jianyong; Yan, Jingbo; Walthall, Joel M.; Mukherjee, Amrita; Heaton, William H.; Wang, Wen-Der; Potet, Franck; Rai, Meena; Kupershmidt, Sabina; Knapik, Ela W.; Hatzopoulos, Antonis K.

2014-01-01

The Bone Morphogenetic Protein antagonist Gremlin 2 (Grem2) is required for atrial differentiation and establishment of cardiac rhythm during embryonic development. A human Grem2 variant has been associated with familial atrial fibrillation, suggesting that abnormal Grem2 activity causes arrhythmias. However, it is not known how Grem2 integrates into signaling pathways to direct atrial cardiomyocyte differentiation. Here, we demonstrate that Grem2 expression is induced concurrently with the emergence of cardiovascular progenitor cells during differentiation of mouse embryonic (ES) stem cells. Grem2 exposure enhances the cardiogenic potential of ES cells by ~20–120 fold, preferentially inducing genes expressed in atrial myocytes such as Myl7, Nppa and Sarcolipin. We show that Grem2 acts upstream to upregulate pro-atrial transcriptional factors CoupTFII and Hey1 and downregulate atrial fate repressors Irx4 and Hey2. The molecular phenotype of Grem2-induced atrial cardiomyocytes was further supported by induction of ion channels encoded by Kcnj3, Kcnj5, and Cacna1D genes and establishment of atrial-like action potentials shown by electrophysiological recordings. We show that promotion of atrial-like cardiomyocyte is specific to the Gremlin subfamily of BMP antagonists. Grem2 pro-atrial differentiation activity is conveyed by non-canonical BMP signaling through phosphorylation of JNK and can be reversed by specific JNK inhibitors, but not by dorsomorphin, an inhibitor of canonical BMP signaling. Taken together, our data provide novel mechanistic insights into atrial cardiomyocyte differentiation from pluripotent stem cells and will assist the development of future approaches to study and treat arrhythmias. PMID:24648383

Repression of Ccr9 transcription in mouse T lymphocyte progenitors by the Notch signaling pathway.

PubMed

Krishnamoorthy, Veena; Carr, Tiffany; de Pooter, Renee F; Emanuelle, Akinola Olumide; Akinola, Emanuelle Olumide; Gounari, Fotini; Kee, Barbara L

2015-04-01

The chemokine receptor CCR9 controls the immigration of multipotent hematopoietic progenitor cells into the thymus to sustain T cell development. Postimmigration, thymocytes downregulate CCR9 and migrate toward the subcapsular zone where they recombine their TCR β-chain and γ-chain gene loci. CCR9 is subsequently upregulated and participates in the localization of thymocytes during their selection for self-tolerant receptor specificities. Although the dynamic regulation of CCR9 is essential for early T cell development, the mechanisms controlling CCR9 expression have not been determined. In this article, we show that key regulators of T cell development, Notch1 and the E protein transcription factors E2A and HEB, coordinately control the expression of Ccr9. E2A and HEB bind at two putative enhancers upstream of Ccr9 and positively regulate CCR9 expression at multiple stages of T cell development. In contrast, the canonical Notch signaling pathway prevents the recruitment of p300 to the putative Ccr9 enhancers, resulting in decreased acetylation of histone H3 and a failure to recruit RNA polymerase II to the Ccr9 promoter. Although Notch signaling modestly modulates the binding of E proteins to one of the two Ccr9 enhancers, we found that Notch signaling represses Ccr9 in T cell lymphoma lines in which Ccr9 transcription is independent of E protein function. Our data support the hypothesis that activation of Notch1 has a dominant-negative effect on Ccr9 transcription and that Notch1 and E proteins control the dynamic expression of Ccr9 during T cell development. Copyright © 2015 by The American Association of Immunologists, Inc.

Regulation of Kisspeptin Synthesis and Release in the Preoptic/Anterior Hypothalamic Region of Prepubertal Female Rats: Actions of IGF-1 and Alcohol.

PubMed

Hiney, Jill K; Srivastava, Vinod K; Vaden Anderson, Danielle N; Hartzoge, Nicole L; Dees, William L

2018-01-01

Alcohol (ALC) causes suppressed secretion of prepubertal luteinizing hormone-releasing hormone (LHRH). Insulin-like growth factor-1 (IGF-1) and kisspeptin (Kp) are major regulators of LHRH and are critical for puberty. IGF-1 may be an upstream mediator of Kp in the preoptic area and rostral hypothalamic area (POA/RHA) of the rat brain, a region containing both Kp and LHRH neurons. We investigated the ability of IGF-1 to stimulate prepubertal Kp synthesis and release in POA/RHA, and the potential inhibitory effects of ALC. Immature female rats were administered either ALC (3 g/kg) or water via gastric gavage at 0730 hours. At 0900 hours, both groups were subdivided where half received either saline or IGF-1 into the brain third ventricle. A second dose of ALC (2 g/kg) or water was administered at 1130 hours. Rats were killed 6 hours after injection and POA/RHA region collected. IGF-1 stimulated Kp, an action blocked by ALC. Upstream to Kp, IGF-1 receptor (IGF-1R) activation, as demonstrated by the increase in insulin receptor substrate 1, resulted in activation of Akt, tuberous sclerosis 2, ras homologue enriched in brain, and mammalian target of rapamycin (mTOR). ALC blocked the central action of IGF-1 to induce their respective phosphorylation. IGF-1 specificity and ALC specificity for the Akt-activated mTOR pathway were demonstrated by the absence of effects on PRAS40. Furthermore, IGF-1 stimulated Kp release from POA/RHA incubated in vitro. IGF-1 stimulates prepubertal Kp synthesis and release following activation of a mTOR signaling pathway, and ALC blocks this pathway at the level of IGF-1R. Copyright © 2017 by the Research Society on Alcoholism.

The Global Redox Responding RegB/RegA Signal Transduction System Regulates the Genes Involved in Ferrous Iron and Inorganic Sulfur Compound Oxidation of the Acidophilic Acidithiobacillus ferrooxidans.

PubMed

Moinier, Danielle; Byrne, Deborah; Amouric, Agnès; Bonnefoy, Violaine

2017-01-01

The chemical attack of ore by ferric iron and/or sulfuric acid releases valuable metals. The products of these reactions are recycled by iron and sulfur oxidizing microorganisms. These acidophilic chemolithotrophic prokaryotes, among which Acidithiobacillus ferrooxidans , grow at the expense of the energy released from the oxidation of ferrous iron and/or inorganic sulfur compounds (ISCs). In At. ferrooxidans , it has been shown that the expression of the genes encoding the proteins involved in these respiratory pathways is dependent on the electron donor and that the genes involved in iron oxidation are expressed before those responsible for ISCs oxidation when both iron and sulfur are present. Since the redox potential increases during iron oxidation but remains stable during sulfur oxidation, we have put forward the hypothesis that the global redox responding two components system RegB/RegA is involved in this regulation. To understand the mechanism of this system and its role in the regulation of the aerobic respiratory pathways in At. ferrooxidans , the binding of different forms of RegA (DNA binding domain, wild-type, unphosphorylated and phosphorylated-like forms of RegA) on the regulatory region of different genes/operons involved in ferrous iron and ISC oxidation has been analyzed. We have shown that the four RegA forms are able to bind specifically the upstream region of these genes. Interestingly, the phosphorylation of RegA did not change its affinity for its cognate DNA. The transcriptional start site of these genes/operons has been determined. In most cases, the RegA binding site(s) was (were) located upstream from the -35 (or -24) box suggesting that RegA does not interfere with the RNA polymerase binding. Based on the results presented in this report, the role of the RegB/RegA system in the regulation of the ferrous iron and ISC oxidation pathways in At. ferrooxidans is discussed.

PICT-1 triggers a pro-death autophagy through inhibiting rRNA transcription and AKT/mTOR/p70S6K signaling pathway.

PubMed

Chen, Hongbo; Duo, Yanhong; Hu, Bo; Wang, Zhiwei; Zhang, Fang; Tsai, Hsiangi; Zhang, Jianping; Zhou, Lanzhen; Wang, Lijun; Wang, Xinyu; Huang, Laiqiang

2016-11-29

PICT-1 was originally identified as a tumor suppressor. Here, we found that PICT-1 overexpression triggered pro-death autophagy without nucleolar disruption or p53 accumulation in U251 and MCF7 cells. Truncated PICT-1 fragments 181-346 and 1-346, which partly or totally lack nucleolar localization, showed weaker autophagy-inducing effects than full-length PICT-1 and a well-defined nucleolar mutant (181-479). Furthermore, PICT-1 partly localizes to the nucleolar fibrillar center (FC) and directly binds to ribosomal DNA (rDNA) gene loci, where it interacts with upstream binding factor (UBF). Overexpression of PICT-1 or the 181-479 mutant, but not the 1-346 or 181-346 mutants, markedly inhibited the phosphorylation of UBF and the recruitment of rRNA polymerase I (Pol I) to the rDNA promoter in response to serum stimulation, thereby suppressing rRNA transcription, suggesting that rRNA transcription inhibition might be an important contributor to PICT-1-induced autophagy. This is supported by the finding that CX-5461, a specific Pol I inhibitor, also induced autophagy. In addition, both CX-5461 and PICT-1, but not the 1-346 or 181-346 mutants, significantly suppressed the activation of the Akt/mTOR/p70S6K signaling pathway. Our data show that PICT-1 triggers pro-death autophagy through inhibition of rRNA transcription and the inactivation of AKT/mTOR/p70S6K pathway, independent of nucleolar disruption and p53 activation.

Role of Akt signaling in resistance to DNA-targeted therapy

PubMed Central

Avan, Abolfazl; Narayan, Ravi; Giovannetti, Elisa; Peters, Godefridus J

2016-01-01

The Akt signal transduction pathway controls most hallmarks of cancer. Activation of the Akt cascade promotes a malignant phenotype and is also widely implicated in drug resistance. Therefore, the modulation of Akt activity is regarded as an attractive strategy to enhance the efficacy of cancer therapy and irradiation. This pathway consists of phosphatidylinositol 3 kinase (PI3K), mammalian target of rapamycin, and the transforming serine-threonine kinase Akt protein isoforms, also known as protein kinase B. DNA-targeted agents, such as platinum agents, taxanes, and antimetabolites, as well as radiation have had a significant impact on cancer treatment by affecting DNA replication, which is aberrantly activated in malignancies. However, the caveat is that they may also trigger the activation of repairing mechanisms, such as upstream and downstream cascade of Akt survival pathway. Thus, each target can theoretically be inhibited in view of improving the potency of conventional treatment. Akt inhibitors, e.g., MK-2206 and perifosine, or PI3K modulators, e.g., LY294002 and Wortmannin, have shown some promising results in favor of sensitizing the cancer cells to the therapy in vitro and in vivo, which have provided the rationale for incorporation of these novel agents into multimodality treatment of different malignancies. Nevertheless, despite the acceptable safety profile of some of these agents in the clinical studies, with regard to the efficacy, the results are still too preliminary. Hence, we need to wait for the upcoming data from the ongoing trials before utilizing them into the standard care of cancer patients. PMID:27777878

Retigabine ameliorates acute stress-induced impairment of spatial memory retrieval through regulating USP2 signaling pathways in hippocampal CA1 area.

PubMed

Li, Cai; Zhang, Ji; Xu, Haiwei; Chang, Mujun; Lv, Chuntao; Xue, Wenhua; Song, Zhizhen; Zhang, Lizhen; Zhang, Xiaojian; Tian, Xin

2018-06-01

Acute stress could trigger maladaptive changes associated with stress-related cognitive and emotional deficits. Dysfunction of ion channel or receptor in the hippocampal area has been linked to the cognitive deficits induced by stress. It is known that Kv7 channel openers, including FDA-approved drug retigabine, show cognitive protective efficacy. However, the underlying molecular mechanisms remain elusive. Here we showed that exposing adult male rats to acute stress significantly impaired the spatial memory, a cognitive process controlled by the hippocampus. Concomitantly, significantly reduced AMPA receptor expression was found in hippocampal CA1 area from acute stressed rats. This effect relied on the down-regulation of deubiquitinating enzyme USP2 and its upstream regulators (PGC-1α and β-catenin), and the subsequent enhancement of mTOR-related autophagy which is regulated by USP2. These findings suggested that acute stress dampened AMPA receptor expression by controlling USP2-related signaling, which caused the detrimental effect on hippocampus-dependent cognitive processes. We also found that retigabine alleviated acute stress-induced spatial memory retrieval impairment through adjusting the aberrance of USP2, its upstream regulators (PGC-1α, E4BP4 and β-catenin) and its downstream targets (mTOR, autophagy and GluA1). Our results have identified USP2 as a key molecule that mediates stress-induced spatial memory retrieval impairment, which provides a framework for new druggable targets to conceptually treat stress-associated cognitive deficits. Copyright © 2018 Elsevier Ltd. All rights reserved.

Modeled microgravity-induced protein kinase C isoform expression in human lymphocytes

NASA Technical Reports Server (NTRS)

Sundaresan, A.; Risin, D.; Pellis, N. R.

2004-01-01

In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited in both microgravity and modeled microgravity (MMG) as reflected by diminished DNA synthesis in peripheral blood lymphocytes and their locomotion through gelled type I collagen. Direct activation of protein kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas the calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 1 g and MMG culture. Human lymphocytes were cultured and harvested at 24, 48, 72, and 96 h, and serial samples were assessed for locomotion by using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta, and -epsilon was assessed by RT-PCR, flow cytometry, and immunoblotting. Results indicated that PKC isoforms delta and epsilon were downregulated by >50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 1-g controls. Events upstream of PKC, such as phosphorylation of phospholipase Cgamma in MMG, revealed accumulation of inactive enzyme. Depressed calcium-independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than PKC, but after ligand-receptor interaction.

Control of flowering time and cold response by a NAC-domain protein in Arabidopsis.

PubMed

Yoo, So Yeon; Kim, Yunhee; Kim, Soo Young; Lee, Jong Seob; Ahn, Ji Hoon

2007-07-25

Plants must integrate complex signals from environmental and endogenous cues to fine-tune the timing of flowering. Low temperature is one of the most common environmental stresses that affect flowering time; however, molecular mechanisms underlying the cold temperature regulation of flowering time are not fully understood. We report the identification of a novel regulator, LONG VEGETATIVE PHASE 1 (LOV1), that controls flowering time and cold response. An Arabidopsis mutant, longvegetative phase 1-1D (lov1-1D) showing the late-flowering phenotype, was isolated by activation tagging screening. Subsequent analyses demonstrated that the phenotype of the mutant resulted from the overexpression of a NAC-domain protein gene (At2g02450). Both gain- and loss-of-function alleles of LOV1 affected flowering time predominantly under long-day but not short-day conditions, suggesting that LOV1 may act within the photoperiod pathway. The expression of CONSTANS (CO), a floral promoter, was affected by LOV1 level, suggesting that LOV1 controls flowering time by negatively regulating CO expression. The epistatic relationship between CO and LOV1 was consistent with this proposed regulatory pathway. Physiological analyses to elucidate upstream signalling pathways revealed that LOV1 regulates the cold response in plants. Loss of LOV1 function resulted in hypersensitivity to cold temperature, whereas a gain-of-function allele conferred cold tolerance. The freezing tolerance was accompanied by upregulation of cold response genes, COLD-REGULATED 15A (COR15A) and COLD INDUCED 1 (KIN1) without affecting expression of the C-repeat-binding factor/dehydration responsive element-binding factor 1 (CBF/DREB1) family of genes. Our study shows that LOV1 functions as a floral repressor that negatively regulates CO expression under long-day conditions and acts as a common regulator of two intersecting pathways that regulate flowering time and the cold response, respectively. Our results suggest an overlapping pathway for controlling cold stress response and flowering time in plants.

Apoptosis in activated T cells: what are the triggers, and what the signal transducers?

PubMed

Häcker, Georg; Bauer, Anette; Villunger, Andreas

2006-11-01

At the end of an immune response, apoptosis drastically reduces the numbers of activated T cells. It has been a matter of intense research how this form of apoptosis is regulated and initiated, and a number of proteins have been identified that contribute to this process. The present, widely accepted model assumes that the interplay of pro- and anti -apoptotic Bcl-2 family members determines the onset of activated T cell death, with the BH3-only protein Bim activating pro-apoptotic Bax/Bak. In the search for up-stream signals, factors from other immune cells have been shown to play a role, and the NFkappaB family member Bcl-3 has been implicated as a signalling-intermediate in T cells. Recent work has tested the interrelation of these factors and has suggested that Bcl-3 acts as a regulator of Bim activation, that the induction of apoptosis through Bim can be complemented by its relative Puma, and that the presence of certain cytokines during T cell activation delays the activation of Bim and Puma. Here we discuss these recent insights and provide a view on how the regulation of activated T cell death is achieved and how extrinsic signals may translate into the activation of the apoptotic pathway.

CIKS, a connection to Ikappa B kinase and stress-activated protein kinase.

PubMed

Leonardi, A; Chariot, A; Claudio, E; Cunningham, K; Siebenlist, U

2000-09-12

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-kappaB and AP-1/ATF families. Activation of NF-kappaB factors is thought to be mediated primarily via IkappaB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKalpha and IKKbeta are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-kappaB essential modulator)/IKKgamma. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKgamma in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-kappaB-dependent reporter. Activation of NF-kappaB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins.

Approach for targeting Ras with small molecules that activate SOS-mediated nucleotide exchange.

PubMed

Burns, Michael C; Sun, Qi; Daniels, R Nathan; Camper, DeMarco; Kennedy, J Phillip; Phan, Jason; Olejniczak, Edward T; Lee, Taekyu; Waterson, Alex G; Rossanese, Olivia W; Fesik, Stephen W

2014-03-04

Aberrant activation of the small GTPase Ras by oncogenic mutation or constitutively active upstream receptor tyrosine kinases results in the deregulation of cellular signals governing growth and survival in ∼30% of all human cancers. However, the discovery of potent inhibitors of Ras has been difficult to achieve. Here, we report the identification of small molecules that bind to a unique pocket on the Ras:Son of Sevenless (SOS):Ras complex, increase the rate of SOS-catalyzed nucleotide exchange in vitro, and modulate Ras signaling pathways in cells. X-ray crystallography of Ras:SOS:Ras in complex with these molecules reveals that the compounds bind in a hydrophobic pocket in the CDC25 domain of SOS adjacent to the Switch II region of Ras. The structure-activity relationships exhibited by these compounds can be rationalized on the basis of multiple X-ray cocrystal structures. Mutational analyses confirmed the functional relevance of this binding site and showed it to be essential for compound activity. These molecules increase Ras-GTP levels and disrupt MAPK and PI3K signaling in cells at low micromolar concentrations. These small molecules represent tools to study the acute activation of Ras and highlight a pocket on SOS that may be exploited to modulate Ras signaling.

Mapping the functional versatility and fragility of Ras GTPase signaling circuits through in vitro network reconstitution

PubMed Central

Coyle, Scott M; Lim, Wendell A

2016-01-01

The Ras-superfamily GTPases are central controllers of cell proliferation and morphology. Ras signaling is mediated by a system of interacting molecules: upstream enzymes (GEF/GAP) regulate Ras’s ability to recruit multiple competing downstream effectors. We developed a multiplexed, multi-turnover assay for measuring the dynamic signaling behavior of in vitro reconstituted H-Ras signaling systems. By including both upstream regulators and downstream effectors, we can systematically map how different network configurations shape the dynamic system response. The concentration and identity of both upstream and downstream signaling components strongly impacted the timing, duration, shape, and amplitude of effector outputs. The distorted output of oncogenic alleles of Ras was highly dependent on the balance of positive (GAP) and negative (GEF) regulators in the system. We found that different effectors interpreted the same inputs with distinct output dynamics, enabling a Ras system to encode multiple unique temporal outputs in response to a single input. We also found that different Ras-to-GEF positive feedback mechanisms could reshape output dynamics in distinct ways, such as signal amplification or overshoot minimization. Mapping of the space of output behaviors accessible to Ras provides a design manual for programming Ras circuits, and reveals how these systems are readily adapted to produce an array of dynamic signaling behaviors. Nonetheless, this versatility comes with a trade-off of fragility, as there exist numerous paths to altered signaling behaviors that could cause disease. DOI: http://dx.doi.org/10.7554/eLife.12435.001 PMID:26765565

A Conserved PHD Finger Protein and Endogenous RNAi Modulate Insulin Signaling in Caenorhabditis elegans

PubMed Central

Hoersch, Sebastian; Jensen, Morten B.; Kawli, Trupti; Kennedy, Lisa M.; Chavez, Violeta; Tan, Man-Wah; Lieb, Jason D.; Grishok, Alla

2011-01-01

Insulin signaling has a profound effect on longevity and the oxidative stress resistance of animals. Inhibition of insulin signaling results in the activation of DAF-16/FOXO and SKN-1/Nrf transcription factors and increased animal fitness. By studying the biological functions of the endogenous RNA interference factor RDE-4 and conserved PHD zinc finger protein ZFP-1 (AF10), which regulate overlapping sets of genes in Caenorhabditis elegans, we identified an important role for these factors in the negative modulation of transcription of the insulin/PI3 signaling-dependent kinase PDK-1. Consistently, increased expression of pdk-1 in zfp-1 and rde-4 mutants contributed to their reduced lifespan and sensitivity to oxidative stress and pathogens due to the reduction in the expression of DAF-16 and SKN-1 targets. We found that the function of ZFP-1 in modulating pdk-1 transcription was important for the extended lifespan of the age-1(hx546) reduction-of-function PI3 kinase mutant, since the lifespan of the age-1; zfp-1 double mutant strain was significantly shorter compared to age-1(hx546). We further demonstrate that overexpression of ZFP-1 caused an increased resistance to oxidative stress in a DAF-16–dependent manner. Our findings suggest that epigenetic regulation of key upstream signaling components in signal transduction pathways through chromatin and RNAi may have a large impact on the outcome of signaling and expression of numerous downstream genes. PMID:21980302

A conserved PHD finger protein and endogenous RNAi modulate insulin signaling in Caenorhabditis elegans.

PubMed

Mansisidor, Andres R; Cecere, Germano; Hoersch, Sebastian; Jensen, Morten B; Kawli, Trupti; Kennedy, Lisa M; Chavez, Violeta; Tan, Man-Wah; Lieb, Jason D; Grishok, Alla

2011-09-01

Insulin signaling has a profound effect on longevity and the oxidative stress resistance of animals. Inhibition of insulin signaling results in the activation of DAF-16/FOXO and SKN-1/Nrf transcription factors and increased animal fitness. By studying the biological functions of the endogenous RNA interference factor RDE-4 and conserved PHD zinc finger protein ZFP-1 (AF10), which regulate overlapping sets of genes in Caenorhabditis elegans, we identified an important role for these factors in the negative modulation of transcription of the insulin/PI3 signaling-dependent kinase PDK-1. Consistently, increased expression of pdk-1 in zfp-1 and rde-4 mutants contributed to their reduced lifespan and sensitivity to oxidative stress and pathogens due to the reduction in the expression of DAF-16 and SKN-1 targets. We found that the function of ZFP-1 in modulating pdk-1 transcription was important for the extended lifespan of the age-1(hx546) reduction-of-function PI3 kinase mutant, since the lifespan of the age-1; zfp-1 double mutant strain was significantly shorter compared to age-1(hx546). We further demonstrate that overexpression of ZFP-1 caused an increased resistance to oxidative stress in a DAF-16-dependent manner. Our findings suggest that epigenetic regulation of key upstream signaling components in signal transduction pathways through chromatin and RNAi may have a large impact on the outcome of signaling and expression of numerous downstream genes.

Salinity effect on the metabolic pathway and microbial function in phenanthrene degradation by a halophilic consortium.

PubMed

Wang, Chongyang; Huang, Yong; Zhang, Zuotao; Wang, Hui

2018-04-25

With the close relationship between saline environments and industry, polycyclic aromatic hydrocarbons (PAHs) accumulate in saline/hypersaline environments. Therefore, PAHs degradation by halotolerant/halophilic bacteria has received increasing attention. In this study, the metabolic pathway of phenanthrene degradation by halophilic consortium CY-1 was first studied which showed a single upstream pathway initiated by dioxygenation at the C1 and C2 positions, and at several downstream pathways, including the catechol pathway, gentisic acid pathway and protocatechuic acid pathway. The effects of salinity on the community structure and expression of catabolic genes were further studied by a combination of high-throughput sequencing, catabolic gene clone library and real-time PCR. Pure cultures were also isolated from consortium CY-1 to investigate the contribution made by different microbes in the PAH-degrading process. Marinobacter is the dominant genus that contributed to the upstream degradation of phenanthrene especially in high salt content. Genus Halomonas made a great contribution in transforming intermediates in the subsequent degradation of catechol by using catechol 1,2-dioxygenase (C12O). Other microbes were predicted to be mediating bacteria that were able to utilize intermediates via different downstream pathways. Salinity was investigated to have negative effects on both microbial diversity and activity of consortium CY-1 and consortium CY-1 was found with a high degree of functional redundancy in saline environments.

Transmembrane voltage potential controls embryonic eye patterning in Xenopus laevis

PubMed Central

Pai, Vaibhav P.; Aw, Sherry; Shomrat, Tal; Lemire, Joan M.; Levin, Michael

2012-01-01

Uncovering the molecular mechanisms of eye development is crucial for understanding the embryonic morphogenesis of complex structures, as well as for the establishment of novel biomedical approaches to address birth defects and injuries of the visual system. Here, we characterize change in transmembrane voltage potential (Vmem) as a novel biophysical signal for eye induction in Xenopus laevis. During normal embryogenesis, a striking hyperpolarization demarcates a specific cluster of cells in the anterior neural field. Depolarizing the dorsal lineages in which these cells reside results in malformed eyes. Manipulating Vmem of non-eye cells induces well-formed ectopic eyes that are morphologically and histologically similar to endogenous eyes. Remarkably, such ectopic eyes can be induced far outside the anterior neural field. A Ca2+ channel-dependent pathway transduces the Vmem signal and regulates patterning of eye field transcription factors. These data reveal a new, instructive role for membrane voltage during embryogenesis and demonstrate that Vmem is a crucial upstream signal in eye development. Learning to control bioelectric initiators of organogenesis offers significant insight into birth defects that affect the eye and might have significant implications for regenerative approaches to ocular diseases. PMID:22159581

Early Induction of NRF2 Antioxidant Pathway by RHBDF2 Mediates Rapid Cutaneous Wound Healing

PubMed Central

Hosur, Vishnu; Burzenski, Lisa M.; Stearns, Timothy M.; Farley, Michelle L.; Sundberg, John P.; Wiles, Michael V.; Shultz, Leonard D.

2017-01-01

Rhomboid family protein RHBDF2, an upstream regulator of the epidermal growth factor (EGF) receptor signaling, has been implicated in cutaneous wound healing. However, the underlying molecular mechanisms are still emerging. In humans, a gain-of-function mutation in the RHBDF2 gene accelerates cutaneous wound healing in an EGFR-dependent manner. Likewise, a gain-of-function mutation in the mouse Rhbdf2 gene (Rhbdf2cub/cub) shows a regenerative phenotype (rapid ear-hole closure) resulting from constitutive activation of the EGFR pathway. Because the RHBDF2-regulated EGFR pathway is relevant to cutaneous wound healing in humans, we used Rhbdf2cub/cub mice to investigate the biological networks and pathways leading to accelerated ear-hole closure, with the goal of identifying therapeutic targets potentially effective in promoting wound healing in humans. Comparative transcriptome analysis of ear pinna tissue from Rhbdf2cub/cub and Rhbdf2+/+ mice at 0h, 15 min, 2h, and 24h post-wounding revealed an early induction of the nuclear factor E2-related factor 2 (NRF2)-mediated anti-oxidative pathway (0h and 15 min), followed by the integrin-receptor aggregation pathway (2h) as early-stage events immediately and shortly after wounding in Rhbdf2cub/cub mice. Additionally, we observed genes enriched for the Fc fragment of the IgG receptor IIIa (FCGR3A)-mediated phagocytosis pathway 24h post-wounding. Although cutaneous wound repair in healthy individuals is generally non-problematic, it can be severely impaired due to aging, diabetes, and chronic inflammation. This study suggests that activation of the NRF2-antioxidant pathway by rhomboid protein RHBDF2 might be beneficial in treating chronic non-healing wounds. PMID:28268192

Early induction of NRF2 antioxidant pathway by RHBDF2 mediates rapid cutaneous wound healing.

PubMed

Hosur, Vishnu; Burzenski, Lisa M; Stearns, Timothy M; Farley, Michelle L; Sundberg, John P; Wiles, Michael V; Shultz, Leonard D

2017-04-01

Rhomboid family protein RHBDF2, an upstream regulator of the epidermal growth factor (EGF) receptor signaling, has been implicated in cutaneous wound healing. However, the underlying molecular mechanisms are still emerging. In humans, a gain-of-function mutation in the RHBDF2 gene accelerates cutaneous wound healing in an EGFR-dependent manner. Likewise, a gain-of-function mutation in the mouse Rhbdf2 gene (Rhbdf2 cub/cub ) shows a regenerative phenotype (rapid ear-hole closure) resulting from constitutive activation of the EGFR pathway. Because the RHBDF2-regulated EGFR pathway is relevant to cutaneous wound healing in humans, we used Rhbdf2 cub/cub mice to investigate the biological networks and pathways leading to accelerated ear-hole closure, with the goal of identifying therapeutic targets potentially effective in promoting wound healing in humans. Comparative transcriptome analysis of ear pinna tissue from Rhbdf2 cub/cub and Rhbdf2 +/+ mice at 0h, 15min, 2h, and 24h post-wounding revealed an early induction of the nuclear factor E2-related factor 2 (NRF2)-mediated anti-oxidative pathway (0h and 15min), followed by the integrin-receptor aggregation pathway (2h) as early-stage events immediately and shortly after wounding in Rhbdf2 cub/cub mice. Additionally, we observed genes enriched for the Fc fragment of the IgG receptor IIIa (FCGR3A)-mediated phagocytosis pathway 24h post-wounding. Although cutaneous wound repair in healthy individuals is generally non-problematic, it can be severely impaired due to aging, diabetes, and chronic inflammation. This study suggests that activation of the NRF2-antioxidant pathway by rhomboid protein RHBDF2 might be beneficial in treating chronic non-healing wounds. Copyright © 2017 Elsevier Inc. All rights reserved.

CGK733-induced LC3 II formation is positively associated with the expression of cyclin-dependent kinase inhibitor p21Waf1/Cip1 through modulation of the AMPK and PERK/CHOP signaling pathways.

PubMed

Wang, Yufeng; Kuramitsu, Yasuhiro; Baron, Byron; Kitagawa, Takao; Tokuda, Kazuhiro; Akada, Junko; Nakamura, Kazuyuki

2015-11-24

Microtubule-associated protein 1A/1B-light chain 3 (LC3)-II is essential for autophagosome formation and is widely used to monitor autophagic activity. We show that CGK733 induces LC3 II and LC3-puncta accumulation, which are not involved in the activation of autophagy. The treatment of CGK733 did not alter the autophagic flux and was unrelated to p62 degradation. Treatment with CGK733 activated the AMP-activated protein kinase (AMPK) and protein kinase RNA-like endoplasmic reticulum kinase/CCAAT-enhancer-binding protein homologous protein (PERK/CHOP) pathways and elevated the expression of p21Waf1/Cip1. Inhibition of both AMPK and PERK/CHOP pathways by siRNA or chemical inhibitor could block CGK733-induced p21Waf1/Cip1 expression as well as caspase-3 cleavage. Knockdown of LC3 B (but not LC3 A) abolished CGK733-triggered LC3 II accumulation and consequently diminished AMPK and PERK/CHOP activity as well as p21Waf1/Cip1 expression. Our results demonstrate that CGK733-triggered LC3 II formation is an initial event upstream of the AMPK and PERK/CHOP pathways, both of which control p21Waf1/Cip1 expression.

A Unique Fungal Two-Component System Regulates Stress Responses, Drug Sensitivity, Sexual Development, and Virulence of Cryptococcus neoformans

PubMed Central

Bahn, Yong-Sun; Kojima, Kaihei; Cox, Gary M.

2006-01-01

The stress-activated mitogen-activated protein kinase (MAPK) pathway is widely used by eukaryotic organisms as a central conduit via which cellular responses to the environment effect growth and differentiation. The basidiomycetous human fungal pathogen Cryptococcus neoformans uniquely uses the stress-activated Pbs2-Hog1 MAPK system to govern a plethora of cellular events, including stress responses, drug sensitivity, sexual reproduction, and virulence. Here, we characterized a fungal “two-component” system that controls these fundamental cellular functions via the Pbs2-Hog1 MAPK cascade. A typical response regulator, Ssk1, modulated all Hog1-dependent phenotypes by controlling Hog1 phosphorylation, indicating that Ssk1 is the major upstream signaling component of the Pbs2-Hog1 pathway. A second response regulator, Skn7, governs sensitivity to Na+ ions and the antifungal agent fludioxonil, negatively controls melanin production, and functions independently of Hog1 regulation. To control these response regulators, C. neoformans uses multiple sensor kinases, including two-component–like (Tco) 1 and Tco2. Tco1 and Tco2 play shared and distinct roles in stress responses and drug sensitivity through the Hog1 MAPK system. Furthermore, each sensor kinase mediates unique cellular functions for virulence and morphological differentiation. Our findings highlight unique adaptations of this global two-component MAPK signaling cascade in a ubiquitous human fungal pathogen. PMID:16672377

Modulation of paracrine signaling by CD9 positive small extracellular vesicles mediates cellular growth of androgen deprived prostate cancer

PubMed Central

Soekmadji, Carolina; Riches, James D.; Russell, Pamela J.; Ruelcke, Jayde E.; McPherson, Stephen; Wang, Chenwei; Hovens, Chris M.; Corcoran, Niall M.; Hill, Michelle M.; Nelson, Colleen C.

2017-01-01

Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by steroid hormones, particularly androgens, and the extracellular environment. Herein, we identify the secretion of CD9 positive extracellular vesicles (EV) by LNCaP and DUCaP PCa cells in response to dihydrotestosterone (DHT) and use nano-LC–MS/MS to identify the proteins present in these EV. Subsequent bioinformatic and pathway analyses of the mass spectrometry data identified pathologically relevant pathways that may be altered by EV contents. Western blot and CD9 EV TR-FIA assay confirmed a specific increase in the amount of CD9 positive EV in DHT-treated LNCaP and DUCaP cells and treatment of cells with EV enriched with CD9 after DHT exposure can induce proliferation in androgen-deprived conditions. siRNA knockdown of endogenous CD9 in LNCaPs reduced cellular proliferation and expression of AR and prostate specific antigen (PSA) however knockdown of AR did not alter CD9 expression, also implicating CD9 as an upstream regulator of AR. Moreover CD9 positive EV were also found to be significantly higher in plasma from prostate cancer patients in comparison with benign prostatic hyperplasia patients. We conclude that CD9 positive EV are involved in mediating paracrine signalling and contributing toward prostate cancer progression. PMID:28881726

NRF2-ome: an integrated web resource to discover protein interaction and regulatory networks of NRF2.

PubMed

Türei, Dénes; Papp, Diána; Fazekas, Dávid; Földvári-Nagy, László; Módos, Dezső; Lenti, Katalin; Csermely, Péter; Korcsmáros, Tamás

2013-01-01

NRF2 is the master transcriptional regulator of oxidative and xenobiotic stress responses. NRF2 has important roles in carcinogenesis, inflammation, and neurodegenerative diseases. We developed an online resource, NRF2-ome, to provide an integrated and systems-level database for NRF2. The database contains manually curated and predicted interactions of NRF2 as well as data from external interaction databases. We integrated NRF2 interactome with NRF2 target genes, NRF2 regulating TFs, and miRNAs. We connected NRF2-ome to signaling pathways to allow mapping upstream NRF2 regulatory components that could directly or indirectly influence NRF2 activity totaling 35,967 protein-protein and signaling interactions. The user-friendly website allows researchers without computational background to search, browse, and download the database. The database can be downloaded in SQL, CSV, BioPAX, SBML, PSI-MI, and in a Cytoscape CYS file formats. We illustrated the applicability of the website by suggesting a posttranscriptional negative feedback of NRF2 by MAFG protein and raised the possibility of a connection between NRF2 and the JAK/STAT pathway through STAT1 and STAT3. NRF2-ome can also be used as an evaluation tool to help researchers and drug developers to understand the hidden regulatory mechanisms in the complex network of NRF2.

Arabidopsis AIP1-2 restricted by WER-mediated patterning modulates planar polarity.

PubMed

Kiefer, Christian S; Claes, Andrea R; Nzayisenga, Jean-Claude; Pietra, Stefano; Stanislas, Thomas; Hüser, Anke; Ikeda, Yoshihisa; Grebe, Markus

2015-01-01

The coordination of cell polarity within the plane of the tissue layer (planar polarity) is crucial for the development of diverse multicellular organisms. Small Rac/Rho-family GTPases and the actin cytoskeleton contribute to planar polarity formation at sites of polarity establishment in animals and plants. Yet, upstream pathways coordinating planar polarity differ strikingly between kingdoms. In the root of Arabidopsis thaliana, a concentration gradient of the phytohormone auxin coordinates polar recruitment of Rho-of-plant (ROP) to sites of polar epidermal hair initiation. However, little is known about cytoskeletal components and interactions that contribute to this planar polarity or about their relation to the patterning machinery. Here, we show that ACTIN7 (ACT7) represents a main actin isoform required for planar polarity of root hair positioning, interacting with the negative modulator ACTIN-INTERACTING PROTEIN1-2 (AIP1-2). ACT7, AIP1-2 and their genetic interaction are required for coordinated planar polarity of ROP downstream of ethylene signalling. Strikingly, AIP1-2 displays hair cell file-enriched expression, restricted by WEREWOLF (WER)-dependent patterning and modified by ethylene and auxin action. Hence, our findings reveal AIP1-2, expressed under control of the WER-dependent patterning machinery and the ethylene signalling pathway, as a modulator of actin-mediated planar polarity. © 2015. Published by The Company of Biologists Ltd.

A Member of the p38 Mitogen-Activated Protein Kinase Family Is Responsible for Transcriptional Induction of Dopa decarboxylase in the Epidermis of Drosophila melanogaster during the Innate Immune Response▿ †

PubMed Central

Davis, Monica M.; Primrose, David A.; Hodgetts, Ross B.

2008-01-01

Drosophila innate immunity is controlled primarily by the activation of IMD (immune deficiency) or Toll signaling leading to the production of antimicrobial peptides (AMPs). IMD signaling also activates the JUN N-terminal kinase (JNK) cascade, which is responsible for immune induction of non-antimicrobial peptide immune gene transcription though the transcription factor AP-1. Transcription of the Dopa decarboxylase (Ddc) gene is induced in response to gram-negative and gram-positive septic injury, but not aseptic wounding. Transcription is induced throughout the epidermis and not specifically at the site of infection. Ddc transcripts are detectible within 2 h and remain high for several hours following infection with either gram-negative or gram-positive bacteria. Using Ddc-green fluorescent protein (GFP) reporter gene constructs, we show that a conserved consensus AP-1 binding site upstream of the Ddc transcription start site is required for induction. However, neither the Toll, IMD, nor JNK pathway is involved. Rather, Ddc transcription depends on a previously uncharacterized member of the p38 mitogen-activated protein kinase family, p38c. We propose that the involvement of DDC in a new pathway involved in Drosophila immunity increases the levels of dopamine, which is metabolized to produce reactive quinones that exert an antimicrobial effect on invading bacteria. PMID:18519585

KRASG12D expression in lung-resident myeloid cells promotes pulmonary LCH-like neoplasm sensitive to statin treatment

PubMed Central

Giblett, Susan; Pritchard, Catrin

2017-01-01

Langerhans cell histiocytosis (LCH) is a rare histiocytic neoplasm associated with somatic mutations in the genes involved in the RAF/MEK/extracellular signal-regulated kinase (ERK) signaling pathway. Recently, oncogenic mutations in NRAS/KRAS, upstream regulators of the RAF/MEK/ERK pathway, have been reported in pulmonary, but not in nonpulmonary, LCH cases, suggesting organ-specific contribution of oncogenic RAS to LCH pathogenesis. Using a mouse model expressing KRASG12D in the lung by nasal delivery of adenoviral Cre recombinase (Cre), here we show that KRASG12D expression in lung-resident myeloid cells induces pulmonary LCH-like neoplasms composed of pathogenic CD11chighF4/80+CD207+ cells. The pathogenic cells were mitotically inactive, but proliferating precursors were detected in primary cultures of lung tissue. These precursors were derived, at least in part, from CD11cdimCD11bintGr1− lung-resident monocytic cells transformed by KRASG12D. In contrast, BRAFV600E expression induced by the same method failed to develop LCH-like neoplasms, suggesting that each oncogene may initiate pulmonary LCH by transforming different types of lung-resident myeloid cells. In vivo treatment of the KRASG12D-induced LCH-like mouse with the cholesterol-lowering drug atorvastatin ameliorated the pathology, implicating statins as potential therapeutics against a subset of pulmonary LCH. PMID:28550040

Downregulation of miR-133 via MAPK/ERK signaling pathway involved in nicotine-induced cardiomyocyte apoptosis.

PubMed

Wang, Lu; Li, Xuelian; Zhou, Yuhong; Shi, Hui; Xu, Chaoqian; He, Hua; Wang, Shuxuan; Xiong, Xuehui; Zhang, Yong; Du, Zhimin; Zhang, Ruixue; Lu, Yanjie; Yang, Baofeng; Shan, Hongli

2014-02-01

Tobacco smoking is a risk factor for many diseases, and nicotine is a major component of tobacco. Our previous work revealed that nicotine can induce myocardial fibrosis. This study aimed to investigate whether nicotine can induce cardiomyocyte apoptosis and to explore the mechanisms involved. Cardiomyocytes were exposed to different nicotine concentrations for 48 h. MTT assay showed that the viability of cardiomyocytes was significantly inhibited by nicotine in a dose- and time-dependent manner. Loss of mitochondrial membrane potential, nuclear and DNA defragmentation determined by TUNEL and ELISA assays, and morphological alterations all revealed the pro-apoptotic property of nicotine. Meanwhile, miR-133, a muscle-specific microRNA, was markedly downregulated by nicotine. Consistently, caspase-9, a target gene for miR-133, was significantly upregulated, leading to an increase in caspase-3, in nicotine-treated cardiomyocytes compared to non-treated cells. Furthermore, ERK1/2 protein levels were considerably downregulated, along with reduction of serum response factor (SRF), which is a downstream target protein of ERK1/2 and an upstream transactivator of miR-133 as well. Our findings therefore revealed that inhibition of the ERK1/2-SRF-miR-133 signaling pathway to increase caspases-9 and -3 is a novel mechanism for nicotine to induce cardiomyocyte apoptosis and these tobacco smokers.

Two chalcones, 4-hydroxyderricin and xanthoangelol, stimulate GLUT4-dependent glucose uptake through the LKB1/AMP-activated protein kinase signaling pathway in 3T3-L1 adipocytes.

PubMed

Ohta, Mitsuhiro; Fujinami, Aya; Kobayashi, Norihiro; Amano, Akiko; Ishigami, Akihito; Tokuda, Harukuni; Suzuki, Nobutaka; Ito, Fumitake; Mori, Taisuke; Sawada, Morio; Iwasa, Koichi; Kitawaki, Jo; Ohnishi, Katsunori; Tsujikawa, Muneo; Obayashi, Hiroshi

2015-07-01

4-Hydroxyderricin (4HD) and xanthoangelol (XAG) are major components of n-hexane/ethyl acetate (5:1) extract of the yellow-colored stem juice of Angelica keiskei. 4-Hydroxyderricin and XAG have been reported to increase glucose transporter 4 (GLUT4)-dependent glucose uptake in 3T3-L1 adipocytes, but the detailed mechanism of this phenomenon remains unknown. This present study was aimed at clarifying the detailed mechanism by which 4HD and XAG increase GLUT4-dependent glucose uptake in 3T3-L1 adipocytes. Both 4HD and XAG increased glucose uptake and GLUT4 translocation to the plasma membrane. 4-Hydroxyderricin and XAG also stimulated the phosphorylation of 5' adenosine monophosphate-activated protein kinase (AMPK) and its downstream target acetyl-CoA carboxylase. In addition, phosphorylation of liver kinase B1 (LKB1), which acts upstream of AMPK, was also increased by 4HD and XAG treatment. Small interfering RNA knockdown of LKB1 attenuated 4HD- and XAG-stimulated AMPK phosphorylation and suppressed glucose uptake. These findings demonstrate that 4HD and XAG can increase GLUT4-dependent glucose uptake through the LKB1/AMPK signaling pathway in 3T3-L1 adipocytes. Copyright © 2015 Elsevier Inc. All rights reserved.

Expression of CdDHN4, a Novel YSK2-Type Dehydrin Gene from Bermudagrass, Responses to Drought Stress through the ABA-Dependent Signal Pathway

PubMed Central

Lv, Aimin; Fan, Nana; Xie, Jianping; Yuan, Shili; An, Yuan; Zhou, Peng

2017-01-01

Dehydrin improves plant resistance to many abiotic stresses. In this study, the expression profiles of a dehydrin gene, CdDHN4, were estimated under various stresses and abscisic acid (ABA) treatments in two bermudagrasses (Cynodon dactylon L.): Tifway (drought-tolerant) and C299 (drought-sensitive). The expression of CdDHN4 was up-regulated by high temperatures, low temperatures, drought, salt and ABA. The sensitivity of CdDHN4 to ABA and the expression of CdDHN4 under drought conditions were higher in Tifway than in C299. A 1239-bp fragment, CdDHN4-P, the partial upstream sequence of the CdDHN4 gene, was cloned by genomic walking from Tifway. Bioinformatic analysis showed that the CdDHN4-P sequence possessed features typical of a plant promoter and contained many typical cis elements, including a transcription initiation site, a TATA-box, an ABRE, an MBS, a MYC, an LTRE, a TATC-box and a GT1-motif. Transient expression in tobacco leaves demonstrated that the promoter CdDHN4-P can be activated by ABA, drought and cold. These results indicate that CdDHN4 is regulated by an ABA-dependent signal pathway and that the high sensitivity of CdDHN4 to ABA might be an important mechanism enhancing the drought tolerance of bermudagrass. PMID:28559903

Expression of CdDHN4, a Novel YSK2-Type Dehydrin Gene from Bermudagrass, Responses to Drought Stress through the ABA-Dependent Signal Pathway.

PubMed

Lv, Aimin; Fan, Nana; Xie, Jianping; Yuan, Shili; An, Yuan; Zhou, Peng

2017-01-01

Dehydrin improves plant resistance to many abiotic stresses. In this study, the expression profiles of a dehydrin gene, CdDHN4 , were estimated under various stresses and abscisic acid (ABA) treatments in two bermudagrasses ( Cynodon dactylon L.): Tifway (drought-tolerant) and C299 (drought-sensitive). The expression of CdDHN4 was up-regulated by high temperatures, low temperatures, drought, salt and ABA. The sensitivity of CdDHN4 to ABA and the expression of CdDHN4 under drought conditions were higher in Tifway than in C299. A 1239-bp fragment, CdDHN4-P, the partial upstream sequence of the CdDHN4 gene, was cloned by genomic walking from Tifway. Bioinformatic analysis showed that the CdDHN4-P sequence possessed features typical of a plant promoter and contained many typical cis elements, including a transcription initiation site, a TATA-box, an ABRE, an MBS, a MYC, an LTRE, a TATC-box and a GT1-motif. Transient expression in tobacco leaves demonstrated that the promoter CdDHN4-P can be activated by ABA, drought and cold. These results indicate that CdDHN4 is regulated by an ABA-dependent signal pathway and that the high sensitivity of CdDHN4 to ABA might be an important mechanism enhancing the drought tolerance of bermudagrass.

Flavonoids from Orostachys japonicus A. Berger induces caspase-dependent apoptosis at least partly through activation of p38 MAPK pathway in U937 human leukemic cells.

PubMed

Lee, Won Sup; Yun, Jeong Won; Nagappan, Arulkumar; Jung, Ji Hyun; Yi, Sang Mi; Kim, Dong Hoon; Kim, Hye Jung; Kim, GonSup; Ryu, Chung Ho; Shin, Sung Chul; Hong, Soon Chan; Choi, Yung Hyun; Jung, Jin-Myung

2015-01-01

Orostachys japonicus A. Berger (A. Berger) is commonly used as a folk remedy for cancer therapy. However, the mechanisms of its anti-cancer activity are poorly investigated in human cancer cells. In this study, we investigated whether flavonoids extracted from Orostachys japonicus A. Berger (FEOJ) might have anticancer effects in human leukemia cells, focusing on cell death mechanisms. U937 human leukemic cancer cells were used. FEOJ induced apoptosis in a dose-dependent manner in human U937 cancer cells. Flow cytometry revealed significant accumulation of cells with sub-G1 DNA content at the concentrations of 200 μg/mL and 400 μg/mL. FEOJ-induced apoptosis was caspase-dependent through loss of mitochondrial membrane potential (MMP, ΔΨm) in human U937 cancer cells, which might be associated with suppression of Bcl-2 and XIAP proteins. FEOJ induced the p38 MAPK signaling pathway, playing at least in part an important role in FEOJ-induced apoptosis. This study suggested that FEOJ may induce caspase-dependent apoptosis in human leukemic cells by regulating MMP (ΔΨm) through suppressing Bcl-2 and X-IAP. In addition, the results indicated that upstream p38 MAPK signaling regulates the apoptotic effect of FEOJ. This study provides evidence that FEOJ might have anti-cancer potential for human leukemic cells.

Regulation of Cdk7 activity through a phosphatidylinositol (3)-kinase/PKC-ι-mediated signaling cascade in glioblastoma

PubMed Central

Desai, Shraddha R.; Pillai, Prajit P.; Patel, Rekha S.; McCray, Andrea N.; Win-Piazza, Hla Y.; Acevedo-Duncan, Mildred E.

2012-01-01

The objective of this research was to study the potential function of protein kinase C (PKC)-ι in cell cycle progression and proliferation in glioblastoma. PKC-ι is highly overexpressed in human glioma and benign and malignant meningioma; however, little is understood about its role in regulating cell proliferation of glioblastoma. Several upstream molecular aberrations and/or loss of PTEN have been implicated to constitutively activate the phosphatidylinositol (PI) (3)-kinase pathway. PKC-ι is a targeted mediator in the PI (3)-kinase signal transduction repertoire. Results showed that PKC-ι was highly activated and overexpressed in glioma cells. PKC-ι directly associated and phosphorylated Cdk7 at T170 in a cell cycle-dependent manner, phosphorylating its downstream target, cdk2 at T160. Cdk2 has a major role in inducing G1–S phase progression of cells. Purified PKC-ι phosphorylated both endogenous and exogenous Cdk7. PKC-ι downregulation reduced Cdk7 and cdk2 phosphorylation following PI (3)-kinase inhibition, phosphotidylinositol-dependent kinase 1 knockdown as well as PKC-ι silencing (by siRNA treatment). It also diminished cdk2 activity. PKC-ι knockdown inhibited overall proliferation rates and induced apoptosis in glioma cells. These findings suggest that glioma cells may be proliferating through a novel PI (3)-kinase-/PKC-ι/Cdk7/cdk2-mediated pathway. PMID:22021906

Regulation of Cdk7 activity through a phosphatidylinositol (3)-kinase/PKC-ι-mediated signaling cascade in glioblastoma.

PubMed

Desai, Shraddha R; Pillai, Prajit P; Patel, Rekha S; McCray, Andrea N; Win-Piazza, Hla Y; Acevedo-Duncan, Mildred E

2012-01-01

The objective of this research was to study the potential function of protein kinase C (PKC)-ι in cell cycle progression and proliferation in glioblastoma. PKC-ι is highly overexpressed in human glioma and benign and malignant meningioma; however, little is understood about its role in regulating cell proliferation of glioblastoma. Several upstream molecular aberrations and/or loss of PTEN have been implicated to constitutively activate the phosphatidylinositol (PI) (3)-kinase pathway. PKC-ι is a targeted mediator in the PI (3)-kinase signal transduction repertoire. Results showed that PKC-ι was highly activated and overexpressed in glioma cells. PKC-ι directly associated and phosphorylated Cdk7 at T170 in a cell cycle-dependent manner, phosphorylating its downstream target, cdk2 at T160. Cdk2 has a major role in inducing G(1)-S phase progression of cells. Purified PKC-ι phosphorylated both endogenous and exogenous Cdk7. PKC-ι downregulation reduced Cdk7 and cdk2 phosphorylation following PI (3)-kinase inhibition, phosphotidylinositol-dependent kinase 1 knockdown as well as PKC-ι silencing (by siRNA treatment). It also diminished cdk2 activity. PKC-ι knockdown inhibited overall proliferation rates and induced apoptosis in glioma cells. These findings suggest that glioma cells may be proliferating through a novel PI (3)-kinase-/PKC-ι/Cdk7/cdk2-mediated pathway.

CCCTC-binding Factor Mediates Effects of Glucose On Beta Cell Survival

PubMed Central

Tsui, Shanli; Dai, Wei; Lu, Luo

2013-01-01

Objectives Pancreatic islet β-cell survival is important in regulating insulin activities and maintaining glucose homeostasis. Recently, Pax6 has been shown to be essential for many vital functions in β-cells, though the molecular mechanisms of its regulation in β-cells remain unclear. The present study investigates the novel effects of glucose- and insulin-induced CTCF activity on Pax6 gene expression as well as the subsequent effects of insulin-activated signaling pathways on β-cell proliferation. Material and methods Pancreatic β-TC-1-6 cells were cultured in DMEM medium and stimulated with high concentrations of glucose (5 to 125 mM) and cell viability was assessed by MTT assays. The effect of CTCF on Pax6 was evaluated in high glucose-induced and CCCTC-binding Factor (CTCF)/Erk suppressed cells by promoter reporter and Western analyses. Results Increases in glucose and insulin concentrations up-regulated CTCF and consequently down-regulated Pax6 in β-cell survival and proliferation. Knocking-down CTCF directly affected Pax6 transcription through CTCF binding and blocked the response to glucose. Altered Erk activity mediated the effects of CTCF on controlling Pax6 expression, which partially regulates β-cell proliferation. Conclusions CTCF functions as a molecular mediator between insulin-induced upstream Erk signaling and Pax6 expression in pancreatic β-cells. This pathway may contribute to regulation of β-cell survival and proliferation. PMID:24354619

Regulation of drought tolerance by the F-box protein MAX2 in Arabidopsis.

PubMed

Bu, Qingyun; Lv, Tianxiao; Shen, Hui; Luong, Phi; Wang, Jimmy; Wang, Zhenyu; Huang, Zhigang; Xiao, Langtao; Engineer, Cawas; Kim, Tae Houn; Schroeder, Julian I; Huq, Enamul

2014-01-01

MAX2 (for MORE AXILLARY GROWTH2) has been shown to regulate diverse biological processes, including plant architecture, photomorphogenesis, senescence, and karrikin signaling. Although karrikin is a smoke-derived abiotic signal, a role for MAX2 in abiotic stress response pathways is least investigated. Here, we show that the max2 mutant is strongly hypersensitive to drought stress compared with wild-type Arabidopsis (Arabidopsis thaliana). Stomatal closure of max2 was less sensitive to abscisic acid (ABA) than that of the wild type. Cuticle thickness of max2 was significantly thinner than that of the wild type. Both of these phenotypes of max2 mutant plants correlate with the increased water loss and drought-sensitive phenotype. Quantitative real-time reverse transcription-polymerase chain reaction analyses showed that the expression of stress-responsive genes and ABA biosynthesis, catabolism, transport, and signaling genes was impaired in max2 compared with wild-type seedlings in response to drought stress. Double mutant analysis of max2 with the ABA-insensitive mutants abi3 and abi5 indicated that MAX2 may function upstream of these genes. The expression of ABA-regulated genes was enhanced in imbibed max2 seeds. In addition, max2 mutant seedlings were hypersensitive to ABA and osmotic stress, including NaCl, mannitol, and glucose. Interestingly, ABA, osmotic stress, and drought-sensitive phenotypes were restricted to max2, and the strigolactone biosynthetic pathway mutants max1, max3, and max4 did not display any defects in these responses. Taken together, these results uncover an important role for MAX2 in plant responses to abiotic stress conditions.

The synthetic α-bromo-2',3,4,4'-tetramethoxychalcone (α-Br-TMC) inhibits the JAK/STAT signaling pathway.

PubMed

Pinz, Sophia; Unser, Samy; Brueggemann, Susanne; Besl, Elisabeth; Al-Rifai, Nafisah; Petkes, Hermina; Amslinger, Sabine; Rascle, Anne

2014-01-01

Signal transducer and activator of transcription STAT5 and its upstream activating kinase JAK2 are essential mediators of cytokine signaling. Their activity is normally tightly regulated and transient. However, constitutive activation of STAT5 is found in numerous cancers and a driving force for malignant transformation. We describe here the identification of the synthetic chalcone α-Br-2',3,4,4'-tetramethoxychalcone (α-Br-TMC) as a novel JAK/STAT inhibitor. Using the non-transformed IL-3-dependent B cell line Ba/F3 and its oncogenic derivative Ba/F3-1*6 expressing constitutively activated STAT5, we show that α-Br-TMC targets the JAK/STAT pathway at multiple levels, inhibiting both JAK2 and STAT5 phosphorylation. Moreover, α-Br-TMC alters the mobility of STAT5A/B proteins in SDS-PAGE, indicating a change in their post-translational modification state. These alterations correlate with a decreased association of STAT5 and RNA polymerase II with STAT5 target genes in chromatin immunoprecipitation assays. Interestingly, expression of STAT5 target genes such as Cis and c-Myc was differentially regulated by α-Br-TMC in normal and cancer cells. While both genes were inhibited in IL-3-stimulated Ba/F3 cells, expression of the oncogene c-Myc was down-regulated and that of the tumor suppressor gene Cis was up-regulated in transformed Ba/F3-1*6 cells. The synthetic chalcone α-Br-TMC might therefore represent a promising novel anticancer agent for therapeutic intervention in STAT5-associated malignancies.

Intact LKB1 activity is required for survival of dormant ovarian cancer spheroids.

PubMed

Peart, Teresa; Ramos Valdes, Yudith; Correa, Rohann J M; Fazio, Elena; Bertrand, Monique; McGee, Jacob; Préfontaine, Michel; Sugimoto, Akira; DiMattia, Gabriel E; Shepherd, Trevor G

2015-09-08

Metastatic epithelial ovarian cancer (EOC) cells can form multicellular spheroids while in suspension and disperse directly throughout the peritoneum to seed secondary lesions. There is growing evidence that EOC spheroids are key mediators of metastasis, and they use specific intracellular signalling pathways to control cancer cell growth and metabolism for increased survival. Our laboratory discovered that AKT signalling is reduced during spheroid formation leading to cellular quiescence and autophagy, and these may be defining features of tumour cell dormancy. To further define the phenotype of EOC spheroids, we have initiated studies of the Liver kinase B1 (LKB1)-5'-AMP-activated protein kinase (AMPK) pathway as a master controller of the metabolic stress response. We demonstrate that activity of AMPK and its upstream kinase LKB1 are increased in quiescent EOC spheroids as compared with proliferating adherent EOC cells. We also show elevated AMPK activity in spheroids isolated directly from patient ascites. Functional studies reveal that treatment with the AMP mimetic AICAR or allosteric AMPK activator A-769662 led to a cytostatic response in proliferative adherent ovarian cancer cells, but they fail to elicit an effect in spheroids. Targeted knockdown of STK11 by RNAi to reduce LKB1 expression led to reduced viability and increased sensitivity to carboplatin treatment in spheroids only, a phenomenon which was AMPK-independent. Thus, our results demonstrate a direct impact of altered LKB1-AMPK signalling function in EOC. In addition, this is the first evidence in cancer cells demonstrating a pro-survival function for LKB1, a kinase traditionally thought to act as a tumour suppressor.

Cross-talk between abscisic acid-dependent and abscisic acid-independent pathways during abiotic stress.

PubMed

Roychoudhury, Aryadeep; Paul, Saikat; Basu, Supratim

2013-07-01

Salinity, drought and low temperature are the common forms of abiotic stress encountered by land plants. To cope with these adverse environmental factors, plants execute several physiological and metabolic responses. Both osmotic stress (elicited by water deficit or high salt) and cold stress increase the endogenous level of the phytohormone abscisic acid (ABA). ABA-dependent stomatal closure to reduce water loss is associated with small signaling molecules like nitric oxide, reactive oxygen species and cytosolic free calcium, and mediated by rapidly altering ion fluxes in guard cells. ABA also triggers the expression of osmotic stress-responsive (OR) genes, which usually contain single/multiple copies of cis-acting sequence called abscisic acid-responsive element (ABRE) in their upstream regions, mostly recognized by the basic leucine zipper-transcription factors (TFs), namely, ABA-responsive element-binding protein/ABA-binding factor. Another conserved sequence called the dehydration-responsive element (DRE)/C-repeat, responding to cold or osmotic stress, but not to ABA, occurs in some OR promoters, to which the DRE-binding protein/C-repeat-binding factor binds. In contrast, there are genes or TFs containing both DRE/CRT and ABRE, which can integrate input stimuli from salinity, drought, cold and ABA signaling pathways, thereby enabling cross-tolerance to multiple stresses. A strong candidate that mediates such cross-talk is calcium, which serves as a common second messenger for abiotic stress conditions and ABA. The present review highlights the involvement of both ABA-dependent and ABA-independent signaling components and their interaction or convergence in activating the stress genes. We restrict our discussion to salinity, drought and cold stress.

Phage display of an intracellular carboxylesterase of Bacillus subtilis: comparison of Sec and Tat pathway export capabilities.

PubMed

Dröge, Melloney J; Boersma, Ykelien L; Braun, Peter G; Buining, Robbert Jan; Julsing, Mattijs K; Selles, Karin G A; van Dijl, Jan Maarten; Quax, Wim J

2006-07-01

Using the phage display technology, a protein can be displayed at the surface of bacteriophages as a fusion to one of the phage coat proteins. Here we describe development of this method for fusion of an intracellular carboxylesterase of Bacillus subtilis to the phage minor coat protein g3p. The carboxylesterase gene was cloned in the g3p-based phagemid pCANTAB 5E upstream of the sequence encoding phage g3p and downstream of a signal peptide-encoding sequence. The phage-bound carboxylesterase was correctly folded and fully enzymatically active, as determined from hydrolysis of the naproxen methyl ester with Km values of 0.15 mM and 0.22 mM for the soluble and phage-displayed carboxylesterases, respectively. The signal peptide directs the encoded fusion protein to the cell membrane of Escherichia coli, where phage particles are assembled. In this study, we assessed the effects of several signal peptides, both Sec dependent and Tat dependent, on the translocation of the carboxylesterase in order to optimize the phage display of this enzyme normally restricted to the cytoplasm. Functional display of Bacillus carboxylesterase NA could be achieved when Sec-dependent signal peptides were used. Although a Tat-dependent signal peptide could direct carboxylesterase translocation across the inner membrane of E. coli, proper assembly into phage particles did not seem to occur.

Full duplex dense-wavelength-division-multiplexing radio-over-fiber system transmission of 75-GHz W-band frequency multiple-input multiple-output orthogonal-frequency-division-multiplexing signals with 3×12 Gbps downstream and 6 Gbps upstream

NASA Astrophysics Data System (ADS)

Fang, Wei Jin; Huang, Xu Guang; Yang, Kai; Zhang, Xiao Min

2012-09-01

We propose and demonstrate a full duplex dense-wavelength-division-multiplexing radio-over-fiber (DWDM-ROF) system for transmitting 75-GHz W-band frequency multiple-input multiple-output orthogonal-frequency-division-multiplexing (MIMO-OFDM) signals with 12 Gbps downstream and 6 Gbps upstream. The downstream transmitting terminal is based on a three-channels sextupling-frequency scheme using an external modulation of a distributed feedback laser diode (DFB-LD) and dual drive Mach-Zehnder modulator (DD-MZM) for carrying downstream signals. MIMO-OFDM algorithms effectively compensate for impairments in the wireless link. Without using costly W-band components in the transmitter, a 12 Gbps downstream transmission system operation at 75 GHz is experimentally validated. For the downstream transmission, a power penalty of less than 3 dB was observed after a 50 km single mode fiber (SMF) and 4 m wireless transmission at a bit error rate (BER) of 3.8×10-3. For the upstream transmission, we use a commercially available 1.5 GHz bandwidth reflective semiconductor optical amplifier (RSOA) to achieve 6 Gbps upstream traffic for 16 QAM-OFDM signals. A power penalty of 3 dB was observed after a 50 km SMF transmission at a BER of 3.8×10-3. The frequency of the local oscillator is reduced due to the frequency sextupling scheme. The cost of the proposed system is largely reduced.

Optimum swimming pathways of fish spawning migrations in rivers

USGS Publications Warehouse

McElroy, Brandon; DeLonay, Aaron; Jacobson, Robert

2012-01-01

Fishes that swim upstream in rivers to spawn must navigate complex fluvial velocity fields to arrive at their ultimate locations. One hypothesis with substantial implications is that fish traverse pathways that minimize their energy expenditure during migration. Here we present the methodological and theoretical developments necessary to test this and similar hypotheses. First, a cost function is derived for upstream migration that relates work done by a fish to swimming drag. The energetic cost scales with the cube of a fish's relative velocity integrated along its path. By normalizing to the energy requirements of holding a position in the slowest waters at the path's origin, a cost function is derived that depends only on the physical environment and not on specifics of individual fish. Then, as an example, we demonstrate the analysis of a migration pathway of a telemetrically tracked pallid sturgeon (Scaphirhynchus albus) in the Missouri River (USA). The actual pathway cost is lower than 105 random paths through the surveyed reach and is consistent with the optimization hypothesis. The implication—subject to more extensive validation—is that reproductive success in managed rivers could be increased through manipulation of reservoir releases or channel morphology to increase abundance of lower-cost migration pathways.

Comparative RNA-Sequence Transcriptome Analysis of Phenolic Acid Metabolism in Salvia miltiorrhiza, a Traditional Chinese Medicine Model Plant

PubMed Central

Song, Zhenqiao; Guo, Linlin; Liu, Tian; Lin, Caicai; Wang, Jianhua

2017-01-01

Salvia miltiorrhiza Bunge is an important traditional Chinese medicine (TCM). In this study, two S. miltiorrhiza genotypes (BH18 and ZH23) with different phenolic acid concentrations were used for de novo RNA sequencing (RNA-seq). A total of 170,787 transcripts and 56,216 unigenes were obtained. There were 670 differentially expressed genes (DEGs) identified between BH18 and ZH23, 250 of which were upregulated in ZH23, with genes involved in the phenylpropanoid biosynthesis pathway being the most upregulated genes. Nine genes involved in the lignin biosynthesis pathway were upregulated in BH18 and thus result in higher lignin content in BH18. However, expression profiles of most genes involved in the core common upstream phenylpropanoid biosynthesis pathway were higher in ZH23 than that in BH18. These results indicated that genes involved in the core common upstream phenylpropanoid biosynthesis pathway might play an important role in downstream secondary metabolism and demonstrated that lignin biosynthesis was a putative partially competing pathway with phenolic acid biosynthesis. The results of this study expanded our understanding of the regulation of phenolic acid biosynthesis in S. miltiorrhiza. PMID:28194403

Zanthoxylum ailanthoides Suppresses Oleic Acid-Induced Lipid Accumulation through an Activation of LKB1/AMPK Pathway in HepG2 Cells

PubMed Central

Kwon, Eun-Bin; Kang, Myung-Ji; Kim, Soo-Yeon; Lee, Yong-Moon; Lee, Mi-Kyeong; Yuk, Heung Joo; Ryu, Hyung Won; Lee, Su Ui

2018-01-01

Zanthoxylum ailanthoides (ZA) has been used as folk medicines in East Asian and recently reported to have several bioactivity; however, the studies of ZA on the regulation of triacylglycerol (TG) biosynthesis have not been elucidated yet. In this study, we examined whether the methanol extract of ZA (ZA-M) could reduce oleic acid- (OA-) induced intracellular lipid accumulation and confirmed its mode of action in HepG2 cells. ZA-M was shown to promote the phosphorylation of AMPK and its upstream LKB1, followed by reduction of lipogenic gene expressions. As a result, treatment of ZA-M blocked de novo TG biosynthesis and subsequently mitigated intracellular neutral lipid accumulation in HepG2 cells. ZA-M also inhibited OA-induced production of reactive oxygen species (ROS) and TNF-α, suggesting that ZA-M possess the anti-inflammatory feature in fatty acid over accumulated condition. Taken together, these results suggest that ZA-M attenuates OA-induced lipid accumulation and inflammation through the activation of LKB1/AMPK signaling pathway in HepG2 cells. PMID:29507591

Zanthoxylum ailanthoides Suppresses Oleic Acid-Induced Lipid Accumulation through an Activation of LKB1/AMPK Pathway in HepG2 Cells.

PubMed

Kwon, Eun-Bin; Kang, Myung-Ji; Kim, Soo-Yeon; Lee, Yong-Moon; Lee, Mi-Kyeong; Yuk, Heung Joo; Ryu, Hyung Won; Lee, Su Ui; Oh, Sei-Ryang; Moon, Dong-Oh; Lee, Hyun-Sun; Kim, Mun-Ock

2018-01-01

Zanthoxylum ailanthoides (ZA) has been used as folk medicines in East Asian and recently reported to have several bioactivity; however, the studies of ZA on the regulation of triacylglycerol (TG) biosynthesis have not been elucidated yet. In this study, we examined whether the methanol extract of ZA (ZA-M) could reduce oleic acid- (OA-) induced intracellular lipid accumulation and confirmed its mode of action in HepG2 cells. ZA-M was shown to promote the phosphorylation of AMPK and its upstream LKB1, followed by reduction of lipogenic gene expressions. As a result, treatment of ZA-M blocked de novo TG biosynthesis and subsequently mitigated intracellular neutral lipid accumulation in HepG2 cells. ZA-M also inhibited OA-induced production of reactive oxygen species (ROS) and TNF- α , suggesting that ZA-M possess the anti-inflammatory feature in fatty acid over accumulated condition. Taken together, these results suggest that ZA-M attenuates OA-induced lipid accumulation and inflammation through the activation of LKB1/AMPK signaling pathway in HepG2 cells.

Stress-mediated translational control in cancer cells.

PubMed

Leprivier, Gabriel; Rotblat, Barak; Khan, Debjit; Jan, Eric; Sorensen, Poul H

2015-07-01

Tumor cells are continually subjected to diverse stress conditions of the tumor microenvironment, including hypoxia, nutrient deprivation, and oxidative or genotoxic stress. Tumor cells must evolve adaptive mechanisms to survive these conditions to ultimately drive tumor progression. Tight control of mRNA translation is critical for this response and the adaptation of tumor cells to such stress forms. This proceeds though a translational reprogramming process which restrains overall translation activity to preserve energy and nutrients, but which also stimulates the selective synthesis of major stress adaptor proteins. Here we present the different regulatory signaling pathways which coordinate mRNA translation in the response to different stress forms, including those regulating eIF2α, mTORC1 and eEF2K, and we explain how tumor cells hijack these pathways for survival under stress. Finally, mechanisms for selective mRNA translation under stress, including the utilization of upstream open reading frames (uORFs) and internal ribosome entry sites (IRESes) are discussed in the context of cell stress. This article is part of a Special Issue entitled: Translation and Cancer. Copyright © 2014 Elsevier B.V. All rights reserved.

Requirement of Xmsx-1 in the BMP-triggered ventralization of Xenopus embryos.

PubMed

Yamamoto, T S; Takagi, C; Ueno, N

2000-03-01

Signaling triggered by polypeptide growth factors leads to the activation of their target genes. Several homeobox genes are known to be induced in response to polypeptide growth factors in early Xenopus development. In particular, Xmsx-1, an amphibian homologue of vertebrate Msx-1, is well characterized as a target gene of bone morphogenetic protein (BMP). Here, using a dominant-negative form of Xmsx-1 (VP-Xmsx-1), which is a fusion protein made with the virus-derived VP16 activation domain, we have examined whether Xmsx-1 activity is required in the endogenous ventralizing pathway. VP-Xmsx-1 induced a secondary body axis, complete with muscle and neural tissues, when overexpressed in ventral blastomeres, suggesting that Xmsx-1 activity is necessary for both mesoderm and ectoderm to be ventralized. We have also examined the epistatic relationship between Xmsx-1 and another ventralizing homeobox protein, Xvent-1, and show that Xmsx-1 is likely to be acting upstream of Xvent-1. We propose that Xmsx-1 is required in the BMP-stimulated ventralization pathway that involves the downstream activation of Xvent-1.

Induction of the SHARP-2 mRNA level by insulin is mediated by multiple signaling pathways.

PubMed

Kanai, Yukiko; Asano, Kosuke; Komatsu, Yoshiko; Takagi, Katsuhiro; Ono, Moe; Tanaka, Takashi; Tomita, Koji; Haneishi, Ayumi; Tsukada, Akiko; Yamada, Kazuya

2017-02-01

The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor which represses transcription of the rat phosphoenolpyruvate carboxykinase gene. In this study, a regulatory mechanism of the SHARP-2 mRNA level by insulin was analyzed. Insulin rapidly induced the level of SHARP-2 mRNA. This induction was blocked by inhibitors for phosphoinositide 3-kinase (PI 3-K), protein kinase C (PKC), and mammalian target of rapamycin (mTOR), actinomycin D, and cycloheximide. Whereas an adenovirus infection expressing a dominant negative form of atypical PKC lambda (aPKCλ) blocked the insulin-induction of the SHARP-2 mRNA level, insulin rapidly activated the mTOR. Insulin did not enhance transcriptional activity from a 3.7 kb upstream region of the rat SHARP-2 gene. Thus, we conclude that insulin induces the expression of the rat SHARP-2 gene at the transcription level via both a PI 3-K/aPKCλ- and a PI 3-K/mTOR- pathways and that protein synthesis is required for this induction.

Photodynamic therapy with redaporfin targets the endoplasmic reticulum and Golgi apparatus.

PubMed

Gomes-da-Silva, Lígia C; Zhao, Liwei; Bezu, Lucillia; Zhou, Heng; Sauvat, Allan; Liu, Peng; Durand, Sylvère; Leduc, Marion; Souquere, Sylvie; Loos, Friedemann; Mondragón, Laura; Sveinbjørnsson, Baldur; Rekdal, Øystein; Boncompain, Gaelle; Perez, Franck; Arnaut, Luis G; Kepp, Oliver; Kroemer, Guido

2018-05-28

Preclinical evidence depicts the capacity of redaporfin (Redp) to act as potent photosensitizer, causing direct antineoplastic effects as well as indirect immune-dependent destruction of malignant lesions. Here, we investigated the mechanisms through which photodynamic therapy (PDT) with redaporfin kills cancer cells. Subcellular localization and fractionation studies based on the physicochemical properties of redaporfin revealed its selective tropism for the endoplasmic reticulum (ER) and the Golgi apparatus (GA). When activated, redaporfin caused rapid reactive oxygen species-dependent perturbation of ER/GA compartments, coupled to ER stress and an inhibition of the GA-dependent secretory pathway. This led to a general inhibition of protein secretion by PDT-treated cancer cells. The ER/GA play a role upstream of mitochondria in the lethal signaling pathway triggered by redaporfin-based PDT Pharmacological perturbation of GA function or homeostasis reduces mitochondrial permeabilization. In contrast, removal of the pro-apoptotic multidomain proteins BAX and BAK or pretreatment with protease inhibitors reduced cell killing, yet left the GA perturbation unaffected. Altogether, these results point to the capacity of redaporfin to kill tumor cells via destroying ER/GA function. © 2018 The Authors.

The stimulus selectivity and connectivity of layer six principal cells reveals cortical microcircuits underlying visual processing.

PubMed

Vélez-Fort, Mateo; Rousseau, Charly V; Niedworok, Christian J; Wickersham, Ian R; Rancz, Ede A; Brown, Alexander P Y; Strom, Molly; Margrie, Troy W

2014-09-17

Sensory computations performed in the neocortex involve layer six (L6) cortico-cortical (CC) and cortico-thalamic (CT) signaling pathways. Developing an understanding of the physiological role of these circuits requires dissection of the functional specificity and connectivity of the underlying individual projection neurons. By combining whole-cell recording from identified L6 principal cells in the mouse primary visual cortex (V1) with modified rabies virus-based input mapping, we have determined the sensory response properties and upstream monosynaptic connectivity of cells mediating the CC or CT pathway. We show that CC-projecting cells encompass a broad spectrum of selectivity to stimulus orientation and are predominantly innervated by deep layer V1 neurons. In contrast, CT-projecting cells are ultrasparse firing, exquisitely tuned to orientation and direction information, and receive long-range input from higher cortical areas. This segregation in function and connectivity indicates that L6 microcircuits route specific contextual and stimulus-related information within and outside the cortical network. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Mitophagy controls beige adipocyte maintenance through a Parkin-dependent and UCP1-independent mechanism.

PubMed

Lu, Xiaodan; Altshuler-Keylin, Svetlana; Wang, Qiang; Chen, Yong; Henrique Sponton, Carlos; Ikeda, Kenji; Maretich, Pema; Yoneshiro, Takeshi; Kajimura, Shingo

2018-04-24

Beige adipocytes are an inducible form of mitochondria-enriched thermogenic adipocytes that emerge in response to external stimuli, such as chronic cold exposure. We have previously shown that after the withdrawal of external stimuli, beige adipocytes directly acquire a white fat-like phenotype through autophagy-mediated mitochondrial degradation. We investigated the upstream pathway that mediates mitochondrial clearance and report that Parkin-mediated mitophagy plays a key role in the beige-to-white adipocyte transition. Mice genetically deficient in Park2 showed reduced mitochondrial degradation and retained thermogenic beige adipocytes even after the withdrawal of external stimuli. Norepinephrine signaling through the PKA pathway inhibited the recruitment of Parkin protein to mitochondria in beige adipocytes. However, mitochondrial proton uncoupling by uncoupling protein 1 (UCP1) was dispensable for Parkin recruitment and beige adipocyte maintenance. These results suggest a physiological mechanism by which external cues control mitochondrial homeostasis in thermogenic fat cells through mitophagy. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Small molecule drug A-769662 and AMP synergistically activate naive AMPK independent of upstream kinase signaling.

PubMed

Scott, John W; Ling, Naomi; Issa, Samah M A; Dite, Toby A; O'Brien, Matthew T; Chen, Zhi-Ping; Galic, Sandra; Langendorf, Christopher G; Steinberg, Gregory R; Kemp, Bruce E; Oakhill, Jonathan S

2014-05-22

The AMP-activated protein kinase (AMPK) is a metabolic stress-sensing αβγ heterotrimer responsible for energy homeostasis, making it a therapeutic target for metabolic diseases such as type 2 diabetes and obesity. AMPK signaling is triggered by phosphorylation on the AMPK α subunit activation loop Thr172 by upstream kinases. Dephosphorylated, naive AMPK is thought to be catalytically inactive and insensitive to allosteric regulation by AMP and direct AMPK-activating drugs such as A-769662. Here we show that A-769662 activates AMPK independently of α-Thr172 phosphorylation, provided β-Ser108 is phosphorylated. Although neither A-769662 nor AMP individually stimulate the activity of dephosphorylated AMPK, together they stimulate >1,000-fold, bypassing the requirement for β-Ser108 phosphorylation. Consequently A-769662 and AMP together activate naive AMPK entirely allosterically and independently of upstream kinase signaling. These findings have important implications for development of AMPK-targeting therapeutics and point to possible combinatorial therapeutic strategies based on AMP and AMPK drugs. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Emerging Role of the Hippo Pathway in Lung Cancers: Clinical Implications.

PubMed

Teoh, Seong Lin; Das, Srijit

2017-11-30

The incidence of lung cancers has increased globally. Increased exposure to tobacco, passive smoking, less consumption of vegetables and fruits and occupational exposure to asbestos, arsenic and chromium are the main risk factors. The pathophysiology of lung cancer is complex and not well understood. Various microRNAs, genes and pathways are associated with lung cancers. The genes involved in lung cancers produce proteins involved in cell growth, differentiation, different cell cycles, apoptosis, immune modulation, tumor spread and progression. The Hippo pathway (also known as the Salvador-Warts-Hippo pathway) is the latest emerging concept in cancers. The Hippo pathway plays an important role in controlling the size of the tissue and organ by virtue of its action on cell proliferation and apoptosis. In the present review, we highlight the mammalian Hippo pathway, role of its core members, its upstream regulators, downstream effectors and the resistance cases in lung cancers. Specific interaction of Mer with cell surface hyaluronan receptor CD44 is vital in cell contact inhibition, thereby activating Hippo pathway. Both transcription co-activators YAP and TAZ (also known as WWTR1, being homologs of Drosophila Yki) are important regulators of proliferation and apoptosis, and serve as major downstream effectors of the Hippo pathway. Mutation of NF2, the upstream regulator of Hippo pathway is linked to the cancers. Targeting YAP and TAZ may be important for future drug delivery and treatment. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Migrations and swimming capabilities of endangered pallid sturgeon (Scaphirhynchus albus) to guide passage designs in the fragmented Yellowstone River

USGS Publications Warehouse

Braaten, P. J.; Elliott, Caroline M.; Rhoten, Jason C.; Fuller, D. B.; McElroy, Brandon J.

2015-01-01

Fragmentation of the Yellowstone River is hypothesized to preclude recruitment of endangered Scaphirhynchus albus (pallid sturgeon) by impeding upstream spawning migrations and access to upstream spawning areas, thereby limiting the length of free-flowing river required for survival of early life stages. Building on this hypothesis, the reach of the Yellowstone River affected by Intake Diversion Dam (IDD) is targeted for modification. Structures including a rock ramp and by-pass channel have been proposed as restoration alternatives to facilitate passage. Limited information on migrations and swimming capabilities of pallid sturgeon is available to guide engineering design specifications for the proposed structures. Migration behavior, pathways (channel routes used during migrations), and swimming capabilities of free-ranging wild adult pallid sturgeon were examined using radiotelemetry, and complemented with hydraulic data obtained along the migration pathways. Migrations of 12–26% of the telemetered pallid sturgeon population persisted to IDD, but upstream passage over the dam was not detected. Observed migration pathways occurred primarily through main channel habitats; however, migrations through side channels up to 3.9 km in length were documented. The majority of pallid sturgeon used depths of 2.2–3.4 m and mean water velocities of 0.89–1.83 m/s while migrating. Results provide inferences on depths, velocities, and habitat heterogeneity of reaches successfully negotiated by pallid sturgeon that may be used to guide designs for structures facilitating passage at IDD. Passage will provide connectivity to potential upstream spawning areas on the Yellowstone River, thereby increasing the likelihood of recruitment for this endangered species.